Sorghum as Dry Land Feedstock for Fuel Ethanol Production

Dec.2010Journal of Northeast Agricultural University(English Edition)vol,17No.483-96Sorghum as Dry Land Feedstock for Fuel Ethanol ProductionWANG Donghai, and WU XiaorongBiological and Agricultural Engineering, Kansas State University, Manhattan, KS 66506, USAPostdoctoral Research Associate, Biological and Agricultural Engineering, Kansas State University, Manhattan, KS 66506, USAAbstract: Dry land crops such as sorghums(grain sorghum, sweet sorghum and forage sorghum) have been identified aspromising feedstocks for fuel ethanol production. The major issue for using the sweet sorghum as feedstock is its stability at roomtemperature. At room temperature, the sweet sorghum juice could lose from 40% to 50% of its fermentable sugars from 7 to 14 daysNo significant sugar content and profile changes were observed in juice stored at refrigerator temperature in two weeks. Ethanolfermentation efficiencies of fresh and frozen juice were high(93%). Concentrated juice(25% sugar)had significantly lowerefficiencies and large amounts of fructose left in finished beer; however, winery yeast strains and novel fermentation techniques maysolve these problems. The ethanol yield from sorghum grain increased as starch content increased. No linear relationship betweenstarch content and fermentation efficiency was found. Key factors affecting the ethanol fermentation efficiency of sorghum includestarches and protein digestibility, amylose-lipid complexes, tannin content, and mash viscosity. Life cycle analysis showed a positivenet energy value (NEV)=25 500 Btw/gal ethanol. Fourier transform infrared(FTIR) spectroscopy and X-ray diffraction(XRD)wereed to determine changes in the structure and chemical composition of sorghum biomasses. Dilute sulfuric acid pretreatment waseffective in removing the hemicellulose from biomasses and exposing the cellulose for enzymatic hydrolysis. Forage sorghum ligninhad a lower syringyl/guaiacyl ratio and its pretreated biomass was easier to hydrolyze. Up to 72% hexose yield and 94%yield were obtained by using a modified steam explosion with 2% sulfuric acid at 140C for 30 min and enzymatic hydrolyKey words: dilute acid pretreatment, dry land feedstock, FTIR, fuel ethanol, sorghum, XRDCLC number: $.2; $514 Document code: A Article ID: 1006-8104(2010)-04-0083-14during 20092. The fuel ethanol can be produced fromIntroductioneither sugar rich feedstocks(sugar cane, sugar beet andsweet sorghum), starch rich feedstocks(cereal grainsAs the world,'s fossil fuel production from the current potatos, cassavas, etc ) or lignocellulosic feedstocksmajor energy sources(coal, crude oil, and natural gas) (agricultural and forest residues, municipal wasteis approaching its peak(-2, people more and more dedicated energy crops, etc. In 2009, approximatelyworry about their future energy supplies. The solution 95% of more than 10 billion gallons of fuel ethanol inis either to develop new types of energy sources or the United States was produced from corn, and%produce substitute fuels from alternative renewable was produced from grain sorghum". The Renewablefeedstocks. The fuel ethanol production is one of these Fuel Standard(RFS)in the Energy Independence andoptions, which experienced a strong growth of 8.1%, Security Act of 2007 specifies that lignocellulosicwhile the world total energy consumption fell off 1. 1% derivereach In hillinn gallons by 2020中国煤化工Received 24 August 2010Supported by National Research Initiative of the USDA Cooperative State Research,CNMHG504-14808)WANG Donghai(1959), male, Ph D, professor, engaged in the research of biofuels and biomaterials. E-mail: dwang(a k-state. eduJournal of Northeast Agricultural University(English Edition)vol17No.42010while the cereal-grain derived ethanol will remain at performance of grain sorghum, which will be15 billion gallons. To keep up with such a challenging used to evaluate the performance of some newlygoal, high-performance crops with excellent agro- developed grain sorghum lines and hybrids for ethanolnomic traits for fuel ethanol production are in great production.need Sorghums(grain sorghum, sweet sorghum and Production of fuel ethanol from lignocellulosicforage sorghum) have been identified as promising biomass using the biological route involves pretreatfeedstocks for bioethanol production because of its ment, enzymatic hydrolysis, and fermentationslow fertilizer requirement, high water efficiency, and Pretreatment is a critical step that breaks up the ligninother favorable agronomic traits)seal, hydrolyzes hemicellulose, and renders celluloseDifferent feedstocks require different approaches in the biomass more accessible to cellulases hydro-or procedures to convert the main components in lysis 2. Ideal pretreatments should be cost-effective,the feedstocks into fuel ethanol. Sweet sorghum can cause no or little carbohydrate degradation or loss, andreadily produce fermentable disaccharide(sucrose) form no or as little inhibitory substances as possibleand monosaccharides(glucose and fructose)in its Despite many other pretreatment techniques available,ice, starch in its grain, and lignocellulose(cellulose dilute acid pretreatment is still considered a relativelyand hemicellulose)in its stalk, which can be used in inexpensive and effective pretreatment method forboth current starch-based ethanol plants and future several types of biomasscellulosic ethanol plants. 