CN107429217A - 使用粗甘油高密度产生生物质和油 - Google Patents
使用粗甘油高密度产生生物质和油 Download PDFInfo
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- CN107429217A CN107429217A CN201680017603.XA CN201680017603A CN107429217A CN 107429217 A CN107429217 A CN 107429217A CN 201680017603 A CN201680017603 A CN 201680017603A CN 107429217 A CN107429217 A CN 107429217A
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- acid
- crude glycerine
- glycerine
- culture medium
- concentration
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 title claims abstract description 227
- 235000011187 glycerol Nutrition 0.000 title claims abstract description 112
- 239000002028 Biomass Substances 0.000 title description 34
- 238000000034 method Methods 0.000 claims abstract description 89
- 244000005700 microbiome Species 0.000 claims abstract description 45
- 239000001963 growth medium Substances 0.000 claims abstract description 44
- 230000000813 microbial effect Effects 0.000 claims abstract description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 46
- 229910052799 carbon Inorganic materials 0.000 claims description 46
- 239000002253 acid Substances 0.000 claims description 33
- 125000001931 aliphatic group Chemical group 0.000 claims description 23
- -1 linoleic acid, leukotrienes Chemical class 0.000 claims description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 claims description 17
- MBMBGCFOFBJSGT-KUBAVDMBSA-N docosahexaenoic acid Natural products CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 claims description 14
- 238000004519 manufacturing process Methods 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 12
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- 238000004458 analytical method Methods 0.000 claims description 3
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- 150000007513 acids Chemical class 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
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- 239000003225 biodiesel Substances 0.000 description 10
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
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Abstract
提供一种培养一种或多种微生物的方法。该方法包括在包含第一浓度水平的粗甘油的培养基中培养一种或多种微生物,一旦甘油的所述第一浓度降低至第一阈值水平,就以足以实现所述第一浓度水平的浓度向所述培养基中馈入额外量的粗甘油,监测所述粗甘油浓度直至所述粗甘油的第一浓度水平降低至所述第一阈值水平。可重复各步骤直至实现所需微生物细胞密度。
