Growth Response of Non-Conventional Yeasts on Sugar-Rich Media: Part 2: Citric Acid Production and Circular-Oriented Valorization of Glucose-Enriched Olive Mill Wastewaters Using Novel Yarrowia lipolytica Strains

Abstract

The global market for citric acid (CA) is one of the biggest and fastest expanding markets in the food industry. The CA production employing microbial bioprocessing with efficient GRAS strains and renewable waste streams is in line with the European Union binding targets for resource efficiency, sustainable consumption-production, and low-carbon technologies. In this work, the potential of three novel wild-type Yarrowia lipolytica strains (namely LMBF Y-46, LMBF Y-47 and ACA-YC 5033) regarding the production of CA and other valuable metabolites was tested on glucose-based media, and the most promising amongst the screened strains (viz. the strain ACA-YC 5033) was cultured on glucose-based media, in which part of the fermentation water had been replaced by olive-mill wastewaters (OMWs) in a novel approach of simultaneous OMW valorization and bioremediation. In the first part of this study, the mentioned strains were cultured under nitrogen-limited conditions with commercial (low-cost) glucose employed as a sole carbon source in shake-flask cultures at an initial concentration (S₀) ≈ of 50 g/L. Variable quantities of secreted citric acid (CA) and intra-cellular compounds (viz. polysaccharides and lipids) were produced. All strains did not accumulate significantly high lipid quantities (i.e., maximum lipid in dry cell weight [DCW] values ≈30% w/w were noted) but produced variable CA quantities. The most promising strain, namely ACA-YC 5033, produced CA up to c. 24 g/L, with a yield of CA produced on glucose consumed (Y_CA/S) ≈ 0.45 g/g. This strain in stirred tank bioreactor experiments, at remarkably higher S₀ concentrations (≈110 g/L) and the same initial nitrogen quantity added into the medium, produced notably higher CA quantities, up to 57 g/L (Y_CA/S ≈ 0.52 g/g). The potential of the same strain (ACA-YC 5033) to bioremediate OMWs and to produce value-added compounds, i.e., yeast cells, CA, and intra-cellular metabolites, was also assessed; under nitrogen-limited conditions in which OMWs had partially replaced tap water and significant glucose concentrations had been added (S₀ ≈ 100 g/L, simultaneous molar ratio C/N ≈ 285 g/g, initial phenolic compounds [Phen₀] adjusted to ≈1.0 g/L; these media were similar to the OMWs generated from the traditional press extraction systems) the notable CA quantity of 60.2 g/L with simultaneous Y_CA/S = 0.66 g/g, was obtained in shake flasks, together with satisfactory phenolic compounds removal (up to 19.5% w/w) and waste decolorization (up to 47.0%). Carbon-limited conditions with Phen₀ ≈ 1.0 g/L favored the production of yeast DCW (up to 25.3 g/L), with equally simultaneous interesting phenolic compounds and color removal. The fatty acid profile showed that cellular lipids were highly unsaturated with oleic, linoleic and palmitoleic acids, accounting for more than 80% w/w. This study proposed an interesting approach that could efficiently address the biotreatment of toxic effluents and further convert them into circular-oriented bioproducts.

Keywords: bioactive compounds; citric acid; microbial oil; polysaccharides; sustainable bioprocessing.

Figure 1

Evolution of biomass (X) (●), citric acid (CA) (■) and glucose (S) (○) (a) and citric acid (CA) (■) vs. glucose (b) during batch bioreactor fermentation of Yarrowia lipolytica ACA-YC 5033 on synthetic glucose-based media.

Figure 1

Evolution of biomass (X) (●), citric acid (CA) (■) and glucose (S) (○) (a) and citric acid (CA) (■) vs. glucose (b) during batch bioreactor fermentation of Yarrowia lipolytica ACA-YC 5033 on synthetic glucose-based media.

Figure 2

Evolution of biomass (X) (●), citric acid (CA) (■) and glucose (S) (○) (a), citric acid (CA) (■) vs. glucose (b) and decolorization (♦) and phenol removal (□) (c) during batch flask fermentation of Y. lipolytica ACA-YC 5033 on media composed of OMWs and glucose, under nitrogen-limited conditions. Initial phenolic compounds into the OMW-based media ≈ 1.0 g/L.

Figure 2

Evolution of biomass (X) (●), citric acid (CA) (■) and glucose (S) (○) (a), citric acid (CA) (■) vs. glucose (b) and decolorization (♦) and phenol removal (□) (c) during batch flask fermentation of Y. lipolytica ACA-YC 5033 on media composed of OMWs and glucose, under nitrogen-limited conditions. Initial phenolic compounds into the OMW-based media ≈ 1.0 g/L.

Figure 3

Evolution of biomass (X) (●) and glucose (S) (○) (a), biomass (X) (●) vs. glucose (b) and decolorization (♦) and phenol removal (□) (c) during batch flask fermentation of Y. lipolytica ACA-YC 5033 on media composed of OMWs and glucose, under carbon-limited conditions. Initial phenolic compounds into the OMW-based media ≈ 1.0 g/L.

Figure 3

Evolution of biomass (X) (●) and glucose (S) (○) (a), biomass (X) (●) vs. glucose (b) and decolorization (♦) and phenol removal (□) (c) during batch flask fermentation of Y. lipolytica ACA-YC 5033 on media composed of OMWs and glucose, under carbon-limited conditions. Initial phenolic compounds into the OMW-based media ≈ 1.0 g/L.

Figure 4

Evolution of biomass (X) (●), citric acid (CA) (■), glucose (S) (○) and ammonium ions (NH4+) (◊) at the relatively early growth steps (up to 250 h) (a), biomass (X) (●), citric acid (CA) (■) and glucose (S) (○) for the whole fermentation period (b) and citric acid (CA) (■) vs. glucose (c) during batch flask fermentation of Y. lipolytica ACA-YC 5033 on media composed of OMWs and glucose, under nitrogen-limited conditions. Initial phenolic compounds into the OMW-based media ≈ 1.0 g/L, S₀ ≈ 100 g/L.

Figure 4

Evolution of biomass (X) (●), citric acid (CA) (■), glucose (S) (○) and ammonium ions (NH4+) (◊) at the relatively early growth steps (up to 250 h) (a), biomass (X) (●), citric acid (CA) (■) and glucose (S) (○) for the whole fermentation period (b) and citric acid (CA) (■) vs. glucose (c) during batch flask fermentation of Y. lipolytica ACA-YC 5033 on media composed of OMWs and glucose, under nitrogen-limited conditions. Initial phenolic compounds into the OMW-based media ≈ 1.0 g/L, S₀ ≈ 100 g/L.