高浓度红薯醪SSF燃料乙醇过程中底物抑制的调节

第34卷第1期太阳能学报Vol 34, No. 12013年1月ACTA ENERGIAE SOLARIS SINICAJan.,2013文章编号:02540096(2013)01010506高浓度红薯醪SSF燃料乙醇过程中底物抑制的调节申渝,张海东,郑旭煦,张贤明(废油资源化技术与装备教育部工程研究中心/重庆市催化理论与应用技术重点实验室,重庆工商大学,重庆4000摘要:红薯干经粉碎、液化处理后得到淀粉含量为230g/kg的高浓度红薯醪,利用工业酵母 Saccharomyces cereviseATCC6508同步糖化发酵(SF)生产燃料乙醇。通过改变糖化酶的添加剂量调节发酵过程中糖化速率控制醪液中葡萄糖浓度的变化。发酵前期严格控制底物抑制对细胞生长速率的影响通过较高的细胞浓度保障发酵后期发酵速率,减轻产物抑制对发酵过程的影响。实验发现在0.2、0.40.6和0.8g/kg(糖化酶/红薯干粉)糖化酶添加量下发酵过程中葡萄糖浓度被严格控制在强烈抑制的浓度水平以下(100g/kg),1.0g/kg剂量下醪液中葡萄糖浓度长时间高于100g/kg,底物抑制严重;发酵后期在02、0.4和0.6g/kg剂量下,由于糖化酶活性降低糖化速率成为发酵速率的限制因素。综合分析糖化酶的最佳剂量为0.8g/kg,该剂量下既能严格控制底物抑制水平同时保持发酵后期较高糖化速率发酵终点乙醇浓度达11823g/kg(16.12%,v/v),发酵时间为72h关键词:同步糖化发酵;高浓度乙醇发酵;底物抑制调节;红薯中图分类号:TK6文献标识码:A0引言杂,且易造成染菌。多级串联连续发酵对减轻底物抑制和产物抑制均有一定效果,被高浓度乙醇发酵当前世界范围内燃料乙醇多以玉米、甘蔗汁等普遍采用7,112),但红薯原料果胶和纤维含量高,高粮食或糖质原料生产;发酵醪液通过精馏得到乙醇浓度醪液粘度较大,不适合连续输送操作。通过添产品,生产过程能耗高、废液排放量大。因此开发替加有机氮源维生素等营养成分可显著改善酵母细代性生产原料降低生产过程能耗和减少废水排放胞对乙醇的耐受能力,减轻发酵后期产物抑是燃料乙醇产业规模化发展面临的关键问题。制13-15,但从成本角度考虑这些方法在工业规模的红薯、木薯等根茎淀粉作物和以玉米为代表的生产装置中不具备实际应用的价值。从过程调控的种子作物相比,对种植土地的要求更低,单位种植面角度考虑,增加细胞浓度、缩短发酵时间是克服产物积淀粉产量更大,更适合作为燃料乙醇的生产原抑制的可行思路。同步糖化发酵( Simultaneous sac-料。薯类作物在我国和东南亚具有广泛的种植基 charification and fermentation,SF)技术操作简单,在础,由于在原料性质和成分上和粮食原料存在较大乙醇发酵中普遍被采用,通过边糖化边发酵的方式控发酵工艺选择微生物菌株适应性等,以指导规细胞浓度,达到弱化产物抑制的效果今力区别,需要系统研究原料预处理、发酵过程优化调能有效避免发酵初期底物抑制,保证发酵后期较高模化工业过程和新工艺的开发26。本文拟通过调节高浓度红薯液化醪液同步糖化高浓度发酵技术可通过提高发酵终点乙醇浓发酵初始糖化酶的添加量,严格控制发酵前期醪液度,进而降低单位产量燃料乙醇的过程能耗,同时减中葡萄糖浓度,减轻底物抑制,保证细胞生长,提高少废水排放展现出巨大的工业应用前景0。发细胞浓度;并利用较高的细胞浓度保证发酵后期强酵初期强烈的底物抑制和发酵后期强烈的产物抑制烈产物抑制水平下系统的发酵速率,减轻产物抑制是制约高浓度发酵技术的瓶颈。补料发酵可消除底对发酵过程的影响。以不增加额外成本为前提,通物抑制,但该工艺需实时监测发酵进度操作流程复过过程调控实现高浓度发酵,为工业过程提供参考。收稿日期:2010-1102基金项目:国家重大科技项目(2009zX07104002);重庆市教委科研项目(KzZH中国煤化工新团队建设项目(KrTD01020)CNMHG通讯作者:申渝(1981—),男,博士、副教授,主要从事生物质燃料方面的研究。106太阳能学报34卷1材料与方法由于实验中发酵温度为30℃,低于糖化酶的最适温度(60℃),糖化速率可能会存在较大差异。经1.1菌种和培养方法工业酿酒酵母 Saccharomyces cerevisiae ATCC测定初始醪液中淀粉含量约为230gkg,完全糖化后6508由大连理工大学生物工程系白凤武教授赠送。的葡萄糖含量约为255g/kg。