CN1193099C - 使用耐热型酵母从木素纤维素生物质中生产乙醇的方法 - Google Patents

使用耐热型酵母从木素纤维素生物质中生产乙醇的方法 Download PDF

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CN1193099C
CN1193099C CNB011164956A CN01116495A CN1193099C CN 1193099 C CN1193099 C CN 1193099C CN B011164956 A CNB011164956 A CN B011164956A CN 01116495 A CN01116495 A CN 01116495A CN 1193099 C CN1193099 C CN 1193099C
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I·B·伯戴希斯
M·M·B·伯戴希斯
J·M·O·多明戈兹
J·C·加西亚
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Abstract

本发明包括以下阶段：将木素纤维素生物质粉碎至15-30mm的大小，对所获得的产物在反应器(2)中于190-230℃下进行1-10分钟的蒸汽***预处理，在旋风分流器(3)中收集预处理过的物料并且在压滤器(9)中通过过滤分离液体和固体部分，将固体部分引入发酵贮器(10)中，添加浓度为15UFP/克纤维素的纤维素酶和12.6国际单位的β-葡萄糖苷酶，所说的纤维素酶和β-葡萄糖苷酶溶解于pH4.8的柠檬酸盐缓冲液中，给发酵贮器(10)接种马克斯克鲁维氏酵母CECT10875耐热型菌株的培养物，该菌株通过化学诱变马克斯克鲁维氏酵母的DER-26菌株来获得，并且将混合物在42℃下摇动72小时。

Description

使用耐热型酵母从木素纤 维素生物质中生产乙醇的方法
技术领域
本发明涉及通过同时进行的糖化和发酵过程从木素纤维素生物质中获得乙醇的改进工艺。更具体说，本发明涉及在初期阶段对木素纤维素生物质进行热水蒸气***预处理，随后同时进行水解(使用市售的纤维素酶)和用新的马克斯克鲁维氏酵母(Kluyveromyces marxianus)耐热型菌株进行发酵的工艺。
背景技术
从生物质中获得乙醇燃料可给能量的提供带来安全性，因为它可代替化石来源的燃料。从生物质中获得乙醇燃料还有利于地区的发展，同时得到与就业有关的益处。与其它可再生能源一样，在运输工业中生产和使用生物乙醇优于得自油的燃料，这是因为减少了污染物的排放并且不增加温室效应。
从可再生原料中生产乙醇可以使用各种大量的底物。用来生产这种乙醇类型的原料是含有糖或淀粉的烃化产物，它们易经历发酵，或者是直接发酵(蔗糖)或者是在水解后(淀粉、菊粉)。诸如甜菜根和甘蔗的作物(第一组)和诸如玉米的谷物(第二组)是目前用来生产生物乙醇的原料的一些实例。也可以使用木素纤维素材料(有机物和森林的残余物以及草本类和木素类作物)作为生产生物乙醇的原料。
对含糖底物和含淀粉底物来说，工艺的总成本取决于原料的价格，其占产品总成本的60-70％。为提高生物乙醇燃料超过油基燃料的竞争能力，就需要开发一种新的工艺从较便宜的底物如某些木素纤维素原料中获得产品。
在被转变成乙醇之前，木素纤维素材料的纤维素部分需要经过水解，变成微生物可发酵的葡萄糖。这个可以通过酸或酶式催化剂来进行的阶段，由于木素提供了纤维素链的化学稳定性和植物组织的保护，从而使得该过程在经济和能量方面存在费用大的问题。
纤维素的酶水解比酸催化剂工艺具有至少三项潜在的优势：
·产率较大，
·设备费用较低，因为它在常压和低温下进行，
·没有因糖的分解而产生可以妨碍发酵的毒性物质。