20-30 dry thm of biomass,The work described in this paper includes theapproximately 40%-45% are fermentable sugar and investigation of chemical, physical, and microbialstarch. If all fermentable sugar in sweet sorghum is characteristics of sweet sorghumunder differentconverted to ethanol, potential ethanol yield could be preprocesses, storage conditions and performance of600-650 gal/acre. The research has been conducted the juice in ethanol fermentation; the evaluation ofon effects of agricultural practices 5, harvest approa- ethanol fermentation performance of some new grainches6), and juice recovery techniques -)on juice yield. sorghum varieties, which may generate the helpfulPerformance of different yeast strains9-0I and fer- information for sorghum breeders and the ethanolmentation techniques i-l6 were also evaluatedindustry, and may lead to a system approach forEthanol production from sorghum grain normally breeding sorghum cultivars for bioethanol conversionfollows similar dry-grind procedures as corn ethanol and grain distillers feed; the study of changes inproduction, which involves the hydrolysis of starch chemical composition and structures in biomassesinto fermentable sugar (glucose and maltose)through from grain sorghum, forage sorghum, sweet sorghumstarch hydrolyzing enzymes(thermal stable a-amylase photoperiod-sensitive sorghum, and brown midriband amyloglucosidase or raw granule hydrolyzing ( BMR)sorghum after pretreatment by using fouriera-amylase) and fermentation of glucose into ethanol transform infrared spectroscopy(FTIR); and X-rayby using yeast( Saccharomyces cerevisiae). Ethanol diffraction(XRD) techniques to analyze the relation-yield from sorghum grain is generally considered ships among composition, microstructure and fer-comparable to that from corn. That is, the higher the mentable sugar yield.starch content in the grain, the higher the ethanolyield it will generate. However, few researches have Materials and Methodsbeen conducted on sorghum grain regarding factorsimpacting the ethanol fermentation performance of Mate中国煤化工sorghum varieties. A laboratory dry-grind procedure Swehum(m81E)washas been developed to evaluate the ethanol production handCNMHGand pressed afterE-mail: xuebaoenglish neau. edu.cnWANG Donghai et al. Sorghum as Dry Land Feedstock for Fuel Ethanol Production85heads and leaves were removed. Juice samples were diluting the juice in mRs broth and incubating thekept in a refrigerator(4℃) and freezer-20℃)Petrifilm plates in a gas Pack jar with an EZ anaerobemediately after harvest. The bacterial load and pH pouch Plates with colony numbers between 25 andvalues of juice stored in the refrigerator and at room 250 were chosen for colony countingtemperature were monitored for 2 weeks to evaluateEthanol fermentation: the ethanol fermentationthe storage stability of the juice under different tem- procedures for sorghum grains were the laboratoryperatures. Seventy sorghum samples with varying dry-grind procedures described by Wu et allchemical compositions and physical properties were The ethanol concentration in the finished beer wassed in this study. Four types of forage sorghum(stems determined by HPLC after distillation as described byand leaves)provided by Texas A&M University were Wu et ak24). The conversion efficiency was calculatedevaluated. FS-l is a photoperiod sensitive BMR forage from the theoretical yield of 56.72 g of ethanol pro-sorghum(4 Evergreen BMR); FS-2 is a photoperiod duced from 100 g of dry starch(assuming l g of starchsensitive,non-BMR sorghum/sudangrass; FS-3 is a could be hydrolyzed into 1.ll g glucose, and eachBMR forage sorghum classified as a medium-early gram of glucose could generate 0.511 g of ethanol)maturing hybrid; and RS is regular forage sorghum(as For sweet sorghum juice, 100 mL of juice(freshthe control).autoclaved, or concentrated)were weighed into250-mL Erlenmeyer flasks and supplemented with0. 