木質(zhì)纖維生產(chǎn)燃料乙醇的生物轉化技術(shù)

第43卷第5期林業(yè)科學(xué)VoL 43. No. 52007年5月SCIENTIA SILVAE SINICAEMay,2007木質(zhì)纖維生產(chǎn)燃料乙醇的生物轉化技術(shù)陳介南王義強何鋼章懷云周再魁(中南林業(yè)科技大學(xué)生物環(huán)境科學(xué)與技術(shù)研究所長(cháng)沙410004摘要:木質(zhì)纖維生物轉化乙醇可以避免以糧食為原料生產(chǎn)燃料乙醇所帶來(lái)的“與農爭地”和“與人爭食”的弊端,兼得經(jīng)濟、生態(tài)、環(huán)保、社會(huì )多重效益。木質(zhì)纖維原料的物理化學(xué)特性決定其具有不同于淀粉和糖類(lèi)原料轉化的難度和復雜性,這也是木質(zhì)纖維生物轉化乙醇成本居高難下、商業(yè)化裹足不前的主要技術(shù)原因。本文以國際(特別是美國)的研究和開(kāi)發(fā)進(jìn)展為背景,探討木質(zhì)纖維生物轉化乙醇的幾個(gè)富有挑戰性的技術(shù)問(wèn)題,并在同步糖化共發(fā)酵(SSCF)概念的基礎上,提出優(yōu)化的并行糖化共發(fā)酵生產(chǎn)模型。關(guān)鍵詞:生物能源;燃料乙醇;木質(zhì)纖維;生物轉化;發(fā)酵工程中圖分類(lèi)號:TQ352文獻標識碼:A文章編號:1001-7488(2007)05-0099-07Bioconversion of lignocellulose to EthanolChen Jienan Wang Yiqiang He Gang Zhang Huaiyun Zhou Zaikui(Institute of Biological and Enironmental Science Technology, Central South University of Forestry and Technology Changsha 410004)Abstract: An overview of recent advances in biological lignocellulose to ethanol technology is presented. The emphasis ofdiscussion is especially on pretreatment, enzymatic/metabolic engineering, C/Cs cofermentation, lignin utilization, and processoptimization, which are of primary importance to the successful application and commercialization of lignoceellulose-basedbioethanol technology. A consolidated lignocellulose-to-ethanol process model is proposed as conceptual improvement to thesimultaneous saccharification and cofermentation( SSCF) model endorsed by U. S. DOE national renewable energy laboratoryNREL)and modified by various industrial applicaKey words: bioenergy ethanol lignocellulose; bioconversion; fermentation生物乙醇( bioethanol)是一種最易普及的生物燃料(相較于生物柴油和燃料電池),憑借其經(jīng)濟、環(huán)保、實(shí)用等多維優(yōu)勢而日益成為替代汽油等傳統能源的新能源之星。迅速普及的汽油醇(10%乙醇、90%汽油)以酒精作為助燃/抗爆劑直接取代 MTBE( methyl tertiary butyl ether)。世界各主要汽車(chē)制造商都能生產(chǎn)以乙醇汽油(乙醇含量達85%)為燃料的汽車(chē)(如美國),甚至飛機也可以使用乙醇燃料(如巴西)。目前全世界超過(guò)90%的乙醇來(lái)自生物質(zhì),但多以糧食[如玉米( Zea mays)、小麥( Triticum aestivum)、高粱( Sorghum vulgare)]和糖類(lèi)作物[如甘蔗( Saccharum officinarum)、甜菜( Beta vulgaris)]為原料( Badger,2002)木質(zhì)纖維是地球上數量最大的一種可再生資源。據估算,如果發(fā)展能源林業(yè)與回收利用廢棄木質(zhì)纖維(可回收部分約占總量的1/3)并舉,則每年可保障替代1/3以上運輸燃料的酒精產(chǎn)量。為此,發(fā)展經(jīng)濟有效而環(huán)境友好的木質(zhì)纖維生物轉化乙醇技術(shù),已成為世界生物能源科技發(fā)展的戰略制高點(diǎn)。美國等國家已開(kāi)始將生物能源研究和開(kāi)發(fā)的戰略重心轉移到以木質(zhì)纖維生產(chǎn)乙醇的方向。針對我國國情,發(fā)展以木質(zhì)纖維生物轉化乙醇為重點(diǎn)的生物能源技術(shù),建立以能源林業(yè)為依托并綜合利用大量農業(yè)廢棄物(如秸稈、皮殼等)( Kim et al.,2004)、林業(yè)殘余物(如鋸木屑、刨花、木材和造紙工業(yè)廢料等)、城鄉垃圾和雜草等低成本木質(zhì)纖維資源的原料保障模式,可以避免以糧食為原料所帶來(lái)的“與農爭地”和“與人爭食”的弊端,兼得經(jīng)濟、生態(tài)、環(huán)保、社會(huì )多重效益,是全面協(xié)調可持續發(fā)展的必然要求。