CN101155928A - 另选原料流在生物质处理和利用中的整合 - Google Patents
另选原料流在生物质处理和利用中的整合 Download PDFInfo
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Abstract
本发明提供用以处理由整合原料组成的生物质以产生可发酵糖的方法。本文描述的方法的一个方面包括预处理步骤,在该步骤中生物质与另选原料流整合,所得的整合原料在相对较高浓度下用相对于生物质干重较低浓度的氨处理。在另一个方面,将高固形物浓度的预处理生物质与另选原料流整合,以进行糖化。
Description
本申请要求2005年4月12日提交的美国临时申请第60/670,437号的权益。
政府权利声明
本发明是在美国能源部授予的04-03-CA-70224号合同项下的美国政府资助下作出的。美国政府在本发明拥有某些权利。
发明领域
本发明涉及生物质处理的一般领域。具体的说,提供了将包括来自工业加工(如谷物或其他种子加工)的低值联产品和废物流在内的另选原料流(alternative feedstream)整合到生物质中的方法,供在生产高价值产品的工艺中进行处理和利用。
发明背景
纤维质和木质纤维质原料和废物,如农作物废弃物、木材、林业废物、造纸污泥及城市和工业固体废物,提供了潜在大量的用于生产化学品、塑料、燃料和饲料的可再生原料。由包含纤维素、半纤维素、葡聚糖和木质素的碳水化合物聚合物组成的纤维质和木质纤维质原料和废物,通常通过多种化学、机械和酶学方法进行处理,以主要释放出己糖和戊糖,它们然后可被发酵成有用的产品。
使用预处理方法,来使由纤维质和木质纤维质材料组成的碳水化合物聚合物更容易被用于其水解的糖化酶类利用。历史上,标准的预处理方法主要利用高温下的强酸,但是由于能量成本高、设备成本高、预处理催化剂回收成本高和与糖化酶类不相容的原因,正在开发替代性方法,如酶法预处理或者使用在更温和温度下的酸或碱,这些预处理过程中发生的生物质碳水化合物聚合物水解减少,故要求有改进的酶系来糖化纤维素和半纤维素。
目前的生物质利用办法通常涉及提供生物质流(往往从一个来源提供)和通过上述标准方法预处理该生物质流。这种办法没有利用到在不同加工步骤将多个生物质流如工艺过程流或废物流整合,没有包括用于所述整合生物质流的经济上健康的处理工艺。
为了实现结合使用着整合生物质原料流的经济上健康的工艺,需要商用工艺,其包括对整合生物质原料流中的木质纤维素碳水化合物的水解。为实现经济可行性,该工艺还必须能用少量的化学品就提供高产出的高浓度糖,且所产生的可发酵糖来源对将糖转化成增值化学品和燃料的发酵微生物的毒性低。
本文描述的方法提出了一些手段,来将所述整合原料流结合到符合上述生产增值化学品和燃料所需的标准的经济上健康工艺中。
发明概述
本发明提供了用以处理由整合原料组成的生物质以生产可发酵糖的方法。
本文描述的方法的一个方面包括预处理步骤,其中将生物质与另选原料流整合,所得的整合原料在相对高浓度下用相对于生物质干重来说低浓度的氨处理。在另一个方面,将高固形物浓度的预处理生物质与另选原料流整合进行糖化。
在一个实施方案中,生物质用包括以下步骤的方法处理:
a)提供生物质;
b)向a)的生物质加入至少一种另选原料流,产生整合原料;
c)使b)的整合原料与包含氨的水溶液接触,形成整合原料-氨水溶液混合物,其中氨以至少足以保持整合原料-氨水溶液混合物的碱性pH的浓度存在,但其中所述氨以相对于整合原料的干重占小于约12重量百分比的比例存在,且其中整合原料的干重相对于整合原料-氨水溶液混合物的重量为至少约15重量百分比的高固形物浓度,以产生预处理的整合原料产物;
d)使c)的产物与糖化酶群(enzyme consortium)在合适的条件下接触,产生可发酵糖产物。
在另一个实施方案中,用包括以下步骤的方法处理和糖化整合的原料:
a)提供生物质;
b)使a)的生物质经历预处理过程,产生预处理的生物质产物;
c)向b)的预处理生物质产物加入至少一种另选原料流,产生第一或第二整合原料;
d)使c)的第一或第二整合原料与糖化酶群在合适的条件下接触,产生可发酵糖产物。
在这个方法的一个方面,b)中的预处理过程包括使生物质与包含氨的水溶液接触,形成生物质-氨水溶液混合物,其中氨以至少足以保持生物质-氨水溶液混合物的碱性pH的浓度存在,但其中所述氨以相对于生物质的干重占小于约12重量百分比的比例存在,且其中生物质的干重相对于生物质-氨水溶液混合物的重量为至少约15重量百分比的高固形物浓度。在这个方法的另一个方面,加入到b)的预处理生物质产物的另选原料流包含种子加工过程中所产生的釜馏物(stillage)。在又一个方面,a)的生物质可以是或者不是本文描述的整合原料。
由本文描述的方法产生的可发酵糖然后可用于生产增值化学品、燃料或其它高价值产品。
发明详述
本发明申请人明确地将所有引用的参考文献的全部内容结合到本公开说明书中。此外,当某个量、浓度或其他数值或参数以某个范围、优选范围或一列优选上限值和优下限值给出时,这应理解为明确地公开了由任何一对任何范围上限或优选值和任何范围下限或优选值所构成的所有范围,而不论范围是否单独公开。在本文叙述了数值范围的地方,除非另有规定,否则该范围意指包括其端点和落入其中的所有整数和分数。不想使本发明的范围受限于定义某个范围时所叙述到的具体数值。
本发明提供处理生物质和另选原料流(通称“整合原料”)以产生可发酵糖的方法。可发酵糖然后可用于生产增值化学品、燃料或其它高价值产品。
另选原料流包括至少一种低值联产品、加工流和/或工业加工废物流。
可将生物质和另选原料流合并,形成供预处理加工的整合原料,以实现对生物质和联产品或加工流的同时预处理。或者,可先预处理非整合的生物质,然后将联产品或加工流加入到预处理的生物质,形成供进行糖化的第二整合原料。在两种情况下都包括预处理步骤,其中将相对高浓度下的非整合生物质或整合原料用相对于初始物料干重来说相对低浓度的氨进行处理,然后将氨处理过的产物用糖化酶群消化,产生可发酵糖。可发酵糖被生物催化剂转化成高价值的目标产品,如化学品、塑料和燃料。
定义:
在本公开说明书中,使用了多种术语。提供了以下定义:
术语“可发酵糖”指能被微生物在发酵过程中用作碳源的寡糖和单糖。
术语“木质纤维质”指包含木质素和纤维素的组合物。木质纤维质材料还可包含半纤维素。
术语“纤维质”指包含纤维素的组合物。
生物质的“干重”是指除去了所有或基本上所有的水的生物质的重量。干重通常按照美国试验与材料协会(ASTM)标准E1756-01(生物质中固形物测定的标准试验方法(Standard Test Method forDetermination of Total Solids in Biomass))或者Technical Association ofthe Pulp and Paper Industry,Inc.(TAPPI)标准T-412om-02(木浆、纸张和纸板的水分(Moisture in Pulp,Paper and Paperboard))来测量。
术语“目标化学品”指通过发酵产生的化学品。化学品以广义使用,包括诸如蛋白质的分子,蛋白质包括例如肽、酶和抗体。
