2010年1月農(nóng)業(yè)機(jī)械學(xué)報(bào)第41卷第1期DoI:10.3969/j.isn.1000-1298.2010.01.021粟酒裂殖酵母發(fā)酵菊芋生產(chǎn)燃料乙醇試驗(yàn)汪倫董英(1.江蘇大學(xué)食品與生物工程學(xué)院,鎮(zhèn)江212013;2.河南科技大學(xué)食品與生物工程學(xué)院,洛陽471003)【摘要】以菊粉為原料,研究了粟酒裂殖酵母的乙醇發(fā)酵性能并考察了溫度、初始pH值和菊粉質(zhì)量濃度對(duì)乙醇發(fā)酵的影響,進(jìn)而研究了粟酒裂殖酵母發(fā)酵菊芋汁和菊芋粉生產(chǎn)乙醇的情況。結(jié)果表明:粟酒裂殖酵母能發(fā)酵菊粉高產(chǎn)乙醇;該菌株最適發(fā)酵溫度為30℃,最適初始pH值為4.0,在此條件下,菊粉質(zhì)量濃度200g/L時(shí),乙醇質(zhì)量濃度達(dá)到74.58g/L,理論轉(zhuǎn)化率為81.24%;直接發(fā)酵菊芋汁和菊芋粉獲得更高的乙醇產(chǎn)率,理論轉(zhuǎn)化率分別達(dá)到84.02%和86.09%關(guān)鍵詞:菊芋粟酒裂殖酵母同步糖化發(fā)酵乙醇發(fā)酵中圖分類號(hào):S216.2文獻(xiàn)標(biāo)識(shí)碼:A文章編號(hào):1000-1298(2010)01010605Ethanol Production from Jerusalem ArtichokeUsing Schizosaccharomyces pombeWang Lunji Dong Ying(1. School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013,China2. School of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471003, ChinAbstractThe ethanol fermentation capability of Schizosaccharomyces pombe from inulin was investigated.Theeffects of temperature, inulin concentration, and initial pH value on the ethanol fermentation werestudied. The ethanol production from Jerusalem artichoke juice and flour were further studied.Experimental results showed that S. pombe demonstrated good ethanol fermentation performance.Theoptimum initial pH value was 4.0, and the optimum temperature was 30C. Under the optimalconditions, the maximum ethanol concentration of 74 58 g/L, equivalent to 81. 24% of the theoreticalyield, was reached from 200 g/L inulin concentration after 72 h of incubation. Higher theoreticalconversion rates of 84. 02% and 86. 09% were obtained when Jerusalem artichoke juice and flour weredirectly used in fermentation, respectively.Key words Jerusalem artichoke, Schizosaccharomyces pombe, Simultaneous saccharification andfermentation, Ethanol production引言有一個(gè)葡萄糖基2),常用來生產(chǎn)高果糖漿,也可以用來生產(chǎn)乙醇-”。