Description
相关申请的交叉引用
本申请要求2015年3月26日提交的美国临时申请号62/138,631的优先权,所述美国临时申请以引用的方式整体并入本文。
背景
异养性微生物发酵是产生高价值油和生物质产品的高效方式。在某些培养条件下,微生物合成细胞内油,其可被提取和用于产生生物燃料(例如生物柴油、生物喷气燃料等)和营养性脂质(例如多不饱和脂肪酸诸如DHA、EPA和DPA)。一些微生物的生物质也由于多不饱和脂肪酸(PUFA)和蛋白质含量较高而具有极大营养价值,并且可用作动物饲料的营养性补充剂。然而,异养性发酵是一种高成本以及高能量和原料消耗的密集型过程。碳原料成本通常占用于产生微生物生物质和油的总成本的极其重大部分。现有微生物发酵主要使用昂贵碳水化合物诸如葡萄糖作为碳源。较便宜的碳替代物正被探究以使得生产过程在经济上有利。除葡萄糖之外,若干其它形式的碳诸如果糖和甘油是微生物的天然碳底物。然而,高度纯化的甘油的成本超过葡萄糖。
发明概述
提供一种培养一种或多种微生物的方法。该方法包括在包含第一浓度水平的粗甘油的培养基中培养一种或多种微生物,一旦甘油的所述第一浓度降低至第一阈值水平,就以足以实现所述第一浓度水平的浓度向所述培养基中馈入额外量的粗甘油,监测所述粗甘油浓度直至所述粗甘油的第一浓度水平降低至所述第一阈值水平。可重复各步骤直至实现所需微生物细胞密度。
也提供一种用于产生一种或多种脂肪酸的方法。方法包括提供能够产生脂肪酸的微生物,提供包含粗甘油的培养基,以及在所述培养基中在足以产生一种或多种脂肪酸的条件下培养所述微生物以提供最终浓度是所述微生物的至少50重量%的一种或多种不饱和脂肪酸。
附图简述
图1是显示在使用来自不同生物柴油厂商的各种粗甘油原料作为唯一碳源进行发酵期间,生物质浓度(g/L)随时间的曲线图。
图2是显示在以不同体积比使用粗甘油和葡萄糖的混合物进行发酵期间,生物质浓度(g/L)随时间的曲线图。甘油意指来自Rothsay的粗甘油;葡萄糖意指750g/L葡萄糖溶液。比率是两种溶液的体积比。
图3是显示在使用粗甘油作为唯一馈入碳源进行破囊壶菌ONC-T18的大规模(200,000L)发酵期间,生物质和脂质浓度随时间的曲线图。
图4是甲醇基生物柴油转酯反应的示意图。
详述
全世界对生物燃料的需求增加以及生物燃料的产能增加已产生巨大剩余的粗甘油作为副产物。精制粗甘油需要过大投资,而未精制粗甘油具有极其少许经济价值。因此,许多生物燃料制造商将粗甘油视为一种类型的工业废水。因此,用以使所述粗甘油转化成较高价值产品的高效方法将具有极大重要性。尽管生物柴油副产物甘油(一种类型的粗甘油)已用作碳源,但由于由粗甘油杂质引起的毒性以及缺乏用于避免所述毒性的适当发酵碳供给策略而尚未实现高密度生物质和油。相比之下,本文提供使用粗甘油培养微生物以及产生油的方法。提供的方法导致较高生物质和油产量,在发酵期间实现172g/L细胞干重,含有68%油。所述生产率与可通过葡萄糖基馈料批式发酵实现的生产率类似。
因此,提供的方法使得能够使用工业副产物甘油(粗制)作为部分或唯一碳底物与其它营养成分组合来使微生物生长以获得高细胞密度(>100g/L)和高油含量(>50%)。来自工业来源的粗甘油需要少许乃至不需要预处理。通过确定和控制方式将粗甘油底物馈入发酵罐中以使任何生物抑制作用都可保持最小,同时可实现极高生物质和油产率。本文也证明为无菌发酵工艺所需的典型灭菌程序可被去除。此外,可减少或消除对粗甘油的预处理,并且甘油可按照在从制造商接收时的原样馈入微生物培养物中。当这种方法以商业规模进行时,这会节约大量能源成本。如本文所用,术语“预处理”是指移除可以物理方式或以生物方式影响培养物生长的非甘油杂质。预处理的实例包括用以沉淀和移除杂质的化学处理,用以匹配培养环境的pH的pH调整,用以移除悬浮固体的过滤或离心。
本文提供一种培养一种或多种微生物的方法。该方法包括在包含第一浓度水平的粗甘油的培养基中培养一种或多种微生物,一旦甘油的所述第一浓度降低至第一阈值水平,就以足以实现所述第一浓度水平的浓度向所述培养基中馈入额外量的粗甘油,监测所述粗甘油浓度直至所述粗甘油的第一浓度水平降低至所述第一阈值水平。任选地,第一浓度水平在1与60g/L之间,或是在1与60g/L之间的任何水平,包括端值。任选地,第一浓度水平在5与60g/L之间,在15与60g/L之间,在5与20g/L之间,或在15与20g/L之间。任选地,第一阈值水平在0与5g/L之间,或是在0与5g/L之间的任何水平,包括端值。因此,第一阈值水平可为例如0、1、2、3、4或5g/L。任选地,重复各步骤直至实现所需微生物细胞密度。任选地,所需微生物细胞密度大于100g/L。任选地,所需细胞密度是50至200g/L、50至150g/L、50至100g/L、100至250g/L、100至150g/L、或80至100g/L。任选地,细胞密度是在50至250g/L之间的任何密度,包括端值。任选地,细胞密度是80至100g/L。任选地,细胞密度是120至230g/L。任选地,微生物细胞密度含有50重量%至80重量%的总脂肪酸。任选地,总脂肪酸包含10至45%DHA。任选地,以总细胞重量计,微生物细胞密度含有5至36%DHA。
