高濃度紅薯醪SSF燃料乙醇過(guò)程中底物抑制的調節

第34卷第1期太陽(yáng)能學(xué)報Vol 34, No. 12013年1月ACTA ENERGIAE SOLARIS SINICAJan.,2013文章編號:02540096(2013)01010506高濃度紅薯醪SSF燃料乙醇過(guò)程中底物抑制的調節申渝,張海東,鄭旭煦,張賢明(廢油資源化技術(shù)與裝備教育部工程研究中心/重慶市催化理論與應用技術(shù)重點(diǎn)實(shí)驗室,重慶工商大學(xué),重慶4000摘要:紅薯干經(jīng)粉碎、液化處理后得到淀粉含量為230g/kg的高濃度紅薯醪,利用工業(yè)酵母 Saccharomyces cereviseATCC6508同步糖化發(fā)酵(SF)生產(chǎn)燃料乙醇。通過(guò)改變糖化酶的添加劑量調節發(fā)酵過(guò)程中糖化速率控制醪液中葡萄糖濃度的變化。發(fā)酵前期嚴格控制底物抑制對細胞生長(cháng)速率的影響通過(guò)較高的細胞濃度保障發(fā)酵后期發(fā)酵速率,減輕產(chǎn)物抑制對發(fā)酵過(guò)程的影響。實(shí)驗發(fā)現在0.2、0.40.6和0.8g/kg(糖化酶/紅薯干粉)糖化酶添加量下發(fā)酵過(guò)程中葡萄糖濃度被嚴格控制在強烈抑制的濃度水平以下(100g/kg),1.0g/kg劑量下醪液中葡萄糖濃度長(cháng)時(shí)間高于100g/kg,底物抑制嚴重;發(fā)酵后期在02、0.4和0.6g/kg劑量下,由于糖化酶活性降低糖化速率成為發(fā)酵速率的限制因素。綜合分析糖化酶的最佳劑量為0.8g/kg,該劑量下既能?chē)栏窨刂频孜镆种扑酵瑫r(shí)保持發(fā)酵后期較高糖化速率發(fā)酵終點(diǎn)乙醇濃度達11823g/kg(16.12%,v/v),發(fā)酵時(shí)間為72h關(guān)鍵詞:同步糖化發(fā)酵;高濃度乙醇發(fā)酵;底物抑制調節;紅薯中圖分類(lèi)號:TK6文獻標識碼:A0引言雜,且易造成染菌。多級串聯(lián)連續發(fā)酵對減輕底物抑制和產(chǎn)物抑制均有一定效果,被高濃度乙醇發(fā)酵當前世界范圍內燃料乙醇多以玉米、甘蔗汁等普遍采用7,112),但紅薯原料果膠和纖維含量高,高糧食或糖質(zhì)原料生產(chǎn);發(fā)酵醪液通過(guò)精餾得到乙醇濃度醪液粘度較大,不適合連續輸送操作。通過(guò)添產(chǎn)品,生產(chǎn)過(guò)程能耗高、廢液排放量大。因此開(kāi)發(fā)替加有機氮源維生素等營(yíng)養成分可顯著(zhù)改善酵母細代性生產(chǎn)原料降低生產(chǎn)過(guò)程能耗和減少廢水排放胞對乙醇的耐受能力,減輕發(fā)酵后期產(chǎn)物抑是燃料乙醇產(chǎn)業(yè)規?；l(fā)展面臨的關(guān)鍵問(wèn)題。制13-15,但從成本角度考慮這些方法在工業(yè)規模的紅薯、木薯等根莖淀粉作物和以玉米為代表的生產(chǎn)裝置中不具備實(shí)際應用的價(jià)值。從過(guò)程調控的種子作物相比,對種植土地的要求更低,單位種植面角度考慮,增加細胞濃度、縮短發(fā)酵時(shí)間是克服產(chǎn)物積淀粉產(chǎn)量更大,更適合作為燃料乙醇的生產(chǎn)原抑制的可行思路。同步糖化發(fā)酵( Simultaneous sac-料。薯類(lèi)作物在我國和東南亞具有廣泛的種植基 charification and fermentation,SF)技術(shù)操作簡(jiǎn)單,在礎,由于在原料性質(zhì)和成分上和糧食原料存在較大乙醇發(fā)酵中普遍被采用,通過(guò)邊糖化邊發(fā)酵的方式控發(fā)酵工藝選擇微生物菌株適應性等,以指導規細胞濃度,達到弱化產(chǎn)物抑制的效果今力區別,需要系統研究原料預處理、發(fā)酵過(guò)程優(yōu)化調能有效避免發(fā)酵初期底物抑制,保證發(fā)酵后期較高?