An Effective Method of Continuous Production of Erythritol from Glycerol by Yarrowia lipolytica MK 1

Erythritol is a sugar alcohol from a group of polyols that occurs in nature as a metabolite or storage compound in seaweeds or fungi, as well as a component of fruits such as pears, melons and grapes (1). It has about 60–80 % of the sweetness of sucrose in a 10 % solution. Its taste profi le resembles that of sucrose, and in many countries it has been safely used in food as a noncariogenic sweetener (2,3). Erythritol is produced in microbiological processes from carbohydrates, especially from glucose, using osmophilic yeast such as Moniliella sp. (4), Trichosporon sp. (5), Aureobasidium sp. (6), Torula sp. (7) or Pseudozyma tsukubaensis (8). Moreover, erythritol may be produced at low pH values also from glycerol by the oleaginous yeast Yarrowia lipolytica (9,10). Various factors aff ecting fermentation have been examined, e.g. diff erent cultivation systems, type of substrate, and addition of salt or supplementation with minerals (9,11–15). Up to now, the batch, fed-batch or repeated-batch cultures have been tested for erythritol biosynthesis from glycerol (9,13). However, the application of a chemostat culture, which is a very promising cultivation system, especially for yeast known to produce well-dispersed, unicellular populations, has never been reported. Continuous cultivation with yeast is successfully used in biotechnology, e.g. in the production of biodiesel (16), bulk chemicals (17), xylitol (18) or citric acid (1).


Introduction
Erythritol is a sugar alcohol from a group of polyols that occurs in nature as a metabolite or storage compound in seaweeds or fungi, as well as a component of fruits such as pears, melons and grapes (1).It has about 60-80 % of the sweetness of sucrose in a 10 % solution.Its taste profi le resembles that of sucrose, and in many countries it has been safely used in food as a noncariogenic sweetener (2,3).Erythritol is produced in microbiological processes from carbohydrates, especially from glucose, using osmophilic yeast such as Moniliella sp.(4), Trichosporon sp.(5), Aureobasidium sp.(6), Torula sp.(7) or Pseudozyma tsukubaensis (8).Moreover, erythritol may be produced at low pH values also from glycerol by the oleaginous yeast Yarrowia lipolytica (9,10).Various factors aff ecting fermentation have been examined, e.g.diff erent cultivation systems, type of substrate, and addition of salt or supplementation with minerals (9,(11)(12)(13)(14)(15).Up to now, the batch, fed-batch or repeated--batch cultures have been tested for erythritol biosynthesis from glycerol (9,13).However, the application of a chemo-stat culture, which is a very promising cultivation system, especially for yeast known to produce well-dispersed, unicellular populations, has never been reported.Continuous cultivation with yeast is successfully used in biotechnology, e.g. in the production of biodiesel (16), bulk chemicals (17), xylitol (18) or citric acid (1).
The overall goal of this study is to improve the production of erythritol on glycerol media with the Y. lipolytica MK1 strain in the classic single-step continuous chemostat culture.The specifi c objective of this research is to investigate the optimal C:N ratio in the feeding medium in order to enhance erythritol production.

Strain
The Yarrowia lipolytica MK1 strain used in this study was isolated from the acetate-negative mutant strain Y. lipolytica Wratislavia K1 aft er its exposure to UV irradiation (19

Media and culture conditions
Pure glycerol (purity of 98 %; Avantor Performance Materials Poland S.A, Gliwice, Poland) was applied in the study.The growth medium for the inoculation culture contained (in g/L): pure glycerol 50, yeast extract 3, malt extract 3 and Bacto TM Peptone 5 (Sigma-Aldrich, St. Louis, MO, USA).
All cultures were made in a 5-litre stirred tank reactor (Biostat B plus; Sartorius, Gött ingen, Germany).Each feeding medium was added to the bioreactor at a rate of 5.0 mL/h and at the same time culture liquid was continuously removed at an equal rate to keep the working volume constant at 1.5 L. The steady state of each culture was achieved aft er about 200 h, when all culture parameters (specifi c growth rate, biomass and product concentrations, culture volume and dissolved oxygen concentration) remained constant.The residence time for the chemostat culture was 100 h and the processes were stable during 400 h.
An inoculum of 0.2 L was added to the bioreactor containing 1.3 L of the medium.The stirring rate and aeration space velocity were 13.3 Hz and 0.6 min -1 , respectively (20).The temperature was maintained at 30 °C and the pH was kept at 3.0 by the addition of 20 % NaOH.The bioreactor containing the specifi c medium (prepared with tap water) was sterilised in an autoclave at 121 °C for 20 min.All cultures were prepared in two biological replicates and standard deviations were calculated.