菊芋適應(yīng)性強(qiáng),耐貧瘠、耐菊芋( Jerusalem artichoke),俗名洋姜,原產(chǎn)北寒、耐旱,特別適合在沙漠、灘涂、鹽堿荒地等非農(nóng)業(yè)美,目前在中國廣泛種植。菊芋塊莖富含菊粉,占其耕地種植,且產(chǎn)量高,價(jià)格低廉。作為一種果糖基能干質(zhì)量的68%-83%。菊粉是果糖經(jīng)β-2,1-糖源植物,菊芋與小麥、玉米等糧食類淀粉質(zhì)原料相苷鍵聚合而成的一種果聚糖,呈直鏈結(jié)構(gòu),末端常含比,具有不占耕地、無需液化等優(yōu)勢,是我國非糧燃中國煤化工收稿日期:2009-04-28修問日期:2009-09-27鎮(zhèn)江市農(nóng)業(yè)攻關(guān)項(xiàng)目(NY2006043)CNMHG作者簡介:汪倫記,博士生,河南科技大學(xué)講師主要從事微生物與發(fā)酵工程研究 E-mail:w, lunji@sohu,com通訊作者:董英教投博士生導(dǎo)師,主要從事食品功能因子和食品生物技術(shù)研究,E-mail;yong@uj,cdu.cn第1期汪倫記等:粟酒裂殖酵母發(fā)酵菊芋生產(chǎn)燃料乙醇試驗(yàn)料乙醇生產(chǎn)及其產(chǎn)業(yè)規(guī)?；l(fā)展可選擇的原料之養(yǎng),定時(shí)稱量,記錄CO2失重,估計(jì)發(fā)酵狀況。發(fā)酵結(jié)束后,全部倒入全玻璃蒸餾器進(jìn)行乙醇蒸餾,測定以菊芋為原料生產(chǎn)乙醇很早就受到國內(nèi)外的關(guān)乙醇含量,對(duì)比S. pombe和S. cerevisiae的乙醇發(fā)酵注。20世紀(jì)50年代以來,國內(nèi)外學(xué)者先后報(bào)道了性能。各種以菊粉為原料生產(chǎn)乙醇的工藝技術(shù),包括12.3溫度對(duì)乙醇發(fā)酵的影響首先酸解或酶解菊粉生成單糖,再利用釀酒酵母發(fā)配制200g/L菊粉發(fā)酵液,接入S. pombe,接種酵生成乙醇的分步糖化發(fā)酵工藝(SHF)和利用產(chǎn)菊量5%,分別在2528、30、33和37℃培養(yǎng)72h,進(jìn)行粉酶的微生物與釀酒酵母進(jìn)行混合培養(yǎng)的同步糖化乙醇發(fā)酵,考察溫度對(duì)乙醇發(fā)酵的影響,確定最佳發(fā)發(fā)酵工藝(SSF)。酵溫度。本文使用粟酒裂殖酵母(S. pombe)進(jìn)行單菌同1.2.4初始pH值對(duì)乙醇發(fā)酵的影響步糖化發(fā)酵菊芋生產(chǎn)燃料乙醇的試驗(yàn)。配制200g/L菊粉溶液,初始pH值分別為3.5、1材料和方法4.0、4.5、5.0、5.5,接種量5%,30℃培養(yǎng)72h,考察pH值對(duì)乙醇發(fā)酵的影響,確定最佳pH值1.1材料1.2.5菊粉質(zhì)量濃度對(duì)乙醇發(fā)酵的影響1.11菌種選擇150200、250和300g/L4種菊粉質(zhì)量濃粟酒裂殖酵母購自中國工業(yè)微生物菌種保藏管度進(jìn)行試驗(yàn),接種量5%,30℃培養(yǎng)72h,研究乙醇理中心;釀酒酵母( Saccharomyces cerevisiae,耐高溫得率,確定最佳原料質(zhì)量濃度。和耐高糖)購自湖北安琪酵母公司;釀酒酵母(S.1.26菊芋汁發(fā)酵cerevisiae)和馬克斯克魯維酵母(K. marrons)為實(shí)鮮菊芋加適量水,粉碎打漿,紗布過濾去除殘驗(yàn)室保存。