Characterisation and development of bacterial biosurfactants for biotechnological applications.

该项目的具体目标：1.首先选择产生强大表面活性物质(即生物乳化剂或生物表面活性剂)的细菌菌株的子集。测定菌株在预优化液体培养基中的生长动力学和生长过程中的产酸能力。回收和部分提纯小量的SA，并通过例如化学分析、核磁共振等方法确定其组成。确定SA的功能性质，如其对pH和温度的耐受性、与不同油底物的相互作用、临界胶束浓度等。利用最初的基于摇瓶的培养和随后的小规模发酵罐研究来优化菌株生产SA。方法：为了筛选产生具有乳化性能的SA的菌株(即生物乳化剂)，将测试糖底物(例如葡萄糖、蔗糖、麦芽提取物)，因为根据我们的经验，这些底物被证明是从海洋细菌菌株中生产出强大的生物乳化剂的合适原料。标准的乳化试验包括将废培养基对着油基质进行乳化，将用于鉴定产生生物乳化剂的菌株，这些乳化剂形成水包油(O/W)和/或油包水(W/O)乳状液。高O/W和W/O乳化活性(与未接种对照相比至少50%)将被用作选择菌株进行进一步评估的标准。对于产生具有表面活性的SA的菌株(即生物表面活性剂)，将使用油基(例如菜籽油)，因为它们被认为有利于生产具有表面活性的SA，而不是乳化特性。在不同石油基质上培养菌株的过程中，将监测位于下水相和上非水(油)相之间的界面半月面的曲率是否有任何变化。将选择使界面半月板完全拉直的菌株--这是生产生物表面活性剂的积极迹象--将通过在生产介质中进行表面和界面测量来进行进一步的评估。被选为生产具有强大表面活性的SA的菌株将在产生这些活性的相同条件下重新生长，这一次是为了确定SA是结合细胞还是释放到培养介质中。这一点很重要，因为在生产过程中，例如在发酵过程中，SA分子在培养基中的物理位置(细胞结合或细胞外释放)可能对其回收过程产生重大影响，进而影响其在经济上是否可行。为了确定这一点，细胞将从培养基中取出，并在测试它们是否具有乳化和/或表面活性之前进行彻底清洗。同样的测试将在无细胞废培养基上进行。只有被发现能产生细胞外释放的具有高乳化和/或表面活性的SA的菌株才会被选作进一步研究。SA的性质将在不同条件下(即对温度、pH、盐度等的耐受性)进行测定。还将确定它们能够乳化和/或降低其表面张力的油基质。用于选择候选SA进行进一步开发的标准将基于它们提供高稳定和乳化活性和/或降低表面张力的能力。该博士项目旨在发现由新的海洋细菌菌株产生的新型SA，并提供关于这些菌株产生的SA的特性的新知识，包括它们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.