“可衍自生物质的”目标化学品是通过以下工艺产生的目标化学品,通过该工艺生物质被水解而释放出可发酵糖,可发酵糖用至少一种生物催化剂发酵产生所需的目标化学品。
术语“增塑剂”和“软化剂”指能造成各聚合物链纵向或横向的内聚性分子间力减少的材料。这种材料可起到例如减少结晶度或者破坏木质素和非木质素碳水化合物纤维(例如纤维素或半纤维素)之间的键的作用。
术语“糖化”指从多糖产生可发酵糖。
术语“预处理生物质”指在糖化前已经历过预处理的生物质。
“生物质”指任何纤维质或木质纤维质材料,包括包含纤维素和任选还包含半纤维素、木质素、淀粉、寡糖和/或单糖的材料。生物质还可包含另外的成分,如蛋白质和/或脂质。根据本发明,生物质可衍自单一来源,或者生物质可包含衍自超过一种来源的混合物;例如,生物质可包含玉米芯和玉米秸秆的混合物,或者草和叶的混合物。生物质包括但不限于生物能源作物、农作物废弃物、城市固体废物、工业固体废物、造纸污泥、庭院废物、木材和林业废物。生物质的实例包括但不限于玉米谷粒、玉米芯、作物残余物如玉米皮、玉米秸秆、草、小麦、小麦秆、干草、大麦、大麦秆、水稻秆、柳枝稷(switchgrass)、废纸、甘蔗渣、高粱、大豆、树木、树枝、根、叶、木屑、锯屑、灌木和矮树、蔬菜、水果、花和动物粪。在一个实施方案中,可用于本发明的生物质包括具有相对较高的碳水化合物值,相对较为密实和/或相对较容易收集、运输、储藏和/或操作的生物质。在本发明的一个实施方案中,有用的生物质包括玉米芯、玉米秸秆和甘蔗渣。
“另选原料流”或“另选原料”包括各种能有益于将生物质转化成可发酵糖以生产增值化学品和/或燃料的整个过程的材料。这种原料流可通过包括谷物或种子加工、食品加工、纸张/木浆加工等在内的工业加工来产生。一种类型的这种材料包括谷物或其它种子加工的联产品和废物,所述谷物或其它种子加工包括玉米干法碾压加工、玉米干法磨粉和玉米湿法磨粉。如本文所述,在一些实施方案中,农业废物和/或工业加工提供了能整合入预处理加工中的纤维质材料。在其它实施方案中,可将工业加工流或其它原料流(如低值流或废物流)并入到预处理后的生物质中,以有益于包括糖化和发酵在内的进一步生物质加工步骤。可构成另选原料流的低值流和废物流的非限制性来源包括农作物废弃物(如玉米皮或其它作物皮壳)、城市固体废物、工业固体废物、造纸污泥、庭院废物、木材和林业废物。
“整合原料”或“整合原料流”指生物质和至少一种另选原料流或原料的组合。
对于本发明的目的,“包含氨的水溶液”指在含水介质中使用氨气(NH3)、包含铵离子(NH4 +)的化合物如氢氧化铵或硫酸铵、降解时即释放出氨的化合物如尿素以及它们的组合。
供预处理的整合原料
本发明的一个方面包括将任何生物质原料与至少一种另选原料流合并,所述另选原料流由低值的联产品或工业加工流如废物流组成。
如上所述,用于生产高价值产品如油、淀粉、蛋白质、糖浆和乙醇的工业加工往往会产生低值的联产品、工艺过程流和废物流。在本发明方法中,这些联产品被捕获以再循环返回高价值产品加工流中,或者被利用来有益于生产增值化学品和燃料的加工过程。
在要求保护的方法的一个方面,可将包含有纤维的谷物或其它种子加工联产品或加工流与其它生物质合并,以提供被预处理的整合原料。
包含有另选原料的目的材料包括低值的联产品、工艺过程流和废物流;它们一起被称之为另选原料流。例如,低值联产品是被出售用作动物饲料的联产品。低淀粉或高油种子加工流的实例包括在大豆、葵花籽、花生和棉籽的加工中分离的皮壳流。含有纤维材料的废物流也可用于制备供预处理的整合原料。供预处理的整合原料可包括在任何种子的加工中产生的含纤维材料,所述种子例如玉米、燕麦、小麦、大麦、大米、卡诺拉油菜籽、葵花籽、棉籽、豌豆、大豆和其它豆类。
美国,大豆通常通过用己烷进行溶剂萃取来加工,以将油回收。将大豆清洁,可进行干燥,然后让其平衡在10-11%水分,以促进种皮或皮壳的松动。然后将它们破碎,通过过筛和抽吸进行脱壳,通过用蒸汽(qv)处理进行调理,以便进行压片。将调理过的果肉压片,用己烷萃取以将油移取。蒸发分离己烷和油水混合物,将己烷回收。(Kirk-Othmer Encylopedia of Chemical Technology,第4版,1997)。在这种加工过程中除去的种皮或皮壳可用作本发明方法中的整合原料流。
葵花籽的加工包括螺旋压榨、用己烷直接萃取,或者预压榨-溶剂萃取。后者在美国最常用。第一步是进行清洁,然后脱壳。将脱壳的种子进行加热调理,然后送入螺旋压榨机,或者在己烷直接溶剂萃取的情况下进行压片。将螺旋压榨饼磨碎以用于饲料中,或者造粒并用己烷萃取以回收剩余的油。(Kirk-Othmer Encylopedia ofChemical Technology,第4版,1997)。在这种加工过程中除去的皮壳可用作本发明方法中的整合原料流。
加工花生以获得花生油是通过螺旋压榨或者预压榨然后进行溶剂萃取来进行的。在螺旋压榨中,将花生去壳,蒸煮,压榨,产生粗油及含大约5%残油的压榨饼。将压榨饼磨碎,并将磨碎的花生壳回掺,以调节蛋白质含量。在预压榨-溶剂萃取中,将蒸煮过的果肉进行低压螺旋压榨,以除去一部分油,然后用己烷进行萃取,以将残油减少至大约1%。通过在除溶剂器中施加夹套或直接蒸汽,回收粕中的残余己烷。通过蒸发回收油水混合物中的己烷。(Kirk-OthmerEncylopedia of Chemical Technology,第4版,1997)。在这种加工过程中除去的皮壳可用作本发明方法中的整合原料流。
在美国,棉籽通过螺旋压榨或溶剂萃取而加工成油和粕。在螺旋压榨中,将种子清洁,剥绒,脱壳,压片,蒸煮,然后进行压榨。螺旋压榨产生出含2.5-4.0%残油的压榨饼。将压榨饼磨成粕,并将磨碎的棉籽壳回掺,以调节蛋白质含量至贸易标准。在溶剂萃取程序中,种子薄片往往通过膨化器(expander)来加工,以快速煮好薄片和形成颗粒物(collet),然后用己烷进行萃取。通过加热将从溶剂萃取器出来的粕除去己烷。(Kirk-Othmer Encylopedia of Chemical Technology,第4版,1997)。在这种加工过程中除去的皮壳可用作本发明方法中的整合原料流。
谷物加工流包括干磨过程中产生的由谷壳组成的纤维流、湿磨产生的含纤维磨粉残余物、在各种工艺过程中产生的废谷物流,以及干法碾压工艺所产生的干酒糟和可溶物(distiller dry grains andsolubles,DDGS)和干酒糟(distiller dry grains,DDG)。DDGS包括纤维、油、蛋白质和酵母,是将乙醇从发酵混合物中除去后剩下的材料。其固形物经过过滤产生出DDG级分,出售用作动物饲料。剩下的液体级分(釜馏物)可再循环回用作工艺过程流,或者经蒸发和与DDG合并形成DDGS联产品。
另选原料流的潜在来源的另一个实例包括根茎加工,如糖用甜菜、马铃薯(白马铃薯或甘薯)等的加工。在生产糖的糖用甜菜加工中,糖通过用水进行逆流提取而移取出来。将排出提取装置的残余甜菜固形物或浆状物压榨除水,使水分含量减少至大约75%。这一压榨浆状物可原样出售给当地的饲料生产作业,但更通常是与糖蜜混合后干燥至大约10%水分,作为牛饲料出售。(Kirk-Othmer Encylopedia ofChemical Technology,第4版,1997)。这一压榨浆状物可用作本发明方法中的整合原料流。
