微藻氮源對紅薯原料燃料乙醇發(fā)酵過(guò)程促進(jìn)效果的研究

第34卷第9期太陽(yáng)能學(xué)報Vol 34. No 92013年9月ACTA ENERGIAE SOLARIS SINICA2013文章編號:02540096(2013)09154205微藻氮源對紅薯原料燃料乙醇發(fā)酵過(guò)程促進(jìn)效果的研究申渝3,張海東2,郭勁松3,陳猷鵬3,鄭旭煦2,張賢明(L.廢油資源化技術(shù)與裝備教育部工程研究中心,重慶工商大學(xué),重慶400072.催化與功能有機分子重慶市重點(diǎn)實(shí)驗室,重慶工商大學(xué),重慶400067;3.重慶綠色智能技術(shù)研究院,中國科學(xué)院,重慶4012)摘要:紅薯干粉碎后經(jīng)雙酶法處理得到葡萄糖含量為210.0g/L的醪液,利用工業(yè)酸酒酵母 Saccharomycescerevise ATCC6508批式發(fā)酵產(chǎn)乙醇。野生水華微藻生物質(zhì)收集后,經(jīng)洗滌、稀鹽酸水解、干燥、粉碎制得微藻氮源( Algal Nitrogen Source,ANs)。實(shí)驗發(fā)現在未添加任何氮源條件下發(fā)酵120h時(shí)乙醇濃度為8914g/L,殘余葡萄糖為7.0g/L,發(fā)酵葡萄糖的乙醇得率為0.4;添加ANS對發(fā)酵過(guò)程的促進(jìn)效果顯著(zhù),當ANS添加量分別為5.010.0和20.0g/L時(shí),發(fā)酵終點(diǎn)時(shí)間(殘余葡萄糖<1.0g/L)分別為96、72和72h,終點(diǎn)乙醇濃度分別為96.198.1和103.0g/L,乙醇得率分別為0.460.47和0.49。蛋白質(zhì)和氨基酸含量分析表明,ANS和酵母粉蛋白質(zhì)含量為32.6%和64.5%(干重),游離氨基酸含量為197%和29.9%(干重),高含量的有機氮源特別是游離氨基酸,對發(fā)酵效率和乙醇得率的提高有重要作用。對比實(shí)驗發(fā)現;添加10.0g/LANS添加3.0g/L酵母粉的發(fā)酵參數相當,因此ANS可替代酵母粉作為紅薯原料乙醇發(fā)酵的氮源添加劑。關(guān)鍵詞:紅薯原料;微藻氮源;燃料乙醇發(fā)酵;發(fā)酵效率;乙醇得率中圖分類(lèi)號:TK6文獻標識碼:A0引言酵母細胞生長(cháng)和代謝的必要營(yíng)養成分,氮源不足會(huì )造成發(fā)酵速率偏低,甚至發(fā)酵停滯,降低乙醇得率和以淀粉為原料生產(chǎn)燃料乙醇工藝成熟、規?；O備生產(chǎn)強度。研究報道表明,添加銨鹽、尿素、氨推廣風(fēng)險低,在一段時(shí)間內仍是燃料乙醇產(chǎn)業(yè)的重基酸、酵母粉等不同種類(lèi)氮源,均可提高酵母細胞對要發(fā)展方向。特別是紅薯、木薯為代表的根莖淀粉乙醇的耐受能力,提高發(fā)酵效率和乙醇得率,特別是作物,可適應山地等貧瘠土壤單位種植面積淀粉產(chǎn)氨基酸和酵母粉等有機氮源,效果尤其顯著(zhù)9-1)量大,可替代傳統玉米等糧食作為燃料乙醇的生產(chǎn)但是有機氮源成本較高乙醇工業(yè)發(fā)酵過(guò)程通常添原料1-4。紅薯在我國大部分地區具有廣泛的種植加氨水或銨鹽作為氮源,盡管如此氮源成本仍是乙基礎由于在原料性質(zhì)和成分上和糧食原料存在較醇生產(chǎn)成本中不可忽略的一部分。大區別需在原料預處理、發(fā)酵過(guò)程和工藝優(yōu)化選富營(yíng)養化水體中以藍藻為代表的微藻大量快速擇微生物菌株適應性等方向作系統的研究工作,并生長(cháng)造成水華現象進(jìn)一步惡化水體質(zhì)量,是當前水以此為基礎開(kāi)發(fā)高效原料預處理和發(fā)酵生產(chǎn)過(guò)程,污染治理中的重要工作之一B,H。盡管采取沉降提升工業(yè)過(guò)程的經(jīng)濟競爭力-7的方式治理可取得短期效果,但由于多數種類(lèi)藍藻紅薯的淀粉含量一般為干重的40%~60%,最具有固氮功能,每爆發(fā)一次水華就會(huì )進(jìn)一步提高水高可達70%,生長(cháng)周期較短,是良好的淀粉作物;但體中氮元素的濃度,周而復始;因此唯有采取集中打紅薯中蛋白質(zhì)含量較低,一般占干重的2%,若作為撈的方法將微藻生物質(zhì)富集的氮元素逐步從水體中燃料乙醇發(fā)酵原料必須額外添加氮源。氮源是移除,才是治理水華的根本。集中打撈的大量水華收稿日期:20110728中國煤化工基金項目:國家重大科技專(zhuān)項(2009ZX07104001);重慶市教委自然科學(xué)基金(KJ307CNMHG市科委自然科學(xué)基金(CSTC2012jB0138)通訊作者:申渝(1981-),男,博士、副教授,主要從事生物質(zhì)燃料、生化反應工程方面的研究。