石油化工2007年第36卷第2期 PETROCHEMICAL TECHNOLOGY107特約述評生物質(zhì)制燃料乙醇陳輝,陸善祥(華東理工大學(xué)聯(lián)合化學(xué)反應(yīng)工程研究所,上海200237)摘要]生物質(zhì)制燃料乙醇具有重要環(huán)保意義,是一科極具前景的石油可替代資源生產(chǎn)工藝目前以植物纖維素為原料生產(chǎn)燃料乙醇的成本仍較高。綜述了近幾年來生物質(zhì)制燃料乙醇在預(yù)處理工藝、水解液發(fā)酵抑制物的脫除方法水解和發(fā)酵工藝、纖維素酶和乙醇發(fā)酵基因工程菌領(lǐng)域的研究進(jìn)展,介紹了國外相關(guān)的大的生物質(zhì)制燃料乙醇項(xiàng)目,述了生物量全利用的意義展望了生物質(zhì)制燃料乙醇的未來發(fā)展方向[關(guān)鍵詞]燃料乙醇生物質(zhì);纖維素;基工程;發(fā)酵文章編號1000-8144(2007)02-0107-11中圖分類號]TQ517.2文獻(xiàn)標(biāo)識碼]A Progress in Production of Fuel-Ethanol from Biomass Resources Chen Hui, Lu Shanxiang (UNILAB of East China University of Science and Technology, Shanghai 200237, China) [Abstract] Production of fuel-ethanol from biomass resources is environment-friendly and is a promising substitutive technology for limited crude oil resource. Current cost for production of ethanol from cellulose plant is still high. The progresses in technology, including pretreatment of materials, removal of fermentation inhibitors in hydrolysate, hydrolysis and fermentation technology, and genetically engineered strains for cellulase production and ethanol fermentation were reviewed. The worlds important projects for utilization of biomass were briefly described and future development for production of fuel-ethanol from biomass was discussed. Keywords fuel ethanol; biomass cellulose genetic engineering; fermentation燃料乙醇是指作為燃料添加到汽油、柴油中的為原料的燃料乙醇生產(chǎn)成本約4000元/t。燃料乙乙醇,可部分替代石油,緩解石油資源短缺。從生物醇的生產(chǎn)成本太高,不足以與汽油競爭,目前各國普質(zhì)光合作用生物質(zhì)制燃料乙醇到乙醇燃燒的過程遍采用補(bǔ)貼或稅收優(yōu)惠的方式支持燃料乙醇的生在自然界形成了CO2的閉合循環(huán),可緩解地球的溫產(chǎn)。植物纖維素資源是地球上最豐富和廉價(jià)的可再室效應(yīng),燃料乙醇還可降低汽車尾氣污染,而燃料生資源,通過生物化學(xué)法和熱化學(xué)法(熱解、氣化、乙醇具有重要的生態(tài)環(huán)保意義汽油中乙醇的體液化和超臨界抽提)可生產(chǎn)燃料乙醇,但生產(chǎn)成本積分?jǐn)?shù)低于10%~15%時(shí),不僅不需對現(xiàn)有汽車發(fā)很高。近年來隨著化工過程、生物技術(shù)、基因工程的動機(jī)進(jìn)行改進(jìn),且汽油還具有辛烷值高和抗爆性好發(fā)展,燃料乙醇的生產(chǎn)成本逐漸降低。從長遠(yuǎn)來看,的優(yōu)點(diǎn),因而燃料乙醇已在世界許多國家廣泛應(yīng)用,生物化學(xué)法具有良好的發(fā)展和應(yīng)用前景。推廣力度不斷加大。本文綜述了近幾年來生物質(zhì)制燃料乙醇在工燃料乙醇生產(chǎn)的主要問題是原料成本高。以含藝基因改造工程菌等領(lǐng)域的研究進(jìn)展,介紹了國外糖作物或谷物為原料時(shí),原料成本占燃料乙醇生產(chǎn)相關(guān)的大的生物質(zhì)制燃料乙醇項(xiàng)目,展望了生物質(zhì)成本的60%左右。