3 g of yeast extract per flask. The inoculation andRVA test: RVA tests were performed with a Model fermentation procedures and conditions were the sameS4A RVA analyzer using Thermocline for Windows as the laboratory dry-grind process. The hydrolyzingver.3.10 software(Newport Scientific, Warriewood, enzymes, Liquozyme(a high-temperature a-amylaseNSw, Australia) using Standard Procedure 1(holding produced by Bacillus licheniformis) and Spirizymeat50℃ for 1 min, heating to95℃at10℃·min1,( a glucoamylase produced by Aspergillus niger),holding at9℃for2min, and cooling to50℃)were provided by Novozymes(Novozymes NorthDSC analysis: DSC analyses of selected sorghum America, Inc, Franklinton, NC). The yeast used forflour samples were conducted on a perkinElmer ethanol fermentation was Ethanol Red from FermentsDiamond dsc by weighing -9 mg of flour into (Lesaffre Yeast Corp, Milwaukee, WI)stainless steel pans on a Perkin Elmer autobalance Pretreatment of biomass: dilute acid pretreatment(Model AD6; Perkin Elmer Life and Analytical was carried out in a Parr pressure reactor(Parr InstruSciences, Shelton, CT, USA). The flour was then ment Company, Moline, IL)with a l-L reaction vesseL.mixed with distilled water to form a slurry with a The ground sorghum biomasses were treated with 2%moisture content of 75%. The temperature program diluted sulfuric acid (w/v)at 10%solid load and 140Cwas holding at 30C for 3 min and then ramped up to for 30 min. Pretreated biomass was washed four timesl80℃at10℃·minwith the hot distilled water and centrifuged to removeBacterial counts: sweet sorghum juice was serial dissolved sugar and sulfuric acid. The supernatantdiluted with sterile water(1: 10 dilution). Bacterial was collected and analyzed for sugar(glucose andloads in juice samples were determined by using a 3M pentoses ) content after being neutralized to pH-6 withPetrifilm aerobic count plate by following the manu- CacO3Enzymatic hydrolysis: pretreated biomass samplesPaul,MN).At the end of the storage period, bacteria were h v凵中国煤化工r(moin the juice stored at room temperature tended to be pH 4CNMH GIde to prevent themostly lactic bacteria, which were enumerated by microbial growth. I he hydrolysis was carried out inhttp://publish.neau.edu.cnJoumal of Northeast Agricultural University(English Edition)VoL.17No.42010125-mL flasks with 50 mL buffer slurry at 5% solidload of pretreated biomasses in a 50C water bath Results and Discussionshaker operating at 140 rmin"for 96 h. The enzymeloading(Accellerase 1000, Genencor Inc, Rochester, Sweet sorghumNY)was I mLg of cellulose. During enzymatic Average dry mass yield for sweet sorghum M8IE inydrolysis, the hydrolysis slurries were sampled and Riley County( KS)was -24 thm". The mass rangesanalyzed periodically up to 96 h. The conversion were from 20 to 26 t- hm". Dry mass yield for the sameefficiency of cellulose was expressed in terms of the sweet sorghum in Doniphan County( Ks)ranged frompercentage of cellulose enzymatically converted to 18 to 32 thm" with an average of 26 thm" .Thereglucose (i.e, enzymatic conversion of cellulose; ECC), was no significant difference in yields at the two testwhich was calculated as described by Varga et alas.XRD tests: forage sorghum samples before and Weitzel et al. reported that stripped stalks gaveafter treatments were analyzed by XRD in a Bruker higher juice yields(58%)than non-stripped stalks didAXS D-8 difractometer. Presence of crystallinity in a (46%-54%)with roller mills. In the present study,sample can be detected by absorption peaks, and cry. all stalks were stripped before pressing. Juice yieldstallinity index(Crl) was used to compare the crystalli- was 57. 4% for Riley samples and 60.9% for Doniphannity among samplessamples. This means that a big portion of fermenFTIR spectroscopy: FtiR measurement was per- table sugar(40%)is still in bagasse. Increasing juiceformed in the original and treated forage sorghums yield or finding ways to make use of residual sugar inusing a Thermo Nicolet Nexus670 FT-IR spectro- bagasse will be of great economical value when sweetphotometer equipped with a Smart Collector Reagent sorghum is used for fuel ethanol productionKBr and samples were dried for 24 h at 50C and Using a screw press could increase the juice yieldthen prepared by mixing 2 mg of sample with 200 mg by -10%. If combined with pith and rind-leafof spectroscopic grade KBr. The analysis was carri- separation, the total sugar yield in juice could reachd out in the wave number range of 400-4 000 cm, 75%, which could generate an extra 400 to 600 L ofwith detector at 4 cm" resolution and 32 scans per ethanol per hectare based on the modest sugar yield(8 000 kg.hm2)and fermentation efficiency(90%).Aimages: images of pretreated surfaces and recent patent application claimed a greater than 95%untreated forage sorghum were examined using a recovery of sugars from sweet sorghum stem usingHitachi S-3500 N scanning electron microscope(SEM) two step emulsifiers and double press operations(Hitachinaka, Ibaraki Pref. Japan). Specimens were If this process is commercialized, ethanol yield permounted on conductive adhesive tape sputter coated acre from sweet sorghum(total of 565 gallons fromwith 4 nm of 60% gold and 40% palladium mixture, a modest sugar yield of 8 000 kg. hm"and grainsand observed using the voltage from 15 to 20 kv.yield of 1 750 kg.hm", approximately 485 gallonsMethods for analyses of crude protein, lipid, and ash from juice and 80 gallons from grain)will be muchwere AOAC 990.03, 920.39 and 942.05, respectively. higher than that of com(464 gallon/acre assuming 160Crude fiber was analyzed by the filter bag technique bushels/acre and 2.9 gallons per bushel), which willbyusingAnkoMA200(http://www.ankom.com/makesweetsorghumamoreattractiveenergycropmedia/documents/Crude Fiber 1 108 A200. pdf)Fermentable sugar in juice was mainly sucroseChemical compositions of untreated and pretreated gluc中国煤化工biomasses were determined by following NREL labora- juiceCNMHGtalks ranged fromtory analytical procedures[27.13.77% to 15. 