然而，由于木素纤维素材料的结构，碳水化合物不易直接被水解酶所接近，因而需要一系列的在先处理来改进水解的产率。纤维素酶严重抑制反应最终产物的积累(主要是指纤维二糖和葡萄糖)，是限制水解产率的另一个因素。
通常来说，从木素纤维素生物质中获得乙醇的方法包括以下阶段：预处理、纤维素的水解和葡萄糖的发酵。预处理阶段的目的是促进酶和微生物的渗入和扩散。
预处理
自1919年以来(当时Beckmann取得关于碱预处理的专利权，该预处理以用氢氧化钠浸渗为基础，改善了稻草可消化性)，开发了很多针对木素纤维素材料的预处理。
在所测试的预处理中，热水工艺似乎对改进木素纤维素材料的可接近性最有效。热水工艺的一个实例可见Shell International Research的西班牙专利ES87/6829，其在气密型反应器中使用了200-250℃的蒸汽来处理预先粉碎的生物质。在此工艺中，将生物质处理之后，将反应器逐渐冷却至常温。然而，尽管其改进了生物质的可接近性，以致最终可被酶所酶解，但包括反应器突然减压的热水处理的变型(称为蒸汽***处理)，据显示是有助于纤维素酶最终作用的最有效的手段之一。蒸汽***是一种热学-力学-化学过程，其结合了热(如蒸汽)、机械力(剪切效果)和化学作用(水解)。结果是改变了细胞壁内的微丝堆积并使纤维断裂，造成纤维素对水解酶的可接近性增加。最佳温度和反应时间条件取决于材料的类型而不同。
Mason于1929年取得了关于不连续蒸汽***处理的专利权(美国专利US1655 618)，用于生产木料板，其结合了蒸汽热处理和木素纤维素纤维的机械结构破坏。在此工艺中，用3.5MPa或更高压力的蒸汽于立式钢筒中处理木制碎片。当处理完成后，将材料从钢筒的底部猛然地卸放出来。该过程将高压和高温对木素纤维素材料的作用与最终突然的减压结合起来。该处理的效果是物理改良(木素纤维素纤维的隔开和断裂)和化学改性(C-O-C键的解聚和断裂)的组合。在蒸汽处理期间，大部分半纤维素被水解成可溶于水的低聚物或游离的糖。
蒸汽***处理有非常不同的用途。例如，美国专利US4 136 207(1979)描述了这种类型的预处理为增加硬质木材(如杨木和桦木)被反刍动物消化性的用途。在此用途中，使用了STAKE技术，在高压管状反应器中连续操作，温度200-250℃并且处理时间不同。
在IOTECH公司开发的间歇式蒸汽***工艺(已知为″闪式水解″)中，将木材粉碎成小颗粒尺寸并且在接近230℃和500psi的温度和压力下处理，并且当达到这些条件时，突然从反应器中卸料。木材的有机酸控制pH并且废气中一直存在有乙酸。公知为IOTECH法中的反应器的设计描述于美国专利US4 461648中。
作为蒸汽***处理木素纤维素材料的另一个用途，加拿大专利CA1 212505描述了结合STAKE和IOTECH蒸汽***工艺来从硬质木材中高产量获得纸浆的用途。
发明内容
在本发明中，使用蒸汽***处理来增加纤维素的可消化性，以便在同时进行的糖化和发酵过程(SSF)中被微生物纤维素酶水解。这种将蒸汽***处理用作SSF过程中的预处理是这种处理的一种新用途，并且是本发明的新颖性之一。
该预处理的基本目的是减少纤维素的结晶度并且离解半纤维素-纤维素复合体。纤维素的可消化性随预处理的深度而增加，并且可消化性的增加与纤维素纤维的可利用表面积(ASA)的增加有直接关系，其有助于纤维素酶的最终酶解。ASA的增加是半纤维素和木素被部分或总体消除的结果。