巴西以甘蔗為原料的燃料乙醇生產(chǎn)成本約0.2美元/L,美國以玉米為原料的燃料收稿日期】2006-09-22[修改稿日期】2006-10-16乙醇生產(chǎn)成本約0.3美元L,歐洲以小麥為原料的[作者簡介陳輝(1974一),女,山東省濰坊市人,碩士,講師聯(lián)系燃料乙醇生產(chǎn)成本約048元,我國以玉米善祥話021-6425101lushx@sohu.com石七工108油 PETROCHEMICAL TECHNOLOGY2007年第36卷制燃料乙醇的發(fā)展方向。結(jié)合,不僅部分溶解纖維素,而且可破壞纖維素的物1原料的種類不斷豐富理結(jié)構(gòu)。 Foody等采用一系列帶V形齒槽的輥?zhàn)虞殙?、剪切潤濕的木質(zhì)纖維素原料(適用于打漿可提供糖的資源都可用作乙醇發(fā)酵的原料。除泵送),漿液先脫除一部分水和水溶性物質(zhì)后,再在了含糖和含淀粉作物,植物纖維素原料中的纖維素160~280℃下對漿液進(jìn)行稀酸水解預(yù)處理?？伤鉃槠咸烟?半纖維素可水解為戊糖和己糖。2.2.2堿預(yù)處理法由于耕地有限,可開發(fā)劣質(zhì)土地種植高產(chǎn)、高糖、耐堿性試劑可采用NaOH、Ca(OH)2、氨等。堿金堿、耐旱的經(jīng)濟(jì)作物。此外,植物纖維素是自然界產(chǎn)屬氫氧化物水溶液(質(zhì)量分?jǐn)?shù)為20%~40%)的量最大的原料,主要有農(nóng)產(chǎn)品廢棄物和草木我國堿性強(qiáng),通過溶解脫除木質(zhì)素提高原料的酶可及度。是農(nóng)業(yè)大國,據(jù)測算,每年農(nóng)田秸桿資源的一半轉(zhuǎn)化NaOH還具有潤脹纖維素的作用,可降低纖維素的為燃料乙醇,燃料乙醇的消費(fèi)量將超過年汽油消費(fèi)結(jié)晶度,易于纖維素酶的水解201量的1.2倍以上2?；诮档统杀竞吞幚韽U物的考Ca(OH)2的堿性較強(qiáng)堿弱,對于玉米秸桿,較慮纖維素原料的種類還擴(kuò)展到含有纖維素的城市優(yōu)化的處理?xiàng)l件為:在氧氣的存在下,55℃處理28廢棄物或工業(yè)低價(jià)值副產(chǎn)品,如建筑垃圾3-51、城市d,可脫除幾乎所有的乙酰基、87.5%的木質(zhì)素,約固體垃圾6-91、酒精廠釜餾物1、玉米谷粒濕磨91.3%的纖維素和51.8%的半纖維素水解為葡萄副產(chǎn)物纖維、軋棉廢棄物、橄欖油提煉殘?zhí)呛湍咎?每克原料僅消耗0.073gCa(OH)2,且預(yù)渣等。處理液不含有乙醇發(fā)酵抑制物2。2纖維素原料的預(yù)處理氨不僅價(jià)格便宜,還可回收循環(huán)利用,因而被廣泛應(yīng)用。氨回收滲濾(ARP)法具有潤脹和脫木質(zhì)纖維素分子內(nèi)和分子間存在氫鍵聚集態(tài)結(jié)構(gòu)素的作用,處理玉米秸桿時(shí),可溶解約50%的半纖復(fù)雜且結(jié)晶度高、反應(yīng)活性低;天然纖維素原料中含維素,保留約92%以上的纖維素,且纖維素基本的有的木質(zhì)素和半纖維素在空間上可阻礙甚至封閉纖晶體結(jié)構(gòu)沒有明顯變化。熱水處理和ARP兩步預(yù)維素分子與酶或化學(xué)試劑的接觸,酶可及度差,增處理:玉米秸桿可在熱水處理過程中脫除84%的半加了水解的難度。通過預(yù)處理可脫除木質(zhì)素和半纖纖維素,并在后續(xù)的ARP過程中脫除75%的木質(zhì)維素,消除空間障礙;降低纖維素的聚合度和結(jié)晶素,預(yù)處理產(chǎn)物中含有79%的纖維素21。度,從而有利于纖維素的有效利用。預(yù)處理可采用2.2.3濕法氧化預(yù)處理法物理法、化學(xué)法、水熱化學(xué)法和生物法,或幾種方法濕法氧化是指水、氧氣弱堿或弱酸在高溫、一相結(jié)合,此外還有電解法15、超聲法16等。定壓力下氧化降解生物質(zhì)的過程 Lissens等23對2.1物理法高木質(zhì)素含量的木材廠廢料進(jìn)行濕法氧化預(yù)處理物理法:可破壞纖維素原料的物理結(jié)構(gòu),降低結(jié)(溫度185~200℃、每100g干基原料加入0~3.3g晶度,包括球磨、剪切、擠壓等,其中最有效的是球Na2CO3、時(shí)間15min、氧氣壓力0.3~1.2MPa),可磨,但由于能耗高而很少采用。溶解79%的半纖維素和49%的木質(zhì)素(生成羧.2化學(xué)法酸)。此方法可處理城市垃圾8、麥桿24和玉米秸化學(xué)法:采用酸、堿、有機(jī)溶劑或氧化劑等化學(xué)桿等草本纖維試劑與纖維素原料進(jìn)行反應(yīng),以降解脫除原料中的2.3水熱化學(xué)法木質(zhì)素和半纖維素并溶解部分纖維素。常用的化學(xué)水熱化學(xué)法的主要特征是較高的溫度和水的存試劑有稀酸、堿性試劑(氨、NaOH、石灰等)、氧化劑在,處理過程常伴隨化學(xué)反應(yīng)水熱處理時(shí)原料中(H2O2和氧氣等)或幾種試劑相結(jié)合。