89%(average 15. 14%+0.94%); thoseE-mail:xuebaoenglish@neau.edu.cnWANG Donghai et al. Sorghum as Dry Land Feedstock for Fuel Ethanol Productionfrom Doniphan county sorghum stalks ranged from During the room temperature (25C) storage,14. 44%to 16.87%(average 15.57%1.02%) there the sugar content and profile of the juice changedwas no significant difference in fermentable sugar dramatically. Approximately 40% of the original sugarcontent between the two locations. Sugar content and disappeared after one week and up to 50% was lostprofile in sweet sorghum juice can be very different after 2 weeks(Fig. 1, left). These results demonstratedamong varieties. Fortunately the juice of the that sweet sorghum juice cannot be stored at the roomsorghum variety used(M81E) in this study had the temperature. When stored in a refrigerator, sugar lossconsistent high sugar content and contained about 70% was much slower, and the amount of loss was notsucrose, 20% glucose, and 10% fructosesignificant even after 14 days(Fig. 1, right)120000120000Day 15600003000010Time(min)Fig1 HPLC chromatograms(Rezex RCM column, 0.60 mLmin'deionized water, 80'C oven, RI detector)of sugar profilesin sweet sorghum juice during storage at room(left)and refrigerator (right) temperatureOriginally, there was essentially no acetic acid and gerated temperature, no significant change in theonly trace amounts of lactic acid and formic acid in organic acid profile was observed during the 2-weekthe juice(Fig. 2A& B). After 3-5 days of the room storage period( Fig 2B)temperature storage, the amounts of lactic acid, acetic The original pH values of juice were around 4.7.acid, and ethanol(Fig. 2A)were noticeable, while that It decreased to around 3. 8 after one week at the roomof formic acid remained the same. Obviously, this was temperature and remained at 3. 8 during the secothe metabolic result of heterofermentative lactic acid week. pH values of refrigerated juice were essentiallybacteria. Formic acid contents remained essentially unchanged during the 2-week storage periodthe same during the 2-week storing period, and the Bacterial counts in juice(original 10 cfurmL )storedamounts of acetic acid and ethanol showed a slight at the room temperature increased from 30- to 300-increase. However, concentrations of lactic acid in- fold (to 3x10 cfumL during the first week, thencreased dramatically in the second week(Fig. 2A). declined from 20-to 200-fold of original levels(3x10This suggested that the activity of heterofermentative cfurmL ) by the end of the 2nd week. Judging by thelactic acid bacteria almost stopped. Instead, the viscous appearance(extracellular polysaccharides),homolactic acid bacteria took over during the second large amount of gas bubbles, and profiles of detectedweek of room temperature storage. The difference in chemicals(ethanol, lactic acid, and acetic acid)(Figs. 1metabolic products of heterofermentative and homo- and 2中国煤化工cid bacteria werefermentative lactic acid bacteria told the story52-33.actitemperature sto-Bacterial count results supported that under refriYHsCNMHGa were dominantpublish neau. edu.cnJournal of Northeast Agricultural University(English Edition)Vol, 17 No 4 2010(95% of the pop-ulation) only after the first week. the end of the 2-week storage period. Activities ofChromatograms in Fig. 2 confirmed such bacterial bacteria in the refrigerated juice did not cause muchactivities. Bacterial counts in the refrigerated juice change in the sugar and organic acid profiles(Fig. Iincreased to about 5-to 10-fold of original counts by right and Fig 2B)A90060000000000000Day 13Time(minTime(min)Fig 2 HPLC chromatograms( Rezex ROA column, 0.60 mL.min"5 mmolL' sulfuric acid, 65 oven, RI detector)showingccumulation of organic acids in juice during storage at()room temperature and (B)refrigerator temperatureFermentation efficiencies of fresh, frozen, and original sugar)remained in the finished beers fromconcentrated(with 20%, 25%, and 30% sugar, w/v) concentrated juice with 25% and 30% sugar(Fig 3).juice was tested. The fermentation efficiency of Residual sugar in the finished beer from concentratedfrozen juice(94.6%1. 