木質(zhì)纖維( lignocellulose)主要由纖維素( cellulose(約占1/3-1/2)、半纖維素( hemicellulose)(約占14-1/3)和木質(zhì)素( lignin)(約占1/5~1/4)組成。纖維素和半纖維素經(jīng)過(guò)糖化和發(fā)酵轉化為乙醇( ethanol),而木質(zhì)素的降解物不含可發(fā)酵糖,只能通過(guò)燃燒供熱(或發(fā)電)或化學(xué)轉化為燃料添加劑(或其他生物制品或工業(yè)原料)加以綜合利用(圖1)。中國煤化工木質(zhì)纖維原料的糖化液中所含的可發(fā)酵糖( fermentableCNMHG素的葡萄糖(D收稿日期:2006-08-08基金項目:國家948項目(2006-4-123)。林業(yè)科學(xué)卷glucose)、甘露糖(D- mannose)和半乳糖(D- galactose)等6碳(C5)H-C-糖,以及源于半纖維素的木糖H-C-OH HO-C-OH(D- xylose)和阿拉伯糖(L-H-C-OH H-C-OHHO-C-Harabinose)等5碳(C5)糖。普通CHrOH CH-OH葡萄糖甘露糖拉伯糖微生物(如酵母)只能發(fā)酵CArabin糖,對C3缺乏代謝能力,而且易H-C-OHH-C-OHC3發(fā)酵受降解產(chǎn)物的抑制木質(zhì)纖維原料的理化特性CH,CH:OH決定了其具有不同于淀粉(如玉米、高粱)和糖類(lèi)(如甘蔗、甜菜)燃燒供熱供電原料轉化的難度和復雜性,因此木質(zhì)纖維生物轉化是復雜且存Ch fermentation燃料添加劑生物乙醇在許多不確定因素的動(dòng)態(tài)過(guò)程Bioethanol( Lin et al.,2006)。將木質(zhì)纖維原料經(jīng)濟有效地轉化成乙醇和圖1木質(zhì)纖維原料的構成及其利用其他生物產(chǎn)品,取決于許多因Fig. 1 Structure and use of lignocellulose material素。本文試圖以國際(特別是美國)的研究和開(kāi)發(fā)進(jìn)展為背景,探討木質(zhì)纖維生物轉化乙醇的幾個(gè)富有挑戰性的技術(shù)問(wèn)題,特別是預處理、酶活性、C/C3糖共發(fā)酵、木質(zhì)素的綜合利用和工藝優(yōu)化1經(jīng)濟有效的快速預處理技術(shù)木質(zhì)纖維材料中的可糖化纖維素和半纖維素在木質(zhì)素包裹下形成穩定的結構,很難被直接水解或酶解纖維素由糖分子的極性基團(- OH groups)通過(guò)氫鍵( hydrogen bonds)相連成長(cháng)鏈,在木質(zhì)素的包裹下形成堅固而穩定的晶體結構,在普通條件下不易被水解或稀酸(稀堿)糖化,酶解糖化是最為經(jīng)濟有效而環(huán)境友好的途徑。半纖維素分子和纖維素類(lèi)似,也是由糖分子構成長(cháng)鏈,但其非晶體(隨機、不規則)結構比較脆弱,不像纖維素那樣強穩,很容易在稀酸(或稀堿)預處理中被糖化。木質(zhì)素在植物體中起結構強化和支撐作用,也是植物抵御病菌侵襲的物理障礙,不容易被微生物降解,特別是在缺氧條件下。富氧條件下木質(zhì)素的生物降解當緩慢(需要許多天),難以達到工業(yè)規模生產(chǎn)的要求。木質(zhì)纖維原料通常需要適當的預處理( pretreatment)來(lái)破除木質(zhì)纖維的晶體結構及木質(zhì)素對纖維素和半纖維素的包裹,增加供酶接觸的有效表面積( Grethelin,985),并消除對微生物有危害的毒性物質(zhì),以利生物降解( Gharpuray et al.,1983)預處理方法可以是物理的、化學(xué)的、生物的,也可以取其交合運用,常用的如表1所示。在選擇預處理方法時(shí),要考慮木質(zhì)纖維原料的特點(diǎn)、經(jīng)濟和環(huán)保性能要求,以及整體工藝設計,避免盲目性。根據文獻,最為看好的是蒸爆( steam explosion)酸解( acidic hydrolysis)和堿濕氧化( alkaline wet oxidation)2C/C5糖共發(fā)酵微生物代謝工程技術(shù)木質(zhì)纖維含有半纖維素,其糖化不僅產(chǎn)生C糖( D-glucose及其異構體),而且有C3糖(D- xylose和Larabinose)。由于普通酵母(yeas)只能發(fā)酵C,對Cs缺乏代謝能力,而且易受降解產(chǎn)物的抑制,半纖維素的利用在相當長(cháng)的時(shí)期被忽視,直接影響了木質(zhì)纖維原料生物轉化乙醇的經(jīng)濟效益。尚無(wú)天然微生物能夠將木質(zhì)纖維糖化液中的C6和C糖同時(shí)快速有效地轉化為乙醇( Ingram et al.,1995),盡管一些微生物(如Neurospora crassa)能夠將纖維素和半纖維素直接轉化成乙醇c1 1004. DL-dtare et al., 1997).因此,開(kāi)發(fā)出能對C和C3糖進(jìn)行快速完全、同步發(fā)酵并且擴中國煤化工成為當務(wù)之急。通過(guò)遺傳重組來(lái)解決C5C5糖共發(fā)酵難題已在一些微CNMHG見(jiàn)的 Saccharomycecerevisiae(Johansson et al., 2001), Zymomonas mobilis( Sprenger, 1993)Fu Escherichia coli( Burchardt et al., 199Dien et al.,2003; Zhou et al.,1999)。這些是研究和應用基礎相對較好的,也是最成功的重組平臺( zaldivar第5期陳介南等:木質(zhì)纖維生產(chǎn)燃料乙醇的生物轉化技術(shù)101表1常用木質(zhì)纖維原料預處理方法Tab. 