Effect of Sludge Concentration and Crude Glycerol Matrix as a Substrate on the Production of Single-Cell Oil by Oleaginous Yeast Yarrowia lipolytica SKY7

BackgroundMicrobial lipids can represent a valuable alternative feedstock for biodiesel production in the context of a viable bio-based economy. This production can be driven by cultivating some oleaginous microorganisms on crude-glycerol, a 10 % (w/w) by-product produced during the transesterification process from oils into biodiesel. Despite attractive, the perspective is still economically unsustainable, mainly because impurities in crude glycerol can negatively affect microbial performances. In this view, the selection of the best cell factory, together with the development of a robust and effective production process are primary requirements.ResultsThe present work compared crude versus pure glycerol as carbon sources for lipid production by three different oleaginous yeasts: Rhodosporidium toruloides (DSM 4444), Lipomyces starkeyi (DSM 70295) and Cryptococcus curvatus (DSM 70022). An efficient yet simple feeding strategy for avoiding the lag phase caused by growth on crude glycerol was developed, leading to high biomass and lipid production for all the tested yeasts. Flow-cytometry and fourier transform infrared (FTIR) microspectroscopy, supported by principal component analysis (PCA), were used as non-invasive and quick techniques to monitor, compare and analyze the lipid production over time. Gas chromatography (GC) analysis completed the quali-quantitative description. Under these operative conditions, the highest lipid content (up to 60.9 % wt/wt) was measured in R. toruloides, while L. starkeyi showed the fastest glycerol consumption rate (1.05 g L−1 h−1). Being productivity the most industrially relevant feature to be pursued, under the presented optimized conditions R. toruloides showed the best lipid productivity (0.13 and 0.15 g L−1 h−1 on pure and crude glycerol, respectively).ConclusionsHere we demonstrated that the development of an efficient feeding strategy is sufficient in preventing the inhibitory effect of crude glycerol, and robust enough to ensure high lipid accumulation by three different oleaginous yeasts. Single cell and in situ analyses allowed depicting and comparing the transition between growth and lipid accumulation occurring differently for the three different yeasts. These data provide novel information that can be exploited for screening the best cell factory, moving towards a sustainable microbial biodiesel production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0467-x) contains supplementary material, which is available to authorized users.

To screen yeasts in relation to the potential to produce single cell oil (SCO) from biodiesel-derived glycerol and to enhance SCO production in Lipomyces starkeyi and Rhodosporidium toruloides yeasts. Yarrowia lipolytica, Cryptococcus curvatus, R. toruloides and L. starkeyi were grown in nitrogen-limited flask cultures. Yarrowia lipolytica strains produced citric acid and mannitol. Lipomyces starkeyi DSM 70296 and R. toruloides NRRL Y-27012 showed potential for SCO production, and were cultivated at increasing the initial glycerol concentrations with the initial nitrogen concentration remaining constant. Significant biomass and SCO production were reported even in cultures with high initial glycerol concentrations (i.e. 180 g l(-1) ). Lipid quantities of c. 12 g l(-1) (lipid in dry cell weight 35-40%) were obtained for both L. starkeyi and R. toruloides, quite high values compared with literature values for oleaginous micro-organisms growing on glycerol. However, these strains exhibited different kinetic profiles in the synthesis of intracellular polysaccharides. Lipomyces starkeyi produced a significant quantity of polysaccharides (c. 7 g l(-1) ). The yeast lipids contained mainly oleic and palmitic and to a lesser extent linoleic and stearic acids. Lipomyces starkeyi and R. toruloides are potential SCO producers from crude glycerol. Very scarce numbers of reports have indicated the production of SCO by L. starkeyi and R. toruloides growing on glycerol. We report here that these yeasts are able efficiently to convert raw glycerol into SCO, while L. starkeyi also synthesizes intracellular polysaccharides in marked quantities.