微生物能够产生一种或多种脂肪酸。因此,提供用于产生一种或多种脂肪酸的方法。方法包括提供能够产生脂肪酸的微生物,提供包含粗甘油的培养基,以及在所述培养基中在足以产生一种或多种脂肪酸的条件下培养所述微生物以提供最终浓度是所述微生物的至少50重量%的一种或多种不饱和脂肪酸。任选地,脂肪酸是多不饱和脂肪酸。多不饱和脂肪酸可为例如α亚麻酸、花生四烯酸、二十二碳六烯酸、二十二碳五烯酸、二十碳五烯酸、γ-亚麻酸、亚油酸、亚麻酸及其组合。
在提供的方法中,监测粗甘油浓度可使用为本领域技术人员所知的多种方法进行。任选地,监测包括测量溶氧水平。任选地,监测包括获得培养基的样品,以及测定所述样品中的甘油浓度。任选地,监测包括使用量热测定、化学反应基量热测定、荧光测定、HPLC测定、酶促测定或其组合分析样品。
存在多种适用于提供的方法中的微生物。本文所述的微生物可为藻类(例如微藻)、真菌(包括酵母)、细菌或原生生物。任选地,微生物包括破囊壶菌目(Thraustochytriales)的破囊壶菌(Thraustochytrid),并且更具体来说,包括破囊壶菌目的破囊壶菌属(Thraustochytrium)。任选地,微生物的群体包括如美国专利号5,340,594和5,340,742中所述的破囊壶菌目,所述美国专利以引用的方式整体并入本文。微生物可为破囊壶菌属种,诸如如美国专利号8,163,515中所述的以ATCC登录号PTA-6245寄存的破囊壶菌属物种(即ONC-T18),所述美国专利以引用的方式整体并入本文。因此,微生物可具有与SEQ ID NO:1至少95%、96%、97%、98%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%、99.9%或更大(例如包括100%)同一的18s rRNA序列。
用于本文所述的方法中的微生物可产生多种脂质化合物。如本文所用,术语脂质包括磷脂、游离脂肪酸、脂肪酸的酯、甘油三酯、固醇和固醇酯、类胡萝卜素(carotenoid)、叶黄素(xanthophyl)(例如氧基类胡萝卜素(oxycarotenoid))、烃和为本领域普通技术人员所知的其它脂质。任选地,脂质化合物包括不饱和脂质。不饱和脂质可包括多不饱和脂质(即含有至少2个不饱和碳-碳键例如双键的脂质)或高度不饱和脂质(即含有4个或更多个不饱和碳-碳键的脂质)。不饱和脂质的实例包括ω-3和/或ω-6多不饱和脂肪酸,诸如二十二碳六烯酸(即DHA)、二十碳五烯酸(即EPA)和其它天然存在的不饱和、多不饱和和高度不饱和化合物,其以10%接种量接种时,这个长久迟滞期得以显著缩短。
提供的方法包括用于根据本领域中已知的方法来培养微生物的额外步骤或可与所述额外步骤联合使用。举例来说,可根据美国专利公布2009/0117194或2012/0244584中所述的方法培养破囊壶菌例如破囊壶菌属某种,所述美国专利公布由于其中使用的方法的各个步骤或组合物而以引用的方式整体并入本文。
使微生物在生长培养基(也称为“培养基”)中生长。多种培养基中的任一种都可适用于培养本文所述的微生物。任选地,培养基为微生物供给各种营养性组分,包括碳源和氮源。用于破囊壶菌培养的培养基可包括多种碳源中的任一种。碳源的实例包括脂肪酸、脂质、甘油、三甘油、碳水化合物、多元醇、氨基糖和任何种类的生物质或废物物流。脂肪酸包括例如油酸。碳水化合物包括但不限于葡萄糖、纤维素、半纤维素、果糖、右旋糖、木糖、乳果糖、半乳糖、麦芽三糖、麦芽糖、乳糖、糖原、明胶、淀粉(玉米淀粉或小麦淀粉)、乙酸盐、肌肉肌醇(例如源于玉米浆)、半乳糖醛酸(例如源于果胶)、L-海藻糖(例如源于半乳糖)、龙胆二糖、葡糖胺、α-D-葡萄糖-1-磷酸酯(例如源于葡萄糖)、纤维二糖、糊精、α-环糊精(例如源于淀粉)和蔗糖(例如来自糖蜜)。多元醇包括但不限于麦芽糖醇、赤藻糖醇和阿东糖醇(adonitol)。氨基糖包括但不限于N-乙酰基-D-半乳糖胺、N-乙酰基-D-葡糖胺和N-乙酰基-β-D-甘露糖胺。
提供的方法中使用的一种或多种培养基包含粗甘油。如本文所用,术语“粗甘油”是指例如生物柴油生产的制造过程的副产物。术语“粗甘油”不同于术语“甘油(glycerol)”或“甘油(glycerin)”,因为这些产品仅仅或基本上包含呈纯净形式的“甘油”。术语“甘油(glycerol)”和“甘油(glycerin)”在整篇本文中可互换使用。因此,粗甘油含有额外于纯净甘油的试剂和/或成分。任选地,粗甘油是生产生物柴油的副产物。粗甘油是通过甘油三酯转酯来生产生物柴油的主要副产物,或是通过水解(皂化)甘油三酯来生产肥皂的副产物。举例来说,典型生物柴油工艺是使脂肪或油(甘油三酯)与醇诸如甲醇反应以形成脂肪酸的酯(生物柴油)和甘油。甲醇基反应实例显示于图4中。由于脂肪/油中的杂质以及反应自身常常用诸如甲醇钠的化学催化剂辅助,所以最终甘油部分含有相对较高含量的杂质。典型粗甘油组合物和物理性质显示于表1中(参考文献:REG Ralston粗甘油分析证书)。相同粗甘油的灰分组成显示于表2中。粗甘油表征的更多实例可见于参考文献Thompson和He,Applied Engineering in Agriculture,第22卷(2):261-265(2006)以及Hu等,J.Agric.Food Chem.,2012,60(23):5915-5921(2012)中,所述参考文献以引用的方式整体并入本文。