关于蒸汽***处理之后底物可接近性增加的研究集中于对与底物有关的一系列因素的研究，例如孔度的分布、结晶度、聚合度或剩余木聚糖的含量，它们决定了其最终的有效性(K.K.Y.Wong等， Biotechnol Bioeng.31，447(1988)；H.L.Chum等， Biotechnol Bioeng.31，643(1988))。他们最初的研究集中于在高温(220-270℃)短时(40-90秒)处理的实验中，突然的减压对纤维素断裂的作用。较近来的研究(Wright，J.D.SERI/TP-231-3310，1988；Schwald等： 蒸汽***技 术，基本原理和工业应用，Facher，Marzetti和Crecenzy(eds.)，308-320页(1989))表明使用较低温度(不超过200-220℃)和较长处理时间(5-10分钟)可产生适宜的溶解速率，还可避免产生一定程度高温分解的可能性，这种高温分解可产生抑制性产物。该应用中所用的条件按照这些规定，并且据测定他们可在剩余物中获得较多的葡萄糖回收率(Ballesteros等： 用于能源、环境、农业和工业的生物 质，Chartier、Beenackers和Grassi(eds.)，第3卷，1953-1958页(1995))。
纤维素的酶水解和葡萄糖的发酵，同时进行的糖化和发酵过程(SSF)
纤维素的酶水解是由已知为纤维素水解酶或纤维素酶类的酶活性混合物来完成。其中的酶之一，叫做内切葡聚糖酶，被吸附在纤维素的表面并且酶解聚合物链的内部，破坏其某一点。然后，第二种酶，叫做外切葡聚糖酶，从链的非还原末端释放两个单元的葡萄糖，叫做纤维二糖。在此反应中产生的纤维二糖可以积聚在介质中并且明显抑制外切葡聚糖酶的活性。第三种酶，β葡萄糖苷酶，使这些两个糖单元***成游离的葡萄糖(其随后被发酵成乙醇)。同样，葡萄糖可以积聚在介质中并且抑制β葡萄糖苷酶的作用，从而引起纤维二糖的积聚，正如我们前面提到，这样会抑制外切葡聚糖酶的活性。
尽管可以产生纤维素酶的微生物有不同类型，包括细菌和不同品种的真菌，但通常使用的是丝状真菌Trichoderma ressei的基因改变菌株，因为它们具有较大的产率。传统的纤维素酶生产方法是间歇式的，使用碳的不溶性来源既作为诱导物又作为底物，用于真菌的生长和酶的产生。在这些体系中，生长的速度和纤维素酶的产生速率受到限制，因为真菌不得不分泌纤维素酶并且对固体进行缓慢的酶水解来获得所必须的碳。通常在间歇式进料的操作中可获得最好的结果，其中将固体底物(如Solka Floc或预处理的生物质)缓慢添加到发酵贮器中，以便它不含太多的底物(Watson等， Biotech.Lett.，6，667，1984)。根据Wright，J.D.(SERI/TP-231-3310，1988)，使用Solka Floc和预处理的农业剩余物的平均生产率为大约50IU/l.h。提高生产率并且增加这些酶的比活性是本发明研究的主要目的所在，其中酶的天然比活性是极其低的。
在由木素纤维素材料生产乙醇的常规方法中，是将纤维素酶添加到在反应器中预处理过的材料中，以便使纤维素糖化成葡萄糖，并且当该反应完成后，在第二个反应器中将葡萄糖发酵成乙醇。该工艺叫做分开糖化和发酵，在获得乙醇的过程中包含两个不同的阶段。使用该方法，纤维素转化成葡萄糖的转化率是低的，这是因为葡萄糖和纤维二糖的积聚造成了对酶复合物作用的抑制，结果获得较大量的非水解的纤维素剩余物，和低产量的乙醇。事实上，根据Wright，J.D.(SERI/TP-231-3310，1988)，这种对最终产品的抑制是分开糖化和发酵工艺的最大缺陷，并且是其成本高的主要原因，因为要尝试解决这个问题就得使用大量的纤维素水解酶。
英国专利GB2 186 289B描述了一种多步分开糖化和发酵以便从豆科草本植物中获得乙醇的工艺。这些步骤是：将植物性材料匀浆、用无机碱水解该材料，让预处理的材料与β纤维素酶反应，过滤反应介质，用微生物体系发酵滤出物来生产乙醇并且分离所产生的乙醇。
对前述方法来说，最感兴趣的选择之一是同时糖化和发酵(SSF)法。在此方法中，酵母与纤维素水解酶一起存在，减少了糖在反应器中的积聚并因此可以获得比分开水解和发酵工艺更大的产率和糖化率。另一个优点是在整个过程中只使用一个发酵贮器，由此减少了所要投资的费用。介质中存在乙醇还使混合物中不太容易侵入不期望的微生物(Wyman，C.E. 生物资源技术，50，3-16，1994)。