实验结果表明发酵温斜面培养基(g):葡萄糖10,蛋白胨2,酵母粉度下,糖化酶对高浓度红薯液化醪液的糖化速率远2,琼脂粉20。接种后置于30℃培养箱静置培养低于最适温度下的速率,如图1所示,0.2、0.4和0.6g/kg剂量下80h内均达到糖化终点;0.8和24h,于4℃冰箱保存。种子培养基(g/L):葡萄糖30,蛋白胨2,酵母粉1.0g/kg剂量下糖化终点时间约为64h和40h,而实2。150mL三角瓶装入50mL种子培养基,灭菌后冷验表明,在60℃最适温度添加1.0g/kg糖化酶情况却至室温,接入斜面种子,摇床培养24h后用作发酵下糖化终点的时间约为4ho16h之前糖化速率和糖种子(温度30℃、转速150r/min)化酶的剂量呈线性正相关,因此糖化酶的剂量会决定发酵初期底物葡萄糖浓度的变化速率,可能影响所有培养基灭菌条件为:温度121℃,20min。1.2高浓度液化红薯醪液制备酵母细胞生长,进而影响整个发酵过程。市售鲜红薯洗净切片后烘干,干红薯片粉碎后过300→02kg04gkg20目筛制得红薯粉,参照文献[18]测得淀粉含量为67.78%。800mL钢制容器中将红薯粉和自来水按1:2.5混合,搅拌加热至85℃保温,用1.0mo/LHCl调烂150pH到6.0,同时按2g/kg(酶粉/红薯粉)加入淀粉酶(北京东华生物酶制剂有限公司,酶活力为400g)保温液化2h,冷却至室温后于-20℃冰箱储存备用。x-06g/kg --0.8g/kg -e-1.0g/kg1.3糖化和同步糖化发酵时间/h高浓度红薯液化醪液解冻后分别称取50g装入20个150mL三角瓶中,加入0.5g尿素,在115℃下灭菌30min。两组样品每组10个分别按0.2、0.4、图1发酵条件下不同糖化酶剂量对高浓度0.6、0.8和1.0g/kg(酶粉/红薯粉)加入糖化酶(北红薯液化醪液糖化过程的影响京东华生物酶制剂有限公司,酶活力为50000/g),Fig 1 Time course of glucose concentration in VHGliquefied sweet potato mash adding different dosages of每个剂量做两个平行样。一组样品接入5mL种子glucoamylase without inoculation at 30C液进行同步糖化发酵,另一组样品不接种,均置于不同糖化酶剂量下高浓度红薯液化醪液同步糖30℃摇床发酵,转速为转速100r/min化发酵过程中主要发酵参数如图2图3所示。从实14分析方法验数据判断在80h内只有0.8g/kg糖化酶添加量时每隔8h按无菌操作流程从每个三角瓶取出约达到发酵终点,80h内0.2,04.6、0.8和1.0g/kg2g醪液,去离子水稀释后测定葡萄糖、乙醇、甘油和剂量下终点乙醇浓度分别为72.449543、11.47细胞浓度。葡萄糖和乙醇浓度用SBA40生物传11.938和101.39g/kg。图2显示在发酵中后期较感分析仪测定(山东省科学院生物研究所)。甘油低糖化酶剂量下由于酶活性存在损耗,造成糖化速浓度用试剂盒测定(北京安迪生物科技有限公司)。率跟不上细胞对葡萄糖的消耗速率,进而限制了发细胞浓度采用显微计数确定同时用亚甲基蓝染色酵速率,这种现象在剂量分别为02和04g/kg时法确定活细胞比例。最为显著。图2显示了不同糖化酶剂量高浓度红薯2结果与讨论液化醪液同步糖化发酵过程中葡萄糖和乙醇浓度变2.1发酵条件下不同糖化酶剂量下的糖化过程化情况。中国煤化工CNMHG1期申渝等:高浓度红薯醪SSF燃料乙醇过程中底物抑制的调节107发酵过程中葡萄糖浓度应严格控制该浓度以下。细胞活性分析表明整个同步糖化过程中所有剂量下活细胞的比例虽呈下降趋势,但均保持在80%以上,且不同剂量之间并无显著差异,如图4所示尽管实验中采用亚甲基蓝染色方法只能确定细胞的活性,不能体现出细胞的代谢能力,仍可以此为依据初步判断1.0g/kg剂量下发酵速率低于0.6和1632时间h0.8g/kg剂量的根本原因是细胞浓度较低(图3)。+0.2g/kg+0.4g/kg -x-06g/kg +0. 