用于本发明的另选原料流除了含有纤维性工艺物料外,还可含有较少量的包括蛋白质、油和/或淀粉在内的其它成分。
本申请人发现,将加工过的种子材料、淀粉和/或油与通常用于预处理的生物质合并,不会不利地影响用本发明方法预处理的整合原料在随后糖化处理中所产生的可发酵糖的产量。此外,预期的从本发明方法的整合原料产生的糖产量可以基于整合原料的总纤维质(包括板纤维质)含量。因此,可将种子加工流加到供进行预处理的其它生物质,将加工流中的纤维与其它生物质一起进行预处理,以进行糖化产生可发酵糖。将这种整合原料用于预处理的有效性,可为生物质处理厂提供丰富、低成本且连续供应的原料。
预处理
在本发明方法中用于对整合原料进行预处理的氨的浓度,最低限度为足以保持整合原料-氨水溶液混合物的pH为碱性的浓度,最大限度为相对于整合原料的干重小于约12重量百分比。这一低浓度的氨足以进行预处理,该低浓度还可以是相对于整合原料的干重小于约10重量百分比。也可使用相对于整合原料的干重小于约6重量百分比或更低的非常低浓度的氨来进行预处理。碱性是指pH大于7.0。特别适合的是整合原料-氨水溶液混合物的pH大于8。在一个实施方案中,氨相对于整合原料的干重小于约8重量百分比。在一个实施方案中,氨相对于整合原料的干重小于约10重量百分比。特别适合的是氨相对于整合原料的干重小于约6重量百分比。
在本发明方法中使用氨比使用其它碱有优势。氨会分配成液相和气相。气态氨比液态碱能更容易扩散通过生物质,导致在更低浓度下即得到更有效的预处理。氨还可通过氨解作用竞争生物质中的乙酰酯的水解,形成乙酰胺(如CL2825(其通过引用结合到本文中)实施例11所示)。乙酰胺比乙酸盐对一些发酵微生物如运动发酵单胞菌(Zymomonas mobilis)的毒性更低(如CL2825(其通过引用结合到本文中)实施例12所示)。因此将乙酰酯转化成乙酰胺而不是乙酸能减少将乙酸除去的需要。氨的使用还能减少发酵过程中用氮源补充所用生长培养基的要求。另外,氨是低成本的材料,因此提供了经济性的工艺。氨在预处理过程中或预处理后还可再循环回预处理反应器中,这因此使更具经济性的工艺成为可能。例如,在预处理后,随着温度降低到适合糖化的温度,可任选在真空存在下使氨气释放出来,且可进行再循环。在连续工艺中,氨可连续进行再循环。
根据本发明,包含氨的水溶液可任选包含至少一种另外的碱,如氢氧化钠、碳酸钠、氢氧化钾、碳酸钾、氢氧化钙和碳酸钙。所述至少一种另外的碱可以以下量加入,该量与氨结合构成的总碱量相对于生物质干重小于约20重量百分比。优选地,第二种碱加上氨的总量小于约15重量百分比。另外的碱可用来例如中和生物质中的酸,给糖化酶提供金属离子,或者给发酵生长培养基提供金属离子。
在本发明方法中,整合原料的干重为占整合原料-氨水溶液混合物的重量的至少约15%至约80%的初始浓度。更合适地,整合原料的干重为占整合原料-氨水溶液混合物重量的约15%至约60%的浓度。将整合原料-氨水溶液混合物中的整合原料的百分比保持得高,是为了使将预处理整合原料糖化所产生的糖进行浓缩以用于发酵的需要减至最低。高整合原料浓度还能减少预处理物料的总体积,使工艺更具经济性。
可将整合原料按从其来源获得时的原样直接使用,或者可给整合原料施加能量,以减少体积、增加暴露表面积和/或增加整合原料中存在的纤维素、半纤维素和/或寡糖对氨和本发明第二步骤中使用的糖化酶的可供性。可用于减少体积、增加暴露表面积和/或增加整合原料中存在的纤维素、半纤维素和/或寡糖对氨和糖化酶的可供性的能量手段,包括但不限于碾碎、压碎、磨碎、切碎、剁碎、盘式精制、超声波和微波。该能量施加可发生在预处理之前或过程中、糖化之前或过程中或者它们的任何组合。
用氨溶液对整合原料的预处理可在任何合适的容器中进行。通常,容器是能经受压力,具有加热装置和内容物混合装置的容器。市售的容器包括例如Zipperclave反应器(Autoclave Engineers,Erie,PA)、Jaygo反应器(Jaygo Manufacturing,Inc.,Mahwah,NJ)和蒸汽枪反应器(Autoclave Engineers,Erie,PA)。可以使用具有类似性能的更大规模的反应器。或者,可将整合原料和氨溶液在一个容器中合并,然后转移到另一个反应器。同样,可将整合原料在一个容器中进行预处理,然后在另一个反应器如蒸汽枪反应器(Autoclave Engineers,Erie,PA)中进行进一步的加工。
在使整合原料与包含氨的水溶液接触前,可给装着整合原料的容器施加真空。通过将整合原料孔隙中的空气抽出,可使氨更好地渗透到整合原料中。施加真空的时间长度和施加给整合原料的负压量将取决于整合原料的类型,可作经验确定,以使整合原料得到最佳的预处理(由糖化后可发酵糖的产生量来测量)。
整合原料与包含氨的水溶液的接触在约4℃至约200℃的温度下进行。使整合原料与氨在4℃下先进行接触,以在此温度下进行浸渍,发现能提高糖化的效率。在另一个实施方案中,所述整合原料接触在约75℃至约200℃的温度下进行。在又一个实施方案中,所述整合原料接触在约90℃至约150℃的温度下进行。
整合原料与包含氨的水溶液的接触进行最多约8小时的时间。更长的预处理时间也可以允许,但出于实用性和经济性的原因,优选更短的时间。
在一个实施方案中,预处理过程可在相对较高的温度下进行相对较短的时间,例如在约100℃至约150℃下进行约5分钟至约2小时。在另一个实施方案中,预处理过程可在相对较低的温度下进行相对较长的时间,例如在约75℃至约100℃下进行约2小时至约8小时。在又一个实施方案中,预处理过程可在室温下(大约22-26℃)进行约24小时的更长时间。也可使用介于这些温度时间组合之间的其它温度时间组合。
对于预处理过程,温度、预处理时间、氨浓度、一种或多种另外的碱的浓度、整合原料浓度、整合原料类型和整合原料颗粒大小是相关的;因此可按需调整这些变量,以获得最佳的产物以待与糖化酶群接触。
可将增塑剂、软化剂或者它们的组合,如多元醇(例如甘油、乙二醇)、多元醇的酯(例如甘油一乙酸酯)、乙二醇醚(例如二甘醇)、乙酰胺、乙醇、乙醇胺,加入到预处理过程(即步骤(a))中。增塑剂可作为氨水溶液的成分加入,作为单独溶液加入,或者作为干成分加入。
预处理反应可在任何合适的容器中进行,如分批反应器或连续式反应器。本领域技术人员会认识到,在较高温度下(100℃以上)需要压力容器。合适的容器可装备有用以搅动生物质-氨水溶液混合物的装置如搅拌器。Lin,K.H.和Van Ness,H.C.(见于Perry,R.H.和Chilton,C.H.(编辑),Chemical Engineer′s Handbook,第5版(1973)第4章,McGraw-Hill,NY)讨论了反应器设计。预处理反应可作为分批过程或者作为连续过程来进行。
本领域技术人员公知,微生物在发酵过程中的生长需要氮源;因此在预处理过程中使用胺能提供氮源,减少或消除发酵过程中用氮源补充使用的生长培养基的需要。如果预处理产物的pH超过糖化酶有活性的pH,或者超过发酵中适合于微生物生长的范围,可用酸来降低pH。为实现所需的pH而使用的酸量,可能会导致所形成的盐的浓度对糖化酶或对微生物生长具有抑制性。为了减少实现所需pH而要求的酸量和减少本预处理工艺中的NH3原材料成本,可将氨气抽出预处理反应器并加以再循环。
