Document Type

Theses, Ph.D

Rights

This item is available under a Creative Commons License for non-commercial use only

Disciplines

1.6 BIOLOGICAL SCIENCES

Publication Details

A thesissubmitted to Technological University Dublin in fulfilment of requirement for the degree of DOCTOR OF PHILOSOPHY

Abstract

Lipase-basedcatalysis in organic solvents offerseveraladvantages and,therefore, havebeen widely researched and applied. Nature provides a vast microbial enzyme pool thatcan be easily bio-discovered through the targeted exploration of existing extremophiles.Among the large number of bacterial lipases, those from species like Geotrichum, Bacillus, Pseudomonas, BurkholderiandStreptomyceshave shownadequate stability forbiosynthetic application. In this study, severalbacterial strains isolated from soils in the Dublin and Laois regionsofIrelandwere screened forpresence ofsolvent stable lipases.Fivebacterialstrains producing most solvent stable lipase(s) were identified asPseudomonas sp. BIM B-86, Sphingomonas sp., Listeria monocytogenes, Pseudomonas reinekeiand Pseudomonas brennerivia 16S rRNA sequencing.Amonst thesefive strains, new lipolytic enzymes from only two strains of Pseudomonassp. i.e. P.reinekeiand P. brenneriwere explored in the present work.Production of lipase(s)from P.reinekei(termed H1)and P. brenneri(termed H3) was enhanced by changing the nitrogen sourcein culture medium to 1%(w/v) L-Lysine and 1% (w/v) peptone respectively. Characterisation of these purified lipases revealed their stability towardsbroad pH range (5.0-9.0) and activation in the presence of organic solvents withlog P≥ 2.0.T50(half-life) of lipase from P. brenneri(H3) andP. reinekei(H1)was found to be 189mins at 60°Cand 89mins at45°Crespectively.Thesepurified lipase were usedin industrially relevant applications, specifically biodieselsynthesis.This study also explored development of an alternative and sustainable lipidssourcefor biodiesel production. Microalgae were selected as cheap, alternate source as theycan grow inwide range of environmental conditionsand can utilise nutrients from wastewaterfor their growth. In this study, two microalgae strains i.e. Chlorella emersoniiand Pseudokirchneriella subcapitatawere examined as potential lipids source for biodiesel production.Both strains were grown in simulated wastewater andunder nitrogen deficient conditionsgeneratedneutral lipid content of 0.61±0.017mg/mg and 0.31±0.006mg/mg biomass respectively. In-situtransesterification of neutral lipids generated from these two microalgae strainsusinglipases from H1 (P. reinekei), H3 (P. brenneri) and porcine pancreas (as a standard) generated FAMEs (Fatty Acid Methyl Esters) with a high proportion of unsaturated fatty acids esters.Blending these FAMEs 3with other FAME sources can producebiodiesel comparable to existing biofuels. Thisstudy is a good illustration for generation/productionof energy rich molecules from waste byproducts exploitingenzymes obtained from extremophilesand wastewater grown microalgae.

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