Chemical and biological conversion of crude glycerol derived from waste cooking oil to biodiesel

Due to its physiological and enzymatic features, Yarrowia lipolytica produces several valuable compounds from a wide range of substrates. Appointed by some authors as an industrial workhorse, Y. lipolytica has an extraordinary ability to use unrefined and complex low-cost substrates as carbon and nitrogen sources, aiding to reduce the waste surplus and to produce added-value compounds in a cost-effective way. Dozens of review papers regarding Y. lipolytica have been published till now, proving the interest that this yeast arouses in the scientific community. However, most of them are focused on metabolic pathways involved in substrates assimilation and product formation, or the development of synthetic biology tools in order to obtain engineered strains for biotechnological applications. This paper provides an exhaustive and up-to-date revision on the application of Y. lipolytica to valorize liquid effluents and solid wastes and its role in developing cleaner biotechnological approaches, aiming to boost the circular economy. Firstly, a general overview about Y. lipolytica is introduced, describing its intrinsic features and biotechnological applications. Then, an extensive survey of the literature regarding the assimilation of oily wastes (waste cooking oils, oil cakes and olive mill wastewaters), animal fat wastes, hydrocarbons-rich effluents, crude glycerol and agro-food wastes by Y. lipolytica strains will be discussed. This is the first article that brings together the environmental issue of all such residues and their valorization as feedstock for valuable compounds production by Y. lipolytica. Finally, it will demonstrate the potential of this non-conventional yeast to be used as a biorefinery platform.

Bioconversion of crude glycerol from waste cooking oils into hydrogen by sub-tropical mixed and pure cultures

The free fatty acids (FFAs) of waste cooking oil (WCO) are readily esterified with crude glycerol in the presence of the solid superacid SO/ZrO2–Al2O3. This reaction lowers the acidity of WCO before biodiesel production. The solid superacid SO/ZrO2–Al2O3 catalyzes both FFA esterification and TAG glycerolysis during the reaction. The conversion of FFA in the WCO with an acid value of 88.4 ± 0.5 mg KOH/g to acylglycerols was 98.4% under optimal conditions (mole ratio of glycerol to FFA = 1.4:1; reaction time = 4 h; reaction temperature = 200°C; catalyst loading = 0.3 wt%) obtained through an orthogonal experiment. The final FAME product with a FAME content of 96.9 ± 0.3 wt% yield was 94.8 wt%, after transesterification of the esterified WCO with methanol, catalyzed by potassium hydroxide. The FAME composition of the products produced by transesterification were identified and quantified by GC–MS. The results suggest that this new glycerol esterification process, using a solid superacid catalyst, affords a promising method to convert oils with high FFA levels, like WCO, to biodiesel. The process has the inherent advantage of easy separation steps for removing excess alcohol and significant savings in energy, when compared to acid catalyzed reactions with methanol to lower acidity.Practical applications: In this work, WCO with a high acid value was esterified with crude glycerol catalyzed by solid super acid, whose formula was expressed as SO/ZrO2–Al2O3. There are distinct advantages to this new esterification process, which include easy separation of the excess crude glycerol by sedimentation or centrifugation, the use of the low cost reactant crude glycerol direct from the byproducts of transesterification, the potential to achieve a very low content of FFAs by post‐refining to improve the yield of the final product, and time and energy saving are found as compared to the traditional methanol esterification process. This new technology provides a promising alternative method for processing feedstocks of high acid value, such as WCO, for the production of biodiesel.

Glycerol is a by-product generated in large amounts during the production of biofuels. This study presents an alternative means of crude glycerol valorization through the production of erythritol and mannitol. In a shake-flasks experiment in a buffered medium, nine Yarrowia lipolytica strains were examined for polyols production. Three strains (A UV’1, A-15 and Wratislavia K1) were selected as promising producers of erythritol or/and mannitol and used in bioreactor batch cultures and fed-batch mode. Pure and biodiesel-derived crude glycerol media both supplemented (to 2.5 and 3.25 %) and not-supplemented with NaCl were applied. The best results for erythritol biosynthesis were achieved in medium with crude glycerol supplemented with 2.5 % NaCl. Wratislavia K1 strain produced up to 80.0 g l−1 erythritol with 0.49 g g−1 yield and productivity of 1.0 g l−1 h−1. Erythritol biosynthesis by A UV’1 and A-15 strains was accompanied by the simultaneous production of mannitol (up to 27.6 g l−1). Extracellular as well as intracellular erythritol and mannitol ratios depended on the glycerol used and the presence of NaCl in the medium. The results from this study indicate that NaCl addition to the medium improves erythritol biosynthesis, and simultaneously inhibits mannitol formation.