表1.REG Ralston粗甘油副产物。
表2 REG的Ralston粗甘油副产物的灰分组成。
任选地,粗甘油是一种或多种培养基中的唯一碳源。任选地,培养基包含一种或多种额外碳源。任选地,不将粗甘油灭菌或预处理。任选地,粗甘油包含甲醇、水、灰分、非甘油有机物质、硫酸钠、牛脂酸甲酯或其组合。任选地,灰分包含钙、铁、镁、钾、钠、锌或其组合。
任选地,在使生物质和/或目标化合物的产量(例如油或总脂肪酸(TFA)含量)增加的条件下培养本文提供的微生物。例如通常在盐水培养基中培养破囊壶菌。任选地,可在具有约0.5g/L至约50.0g/L的盐浓度的培养基中培养破囊壶菌。任选地,在具有约0.5g/L至约35g/L(例如约18g/L至约35g/L)的盐浓度的培养基中培养破囊壶菌。任选地,可使本文所述的破囊壶菌在低盐条件下生长。举例来说,可在具有约0.5g/L至约20g/L(例如约0.5g/L至约15g/L)的盐浓度的培养基中培养破囊壶菌。培养基任选包括NaCl。任选地,培养基包括天然或人工海盐和/或人工海水。
培养基可包括非含氯钠盐作为钠的来源。适于根据本发明方法使用的的非氯钠盐的实例包括但不限于苏打灰(碳酸钠和氧化钠的混合物)、碳酸钠、碳酸氢钠、硫酸钠及其混合物。参见例如美国专利号5,340,742和6,607,900,其各自的整个内容以引用的方式并入本文。总钠中的重大部分例如可由非氯盐供给以使培养基中总钠的小于约100%、75%、50%或25%由氯化钠供给。
任选地,培养基具有小于约3g/L、500mg/L、250mg/L或120mg/L的氯离子浓度。举例来说,用于提供的方法中的培养基可具有在约60mg/L与120mg/L之间,并且包括约60mg/L和120mg/L的氯离子浓度。
用于破囊壶菌培养的培养基可包括多种氮源中的任一种。示例性氮源包括铵溶液(例如含NH4的H2O)、铵或胺盐(例如(NH4)2SO4、(NH4)3PO4、NH4NO3、NH4OOCH2CH3(NH4Ac))、蛋白胨、胰蛋白胨、酵母提取物、麦芽提取物、鱼粉、谷氨酸钠、大豆提取物、酪蛋白氨基酸和酒糟。适合培养基中的氮源的浓度通常在约1g/L与约25g/L之间的范围内,并且包括约1g/L和约25g/L。
培养基任选包括磷酸盐诸如磷酸钾或磷酸钠。培养基中的无机盐和微量营养物可包括硫酸铵、碳酸氢钠、原钒酸钠、铬酸钾、钼酸钠、亚硒酸、硫酸镍、硫酸铜、硫酸锌、氯化钴、氯化铁、氯化锰、氯化钙和EDTA。可包括维生素,诸如盐酸吡哆辛、盐酸硫胺、泛酸钙、对氨基苯甲酸、核黄素、烟酸、生物素、叶酸和维生素B12。
可使用酸或碱,当适当时,和/或使用氮源,将培养基的pH调整至在3.0与10.0之间,并且包括3.0和10.0。任选地,可将培养基灭菌。
通常,用于培养微生物的培养基是液体培养基。然而,用于培养微生物的培养基可为固体培养基。除如本文讨论的碳源和氮源之外,固体培养基可含有一种或多种提供结构支撑和/或使培养基呈固体形式的组分(例如琼脂或琼脂糖)。
任选地,对所得生物质进行巴氏杀菌(pasteurized)以使生物质中存在的不合需要的物质失活。举例来说,可对生物质进行巴氏杀菌以使化合物降解性物质失活。生物质可存在于发酵培养基中或从发酵培养基分离以进行巴氏杀菌步骤。巴氏杀菌步骤可通过将生物质和/或发酵培养基加热至高温来进行。举例来说,可将生物质和/或发酵培养基加热至约50℃至约95℃(例如约55℃至约90℃或约65℃至约80℃)的温度。任选地,可将生物质和/或发酵培养基加热约30分钟至约120分钟(例如约45分钟至约90分钟或约55分钟至约75分钟)。可使用适合加热手段诸如像通过直接蒸汽喷射来进行巴氏杀菌。
任选地,不进行巴氏杀菌步骤。换句话说,本文教导的方法任选缺乏巴氏杀菌步骤。
任选地,可根据多种方法收集生物质,所述方法包括当前为本领域技术人员所知的那些。举例来说,生物质可使用例如离心(例如用固体排出离心机)或过滤(例如交叉流过滤)从发酵培养基收集。任选地,收集步骤包括使用沉淀剂来达成对细胞生物质的加速收集(例如磷酸钠或氯化钙)。
任选地,用水洗涤生物质。任选地,可使生物质浓缩直至约20%固体。举例来说,可使生物质浓缩至约5%至约20%固体、约7.5%至约15%固体、或约固体至约20%固体、或所叙述范围内的任何百分比。任选地,可使生物质浓缩至约20%固体或更少、约19%固体或更少、约18%固体或更少、约17%固体或更少、约16%固体或更少、约15%固体或更少、约14%固体或更少、约13%固体或更少、约12%固体或更少、约11%固体或更少、约10%固体或更少、约9%固体或更少、约8%固体或更少、约7%固体或更少、约6%固体或更少、约5%固体或更少、约4%固体或更少、约3%固体或更少、约2%固体或更少、或约1%固体或更少。
提供的方法任选包括从生物质或微生物分离多不饱和脂肪酸。可使用多种方法中的一种或多种进行多不饱和脂肪酸的分离,所述方法包括当前为本领域技术人员所知的那些。举例来说,分离多不饱和脂肪酸的方法描述于美国专利号8,163,515中,所述美国专利以引用的方式整体并入本文。任选地,在分离多不饱和脂肪酸之前不将培养基灭菌。任选地,灭菌包括增加温度。任选地,由微生物产生以及根据提供的方法分离的多不饱和脂肪酸是中链脂肪酸。任选地,一种或多种多不饱和脂肪酸选自由α亚麻酸、花生四烯酸、二十二碳六烯酸、二十二碳五烯酸、二十碳五烯酸、γ-亚麻酸、亚油酸、亚麻酸及其组合组成的组。