shenyu@ctbu.edu.cn期申渝等:微藻氮源對紅薯原料燃料乙醇發(fā)酵過(guò)程促進(jìn)效果的研究1543微藻生物質(zhì)中蛋白質(zhì)含量可達干重的20%~35%,比。然后,用1.0mo/L的HCl將初始pH調節至可作為有效的蛋白質(zhì)資源開(kāi)發(fā)利用,同時(shí)可避免處5.0,15℃滅菌20min,冷卻至室溫,按5%的接種量理不當而對環(huán)境造成二次污染。接入液體種子,于30℃恒溫搖床發(fā)酵,轉速為本文將水華微藻生物質(zhì)加工處理后作為紅薯原150r/min。各樣品做兩次重復樣料乙醇發(fā)酵過(guò)程的外加氮源,并考查其對發(fā)酵速率,1.5分析方法乙醇得率以及對發(fā)酵過(guò)程的影響,為水華微藻生物紅薯粉、微藻生物質(zhì)和酵母粉中總蛋白含量采質(zhì)的資源化開(kāi)發(fā)做出嘗試用微量凱氏定氮儀分析。還原糖含量采用DNs1材料與方法法分析。原料中淀粉含量按照文獻[18】所述方法處理之后,通過(guò)測定葡萄糖含量折算成淀粉含量。11菌種和培養方法按無(wú)菌操作流程從每個(gè)三角瓶取出1m醪液,普通工業(yè)釀酒酵母 Saccharomyces cerevisiae去離子水稀釋后測定葡萄糖、乙醇和細胞濃度。葡ATC6508由大連理工大學(xué)生命科學(xué)與技術(shù)學(xué)院白萄糖和乙醇濃度用SBA40Cc生物傳感分析儀測定鳳武教授課題組贈送,菌種培養保存和種子培養方(山東省科學(xué)院生物研究所)。細胞濃度采用顯微法參考文獻[15]。計數確定1.2微藻氮源制備游離氨基酸和總氨基酸含量分析參考文獻[19]野生水華微藻生物質(zhì)收集后,用自來(lái)水洗滌所述流程。稱(chēng)取約0.20g樣品,懸浮于1mL去離子2~3遍,過(guò)濾后置于85℃烘箱內烘干,粉碎,裝入塑水中,沸水浴中溶解100min,然后離心收集上清料袋置于4℃冰箱保存。稱(chēng)取約200g藻粉,放入(1000o/min,5min)用于測定游離氨基酸。氨基酸1000mL不銹鋼容器內,同時(shí)加入300mL0.1moL濃度采用氨基酸分析儀(日立L8900)。HCl溶液,置于沸水浴中加熱攪拌水解2h;用2結果與討論碎后制得微藻氮源( Algal Nitrogen Source,ANs)裝2.1原料主要成分分析入塑料袋置于4℃冰箱保存備用實(shí)驗中所用的主要原料中蛋白質(zhì)還原糖和淀1.3紅薯糖液制備粉含量如表1所示。紅薯粉中還原糖含量約占干重市售鮮紅薯洗凈切片后烘干,干紅薯片粉碎后的758%,其中淀粉含量約為67.5%,占總還原糖過(guò)20目篩制得紅薯粉。8000mL鋼制容器中將紅薯的890%,蛋白質(zhì)含量較低,約為干重的25%。微粉和自來(lái)水按1:2.5的比例混合,攪拌加熱至85℃藻生物質(zhì)中蛋白質(zhì)和還原糖的含量相當,分別為干保溫,用1.0mo/LHCl調節pH值至6.0,同時(shí)按重的326%和383%。酵母粉中蛋白質(zhì)含量約占20g/kg(酶粉/紅薯粉)加入淀粉酶(北京東華生物干重的64.5%。從分析原料的成分可知野生水華酶制劑有限公司酶活力為4000U1g)攪拌并保溫液微藻生物質(zhì)蛋白含量極高,約為酵母粉的50%,是化2h后將溫度降至55℃,按4.0g/kg(酶粉/紅薯良好的蛋白質(zhì)原料。粉)的比例加入糖化酶(北京東華生物酶制劑有限表1紅薯粉、水華微藻生物質(zhì)和酵母粉中主要成分公司,酶活力為50000g),攪拌保溫4h。冷卻至Table 1 citions of sweet potato powder, bloom室溫后用帆布過(guò)濾收集糖液,置于-20℃冰箱儲存algae biomass and yeast extract powder備用。含量/%干重紅薯粉水華微藻生物質(zhì)酵母粉1.4乙醇發(fā)酵蛋白質(zhì)2.5±0.2326±2864.5±2.1紅薯糖液室溫解凍后,用自來(lái)水將葡萄糖濃度還原糖758±2338.3±1.62.4±0.1稀釋到210.0g/L。250mL三角瓶5個(gè),分別裝入淀粉150mL紅薯糖液,其中3個(gè)分別添加5.0、10.0和中國煤化工20.0g/L微藻氮源1個(gè)添加3.0g/L酵母粉(北京22乙醇發(fā)CNMHG奧博星生物技術(shù)有限公司),1個(gè)不添加氮源作為對在實(shí)驗條件完全相同的前提下,添加不同劑量1544太陽(yáng)能學(xué)報34卷的微藻氮源、3.0gL酵母粉和未添加氮源的5組乙條件下生產(chǎn)強度的最高值為1.62g/(Lh),而添加醇發(fā)酵實(shí)驗中乙醇和葡萄糖濃度變化情況如圖1所20.0gL微藻氮源(ANS)后最高生產(chǎn)強度可達示。實(shí)驗表明不添加任何氮源條件下,在120h時(shí)未2.50g/(L·h),提高了54%。