1% for Riley county juice and juice with high sugar contents were similar to those94.3%2.7% for Doniphan county juice)was a little (1.8%-8.5%, w/v)reported by Laopaiboon et al.inhigher than the that of the autoclaved juice(93.8% high gravity sweet sorghum juice fermentation. This0.8% Riley county juice and 91.6%1 1% for indicated that normal yeast used for ethanol productionDoniphan county juice). Fermentation efficiencies (brewing and distillers yeast might not be able toof concentrated juice decreased as the sugar concen- convert all the fermentable sugar in concentrated sweettration in the juice increased(93. 3%*3.0%, 86.4%* sorghum juice into ethanol. A major portion of the3.9%, and 72.4%*7.5% for 20%, 25%, and 30% residual sugars in finished beer was fructose; there wassugar Riley juice; 93.8%# 1.9%0, 89.4%*3.1%, and essentially no sucrose and glucose in finished beer(Fig 3)77.0%+4.4%for 20%, 25%, and 30% sugar Doniphan This indicated that, among the three fermentable sugarjuice)and were significantly lower than those of the in the concentrated sweet sorghum juice, sucrosefrozen or autoclaved juice except the juice with 20% and glucose were consumed first by the yeast, but asugar content. The lower fermentation efficiencies considerable amount of fructose(1.0%0-5.1%, w/vfrom high sugar concentrated juice could be due to was still in the finished beers from concentrated juicethe stress of initially high sugar and later high ethanol (25%and 30%sugar). Most brewery yeasts, strainsconcentrations on yeast.of S. cerevisiae, utilize sugar in the order of sucrose,There were essentially no or very little fermentable glucose, fructose, maltose, and matotrioses5.Becausesugar left in the finished beer of normal sweet sorghum of S. cerevisiae's preference in sugar utilizationice(fresh, frozen, or autoclaved )and concentrated sucrose and glucose are consumed and convertedjuice with 20% sugar(0. 22%-0.35%, w/v). Significant befo中国煤化工 tures like sweetamounts of residual sugar(1.0%-1.7% in the 25% sorglCNMH Fermentation 6).Ifjuice and 4. 1%-5.1% in the 30% juice; -4%-17% of the concentrations of sucrose and glucose are not tooE-mail:xuebaoenglish@neau.edu.cnWANG Donghai et al. Sorghum as Dry Land Feedstock for Fuel Ethanol Production89high, as those presented in the original sweet sorghum contains approximately equal amounts of glucose andce(15% sugar) and the less concentrated juice fructose, but the residual fructose concentrations in(<25% sugar, w/v), the yeast, although under stress the finished wine are very low(0.15% to 0. 7%)of moderate ethanol concentration, still can manage With over 25% sugar, normal brewery yeast willto convert the fructose in the broth into ethanol after always leave significant amounts of residual sugar inall the sucrose and glucose have been utilized. The finished beer s1. Some ethanol, osmo-tolerant yeastfinal fermentation efficiencies are reasonably high strains could efficiently ferment high sucrose and(86%93%) However, when concentrated juice has> fructoseImmobilized yeast cells had shown25%sugar, ethanol concentrations in the fermentation much improved ethanol tolerance and fermentedbroth will be high enough after sucrose and glucose high gravity mashes(300-320 g sugars.L) with higare consumed to completely repress the activity of efficiencyyeast to further ferment fructose into ethanol. whensucrose is utilized by yeast, it is hydrolyzed into Grain sorghum resultsglucose and fructose by invertase. Fructose will stay In the normal ethanol dry-grind process, starches inin the broth as long as there is still glucose. Therefore, the feedstock are hydrolyzed by starch hydrolyzingthe residual fructose concentration in finished beer enzymes into glucose, which is then converted intocould be higher than the initial fructose concentration ethanol by yeast. Therefore, the higher the starchin the concentrated juicecontent in the grains, the higher the expected ethanolyield. Starch content in 70 sorghum grain samples150000ranged from 64% to 74%(db). Ethanol yield fromthese sorghum grains should be proportional to their120000starch contents. Even though the ethanol yields90000showed a good linear relationship with starch contentsthe overall results revealed that starches in different60000sorghum varieties did not contribute equally in the30000ethanol production. Variations in ethanol yields couldbe as large as 7. 