为了从整合原料获得足够数量的糖,可将整合原料用氨水溶液预处理一次或超过一次。同样,糖化反应可进行一次或多次。如有需要,可重复进行预处理过程和糖化过程,以获得更高产量的糖。为评估预处理过程和糖化过程单独或一起的性能,可确定可从起始整合原料获得的糖的理论产量,并与测量产量进行比较。
在预处理后,产物包含氨、部分降解整合原料和可发酵糖的混合物。在进行进一步的加工之前,可通过施加真空将氨从预处理的整合原料除去。除去氨能降低pH,从而用较少的中和酸就获得所需的糖化和发酵pH。这能导致预处理混合物中盐负荷较低。通常会有一些氨剩余,这是给发酵提供氮源所需要的。
在一个优选的实施方案中,将包含可溶性和不溶性级分两者的整个预处理混合物用于糖化反应中。在另一个实施方案中,在糖化前,可将包含氨和溶解糖的含水级分与混合物中剩下的不溶性颗粒物相分离。用以将可溶性级分与不溶性级分相分离的方法包括但不限于倾析和过滤。不溶性颗粒物可再循环到预处理反应器。不溶性颗粒物可任选用含水溶剂(例如水)进行洗涤,以在再循环到预处理反应器前先除去吸附的糖。不溶性级分然后可如上文对预处理的描述用氨水溶液再作处理,接着用糖化酶群糖化。可溶性级分在糖化前还可用合适的工艺如蒸发进行浓缩。
第二整合原料-预处理后进行合并
在本发明方法的另一个方面,将另选原料流如种子加工流与预处理的生物质合并,以提供待糖化的第二整合原料。预处理的生物质是已如本文所述进行过预处理的材料(用生物质取代整合原料),或者将另选原料流如上述种子加工流与预处理的整合原料合并,以提供待糖化的第二整合原料。
对于为种子加工流的另选原料流,可与预处理材料合并的种子加工流包括通常纤维含量低的材料,如上文描述的釜馏物流或玉米浆。特别有用的是能提供对糖化和/或对发酵糖产物有益的特性或成分的种子加工流。种子加工流可提供的一个益处在于调节预处理的材料的pH。在预处理过程在碱性pH下进行的情况中,酸性的种子加工流特别有用。在糖化前降低pH以匹配糖化酶群的最适pH通常是有用的。例如,上述从干法碾压和乙醇发酵产生的釜馏物,其pH通常在3-5之间。将釜馏物加入到预处理的材料会降低糖化过程的pH。
在将另选原料流与预处理的生物质(该生物质可以是或不是预处理的整合生物质)整合的这种方法中,预处理方法是非限制性的。预处理的生物质可能已经历过其它常规预处理方法,如酸水解或者本领域公知的其它预处理方法(参见美国专利第5,916,780号)。
在这种方法的一个方面,另选原料流由釜馏物组成。釜馏物通常含有来自酵母发酵的对发酵糖产物有益的氨基酸和其它营养物。因此,从整合原料糖化产生的发酵糖产物可含有可发酵糖以及衍自种子加工流的其它营养物。营养物的存在可导致糖化产物在其用于生物催化剂发酵前对要另外加入的营养物的需求减少。
糖化
在本发明方法中,以下原料可被糖化:1)预处理的整合原料;2)在预处理后与至少一种另选原料流合并形成待糖化第二整合原料的预处理的整合原料;或3)与至少一种预处理后的另选原料流合并形成第二整合原料的预处理的非整合生物质。在本发明方法中,上述编号1-3的整合原料中的一者或多者在糖化酶群存在下被水解而释放寡糖和/或单糖到水解液中。本发明方法中的糖化是对整合原料进行的,不管整合出现在预处理前、预处理后或前后两者。Lynd,L.R.等(Microbiol.Mol.Biol.Rev.(2002)66:506-577)综述了糖化酶和生物质处理方法。
在本发明方法的一个方面,在糖化前,可将包含氨和溶解糖的含水级分与混合物中剩下的不溶性颗粒物相分离。用以将可溶性级分与不溶性级分相分离的方法包括但不限于倾析和过滤。不溶性颗粒物可再循环到预处理反应器。不溶性颗粒物可任选用含水溶剂(例如水)进行洗涤,以在再循环到预处理反应器前先除去吸附的糖。不溶性级分然后可如上文对预处理所描述用氨水溶液再作处理,接着用糖化酶群糖化。可溶性级分在糖化前还可用合适的工艺如蒸发进行浓缩。
糖化前,可对预处理的产物进行处理以改变pH、组成或温度,使得糖化酶群的酶会有活性。pH可通过如上文所述加入另选原料流,或者通过加入固体或液体形式的酸来改变。或者,可以利用可从发酵回收的二氧化碳(CO2)来降低pH。例如,可从发酵罐收集CO2,并例如通过鼓泡方式送进预处理产物中,同时监测pH,直到达到所需的pH。可使温度达到与糖化酶活性相容的温度,这在下文提到。可加入糖化用酶的活性所要求的任何辅因子
糖化酶群包括一种或多种主要选自(但非排他)“糖苷酶”的酶类,糖苷酶能水解二糖、寡糖和多糖的醚键,见于酶分类“水解酶类”(EC3.)大类下的EC 3.2.1.x(Enzyme Nomenclature 1992,Academic Press,San Diego,CA及其Supplement 1(1993)、Supplement 2(1994)、Supplement 3(1995,Supplement 4(1997)和Supplement 5[分别见Eur.J.Biochem.(1994)223:1-5、Eur.J.Biochem.(1995)232:1-6、Eur.J.Biochem.(1996)237:1-5、Eur.J.Biochem.(1997)250:1-6和Eur.J.Biochem.(1999)264:610-650,10])。可用于本发明方法的糖苷酶可通过其水解的整合原料来分类。可用于本发明方法的糖苷酶包括纤维素水解糖苷酶(例如纤维素酶、内切葡聚糖酶、外切葡聚糖酶、纤维二糖水解酶、β-葡萄糖苷酶)、半纤维素水解糖苷酶(例如木聚糖酶、内切木聚糖酶、外切木聚糖酶、β-木糖苷酶、阿拉伯木聚糖酶、甘露聚糖酶、半乳糖酶、果胶酶、葡糖醛酸酶)和淀粉水解糖苷酶(例如淀粉酶、α-淀粉酶、β-淀粉酶、葡糖淀粉酶、α-葡萄糖苷酶、异淀粉酶)。另外,向糖化酶群加入其它酶活性如肽酶(EC 3.4.x.y)、脂肪酶(EC3.1.1.x和3.1.4.x)、木质素酶(EC 1.11.1.x)和阿魏酸酯酶(EC 3.1.1.73),以帮助将多糖从整合原料的其它成分中释放出来,也可以是有益的。本领域公知,能产生多糖水解酶的微生物往往显示出由具有不同底物特异性的几种酶或一组酶催化的活性,如纤维素降解。因此,微生物的“纤维素酶”可包括一组酶,它们都可促成纤维素降解活性。市售的或非市售的酶制剂如纤维素酶,取决于获得该酶所采用的纯化方案,可能包括多种酶。因此,本发明方法的糖化酶群可包含酶活性如“纤维素酶”,但要认识到这一活性可能由超过一种酶来催化。
糖化酶可市售获得,如SpezymeCP纤维素酶(GenencorInternational,Rochester,NY)和Multifect木聚糖酶(Genencor)。另外,糖化酶可用生物法生产,包括使用重组微生物。
本领域技术人员会知道如何确定有效量的用于酶群的酶,并调节条件以获得最适酶活性。本领域技术人员还会知道如何使酶群中所要求的酶活性类别最优化,以使给定的预处理产物在选定条件下得到最佳糖化。
上文提到,可用来形成整合原料的另选原料流,如高纤维加工流和其它种子加工流,可能包括淀粉成分。这一淀粉可用淀粉水解糖苷酶(例如淀粉酶、α-淀粉酶、β-淀粉酶、葡糖淀粉酶、α-葡萄糖苷酶、异淀粉酶)分解成可发酵糖。因此,当将包含淀粉的另选原料流在预处理前或预处理后进行整合时,在本发明方法的糖化过程中加入淀粉水解酶以提高可发酵糖产量是特别合适的。