通過(guò)顯微計數分析每達到發(fā)酵終點(diǎn),殘余葡萄糖濃度為7.0g/L;而添加發(fā)酵樣品最后的細胞濃度表明,未添加氮源的樣品微藻氮源和酵母粉對發(fā)酵過(guò)程的促進(jìn)作用非常顯著(zhù),終點(diǎn)細胞濃度為1.65×10個(gè)/m,而添加3.0g/L添加5.0g/L微藻氮源后發(fā)酵時(shí)間縮短到96h,終點(diǎn)乙酵母粉以及分別添加5.0、10.0和20.0g/L微藻氮醇濃度為96.1g/L,而添加10.0和20.0g/L微藻氮源源的樣品終點(diǎn)細胞濃度分別為1.95×103、1.76以及3.0g/L酵母粉之后發(fā)酵時(shí)間均縮短到72h,終點(diǎn)10°2.01×103和2.35×108個(gè)/mL。由于乙醇屬于乙醇濃度分別為981、103.0和97.6g/L。初級代謝產(chǎn)物,其生成速率和細胞生長(cháng)直接關(guān)聯(lián),因此,外加氮源提高了酵母細胞的生長(cháng)速率是導致發(fā)酵速率和生產(chǎn)強度提升的直接原因之一。802.3微藻氮源和酵母粉氨基酸分析紅100文獻報道中普遍認為相對于銨鹽、硝酸鹽等無(wú)機氮源以蛋白質(zhì)和游離氨基酸為代表的有機氮源對酵母細胞乙醇發(fā)酵過(guò)程的促進(jìn)作用更加明豐富的有機氮源不但可為細胞的生長(cháng)代0謝提供必要的營(yíng)養元素,同時(shí)還可提升酵母細胞對發(fā)酵時(shí)間/h不添加氮源→5 g/L AHBAP亠10 g/L AHBAP乙醇的耐受能力,進(jìn)而在乙醇抑制的條件下提升細胞的發(fā)酵速率。實(shí)驗分析了微藻氮源(ANS)和圖1葡萄糖含量為20.0/L的紅薯醒液乙醇發(fā)酵添加酵母粉(YE)的游離氨基酸含量分析結果表明,微微藻氮源、酵母粉和不添加氮源對發(fā)酵過(guò)程的影響藻氮源中游離氨基酸含量為19.7%(干重),酵母粉Fig1 Eifect of algal nitrogen source adding on parameters of中游離氨基酸含量為29.9%(干重)。以游離氨基residue glucose and ethanol concentrations in fuel ethanol酸作為氮源,可直接為細胞生長(cháng)提供氨基酸成分,減fermentation using sweet potato medium少胞內氨基酸合成,進(jìn)而減少合成氨基酸過(guò)程中產(chǎn)圖2中進(jìn)一步分析各批次發(fā)酵過(guò)程中生產(chǎn)強度生的NADH,在這種情況下乙醇發(fā)酵主要副產(chǎn)物甘(即單位體積發(fā)酵液中產(chǎn)物乙醇的生成速率)的變油的產(chǎn)量有所降低,從而提高乙醇的收率叫。通過(guò)化情況。由圖2可清晰反映出添加氮源之后對設備比較計算不同發(fā)酵樣品中產(chǎn)物乙醇對葡萄糖的得生產(chǎn)強度的提升效果。整個(gè)發(fā)酵過(guò)程中未添加氮源率,表明未添加氮源樣品乙醇得率為0.44,而添加3.0■不添加氮源口5gL3.0g/L酵母粉以及分別添加5.0、10.0和20.0g/LB 1Og/L 020g/L BYE 3g/L微藻氮源樣品的乙醇得率分別為0.47、0.46、0.47和0.49,均有一定幅度的提高。蛋白質(zhì)和氨基酸含量數據表明,微藻氮源和酵901.5母粉蛋白質(zhì)含量為32.6%和64.5%(千重),游離氨基酸含量為19.7%和29.9%(干重),高含量的有機氮源特別是游離氨基酸對發(fā)酵效率和乙醇得率的提高具有重要作用。乙醇發(fā)酵實(shí)驗證明,添加816243240567296120發(fā)酵時(shí)間h定劑量的微藻氮源可對發(fā)酵速率和乙醇得率起到圖2葡萄糖含量為210.0g/L的紅薯醪液乙醇發(fā)酵添加促進(jìn)和提升作用,因此微藻氮源可替代酵母粉作為微藻氮源、酵母粉和不添加氮源條件下不同時(shí)刻的生產(chǎn)強度酵母細胞培養和膨酤訇中國煤化本文只分析Fig 2 Productivities of batches adding algal nitrogen source了微藻氮源中并討論其對(ANS), yeast extract(YE)and no nitrogen source adding at乙醇發(fā)酵過(guò)程CNMH所含有的維生different times in ethanol fermentation using sweet potato mash素、礦物質(zhì)等其他成分同樣可能對酵母細胞生長(cháng)和發(fā)申渝等:微藻氮源對紅薯原料燃料乙醇發(fā)酵過(guò)程促進(jìn)效果的研究1545酵產(chǎn)生促進(jìn)作用,相關(guān)研究尚需進(jìn)一步系統深入production at laboratory, pilot and industrial scales[J]Bioresource Technology, 2011, 102(6 ): 4573-45793結論[8] Bradbury J H, Hammer BT. et al. pre以酸水解后的野生水華微藻生物質(zhì)為氮源營(yíng)quantity and quality and trypsin inhibitor content of養,研究其對紅薯原料發(fā)酵過(guò)程的影響。