的乙?；纱姿?醋酸可進(jìn)一步進(jìn)行催化水解,因2.2.1稀酸水解預(yù)處理法而水熱化學(xué)法又常稱為自動水解法。稀酸水解預(yù)處理法主要是脫除原料中易于水解水熱化學(xué)法包括水熱處理、蒸汽爆破(SE)、超的半纖維素和部分纖維素,以提高原料的酶可及度。臨界水處理26等過程,其中最常用的是SE法。SE Eyini等以米糠為原料,比較了稀酸、酶解和微生法:原料用高壓飽和蒸汽處理一段時(shí)間后快速泄壓,物水解預(yù)處理的效果,其中,稀酸水解預(yù)處理法產(chǎn)生產(chǎn)生爆裂作用,從而減小原料尺寸和纖維素的結(jié)晶的還原糖量最多。將稀酸水解預(yù)處理法與物理潔相度,高溫時(shí)有利于半纖維素的降解木質(zhì)素的轉(zhuǎn)化第2期陳輝等生物質(zhì)制燃料乙醇109使組分易于分離,提高纖維素的酶可及度。由于 SE mmol/L)時(shí)纖維素降解生成的發(fā)酵抑制物的量增法中包含原料結(jié)構(gòu)的物理破壞(爆裂)和化學(xué)反應(yīng),加,接種量為0.6g/L時(shí),乙醇相對產(chǎn)率可增至因此可用物理和化學(xué)參數(shù)表征SE過程中的變化,95%3種表面活性劑添加到SE法的預(yù)水包括O與C比、H與C比、色度、元素分析、有機(jī)溶由于法在高溫、高壓條件下操作,故能耗較液含量、纖維素的結(jié)晶度及熱重分析等2。SE法高。陳輝38與水熱處理法相比,SE法效果較好,纖維素的回收解過程中,纖維素可在相對較低的強(qiáng)度(185℃、率達(dá)95%以上,且原料粒徑對酶解和發(fā)酵過程沒有min)下得到較好的處理效果與不加表面活性劑相明顯的影響,可使用大粒徑原料[28。比,該法的酶解率提高了24%。將氨與表面活性劑一般的水熱處理法的效果較差,但在控制pH復(fù)配后,進(jìn)一步促進(jìn)了SE過程,酶解率達(dá)到93%。的條件下可促進(jìn)水解過程。玉米秸桿在溫度1602.4生物法℃、pH>4.0時(shí)進(jìn)行水熱處理,20min內(nèi)可溶解生物法:利用可降解木質(zhì)素的微生物或酶選擇50%的纖維(其中80%為可溶性低聚糖,20%為單性地脫除原料中的木質(zhì)素?？山到饽举|(zhì)素的降解菌糖),降解損失率低于1%,且由于單糖量較少故降株多為白腐菌(如 Basidiomycete, Subvermispora解產(chǎn)物的生成量也較少,低聚糖的量較多易于發(fā)酵DSqualens,pOstreatus,,《DSquIensPOsreatsCVersco)《)[39)。生物法生成乙醇291。條件溫和,能耗低,無污染。與單純的醇解效果相S法適用于農(nóng)作物秸桿等草本纖維素原比,將醇解與白腐菌相結(jié)合的醇解效果可節(jié)約15%料14,但只采用SE法處理原料的苛刻度較高。在的電能39?？山到饽举|(zhì)素的酶主要為酯酶。2.55MPa、3min時(shí),甘蔗渣達(dá)到最好的脫木質(zhì)素效 Anderson等40用酯酶預(yù)處理草類纖維,但酶處理液g30]SE法與稀酸水解預(yù)處理法結(jié)合可促進(jìn)草本中含有抑制發(fā)酵細(xì)菌生長和乙醇生成的物質(zhì),脫除纖維素原料的水解,玉米秸桿在200℃5mn、質(zhì)量酶處理液中的酚類物質(zhì)可提高乙醇的產(chǎn)率。分?jǐn)?shù)為2%H2SO4條件下,纖維素的轉(zhuǎn)化率比只采用SE法可提高4倍,且預(yù)處理水解液沒有明顯的毒素3發(fā)酵抑制物及其脫除抑制作用3。SE法與稀酸水解預(yù)處理法結(jié)合也適纖維素在預(yù)水解過程中發(fā)生了化學(xué)反應(yīng),生成用于木質(zhì)素含量高的原料有效阻斷木質(zhì)素與纖維了對酶具有毒性或?qū)Πl(fā)酵過程具有抑制作用的物素酶間的作用,使水解產(chǎn)率提高5%~20%,纖維素質(zhì)。這些物質(zhì)可采用物理、生物或化學(xué)手段脫除,也酶的利用率提高50%32可在控制水解過程中減少抑制物和毒素的生成。兩步稀硫酸浸漬SE法:首先在低強(qiáng)度(180℃、 Martin等41采用SE法預(yù)處理土豆桿,水解液中抑10min、質(zhì)量分?jǐn)?shù)為0.5%H2SO4)下用SE法水解制物的含量較低,但隨處理時(shí)間的延長,抑制物的含提取半纖維素,然后在高強(qiáng)度(200℃、2min、量分量增加。近年來構(gòu)建了許多耐受抑制物的工程菌,數(shù)為2%H2SO4)下用SE法水解部分纖維素并使纖可省略脫除過程,降低成本。維素更易于酶解(此時(shí)乙醇的產(chǎn)率可達(dá)到最大3.1脫除方法值) Soderstrom等34進(jìn)一步將稀硫酸浸漬SE發(fā)酵抑制劑的種類和數(shù)量與生物質(zhì)的種類和預(yù)法與SO2注入SE法相結(jié)合,第一步在低強(qiáng)度180處理方法有關(guān),其抑制機(jī)理也各有不同。 