1 Lignocellulose pretreatment methe預處理方法描述特點(diǎn)參考文獻Pretreatment methodReference碎機械研磨碾壓,或打碎,效果與顆粒大小和分布有關(guān)工藝要求不高,但能耗大,效率低 Simple,蒸爆高溫(200℃)高壓蒸汽將小塊化原料爆破數十秒至幾分能耗高,要求特殊設備,有時(shí)糖化效果不elevated temperatures(200 C)and t High energy input, specicalized equipmentpressures for 30 seconds to a few minutesvariable saccharification efficiency酸/堿水解稀酸(1%H2SO4)或稀堿(2%-5%NaOH)和高溫(100較為經(jīng)濟有效,適合各種木質(zhì)纖維原料200℃)下進(jìn)行,時(shí)間不- Dilute acid(1%H2SO4)or但影響后繼反應環(huán)境 Efective, suitable for Grethelin(2%-5% NaOH)hydrolysis at elevated temperatures most lignocellulosic feedstocks, but affects(100-200C), with various reaction timessubsequent process environment堿濕氧化弱堿Na2CO3溶液加溫加壓加氧處理10min左右避免產(chǎn)生大量對發(fā)酵微生物有抑制作用 Linke et alAlkalineOxidation in weak alkaline solution at elevated temperatures的降解物 Avoid inhibitory products such2002: Schmidtoxidation (150-200 C)for 10-15 min, usually with added oxygen furfuraleral.,1998Ammonia/10%氨溶液浸泡24-48h,脫除大部分木質(zhì)素10%可能損失部分半纖維素(特別是氨濃度較氨處理lapagliatreatment ammonia/urea treatment for 24-48 h, most lignin removed高時(shí)) Partial loss of hemicellulose, especiallyat high ammonia/ urea concentrations使用白院菌等降解本質(zhì)素的做生物Uwm,數周)Sm:m)Hawada ef al. 1995eral,,2001)。重組S. cereuisiae發(fā)酵路徑遇到的一個(gè)棘手問(wèn)題是C(如L- arabinose)轉運( transport)不好,使發(fā)酵達不到理想效果。目前美國能源部的國家可再生能源實(shí)驗室(NREL)已經(jīng)在攻克這一難題。美國佛羅里達大學(xué)的 Ingram教授領(lǐng)導的實(shí)驗室于20世紀80年代就嘗試了以外源基因補充(或改進(jìn))受體微生物的發(fā)酵代謝途徑( Ingram et al.,1987)。他們將 Zymomonas mobilis的C發(fā)酵基因轉入具備C發(fā)酵途徑的 Escherichia coli,除了成功使大腸桿菌獲得在富氧( aerobic)和缺氧( anaerobic條件下發(fā)酵 pyruvate的功能外,這個(gè)由 lactose啟動(dòng)子( promoter)加 pyruvate decarboxylase(pd)和 alcohol dehydrogenase(adh)基因串聯(lián)組合的操作元( operon)還使 pyruvate的代謝具有向發(fā)酵 ethanol方向傾斜的選擇性,并使微生物生長(cháng)明顯優(yōu)于對照。這一操作元在 Clostridium cellulolyticum中也得到較好的表達( Guerdon et al.,2002; Jennet2000)。相對于酵母發(fā)酵(以小時(shí)計)來(lái)說(shuō),細菌( bacteria)發(fā)酵有速度快(以分鐘計)的優(yōu)勢( Badger,2002)。NREL資助的研究( Zhang et al.,1998)成功地把C糖代謝所需的7個(gè)外源酶基因(包括 araBAD Operon, Lee et al.,1986)以3個(gè)操作元串聯(lián)在一個(gè)質(zhì)粒載體( vector)轉入2 ymomonas mobilis并獲得了80%~90%的CC共轉化率。從遺傳重組的 Zymomonas mobilis表型分析可以推導,這3個(gè)操作元( Pea xylA/xylB, P-araBAD和Pal/tkt)通過(guò)ED途徑( entner- doudoroff pathway)( Conway,1992)與內源控制 pyruvate發(fā)酵的操作元(Pl-pdaadh)( Ingram et al.,1987)并聯(lián)成完整的CC5發(fā)酵代謝體系(圖2)。ED途徑是 glucose→ pyruvate的3個(gè)已知代謝途徑中(其他是 glycolysis和 pentose phosphate pathway)較簡(jiǎn)單而有效的一個(gè)。3提高酶活性的微生物酶工程技術(shù)木質(zhì)纖維的生物降解是由一系列纖維素酶、半纖維素酶和木質(zhì)素酶組成的復雜系統來(lái)執行的。一般纖維素酶生產(chǎn)菌也生產(chǎn)半纖維素酶(及或多或少的木質(zhì)素酶)(如 Phanerochaete chrysosporium)( Broda et al.,1994)。