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Studies on Upgradation of Waste Fish Oil to Lipid-Rich Yeast Biomass in Yarrowia lipolytica Batch Cultures.

ABSTRACT: The aim of the study was to evaluate the possibility to utilize a fish waste oil issued from the industrial smoking process in nitrogen-limited Yarrowia lipolytica yeast batch cultures. The waste carbon source was utilized by the yeast and stimulated the single cell oil production via an ex novo pathway. The yeast biomass contained lipids up to 0.227 g/g d.m.. Independently from culture conditions, high contents of very long chain fatty acids were quantified in yeast biomass including docosahexaenoic (DHA), eicosapentaenoic acid (EPA), eicosenic and erucic acids. The pH regulation did not influence the cellular lipids yield (0.234 g/g d.m.). Meanwhile, the intensification of the oxygenation of medium by changing the mixing speed (maximum concentration of lipids produced 4.64 g/dm³) and decreasing the amount of inoculum had a positive effect on the culture parameters in waste fish oil medium. Further work on upgradation of the original waste is advisable, especially because the oil indicated high content of polyphenols and lower susceptibility to oxidation than microbial oil derived from control olive oil medium.

SUBMITTER: Fabiszewska AU

PROVIDER: S-EPMC7922964 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. Results:In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. Conclusions:This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

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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Studies on Upgradation of Waste Fish Oil to Lipid-Rich Yeast Biomass in Yarrowia lipolytica Batch Cultures.

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