Oxidation of 1,6-hexanediol to adipic acid using resting cells of Gluconobacter oxydans and screening of the responsible enzyme(s)

Abstract

Adipic acid (AA) has been a valuable industry chemical over the last century, whose production process carbon footprint resides far away from today’s environment-friendly ideals. Therefore, a biological approach for adipic acid production under mild conditions has been studied in this thesis. Wild-type strains of Gluconobacter oxydans have been cultivated to obtain resting cells that were used for the oxidation of 1,6-hexanediol (1,6-HD) to adipic acid (AA). In a previous study, in order to overcome the limitations on AA production caused by cultivation conditions, several parameters were optimised including; carbon source, different G. oxydans species (DSM 50049, DSM 2003 and DSM 2343), and the effect of the incubation time, all performed on a laboratory bench scale. In this thesis, the optimal conditions are used to scale up the process and test the effect of aeration, not only on cell activation but also the production of AA, results show that it is a limiting factor in both cases.
22 L of active cells were produced in a 30 L bioreactor under the optimum conditions. These cells were then used for the biotransformation experiment, which consisted of four feeds of 5 g/L of 1,6-HD each additional to the starting 10 g/L. After 76 hours of reaction, 100% yield was achieved. Although a high concentration of product is reached, overall productivity decreases with each feed, starting with 1.31 g AA/L*h in the first 10 hours and ending with 0.30 g AA/L*h in the last 28 hours. The AA purification process followed is simple and cost-effective, avoiding the use of organic solvents and reaching an overall recovery yield of 74%.
In addition, 9 unknown genes of dehydrogenases from G. oxydans DSM50049 were amplified, and cloned in a pET30a vector, and four of them were expressed in E. coli Bl21 to identify the enzyme(s) responsible for AA production.
All in all, the process has been proven to be successful in the 30 L scale up for cell activation and in the fed-batch biotransformation. On the other hand, the enzyme activity was tested against 10 g/L of 1,6-HD, however, further experiments are needed in order to confirm or deny the role of these enzymes in the oxidation of 1,6-HD.

Popular Abstract

What do clothes, carpets, tire cords, conveyor belts, and brushes have in common? That they are usually made of nylon. Nylon is known to be a durable fibre used in textiles and plastics, normally made of petroleum.
If we take a closer look at the nylon’s composition, we can see that it is generally made of two components, one of them is adipic acid (AA). Every year, millions of kilograms of adipic acid are produced mainly for the production of nylon.
With the growing interest in getting rid of petroleum-based products, greener ways of obtaining these components have been invented. In this study, the route performed consisted in culturing bacteria that can produce adipic acid. The bacteria used, called Gluconobacter oxydans can be found in fruit, cider, beer, wine… and it has the ability to produce adipic acid if supplied with a certain substrate, 1,6-hexanediol (1,6-HD), which can potentially be made from sugar-based building blocks.
In order to perform the experiments, the optimal conditions to culture the bacteria are screened on a small scale (approximately 50 mL, less than a glass of water). When these conditions are settled, the process is scaled up to 22 litres, obtaining a huge number of bacteria.
To use the bacterial cells for the production of AA, the 22 L was split into batches of 4L. This 4 L is centrifuged and the cells in it are resuspended in 1 L of 1,6-HD, then the reaction starts. The 1,6-HD is converted by the cells to AA but not immediately, the cells need approximately 24 hours to convert all the substrate into product. During this time, several samples are taken to monitor the conversion. One experiment consisted in a fed-batch, which is a technique where substrate is supplied to the cells several times. The product is accumulated and the final amount is much more than if the substrate is given to the cells once. But we found out that after supplying 5 times substrate to the same cells, the cells get “tired”, and every time take a longer time to produce AA. Then AA is purified through a simple process that avoids the use of organic solvents.
For a better understanding of the reaction, we wanted to know why the cells lose activity. Those responsible for carrying out the reaction are the enzymes. There are a lot of enzymes in a cell, and we wanted to figure out which ones were working for this reaction. For this reason, a number of unknown genes encoding for enzymes from G. oxydans were selected and expressed. It is not yet clear what enzymes they are, but work is underway to find it out.

• author: Amorrortu Gallo, Jorge and Fernandez Ros, Nídia
• supervisor: Mahmoud Sayed; Mohamed Ismail
• organization: Biotechnology (MSc); Biotechnology (M.Sc.Eng.)
• course: KBTM01 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: Gluconobacter oxydans, 1 6-hexanediol, adipic acid, microbial oxidation, reaction optimization, scale-up, fed-batch, enzyme, biotechnology
• language: English
• id: 9099937
• date added to LUP: 2022-09-14 12:49:02
• date last changed: 2022-09-14 12:49:02