Online flow cytometry, an interesting investigation process for monitoring lipid accumulation, dimorphism, and cells’ growth in the oleaginous yeast Yarrowia lipolytica JMY 775

Less than 30 species of the 600 identified yeast species are known for their oleaginous property (Ratledge 2004). Thus, there is a great attention to the field of lipid production and metabolism from various unicellular organisms, particularly oleaginous yeasts which are suited to this production (Fickers et al. 2005). Furthermore, they are suitable for large-scale fermentation and are free of endotoxins. Hence, oils sourced from microbial cultures have similar lipid profiles in type and in composition to those produced by plants (Beopoulos et al. 2011). Yarrowia lipolytica is among the best used microbial cultures and the most interesting. In addition, thanks to its non-pathogenicity, spit of its dimorphic property which confers to it the ability to appear under a mycellar shape or yeast cell shape is the most studied non-conventional yeast. It has also the ability to store more than 20%, and even more than 40% for some strains, under particular conditions (Beopoulos et al. 2009).

Lipids are accumulated in Y. lipolytica by two antagonist ways: first, de novo biosynthesis of fatty acids, which is an anabolic pathway, corresponding to the production, under defined conditions, of fatty acids precursors, such as acetyl-CoA and malonyl-CoA, leads their integration in the lipids biosynthetic pathway (phospholipids, sphingolipids, triacylglycerol, etc). This pathway is so called the Kennedy pathway (Beopoulos et al. 2009; Papanikolaou and Aggelis 2011) and second, ex novo pathway corresponding to the incorporation of fatty acids, triglycerides, or alkanes, from the culture medium to the interior of the cell. This pathway requires the hydrolysis of the hydrophobic substrates, the transport and the incorporation of free fatty acids within the cell, and reassembly of stored fatty acids under triacylglycerides form, or sterol esters (Beopoulos et al. 2009). The accumulation and storage of fat are performed in specific compartments, called lipid bodies (Fujimoto et al. 2008).

Lipid accumulation in Y. lipolytica is influenced by several external factors, namely, dissolved oxygen, pH, presence of inorganic salts, and also temperature (Subramaniam et al. 2010). To rapidly determine physiological properties on single cell or to detect and identify the different sub-populations in a media, flow cytometry seems to be the appropriate tool for research on microorganisms. By this emerging tool, thousands of cells or molecules per second can be automatically analysed, thus making it a versatile and a fast technique even described as an automated microscopy (Ueckert et al. 1995; Díaz et al. 2010). Actually, several cellular parameters can be simultaneously measured, using modern laser-based flow cytometers. In flow cytometer when laser light beam hits a stream of cells or particles, it is scattered in different directions. This light is measured in forward scatter (FSC), whose detection can be viewed as an approximation of the detected cell or the particles and inside scatter (SSC) that is an approximation to granularity or shape (Bradner and Nevalainen 2003).

Thus, the objective of this study is to use for the first time flow cytometry for a better understanding and control of mechanisms and parameters modulating the different phenomena observed in Y. lipolytica culture. Mainly, this study allowed a fast investigation using this process, the growth dynamic, lipid accumulation, and cell dimorphism. The study of these phenomena and of the profile of their progress was possible thanks to the combination of the conventional analyses tools with flow cytometry as an-online culture monitoring.

Yarrowia lipolytica is able to use free fatty acids or to store them as triacylglycerol’s (TAG) and stearyl esters (STE) into lipid bodies depending on the environmental conditions. A lipid body is a hydrophobic core formed from neutral lipids, mainly TAG and in a lesser amount as STE (Papanikolaou et al. 2006; Beopoulos et al. 2009; Nicaud 2012). As described in earlier studies, a mixture of hydrophilic and hydrophobic substrates had been used, i.e, glucose and oleic acid. Hydrophobic substance assimilation by Y. lipolytica requires usually morphological and physiological adaptations. For example, Mlíčková et al. (2004) found that cells growing on glucose had smooth surfaces. In agreement to this present study, cells growing on a medium enriched with fatty acids had small protrusions scattered across their surfaces, especially those which had taken a mycelar form. The number of protrusions increased with the time of incubation in oleic acid, so at the beginning of the experimentation, protrusion number increased from 48 h after the induction. The lipid droplets on cells’ surfaces were also observed and their number increased when protrusion were several. These findings are in agreement with the observations reported in numerous previous studies (Mlíčková et al. 2004; Fickers et al. 2005; Beopoulos et al. 2008, 2009; Coelho et al. 2010).