渣,菊芋汁80℃加熱15min滅酶,以防褐變,蒸發(fā)濃1.12菊芋粉和菊粉縮至總糖質(zhì)量濃度為200g/L,接種發(fā)酵,接種量菊芋購自安徽省滁州市,鮮菊芋經(jīng)洗凈、切片、5%,溫度30℃,轉(zhuǎn)速200r/min。晾曬、干燥、粉碎后置于干燥環(huán)境中保存?zhèn)溆?菊粉,12.7菊芋粉發(fā)酵購自北京威德生物科技有限公司。以100目篩分菊芋粉為底物進(jìn)行乙醇發(fā)酵,菊1.L3培養(yǎng)基芋粉質(zhì)量濃度200g/L,溫度30℃,轉(zhuǎn)速200r/min(1)斜面培養(yǎng)基。豆芽汁蔗糖瓊脂培養(yǎng)基,接接種量5%。種一環(huán)菌種于豆芽汁蔗糖瓊脂斜面培養(yǎng)基,30℃,培1.3分析方法養(yǎng)72h,儲(chǔ)于4℃冰箱備用。(1)乙醇質(zhì)量濃度的測定:HP4890D氣相色(2)種子培養(yǎng)基S.pmbe和K. marxianus:菊粉譜工作站,填料 Porapak-Q粒度80-100目,長2m20g/L,酵母膏15g/L,蛋白胨20g/L,pH值4.6;外徑3mm,內(nèi)徑2mm。氣化室、色譜柱及熱導(dǎo)檢測s. cerevisiae:豆芽汁蔗糖培養(yǎng)基。12l℃滅菌20min器的溫度分別為150、120、150℃。備用。理論轉(zhuǎn)化率山為(3)發(fā)酵培養(yǎng)基三角瓶發(fā)酵:菊粉(按試驗(yàn)要求配制所需質(zhì)量x100%0.51Cf濃度,g/L),酵母膏15g/L,蛋白胨20g/L,除特別指式中Cg-乙醇的質(zhì)量濃度,g/L出外初始pH值均用稀鹽酸調(diào)節(jié)至4.0,115℃滅菌C——菊粉或菊芋粉質(zhì)量濃度,gLf一威德菊粉或菊芋粉中初始總糖質(zhì)量分5L發(fā)酵罐:菊芋汁、菊芋粉配成的培養(yǎng)基,用稀數(shù)(總糖質(zhì)量分?jǐn)?shù)定義為原料中菊粉和鹽酸調(diào)節(jié)初始pH值至4.0,115℃滅菌35min。還原糖質(zhì)量分?jǐn)?shù)總和,試驗(yàn)測定菊芋粉12試驗(yàn)方法中總糖質(zhì)量分?jǐn)?shù)為75%,威德菊粉為1.2.1種子液的制備90%)酵母接入種子培養(yǎng)基中,30℃振蕩培養(yǎng)48h,使其中中國煤化工5細(xì)胞濃度達(dá)到1×103個(gè)/mL以上。1.2.2S. pombe的乙醇發(fā)酵性能CNMHG還原糖測定采用DN噌四疋以還原糖計(jì),定量待每個(gè)三角瓶裝入100mL、200g/L菊粉發(fā)酵液,測樣品于0.05mol/LHCl中,沸水浴中回流水解分別接入乙醇發(fā)酵菌種,接種量5%,30靜止培1h,用0.05mo/ L NaOH調(diào)成中性。108農(nóng)業(yè)機(jī)械學(xué)報(bào)2010年(3)酶活測定:0.5mL酶液(若必要,適當(dāng)22乙醇發(fā)酵進(jìn)程中菊粉酶酶活和S. pombe增長稀釋)加入到4.5mL質(zhì)量分?jǐn)?shù)2%的蔗糖溶液(pH在以菊粉為原料發(fā)酵生成乙醇的過程中,菊粉值4.8的0.2mo/L醋酸緩沖液配制),5℃反應(yīng)酶控制菊粉生成果糖,因此發(fā)酵過程中菊粉酶的活10min,沸水滅活。DNS法測還原糖含量。外切菊性會(huì)影響對(duì)菊粉的水解,進(jìn)而可能影響乙醇產(chǎn)量和粉酶活力定義為:以蔗糖為底物,每分鐘水解1μmol發(fā)酵進(jìn)程。為此,對(duì) S pombe利用菊粉(200g/)生蔗糖所需的酶量為一個(gè)酶活力單位。產(chǎn)乙醇過程中菊粉酶酶活性進(jìn)行了研究,結(jié)果如(4)酵母濃度的測定3-16:采用血球板計(jì)數(shù),圖1所示。