因木質(zhì)素不含可發(fā)酵糖,其降解是個(gè)綜合利用的問(wèn)題,不是糖化的目標。在工業(yè)規模的木質(zhì)纖維生物轉化中,大部分半纖維素在預處理階段就被糖化,而且許多中國煤化工性,故纖維素水是酶解糖化階段的重點(diǎn)對象。纖維素酶按存在狀態(tài)可分為自由合酶( complexed即 cellulosomes)系統。好氧真菌(最著(zhù)名的是 Trichoderma reeseiCNMHGa5和 Thermobifida)般產(chǎn)生自由纖維素酶,而絡(luò )合纖維素酶系統則主要源于厭氧細菌(如 Clostridium和 Ruminococcus)和少數厭氧真菌(如 Piromyces)林業(yè)科43卷木糖阿拉伯糖-arabinoseED途徑Operon磷酸戊糖途徑5-磷酸酸木酮糖操縱元OperonGlycolysis6確酸果糖NADH3-確酸甘油P圖2木質(zhì)纖維生物轉化乙醇中C/C3共發(fā)酵的可能代謝途徑Fig. 2 Probable pathway for C/Cs cofermentation of lignocellulose to ethanolrag: glyceraldehyde-3-phosphate dehydrogenase promoter; ayLA xylose isomerase; rydB: xylulokinase: araA L-arabinose isomerase;al: transaldolase; tk: transketolase(Zhang et alpromoter: pdc: pyruvate decarboxylase; adh alcohol deh個(gè)纖維素酶系統按作用方式可大致分為:內切葡萄糖苷酶( endoglucanases)(EC3.2.1.4);外切葡萄糖苷酶( exoglucanases),包括 cellodextrinases(EC3.2.1.74)和 cellobiohydrolases(EC3.2.1.91)和(外切型)葡萄單糖酶( glucosiEC3.2.1.21)(圖3)。纖維素酶成本的居高難下始終是葡萄糖苷酶(胞內物特酶)個(gè)困擾木質(zhì)纖維生物轉化乙醇技術(shù)產(chǎn)業(yè)萄糖苷酶化的主要障礙之一( McAloon et al內切葡萄糖苷酶纖維二糖水解Endoglucanase2000)。遺傳重組技術(shù)不僅用于控制(或纖維素纖維二糖(1省萄糖Cellulose調節)發(fā)酵代謝途徑( metabolic pathway)(Glucose )nlobiose( Ingram et al.,1998),而且也是纖維素酶內切葡萄糖苷酶+纖維二糖水解酶EndoglucCellobiohydrolase優(yōu)化(增強活性)的一個(gè)重要技術(shù)手段內切葡萄糖苷酶(葡萄糖苷酶)xoglucosidase(B-glucosidase,1992)應用遺傳重組技術(shù)開(kāi)發(fā)優(yōu)質(zhì)纖維素圖3纖維素酶降解系統酶、半纖維素酶和木質(zhì)素酶工程菌,高活Fig. 3 Biodegradation of lignocell性纖維素酶酵母工程菌和產(chǎn)朊假絲酵母工程菌等GEM材料已取得顯著(zhù)成績(jì),包括將纖維素酶編碼序列克隆到細菌、酵母、霉菌和植物中,以產(chǎn)生新的更優(yōu)質(zhì)纖維素酶( Lynd et al.,2002)。一個(gè)重要進(jìn)展是細胞表面工程技術(shù)( Murai et al.,1998),即在酵母細胞表層蛋白(a-凝聚素等基因上植入纖維素酶等基因,使菌體和酵素在反應器內始終保持高濃度,持續反復利用,從而降低成本,提高糖化效率。歸納起來(lái),微生物酶工程技術(shù)策略可分為:1)選用抗逆高產(chǎn)工程菌(如NREI/ GENENCOR采用嗜熱耐酸性的 Acidothermus cellulolyticus),改進(jìn)纖維素酶的就地生產(chǎn)(on- site production)( Thygesen et al.,2003);2)用異源纖維素酶(如源于 bacteria, fungi, and plants)重組更有效的纖維素酶系統(例如,在最常用的 Trichodermaree維素酶系統的基礎上添加 Aspergillus的 B-glucosidases,從而克服 glucose的抑制)( Gunata et al.,199Yan et al.,1997);3)將外源纖維素酶基因轉入優(yōu)質(zhì)發(fā)酵工mobi),使之至少能供應木質(zhì)纖維直接轉化乙醇所需的部中國煤化工2004: Zhou et al1999);4)將外源發(fā)酵基因轉入纖維素酶生產(chǎn)菌(如 TrichodermCNMHGcum),實(shí)現酶解和發(fā)酵工程菌的一體化( Guedon et al.,2002);5)運用代謝工程策略增加欲求的( gene transfer)、強化有利的( gene overexpression)和排除(或弱化)不利的( gene suppression/ gene knockout)( Kamionka et al.,2005)代謝途徑第5期陳介南等:木質(zhì)纖維生產(chǎn)燃料乙醇的生物轉化技術(shù)103( Lynd et al.,2002)。此外,應用納米技術(shù)進(jìn)行分子設計,可以“對號入座”制造與纖維素酶結構和功能類(lèi)似的納米催化劑,獲得新的或更加穩定轉化的催化途徑,并實(shí)現催化劑的固定重復循環(huán)使用。同時(shí),通過(guò)納米傳感器和無(wú)線(xiàn)網(wǎng)絡(luò )對酶解/發(fā)酵過(guò)程進(jìn)行智能化在線(xiàn)監控,可以實(shí)時(shí)精確地優(yōu)化動(dòng)態(tài)反應條件,提高酶解/發(fā)酵效率。