實(shí)驗表明sweet potato cultivars from the highlands of Papua New添加微藻氮源可提升以紅薯為原料的酵母發(fā)酵產(chǎn)乙uinea[J]. Joumal of Agricultural and FoodChemistry,1985,33(3):281-285.醇速率,大幅縮短發(fā)酵時(shí)間;添加微藻氮源后可促進(jìn)(9」 Albers E, Larsson C, Liden g,etal. Influence of the酵母細胞生長(cháng),提高細胞濃度,這是系統發(fā)酵速率提nitrogen source on Saccharomyces cerevisiae anaerobi升的原因之一;同時(shí)添加微藻氮源后可增加產(chǎn)物乙growth and product formation[ J]. Applied Environmental醇得率。and Microbiology, 1996, 62(9): 3187--3195[參考文獻][10] Bach B, Sauvage F X, Dequin S, et al. Role ofgamma-aminobutyric acid as a source of nitrogen and[1] Srichuwong S, Fujiwara M, Wang X H, et alsuccinate in wine [J]. American Journal Enology anSimultaneous saccharification and fermentation( SSF )ofViticulture,2009,60(4):508-516.very high gravity(VHG)potato mash for the production [11] De Cruz S H, Maffud C E, Emandes Jose R, et alof ethanol[ J]. Biomass and Bioenergy, 2009, 33 (5)tructural complexity of the nitrogen source and influenceon yeast growth and fermentation[J]. Journal of Institut[2] Dai D, Hu ZY, PuG Q, et al. Energy efficiency andof brewing,2002,l08(1):54-61potentials of cassava fuel ethanol in Guangxi region of [12] Thomas K C, Ingledew W M. Fuel alcohol productionChina[ j]. Energy Convers Manage, 2006, 47(14)Effects of free amino nitrogen on fermentation of very-1686-1699high-gravity wheat mashes[J]. Applied Environmental[3 Sriroth K, Piyachomkwan K, Wanlapatit S, et al. Theand Microbiology, 1990, 56(7): 2046--2050promise of a technology revolution in cassava bioethanol:[13]沈銀武,劉水定,吳國樵,等.富營(yíng)養湖泊滇池水華From thai practice to the world practice [J]. Fuel藍藻的機械清除[J].水生生物學(xué)報,2004,2(2):2010,89(7):1333-1338.131-136.[4] Nguyen T L T, Shabbir H, Gheewala S G. Full chain [13] Shen Yinwu, Liu Yongding, Wu Guoqiao, et alenergy analysis of fuel ethanol from cassava in thailandMechanical removal of heavy cyanobacterial bloom in the[J]. Environmental Science and Technology, 2007, 41per-eutrophic lake dianchi[ J]. Acta Hydrobiologica(11):4135-4142Sinica,2004,2(2):131-136[5]靳艷玲,甘明哲,方楊,等.