4% among sorghum varieties with10similar starch contents( Fig. 4)Fig, 3 Profile of residual sugar in finished beer from con-150y=02247x20311centrated juice with 20%, 25%, and 30% sugar contents145R08197(HPLC conditions: Rezex RCM column, 0.60 mL. mindeionized water, 80C oven, Ri detector)**Using yeast strains with enhanced fructose metabo-lism capacity or ethanol tolerance, or employing,,120novel fermentation processes that alleviate the re64666870pression effects of high ethanol and sugar conStarch content (%)Fig 4 Relationship between ethanol yield and starch con-centrations may help to solve the residual fructoseproblem. The winemaking yeast strains, especiallythose used for making dry wine, are more effectiveH中国煤化工 sorghum varietiesin turning fructose into ethanol than most baker's haveNMHGT non-waxy tralyeasts or brewery yeast 7]. The grape juice usuallyvarietese stren level. These differencestp: //publish neau.edu.cnJoumal of Northeast Agricultural University(English Edition)vol.17No.42010can be explained by the adverse effects of amylose Tannin sorghum mashes were much more viscous thanon the pasting and gelatinization of"regular"starch mashes of other sorghums and stayed viscous during(a mix of amylose and amylopectin types). The poor the first 24 h of fermentation. Generally, the higherpasted and gelatinized starches subsequently restrict the tannin content is, the thicker the finished mash ishydrolytic enzymes to access the starch molecules, the slower the fermentation proceeds, and the lowerresulting in incomplete hydrolysis of starch to glucose. the fermentation efficiency is. Viscous mashes notBefore enzymatic hydrolysis, DSC thermogram of only lead to slow and incomplete the hydrolysis ofnormal sorghum(25%amylose)showed a prominent starch, but also cause other processing problems suchamylose-lipid complex peak at 90-105Cl42. After as a higher energy consumption for transporting andenzymatic hydrolysis, the amylose-lipid complexes mixing, lower heat exchange efficiency, and morepeaks enlarged and shifted from 105C to 120C. difficult cleaning in ethanol plants. Fig. 5 clearlyThis result indicated that additional amylose-lipid shows the performance of waxy, normal and highcomplexes formed during the mashing process, which tannin sorghum in dry-grind ethanol process.rendered hydrolyzing enzymes difficult to act on theseamyloses and converted very slowly into glucose.onsequently, sorghums with higher amylose content850showed a slower starch hydrolyzing rate and lowerthanol yields, which further confirmed that low士waxy◆ Normal75amylose grains(waxy and heterowaxy) were preferredTAnninvarieties for ethanol productionThe protein content in cereal grains are usuallynegatively proportional to their starch content,Fig. 5 Fermentation features of wary, normal, and tannintherefore, ethanol yields decreased as protein content sorghumsincreased. However, at the same protein level, theethanol fermentation efficiency varied as much as Particle size of the ground sorghum meal also plays8%, indicating factors other than protein content an important role in the starch to ethanol conversionimpacting ethanol yield. Nine sorghum genotypes process. The fermentation efficiencies and ethanol(hybrids or breeding lines) covering a broad range of yields of the finely-ground samples were approximatelyethanol fermentation efficiencies were selected, and 5% higher than coarsely-ground samples. The resultsused to study the effect of protein quality on ethanol indicated that gelatinization temperature of largerfermentation efficiency. The results showed that particles was 5-10C higher than those of the smallerthere was a strong linear relationship between protein particles. This definitely affected the starch pasting,digestibility and fermentation efficiency (R2=0.91). It gelatinization, and hydrolysis process resulting inis possible that the sorghum samples with high protein lower fermentation efficiency for the larger particlesdigestibility provided more free-amino acid for yeastThe sale of DG accounts for 15%-20% of thegrowth during the fermentationannual revenue of an ordinary dry-grind ethanol plantBecause of tannins'abilities to interact with pro- The quality of DG will directly affect its feeding andteins (including hydrolytic enzymes), tannins have market values. Most of DG from dry-grind ethanolbeen recognized as having adverse effects on starch plants is used as animal feed for ruminants such asdigestion. The results confirmed tannins' negative dairy中国煤化工ontent is the mosteffects on the action of amylase. Tannin sorghums impCNMHGliquefied much slower than other sorghum samples. plantaumann: MaUw,n contents of theirE-mail:xuebaoenglish@neau.edu.cnWANG Donghai et al. Sorghum as Dry Land Feedstock for Fuel Ethanol Productionfeedstock and dG to guarantee it meets customers' (amide Ii)". These bands were well defined inrequirements. Another important issue related with untreated biomasses, especially in FS-3, which hadDG is mycotoxins. FAO has estimated -25% of the the highest protein content(7.46%). The protein bandsworlds grain supply is contaminated by various kinds disappeared after treatment, suggesting proteins wereof mycotoxins. These toxins can negatively impact the removed during the pretreatmentperformance of the fermentation yeast in the ethanolconversion process, and can also be concentrated ins+++DG(up to 3-fold). FDA has set limits for some of thecommon mycotoxins such as aflatoxins, vomitoxin,fumonisins, and zearalenone in animal feeds/7.Theincidence and contamination level of mycotoxinproducing fungi and mycotoxins are generally lower insorghum than in corns.Time(h)Sorghum biomass resultsFig. 