4環(huán)境友好的綜合利用技術(shù)木質(zhì)素不含糖,目前還沒(méi)有發(fā)現將其轉化為乙醇的有效方法,其主要用途是燃燒發(fā)電或供熱。開(kāi)發(fā)高附加值的木質(zhì)素綜合利用技術(shù),通過(guò)增加收益來(lái)降低(抵消一部分)木質(zhì)纖維生物轉化乙醇的生產(chǎn)成本,是使經(jīng)濟和環(huán)保相得益彰的必然選擇。由木質(zhì)素生產(chǎn)高 octane的燃料添加劑( Ragauskas et al.,2006; Shabtai etal.,1999技術(shù)已取得進(jìn)展,將纖維素(和半纖維素)直接化學(xué)轉化為高附加值產(chǎn)品(特別是燃料成份)的技術(shù)也在探索中( Miller et al.,1999 Huber et al.,2005; Ragauskas et al.,2006; Rostrup- Nielsen,2005),對促進(jìn)木質(zhì)纖維資源綜合有效利用及提高生物乙醇技術(shù)的市場(chǎng)競爭力將大有助益。如圖4所示,木質(zhì)纖維原料可以生成多種高附加值的主產(chǎn)品和副產(chǎn)品(包括燃料添加劑、生物塑料、化學(xué)品等)。盡管酒精燃燒時(shí)產(chǎn)生的熱木質(zhì)纖維能比汽油低(酒精為22.1MJ·L'、汽油為34.3MJ·L1)( Nordin,1994),但酒精的辛烷值纖維素半纖維素比汽油高許多,是抗爆劑,又是CelluloseHemicellulose助燃劑,所以用汽油醇作燃料不用再添加四乙基鉛或MIBE,就生物塑料化學(xué)制品可成為高標號燃料油,可減少空熱電聯(lián)產(chǎn)燃料添加劑清潔燃料氣中鉛的污染。Fuel additives5木質(zhì)纖維生物轉化乙醇工藝的優(yōu)化氧化劑木質(zhì)纖維生物轉化乙醇技Methyl tertiary butyl ether術(shù)的發(fā)展是一個(gè)漸進(jìn)的過(guò)程。高標 Premium93最初是從化學(xué)轉化演變而來(lái),即用酶解替代酸解,再進(jìn)行發(fā)酵,圖4木質(zhì)纖維的綜合利用即所謂的分步糖化發(fā)酵Fig. 4 Lignocellulose utilization( saccharification and fermentation,簡(jiǎn)稱(chēng)SF)法( Wilke et al.,1976)。SF法存在纖維素酶受葡萄糖( glucose)和纖維二糖( cellobiose)終產(chǎn)物抑制,酶解效果差、酶制劑用量大的弊端。因此,同步糖化發(fā)酵( simultaneoussaccharification and fermentation,簡(jiǎn)稱(chēng)SSF)法( Gauss et al.,1976)便應運而生。SSF法使水解( hydrolysis)與發(fā)酵( fermentation)兩步合一,消除了SF法的弊端,提高了糖化效率隨著(zhù)遺傳重組微生物技術(shù)的應用( Ingram et al.,1991; Zhang et al.,1998),以前無(wú)法實(shí)現的C/C3糖共發(fā)酵成為可能。日前相對比較成熟的是美國能源部的國家可再生能源實(shí)驗室(NREL)推薦的同步糖化共發(fā)酵( simultaneous saccharification and cofermentation,簡(jiǎn)稱(chēng)SSCF)法。SSCF法把源于半纖維素的木糖等C3糖和源于纖維素的葡萄糖等C糖一道轉化成乙醇,從而提高了轉化效率降低了生產(chǎn)成本。在 NREL Model的應用中,通常用稀酸(≤1%H2SO)進(jìn)行預處理,然后漿液的一小部分供送生產(chǎn)纖維素酶的微生物培養罐,其余流向發(fā)酵罐,同時(shí)加入纖維索酶和發(fā)酵微生物,實(shí)現同步糖化共發(fā)酵。通常絕大部分半纖維素在預處理過(guò)程中便被糖化,所釋V凵中國煤化工發(fā)酵。圖5是基于SSCF概念( NrEL Model)改進(jìn)的并行糖化共發(fā)酵生產(chǎn)模型(工藝CNMHG將預處理釋放的C糖化液并聯(lián)發(fā)酵,以及對預處理后的殘留物(主要含纖維素和木質(zhì)素及少量半纖維素)進(jìn)行同步糖化共發(fā)酵結論木質(zhì)纖維原料Lignocellulos木質(zhì)纖維生物轉化乙醇是Cs糖發(fā)酵個(gè)動(dòng)態(tài)發(fā)展、不斷完善的技術(shù)。乙醇回收技術(shù)進(jìn)步的核心方向(或最終目酶生產(chǎn)hanol recoveryEnzyme production的)是要降低生產(chǎn)成本,提高生物乙醇的市場(chǎng)(價(jià)格)競爭力基本策略是生物的(如工程菌和纖維素酶)和工藝的(如預處理,預處理綜合利用,和系統集成)優(yōu)化并同步糖化共發(fā)酵SSCE舉,特別是生物的改進(jìn)尚有較大固殘余物處理的空間,而纖維素酶活性的提高Solid residual treatment是關(guān)鍵。優(yōu)質(zhì)高產(chǎn)抗逆C/C5木質(zhì)纖維生物轉化乙醇的并行糖化共發(fā)酵生產(chǎn)流程共發(fā)酵工程菌和產(chǎn)酶工程菌的Fig. 5 Optimized SSCF process for bioconversion of lignocellulose to ethanol開(kāi)發(fā)所取得的進(jìn)展,為木質(zhì)纖維consolidated from NREL model and its various industrial modifications生物轉化乙醇技術(shù)奠定了發(fā)展基礎。