The findings showed that in all the bioreactors, the step corresponding to a fast medium’s fat consumption coincides with the step of glucose depletion. (Fickers et al. 2005) reported that few lipid bodies could be observed in Y. lipolytica when grown in glucose medium, whereas lipid body accumulation was observed during culture in fatty acid or triglyceride medium. This suggests that when growing on a mixed medium composed of sufficient quantities of glucose and oleic acid, yeast cells began to metabolize first glucose as preferential carbon source (Papanikolaou et al. 2006; Weinhandl et al. 2014). After that, when glucose decreases greatly, cell fatty acid accumulation was triggered. This is clearly shown by cells’ growth and oleic acid accumulation which slowed after 30 h of culturing, both in control (F1) and in pH-drop bioreactor (F3). Nevertheless, growth and lipid accumulation appeared to extend in F3. Thus, we can suggest that stationary phase may occur probably when cells metabolized by the oxidation of their intracellular accumulated lipids (using them as carbon source instead of glucose). This process was independent of the nitrogen concentration in the culture medium as already reported by Papanikolaou et al. (2002). However, low oxygenation rate in F3 enabled to slow oxidation of carbon sources, and leads to extend the time of intracellular lipid accumulation and cell growth phase. Previous studies showed that the improvement of the oxygen availability either by acting on saturation level (5–15%) (Papanikolaou et al. 2002), on air pressure (6 bar) (Lopes et al. 2009), or on the aeration inside the bioreactor (Coelho et al. 2010) and have a significant effect in cells’ growth rate. Thus, as oxygenation is a very important parameter, it is useful to know how to manage this parameter in a bioprocess. In this context, Coelho et al. (2010) reported that oxygen transfer rate (OTR) allows the analysis of the oxygen impact on a bioprocess. For a specific bioreactor and medium, OTR could be increased by increasing agitation and aeration rate (Coelho et al. 2010). This statement was observed in this study. Furthermore, an increase of lipase production and lipolysis activity in Y. lipolytica was previously described (Alonso et al. 2005; Coelho et al. 2010). We suggest that these may be verified by reducing agitation in the F3 bioreactor.

The developed online flow cytometry process allowed an efficient monitoring and in a real-time manner of the lipid accumulation during the primary anabolic growth phases under the influencing medium’s pH, incubation temperature, and aeration parameters. Accordingly, Papanikolaou et al. (2002) reported that at pH 6.0 and a temperature of 28 °C, high quantities of dry biomass may be produced in shorter time, whereas significant quantities of lipids substrate were rapidly consumed by yeast cells. Indeed, under optimal conditions of aeration (600 rpm), temperature (28 °C), and pH 6.0, cells’ growth was faster in F1 bioreactor. This may be explained by the fast consumption of both glucose and oleic acid, as carbon sources by the cells. These findings are supported by the results previously reported by Papanikolaou et al. (2002).

pH and incubation temperature have been reported as significant factors. They are in relation with the process of single-cell oil accumulation. For example, the influence of pH was already studied when Y. lipolytica was growing on stearin (10 g/L) in the presence of (NH₄)₂SO₄ and at 28 °C. Thus, it seems that pH has a crucial effect on single-cell oil accumulation, and also on cells’ growth. Therefore, in their investigation related to the effect of medium pH on lipid production by Rhodosporidium toruloides strain, (Dias et al. 2016) found that yeast biomass concentration and lipid amount were the highest at pH 4.0. Using the same operating process, we observed that fat accumulation by Y. lipolitica JMY755 was more efficient at pH 6.0 in contrast to the results by Papanikolaou et al. (2002) reporting that at pH 5.0 and 7.0, the cell growth was narrowed. Therefore, in this report, under the unregulated-pH conditions (F2), the consumption of fatty acids was slowed when pH decreased under the value of 5.0. Thus, after 48 h of culturing, cells in F2 looked barely fluorescent and medium’s oil rate starts dropping as in F1, but with a lower rate. The minimal level of medium fatty acids was attainted in F2 after 36 h of culturing. This may be probably caused by the important pH drop due to the critical pH value (pH ≤4.0) at 24 h of culturing. Thus, under critical pH value, glucose consumption was completely inhibited, while intracellular lipid assimilation was affected by the significant decreasing of pH, hence, affecting well cell growth. The optimization of culture parameters allowed a faster cell growth process and energetic metabolism. In these conditions, glucose as a preferred energy source was rapidly metabolized in the oxidation process, supported by better availability of oxygen (in both F1 and F2). Thus, glucose was rapidly used by the biomass and the oleic acid consumption started to operate as soon as glucose was used. Several experiments, in which fatty acids were added into the growth medium, showed an important fatty acid biosynthesis through de novo mechanism especially when cells assimilated high sugar quantities (Papanikolaou et al. 2006). A decrease of lipid storage in Y. lipolytica occurs when growth was done on low substrate fat concentration media. Oleaginous microorganisms growing on fats commonly consume their own storage lipids when the flow rate of exocellular fatty acids is considerably decreased in the culture medium (Aggelis et al. 1995). Lipid accumulation from fats was reported as a result of an unbalanced uptake and assimilation rate of aliphatic chains. When this uptake is higher than that of the assimilation rate, lipid is accumulated inside of the microbial cell. In contrary, when the assimilation rate is higher than the substrate fat, the growth needs are covered by the degradation of storage lipid (Papanikolaou and Aggelis 2003). These findings were verified in our study using online cytometric process. In addition, this process allowed to observe that at the end of culture, cells derived from control bioreactor (F1) were not fluorescent; in contrast to those from F3. It seems that, in limited-oxygen bioreactor (F3), cells still fluorescent after 48 h of culturing.