從圖中可以看出,在乙醇發(fā)酵過程中胞活細(xì)胞濃度測定采用美蘭染色外菊粉外切酶酶活性很低,只有2U/mL左右,整個(gè)發(fā)酵過程酶活變化幅度很小,但低菊粉酶活性對(duì)乙2結(jié)果與討論醇發(fā)酵沒有影響,S. pombe經(jīng)過短暫的延滯期后,快2.1S. pombe的乙醇發(fā)酵性能速分解菊粉生成乙醇,至72h乙醇發(fā)酵結(jié)束,此時(shí)s. pombe和 S. cerevisiae發(fā)酵菊粉產(chǎn)乙醇對(duì)比試乙醇質(zhì)量濃度達(dá)到n2.97gL(圖2)。而葛向陽驗(yàn)結(jié)果如表1所示。由表1可知,. pombe乙醇發(fā)等利用A.ngsL-09和 S. cerevisiae Z-06發(fā)酵酵能力高于. cerevisiae,其乙醇質(zhì)量濃度達(dá)到菊芋生產(chǎn)燃料乙摩的試驗(yàn)中發(fā)現(xiàn),菊粉外切酶是整73.64g/L,理論轉(zhuǎn)化率為80.21%;而S. cereuLsIO個(gè)過程中最關(guān)鍵一環(huán),高活性菊粉外切酶能縮短發(fā)的乙醇質(zhì)量濃度和理論轉(zhuǎn)化率只達(dá)到40g/L和酵周期、降低殘?zhí)?、提高乙醇產(chǎn)量和轉(zhuǎn)化率。對(duì)此有40%左右,且其發(fā)酵時(shí)間比S. pombe長。出現(xiàn)這種待進(jìn)行進(jìn)一步的深入研究?，F(xiàn)象的原因是由于S. cerevisiae不能直接發(fā)酵菊粉產(chǎn)乙醇,只有K. martianus分泌胞外的菊粉外切酶先把菊粉分解成果糖,然后S. cerevisiae再發(fā)酵果糖生成乙醇,在這個(gè)過程中,K. marxianus分泌菊粉外S.pomb細(xì)胞含量切酶與S. cerevIsIae發(fā)酵果糖無法達(dá)到協(xié)調(diào)一致,而賬1×10粉酶活力S. pombe發(fā)酵菊粉生產(chǎn)乙醇過程中糖化和發(fā)酵同步發(fā)酵時(shí)間完成。 Ohta, Nakamura和 Szambelan1等人利圖1乙醇發(fā)酵過程中菊粉酶活性和S. pombe細(xì)胞含量用高產(chǎn)菊粉外切酶的微生物,如 Aspergillus niger和Fig 1 Activity of inulinase and S. pombeKluyveromyces fragilis和乙醇發(fā)酵性能優(yōu)良的concentration in the brothS. cerevisiae進(jìn)行混合發(fā)酵菊粉產(chǎn)乙醇,并達(dá)到較高由圖1和圖2還可看出,在乙醇質(zhì)量濃度快速乙醇產(chǎn)量,理論轉(zhuǎn)化率也達(dá)到80%以上。但由于糖增長時(shí),同時(shí)伴隨著S. pombe的快速增長,細(xì)胞濃度化和乙醇發(fā)酵分別由兩種微生物完成,造成發(fā)酵周初始細(xì)胞含量為0.53×10個(gè)/mL,48h細(xì)胞含量為期較長,需要5d以上,而S. pombe僅需72h。以菊5.67×10°個(gè)/mL,達(dá)到最高,96h發(fā)酵結(jié)束后下降粉為原料進(jìn)行燃料乙醇的工業(yè)化生產(chǎn)中,S.p0mbe到4.12x10個(gè)/mL。發(fā)酵后期細(xì)胞含量的下降可的同步糖化發(fā)酵比混菌發(fā)酵的同步糖化發(fā)酵相比,能由于發(fā)酵液中可發(fā)酵糖的耗盡和乙醇對(duì)酵母細(xì)胞減少了發(fā)酵環(huán)節(jié),縮短發(fā)酵周期的抑制作用襄1S. pombe或S. cerevisiae的菊粉乙醇發(fā)酵Tab 1 Ethanol production from inulin by SSF usingS pombe or S cerevisiae總糖質(zhì)量濃度菌株乙醇質(zhì)量理論轉(zhuǎn)發(fā)酵圖N濃度/gL1化率/%時(shí)間/hK. martian +s