就工藝而言,本文根據美國能源部可再生能源實(shí)驗室(NREL)所推薦的SSCF Model進(jìn)行概念性改進(jìn),提出了并行糖化共發(fā)酵生產(chǎn)模型。目前類(lèi)似的生物轉化模型已接近產(chǎn)業(yè)化(實(shí)用)階段,但其運行成本和效率取決于多方面因素,應該避免盲目地照搬硬套。不過(guò),可以謹慎樂(lè )觀(guān)地預期,隨著(zhù)汽油價(jià)格的飆升和生產(chǎn)成本的下降,木質(zhì)纖維生物轉化乙醇技術(shù)將越來(lái)越顯現其生命力和市場(chǎng)競爭力。參考文獻Badger P C. 2002. Ethanol from cellulose: a general review/ Janick J, Whipkey A. Trends in New Crops and New Uses. ASHS Press, Alexandria, VA, 17Basaglia M, Concheri G, Cardinali S, et al. 1992. Enhanced degradation of ammonium-pretreated wheat straw by lignocellulolytic Streptomyces spp. CanadianJournal of micorbiology, 38(10): 1022-1025Broda P, Birch P, Brooks P, et al. 1994. Phanerochaete chrysosporium and its natural substrate. FEMS Microbiol Rev, 13: 189-196Burchardt G, Ingram L 0. 1992. Conversion of xylan to ethanol by ethanologenic strains of Escherichia coli and Klebsiella oxytoca. Appl Environ Microbiol, 58:Conway T. 1992. The entner-doudoroff pathway history, physiology and molecular biology. FEMS Microbiol Rev, 9(1): 1-27Crawford D L. 1986. The role of actinomycetes in the decomposition of lignocellulose, FEMS Symp, 34: 715-728Deshpande V, Keskar S, Mishra C, et al. 1986. Direct conversion of cellulose/hemicellulose to ethanol by Neurospora crassa. Enzyme Microb Technol, 8: 149-152Dien B S, Iten L B, Nichuls NN, et al. 2003. Conversion of lignocellulose to ethanol using a recombinant E. coli strain. Society of Industrial MicrobiologyFoody P. 1980. Optimization of steam explosion pretreatment. Final Report to DOE, Contract ACO2-79ET23050auss W F, Suzuki S, Takagi M. 1976. Manufacture of alcohol from cellulosic materials using plural ferments. United States Patent 3,990,944,NovemberGharpuray MM, Lee Y H, Fan L T. 1983. Structural modification of lignocellulosics by pretreatments to enhance enzymatic hydrolysis. Biotechnology andBioengineering, 25( 11): 157-172Grethelin H E, 1985. The effect of pore size distribution on the rate of enzymatic hydrolysis of cellulosic substrates. Bio/Technology, 3: 155-160udon E, Desvaux M, Petitdemange H. 2002. Improvement of cellulolytic properties of clostridium cellulolyticum by metabolic engineering, Appl EnvironMicrobiol, 68(1): 53-58unta Z, Vallier M J. 1999. Production of a highly glucose-tolerant extracellular B-glucosidase by three Aspergillus strains. Biotechnol Lett, 21: 219-223Hatakka A 1. 