根据本文所述的方法产生的包括多不饱和脂肪酸(PUFA)和其它脂质的油可用于利用它们的生物、营养或化学性质的多种应用中的任一种中。因此,提供的方法任选包括从具有阈值体积的收集部分分离油。任选地,油用于生产燃料例如生物燃料。任选地,油可用于药物、食物补充剂、动物饲料添加剂、化妆品等中。根据本文所述的方法产生的脂质也可作为中间体用于生产其它化合物。
举例来说,由使用提供的方法培养的微生物产生的油可包含脂肪酸。任选地,脂肪酸选自由α亚麻酸、花生四烯酸、二十二碳六烯酸、二十二碳五烯酸、二十碳五烯酸、γ-亚麻酸、亚油酸、亚麻酸及其组合组成的组。任选地,油包含甘油三酯。任选地,油包含选自由棕榈酸(C16∶0)、肉豆蔻酸(C14∶0)、棕榈油酸(C16∶1(n-7))、顺式十八碳烯酸(C18∶1(n-7))、二十二碳五烯酸(C22∶5(n-6))、二十二碳六烯酸(C22∶6(n-3))及其组合组成的组的脂肪酸。
任选地,可将根据本文所述的方法产生的脂质并入最终产品(例如食物或饲料补充剂、婴儿配制食品、药物、燃料等)中。脂质可被并入其中的适合食物或饲料补充剂包括饮料诸如奶、水、运动饮料、能量饮料、茶和果汁;甜食诸如糖果、果冻和饼干;含脂肪食物和饮料诸如乳制品;加工食品诸如软质米饭(或粥);婴儿配制食品;早餐谷物;等。任选地,可将一种或多种产生脂质并入膳食补充剂诸如像维生素或复合维生素中。任选地,根据本文所述的方法产生的脂质可被包括在膳食补充剂中,并且任选地可直接并入食物或饲料的组分(例如食物补充剂)中。
通过本文所述的方法产生的脂质可被并入其中的饲料的实例包括宠物食物诸如猫食物;狗食物等;用于观赏鱼、养殖鱼或甲壳动物等的饲料;用于农场饲养动物(包括家畜和在水产养殖业中饲养的鱼或甲壳动物)的饲料。根据本文所述的方法产生的脂质可被并入其中的食物或饲料物质优选对于作为预定接受者的生物体而言是适口的。这个食物或饲料物质可具有当前关于食物物质已知的任何物理性质(例如固体、液体、软质)。
任选地,可将一种或多种产生化合物(例如PUFA)并入营养食品或药物中。这种营养食品或药物的实例包括各种类型的片剂、胶囊、饮剂等。任选地,营养食品或药物适于表面施加。剂型可包括例如胶囊、油剂、颗粒剂、细粒剂、散剂、片剂、丸剂、糖锭等。
可将根据本文所述的方法产生的油或脂质与多种其它试剂中的任一种组合并入如本文所述的产品中。举例来说,可使所述化合物与一种或多种粘合剂或填充剂、螯合剂、色素、盐、表面活性剂、保湿剂、粘度调节剂、增稠剂、润滑剂、芳香剂、防腐剂等或其任何组合进行组合。
公开的是可用于所公开方法和组合物,可与所公开方法和组合物联合使用,可用于为所公开方法和组合物作准备,或是所公开方法和组合物的产物的材料、组合物和组分。本文公开这些和其它材料,并且应了解当公开这些材料的组合、子组、相互作用、群组等时,尽管可未明确公开对这些化合物的各各种个别以及集合组合和排列的特定提及,但各自被明确涵盖以及描述于本文中。举例来说,如果公开和讨论方法,并且讨论可对许多分子,包括对所述方法进行的许多修改,那么除非相反地明确指示,否则明确涵盖所述方法和所述修改的各个和每个可能组合和排列。同样,也明确涵盖和公开这些的任何子组或组合。这个观念适用于本公开的所有方面,包括但不限于使用所公开组合物的方法中的步骤。因此,如果存在可被进行的多种额外步骤,那么应了解这些额外步骤各自可与所公开方法的任何特定方法步骤或方法步骤的组合一起进行,并且各所述组合或组合的子组被明确涵盖并应视为被公开。
如整篇所用,范围(例如1-10)和提及约某一给定值(例如约1或约10)包括一个或多个所叙述值(例如1和/或10)。
本文引用的出版物和引用它们所针对的素材据此以引用的方式整体明确并入本文。
以下实施例意图进一步说明本文所述的方法和组合物的某些方面,并且不意图限制权利要求的范围。
实施例
实施例1.粗甘油发酵
从3个不同生物柴油厂商(Rothsay(Winnipeg,Canada)、BIOX(Hamilton,Canada)和REG Newton LLC(Newton,Iowa))获得4种粗甘油物流。基于由相应厂商提供的信息将它们的组成列于表3中。明确的是在不同粗甘油之间展现极大可变性。
表3.来自生物柴油的粗甘油副产物的粗甘油组分。
*MONG:非甘油有机物质
为进行实验室规模发酵实验,使用氢氧化钠溶液将Rothsay和BIOX 65粗甘油的pH调整至5.5。未对BIOX 98和REG Newton粗甘油进行pH调整。接着高压灭菌所有粗甘油物流,并且其作为唯一馈入碳源用于在2L发酵罐中进行破囊壶菌ONC-T18的单独粗甘油基馈料批式发酵。所有初始发酵培养基都含有(每升):60g甘油;2g大豆蛋白胨;1.65g氯化钠;4g硫酸镁七水合物;2.2g磷酸二氢钾;2.4g磷酸氢二钾;20g硫酸铵;0.1g氯化钙二水合物;0.003g氯化铁;0.003g硫酸铜五水合物;0.0015g钼酸钠脱水物;0.003g硫酸锌七水合物;0.0015g氯化钴六水合物;0.0015g氯化锰四水合物;0.0015g硫酸镍六水合物;0.00003g维生素B12;0.00003g生物素;盐酸硫胺0.006g。2L发酵罐用于Rothsay、BIOX 65和BIOX 98发酵,而5L发酵罐用于REG Newton发酵。将所有发酵的pH都用氢氧化钠和磷酸溶液控制在4.5±0.2下。将所有发酵的温度都控制在28℃下。所有发酵都以馈料批式模式操作,使用相应粗甘油作为唯一碳原料。在适当时间,将一定剂量的粗甘油自动泵送至发酵罐中以致使培养基中甘油浓度达到60g/L(对于2L操作)或20g/L(对于5L操作)。
