Production of 2,5-Furandicarboxylic Acid from 5-Formyl-2-furancarboxylic Acid using resting cells of Gluconobacter oxydans and finding the responsible enzyme(s).

Abstract

2,5-Furandicarboxylic acid (FDCA) is a promising bio-based chemical that may serve as a green alternative to polyethylene terephthalate as a raw material in manufacturing plastics. A novel method to produce FDCA from the oxidation of 2-formyl-2-furancarboxylic acid (FFCA) using resting cells of Gluconobacter oxydans was tested in this project. The different subspecies of G. oxydans including DSM 2343, 50049, and 2003 were tested to check whether they can carry out the oxidation of FFCA to FDCA. Strains 50049 and 2003 were able to convert 5 g/L of FFCA to FDCA within 24 h and were therefore compatible for the process but 2003 had longer lag phase which resulted in growth related issues. Various cell parameters like effect of cultivation time on cell activity were tested by collecting cells at various growth phases, where cells collected at 16 h. gave complete conversion of FFCA with a yield of 96 %. Key factor parameter is pH that was tested using 0.1M acetate buffer at pH 5.0 and 0.1M sodium phosphate buffer at pH 6.4, 7.0 and 8.0. The optimum pH was around pH 5. Optimal FFCA concentration for the reaction was tested by using different concentrations of FFCA (5, 10, 15, 20 g/L) where up to 10 g/L gave optimum results. FFCA concentration above 10g/L had inhibitory effects on the oxidation. Various Cell concentrations (4, 8, 16, 32 mL) were used in the reaction to check if the increase in cell density helps with improving the inhibition found with an increase in FFCA concentration.
Once the optimal conditions were achieved, these were used to scale up the process to produce FDCA in a 3 L bioreactor. G. oxydans cells with a wet weight of 21.7g were used in the fed-batch biotransformation of FFCA to FDCA. Three feeds of 5 g/L FFCA were successfully converted to FDCA but as the no. of feeds increased there was a decrease in the activity of the cells which led to a 55% decrease in productivity which was later found to be due to product accumulation in the bioreactor. 80% of the FDCA was recovered using various downstream processing steps.
Putative oxidoreductases from G. oxydans were expressed to understand their role in the oxidation of FFCA to FDCA. Four genes were selected and introduced into a suitable plasmid which was then transformed into competent cells for protein expression. Further research into optimizing the bottleneck steps like ligation and protein expression must be done in order characterize the enzymes.

Popular Abstract

Plastics are a vital part of our life and is used in almost every sector in the world. 500 million tons of plastic is produced annually. Major problems with plastics are that they are produced from fossil fuels, they are also non-biodegradable, and end up in ocean or landfills. Plastic bottles and containers are made from a variety of polymers like polyethylene terephthalate (PET), Polyvinyl chloride (PVC), polypropylene etc.
Alternative for plastics is being explored for the past decade. Polyethylene furanoate (PEF) is a biobased polymer. PEF is better than PET because it is recyclable and in turn reduce waste, it has better mechanical and thermal properties. PEF is a large molecule consisting of building blocks called FDCA and ethylene glycol.
The earlier methods used to produce Furandicarboxylic Acid (FDCA) have very harsh conditions that is why there is an increase in the need to find a greener pathway. In this study, the bacterium G. oxydans will help to convert Formylfurancarboxylic Acid (FFCA) to the building block FDCA. Experiments were performed to find the optimum conditions for the reaction and the growth of bacteria. These parameters were pH 5.0, 0.1M Acetate buffer, substrate concentration up to 10g/L and 16h cultivation time for the bacteria.
These conditions were implemented for scaling up the reaction in a 3L bioreactor using fed batch mode. In this experiment, the bacteria were supplied with FFCA and once all the FFCA is converted to FDCA, more FFCA was supplied thus accumulating the product FDCA in the reactor. Here 15g of FFCA was converted to FDCA in three feeds of 5g FFCA each. We get 100% yield and selectivity from this reaction. After the reaction took place for a certain amount of time it was noticed that the addition of more FFCA was inhibiting the cell activity to produce FDCA.
We also wanted to find what was causing the conversion in the bacteria. These proteins in the bacteria are called enzymes and there are thousands of enzymes in the cells, and we wanted to find out the enzymes responsible for the oxidation of FFCA to FDCA. This will help us understand how to improve the reaction conditions for the bacterium to carry out the oxidation. Several unknown enzymes were chosen and tested to find out their function.

• author: Sivasubramanian, Anirudh
• supervisor: Mahmoud Sayed; Mohamed Ismail
• organization: Biotechnology (MSc); Biotechnology (M.Sc.Eng.)
• alternative title: Microbial Oxidation of FFCA to FDCA and to find the responsible enzyme(s).
• course: KBTM01 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: Furan dicarboxylic acid, FDCA, 2-Formyl 2-Furan carboxylic acid, FFCA, G. oxydans, resting cell Biocatalysis, Fed-batch, recyclable polymer, PEF
• language: English
• id: 9099904
• date added to LUP: 2022-09-13 14:36:08
• date last changed: 2022-09-13 14:36:08