Analytical methods
For dry biomass quantifi cation 10 mL of culture broth were centrifuged (centrifuge model 5804/5804 R; Eppendorf, Hamburg, Germany) at 2700×g for 5 min.The biomass was washed with distilled water, fi ltered through a membrane with a pore size of 0.45 μm and determined gravimetrically aft er drying at 105 °C, expressed in grams of cell dry mass per litre.
The total intracellular lipids were determined by the Soxhlet method (21).Fat was extracted using a Büchi B-811 universal extraction system (Büchi Labortechnic AG, Flawil, Switzerland).The fatt y acid profi le was determined in the fat extracted from yeast biomass according to the method described by Ackman (22) with some modifi cations: 0.2 g of dry biomass was treated with a mixture of 1 mL of benzene and 1 mL of BF 3 -methanol (Sigma--Aldrich).Next, the sample was shaken at 140 rpm for 5 min, heated for 15 min at 50 °C and cooled.Aft er the addition of 0.5 mL of H 2 O the sample was centrifuged at 2700×g for 5 min and the upper layer was evaporated in a nitrogen atmosphere.Dry residue was dissolved in 0.1 mL of CH 2 Cl 2 (Sigma-Aldrich).Fatt y acid methyl esters were separated by gas chromatography (Philips, Cambridge, UK) according to the methodology described by Kita et al. (23).
The concentrations of glycerol, erythritol, mannitol, arabitol, citric acid and α-ketoglutaric acid were measured in the culture supernatants by high-performance liquid chromatograph (Thermo Scientifi c, Waltham, MA, USA) using an HyperRez Carbohydrate H+ column (Thermo Scientifi c) coupled to a UV detector (λ=210 nm; Dionex, Sunnyvale, CA, USA) and a refractive index detector (Shodex, Ogimachi, Japan).The column was eluted with 25 mM trifl uoroacetic acid at 65 °C and a fl ow rate of 0.6 mL/min.Protein concentration in the biomass harvested at the end of the cultivation was analysed using the Kjeldahl method (24).The amino acid profi le was determined using an AAA400 Automatic Amino Acids Analyser (IN-GOS Ltd., Prague, Czech Republic).