Helle℃、10min、質(zhì)量分?jǐn)?shù)為0.5%H2SO4)下預(yù)處理云等2研究了抑制物對木糖發(fā)酵產(chǎn)生乙醇工程菌S杉,第二步在高強(qiáng)度(210℃、5min、質(zhì)量分?jǐn)?shù)為3% Cerevisiae的影響,醋酸、氨、糠醛對工程菌和其母株SO2)下預(yù)處理云杉,發(fā)酵乙醇的產(chǎn)率可達(dá)到理論值均具有抑制作用。 Klinke等4研究了纖維素高溫的66%,間接發(fā)酵(SHF)的乙醇產(chǎn)率可達(dá)到理論值物理預(yù)處理的水解產(chǎn)物,其中糖降解產(chǎn)物呋喃和木的71%。質(zhì)素降解產(chǎn)物單酚是最主要的發(fā)酵抑制物,會抑制氨纖維爆破預(yù)處理(AFEX)法與SE法相似只酒精酵母的生長和生成乙醇的速率,但不會影響燃是前者采用液氨為介質(zhì)。AFEX法可提高纖維素的料乙醇的產(chǎn)量。 Cantarella研究了S法預(yù)處酶解,纖維素轉(zhuǎn)化率從未處理的16%增至93%,乙理過程中產(chǎn)生的毒素(如甲酸、醋酸、乙酰丙酸、糠醇產(chǎn)率可提高2.2~2.5倍3536醛、羥甲基糠醛(HMF)、丁香醛、羥基苯甲醛、香蘭 Fenton試劑(H2O2和Fe2+)的氧化反應(yīng)也能加醛)對纖維素酶活性的影響,實(shí)驗(yàn)結(jié)果表明,甲酸可強(qiáng)SE法的預(yù)水解過程,但在使用高濃度HO(50使酶先活香蘭醛可部影響酶的活性其他毒素對石油化工110 PETROCHEMICAL TECHNOLOGY2007年第36卷酶活性的影響不大。ZM4/AcR細(xì)胞內(nèi)的pH和能量狀態(tài)基本上不受醋酸抑制物的脫除可采用吸附法、離子交換鈉的影響,因而具有很高的耐受性。法546、萃取法、生物法、過量堿中和法7等?？啡┖虷MF是生物質(zhì)降解產(chǎn)物中最主要的兩吸附法需使用選擇性吸附劑,較好的吸附劑有種發(fā)酵抑制物,高耐受毒素的 Cerevisiae和P木炭、聚合樹脂等。Weil等研究了苯乙烯基 Stipitis可將HMF轉(zhuǎn)化為2,5-雙-(羥甲基呋喃),聚合物XAD-4吸附劑對糠醛脫除效果的影響,實(shí)糠醛轉(zhuǎn)化為糠醇7驗(yàn)結(jié)果表明,糠醛的質(zhì)量濃度可從1~5g/L降至0.01g/L。吸附劑可用乙醇脫附和再生。4纖維素酶和發(fā)酵生產(chǎn)乙醇的微生物生物法需使用可代謝抑制物或毒素的微生物或4.1纖維素酶酶。 Ligniaria NRRL30616可脫除玉米秸桿稀酸纖維素酶是混合酶,主要包括內(nèi)切葡聚糖酶、外水解液中的發(fā)酵抑制物 Reesi能降解蒸汽預(yù)切葡聚糖酶(CBH)和B-葡萄糖苷酶,三者協(xié)同作處理柳樹過程中半纖維素水解所產(chǎn)生的抑制物,從用。纖維素的酶分子普遍具有類似的結(jié)構(gòu),由催化而將燃料乙醇的產(chǎn)量提高4倍,產(chǎn)率提高3倍。結(jié)構(gòu)域、連接橋和纖維素結(jié)合結(jié)構(gòu)域(CBD)3部分生物酶對底物具有專一性,因而應(yīng)用較少。如漆酶組成。最近的研究表明,些區(qū)域的功能不是惟一可脫除單酚和苯酚,但對大分子苯系化合物僅具有的,CBD區(qū)域?qū)Χ嗑厶墙Y(jié)構(gòu)也具有破壞作用,因而降低相對分子質(zhì)量的作用,不能將其轉(zhuǎn)化為不具發(fā)也可促進(jìn)水解的進(jìn)行。aaj -Kolstad-等589的研酵抑制性的物質(zhì)。究結(jié)果表明,CBD中的少量非催化性蛋白(CBP21)過量堿中和法是采用堿性物質(zhì)脫除水解液中抑對殼質(zhì)的降解具有決定作用。CBP21會改變底物制物的方法,對于稀酸水解液,Ca(OH)2過量堿中的結(jié)構(gòu),促進(jìn)纖維素水解的進(jìn)行。纖維素酶的CBD和是最常用的脫除方法C核磁共振分析結(jié)果顯參與了酶與木質(zhì)素的結(jié)合,但無CBD的纖維素酶仍示21,過量堿中和法脫除的主要是脂肪酸和芳香性對木質(zhì)素表現(xiàn)出親合力酸或酯及其他芳香和脂肪化合物,酮和醛官能團(tuán)沒可發(fā)酵產(chǎn)生纖維素酶的微生物有木霉有明顯的變化。 O'Brien等比較了a(H)2過(Trichoderma)、曲霉( Aspergillus)、青霉(Penicillum量堿中和法和強(qiáng)堿陰離子中和(AEN)法對玉米纖和 Humicola)、鐮刀菌( Fusarium)、鏈霉菌屬維稀酸水解液中抑制物的脫除效果,實(shí)驗(yàn)結(jié)果表明,( Streptomyces)、芽孢桿菌屬( Bacillus)纖維單胞菌AEN法的脫除 Cantarella率更高。