BackgroundIntegrating waste management with fuels and chemical production is considered to address the food waste problem and oil crisis. Approximately, 600 million tonnes crude glycerol is produced from the biodiesel industry annually, which is a top renewable feedstock for succinic acid production. To meet the increasing demand for succinic acid production, the development of more efficient and cost-effective production methods is urgently needed. Herein, we have proposed a new strategy for integration of both biodiesel and SA production in a biorefinery unit by construction of an aerobic yeast Yarrowia lipolytica with a deletion in the gene coding succinate dehydrogenase subunit 5.ResultsRobust succinic acid production by an engineered yeast Y. lipolytica from crude glycerol without pre-treatment was demonstrated. Diversion of metabolic flow from tricarboxylic acid cycle led to the success in generating a succinic acid producer Y. lipolytica PGC01003. The fermentation media and conditions were optimized, which resulted in 43 g L−1 succinic acid production from crude glycerol. Using the fed-batch strategy in 2.5 L fermenter, up to 160 g L−1 SA was yielded, indicating the great industrial potential.ConclusionsInactivation of SDH5 in Y. lipolytica Po1f led to succinic acid accumulation and secretion significantly. To our best knowledge, this is the highest titer obtained in fermentation on succinic acid production. In addition, the performance of batch and fed-batch fermentation showed high tolerance and yield on biodiesel by-product crude glycerol. All these results indicated that PGC01003 is a promising microbial factorial cell for the highly efficient strategy solving the environmental problem in connection with the production of value-added product.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0597-8) contains supplementary material, which is available to authorized users.

Highly efficient production of FAMEs and β-farnesene from a two-stage biotransformation of waste cooking oils

Biodiesel is a renewable alternative fuel and glycerol as a main byproduct of the manufacturing process. Lipids could be produced from crude glycerol by using yeasts. The ability of 107 yeast strains to utilize glycerol was screened and 92 of these were selected. 60 strains were determined as a potential for lipid production by Sudan Black B staining. After secondary screening 25 of them showed specific growth rates (OD 600), high biomass production and lipid content. These strains were identified as Pichia cactophila, P. fermentans, P. anomala, Rhodotorula mucilaginosa, R. dairenensis, Clavispora lusitaniae, Saccharomyces cerevisiae, Wickerhamomyces anomalus, Candida glabrata, C. inconspicua, C. albicans, Yarrowia lipolytica with molecular identifications based on ITS and D1/D2 26S rDNA sequences. The results showed that P. cactophila accumulated lipid up to 64.94%, the highest lipid content. C16:0, C18:0, C18:1 and C18:2 essential fatty acids for biodiesel production were detected by GC–MS in the lipids accumulated by all strains. P. cactophila and C. lusitaniae were reported for the first time as lipid-producing yeasts. The results suggest that selected 25 isolates have the ability to grow on crude glycerol and especially P. cactophila produce lipid that has potential use as a feedstock for second generation biodiesel production.

Continuous energy crises and increasing demand for conventional fuels has resulted in the need for biofuels on a commercial scale. Transesterification of oils to yield biodiesel – one of the principal biofuels currently produced in large‐scale operations – is coupled with significant production of a glycerol‐rich water (so‐called “crude” or “raw” glycerol), as an important side‐product of the process. The increasing demand for biodiesel leads to abundant quantities of this glycerol‐rich material on the market. Therefore, glycerol valorization has much to offer in the cost reduction of biodiesel production. To this end, various chemical or biotechnological strategies have been developed to obtain added‐value products using crude glycerol as substrate. This review combines an account of our attempts to achieve a biotechnological valorization of raw glycerol with a review of appropriate literature.