；I(yè)過(guò)程和新工藝的開(kāi)發(fā)26。本文擬通過(guò)調節高濃度紅薯液化醪液同步糖化高濃度發(fā)酵技術(shù)可通過(guò)提高發(fā)酵終點(diǎn)乙醇濃發(fā)酵初始糖化酶的添加量,嚴格控制發(fā)酵前期醪液度,進(jìn)而降低單位產(chǎn)量燃料乙醇的過(guò)程能耗,同時(shí)減中葡萄糖濃度,減輕底物抑制,保證細胞生長(cháng),提高少廢水排放展現出巨大的工業(yè)應用前景0。發(fā)細胞濃度;并利用較高的細胞濃度保證發(fā)酵后期強酵初期強烈的底物抑制和發(fā)酵后期強烈的產(chǎn)物抑制烈產(chǎn)物抑制水平下系統的發(fā)酵速率,減輕產(chǎn)物抑制是制約高濃度發(fā)酵技術(shù)的瓶頸。補料發(fā)酵可消除底對發(fā)酵過(guò)程的影響。以不增加額外成本為前提,通物抑制,但該工藝需實(shí)時(shí)監測發(fā)酵進(jìn)度操作流程復過(guò)過(guò)程調控實(shí)現高濃度發(fā)酵,為工業(yè)過(guò)程提供參考。收稿日期:2010-1102基金項目:國家重大科技項目(2009zX07104002);重慶市教委科研項目(KzZH中國煤化工新團隊建設項目(KrTD01020)CNMHG通訊作者:申渝(1981—),男,博士、副教授,主要從事生物質(zhì)燃料方面的研究。106太陽(yáng)能學(xué)報34卷1材料與方法由于實(shí)驗中發(fā)酵溫度為30℃,低于糖化酶的最適溫度(60℃),糖化速率可能會(huì )存在較大差異。經(jīng)1.1菌種和培養方法工業(yè)釀酒酵母 Saccharomyces cerevisiae ATCC測定初始醪液中淀粉含量約為230gkg,完全糖化后6508由大連理工大學(xué)生物工程系白鳳武教授贈送。的葡萄糖含量約為255g/kg。實(shí)驗結果表明發(fā)酵溫斜面培養基(g):葡萄糖10,蛋白胨2,酵母粉度下,糖化酶對高濃度紅薯液化醪液的糖化速率遠2,瓊脂粉20。接種后置于30℃培養箱靜置培養低于最適溫度下的速率,如圖1所示,0.2、0.4和0.6g/kg劑量下80h內均達到糖化終點(diǎn);0.8和24h,于4℃冰箱保存。種子培養基(g/L):葡萄糖30,蛋白胨2,酵母粉1.0g/kg劑量下糖化終點(diǎn)時(shí)間約為64h和40h,而實(shí)2。150mL三角瓶裝入50mL種子培養基,滅菌后冷驗表明,在60℃最適溫度添加1.0g/kg糖化酶情況卻至室溫,接入斜面種子,搖床培養24h后用作發(fā)酵下糖化終點(diǎn)的時(shí)間約為4ho16h之前糖化速率和糖種子(溫度30℃、轉速150r/min)化酶的劑量呈線(xiàn)性正相關(guān),因此糖化酶的劑量會(huì )決定發(fā)酵初期底物葡萄糖濃度的變化速率,可能影響所有培養基滅菌條件為:溫度121℃,20min。1.2高濃度液化紅薯醪液制備酵母細胞生長(cháng),進(jìn)而影響整個(gè)發(fā)酵過(guò)程。市售鮮紅薯洗凈切片后烘干,干紅薯片粉碎后過(guò)300→02kg04gkg20目篩制得紅薯粉,參照文獻[18]測得淀粉含量為67.78%。800mL鋼制容器中將紅薯粉和自來(lái)水按1:2.5混合,攪拌加熱至85℃保溫,用1.0mo/LHCl調爛150pH到6.0,同時(shí)按2g/kg(酶粉/紅薯粉)加入淀粉酶(北京東華生物酶制劑有限公司,酶活力為400g)保溫液化2h,冷卻至室溫后于-20℃冰箱儲存備用。x-06g/kg --0.8g/kg -e-1.0g/kg1.3糖化和同步糖化發(fā)酵時(shí)間/h高濃度紅薯液化醪液解凍后分別稱(chēng)取50g裝入20個(gè)150mL三角瓶中,加入0.5g尿素,在115℃下滅菌30min。