Kolmar, JF; (2017) Microscale tools for rapid evaluation of two-liquid phase bio-oxidations of volatile alkanes. Doctoral thesis , UCL (University College London).

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Abstract

The direct ω-oxyfunctionalisation of aliphatic alkanes in a regio- and chemoselective manner remains difficult to perform by industrial organic chemistry. Monooxygenases such as the AlkBGT enzyme complex from Pseudomonas putida efficiently catalyse these readily available substrates to fatty alcohols, aldehydes and acids under mild conditions. However, numerous challenges remain to achieve industrially competitive space-time-yields for bio-oxidations. The ability to rapidly screen bioconversion reactions for characterisation and optimisation is of major importance in bioprocess development and biocatalyst selection; studies at lab scale are time consuming and labour intensive with low experimental throughput. This study developed and validated a robust high-throughput microwell platform for whole-cell two-liquid phase bio-oxidations of highly volatile alkanes. Using microwell plates machined from polytetrafluoroethylene and a sealing clamp, highly reproducible results were achieved with no significant variability such as edge effects determined. Further, the developed platform was extended by a fed-batch implementation revealing the large impact of feeding conditions on the resting cell bio-oxidation of volatile alkanes. The unpredictable nature and large differences between varying alkane substrates show the importance of being able to test fed-batch conditions early in development. Lastly, six co-solvents were screened to relieve organic phase toxicity and improve control over the product spectrum in the bio-oxidation of hydrocarbons. In combination with computational and statistical tools, it was shown that polar surfactants allow the extraction of the alcohol product, increasing the alcohol yield and reducing phase toxicity. Specifically, the solubility of co-solvents reaction substrate and product was revealed to be the determining factor for product selectivity. Overall, the developed microwell plate greatly improves experimental throughput, accelerating the screening procedures specifically for biocatalytic processes in non-conventional media. Its simplicity, robustness and standardisation ensure high reliability of results. The accelerated data collection on biological as well as process options allows obtaining key process design data early on, de-risking and speeding up the translation of new processes from laboratory to pilot-plant scale.

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