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Optimizing the catalytic activities of methanol and thermotolerant Kocuria flava lipases for biodiesel production from cooking oil wastes.

ABSTRACT: In this study, two highly thermotolerant and methanol-tolerant lipase-producing bacteria were isolated from cooking oil and they exhibited a high number of catalytic lipase activities recording 18.65 ± 0.68 U/mL and 13.14 ± 0.03 U/mL, respectively. Bacterial isolates were identified according to phenotypic and genotypic 16S rRNA characterization as Kocuria flava ASU5 (MT919305) and Bacillus circulans ASU11 (MT919306). Lipases produced from Kocuria flava ASU5 showed the highest methanol tolerance, recording 98.4% relative activity as well as exhibited high thermostability and alkaline stability. Under the optimum conditions obtained from 3D plots of response surface methodology design, the Kocuria flava ASU5 biocatalyst exhibited an 83.08% yield of biodiesel at optimized reaction variables of, 60 ^○C, pH value 8 and 1:2 oil/alcohol molar ratios in the reaction mixture. As well as, the obtained results showed the interactions of temperature/methanol were significant effects, whereas this was not noted in the case of temperature/pH and pH/methanol interactions. The obtained amount of biodiesel from cooking oil was 83.08%, which was analyzed by a GC/Ms profile. The produced biodiesel was confirmed by Fourier-transform infrared spectroscopy (FTIR) approaches showing an absorption band at 1743 cm^-1, which is recognized for its absorption in the carbonyl group (C=O) which is characteristic of ester absorption. The energy content generated from biodiesel synthesized was estimated as 12,628.5 kJ/mol. Consequently, Kocuria flava MT919305 may provide promising thermostable, methanol-tolerant lipases, which may improve the economic feasibility and biotechnology of enzyme biocatalysis in the synthesis of value-added green chemicals.

SUBMITTER: Najjar A

PROVIDER: S-EPMC8249636 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Oleaginous yeasts are fast emerging as a possible feedstock for biodiesel production. Yarrowia lipolytica, a model oleaginous yeast is known to utilize a variety of hydrophobic substrates for lipid accumulation including waste cooking oil (WCO). Approaches to increase lipid content in this yeast include metabolic engineering which requires manipulation of multiple genes in the lipid biosynthesis pathway. A classical and cost-effective approach, namely, random chemical mutagenesis on the yeast can lead to increased production of biodiesel as is explored here.In this study, chemical mutagenesis using the alkylating agent, N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) as well as an additional treatment with cerulenin, a fatty acid synthase inhibitor generated 800 mutants of Y. lipolytica NCIM 3589 (761 MNNG treated and 39 MNNG + cerulenin treated). A three-stage screening using Sudan Black B plate technique, Nile red fluorimetry and total lipid extraction using solvent was performed, which enabled selection of ten high lipid yielding mutants. Time course studies of all the ten mutants were further undertaken in terms of biomass, lipid yield and lipid content to select three stable mutants (YlB6, YlC7 and YlE1) capable of growing and accumulating lipid on WCO, with lipid contents of 55, 60 and 67% as compared to 45% for the wild type. The mutants demonstrated increased volumetric lipid productivities (0.062, 0.044 and 0.041 g L-1 h-1) as compared to the wild type (0.033 g L-1 h-1). The fatty acid profile of the three mutants consisted of a high content of C16 and C18 saturated and monounsaturated fatty acids and was found to be suitable for biodiesel production. The fuel properties, namely, density, kinematic viscosity, total acid number, iodine value of the three mutants were evaluated and found to lie within the limits specified by internationally accepted standards. Additionally, it was noted that the mutants demonstrated better cetane numbers and higher heating values than the wild type strain.The chemical mutagenesis strategy adopted in this study resulted in the successful isolation of three stable high SCO yielding mutants. The mutants, namely, YlB6, YlC7 and YlE1 exhibited a 1.22, 1.33 and 1.49-fold increase in lipid contents when grown on 100 g L-1 waste cooking oil than the parental yeast strain. The fatty acid methyl ester (FAME) profiles of all the three mutants was determined to be suitable for biodiesel suggesting their potential applicability while simultaneously addressing the management of waste cooking oil.

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Optimizing the catalytic activities of methanol and thermotolerant Kocuria flava lipases for biodiesel production from cooking oil wastes.

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