13TSB_CRD: HIGH PRODUCTIVITY HOMOFERMENTATIVE PROCESS for BUTANOL (HIPHOP)

13TSB_CRD：丁醇的高产均质发酵工艺 (HIPHOP)

• 批准号: BB/L011492/1
• 负责人: Gillian Stephens
• 金额: $ 18.93万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL011492%2F1
• 关键词: 13TSB_CRD; PRODUCTIVITY; HOMOFERMENTATIVE; PROCESS; BUTANOL

The acetone-butanol-ethanol (ABE) fermentation uses anaerobic bacteria from the genus Clostridium to ferment sugars orstarch to solvent mixtures in a typical A:B:E ratio of 3:6:1. Historically, the fermentation was used to manufacture solventsand chemicals, but fell out of favour when the oil industry developed cheaper ways to make these chemicals. With growingconcerns about oil security and global warming, the ABE fermentation is now undergoing a massive revival. Today, butanolis the preferred product, since it can be used as a biofuel, a solvent and an intermediate to manufacture a wide range ofchemicals.At present, butanol fermentations are inefficient because the accumulating butanol poisons the bacteria, ultimately causingthe fermentation to stop. Furthermore, butanol-producing Clostridium species are genetically unstable, so the fermentationcan only be run for a short time before shutting down and restarting from fresh cells. There are also problems withrecovering the butanol, because the product stream is a dilute mixture of butanol, acetone and ethanol in water. Distillationprovides the only easy way to recover the products, but requires a lot of energy.We will use synthetic biology to produce new Clostridium strains that produce butanol without forming acetone and ethanol- homofermentative strains. Scientists at Green Biologics have already developed homofermentative mutants usingtraditional mutagenesis techniques and have sequenced their DNA, to identify the mutated genes. In this project, we willselect the most important mutations and recreate them in a commercial production strain to develop a genetically stable,high productivity butanol-producing organism.The new organisms will produce much cleaner product streams, allowing development of new separation processes, basedon liquid-liquid extraction. This involves mixing the growing culture with a water-immiscible solvent that dissolves thebutanol more efficiently than water. As a result, the butanol will transfer into the solvent phase, which can easily beseparated by allowing the two immiscible liquid phases to settle out (like oil and water). This provides a very neat way tosolve problems with butanol toxicity, because the butanol is removed from the immediate environment surrounding thecells, so the cells are not exposed to the poisonous product. This allows butanol production to continue until the solventphase is saturated, so that the cells can form very high butanol concentrations.In situ solvent extraction depends on finding a water-immiscible liquid that is not only a good solvent for butanol but is alsonot poisonous to the cells. Most conventional solvents struggle to extract butanol from water and are just as poisonous asbutanol itself. However, scientists at the University of Nottingham have discovered that a new class of solvents called ionicliquids (ILs) can extract butanol from water and are not poisonous to living cells. ILs are made from salts that are molten atroom temperature, and so are non-volatile, unlike conventional solvents. Therefore, the butanol can easily be recovered,simply by separating the IL phase and then boiling off the butanol, leaving the IL behind for re-use.Overall, this project brings together synthetic biology and innovative bioseparations to develop a single-product, highproductivity butanol fermentation, together with a simple, low energy process for product purification. The last part of theproject will bring these technologies together to develop a continuous process for butanol production with stable operationover long periods. This process will exploit the genetic stability of the new, engineered strains, the simplified butanolseparation and the relief of product inhibition by in situ butanol recovery. The new process will provide significantly greaterproductivity than conventional batch fermentations, thus transforming the economics of butanol production

丙酮-丁醇-乙醇(ABE)发酵使用梭状芽孢杆菌属的厌氧细菌发酵糖或淀粉与溶剂的混合物，典型的A：B：E比例为3：6：1。历史上，这种发酵被用来制造溶剂和化学品，但当石油工业开发出更便宜的方法来制造这些化学品时，这种发酵就不再受欢迎了。随着人们对石油安全和全球变暖的担忧与日俱增，安倍发酵现在正在经历大规模的复兴。今天，丁醇是首选产品，因为它可以用作生物燃料、溶剂和中间体来制造各种化学物质。目前，丁醇发酵效率低下，因为积累的丁醇会毒害细菌，最终导致发酵停止。此外，生产丁醇的梭状芽孢杆菌在遗传上是不稳定的，因此发酵只能运行一小段时间，然后关闭并从新鲜细胞重新启动。回收丁醇也有问题，因为产品流是丁醇、丙酮和乙醇在水中的稀释混合物。蒸馏提供了唯一简单的方法来回收产品，但需要大量的能量。我们将使用合成生物学来产生新的梭状芽孢杆菌菌株，这些菌株可以产生丁醇，而不会形成丙酮和乙醇均一发酵菌株。绿色生物公司的科学家已经使用传统的突变技术开发了同种发酵突变株，并对其DNA进行了测序，以识别突变基因。在这个项目中，我们将选择最重要的突变并在商业生产菌株中重建它们，以开发基因稳定、高产的丁醇生产有机体。新生物将产生更清洁的产品流，允许开发基于液-液提取的新分离工艺。这包括将正在生长的培养物与一种不溶于水的溶剂混合，这种溶剂比水更有效地溶解丁醇。因此，丁醇将转移到溶剂相，通过让两个不相容的液体相(如油和水)沉淀出来，可以很容易地分离出溶剂相。这提供了一种非常巧妙的方法来解决丁醇毒性问题，因为丁醇被从细胞周围的直接环境中移除，因此细胞不会暴露在有毒产品中。这使得丁醇的生产可以继续进行，直到溶剂相饱和，从而细胞可以形成非常高的丁醇浓度。原位溶剂提取依赖于找到一种不溶于水的液体，该液体不仅是丁醇的良好溶剂，而且对细胞也没有毒性。大多数传统溶剂很难从水中提取丁醇，而且它们本身也同样有毒。然而，诺丁汉大学的科学家们发现，一种名为离子液体(ILS)的新型溶剂可以从水中提取丁醇，对活细胞无毒。ILS是由在室温下熔化的盐制成的，因此与传统溶剂不同，它是不挥发的。因此，丁醇可以很容易地回收，只需分离IL相，然后将丁醇煮沸，留下IL供重复使用。总的来说，该项目结合了合成生物学和创新的生物分离技术，开发了一种单一产品、高生产率的丁醇发酵，以及一种简单、低能量的产品纯化工艺。该项目的最后一部分将把这些技术结合在一起，开发一种长期稳定运行的丁醇生产连续工艺。这一过程将利用新的工程菌株的遗传稳定性，简化的丁醇分离，并通过原位丁醇回收解除产物抑制。新工艺将提供比传统间歇发酵显著更高的生产率，从而改变丁醇生产的经济性。