在同时水解和发酵工艺中，发酵和糖化必须相容并且具有相近的pH、温度和最佳底物温度。SSF工艺中的一个问题是糖化和发酵的最佳温度不同。由于最佳糖化温度为45-50℃范围，因此推荐在同时SSF工艺中使用耐热型酵母。
近些年来，人们对能够在40℃以上温度生长的酵母的不同菌株进行了研究并且撰写了文章，但关于使用这些微生物高产率乙醇发酵的文献并不太多。Szczodrak和Targonski( 生物技术和生物工程，第31卷，300-303页，1988)从12个科的酵母中试验了总共58种菌株，测试它们在40-46℃下生长和发酵糖的能力。从酵母属、克鲁维氏酵母属和豆孢酵母属中根据它们分别在40、43和46℃下发酵葡萄糖、半乳糖和甘露糖的能力选择了几种菌株。发现脆弱豆孢酵母和脆壁克鲁维氏酵母的两株菌株的乙醇产率最大，其在43和46℃下从140g/l葡萄糖中分别生产了56和35g/l的乙醇。
在本发明中，我们使用了通过化学诱变并且接下来筛选获得的马克斯克鲁维氏酵母的新菌株，CECT 10875，其能够在42℃下将纤维素水解产生的葡萄糖发酵成乙醇，并且与原始菌株相比，其产率得到改进。
近年来对SSF工艺的研究导致了乙醇产量的明显改进，这也是很多专利的目标。这些研究主要是基于微生物的筛选、酶的最佳浓度和不同的底物预处理，但大部分是针对间歇式工艺。例如，MAXOL & C.B.的专利WO96/37627描述了一种从植物材料中生产乙醇的间歇式SSF方法，其中糖化过程使用半纤维素酶和市售纤维素酶的混合物，并且使用来自假丝酵母属、克鲁维氏酵母属、毕赤氏酵母属和酵母属的酵母或其混合物来发酵所产生的不同糖。在此方法中，用酸或碱对植物材料进行预处理，但其缺点是材料预先要被粉碎成大约1mm大小，这代表了高能量消耗。所用的植物来源的材料是非常杂的，并且当使用与本发明类似的材料时，如饲料稻草，所获得的产量比本发明所述使用耐热酵母工艺所获得的产率低差不多10倍。这种低产率可归因于此方法使用的温度为35℃，这个温度对用所选的酵母进行发酵来说是足够的，但超出了糖化过程所需的最佳范围。
获得乙醇的SSF工艺的其它实例可见Wyman等的文章( Biotech Bioeng. Symp.，21-238页，1998)和Spindler等的文章( Appl.Biochem.Biotechnol.，28/29，773页，1991)，其使用的介质是克劳森酒香酵母(Bretanomyces clousenii)和酿酒酵母的混合物，它们发酵由水解纤维素所产生的纤维二糖和葡萄糖。该过程在37℃下进行，处理时间为7天，可以认为其对这样类型的方法来说是非常高了。
本发明描述的SSF方法是对先前所述方法的改进，因为本发明引入使用了耐热型菌株，该菌株可以允许在42℃下进行水解和发酵过程，该温度接近于纤维素水解复合物的最佳温度。它还显著缩短了处理时间。
对连续SSF***来说，惯常的设计是连续摇动的罐式反应器，以串联或级联的形式排列。这种类型***的最大缺陷是其成本昂贵，因为它需要长的处理时间和剧烈的摇动，会导致酶变性并且必需要经常更换它们。Nguyen，Q.A.的专利WO98/30710描述了一种塔式生物反应器***，其以流动-活塞反应器技术为基础，使发酵贮器的体积和摇动所需要的能量大大减少。该***允许使用高含量的悬浮固体混合料，如预处理的木素纤维素材料，因为在前述的***中它们仅可适用于含水混合料。虽然如此，文献中仍没有包含关于获得高产率和生产率的连续SSF法发展的描述。
本发明所包括的工艺是从木素纤维素生物质中获得乙醇的间歇式工艺，其包括蒸汽***预处理以及使纤维素变成乙醇的同时进行的糖化(通过市售纤维素酶)和发酵(使用新的耐热型酵母，具体说是马克斯克鲁维氏酵母CECT10875)。该过程在42℃下进行。摇动在150rpm下进行并且处理时间为72小时。在预处理之后，1,000g的纤维素含量为30-40％的生物质(不易受酶水解)得到270-360g容易被水解的纤维素。通过SSF工艺将该纤维素转变成90-120g的乙醇。