cerevisiae168thermal-tolerance發(fā)酵時(shí)間hK. marxianus +S cerevisiae41.23(sugar-tolerance)2.3Ha中國煤化工CNMHGK. marxian +S. cerevisiaeS pombe80.21溫度是微生物生長的重要環(huán)境條件之一,不但影響S. pombe生長和繁殖,同時(shí)也影響乙醇發(fā)酵酶第1期汪倫記等:粟酒裂殖酵母發(fā)酵菊芋生產(chǎn)燃料乙醇試驗(yàn)系的分泌和活性,所以研究溫度對(duì)S. pombe在乙醇增加,但理論轉(zhuǎn)化率卻降低,300g/L和250g/L菊發(fā)酵中的影響,對(duì)提高發(fā)酵生產(chǎn)的效率具有十分重粉溶液乙醇質(zhì)量濃度達(dá)到91.97gL和84.79g/L要的意義。由圖3可知,S. pombe最適發(fā)酵溫度為而其理論轉(zhuǎn)化率只有66.79%和73.89%,200g/L30℃,乙醇質(zhì)量濃度達(dá)74.12g/L,28℃和33℃也具菊芋粉溶液發(fā)酵產(chǎn)乙醇其理論轉(zhuǎn)化率達(dá)81.24%有較高的乙醇質(zhì)量濃度,分別為64.12gL和乙醇質(zhì)量濃度達(dá)到74.58g/L,達(dá)到較高轉(zhuǎn)化率;6749gL,而在25℃和37℃進(jìn)行發(fā)酵時(shí),乙醇質(zhì)量150g/L菊芋粉溶液發(fā)酵產(chǎn)乙醇雖然理論轉(zhuǎn)化率達(dá)濃度只有42.98gL和46.41g/L到88.91%,但乙醇質(zhì)量濃度偏低,只有61.22g/L2菊粉質(zhì)量濃度對(duì)乙醇發(fā)酵的影響Tab 2 Effect of inulin concentration on ethanol production菊粉質(zhì)量濃度/g“L1乙醇質(zhì)量濃度理論轉(zhuǎn)化率/%61.22圖3培養(yǎng)溫度對(duì)乙醇發(fā)酵的影響91.9756.79Fig 3 Effect of temperatures on ethanol production2.6菊芋汁乙醇發(fā)酵研究2.4不同初始pH值對(duì)乙醇發(fā)酵的影響S. pombe能發(fā)酵菊粉產(chǎn)乙醇,但菊粉的生產(chǎn)成pH值是影響微生物生長的另一重要因素通過本較高,無法應(yīng)用于工業(yè)化生產(chǎn)。因此,對(duì)S. pombe影響酶的活性而影響發(fā)酵。由圖4可知,粟酒裂殖發(fā)酵菊芋汁的效果進(jìn)行了研究結(jié)果如圖5所示。酵母最適發(fā)酵初始P值為40左右,乙醉質(zhì)量濃由圖5可知,s. mbe能快速發(fā)酵菊芋汁產(chǎn)乙醇,度達(dá)到74.19g/L。發(fā)酵結(jié)束后發(fā)酵液的pH值比56h乙醇發(fā)酵結(jié)束,乙醇質(zhì)量濃度達(dá)到85.69g/L初始時(shí)有不同幅度的下降。高的初始pH值導(dǎo)致發(fā)理論轉(zhuǎn)化率為84.02%。與發(fā)酵菊粉產(chǎn)乙醇相比酵液褐變同時(shí)溶液變粘稠流動(dòng)性變差乙醇產(chǎn)量可獲得更高的理論轉(zhuǎn)化率且達(dá)到最大乙醇質(zhì)量濃也隨初始PH值的升高而下降,這可能是由于分解度的發(fā)酵時(shí)間縮短了16h。菊粉的酶和發(fā)酵糖產(chǎn)乙醇的酶系受pH值升高而導(dǎo)致酶活下降的緣故總物質(zhì)敏濃度→乙醇質(zhì)量濃度401624pH值圖5菊芋汁乙醇發(fā)酵圖4不同初始pH值對(duì)乙醇發(fā)酵的影響Fig 5 Ethanol production from Jerusalem artichokeFig. 