Eff ect of C:N ratio on the growth and erythritol production by Y. lipolytica MK1 in a chemostat culture
Continuous cultures undoubtedly have many advantages, among which the most important include the possibility to set the physiological state of the cells by selecting the fl ow rate of the feeding medium and the composition of the culture medium, physical and chemical homogeneity of the culture, automation of the process, higher process effi ciency, maximisation of apparatus utilisation, and even spread of the workload (25,26).However, the disadvantages of these processes are the diffi culty in ensuring aseptic conditions and the possibility of degeneration or mutation of strains (27,28).Some of the well-described continuous processes using Y. lipolytica yeast include the production of lipids from molasses and glycerol-containing medium (29) and the production of citric acid using glycerol as a carbon source (1).In the literature, there is a lack of information about erythritol production using chemostat cultivation.
In this study, three medium compositions were tested.The PM1 and PM2 had a C:N ratio of 160:1 and 80:1, respectively.The PM3 also had the C:N ratio of 80:1, but in this case the increased carbon and nitrogen concentrations were tested.Fig. 1a shows the infl uence of time and diff erent medium composition on biomass production in chemostat cultures.All tested cultures were carried out for about 600 h and the steady state was achieved aft er about 200 h of cultivation, which is equal to almost fi ve replacements of the culture medium in the bioreactor under steady state.The biomass concentration under steady state was the highest (24.3 g/L) in the culture with PM3 where the C:N ratio of 80:1 and high ammonium sulfate concentration were applied (Fig. 1a).The lower content of nitrogen in the PM2 with the same C:N ratio resulted in lower biomass production (21.8 g/L) (Fig. 1a).In PM1, biomass concentration under steady state reached 11.6 g/L (Fig. 1a).
Figs. 1b and c show the time courses of erythritol production and glycerol utilisation, respectively.Additionally, Table 1 summarises the parameters of erythritol production.High concentrations of erythritol were observed when PM2 and PM3 were applied, i.e. 113.1 and 112.6 g/L, respectively (Fig. 1b; Table 1).However, the highest productivity (1.1 g/(L•h)) and erythritol production yield (0.57 g/g) were observed in the PM2 culture (Table 1).The increased concentration of carbon and nitrogen in PM3 without changes in the C:N ratio (80:1) did not improve productivity and yield of erythritol formation when compared to the PM2 (80:1) (Table 1).The application of the PM1 with the highest C:N ratio resulted in a signifi cantly lower concentration of erythritol produced (83 g/L) (Fig. 1b, Table 1), and its production parameters were also not satisfactory (Table 1).
Interestingly, there was a signifi cant eff ect on a residual glycerol level when the C:N ratio was changed from 160:1 to 80:1 (Fig. 1c).The residual glycerol concentration was the lowest in the PM2 culture and reached only about 1.9 g/L under steady state (Fig. 1c).Its comparable concentration (45 g/L) was observed in the chemostat PM1 and PM3 cultures (Fig. 1c), which indicates cost-ineff ectiveness of the process and great loss of the substrate.Batch or fed-batch cultures on glycerol are commonly used as systems for erythritol production, and therefore glycerol seems to be an effi cient substrate for this purpose (9,14).As reported before, in batch culture the same strain, Y. lipolytica MK1, was able to produce up to 82.2 g/L of erythritol from glycerol with yield of 0.55 g/g and productivity of 0.84 g/(L•h) (19).Erythritol production parameters obtained in this study in continuous cultivation were higher than those observed by Rywińska et al. (11) in a batch culture on crude glycerol using the parental strain Y. lipolytica Wratislavia K1.The application of crude glycerol at 13.3 Hz and 0.6 min -1 resulted in erythritol concentration of 58.2 g/L, yield of 0.38 g/g and productivity of 0.78 g/(L•h).On the other hand, Rymowicz et al. (9) reported that in the fed-batch cultivation the application of crude glycerol (300 g/L) with Y. lipolytica Wratislavia K1 resulted in a higher concentration of erythritol (170 g/L) with the same yield of erythritol production of 0.56 g/g and lower productivity of 1.0 g/(L•h) than in the present study.Bett er results were obtained by Tomaszewska et al. (13) when using the parental strain Y. lipolytica Wratisla-via K1 in the fed-batch system with pulsed addition of glycerol (325 g/L).Under these conditions, the yeast produced 201.2 g/L of erythritol aft er 168 h of cultivation, which corresponded to a yield of 0.62 g/g and productivity of 1.2 g/(L•h) (13).It is worth noting that erythritol has been produced commercially from glucose (3).The available data obtained when using diff erent microorganisms on glucose media showed that the highest value of erythritol productivity in the fed-batch culture was 2.86 g/(L•h) with Pseudozyma tsukubaensis (8), whereas the highest value of erythritol production yield, 0.63 g/g, was noted by Lin et al. (4) with Moniliella sp.N61188-12, when compared to the present results.Similar productivity (1.2 g/(L•h)) was obtained by Kohl et al. (30), who used Candida magnoliae in fed-batch fermentation with simultaneous glucose and yeast extract feeding.However, another mutant of Candida magnoliae in the medium with 20 % glucose produced erythritol with lower productivity (0.54 g/(L•h)) and yield (0.43 g/g) than in this study (26).
The high osmotic pressure caused by salt addition to the medium and a high concentration of glycerol are the key factors that enhance erythritol production by Y. lipolytica and at the same time reduce the formation of mannitol and arabitol (31,32).In all tested chemostat cultures, regardless of C:N ratio, the concentration of mannitol was low and did not exceed 2 g/L (Table 1).Moreover, the concentration of arabitol remained at a very low level (0.3 g/L) when PM1 and PM2 where used.Arabitol concentration increased to 4.4 g/L in PM3 (Table 1).Organic acids, such as citric and α-ketoglutaric acids, were also determined in the culture broths, although in small quantities (Table 1).It is worth noting that selectivity of erythritol biosynthesis in all performed chemostat cultures was very satisfactory.
One limiting factor of chemostat culture is dissolved oxygen concentration (29).During chemostat cultures with PM2 and PM3 the dissolved oxygen concentration was lower than in PM1 and was about 20 % in PM2 and PM3 and 45 % in PM1 (data not shown).Low dissolved oxygen concentrations have been shown to induce the yeast-to-mycelium transition in Y. lipolytica.Bellou et al. (33) demonstrated that mycelial and pseudomycelial forms predominated over the yeast form when the dissolved oxygen concentration was low, regardless of the carbon and nitrogen sources used.

