Use of Plant Cell Cultures in Biocatalyst Development for Bio-desulfurization

Abstract

An impurity such as sulfur reduces the quality of fossil fuels and contributes significantly to air pollution, which has serious harmful effects on the environment. Therefore, this research project focuses on the development of biocatalysts based on plant cell cultures for the desulfurization of crude oil. Microbial biodesulfurization has been investigated for a few decades now. Yet there is no process based on biodesulfurization. There are many reasons for this, the main ones involve the difficulty of separating cells from the crude oil and hence the problem of consumption of the hydrocarbons by the cells leading to the loss of value of the crude oil, and high temperatures of the crude oil not being suitable for microbial cultures. The biogenesis of sulfur compounds in fossil fuels is caused by the death and decomposition under high temperature and pressure in the earth’s crust of the large populations of plants and animals over geological time. Plants produce complex sulfur compounds and therefore must have enzymes that can degrade sulfur compounds. Use of plant cell cultures for biodesulfurization was the novelty of this project. One of the pathways of bacterial biodesulfurization was elucidated as the 4S pathway. Using the identified enzymes in the 4S pathway, equivalent enzymes in various plant species were searched using databases such as KEGG, and molecular docking software SwissDock. Specially for Nicotiana tabacum (tobacco) as the chosen plant for this research, feruoyl coenzyme A 3-O-methyltransferase (1sus) was 7.8 times more efficient than 2'-hydroxybiphenyl-2-sulfinate desulfinase (DszB) enzyme used in the last step in 4S pathway in a biodesulfurizing micoorganism like Rhodococcus erythropolis. Three plant species, Arabidopsis thaliana, Nicotiana tabacum (tobacco), Armoracia rusticana (horseradish) were cultured. Nicotiana tabacum was the chosen plant for the biodesulfurization experiments due to the good growth characteristics and minimal contamination problems. Dibenzothiophene (DBT) was the model sulfur compound used for testing the biodesulfurisation ability of the cultures. The control experiments and biodesulfurization experiments were performed in aqueous medium in shake flasks using Murashige-Skoog (MS) and sulfur-free Murashige-Skoog (SFM) media supplemented either 100 or 200ppm DBT. The MS with 100ppm DBT experiment had the highest maximum biomass concentration at 24.46 g/l by day 15, 95% of DBT was degraded by day 15 and the average concentration of 2-Hydroxyblphen (2-HBP) produced was 66.3ppm. In advance of adding crude oil to the biodesulfurization experiments some tests were done and confirmed that cells could stay alive after contacting with crude oil, this was achieved by looking the cells treated with fluorescein diacetate staining (FDA) under UV microscope. The cells were allowed to have contact with crude oil for 24 h then washed and grown in MS normal medium for around 2 weeks. For the biodesulfurization experiments involving crude oil, the MS with 100ppm DBT and SFM with DBT at 100, 200, 300 and 400ppm were performed in airtight vessels for safety reasons. The 100ppm DBT concentration in SFM had the highest maximum biomass concentration at 26.7 g/l in day 21 and 97.7% of DBT degradation, and fluctuated concentrations of 2-HBP with an average of 46.31ppm. A kinetic model was used for some of the batch experiments; although preliminary attempt prediction was promising, nevertheless the kinetic model needs further refinement with further experiments in order to obtain the model parameters more accurately.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ferda Mavituna (Supervisor) & Robin Curtis (Supervisor)