为实现前面的描述，并且更好地理解本发明的特征，以下将详细描述优选的实施方案，其以一组定向性但非限制性的说明书附图为基础：
附图描述
图1显示了本发明的工艺流程。
具体实施方式
本发明中所用的原料是主要含有纤维素的物料，如林业和农业残余物、纸浆、木素纤维素作物生物质和家庭垃圾的有机部分。正常来说，这种材料已被风干并且含有10-15％水分。
尽管在预处理前需要将材料粉碎，但所需要的颗粒大小(15-30mm)相当地大于其它反应器设计中所用的粒度，从而减少了粉碎相关的能量消耗。这种蒸汽热处理导致冷凝并且产生湿的木素纤维素物质。由于温度足够高到可热力学迫使液体水的离解，因而发生自身水解，产生酸性介质，克服了水解的能量屏障。由于蒸汽相的扩散大于液体相的扩散，因而保证了蒸汽引入木素纤维素材料的结构中。蒸汽首先渗入，然后冷凝。这种毛细管水由于高压而处于平衡。当材料被减压时，毛细管水迅速蒸发，其具有分离和破坏一些纤维的机械作用，可能对最弱的区域(非晶形纤维素)的作用最大。这种机械作用显然是由内部水分的迅速蒸发引起的。这种蒸发产生了剪切力，造成纤维分离。
本发明中预处理所用的设备由三个装置组成：蒸汽蓄积器1、蒸汽***反应器2和卸料旋风分流器3，其特征如下所述。参见图1。
蒸汽蓄积器1为蒸汽***反应器2提供245℃和a-3MPa压力的蒸汽。它由压力接收器组成，其中装配有许多电阻6。在蒸汽出口处，有一大气压通气口，被两个活阀8关闭，以便在初期的装料过程中有空气脱逸(当其被设定至操作压力和温度时)。在压力刻度测量之处设置作用于电阻6上的压力开关7，并且每个压力开关作用于一个电阻6上，根据是否达到所设定的值来关或开之。
蒸汽***反应器2是将木素纤维素生物质在其中压缩和突然减压的反应室。它由3″直径的不锈钢316垂直管构成，两端为两个3″直径的不锈钢316节流阀。反应室顶部的入口阀4通过手来打开和关闭，并且用于向反应器2中装填粉碎木素纤维素生物质。出口阀5位于反应室的底部，由触发和弹簧装置在1秒种之内打开。由此将蒸汽和生物质混合物猛然排放出去，并且经过管道被带往旋风分流器3。反应室2、阀门和卸料管道用70mm厚的石纤维保温，以便在压缩-膨胀过程中尽可能地减少蒸汽的冷凝。
卸料旋风分流器3由不锈钢316制成并且具有直径为16″的圆柱形部分和一个圆锥形部分，其中圆锥形部分由圆柱形部分向下并且呈60度角，末端为DN-80和PN-16凸缘颈，其上有一热固型阀门，通过其将反应器中膨胀的物料除去。旋风分流器圆柱部分上边缘的末端为一16″凸缘，其上装配有锁定拉环(locking tab)和紧固件，该紧固件压制有眼螺栓，而所说的有眼螺栓将起旋风分流器盖作用的堵头盲板(blind flange)拴紧。旋风分流器装有温度计和压力计。
在SSF中使用发酵贮器10，用不锈钢制成并且装配有机械摇动、pH和温度控制。在入口处有一压滤器9。
工艺如下：
将粉碎的物料引入蒸汽***装置2中，并且通过注射入来自蒸汽蓄积器1的饱和蒸汽进行1-3MPa压力和190-230℃的处理，并且根据所用的原料进行1-10分钟。当预处理结束后，让排出的蒸汽和木素纤维素生物质的混合物以水平地并且切线方向地进入卸料旋风分流器3，在其中除去挥发性成分并且将其过滤，以便将液体部分与固体部分分离。液体部分基本上含有大部分的半纤维素糖(木糖、***糖、甘露糖和半乳糖)、这些糖的降解产物(糠醛、羟甲基糠醛)、有机酸(主要是乙酸)和因木素溶解而产生的酚类化合物。固体部分基本上含有纤维素和木素，并且将其用作纤维素水解生产葡萄糖的原料。该葡萄糖是发酵生产乙醇的底物。