在整个发酵期间进行所述碳馈入,直至已达到某一生物质浓度和细胞内油含量。获得在140g/L至165g/L的范围内的最终生物质浓度(图1),含有在65%至78%的范围内的细胞内脂质(表4)。考虑到测试粗甘油原料之间的品质变化以及应用于它们的最小程度预处理,这些结果是对所开发的用于使用粗甘油副产物来进行商业上可行的生物质和脂质生产的高细胞/脂质密度馈料批式发酵工艺具有稳健性的良好证明。
表4.使用各种粗甘油原料作为唯一碳源的发酵结果的概述。
实施例2.混合碳源发酵
足够和不间断数量的碳原料供给对于发酵基生产工厂的运转具有关键重要性。在当主要碳原料处于供给短缺时的情况下,能够利用补充碳原料对于工厂的持续运转可为重要的。因此,使用葡萄糖和粗甘油的混合物进行破囊壶菌ONC-T18的发酵。尽管葡萄糖是用于破囊壶菌高密度发酵以产生脂质的熟知碳源,仍不明确的是如果葡萄糖与第二碳源一起用于同一发酵中,那么是否将发生碳分解代谢物阻遏。使用与实施例1中所示相同的初始培养基配方来准备两个2L发酵罐。获得来自Rothway、具有实施例1中所示的规格的粗甘油;并且也制备750g/L葡萄糖溶液。接着将粗甘油和葡萄糖溶液以80∶20体积比物理混合作为用于第一发酵罐的碳馈料,并且以20∶80体积比混合作为用于第二发酵罐的馈料。通过实施例1中所述的相同碳馈入策略来进行发酵。如由图2和表5中所示的数据所证明,使用粗甘油和葡萄糖作为混合碳馈料产生的生物质和脂质结果与当粗甘油用作唯一碳馈料时的那些结果类似。在这个实施例中未发现碳分解代谢物阻遏情况属实,所述阻遏是当两种碳均存在于生长培养基中时,存在优选碳源会抑制第二碳源的代谢的现象。在发酵期间,在整个培养过程中,甘油与葡萄糖两者均同时被消耗。这个实施例证明在商业培养破囊壶菌期间,粗甘油可用作主要碳源或次要碳源,并且第二碳原料可用于同一发酵中。所述碳使用灵活性确保大规模发酵工艺运转的稳定性。
表5.以不同体积比使用粗甘油和葡萄糖的混合物的发酵结果的概述。
实施例3.混合粗甘油发酵
类似于实施例2,对于持续大规模工厂运转,也将有利的是能够使用来自不同生物柴油制造过程的粗甘油的混合物。为此,以相等体积比混合4种粗甘油物流以形成粗甘油混合物。各物流的规格列于表6中。在混合之后,粗甘油混合物不经任何进一步处理(例如pH调整、过滤)或灭菌即用作碳馈料。以具有与实施例1中所示相同的配方的初始培养基为这个实验准备30L发酵罐,例外之处是在灭菌程序之前不在初始培养基中批式加入甘油。紧接在接种之后,根据实施例1中所述的馈入策略馈入粗甘油混合物,其中各次给料致使培养基中甘油浓度达到20g/L。截至70小时,生物质达到171.54g/L,具有68.00%脂质。尽管未灭菌即使用,但发酵不含污染。
表6.来自不同生物柴油制造过程的用以制备粗甘油混合物的粗甘油副产物的组成。
*MONG:非甘油有机物质
实施例4.粗甘油用于商业生物质和脂质生产
为证明所开发的粗甘油基发酵工艺用于商业生物质和脂质生产的可行性,进行两个大规模(200,000L)发酵。REG Ralston粗甘油不经任何预处理或灭菌即用作唯一馈入碳源。培养基配方与实施例1中所述相同,例外之处是仅30g/L葡萄糖被批式加入初始培养基中,1g/L大豆蛋白胨用于第一批次中,并且1g/L玉米浸渍固体(替代大豆蛋白胨)用于第二批次中。在pH 4.5±0.3下进行发酵,其中将温度控制在29±1℃下。两个发酵中的一者的生物质和脂质时间曲线显示于图3中;而来自两个大规模操作的全过程量度列于表7中。平均来说,可使用生物柴油粗甘油副产物作为唯一馈入碳源,在不进行任何预处理或灭菌程序下,通过200,000L发酵罐产生180.76g/L生物质,具有126.53g/L脂质。这些结果证明能够使用粗甘油作为用于大规模商业生产生物质和脂质的碳原料。
表7.使用粗甘油作为唯一馈入碳源在不进行预处理或灭菌下的大规模(200,000L)发酵。
序列表
<110> 玛拉可再生能源公司(Mara Renewables Corporation)
<120> 使用粗甘油高密度产生生物质和油
<130> 095523-0972128 (011WO1)
<150> 62/138,631
<151> 2015-03-25
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1723
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成构建体
<400> 1
gtagtcatac gctcgtctca aagattaagc catgcatgtg taagtataag cgattatact 60