可在本发明方法中用来形成整合原料的另选原料流可能包含蛋白质成分。蛋白质可用肽酶来分解。因此,当将包含蛋白质的谷物加工流在预处理前或预处理后进行整合时,在本发明方法的糖化过程中加入蛋白质水解酶以用氨基酸增强可发酵糖产物可以是合适的。
优选地,糖化反应在或接近糖化酶的温度和pH最佳范围下进行。本发明方法中糖化酶群所用的温度最佳范围是15℃至约100℃。另一个最佳范围是20℃至约80℃。pH最佳范围可为约2至约11。在另一个实施方案中,本发明方法中糖化酶群所用的pH最佳范围是约4至约10。
糖化可进行约数分钟至约120小时的时间,优选约数分钟至约48小时。反应时间将取决于酶浓度和比活,以及所用底物和环境条件,如温度和pH。本领域技术人员能容易确定具体的底物和糖化酶群所要用的温度、pH和时间方面的最佳条件。
糖化可以按分批工艺或按连续工艺进行。糖化也可一步进行,或者分多步进行。例如,糖化所需的不同的酶可能显示不同的pH或温度最佳范围。可用一种(类)酶在一个温度和pH下进行初级处理,然后用另一种(类)酶在另一温度和/pH下进行二级或三级(或更多级)处理。另外,可在相同pH和/或温度下用不同的酶在连续步骤中进行处理,如使用半纤维素酶后接着使用纤维素酶;
糖化后糖从整合原料或第二整合原料溶出的程度,可通过测量单糖和寡糖的释放来监测。单糖和寡糖的测量方法是本领域公知的。例如,可用1,3-二硝基水杨酸(DNS)测定法(Miller,G.L.,Anal.Chem.(1959)31:426-428)测定还原糖的浓度。或者,可用本文在“一般方法”一节中所描述的适当柱,通过HPLC测量糖。
从整合原料或第二整合原料释放出的可发酵糖,可由合适的微生物利用来产生目标化学品。在糖化后但在发酵前,可将糖化混合物例如通过蒸发进行浓缩,以提高可发酵糖的浓度。任选地,可用分批方法或连续方法使糖化产物中的液体与固形物相分离。任选地,在发酵前可将该液体或整个糖化产物进行灭菌。取决于发酵过程中所用的微生物和糖化过程中所用的pH,可将pH调节至适合发酵的pH。另外,可给糖化混合物补充以微生物生长所需的另外的营养物。补充物可以包括例如酵母膏、特定氨基酸、磷酸盐、氮源、盐和痕量元素。还可包括特定生物催化剂生产特定产物所需的成分,如用以维持质粒的抗生素或酶催化体系中所需的辅因子。而且还可包括另外的糖,以增加总糖浓度。糖化混合物可用作发酵液的成分,例如占最终培养基的约90%至约10%。
取决于可用于发酵微生物的条件,还可对温度和/或顶空气体进行调节。发酵可以是好氧发酵或厌氧发酵。发酵可在糖化后进行,或者可通过同步糖化发酵(SSF)法与糖化同时进行。SSF可以使糖化所产生的糖水平保持低下,从而减少对糖化酶的潜在产物抑制作用,减少可供给污染性微生物的还原糖量,和提高预处理生物质向单糖和/或寡糖的转化。
可通过发酵产生的目标化学品包括例如酸、醇、烷烃、烯烃、芳族化合物、醛、酮、生物聚合物、蛋白质、肽、氨基酸、维生素、抗体和药物。醇包括但不限于甲醇、乙醇、丙醇、异丙醇、丁醇、乙二醇、丙二醇、丁二醇、甘油、赤藓糖醇、木糖醇和山梨糖醇。酸包括乙酸、乳酸、丙酸、3-羟基丙酸、丁酸、葡糖酸、衣康酸、柠檬酸、琥珀酸和乙酰丙酸。氨基酸包括谷氨酸、天冬氨酸、甲硫氨酸、赖氨酸、甘氨酸、精氨酸、苏氨酸、苯丙氨酸和酪氨酸。另外的目标化学品包括甲烷、乙烯、丙酮和工业用酶。
糖向目标化学品的发酵可通过一种或多种适当的生物催化剂在单步或多步发酵中进行。生物催化剂可以是选自细菌、丝状真菌和酵母的微生物。生物催化剂可以是野生型微生物或重组微生物,包括埃希氏菌属(Escherichia)、发酵单胞菌属(Zymomonas)、酵母菌属(Saccharomyces)、假丝酵母属(Candida)、毕赤酵母属(Pichia)、链霉菌属(Streptomyces)、芽孢杆菌属(Byacillus)、乳酸杆菌属(Lactobacillus)和梭菌属(Clostridium)。在另一个实施方案中,生物催化剂可选自重组大肠杆菌(Escherichia coli)、运动发酵单胞菌(Zymomonas mobilis)、脂肪嗜热芽孢杆菌(Bacillus stearothermophilus)、酿酒酵母(Saccharomyces cerevisiae)和树干毕赤氏酵母(Pichia stipitis)。
许多用于发酵生产目标化学品的生物催化剂已有描述,其它的可被发现、通过突变产生或通过重组手段工程改造。任何能利用本发明方法产生的可发酵糖的生物催化剂,都可用来通过本发明方法中的发酵制造已知它能生产的目标化学品。
通过产溶剂梭菌发酵碳水化合物产生丙酮、丁醇和乙醇(ABE发酵)是公知的(Jones和Woods(1986)Microbiol.Rev.50:484-524)。US5192673描述了用丙酮丁醇梭菌(Clostridium acetobutylicum)的突变株产生高水平的丁醇,同时还产生丙酮和乙醇的发酵方法。US 6358717描述了用拜氏梭菌(Clostridium beijerinckii)的突变株来产生高水平的丁醇,同时还产生丙酮和乙醇。大肠杆菌(E.coli)的遗传修饰株也已被用作生物催化剂进行乙醇生产(Underwood等,(2002)Appl.Environ.Microbiol.68:6263-6272)。US 2003/0162271A1描述了乙醇产量提高的运动发酵单胞菌遗传修饰株。
乳酸已通过以下菌株来发酵生产:大肠杆菌的重组株(Zhou等,(2003)Appl.Environ.Microbiol.69:399-407)、芽孢杆菌的天然株(US20050250192)和米根霉(Rhizopus oryzae)(Tay和Yang(2002)Biotechnol.Bioeng.80:1-12)。已将大肠杆菌的重组株用作生物催化剂发酵生产1,3-丙二醇(US 6013494、US 6514733)和己二酸(Niu等,(2002)Biotechnol.Prog.18:201-211)。已用重组的梭菌(Cheryan等,(1997)Adv.Appl.Microbiol.43:1-33)和新鉴定的酵母株(Freer(2002)World J.Microbiol.Biotechnol.18:271-275)来发酵生产乙酸。US 6159738公开了通过重组的大肠杆菌和其它细菌生产琥珀酸,Lin等,(2005)Metab.Eng.7:116-127)则公开了用突变重组的大肠杆菌来生产。已经通过突变的Torulopsis glabrata酵母(Li等,(2001)Appl.Microbiol.Technol.55:680-685)和通过突变的大肠杆菌(Yokota等,(1994)Biosci.Biotech.Biochem.58:2164-2167)来生产丙酮酸。已用大肠杆菌的重组株作为生物催化剂来生产对羟基肉桂酸(US20030170834)和奎尼酸(US20060003429)。