8g/kg -o-1.0g/kg葡萄糖浓度乙醇浓度图2不同糖化酶剂量高浓度红薯液化醪液同步糖化发酵过程中葡萄糖和乙醇浓度变化情况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全糖化后稀释0.6g/kg·0.8gkg时间h15图4不同糖化酶剂量高浓度红薯液化醪液同步糖化发酵过程中活细胞比例变化情况g 4 Time courses of yeast cells vibialities in SSFprocesses at different dosages of glucoamylase全糖化后稀释研究表明乙醇发酵过程中葡萄糖底物对酵母细胞的抑制作用,主要是通过提高环境中渗透压的方式对细胞产生作用,酵母细胞对环境中渗透压变化图3不同糖化酶剂量高浓度红薯液化醪液的胞内反馈主要途径包括加速甘油通路,由于甘油同步糖化发酵过程中细胞浓度变化情况作为代谢产物在胞内外存在浓度平衡,因此醪液中Fig 3 Time courses of yeast cells concentration in SSF甘油浓度的变化可间接反映细胞所受到的渗透压作processes at different dosages of glucoamylase用情况9。本实验数据(图5)也充分证实了该观2.2不同糖化酶剂量下同步糖化发酵点,醪液中甘油含量变化和糖化酶剂量有直接关系,细胞生长情况表明糖化酶的剂量会对整个同步-+0.2g/kg +0. 4g/kg糖化发酵过程中细胞浓度的变化产生重要影响,主米06gkg要表现在:首先,发酵前期(0~40h)细胞生长速率和糖化酶的剂量呈负相关,这个阶段醪液中乙醇浓度较低对细胞生长的抑制作用不明显,而葡萄糖浓赵烂度较高且变化幅度较大,对细胞生长起主要影响作用;其次,发酵中后期(40~80h)0.2g/kg剂量下细胞生长速率低于0.4g/kg剂量,主要是由于葡萄糖时间h底物供给不足造成的;最后,整个发酵过程中10g/kg剂量下细胞浓度显著低于其他剂量,因为该图5不同糖化酶剂量高浓度红薯液化醪液剂量下醪液中葡萄糖浓度在10~40h时高于同步中国煤化工七情况100g/kg,前期实验表明红薯糖化液中葡萄糖浓度高Fig 5 TiCNMH Gation in SSF于100g/kg时会对该菌株产生强烈底物抑制,因此processes at different dosages of glucoamylase108太阳能学报34卷剂量越高甘油含量越高,1.0g/kg剂量下甘油含量远的实验数据归纳于表1。数据显示80h时发酵醪液高于其他剂量,证明了该剂量下酵母细胞受到的渗中酵母细胞浓度严重偏低,尽管和0.4gkg剂量下透压抑制远强于其他剂量。分析1.0g/kg剂量下甘乙醇浓度相近,细胞浓度却只有该剂量下的44%,油浓度变化情况还发现,尽管底物强烈抑制出现在这表明差异主要是由底物抑制差异造成的。实验还10~40h阶段但甘油浓度快速增加却出现在30h发现采用全糖发酵7d后仍不能到达发酵终点且在之后,这也佐证了酵母细胞对抑制因素反映的滞后发酵5d后细胞活性大幅降低(活细胞比例<30%),性是普遍存在的22出现发酵停滞现象。显然,高浓度红薯醪液不宜采23不同浓度底物抑制发酵过程比较用全糖发酵,更适合采用同步糖化发酵,实验证明在为了进一步比较红薯醪液不同浓度底物抑制对初始糖化酶添加量为0.8g/kg时,发酵时间为72h细胞生长及发酵过程的影响,又在相同发酵条件下终点乙醇浓度为118.23g/kg(16.12%,v/v),生产强采用完全糖化后(葡萄糖含量为256.2g/kg)高浓度度为1.64g/(kgh)。红薯醪液进行发酵,实验结果和不同糖化酶剂量下表1不同糖化酶剂量同步糖化和全糖发酵实验结果比较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千重葡萄糖浓度/gkg13.203.7043.10细胞浓度/10g12.352.43乙醇浓度/g·kg172.44l18.23得率(理论得率)/%73.2487.0877.81生产强度/g(kgh)11.120.91注:*发酵完成时间为72h。3结论gravity(VHG )potato mash for the production of ethanol[J]. 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Biotechnology Progress, 1999Microbiology and Biotechnology15(4):667-680.2002,60(1):6-72.中国煤化工CNMHG110太阳能学报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中国煤化工CNMHG