gtgagactgc gaacggctca ttatatcagt tatgatttct tcggtatttt ctttatatgg 120
atacctgcag taattctgga attaatacat gctgagaggg cccgactgtt cgggagggcc 180
gcacttatta gagttgaagc caagtaagat ggtgagtcat gataattgag cagatcgctt 240
gtttggagcg atgaatcgtt tgagtttctg ccccatcagt tgtcgacggt agtgtattgg 300
actacggtga ctataacggg tgacggggag ttagggctcg actccggaga gggagcctga 360
gagacggcta ccacatccaa ggaaggcagc aggcgcgtaa attacccaat gtggactcca 420
cgaggtagtg acgagaaata tcaatgcggg gcgcttcgcg tcttgctatt ggaatgagag 480
caatgtaaaa ccctcatcga ggatcaactg gagggcaagt ctggtgccag cagccgcggt 540
aattccagct ccagaagcgt atgctaaagt tgttgcagtt aaaaagctcg tagttgaatt 600
tctggggcgg gagccccggt ctttgcgcga ctgcgctctg tttgccgagc ggctcctctg 660
ccatcctcgc ctcttttttt agtggcgtcg ttcactgtaa ttaaagcaga gtgttccaag 720
caggtcgtat gacctggatg tttattatgg gatgatcaga tagggctcgg gtgctatttt 780
gttggtttgc acatctgagt aatgatgaat aggaacagtt gggggtattc gtatttagga 840
gctagaggtg aaattcttgg atttccgaaa gacgaactac agcgaaggca tttaccaagc 900
atgttttcat taatcaagaa cgaaagtctg gggatcgaag atgattagat accatcgtag 960
tctagaccgt aaacgatgcc gacttgcgat tgcggggtgt ttgtattgga ccctcgcagc 1020
agcacatgag aaatcaaagt ctttgggttc cggggggagt atggtcgcaa ggctgaaact 1080
taaaggaatt gacggaaggg caccaccagg agtggagcct gcggcttaat ttgactcaac 1140
acgggaaaac ttaccaggtc cagacatagg taggattgac agattgagag ctctttcttg 1200
attctatggg tggtggtgca tggccgttct tagttggtgg agtgatttgt ctggttaatt 1260
ccgttaacga acgagacctc ggcctactaa atagcggtgg gtatggcgac atacttgcgt 1320
acgcttctta gagggacatg ttcggtatac gagcaggaag ttcgaggcaa taacaggtct 1380
gtgatgccct tagatgttct gggccgcacg cgcgctacac tgatgggttc aacgggtggt 1440
catcgttgtt cgcagcgagg tgctttgccg gaaggcatgg caaatccttt caacgcccat 1500
cgtgctgggg ctagattttt gcaattatta atctccaacg aggaattcct agtaaacgca 1560
agtcatcagc ttgcattgaa tacgtccctg ccctttgtac acaccgcccg tcgcacctac 1620
cgattgaacg gtccgatgaa accatgggat gaccttttga gcgtttgttc gcgagggggg 1680
tcagaactcg ggtgaatctt attgtttaga ggaaggtgaa gtc 1723
Claims (32)
1.一种培养一种或多种微生物的方法,其包括:
(a)在包含第一浓度水平的粗甘油的培养基中培养一种或多种微生物;
(b)一旦甘油的所述第一浓度降低至第一阈值水平,即以足以实现所述第一浓度水平的浓度向所述培养基中馈入额外量的粗甘油;
(c)监测所述粗甘油浓度直至所述粗甘油的第一浓度水平降低至所述第一阈值水平;以及
(d)重复步骤(b)(c)和(d)直至实现所需微生物细胞密度。
2.如权利要求1所述的方法,其中所述一种或多种微生物属于破囊壶菌属或裂殖壶菌属。
3.如权利要求1-2中任一项所述的方法,其中所述一种或多种微生物是ONC-T18。
4.如权利要求1-3中任一项所述的方法,其中所述微生物能够产生一种或多种脂肪酸。