已将产丙酸丙酸杆菌(Propionibacterium acidipropionici)的突变株用于发酵生产丙酸(Suwannakham和Yang(2005)Biotechnol.Bioeng.91:325-337),已通过酪丁酸梭菌(Clostridium tyrobutyricum)生产丁酸(Wu和Yang(2003)Biotechnol.Bioeng.82:93-102)。已通过梭菌17crl株从苏氨酸发酵生产丙酸盐和丙醇(Janssen(2004)Arch.Microbiol.182:482-486)。已用酵母样出芽短梗霉(Aureobasidium pullulans)(Anantassiadis等,(2005)Biotechnol.Bioeng.91:494-501)和用黑曲霉(Aspergillis niger)突变株(Singh等,(2001)Indian J.Exp.Biol.39:1136-43)来生产葡糖酸。5-酮基-D-葡糖酸通过氧化葡萄糖酸杆菌(Gluconobacter oxydans)的突变株生产(Elfari等,(2005)Appl Microbiol.Biotech.66:668-674),衣康酸通过土曲霉(Aspergillus terreus)的突变株生产(Reddy和Singh(2002)Bioresour.Technol.85:69-71),柠檬酸通过黑曲霉(Aspergillus niger)突变株生产(Ikram-U1-Haq等,(2005)Bioresour.Technol.96:645-648),木糖醇通过季也蒙假丝酵母(Candidaguilliermondii) FTI 20037生产(Mussatto和Roberto(2003)J.Appl.Microbiol.95:331-337)。同时还含有大量的3-羟基丁酸和3-羟基戊酸的含4-羟基戊酸盐的生物聚酯,通过重组的恶臭假单胞菌(Pseudomonas putida)和真养雷氏菌(Ralstonia eutropha)(Gorenflo等,(2001)Biomacromolecules 2:45-57)生产。L-2,3-丁二醇通过重组大肠杆菌生产(Ui等,(2004)Lett.Appl.Microbiol.39:533-537)。
已用棒状杆菌属(Corynebacterium)、短杆菌属(Brevibacterium)和沙雷氏菌属(Serratia)的营养缺陷型菌株和氨基酸类似物抗性菌株,实现氨基酸的发酵生产。例如,日本专利出版物第8596/81号描述了使用对组氨酸类似物具有抗性的菌株来生产组氨酸,EP136359描述了使用一重组菌株来生产组氨酸。例如,日本专利出版物第4505/72号和第1937/76号描述了使用对色氨酸类似物具有抗性的菌株来生产色氨酸。例如,日本专利出版物第38995/72号、第6237/76号和第32070/79号描述了使用对异亮氨酸类似物具有抗性的菌株来生产异亮氨酸。日本专利出版物第10035/81号描述了使用对苯丙氨酸类似物具有抗性的菌株来生产苯丙氨酸。已描述了使用对酪氨酸具有抗性的其生长需要苯丙氨酸的菌株(Agr.Chem.Soc.Japan(1)R79-R87(1976)或者使用重组菌株(EP263515、EP332234)来生产酪氨酸,已描述了使用对L-精氨酸类似物具有抗性的菌株来生产精氨酸(Agr.Biol.Chem.(1972)36:1675-1684,日本专利出版物第37235/79号和第150381/82号)。还用大肠杆菌ATCC31882、31883和31884菌株发酵生产苯丙氨酸。US 6962805描述了用重组棒状杆菌生产谷氨酸。Okamoto和Ikeda(2000)J.Biosci Bioeng.89:87-79描述了通过大肠杆菌的突变株生产苏氨酸。甲硫氨酸由百合棒杆菌(Corynebacterium lilium)的突变株生产(Kumar等,(2005)Bioresour.Technol.96:287-294)。
也通过生物催化剂生产了有用的肽、酶和其它蛋白质(例如见US6861237、US6777207、US6228630)。
CL2825中(实施例9,其通过引用结合到本文中)例示了将生物质预处理和糖化成可发酵糖,然后将糖发酵成目标化学品,即用运动发酵单胞菌作为将糖发酵成乙醇的生物催化剂,从预处理的玉米芯生产乙醇。本发明的方法也可用来从生物质生产1,3-丙二醇。生物质按本发明进行预处理和糖化;在糖化后(或糖化过程中),用大肠杆菌按CL2825(实施例10)所述生产1,3-丙二醇。
通过生物催化剂发酵产生的目标化学品可用本领域公知的多种方法回收。可通过离心、过滤、微过滤和纳滤,使产物与其它发酵成分相分离。产物可通过离子交换、溶剂萃取或电渗析来提取。可使用絮凝剂来帮助产物分离。作为具体实例,可用本领域公知的针对ABE发酵的方法(参见例如Durre,Appl.Microbiol.Biotechnol.49:639-648(1998),Groot等,Process.Biochem.27:61-75(1992)及其中的参考文献)从发酵培养基分离生物法产生的1-丁醇。例如,可通过离心、过滤、倾析等方法从发酵培养基除去固形物。然后,可使用诸如蒸馏、共沸蒸馏、液-液萃取、吸附、气提、膜蒸发或渗透汽化的方法,从发酵培养基分离出1-丁醇。1,3-丙二醇从发酵培养基的纯化可例如通过使反应混合物进行有机溶剂萃取、蒸馏和柱色谱来实现(U.S.5,356,812)。对这个方法特别良好的有机溶剂是环己烷(U.S.5,008,473)。氨基酸可通过例如离子交换树脂吸附和/或结晶的方法从发酵培养基收集。
实施例
一般方法和材料
使用了以下缩写:“HPLC”指高效液相色谱,“C”指摄氏度、“kPa”指千帕、“m”指米、“mm”指毫米、“kW”指千瓦、“μm”指微米、“μL”指微升、“mL”指毫升、“L”指升、“min”指分钟、“mM”指毫摩尔、“cm”指厘米、“g”指克、“kg”指千克、“wt”指重量、“hr”指小时、“temp”或“T”指温度、“theoret”指理论值、“pretreat”指预处理、“DWB”指生物质干重。
硫酸、氢氧化铵、乙酸、乙酰胺、酵母膏、2-吗啉代乙磺酸(MES)、磷酸钾、葡萄糖、木糖、胰蛋白胨、氯化钠和柠檬酸获自Sigma-Aldrich(St.Louis,MO)。
预处理和酶水解反应器(PEHR)
尺寸和特征如图2所示和上文所述的生物质处理设备称为PEH反应器,用于以下实施例中。简单说来,9L的PEH反应器(在美国科罗拉多州Golden市的NREL制造,详细描述于共同待审的申请案CL3447中)具有大约15cm×51cm的不锈钢反应容器,该容器带有用以引入加工用反应物的注入枪。注入枪用回转接头连接到在容器一个末端上的盖子中的开口,容器还有另一个开口供作容器检查口(vessel access)。在容器内壁纵向有四个挡板,垂直连接到壁上。挡板和22个在容器中自由浮动的3.2cm×3.2cm陶瓷摩擦介质筒(Advanced Ceramics,East Palestine,OH),在容器旋转时使生物质和反应物发生机械混合,促进反应物向生物质中的同化吸收。PEH反应器放在提供旋转机构的Bellco Cell-Production Roller Apparatus(BellcoTechnology,Vineland,NJ)上,该反应器及滚筒装置安装在提供热量的温控箱中。温控箱有一铝框用来支撑包围着Bellco Cell ProductionApparatus的软木保温垫,该滚筒装置连接有加热器,该加热器由通过注入枪中心插入的热电偶控制。可给反应容器施加真空和压力,施加方式是将外源真空和压力连接到盖子中连接着注入枪的开口。