兩組樣品每組10個(gè)分別按0.2、0.4、圖1發(fā)酵條件下不同糖化酶劑量對高濃度0.6、0.8和1.0g/kg(酶粉/紅薯粉)加入糖化酶(北紅薯液化醪液糖化過(guò)程的影響京東華生物酶制劑有限公司,酶活力為50000/g),Fig 1 Time course of glucose concentration in VHGliquefied sweet potato mash adding different dosages of每個(gè)劑量做兩個(gè)平行樣。一組樣品接入5mL種子glucoamylase without inoculation at 30C液進(jìn)行同步糖化發(fā)酵,另一組樣品不接種,均置于不同糖化酶劑量下高濃度紅薯液化醪液同步糖30℃搖床發(fā)酵,轉速為轉速100r/min化發(fā)酵過(guò)程中主要發(fā)酵參數如圖2圖3所示。從實(shí)14分析方法驗數據判斷在80h內只有0.8g/kg糖化酶添加量時(shí)每隔8h按無(wú)菌操作流程從每個(gè)三角瓶取出約達到發(fā)酵終點(diǎn),80h內0.2,04.6、0.8和1.0g/kg2g醪液,去離子水稀釋后測定葡萄糖、乙醇、甘油和劑量下終點(diǎn)乙醇濃度分別為72.449543、11.47細胞濃度。葡萄糖和乙醇濃度用SBA40生物傳11.938和101.39g/kg。圖2顯示在發(fā)酵中后期較感分析儀測定(山東省科學(xué)院生物研究所)。甘油低糖化酶劑量下由于酶活性存在損耗,造成糖化速濃度用試劑盒測定(北京安迪生物科技有限公司)。率跟不上細胞對葡萄糖的消耗速率,進(jìn)而限制了發(fā)細胞濃度采用顯微計數確定同時(shí)用亞甲基藍染色酵速率,這種現象在劑量分別為02和04g/kg時(shí)法確定活細胞比例。最為顯著(zhù)。圖2顯示了不同糖化酶劑量高濃度紅薯2結果與討論液化醪液同步糖化發(fā)酵過(guò)程中葡萄糖和乙醇濃度變2.1發(fā)酵條件下不同糖化酶劑量下的糖化過(guò)程化情況。中國煤化工CNMHG1期申渝等:高濃度紅薯醪SSF燃料乙醇過(guò)程中底物抑制的調節107發(fā)酵過(guò)程中葡萄糖濃度應嚴格控制該濃度以下。細胞活性分析表明整個(gè)同步糖化過(guò)程中所有劑量下活細胞的比例雖呈下降趨勢,但均保持在80%以上,且不同劑量之間并無(wú)顯著(zhù)差異,如圖4所示盡管實(shí)驗中采用亞甲基藍染色方法只能確定細胞的活性,不能體現出細胞的代謝能力,仍可以此為依據初步判斷1.0g/kg劑量下發(fā)酵速率低于0.6和1632時(shí)間h0.8g/kg劑量的根本原因是細胞濃度較低(圖3)。+0.2g/kg+0.4g/kg -x-06g/kg +0. 8g/kg -o-1.0g/kg葡萄糖濃度乙醇濃度圖2不同糖化酶劑量高濃度紅薯液化醪液同步糖化發(fā)酵過(guò)程中葡萄糖和乙醇濃度變化情況0.2g/kg→0.4g/kFig 2 Time courses of glucose and ethanol concentration in SSF80060kg→08gkgprocesses feeding different dosage of glucoamylase全糖化2.5◆0.2gkg0.4gkg全糖化后稀釋0.6g/kg·0.8gkg時(shí)間h15圖4不同糖化酶劑量高濃度紅薯液化醪液同步糖化發(fā)酵過(guò)程中活細胞比例變化情況g 4 Time courses of yeast cells vibialities in SSFprocesses at different dosages of glucoamylase全糖化后稀釋研究表明乙醇發(fā)酵過(guò)程中葡萄糖底物對酵母細胞的抑制作用,主要是通過(guò)提高環(huán)境中滲透壓的方式對細胞產(chǎn)生作用,酵母細胞對環(huán)境中滲透壓變化圖3不同糖化酶劑量高濃度紅薯液化醪液的胞內反饋主要途徑包括加速甘油通路,由于甘油同步糖化發(fā)酵過(guò)程中細胞濃度變化情況作為代謝產(chǎn)物在胞內外存在濃度平衡,因此醪液中Fig 3 Time courses of yeast cells concentration in SSF甘油濃度的變化可間接反映細胞所受到的滲透壓作processes at different dosages of glucoamylase用情況9。