6 Hexose yields from dilute acid pretreated sorghumbiomass during enzymatic hydrolysis at 45C and pH 4.8The pretreatment of sorghum biomasses with 2% (enzyme loads per gram of cellulose: cellulase 15 FPU arsulfuric acid at 140C for 30 min was very effective B-glucosidase 50 CBU)in removing hemicellulose and exposing cellulosefor enzyme hydrolysis. The pentose yield ranged There are two types of aromatic components(guaifrom 79.3% for FS-2, 83.9% for RS, 86.6% for FS-l, cyl and syringyl rings) in forage sorghum ligninsto 93% for FS-3. The high amount of pentose yield These rings have bands at 1 510 cm" showing aro-during pretreatment means the high efficiency in matic skeletal vibrations of the benzene ring 43. 451removing hemicellulose from treated biomasses. The and sometimes shifted toward a higher wave numberresults also indicated that dilute acid pretreatment(1 510 cm")in softwoods. All the original untreatedmore effective with FS-3 and FS-I than with RSbiomass samples showed guaiacyl ring-related bandsFS-2. Results of enzymatic hydrolysis showed glucose at 1 516-1 517 cm, which were more distinct in theyields of 43% for FS-2 and 79% for FS-3 after 72 h pretreated samples. That agrees with what pretreathydrolysis at4℃(Fig6ment supposes to achieve: removing as much hemiFTIR spectra(fingerprint region) of the forage celluloses, pectins, proteins, and other extractivessorghum samples before and after dilute acid pretrea- possible. The result is as below: cellulose and lignintments are shown in Fig. 7. All the spectra of untreated contents in the remaining solid increased significantlyoriginal biomasses showed a distinct hemicelluloseCellulose-related Ftir bands are around 1 430band at 1 732 cm( Fig. 7A). This peak is related to 1 370, 1 162, 1 098 and 900 cm" 43,45). The absorbancesaturated alkyl esters from hemicellulosel4-4, which at 900 cm is associated with the anti- symmetric outwas not discernible in the spectra of the pretreated of-phase ring stretch of amorphous cellulose andbiomasses(Fig. 7B). This indicated that hemicellulose the 1 098 cm" band is related to the C-o vibrationwas effectively removed during dilute sulfuric acid of crystalline cellulose 4647. For reasons similar topretreatment. Similar changes happened to the pectins the lignin situation, the intensity of both crystallinend some phenolics in the 1 245 cm region'098-1 109 cm" )and amorphous(897-900 cm)again confirmed solubilization of phenolicsfV凵中国煤化工 treatment. C-Hremoval of esters from the treated biomasses proteins deforCNMHis indicated inare seen at about 1 653 cm '(amide I)and 1 549 cm bands at 13/2 cII nis peaK appeared aroundhttppublishneau.edu.cnJournal of Northeast Agricultural University( English Edition)Vol 17 No 4 201 370-1 375 cm" in all untreated samples with enzymatic hydrolysis were totally different from onea weak signal in FS-1. After treatment, the band anotherresidues(except RF-2)showed distinctdecreased in intensity and shifted to 1 366 cm". The lignin1 514 and 1 452 cm") together withmainly antisymmetric stretching C-0-C glycoside in cellulose bands ranging from barely discernable tocellulose was around the 1 162 cm"region, 47. The obvious(1 430, 1 367, 1 162, and 1").Theantisymmetric C-O-C vibration bands around 1 159. only thing that was contradictory to the hydrolysis1 162 cm 'and C-o stretching peaks around 1 058 results were shown in Fig. 6. Residues from the leastand 1 035 cm were all well defined in all treated digestible sample(FS-2)showed neither lignin bandssamples4). That confirmed that cellulose was fully nor any cellulose bands, but residues from the mostexposed for further enzymatic hydrolysis after pre- easily digested sample(FS-3)had the most distincttreatment. The FTiR spectra of the solid residues after and intense lignin and cellulose bands0000.030002000151800Wave numbers(cm")0.200.10Wave numbers(cm)Fig. 7 FTIR spectra of original(A)and dilute sulfuric acid pretreated (B) sorghum biomasses in the fingerprint regionFig 8 shows XRD diffraction patterns of untreat- FS-1 predominated, probably because of the presenceed, pretreated, and enzymatic hydrolyzing residue of high amounts of amorphous cellulose and othersamples. The intensity ratio of crystalline to amor- amorphous components (hemicellulose, pectinphous diffractions is approximately equal to the mass lignin中国煤化工Fs2 and rs. theratio of crystalline to amorphous parts of a polymerC MH Gms that untreatedThe amorphous XRD patterns in untreated FS-3 and FS-2 and rs had the high crystalline cellulose content,E-mail: xuebaoeau. edu. cnWANG Donghai er al. Sorghum as Dry Land Feedstock for Fuel Ethanol Productionwhich might contribute to its difficulty being converted to the results derived from FTIR spectra of FS-2 andinto amorphous cellulose during pretreatment and RS. Less intense crystalline peaks in FS-3 and FS-1being hydrolyzed into glucose by cellulases(Fig. 6). suggests that pretreatment was effective in