等3比較了不同屬( Cellulomonas)等。纖維素酶的生產(chǎn)有固態(tài)發(fā)酵抑制物脫除方法,實(shí)驗(yàn)結(jié)果表明,Ca(OH)2過量堿法和液態(tài)發(fā)酵法。一般采用液態(tài)發(fā)酵法,纖維素中和法效果最好,水解液脫除抑制物后發(fā)酵生產(chǎn)乙酶較易于提取,其生產(chǎn)成本可隨著規(guī)模的擴(kuò)大而醇的產(chǎn)率可達(dá)到理論產(chǎn)率的92%。降低,但由于纖維素酶的生產(chǎn)需固體纖維素的誘由于水解液中抑制物主要在發(fā)酵初期起作用,導(dǎo),生產(chǎn)周期長,效率低,且纖維素酶解耗酶量大,隨發(fā)酵的進(jìn)行,細(xì)胞繁殖彌補(bǔ)了抑制物造成的細(xì)胞因而較高的纖維素酶生產(chǎn)成本大幅度降低了生物死亡,維持了一定的細(xì)胞濃度水平,因而可采用高接質(zhì)制燃料乙醇的經(jīng)濟(jì)性近年來里氏木霉纖維素種量消除發(fā)酵初期抑制物的作用。此外還可采酶的生產(chǎn)成本大幅度降低(不到原來生產(chǎn)成本的用固定化酵母法。酵母膠囊化后可降低失活速率,1/10),乙醇酶的生產(chǎn)成本降至0.026~0.053但由于水解液的毒性太大膠囊化的酵母也會逐漸美元/L61],因而生物質(zhì)制燃料乙醇的經(jīng)濟(jì)性可望喪失活性。大幅度提高。3.2耐毒微生物為改善纖維素酶的性能、降低纖維素酶用量,在近年來,由于對發(fā)酵抑制物毒理認(rèn)識水平的提產(chǎn)酶菌株的改良和構(gòu)建方面展開了許多研究。文獻(xiàn)高,采用馴化或基因改造等生物技術(shù)改良菌種,可提[62]報(bào)道,將定點(diǎn)突變、定點(diǎn)飽和誘變、易錯聚合酶高某些發(fā)酵微生物對抑制物的耐受性。鏈反應(yīng)技術(shù)和DNA重組技術(shù)相結(jié)合,產(chǎn)生了變種TKim等[6研究了抑制物對 Mobilis ZM44及其 Reesi纖維二糖水解酶基因Ce17A。將此變異基因耐毒素變異菌株ZM4/AcR發(fā)酵生產(chǎn)乙醇的抑制作在 Serevisiae中表達(dá)并篩選以提高熱穩(wěn)定性和高用P核磁共振分析結(jié)果顯示,細(xì)胞內(nèi)酸化和能量溫活性。其中一個變種被認(rèn)定在 Reesi中代替了新勵具酷酸撲顯示制性的兩種更機(jī)出的C17重整菌酶對理種桿的第2期陳輝等.生物質(zhì)制燃料乙醇111水解效果超過親本。 Bower等3將細(xì)菌6054721, Cerevisiae EF101473, Cerevisiae Cellulolyticus的內(nèi)切葡聚糖酶GH5A基因融合到真42445 Cerevisiae TMB3400等;構(gòu)建了可菌ees的纖維二糖水解酶CBH上,融合蛋白在發(fā)酵阿拉伯糖的工程菌有 Becker構(gòu)建的S Reesi中表達(dá)得到的菌株酶對預(yù)處理玉米秸桿的 Cerevisiae工程菌;構(gòu)建了具有特定耐受性的發(fā)酵木糖化水解更有效。糖生產(chǎn)乙醇的工程菌有:有氧發(fā)酵可耐受稀酸水解轉(zhuǎn)基因植物纖維素酶是一個較新的研究方向液的 Indicus[76,耐高溫并具有較高乙醇耐受性已將 Cellulolytics內(nèi)切葡聚糖酶和 Reesi的纖的F-71m,可有氧發(fā)酵的 Opuntiae1605781耐維二糖水解酶基因表達(dá)在馬鈴薯和西紅柿中。這些受抑制物的 Mathranii A33M3等植物纖維素酶與微生物纖維素酶相似,生產(chǎn)成本4.3可直接利用纖維素原料的微生物大幅度降低。以內(nèi)切葡聚糖為例,每千克酶的成本纖維素原料的水解糖化和發(fā)酵產(chǎn)生乙醇可在同由原來的5美元降至1.40美元。Hood等5一株微生物中完成。如可利用纖維素直接生產(chǎn)乙醇多糖降解酶表達(dá)在農(nóng)作物的種子組織中轉(zhuǎn)基因作的 Thermocellum791,可發(fā)酵微晶纖維素和多種糖物的種子組織可作為酶源,從而可降低燃料乙醇的生產(chǎn)乙醇的 Trichoderma A1100,可發(fā)酵纖維素和生產(chǎn)成本。己糖生產(chǎn)乙醇的 Marxianus,在產(chǎn)醇酵母細(xì)胞4.2可代謝戊糖生產(chǎn)乙醇的基因工程菌表面共表達(dá)3種纖維素酶組分可構(gòu)建發(fā)酵無定形纖半纖維素在生物質(zhì)中占有相當(dāng)大的比例,其水維素的工程菌82,在可發(fā)酵木糖的釀酒酵母細(xì)胞表解產(chǎn)物主要是木糖,以農(nóng)作物秸桿和草為原料時(shí)水面共表達(dá)兩種木聚糖分解酶(半纖維素酶和半纖維解產(chǎn)物中還有一定量的阿拉伯糖(可占戊糖質(zhì)量的素二糖酶)可構(gòu)建發(fā)酵木聚糖的工程酵母310%~20%),故發(fā)酵戊糖生產(chǎn)乙醇的效率也是決4.4提高特定耐受性的微生物定該過程經(jīng)濟(jì)性的重要因素。