Citric acid was produced by five species of the yeast Candida after growth on a medium containing soy biodiesel-based crude glycerol. After growth on a medium containing 10 g L−1 or 60 g L−1 crude glycerol for 168 hr at 30°C, Candida parapsilosis ATCC 7330 and C. guilliermondii ATCC 9058 produced the highest citric acid levels. On 10 g L−1 or 60 g L−1 crude glycerol for 168 hr at 30°C, the citric acid level produced by C. parapsilosis ATCC 7330 was 1.8 g L−1 or 11.3 g L−1, respectively, while C. guilliermondii ATCC 9058 produced citric acid concentrations of 3.0 g L−1 or 10.4 g L−1, respectively. Biomass production by C. guilliermondii ATCC 9058 on 10 g L−1 or 60 g L−1 crude glycerol for 168 hr at 30°C was highest at 1.2 g L−1 or 6.9 g L−1, respectively. The citric acid yields observed for C. guilliermondii ATCC 9058 after growth on 10 g L−1 or 60 g L−1 crude glycerol (0.35 g g−1 or 0.21 g g−1, respectively) were generally higher than for the other Candida species tested. When similar crude glycerol concentrations were present in the culture medium, citric acid yields observed for some of the Candida species utilized in this study were about the same or higher compared to citric acid yields by Yarrowia lipolytica strains. Based on the findings, it appeared that C. guilliermondii ATCC 9058 was the most effective species utilized, with its citric acid production being similar to what has been observed when citric acid-producing strains of Y. lipolytica were grown on crude glycerol under batch conditions that could be of significance to biobased citric acid production.

A cost efficient way to obtain lipid accumulation in the oleaginous yeast Rhodotorula glutinis using supplemental waste cooking oils (WCO)

It is important to utilize crude glycerol, the main byproduct of biodiesel production, to manufacture high value-added chemicals. Since crude glycerol typically contains less than 65 wt % glycerol, purification is the first step for its utilization. Owing to the wide variety of triglycerides, alcohols, catalysts, and separation processes used in biodiesel production, crude glycerol composition varies widely, leading to different crude glycerol purifications. In the present work, we develop a universal procedure for crude glycerol purification, including as key steps initial microfiltration of the crude glycerol, saponification, acidification, phase separation, and biphasic extraction of upper- and lower-layer products. The procedure was utilized to purify crude glycerol samples from two biodiesel production companies, experimentally upgrading both samples to >94 wt % purity. On an Aspen Plus software platform, the purification procedure was simulated using a process model based on two submodels to obtain ...

Organic acids like succinic and citric acids are of great interest as platform organic products that play important roles as precursors for a wide range of bio-based materials. Succinic and citric acids can be successfully produced biotechnologically from renewable resources of both hydrophilic and hydrophobic nature through efficient microbiological conversion. Yarrowia lipolytica represents one of the most versatile microbial factories in terms of organic acids production, as it easily develops and produced metabolites starting from glucidic-based and/or lipid-based substrates. The purpose of this work was to investigate the ability of Y. lipolytica to adapt to hydrophilic and hydrophobic sources and to biosynthesize important platform chemicals like succinic and citric acids. The selected strain was monitored during a batch cultivation for 192 h on 100 g/L carbon source: pure glycerol as a hydrophilic source, sunflower waste cooking oil as a hydrophobic source, and crude glycerol deriving from biodiesel production as a mixture of hydrophilic and hydrophobic sources. Cellular viability, biomass accumulation, and metabolites formation in terms of succinic acid and citric acid was monitored, and the highest results were registered for cultivations performed on waste cooking oil [10.35 ± 0.29 (log 10 ) CFU/mL, 8.15 g/L cell dry weight, 3.50 ± 0.04 g/L citric acid, and 21 ± 0.16 g/L succinic acid]. The results obtained in this work outline the industrial potential of the oleaginous yeast strain of Y. lipolytica to bioconvert the lipidic residual biomass with negative environmental implications into valuable organic compounds with wide-range applicability. • Sunflower waste cooking oil is a renewable biomass for biotechnological approaches. • Y. lipolytica produces up to 21 g/L succinic acid and 3.5 g/L citric acid from SWCO. • Y. lipolytica produces up to 14 g/L succinic acid and 4.9 g/L citric acid from CG. • Y. lipolytica produces organic acids from both hydrophilic and hydrophobic sources.