Assessing an effective feeding strategy to optimize crude glycerol utilization as sustainable carbon source for lipid accumulation in oleaginous yeasts

Abstract

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.

Microtiter plate cultivation of oleaginous fungi and monitoring of lipogenesis by high-throughput FTIR spectroscopy

“…Indeed, the homogenization of lipid-rich fungal biomass used for FTIR required long time, while that used for GC analysis required the use of hydrochloric acid and harsh mechanical pretreatment (bead beating) [34]. Ami et al [8] and Signori et al [10] observed similar cell wall changes in FTIR spectra during oleaginous yeast lipogenesis.…”

Bioprospecção de leveduras oleaginosas do Agreste Pernambucano: síntese de lipídios para a produção de biodiesel a partir do resíduo glicerol bruto

“…O gênero Yarrowia é conhecido na literatura por sua capacidade de acumular lipídios (Chen et al, 2009;Signori et al, 2016). Este estudo comprovou que todos os isolados de Yarrowia coletados nos laticínios da bacia do Agreste-PE apresentaram potencial de crescimento e de acúmulo de lipídios, utilizando o glicerol bruto como fonte de carbono.…”

“…Muitas vantagens estão envolvidas no uso de leveduras oleaginosas para a produção de lipídios, como a alta taxa de crescimento em biorreatores, a capacidade de assimilar diversas fontes de carbono e não serem afetadas por limitações de espaço, clima ou variações de luz (Ami et al, 2014). Entre as leveduras mais citadas na literatura, como oleaginosas, figuram as do gênero Rhodotorula e da espécie Yarrowia lipolytica (Chen et al, 2009;Signori et al, 2016).…”

Lipids detection and quantification in oleaginous microorganisms: an overview of the current state of the art

“…These methods, based on a vital dye, offer a rapid and inexpensive approach for measuring lipid content and can be applied in various systems, in situ and in vitro [35]. Other non-destructive methods, such as infrared (IR), nuclear magnetic resonance (NMR), and Raman spectroscopy, have also been used to some extent for lipid determination in oleaginous microorganisms [38][39][40][41][42]. Techniques for lipid content determination in microalgae have been thoroughly described in several recent reviews [31,[33][34][35] and are also commonly applied to lipid quantification in other oleaginous microorganisms.…”

“…IR spectroscopy has gained ground as a fast and non-invasive lipid quantification method that can be applied on small amounts of whole cells for real-time monitoring and screening of cultures [40,105]. Flow-cytometry and Fourier transform infrared spectroscopy (FTIR) supported by multivariate analysis, have been proposed as fast and reliable means to monitor and quantify lipids over time in the oleaginous yeasts Cryptococcus curvatus, Rhodosporidium toruloides, and Lipomyces starkeyi [38,40].…”