1983. Pretreatment of wheat straw by white rot fungi for enzymic saccharification of cellulose. Appl Microbiol Biotechnol, 18(6):Hogsett D A, Ahn H J, Bemardez T D, et al. 1992. Direct microbial conversion: persobstacles. ADpl Biochem Biotechnol, 35: 527-542Huber G W, Chheda J N, Barrett J, Dumesic J A. 2005, Production of liquid alkanes中國煤化工 carbohydrates,senm308:1446-1450CNMHGngram L O, Conway T, Alterthum F. 1991. Ethanol production by Escherichia coli strainstor-eun genes. United States PatentIngram L O, Conway T, Clark D P, ef al. 1987. Genetic engineering of ethanol production in Escherichia coli. Appl Environ Microbiol, 53: 2420-2425第5期陳介南等:木質(zhì)纖維生產(chǎn)燃料乙醇的生物轉化技術(shù)Ingram L O, Doran J B. 1995. Comversion of cellulosic materials to ethanol. FEMS Microbiology Reviews, 16(2-3): 235-241ngram L O, Gomez P F, Lai X, et al. 1998. Metabolic engineering of bacteria for ethanol production. Biotechnol Bioeng, 58: 204-214Jenner K, Tardif C, Young D, ef al. 2000. Gene transfer to clostridium cellulolyticum ATCC 35319. Microbiology, 146: 3071-3080Johansson B, Christenson C, Hobley T, et al. 2001. Xylulokinase overexpression in two strains of Saccharomyces cerso expressing xylose reductase andxylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate. Appl Environ Microbiol, 67(9): 4249-4255Kim S, Dale B E. 2004. Global potential bioethanol production from wasted crops and crop residues. Biomass and Bioenergy, 26: 361-375Kamionka A. Bertram R, Hillen W. 2005. Tetracycline-dependent conditional gene knockout in Bacillus subtilis. Appl Environ Microbiol, 71(2): 728-733Kirk T K, Farrell R L. 1987. Enzymatic"combustion": the microbial degradation of lignin. Annu Rey Microbiol, 41: 465-505Klinke H B, Ahring B K, Schmidt A S, ef al. 2002, Characterization of degradation products from alkaline wet oxidation of wheat straw. BioresourceTechnology, 82: 15Ladisch M R, Lin K W, Voloch M, ef al. 1983. Process considerations in the enzymatic hydrolysis of biomass. Enzyme Microb Technol, 5(2): 82-10Lee N, Gielow W, Martin R, et aL. 1986. The Organization of the araBAD Operon of Escherichia coli. Gene, 47: 231-244Lin Y. Tanaka S. 2006. Ethanol fermentation from biomasss: current state and prospects. Appl Microbiol Biotechnol, 69(6): 627-642Lynd LR. Weimer P J. van Zyl WH, e al. 2002. Microbial cellulose utization: fundamentals andlogy. Microbiol Mol Biol Rev, 66(3): 506-577MeAloon A, Taylor F, Yee W, et al. 2000, Determining the cost of producing ethanol from com starch and lignocellulosic feedstocks. National ReLaboratory NREL/TP-580-28893, 43Miller J E, Evans L, Littlewolf A, et al. 1999. Batch microreactor studies of lignin and lignin model compound depolymerization by bases in alcohol