根據(jù)過程需要,在耐熱性、有氧發(fā)酵、抑制物耐葡萄糖的代謝途徑與木糖不同,且往往對木糖受能力等方面也進(jìn)行了工程菌的構(gòu)建?；谕瑫r(shí)糖的代謝產(chǎn)生抑制作用。 Lusitaniaes可發(fā)酵木質(zhì)纖化和發(fā)酵法的需要,化學(xué)誘變 Marxianus DER-維素水解液中的葡萄糖生成乙醇,在水解液中無葡26得到了耐熱菌工程菌CECT108753584有氧發(fā)萄糖時(shí)可同時(shí)發(fā)酵纖維二糖和木糖生成乙醇。酵的變異酵母可在發(fā)酵的同時(shí)細(xì)胞生長并保持活 Cerevisiae TMB3001, CPB CR1, CPB CR2也是先性,生產(chǎn)效率高、乙醇的產(chǎn)率高釀酒酵母中表達(dá)發(fā)酵葡萄糖后再利用木糖671。利用分子生物學(xué)可ADH6可提高乙醇的生產(chǎn)力,毒素的耐受力強(qiáng)。構(gòu)建發(fā)酵戊糖和己糖生產(chǎn)乙醇的基因工程菌。常用的釀酒酵母 Cerevisiae不能發(fā)酵木糖生成乙醇但5水解和發(fā)酵工藝能利用木酮糖,可將戊糖代謝為木酮糖的關(guān)鍵酶引發(fā)酵法制乙醇的生產(chǎn)工藝有3種:分別水解發(fā)到酵母中。 Coli, Stipititis, Mobilis可利用酵SHF(法)、同時(shí)糖化發(fā)酵SSF)法、直接發(fā)酵法。木糖發(fā)酵生產(chǎn)乙醇,但需脫除發(fā)酵液中的抑制物,且5.1SHF法乙醇產(chǎn)率較低,可在這類菌株中引入高效產(chǎn)生乙醇SHF法:先水解糖化再發(fā)酵的二步發(fā)酵法。纖的關(guān)鍵酶。 Coli厭氧發(fā)酵產(chǎn)物中的乙醇含量低,維素原料的水解糖化方法主要有稀酸水解788、濃pH控制在6.0~8.0,其基因改造主要是適應(yīng)稀酸酸水解、酶解。濃酸水解目前采用的不多,主要有日水解液條件,并控制代謝產(chǎn)物向乙醇方向進(jìn)行。P本JBC公司和 Masada美國 OxyNol LLC公司的 Stipitis中含有代謝木糖的木糖還原酶和木糖醇脫氫 Masada CES OxyNol過程酶解法需進(jìn)行原料的酶,但對木糖的代謝卻受葡萄糖的抑制,對乙醇和纖預(yù)處理,使用纖維素酶催化水解。維素水解抑制物的耐受性差,且需控制微好氧條件發(fā)酵菌種有時(shí)不能同時(shí)利用水解液中的混合發(fā)酵。 Mobilis工程菌發(fā)酵葡萄糖和木糖的動力糖,或?qū)σ种莆锩舾?因而可對水解液進(jìn)行分離,或?qū)W研究表明,醋酸能強(qiáng)烈抑制發(fā)酵過程8重整菌采用葡萄糖和木糖兩步發(fā)酵法,或采用一步發(fā)酵法。 Mobilis8b較能耐受醋酸,pH=6時(shí)乙醇產(chǎn)率可達(dá)一步發(fā)酵法可用混合酵母或可發(fā)酵混合糖的菌到85%。種?？刹捎蒙V分離法分離水解液中的糖,吸附劑近年來,構(gòu)建了可發(fā)酵木糖的工程菌有: Coli可采用 Dowex99和聚乙烯吡啶(pvp)等pvpko1170, Stipitis CBS5773, Stipitis CBS模擬移動床可有效分離玉米秸桿水解液中的糖(阿石油化工112 PETROCHEMICAL TECHNOLOGY2007年第36卷拉伯糖、甘露糖、木糖、半乳糖、葡萄糖、纖維二糖)汽化工藝結(jié)合了膜分離和蒸發(fā)過程,可大幅度降低和雜質(zhì)(硫酸、醋酸、糠醛、羧甲基糠醛)。能耗,提高生物質(zhì)制燃料乙醇的經(jīng)濟(jì)性,是最有應(yīng)用5.2S法前景的分離技術(shù)。膜材料可采用殼聚糖衍生SSF法將酶解和發(fā)酵放在同一個反應(yīng)器中進(jìn)物01、聚二甲基硅氧烷102等。其他脫水技術(shù)還行,可克服酶解過程中的反饋抑制作用,簡化了生產(chǎn)有分子篩吸附法等,常需與蒸餾過程相結(jié)合。工藝,但存在的主要問題是酶解和發(fā)酵溫度條件的7生物質(zhì)制燃料乙醇的項(xiàng)目不一致性。酶解適宜的溫度約為50℃,而發(fā)酵的控制溫度是37~40℃。解決的方法:一是采用非等溫較大的生物質(zhì)制燃料乙醇的項(xiàng)目有美國的可再過程,二是利用耐熱酵母的等溫過程。等溫過程一生能源實(shí)驗(yàn)室(NREL)開發(fā)的NREL過程、加拿大般比非等溫過程的乙醇產(chǎn)率高21。近年來耐熱酵的 Iogen過程、印度的 IT Delhi過程等。母構(gòu)建取得了很大進(jìn)展,等溫SSF法存在的問題已NREL過程是專門立項(xiàng)的“生物乙醇”項(xiàng)目解決。