本實(shí)驗數據(圖5)也充分證實(shí)了該觀(guān)2.2不同糖化酶劑量下同步糖化發(fā)酵點(diǎn),醪液中甘油含量變化和糖化酶劑量有直接關(guān)系,細胞生長(cháng)情況表明糖化酶的劑量會(huì )對整個(gè)同步-+0.2g/kg +0. 4g/kg糖化發(fā)酵過(guò)程中細胞濃度的變化產(chǎn)生重要影響,主米06gkg要表現在:首先,發(fā)酵前期(0~40h)細胞生長(cháng)速率和糖化酶的劑量呈負相關(guān),這個(gè)階段醪液中乙醇濃度較低對細胞生長(cháng)的抑制作用不明顯,而葡萄糖濃趙爛度較高且變化幅度較大,對細胞生長(cháng)起主要影響作用;其次,發(fā)酵中后期(40~80h)0.2g/kg劑量下細胞生長(cháng)速率低于0.4g/kg劑量,主要是由于葡萄糖時(shí)間h底物供給不足造成的;最后,整個(gè)發(fā)酵過(guò)程中10g/kg劑量下細胞濃度顯著(zhù)低于其他劑量,因為該圖5不同糖化酶劑量高濃度紅薯液化醪液劑量下醪液中葡萄糖濃度在10~40h時(shí)高于同步中國煤化工七情況100g/kg,前期實(shí)驗表明紅薯糖化液中葡萄糖濃度高Fig 5 TiCNMH Gation in SSF于100g/kg時(shí)會(huì )對該菌株產(chǎn)生強烈底物抑制,因此processes at different dosages of glucoamylase108太陽(yáng)能學(xué)報34卷劑量越高甘油含量越高,1.0g/kg劑量下甘油含量遠的實(shí)驗數據歸納于表1。數據顯示80h時(shí)發(fā)酵醪液高于其他劑量,證明了該劑量下酵母細胞受到的滲中酵母細胞濃度嚴重偏低,盡管和0.4gkg劑量下透壓抑制遠強于其他劑量。分析1.0g/kg劑量下甘乙醇濃度相近,細胞濃度卻只有該劑量下的44%,油濃度變化情況還發(fā)現,盡管底物強烈抑制出現在這表明差異主要是由底物抑制差異造成的。實(shí)驗還10~40h階段但甘油濃度快速增加卻出現在30h發(fā)現采用全糖發(fā)酵7d后仍不能到達發(fā)酵終點(diǎn)且在之后,這也佐證了酵母細胞對抑制因素反映的滯后發(fā)酵5d后細胞活性大幅降低(活細胞比例<30%),性是普遍存在的22出現發(fā)酵停滯現象。顯然,高濃度紅薯醪液不宜采23不同濃度底物抑制發(fā)酵過(guò)程比較用全糖發(fā)酵,更適合采用同步糖化發(fā)酵,實(shí)驗證明在為了進(jìn)一步比較紅薯醪液不同濃度底物抑制對初始糖化酶添加量為0.8g/kg時(shí),發(fā)酵時(shí)間為72h細胞生長(cháng)及發(fā)酵過(guò)程的影響,又在相同發(fā)酵條件下終點(diǎn)乙醇濃度為118.23g/kg(16.12%,v/v),生產(chǎn)強采用完全糖化后(葡萄糖含量為256.2g/kg)高濃度度為1.64g/(kgh)。紅薯醪液進(jìn)行發(fā)酵,實(shí)驗結果和不同糖化酶劑量下表1不同糖化酶劑量同步糖化和全糖發(fā)酵實(shí)驗結果比較Table 1 Fermentation results of at different dosages of glucoamylase in 80 hours糖化酶加入量完全糖化0.20.40.60.8(kg千重葡萄糖濃度/gkg13.203.7043.10細胞濃度/10g12.352.43乙醇濃度/g·kg172.44l18.23得率(理論得率)/%73.2487.0877.81生產(chǎn)強度/g(kgh)11.120.91注:*發(fā)酵完成時(shí)間為72h。3結論gravity(VHG )potato mash for the 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Biotechnology Progress, 1999Microbiology and Biotechnology15(4):667-680.2002,60(1):6-72.