solventsFuel,(78):1363-1366Murai T,Ueda M, Kawaguchi T, ef al. 1998. Assimilation of cellooligosaccharides by a cell surface-engineered yeast expressing-glucosidase andcarboxymethylcellulase from aspergillus aculeatus. Appl Environ Microbiol, 64(12): 4857-486INordin A. 1994. Chemical elemental characteristics of biomass fuels. Biomass and Bioenergy, 6: 339-347Panttila M E, Andre L, Saloheimo M, et al. 2004. Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyceces cererisiae. Yeast, 3(3): 175Phadtare S U, Rawat U B, Rao M B. 1997. Purification and characterisation of xylitol dehydrogenase from NeurosporaFEMS Microbiology Letters, 146:Ragauskas A ], Williams C K, Davison B H, et al. 2006. The path forward for biofuels and biomaterials. Science, 311: 484-489Rostrup-Nielsen J R. 2005. Making fuels from biomass. Science, 308: 1421-1422Sawada T, Nakamura Y, Kobayashi F, et al. 1995. Effects of fungal pretreatment and steam explosion pretreatment on enzymatic saccharification of plantbiomass, Biotechnology and BioengineeringShabtai J, Zmiercnak W, Kadangode S, et al. 1999. Lignin conversion to high-octane fuel additives. Proceedings of the Fourth Biomass Conference of theAmericas, Oakland, CA, 811-818Schmidt A S, Thomsen A B. 1998. Optimization of wet oxidation pretreatment of wheat straw. Bioresource Technol, 64: 139-151Sprenger G A. 1993. Approaches to broaden the substrate and product range of the ethanologenic bacterium Zymomonas mobilis by genetic engineering. JBiotechnol,27(3):225-237Thygesen A, Thomsen A B, Schmidt A S, et al. 2003. Production of cellulose and hemicellulose-degrading enzymes by filamentous fungi cultivated on wet.oxidised wheat straw. Enzyme and Microbial Technology, 32: 606-615Wilke C R, Yang R DStocker U. 1976OHYan T R, Lin C L, 1997, Purification and characterization of a glucose-tolerant B-glucosidase from Aspergillus niger CCRC 31494. Biosci Biotechnol Biochem6l:965-970Yang HH, Emand M J, Kirk T K. 1980. Factors influencing fungal degradation of lignin in a representative lignocellulosic, thermomechanical pulp.Biotechnology and Bioengineering, 22(1): 65-77zaldivar ], Nielsen J, Olsson L. 2001. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl MicrobiolZhang M, Chou Y C, Picataggio S K, et al. 1998. Single Zymomonas mobilis strain for xylose and arabinose fermentation. United States Patent 5,843, 760Zhou S, Yomano L P, Saleh A Z, et al. 1999. Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase( Encoded by celL)inEscherichia coli B. Appl Environ Microbiol, 65(6): 2439-2445中國煤化工任編石紅青)CNMHG