分析方法
糖、乙酰胺、乳酸和乙酸含量的测量
糖化醪中的可溶性糖(葡萄糖、纤维二糖、木糖、半乳糖、阿拉伯糖和甘露糖)通过HPLC(Agilent Model 1100,Agilent Technologies,Palo Alto,CA)测量,使用的是带有适当保护柱的Bio-Rad HPX-87P和Bio-Rad HPX-87H柱(Bio-Rad Laboratories,Hercules,CA)。测量样品pH,如有必要,用硫酸调节到5-6。然后使样品通过0.2μm针头过滤器直接进入HPLC小瓶中。HPLC运行条件如下:
HPX-87P(对于碳水化合物):
注入体积:10-50μL,取决于浓度和检测限
流动相:HPLC级水,0.2μm过滤并脱气
流速:0.6 mL/分钟
柱温度:80-85℃,保护柱温度<60℃
检测器温度:尽量接近主柱温度
检测器:折光指数检测器
运行时间:35分钟的数据收集加上15分钟的后运行(对较后洗脱出的化合物作可能的调整)
Biorad Aminex HPX-87H(对于碳水化合物)
注入体积:5-10μL,取决于浓度和检测限
流动相:.01N硫酸,0.2μm过滤并脱气
流速:0.6mL/分钟
柱温度:55℃
检测器温度:尽量接近柱温度
检测器:折光指数检测器
运行时间:25-75分钟的数据收集
运行后,从每种化合物的标准曲线确定出样品中的浓度。
实施例1
在PEH反应器中对含玉米芯和不同废糟样品
的合并生物质进行预处理和糖化
从以下物料制备废糟样品:
1.#2黄色顶陷整粒玉米谷粒(购自Agway)
2.用美国伊利诺大学(University of Illinois)开发的Quick Germ方法(Singh和Eckoff(1996)Cereal Chem.74:462-466)去胚乳的玉米谷粒。原料获自伊利诺大学的Vijay Singh。
3.通过用以除去胚乳和皮壳纤维的Quick Fiber方法(US6254914)加工的玉米谷粒。原料获自伊利诺大学的Vijay Singh。
4.制啤酒的粗碾谷粒获自Cargill(Minneapolis,MN)。
废糟指谷物加工(淀粉被转化成糖)后的残余固形物。废糟主要按基本威士忌方法(basic whiskey process)生产。将不同的原料用淀粉降解酶处理产生糖,将所得的糖化醪过滤以回收滤饼固形物或废糟。
将原料在Foss(北美总部:美国明尼苏达州Eden Prarie)Cyclotec1093样品研磨机(上述原料1和2)中研磨至250μm,或者在掺合机(上述原料3和4)中研磨,然后与2L带夹套、搅拌下的玻璃反应容器中的水和200mM CaCl2·H2O合并。用1N NaOH将混合物的pH调节至6.5,加入一半总量的α-淀粉酶(Spezyme HPA,Genencor International,Palo Alto,CA)。然后将反应容器加热到95℃,20分钟后加入余下的α-淀粉酶。在95℃下保持指定的时间后,将容器冷却到70℃,用1MHCl将混合物的pH调节至4.5。加入葡糖淀粉酶(Gzyme 480,Genencor),将温度再降低到50℃,保持过夜。此时将反应器冷却至<40℃,将其内容物滤过孔径大小为10μm的Dacron滤布。用水洗涤滤饼,将最终滤饼或者说废糟在105℃下干燥过夜,室温保藏以备在预处理实验中使用。每种原料的具体反应条件在下表1中列出。
表1.废糟样品的处理
原料 | 加入的原料(g) | 加入的水(g) | 加入的α-淀粉酶(ml) | 95℃下的时间 | 加入的葡糖淀粉酶(ml) | 50℃下的时间(hr) |
1 | 375 | 1095 | 3 | 150 | 3 | 17 |
2 | 505 | 1095 | 3 | 150 | 3 | 23 |
3 | 1180 | 500 | 6 | 120 | 3 | 17.5 |
4 | 1160 | 500 | 6 | 120 | 3 | 18 |
用颚板间距大约为0.95cm的颚式破碎机(2.2kW电动机)接着是脱块器(delumper)(1.5kW电动机,Franklin Miller Inc.)对整个的玉米芯进行处理,然后用装备有1.9cm美国标准筛的Sweco筛子过筛。将破碎的玉米芯与一种类型的表3所列废糟一起装载入PEH反应器中。废糟大约占反应器中生物质总干重的10%。总干生物质装载量大约为473g。在实验开始前,将每个装料的反应器在滚筒保温箱中预加热到95℃,不进行旋转。给反应容器施加真空(大约85kPa表压),将容器密封。当每个反应容器当中的温度再次稳定在95℃时,以19rpm开始旋转。加入稀氢氧化铵溶液,使氨浓度为4g氨/100g生物质干重,固形物浓度为30g生物质干重/100g生物质-氨混合物总重量。注入后,将容器上的真空解除到大气压。使反应器保持在95℃30min,然后使温度降低到85℃,让反应器在此温度下保持4hr,同时旋转。时间结束时,给反应容器施加真空(大约85kPa表压)30分钟以除去氨,使每个反应器的内容物温度降低到大约50℃。然后向每个反应器中注入二氧化碳以解除真空,将反应器用CO2加压到138kPa表压,在50℃和压力下旋转30分钟。
之后,将反应器减压,打开,通过注入75ml的1M柠檬酸缓冲液(pH 4.8,其中加入和溶解有一水柠檬酸)将其内容物的pH调节至大约5.5。柠檬酸缓冲液加热到50℃后注入每个反应器中,然后通过在50℃、19rpm下使反应器保温1小时进行平衡。将反应器从保温箱中取出,打开,测定样品的pH。如果pH高于5.5,则加入另外的固体一水柠檬酸,将反应器在50℃旋转下再保温1小时。按需多次重复这个过程,以达到每个反应器的pH为~5.5。一旦达到所需的pH,向反应器中加入28.4mg/g纤维素的SpezymeCP纤维素酶(Genencor)和10.1mg活性蛋白质/g纤维素的Diversa D2混合酶(含β-葡萄糖苷酶、木聚糖酶、β-木糖苷酶和阿拉伯呋喃糖苷酶)。反应器保持在50℃和19rpm下的保温箱中72hr。在该预处理和糖化之后,按“一般方法”中所描述测定糖产量。表2显示了基于两种进料的总葡聚糖和木聚糖的葡萄糖和木糖产量。玉米芯加废糟组合生物质样品的产量与玉米芯单独样品的产量相似。
表2.合并的生物质进料在预处理和糖化后的糖产量
单体葡萄糖 | 总葡萄糖产量 | 单体木糖产量 | 总木糖产量 | |
仅含玉米芯(2份平均值) | 68.2% | 85.6% | 41.8% | 88.9% |
玉米芯+Quick Germ废糟 | 67.9% | 86.5% | 49.0% | 86.5% |
玉米芯+Quick Fiber废糟 | 69.5% | 88.3% | 54.6% | 87.3% |
玉米芯+制啤酒的粗碾谷粒废糟 | 65.6% | 79.5% | 48.3% | 83.2% |
实施例2
