Characterisation and development of bacterial biosurfactants for biotechnological applications.

项目具体目标：1。首先选择一个子集的细菌菌株产生强大的表面活性剂（即生物乳化剂或生物表面活性剂）.确定菌株在预优化的液体培养基中生长期间SA的生长动力学和生产。为了回收和部分纯化小规模量的SA，并通过例如，化学分析、NMR等。测定SA的功能特性，如其pH和温度耐受性，与不同油基质的相互作用，临界胶束浓度等.为了优化菌株的SA生产，首先使用基于烧瓶的培养物，然后进行小规模发酵罐研究。方法学：为了筛选能产生具有乳化特性的SA（即生物乳化剂）的菌株，我们将测试糖基质（例如葡萄糖、蔗糖、麦芽提取物），因为根据我们的经验，这些物质已被证明是从海洋细菌菌株中产生强大生物乳化剂的合适原料。将使用标准乳化试验（其涉及将用过的培养基相对于油基质乳化）来鉴定产生形成水包油（O/W）和/或油包水（W/O）乳液的生物乳化剂的菌株。高O/W和W/O乳化活性（相对于未接种对照至少50%）将用作选择菌株进行进一步评价的标准。对于产生具有表面活性剂特性（即生物表面活性剂）的SA的菌株，将使用油基质（例如菜籽油），因为它们被认为有利于产生具有表面活性剂而非乳化特性的SA。在不同油基质上培养菌株期间，将监测位于下部水相和上部非水（油）相之间边界处的界面弯月面曲率的任何变化。将选择完全拉直界面弯月面的菌株--这是生物表面活性剂产生的积极迹象--用于在生产培养基中使用表面和界面测量进行进一步评估。选择用于产生具有推定的强大表面活性特性的SA的菌株将在产生这些活性的相同条件下再生长，这次是为了确定SA是细胞结合的还是细胞外释放到培养基中。这是重要的，因为SA分子在其生产过程中（例如在发酵过程中）在培养基中的物理位置（细胞结合或细胞外释放）可能对涉及其回收的过程产生重大影响，进而影响其在经济上是否可行。为了确定这一点，将细胞从培养基中取出，并在检测前彻底清洗细胞是否具有乳化和/或表面活性剂活性。将在无细胞用过的培养基上进行相同的检测。只有发现产生具有高乳化和/或表面活性剂品质的胞外释放SA的菌株将被选择用于进一步的研究。SA的性质将在各种条件下确定（即对温度、pH、盐度等的耐受性）。并且还将确定它们能够润湿和/或降低它们的表面张力的油基质。用于选择候选SA进行进一步开发的标准将基于其赋予高稳定和乳化活性和/或表面张力降低的能力。该博士项目旨在发现由新型海洋细菌菌株产生的新型SA，并提供有关菌株产生的SA特性的新知识，包括其潜在的生物技术用途。

Specific objectives of the project:1. To first select a subset of bacterial strains that produce powerful surface-active agents (i.e. bioemulsifiers or biosurfactants).2. Determine the growth kinetics and production of the SAs during growth of the strains in pre-optimised liquid medium.3. To recover and partially purify small-scale quantities of the SAs and determine their composition by, for e.g., chemical assays, NMR etc.4. To determine the functional properties of the SAs, such as their pH and temperature tolerances, interaction with different oil substrates, critical micelle concentration etc.5. To optimise the production of the SAs by the strains using initially flask-based cultures followed by small-scale fermenter studies.6. To explore protocols and fine-tune the experimental conditions of the SAs for subsequent testing to evaluate their potential for use in commercial formulations and/or biotechnological applications.Methodology:To screen for strains that produce SAs with emulsifying properties (i.e. bio-emulsifiers), sugar substrates (e.g. glucose, sucrose, malt extract) will be tested since these have been shown, from our experience, to be suitable feedstocks for yielding powerful bio-emulsifiers from marine bacterial strains. A standard emulsification assay, which involves emulsifying the spent culture medium against an oil substrate, will be used to identify strains that produce bio-emulsifiers that form oil-in-water (O/W) and/or water-in-oil (W/O) emulsions. High O/W and W/O emulsification activities, of at least 50% relative to uninoculated controls, will be used as the criterion for the selection of strains for further evaluation.For strains that produce SAs with surfactant properties (i.e. bio-surfactants), oil substrates (e.g. rapeseed oil) will be used because they are recognized to favour the production of SAs with surfactant, rather than emulsifying, properties. During incubation of the strains on different oil substrates, the interfacial meniscus, which sits at the boundary between the lower aqueous and upper non-aqueous (oil) phases, will be monitored for any change to its curvature. Strains that completely straighten the interfacial meniscus - a positive indication of bio-surfactant production - will be selected for further evaluation using surface and interfacial measurements in the production medium.Strains selected for producing SAs with putative powerful surface-active properties will be regrown under the same conditions that yielded these activities, this time to determine if the SAs are cell-bound or released extracellularly into the culture medium. This is important because the physical location (cell-bound or extracellularly released) of the SA molecules in the culture medium during their production, such as in a fermentation process, can have a significant impact to the processes involved in their recovery and, in turn, whether it is economically feasible. To determine this, the cells will be removed from the culture medium and thoroughly washed prior to testing if they possess emulsifying and/or surfactant activities. The same will be tested on the cell-free spent medium. Only strains that are found to produce extracellulary-released SAs exhibiting high emulsifying and/or surfactant qualities will be selected for further study.Properties of the SAs will be determined for various conditions (i.e. tolerances to temperature, pH, salinity, etc.) and the oil substrates they are able to emulsify and/or reduce their surface tension will also be determined. Criteria that will be used for selecting candidate SAs for further development will be based on their ability to confer high stabilization and emulsification activities and/or surface tension reduction. This PhD project aims to discover new types of SAs produced by novel marine bacterial strains, and provide new knowledge on the properties of the SAs the strains produce, including their potential biotechnological use.