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 the strain ACA-YC 5033) was cultured on glucose-based media, in which part of the fermentation water had been replaced by olive-mill wastewaters (OMWs) in a novel approach of simultaneous OMW valorization and bioremediation.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 (YCA/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 (YCA/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 YCA/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.
ABSTRACT
Sugar-rich waste streams, generated in very high quantities worldwide, constitute an important source of environmental pollution. Their eco-friendly conversions into a plethora of added-value compounds through the use of microbial fermentations is currently a very "hot" scientific topic. The aim of this study, was to assess the potential of single cell oil (SCO), microbial mass and citric acid (CA) production by non-conventional yeast strains growing on expired ("waste") glucose. Six yeast strains (viz. Rhodosporidium toruloides DSM 4444, Rhodotorula glutinis NRRL YB-252, R. toruloides NRRL Y-27012, Yarrowia lipolytica LFMB Y-20, Y. lipolytica ACA-DC 50109 and Lipomyces starkeyi DSM 70296) were initially grown in shake flasks with expired glucose used as substrate under nitrogen limitation, in order to "boost" the cellular metabolism towards the synthesis of SCO and CA, and their growth response was quantitatively evaluated. Initial glucose concentration (Glc0) was adjusted at c. 50 g/L. Besides Y. lipolytica, all other yeast strains produced noticeable SCO quantities [lipid in dry cell weight (DCW) ranging from 25.3% w/w to 55.1% w/w]. Lipids of all yeasts contained significant quantities of oleic acid, being perfect candidates for the synthesis of 2nd generation biodiesel. The highest DCW production (=13.6 g/L) was obtained by L. starkeyi DSM 70296, while both Y. lipolytica strains did not accumulate noticeable lipid quantities, but produced non-negligible CA amounts. The most promising CA-producing strain, namely Y. lipolytica ACA-DC 50109 was further studied in stirred-tank bioreactor systems, while the very promising DCW- and SCO-producing L. starkeyi DSM 70296 was further studied in shake flasks. Both strains were grown on media presenting higher Glc0 concentrations and the same initial nitrogen quantity as previously. Indeed, L. starkeyi grown at Glc0 = 85 g/L, produced DCWmax = 34.0 g/L, that contained lipid =34.1% w/w (thus SCO was =11.6 g/L). The strain ACA-DC 50109 in stirred tank bioreactor with Glc0 ≈ 105 g/L produced CA up to 46 g/L (yield of CA produced on glucose consumed; YCA/Glc ≈ 0.45 g/g). Finally, in fed-batch bioreactor experiment, the significant CA quantity of 82.0 g/L (YCA/Glc = 0.50 g/g) was recorded. Concluding, "waste" glucose proved to be a suitable substrate for a number of non-conventional yeast strains. Y. lipolytica ACA-DC 50109 produced significant quantities of CA while L. starkeyi DSM 70296 was a very interesting DCW- and SCO-producing candidate. These strains can be used as potential cell factories amenable to convert glucose-based residues into the mentioned metabolic compounds, that present high importance for food, chemical and biofuel facilities.
ABSTRACT
In the present report and for the first time in the international literature, the impact of the addition of NaCl upon growth and lipid production on the oleaginous yeast Rhodosporidium toruloides was studied. Moreover, equally for first time, lipid production by R. toruloides was performed under nonaseptic conditions. Therefore, the potentiality of R. toruloides DSM 4444 to produce lipid in media containing several initial concentrations of NaCl with glucose employed as carbon source was studied. Preliminary batch-flask trials with increasing amounts of NaCl revealed the tolerance of the strain against NaCl content up to 6.0% w/v. However, 4.0% w/v of NaCl stimulated lipid accumulation for this strain, by enhancing lipid production up to 71.3% w/w per dry cell weight. The same amount of NaCl was employed in pasteurized batch-flask cultures in order to investigate the role of the salt as bacterial inhibiting agent. The combination of NaCl and high glucose concentrations was found to satisfactorily suppress bacterial contamination of R. toruloides cultures under these conditions. Batch-bioreactor trials of the yeast in the same media with high glucose content (up to 150 g/L) resulted in satisfactory substrate assimilation, with almost linear kinetic profile for lipid production, regardless of the initial glucose concentration imposed. Finally, fed-batch bioreactor cultures led to the production of 37.2 g/L of biomass, accompanied by 64.5% w/w of lipid yield. Lipid yield per unit of glucose consumed received the very satisfactory value of 0.21 g/g, a value among the highest ones in the literature. The yeast lipid produced contained mainly oleic acid and to lesser extent palmitic and stearic acids, thus constituting a perfect starting material for "second generation" biodiesel.