在PEH反应器中含玉米芯、废糟和另外成分
的合并生物质的预处理和糖化
将按实施例1的描述制备的破碎玉米芯和威士忌废糟,按实施例1的描述在PEH反应器中进行合并。另外加入其它谷物成分。在一个样品中,以5g/100g生物质总干重的水平加入淀粉(SigmaS4126,批号093K0033)。在另一个样品中,以约2g/100g总干生物质的水平加入玉米油(Sysco Classic玉米油,批号4119095)。将样品按实施例1的描述进行预处理和糖化。结果在表3中显示。这些结果与表2中的对照数据(仅含玉米芯)相比是良好的。
表3.玉米芯、废糟和另外谷物成分的预处理和糖化产生的糖产量
单体葡萄糖 | 总葡萄糖产量 | 单体木糖产量 | 总木糖产量 | |
玉米芯+威士忌废糟+淀粉 | 70.4% | 90.2% | 48.4% | 96.1% |
玉米芯+威士忌废糟+油 | 79.2% | 87.5% | 54.9% | 101.4% |
实施例3
在PEH反应器中含玉米芯和玉米纤维
的合并生物质的预处理和糖化
将破碎的玉米芯和Cargill Bran 80(Cargill,Minnetonka,MN)玉米纤维合并,使得纤维占总干生物质的大约10%。将合并的生物质按实施例1的描述进行预处理和糖化。所得的糖产量在表4中显示。玉米芯加玉米纤维组合生物质的产量与单独玉米芯样品的产量相似。
表4:玉米芯和玉米纤维组合样品的预处理产生的糖产量
单体葡萄糖 | 总葡萄糖产量 | 单体木糖产量 | 总木糖产量 | |
玉米芯+Cargill Bran 80 | 66.4% | 82.3% | 47.0% | 83.5% |
Claims (39)
1.一种处理由整合原料组成的生物质的方法,所述方法包括:
a)提供生物质;
b)向a)的生物质加入至少一种另选原料流,产生整合原料;
c)使b)的整合原料与包含氨的水溶液接触,形成整合原料-氨水溶液混合物,其中氨以至少足以保持整合原料-氨水溶液混合物的碱性pH的浓度存在,但其中所述氨以相对于整合原料的干重占小于约12重量百分比的比例存在,且其中整合原料的干重相对于整合原料-氨水溶液混合物的重量为至少约15重量百分比的高固形物浓度,以产生预处理的整合原料产物;
d)使c)的产物与糖化酶群在合适的条件下接触,产生可发酵糖产物。
2.权利要求1的方法,其中所述整合原料-氨水溶液混合物的pH大于8。
3.权利要求1的方法,其中所述整合原料的干重为占至少约15%至约80%的初始浓度。
4.权利要求3的方法,其中所述整合原料的干重为占至少约15%至约60%的初始浓度。
5.权利要求1的方法,其中所述氨以相对于整合原料的干重占小于约10重量百分比的比例存在。
6.权利要求1的方法,其中所述氨以相对于整合原料的干重占约6%或更低重量百分比的比例存在。
7.权利要求1的方法,其中步骤(c)在约4℃至约200℃的温度下进行。
8.权利要求7的方法,其中步骤(c)在约75℃至约150℃的温度下进行。
9.权利要求8的方法,其中步骤(c)在大于90℃至约150℃的温度下进行。
10.权利要求1的方法,其中步骤(c)进行最多约8小时的时间。
11.一种处理由整合原料组成的生物质的方法,所述方法包括:
a)提供生物质;
b)使a)的生物质经历预处理过程,产生预处理的生物质产物;
c)向b)的预处理生物质产物加入至少一种另选原料流,产生第一或第二整合原料;
d)使c)的第一或第二整合原料与糖化酶群在合适的条件下接触,产生可发酵糖产物。
12.权利要求11的方法,其中(b)中的预处理过程包括使生物质与包含氨的水溶液接触,形成生物质-氨水溶液混合物,其中氨以至少足以保持生物质-氨水溶液混合物的碱性pH的浓度存在,但其中所述氨以相对于生物质的干重占小于约12重量百分比的比例存在,且其中生物质的干重相对于生物质-氨水溶液混合物的重量为至少约15重量百分比的高固形物浓度。
13.权利要求12的方法,其中所述生物质-氨水溶液混合物的pH大于8。
14.权利要求12的方法,其中所述生物质的干重为占至少约15%至约80%的初始浓度。
15.权利要求12的方法,其中所述生物质的干重为占至少约15%至约60%的初始浓度。
16.权利要求12的方法,其中所述氨以相对于生物质的干重占小于约10重量百分比的比例存在。
17.权利要求16的方法,其中所述氨以相对于生物质的干重占约6%或更低重量百分比的比例存在。
18.权利要求12的方法,其中步骤(b)在约4℃至约200℃的温度下进行。
19.权利要求18的方法,其中步骤(b)在约75℃至约150℃的温度下进行。
20.权利要求19的方法,其中步骤(b)在大于90℃至约150℃的温度下进行。
21.权利要求12的方法,其中步骤(b)进行最多约8小时的时间。
22.权利要求11的方法,其中步骤a)的生物质为整合原料。
23.权利要求11的方法,其中所述另选原料流由釜馏物组成。
24.权利要求1或11的方法,其中所述生物质选自生物能源作物、农作物废弃物、城市固体废物、工业固体废物、庭院废物、木材和林业废物。
25.权利要求1或11的方法,其中所述生物质选自柳枝稷、废纸、造纸污泥、玉米谷粒、玉米芯、玉米皮、玉米秸秆、草、小麦、小麦秆、干草、水稻秆、甘蔗渣、高粱、大豆、树木、树枝、根、叶、木屑、锯屑、灌木和矮树、蔬菜、水果、花和动物粪。
26.权利要求1或11的方法,其中所述生物质选自玉米芯、玉米秸秆、玉米皮、甘蔗渣、柳枝稷、小麦秆、干草、大麦、大麦秆、水稻秆和草。
27.权利要求26的方法,其中所述生物质选自玉米芯、玉米秸秆和甘蔗渣。
28.权利要求1或12的方法,其中氨选自氨气、氢氧化氨、尿素和它们的组合。
29.权利要求1或12的方法,其中所述包含氨的水溶液还包含至少一种另外的碱。
30.权利要求1或12的方法,其中氨进行再循环。
31.权利要求1或11的方法,其中所述另选原料流选自联产品、工艺过程流和废物流
32.权利要求31的方法,其中所述另选原料流是玉米、燕麦、小麦、大麦、大米、卡诺拉油菜籽、葵花籽、棉籽、豌豆或大豆和其它豆类的种子加工流。
33.权利要求1或11的方法,其中所述糖化酶群包含至少一种糖苷酶。
34.权利要求1或11的方法,其中所述糖化酶群包含至少一种选自以下的酶:纤维素水解糖苷酶、半纤维素水解糖苷酶、淀粉水解糖苷酶、肽酶、脂肪酶、木质素酶和阿魏酸酯酶。
35.权利要求1或11的方法,其中所述糖化酶群包含至少一种选自以下的酶:纤维素酶、内切葡聚糖酶、外切葡聚糖酶、纤维二糖水解酶、β-葡萄糖苷酶、木聚糖酶、内切木聚糖酶、外切木聚糖酶、β-木糖苷酶、阿拉伯木聚糖酶、甘露聚糖酶、半乳糖酶、果胶酶、葡糖醛酸酶、淀粉酶、α-淀粉酶、β-淀粉酶、葡糖淀粉酶、α-葡萄糖苷酶、异淀粉酶。
36.权利要求1或11的方法,其中步骤(d)在约15℃至约100℃的温度下和约2至约11的pH下进行。
37.权利要求11的方法,其中a)的生物质由至少一种另选原料流组成。
38.权利要求23的方法,其中所述釜馏物用来降低预处理物料的pH,使得它更易于糖化。
39.权利要求1或11的方法,其中所述可发酵糖用来产生增值的化学品、燃料或其它高价值产品。
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