(DOE Bioethanol Program)NRl過程(199)5ssF法與SHF法相比,雖SHF法乙醇的產(chǎn)率是以玉米秸桿為原料,通過稀酸預(yù)處理(190℃、10高,但SSF法耗時(shí)短,燃料乙醇的產(chǎn)量高3 SSF min),同時(shí)糖化和共發(fā)酵(木糖和葡萄糖)生產(chǎn)燃料法有連續(xù)或半連續(xù)工藝,半連續(xù)的SSF法可減少酶乙醇。預(yù)處理產(chǎn)物快速閃蒸分離,殘留在液相中的的用量醋酸通過連續(xù)的離子交換去除,漿料以石膏過量堿5.3直接發(fā)酵法中和脫毒。預(yù)水解液和漿料再進(jìn)行混合生產(chǎn)燃料乙直接發(fā)酵法是直接發(fā)酵纖維素生產(chǎn)乙醇,這種醇。發(fā)酵菌為可同時(shí)利用木糖和葡萄糖的重整Z方法設(shè)備簡單,但也存在一些問題。如纖維素發(fā)酵 Mobilis該方法的發(fā)酵溫度較低(30℃),發(fā)酵時(shí)間速率慢、容積生產(chǎn)力低;發(fā)酵產(chǎn)物中含有乙酸、酸長達(dá)7d。乙醇采用蒸餾和分子篩吸附提純。蒸餾等有機(jī)酸和氫氣等,乙醇產(chǎn)率低。常用的解決方法塔底出料為固體木質(zhì)素/細(xì)胞,可燃燒生產(chǎn)蒸汽,是與不分解纖維素的嗜熱菌混菌培養(yǎng),利用游離單蒸汽用來發(fā)電,塔底出料液體蒸發(fā)濃縮回收水。糖產(chǎn)生乙醇。如耐熱 Clostridia可在厭氧條件下用這一過程每升的燃料乙醇生產(chǎn)成本約為0.395美于混菌培養(yǎng)并提高燃料乙醇的產(chǎn)量。元,若提高預(yù)處理過程效率,提高發(fā)酵溫度5.4發(fā)酵過程的促進(jìn)(55℃),構(gòu)建可利用多種糖的產(chǎn)醇重組菌,提高發(fā)酵過程可用磁場添加表面活性劑等促進(jìn)方纖維素酶的生產(chǎn)能力和酶活性,每升燃料乙醇的法提高乙醇產(chǎn)率和產(chǎn)量。強(qiáng)磁場不會改變微生物細(xì)成本可降至0.248美元。最近有報(bào)道稱NREL的胞的宏觀特征,僅影響生物量和燃料乙醇的產(chǎn)合作伙伴 Novozymes公司降低了纖維素酶的生產(chǎn)量,可使生物量提高2.5倍,乙醇濃度提高3.4成本,每升燃料乙醇消耗纖維素酶的成本由最初倍。在SSF過程中加入吐溫20(約2.5g/L),反的1.32美元降至0.079美元,美國 Genencor公司應(yīng)速率加快,乙醇產(chǎn)率提高了8%,同等乙醇產(chǎn)率下甚至降至0.026~0.053美元03。生物法低成本酶的用量可減少50%,SF過程結(jié)束時(shí)酶的活性有生產(chǎn)燃料乙醇面臨突破。所提高8。ogen公司是加拿大工業(yè)纖維素酶和半纖維素通過完善酶解發(fā)酵工藝,也可提高乙醇產(chǎn)率,降酶的主要生產(chǎn)商,2003年建造了40/d的纖維素制低生產(chǎn)成本濃縮SE預(yù)水解液時(shí),抑制物的濃度燃料乙醇的裝置(并與其酶的生產(chǎn)相結(jié)合)。此后,也同時(shí)增加,降低了可發(fā)酵性。在濃縮物中添加富 logen公司和加拿大石油公司合作投產(chǎn)了世界上最含葡萄糖的纖維素水解液,可顯著改善發(fā)酵效大的以纖維素廢料為原料生產(chǎn)燃料乙醇的工業(yè)裝果纖維素酶和纖維二糖酶的成本高,可采用超置,可使12~15kt/a谷物秸桿轉(zhuǎn)化為(3~4)×106濾回收0。L/a燃料乙醇,預(yù)計(jì)采用新技術(shù)后生產(chǎn)費(fèi)用可降至6乙醇濃縮提純工藝0.24美元/ Iogen過程首先將纖維素原料進(jìn)行稀酸催化的SE預(yù)處理,然后用纖維素酶水解糖從生物質(zhì)發(fā)酵液中提純乙醇,傳統(tǒng)的工藝是采化最后進(jìn)行SHF發(fā)酵生產(chǎn)乙醇。預(yù)處理產(chǎn)物用蒸餾的方法,由于乙醇與水可形成共沸物,因此需用堿調(diào)節(jié)pH=5,加入纖維素酶發(fā)酵原液在50℃水萃取精餾、恒沸精餾以制取無水乙醇,能耗高。滲透解5~7d。水解固相過濾分離,濾液與水解液以酒第2期陳輝等生物質(zhì)制燃料乙醇113精酵母發(fā)酵葡萄糖生產(chǎn)乙醇,蒸餾提純乙醇。木半纖維素可發(fā)酵生產(chǎn)乙醇、木糖醇、丁二醇等121質(zhì)素用于燃燒提供熱量和蒸汽。 Iogen過程最大木質(zhì)素可直接燃燒轉(zhuǎn)化為能量,或熱解生產(chǎn)合成氣、的優(yōu)勢在于其纖維素酶的現(xiàn)場利用,以原液方式燃料油,或用于生產(chǎn)木質(zhì)素磺酸鹽等化學(xué)品;高純度加入,省去了酶的制備費(fèi)用,且一部分水解液可用的纖維素可降低纖維素酶的用量,提高葡萄糖產(chǎn)率。