The present study showed also that different parameters may play significant roles in Y. lipolytica dimorphism. For example, at 24 h of culturing, almost of the cells presented a pseudo-mycelial form with no yeast-like form. Since the 1990s, the undertaken dimorphism studies on Y. lipolytica, showed its ability to grow as a yeast-like or hyphal forms, depending on growth conditions (Fickers et al. 2005). Furthermore, Y. lipolytica was reported as a model for dimorphism (Barth and Gaillardin 1997; Nicaud 2012). Thus, the cytograms of this study revealed clearly homogeneous populations within each bioreactor. Microscopic observations enable to note that at the end of culturing (48 h), cells in the F1 took a yeast-like form, while in the F2, a transitional situation was observed. However, in F3, cells appeared still filamentous with elongated and homogeneous forms but more voluminous than at the beginning of experiment. (Makri et al. 2010) reported that during biomass production, phase short and true mycelia and pseudo-mycelia may predominate. Indeed, large obese cells with discernible lipid globules appeared in the early stationary phase, namely, in the lipogenic phase. At this phase, the lipid globule sizes were diminished during the late stationary phase, namely, at the citric acid production phase (Makri et al. 2010). This phase appeared earlier in F1 and F2 bioreactors than in F3. Moreover, it has been reported that during the exponential growth phase, when mycelial form dominated over yeast form, high respiration activity may be observed (Makri et al. 2010). A gradual transition to yeast-like cells form in lipogenic and in citric acid production phases appeared in the F2 bioreactor. The decline of pH may explain part of these findings.

On another hand, cell size was affected by the different percentages of the accumulated lipids among lipogenic and citric acid production phase as reported by Makri et al. (2010). However, many inconsistencies were reported by earlier studies about Y. lipolytica’s dimorphism. For example, (Rodriguez and Domínguez 1984) reported that elongation may operate only upon entering the stationary phase, and during the growth phase, Y. lipolytica may stay in yeast-like mode (Chen et al. 1997; Makri et al. 2010). The differences may be explained by the effect of temperature (Makri et al. 2010) due to heat shock (Guevara-Olvera et al. 1993), to the atmosphere composition and in the presence of specific compounds in the culture media (Barth and Gaillardin 1997; Cervantes-Chávez et al. 2009), as well as the nature of carbon substrate (Rodriguez and Domínguez 1984; Guevara-Olvera et al. 1993; Ruiz-Herrera and Sentandreu 2002), and to the atmosphere of growth media and pH (Ruiz-Herrera and Sentandreu 2002). Other studies (Barth and Gaillardin 1997; Zinjarde et al. 1998) also suggest that the capacity of dimorphism is strain specific. In this context, (Szabo and Štofanı́kova 2002) confirmed that external pH regulates dimorphism predominantly via modulation of the availability and/or the use of the organic sources of nitrogen in Y. lipolytica. Though, we could be confronted once again to some inconsistencies to some of the reported studies concerning the effect of this parameter. Therefore, when some of the studies conclude that mycelium formation was maximal at pH near neutrality and decreased as pH was lowered to converge to zero at pH 3.0 (Ruiz-Herrera and Sentandreu 2002), other studies reported that under particular conditions, mycelial form may be observed on cells subjected to pH shock (Kar et al. 2011) or on cells exposed to acidic pH (Szabo 1999).

Online flow cytometry process appears as an efficient tool for monitoring a bioreactor culture of Y. lipolytica. This study showed the great potential of this emerging and versatile technique, not only for the study of the involved parameters cell culture evolution, but also into study of the various phenomena induced by modifications in each culture condition. Thus, it was proposed to offer a considerable time saving for obtaining and processing results in comparison with the conventional techniques.