convertingAfter pretreatment, the main peak relative to plane some crystalline cellulose to amorphous cellulose,002 is easily observed in all treated samples, showing which is more easily hydrolyzed by cellulases duringthat the amount of cellulose increased because of enzymatic hydrolysis. XRD patterns of samples afterthe removal of lignin and hemicellulose. Crystalline enzymatic hydrolysis showed that the cellulose contentpeaks in RS and FS-2 were more intense than other decreased. Crystallinity index( Crf)may also be usedtwo samples, suggesting that celluloses in RS and to describe the crystalline and amorphous state of aFS-2 were in a more organized crystalline state even sample/26. Crl data in Fig 9 agrees with both FTIRafter pretreatment, which provides additional proof spectra and XRD pattems shown in Fig 8.450505025和505052-Theta-scale2-Theta-scale352-Theta-scaleFig 8 X-ray diffraction patterns of sorghum biomass samplesa)FS-2; b)RS; c)FS-1; d)FS-3. The labeled peaks are the principal 002 peak(100% intensity)and 101 peak of native celluloseg UntreatedSEM images revealed significant difference insurface appearance and intemal structures among theE Hydrolyzedtreated samples seemed to have deposits on the surface( Fig. 10 Al, B1). This surfacelayer can include waxes, hemicellulose, lignin, andother binding materials". As the surface layer wasremoved during treatment, internal structure andFS-Ifibers中国煤化工oth surfacFig9 Crystallinity index of untreated, pretreated and expoCNMHGhydrolyzed sorghum biomassesa hydrolysis, FTIRhttp://publish.neau.edu.cnJourmal of Northeast Agricultural University(English Edition)vol.17No.42010spectra, and XRD pattern data and a previous report particles existed as aggregates of crystalline celluloseby reddy et al. I4, in which showed that cellulose entities27Mar-07SE 27. Mar-07B2Fig 10 SEM images of untreated(Al and Bl)and dilute acid pretreated (A2 and B2)sorghum biomassesresidual fructose will be expected in finished beer.ConclusionGrain sorghum can be used for fuel ethanol production just like corn using the drygrind process. HoOur studies showed that sorghums(sweet sorghum, ever, not all the starches in the sorghum grain makegrain sorghum, and forage sorghum)as dry land crops equal contribution to the ethanol yield. Starch andcan achieve high dry mass yield and can contribute protein quality, amylose lipid complexation, andtannin content are major factors impacting ethanoljuice is an excellent feedstock for ethanol production. fermentation efficiency and ethanol yield. Life cycleFermentation efficiency can easily reach 93%. The analysis indicated that dry-grind sorghum ethanolmajor issue for using sweet sorghum as feedstock had a positive net energy value of 25 500 BTU/galis the storage stability of juice. A couple of day s This study on sorghum biomass showed that diluteexposure at the room temperature could completely sulfuric acid pretreatment ( 140C for 30 min)wasruin its commercial value due to a significant loss of effective in removing most of hemicellulose, pectin,its fermentable sugar. No significant sugar content and proteins from sorghum biomasses. Pentose yieldsand profiles changes were observed in juice stored from the pretreatment ranged from 80% to 94%at refrigerator temperature in two weeks. New fer- FTIR spectra of the pretreated sorghum biomassesmentation yeast or novel fermentation techniques may indicated that the treated biomass had higher celluloseneed for high gravity ethanol fermentation using con- and中国煤化工 ntreated ones ancentrated juice(25% sugar), otherwise, significantly the crd data revealedCNMHGlow fermentation efficiencies and large amounts of crystovium waples varied, whichE-mail:xuebaoenglish@neau.edu.cnWANG Donghai et al. Sorghum as Dry Land Feedstock for Fuel Ethanol Productionmight be used to predict the easiness of a sample inand juice expression systems for sweet sorghum [] TRANSpretreatment and hydrolysis. Sorghum samples withASAE,1985,28:268-274.lower crystallinity(FS-3 and FS-1)gave higher hexose 12 Gibbons W R, Westby C A, Dobbs T L Intermediate-scale, semi-elds than those with higher crystallinity(FS-2 andcontinuous solid-phase fermentation process for production of fuelRS) during enzymatic hydrolysis with cellulasesweet sorghum [] Appl Environ Microbiol, 1986,Overall, infor-mation from our study could be used to51(1):115-122assist the development of new or improved sorghum 13 Kundiyana D, Bellmer D, Huhnke R, et al. 2006."Sorganolhybrids for ethanol production and to encourage theproductionofethanolfromsweetsorghum.(2010-08-05).http://sorghum production across the drier sorghum-growingasae.frymulti. com/azdez. asp?JID-5&AID-21449& CId-por2006&states and revive rural economy.T=214 Laopaiboon L, Thanonkeo P, Jaisil P, ef al. Ethanol productionReferencesfrom sweet sorghum juice in batch and fed-batch fermentation byI Kharecha P A, Hansen J E. 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