于酶的生產(chǎn)105陳洪章等22將SE法處理的麥草用水抽提出其中 IIT Delhi過程5以稻草為原料,預(yù)處理采用自的半纖維素,再用乙醇萃取木質(zhì)素后得到纖維素,纖動水解和乙醇抽提脫木質(zhì)素兩步法。自動水解過程維素的酶解率可達(dá)到90%以上。 Ligno過程以乙醇中約有70%的半纖維素水解,水解液中含以木糖為為溶劑從木材中分離木質(zhì)素和半纖維素以獲得具有主的多種糖,此水解液用于培養(yǎng) Utilis用作動物酶可及度高的纖維素,乙醇和水可循環(huán)利飼料)。自動水解的固體殘?jiān)诖呋瘎┑拇嬖谙掠?124 Pure Vision過程[15是將木質(zhì)纖維素原(170℃、30min)用體積分?jǐn)?shù)50%的乙醇抽提脫木料用水、稀堿、堿性氧化劑在較高溫度下采用串聯(lián)、質(zhì)素。兩步法預(yù)處理后的纖維素(76%)進(jìn)行SSF。逆流的方法洗滌,然后在獨(dú)立的反應(yīng)器中連續(xù)進(jìn)行ssF過程采用 Reesi纖維素酶、 Wentiiβ葡萄SE,其液相組分中主要含有木質(zhì)素、半纖維素和其糖苷酶、 Acidothermophilum為發(fā)酵菌種。SS過程他抽提物,固相組分主要是高反應(yīng)活性的高純度纖采用間歇加料,伴有程序真空回收過程。由于溶劑維素。預(yù)處理過程所需能耗高,燃料乙醇的生產(chǎn)成本約為9結(jié)語0.544美元/L。近年來,生物質(zhì)制燃料乙醇的研究和應(yīng)用取得8生物質(zhì)全利用整體工程了很大的進(jìn)展,燃料乙醇的生產(chǎn)成本大幅度降低,但規(guī)模經(jīng)濟(jì)可降低生產(chǎn)成本,但植物纖維素原料燃料乙醇的生產(chǎn)僅是整個生物質(zhì)加工工程的一部的運(yùn)輸往往限制其規(guī)模的擴(kuò)大1事實(shí)上,生分,從可再生的生物質(zhì)獲取多種產(chǎn)品和能量將是未物質(zhì)制燃料乙醇的能耗高于乙醇燃燒生成的能量,來不可阻擋的發(fā)展方向。近期的發(fā)展方向?yàn)?如以玉米粒為原料的能耗比產(chǎn)能高29%,以軟枝草(1)繼續(xù)降低纖維素原料制燃料乙醇的成本。為原料的能耗比產(chǎn)能高50%以上,以木材為原料的目前,纖維素原料制燃料乙醇的成本仍然很高,其制能耗更高(為57%以上)107。因而,單一乙醇產(chǎn)品約因素主要是預(yù)處理過程和纖維素酶,因而有待在的經(jīng)濟(jì)性差, Hamelinck的技術(shù)經(jīng)濟(jì)評估認(rèn)為,基于稀技術(shù)和菌種上進(jìn)一步突破。酸水解工藝,生產(chǎn)效率為35%,結(jié)合木質(zhì)素發(fā)電后生(2)擴(kuò)大乙醇的利用范圍。由于乙醇易于反應(yīng)產(chǎn)效率可達(dá)到60%。改進(jìn)預(yù)處理和生物技術(shù)后生生產(chǎn)乙烯,因而目前95%來自石油的合成聚合物可產(chǎn)效率可達(dá)48%和68%108因而生物質(zhì)制燃料乙從生物質(zhì)資源中獲得1261醇生產(chǎn)過程應(yīng)形成多元化產(chǎn)品或產(chǎn)能的整體工程,提(3)提高生物質(zhì)整體化工程的技術(shù)經(jīng)濟(jì)性。未高整體經(jīng)濟(jì)性,以降低燃料乙醇的生產(chǎn)成本。來的生物質(zhì)加工廠的經(jīng)濟(jì)效益來自多種產(chǎn)品而不是整體化工程是生物質(zhì)和能量全利用1的過僅依靠一種產(chǎn)品。正如石油煉廠的多模式一樣,生程:一要結(jié)合多種加工方法,如發(fā)酵、蒸汽重整、氣物質(zhì)加工廠也應(yīng)面向市場,因地制宜,具有足夠的靈化、液化、熱解、水解、快速裂解10、電解二要活性2充分利用生物質(zhì),形成多元化產(chǎn)品。日本充分利用參考文獻(xiàn)發(fā)酵廢渣,進(jìn)行水熱催化氣化生成乙醇,乙醇產(chǎn)率由25%增至30%~36% Wilkening等13利用1錢伯章世界乙醇產(chǎn)業(yè)開始步入黃金發(fā)展期中國石化200餾物清液生產(chǎn)甲烷氣。多元化產(chǎn)品可以燃料(如(4):26-27CH4、氫氣、合成氣、乙醇、生物油等)為中心,還2張曉陽論國內(nèi)發(fā)展燃料乙醇的優(yōu)勢及前景中國能源,200可副產(chǎn)天然纖維、甜味劑、單細(xì)胞蛋白、乳酸、微1(1):106~120生物酶16、活性炭7、優(yōu)質(zhì)蛋白飼料8、土壤改3 Nagata Alcohol Fermentation from Construction Waste Jap良劑119、合劑120等 Ins 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