4 Effect of initial PHjuice using S pombe2.5原料質(zhì)量濃度對(duì)乙醇發(fā)酵的影響2.7菊芋粉乙醇發(fā)酵研究原料質(zhì)量濃度對(duì)乙醇發(fā)酵有重要的影響,乙醇不需補(bǔ)充添加任何營養(yǎng)成分,S. pombe能直接發(fā)酵是典型的產(chǎn)物抑制,在乙醇體積分?jǐn)?shù)達(dá)到4%發(fā)酵菊芋汁產(chǎn)乙醇。在此研究的基礎(chǔ)上,對(duì)以菊芋左右時(shí),就開始產(chǎn)生抑制,發(fā)酵速度逐漸降低直至停粉(200g/L)為原料的乙醇發(fā)酵進(jìn)行了試驗(yàn),結(jié)果如止9),因此原料質(zhì)量濃度過高會(huì)導(dǎo)致原料利用不完圖6所示。由圖6可知,S. pombe也能快速發(fā)酵菊全。為盡量提高原料的利用率,常常采用較低質(zhì)量芋粉產(chǎn)乙醇,56h乙醇發(fā)酵結(jié)束,此時(shí)乙醇質(zhì)量濃度濃度的原料進(jìn)行乙醇發(fā)酵但質(zhì)量濃度的降低會(huì)造和理論轉(zhuǎn)化率分別為65.86g/L和86.09%。發(fā)酵成發(fā)酵結(jié)束后發(fā)酵液乙醇含量低,增大了蒸餾的能結(jié)束凵中國煤化工僅有,0E,耗。因此對(duì)不同質(zhì)量濃度菊粉溶液進(jìn)行了發(fā)酵產(chǎn)95.3乙醇的試驗(yàn),對(duì)比不同質(zhì)量濃度菊粉溶液的乙醇產(chǎn)CNMHGpombe同步糖化量和理論轉(zhuǎn)化率,以確定合適的發(fā)酵質(zhì)量濃度。從發(fā)酵菊粉產(chǎn)乙醇不僅省去了菊粉酶解步驟,而且可表2可知,乙醇質(zhì)量濃度隨菊粉質(zhì)量濃度的增加而以防止因菊粉降解物糖的積累抑制乙醇發(fā)酵的現(xiàn)農(nóng)業(yè)機(jī)械學(xué)報(bào)2010年化和糖化設(shè)備的投資和能耗,乙醇的生產(chǎn)成本也得到降低?？偺琴|(zhì)敏濃度1303結(jié)論乙醇質(zhì)量濃度(1)S. pombe能同步糖化發(fā)酵菊粉產(chǎn)乙醇且乙醇發(fā)酵性能優(yōu)良,在pH值4.0、溫度30℃的優(yōu)化發(fā)發(fā)酵時(shí)間h酵條件下,200g/L菊粉溶液發(fā)酵產(chǎn)乙醇其理論轉(zhuǎn)化圖6菊芋粉乙醇發(fā)酵率達(dá)到81.24%,乙醇質(zhì)量濃度達(dá)到74.58g/L。發(fā)Fig 6 Ethanol production from Jerusalem artichoke酵過程中低菊粉酶活性對(duì)乙醇發(fā)酵沒有影響。four using S pombeS. pombe發(fā)酵菊芋汁和菊芋粉獲得更高的乙醇產(chǎn)象,有利于維持較高的乙醇生產(chǎn)強(qiáng)度;與混菌培率,理論轉(zhuǎn)化率分別達(dá)到84.02%和86.09%。養(yǎng)的同步糖化發(fā)酵工藝相比,糖化和發(fā)酵都由粟酒(2)S. pombe發(fā)酵菊粉產(chǎn)乙醇的糖化與發(fā)酵同裂殖酵母完成更易于進(jìn)行產(chǎn)菊粉酶條件和乙醇發(fā)步完成的特性使其比利用S. cerevisiae或 Zymomonas酵條件的同步優(yōu)化,而與淀粉質(zhì)原料相比,菊芋粉不 mobile作為乙醇發(fā)酵菌株更有優(yōu)勢,能避免糖化與需蒸煮和預(yù)先糖化,可直接進(jìn)行發(fā)酵從而減少了液發(fā)酵分步進(jìn)行;這可以降低乙醇發(fā)酵的成本和縮短化和糖化大量的能耗。在工業(yè)化生產(chǎn)時(shí),減少了液乙醇發(fā)酵時(shí)間。考文獻(xiàn)I Fleming S E, Groot Wassink J W. 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