中國煤化工CNMHG110太陽(yáng)能學(xué)報34卷SUBSTRATE INHIBITION REGULATED BYGLUCOAMYLASE DOSAGES IN SSF OF VHG SWEET POTATO MASH FORETHANOL PRODUCTION USING SACCHAROMYCES CEREVISIAEshen Yu, Zhang Haidong, Zheng Xuxu, Zhang XianmingEngineering Research Centre for Waste Oil Recovery Technology and equipment of MOe and Key Laboratory of catalysis Science andTechnology of CQEC, Chongqing Technology and Business University, Chongging 400067, China)Abstract: Very high gravity(VHG) sweet potato mash containing 230g/ kg liquefied starch was used for simultane-ous saccharification and fermentation( SSF)of fuel ethanol. Different dosages of glucoamylase were added in initialmash to regulate the saccharification rates during SSF processes. The glucose concentration in broth was strictlycontrolled bellow the high inhibition limitation level (100g/kg glucose in broth)in whole SSF runnings at the dosa-ges of 0. 2, 0. 4, 0. 6 and 0. 8gkg dry matter glucoamylase, while saccharifying rates decreased under the substrate consumption rates which limited the ethanol production rates in later SSF stage at the dosages of 0. 2, 0.4 and0. 6g/kg dry matter glucoamylase. Data analysis indicated that 0. 8g/kg is the optimum glucoamylase adding dos-ge, under such condition, the substrate inhibition is controlled strictly under the high inhibition limitation leveland saccharifying rate is kept high enough for the substrate providing throughout the SSF running, and 11823g/kg(16. 12%,v/v)ethanol equivalent to 90. 73% of theoretical yield is achieved in 72 hoursKeywords: simultaneous saccharification and ermentation( SSF); very high gravity(VHG ) ethanol fermentation;substrate inhibition regulating; sweet potato中國煤化工CNMHG