鮮甘薯發(fā)酵生產(chǎn)高濃14]劉麗萍.滇池水華特征及成因分析[門(mén)].環(huán)境科學(xué)度乙醇的技術(shù)[J].應用與環(huán)境生物學(xué)報,2009,15研究,1999,21(5):36-37(3):410413[14] Liu Liping. Characteristics of blue algal bloom inYang, et al. Veryedianchi lake and analysis on its cause[ J]. Research ofhigh gravity fermentation of ethanol with fresh sweetEnvironmental Sciences, 1999, 21(5): 36-37potato[J]. Chinese Jourmal of Applied& Environmental[15]申渝,張海東,鄭旭煦,等.高濃度紅薯醪SSF燃Biology,2009,15(3):410-413料乙醇過(guò)程中底物抑制的調節[J].太陽(yáng)能學(xué)報,[6]羅希,何華坤,劉莉,等.高濃度發(fā)酵制備紅薯2013,34(1)105-109燃料乙醇的研究[門(mén)].釀酒科技,2008,17(7):32-35.[15] Shen yu, Zhang haidong, Zheng Xuxu,etl. Substrate[6 Luo Xi, He Huakun, Liu Li, Wang Hongxia, et alinhibition regulated by glucoamylase dosages in ssf ofesearch onhigh gravity fermentation for the productiong sweet potato mash for ethanol productionof fuel alcohol by sweet potatoes [J]. Liquor-Makingsaccharomyces cerevisiae[J]. Acta EnergiaeScience Technology, 2008, 7(7): 32-35[7 Zhang L, Zhao H, Gan M Z, et al. Application of [16] Roulston中國煤化工simultaneous saccharification and fermentation( SSFCNMHG What govemsroteincumausur preferences,from viscosity reducing of raw sweet potato for bioethanol1546太陽(yáng)能學(xué)報34卷Monographs,2000,70(4):617-643under nitrogen starvation[ J]. The Joumal of Bic[17] Breuila C, Saddlera J N. Comparison of theChemistry,2005,280(35):31582-315863, 5-dinitrosalicylic acid and Nelson-Somogyi methods [20] Palnitkar S, Lachke A. Effect of nitrogen sources onof assaying for reducing sugars and determining cellulaseoxidoreductive enzymes and ethanol production durinJ. Enzyme and Microbial Technology, 1985D-xylose fermentation by Candida shehatae [ JJ7(7):327-32Canadian Journal of Microbiology, 1992, 38 (3): 258-[18 Hirokawa T, Hata M, Takeda H. Correlation betweenthe starch level and the rate of starch synthesis during [21] Broa C, Regenberga B, Forsterb J, et al. In silicoaided metabolic engineering of Saccharomyces cerevisiaePlant and