5.如权利要求4所述的方法,其中所述脂肪酸是多不饱和脂肪酸。
6.如权利要求5所述的方法,其中所述多不饱和脂肪酸选自由α亚麻酸、花生四烯酸、二十二碳六烯酸、二十二碳五烯酸、二十碳五烯酸、γ-亚麻酸、亚油酸、亚麻酸及其组合组成的组。
7.如权利要求4所述的方法,其中所述方法进一步包括分离所述脂肪酸。
8.如权利要求1-7中任一项所述的方法,其中所述第一浓度水平在1与60g/L之间。
9.如权利要求1-8中任一项所述的方法,其中所述第一阈值水平在0与5g/L之间。
10.如权利要求1-9中任一项所述的方法,其中所述监测包括测量溶氧水平。
11.如权利要求1-10中任一项所述的方法,其中所述监测包括获得所述培养基的样品,以及测定所述样品中的所述甘油浓度。
12.如权利要求12所述的方法,其中所述监测包括使用量热测定、化学反应基量热测定、荧光测定、HPLC测定、酶促测定或其组合分析所述样品。
13.如权利要求1-10中任一项所述的方法,其中所述细胞密度在50g/L与250g/L之间。
14.如权利要求1-13中任一项所述的方法,其中所述微生物细胞密度含有50重量%至80重量%的总脂肪酸。
15.如权利要求1-13中任一项所述的方法,其中所述总脂肪酸包含10至45%DHA。
16.如权利要求1-13中任一项所述的方法,其中以总细胞重量计,所述微生物细胞密度含有5至36%DHA。
17.如权利要求1-16中任一项所述的方法,其中所述培养基包含一种或多种额外碳源。
18.如权利要求1-16中任一项所述的方法,其中不将所述粗甘油灭菌。
19.如权利要求1-16中任一项所述的方法,其中所述粗甘油包含甲醇、水、灰分、非甘油有机物质、硫酸钠、牛脂酸甲酯或其组合。
20.如权利要求19所述的方法,其中所述灰分包含钙、铁、镁、钾、钠、锌或其组合。
21.如权利要求1-20中任一项所述的方法,其中所述粗甘油是生物柴油副产物。
22.一种用于产生一种或多种脂肪酸的方法,其包括:
(a)提供能够产生脂肪酸的微生物;
(b)提供包含粗甘油的培养基;以及
(c)在所述培养基中在足以产生所述一种或多种脂肪酸的条件下培养所述微生物以提供最终浓度为所述微生物的至少50重量%的所述一种或多种不饱和脂肪酸。
23.如权利要求22所述的方法,其中所述微生物属于破囊壶菌属或裂殖壶菌属。
24.如权利要求22所述的方法,其中所述微生物是ONC-T18。
25.如权利要求22-24中任一项所述的方法,其中所述脂肪酸包含多不饱和脂肪酸。
26.如权利要求25所述的方法,其中所述多不饱和脂肪酸选自由α亚麻酸、花生四烯酸、二十二碳六烯酸、二十二碳五烯酸、二十碳五烯酸、γ-亚麻酸、亚油酸、亚麻酸及其组合组成的组。
27.如权利要求22-26中任一项所述的方法,其进一步包括分离所述脂肪酸。
28.如权利要求22-27中任一项所述的方法,其中所述培养基包含一种或多种额外碳源。
29.如权利要求22-28中任一项所述的方法,其中不将所述粗甘油灭菌。
30.如权利要求22-29中任一项所述的方法,其中所述粗甘油包含甲醇、水、灰分、非甘油有机物质、硫酸钠、牛脂酸甲酯或其组合。
31.如权利要求30所述的方法,其中所述灰分包含钙、铁、镁、钾、钠、锌或其组合。
32.如权利要求22-20中任一项所述的方法,其中所述粗甘油是生物柴油副产物。
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AU2016238396A1 (en) | 2017-09-28 |
CA2980679C (en) | 2022-11-15 |
AR104042A1 (es) | 2017-06-21 |
US20160281054A1 (en) | 2016-09-29 |
HK1246346A1 (zh) | 2018-09-07 |
EP3274441A1 (en) | 2018-01-31 |
WO2016151545A1 (en) | 2016-09-29 |
KR20170131565A (ko) | 2017-11-29 |
CA2980679A1 (en) | 2016-09-29 |
MX2017011516A (es) | 2018-01-11 |
JP7346792B2 (ja) | 2023-09-20 |
NZ735277A (en) | 2024-03-22 |
CL2017002380A1 (es) | 2018-03-16 |
JP6988031B2 (ja) | 2022-01-05 |
US11578304B2 (en) | 2023-02-14 |
AU2016238396B2 (en) | 2021-07-08 |
ZA201706258B (en) | 2021-01-27 |
SG11201707234SA (en) | 2017-10-30 |
EP3274441A4 (en) | 2018-10-24 |
JP2018512135A (ja) | 2018-05-17 |
JP2022002522A (ja) | 2022-01-11 |
KR102300392B1 (ko) | 2021-09-08 |
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