在离开压滤器9之后，将物料以8-15％(w/v)的固/液比引入发酵贮器10中，其中所说的固/液比根据构成木素纤维素生物质的物料而不同。当将物料引入发酵贮器中并且被充分稀释后，加入浓度为15滤纸单位(Unit of Filter Paper，UFP)/克纤维素的市售纤维素水解化合物(如CELLUCLAST 1.5L，NOVO-NORDISK公司出品)和12.6国际单位/克纤维素的β葡萄糖苷酶(如NOVOZYME 188，NOVO-NORDISK公司出品)，两者均再悬浮于pH4.8的柠檬酸盐缓冲剂中。按照国际IUPAC(纯化学和应用化学国际联盟)描述、由Ghose，T.K.( 纯化学和应 用化学，第59卷，第2号，257-268页，1987)描述的方法测定酶的活度。
由于前述的最终产品抑制纤维素水解复合物，因此诸如本发明所述的SSF工艺(其中当葡萄糖产生时即从介质中去除)，可对水解的产率带来明显的改进。出于此目的，本发明使用一种新的马克斯克鲁维氏酵母耐热型菌株(CECT10875)，由于它使酶复合物的作用与发酵相容在接近两种情况的最佳温度，因而提供了本质的优点。这种新的菌株通过化学诱变DER-26马克斯克鲁维氏酵母菌株获得，所说的DER-26马克斯克鲁维氏酵母菌株属于可再生能源CIEMAT部门的收集品。按照 应用生物化学和生物技术，第39/40卷，201-211页(1993)中所述，用不同剂量的烷基化剂甲磺酸乙酯处理这种原始菌株，然后根据在42-45℃范围内生长和将葡萄糖发酵成乙醇的能力进行筛选。这种菌株于1998年7月2日保藏于西班牙典型培养物保藏中心(Universidad de Valencia，Edificio de Investigacion，Campus de Burjasot，46100 Burjasot，Valencia)，保藏号为10875。
在属于本发明的SSF方法中，将马克斯克鲁维氏酵母CECT 10875在42℃下生长16小时的培养物的悬浮液以10％浓度(v/v)接种于前述的含有预处理过的生物质和纤维素水解复合物的发酵贮器(10)中。在42℃下，将该混合物于150rpm下摇动72小时。在此时间之后，显示乙醇的浓度不再增加，所以认为72小时后该过程结束，通过HPLC测定乙醇的最终浓度和介质中的剩余糖的浓度。

Claims (6)

1、从木素纤维素生物质中生产乙醇的方法，其特征在于所说的方法包括以下步骤：
将木素纤维素生物质粉碎成颗粒大小为15-30mm；
对粉碎的木素纤维素生物质进行蒸汽***预处理，根据所用材料的类型保持1-3MPa的压力和190-230℃的温度1-10分钟的时间，之后引发迅速减压；
收集预处理过的物料并且通过过滤分离液体和固体部分，并且将固体部分引入发酵贮器(10)中；
向发酵贮器(10)添加浓度为15UFP/克纤维素的纤维素酶和12.6国际单位/克纤维素的β-葡萄糖苷酶；
给发酵贮器(10)接种马克斯克鲁维氏酵母CECT10875耐热型酵母培养物的悬浮液；
将混合物在42℃下摇动72小时；
当反应完成之后，测定混合物中乙醇和剩余糖的浓度。
2、根据权利要求1的从木素纤维素生物质中生产乙醇的方法，其特征在于木素纤维素生物质的含水量为10-15％。
3、根据权利要求1的从木素纤维素生物质中生产乙醇的方法，其特征在于引入发酵贮器中的固体部分的固/液比为在重量体积比8-15％之间变化。
4、根据权利要求1的从木素纤维素生物质中生产乙醇的方法，其特征在于纤维素酶和β-葡萄糖苷酶溶解于pH4.8的柠檬酸盐缓冲液中。
5、根据权利要求1的从木素纤维素生物质中生产乙醇的方法，其特征在于马克斯克鲁维氏酵母CECT10875的接种物的浓度为10％体积比。
6、根据权利要求1的从木素纤维素生物质中生产乙醇的方法，其特征在于将混合物在150rpm下摇动。
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