Cell Physiology, 1982, 23(5):813-820for improved bioethanoloduction [J]. Metabolic[19 Onodera J, Ohsumi Y. Autophagy is requiredEngeering, 2006, 8(2): 102-111maintenance of amino acid level and protein syntheFERMENTATION PROMOTION BY ALGAL NITROGEN SOURCEFEEDING IN FUEL ETHANOL PRODUCTION FROMSWEET POTATO MASHShen yu 3, Zhang Haidong", Guo Jinsong, Chen Youpeng', Zheng Xuxu, Zhang Xianming(1. Engineering Research Centre for Waste Oil Recovery Technology and Equipment, Ministry of Education, Chongqing Technology and Business UniversiyChongqing 400067, China; 2. Chongqing Key Lab of Catalysis and Functional Organic Molecules, Chongqing Technology and Business UniuersityChongqing 400067, China; 3. Chongqing Institute of Green and intelligent Technology, Chinese Academy of Sciences, Chongging 401122,ChinAbstract: Sweet potato medium contained 210. 0g/L glucose was produced from sweet potato powder based twoenzyme method, and was used for fuel ethanol fermentation by Saccharomyces cerevise ATCC 6508. Bloom algaebiomass was collected and processed into algal nitrogen source(ANS)after processing steps including wash, acidhydrolysis, drying and milling. It was indicated that the residue glucose was 7. 0g/L in batch without nitrogensource feeding after 120h fermentation, and the yield of ethanol and final ethanol concentration were 0. 44 ane89. 14g/L While the fermentation times were reduced to 96, 72 and 72h when 5.0, 10.0 and 20. 0g/L anS wasadded, 96. 1, 98. 1 and 103. Og/L final ethanol concentrations were attained, ethanol yields amounted to 0.46047 and 0. 49 respectively. Protein and amino acid analysis proved that the protein content in ANS and yeast extractwere 32. 6 and 64.5%, and free amino acid content were 19. 7 and 29. 9%, high content of organic nitrogen matterwas expected to be responsible for fermentation rate promotion. The study also indicated ANS was a reliablealternative nitrogen source for yeast extract in ethanol fermentation using sweet potato mash as the medium.Keywords: sweet potato mass; algal nitrogen source(ANS); fuel ethanol fermentation; fermentation efficiencyethanol yield中國煤化工CNMHG