The nutritional value of Y. lipolytica MK1 biomass
For more in-depth investigation of the long-term chemostat culture, at the end of the cultivation the protein content of Y. lipolytica MK1 biomass was analysed (Table 1).The European Feed Manufacturers' Federation authorised the sale of Y. lipolytica fodder yeast produced from glycerol and registered it under catalogue number 00 575--EN (19).Therefore, the biomass from the chemostat culture was collected and nutritional characteristics were analysed for potential use as feed.The highest mass fraction of protein in the biomass reached 33.7 % (Table 1) and was observed in the PM3, which also gave the highest biomass concentration (Fig. 1a).Yeast biomass that contains below 65 % protein is referred to as single-cell biomass.Protein content of the biomass recommended for fodder yeasts in the standards established by the FAO/ WHO is 40-52 % (34).Protein content in the culture with the PM2 was also high and reached 29.5 %.In comparison with the present work, a similar protein mass fraction ((35.2-36.5)%) was noted by Juszczyk and Rymowicz (35) in the Y. lipolytica biomass produced using glycerol waste.
For comparison, the mass fraction of protein in the Y. lipolytica Wratislavia K1 biomass aft er the erythritol biosynthesis on pure glycerol in repeated-batch culture was lower and reached 16.9-23.4% when 40 % of medium was replaced (19).
The PM2 gave the highest erythritol concentration, productivity and yield, with a low concentration of residual glycerol and by-products.Therefore, the biomass from the chemostat culture produced with the PM2 medium was also tested for amino acid, lipid, essential saturated and unsaturated fatt y acid content.The amino acid composition in yeast biomass is strongly dependent on the yeast strain, medium composition, growth phase and cultivation time (25,35).The amino acid profi le showed that aspartic and glutamic acids were the main components of the proteins, constituting 11.6 and 11.3 %, respectively (Table 2).The other amino acids were identifi ed as leucine, lysine, valine, alanine and threonine, in mass fractions of 10.5, 8.7, 8.0, 8.0 and 7.4 %, respectively.In contrast, the biomass of Y. lipolytica derived from the culture on raw glycerol was rich in lysine (8.26 g per 100 g of protein) and threonine (5.39 g per 100 g of protein) (34).Moreover, proteins in the Y. lipolytica MK1 biomass were poor in sulphuric amino acids (methionine and cysteine), which was also observed when other strains of Y. lipolytica were cultivated on pure as well as on raw glycerol (34,35).
The lipid content and the mass fractions of saturated, monounsaturated and polyunsaturated fatt y acids in the Y. lipolytica MK1 biomass were analysed.Lipid content in the biomass was 20.9 % (Table 3).The main fatt y acid in the yeast biomass was oleic acid (C18:1n9; 38.2 %), as observed when using other oleaginous yeasts (31,32).Oleic acid was described as the main monounsaturated fatt y  3), and these results are in agreement with other studies performed with Y. lipolytica grown on glycerol (16).In general, diff erences in fatt y acid profi les seem to result not from culture type, but from substrate type.For example, when glycerol was used as a carbon source by Y. lipolytica UFLA CM-Y9.4,stearic (C18:0) and palmitic (C16:0) acids were predominant in the mass fractions of 87.64 and 74.67 %, respectively (36).In turn, using industrial fats in the process with yeast resulted in a different total fatt y acid composition, which was characterised by a high concentration of cellular stearic acid (37).

Conclusions
A growing interest is observed today in the management of glycerol, which seems to be an effi cient substrate for erythritol production.One possible application of glycerol shown in this work is effi cient production of erythritol using Yarrowia lipolytica MK1 strain in a chemostat culture.The biosynthesis of erythritol in such a system is of high selectivity with low concentrations of by--products.Moreover, the strain preserves its high stability throughout the long-lasting continuous chemostat process.The chemical composition of the biomass was analysed.The mass fractions of lysine and threonine in the biomass protein amino acid profi le were higher than those required by the FAO/WHO for fodder yeast.

Table 1 .
Parameters of erythritol production by Yarrowia lipolytica MK1 in a chemostat culture in media with diff erent composition a average volumetric erythritol production rate under steady state, b average erythritol production yield under steady state, c amino acid composition is given in Table2

Table 2 .
Mass fractions of amino acids in Yarrowia lipolytica MK1 biomass derived from a chemostat culture with PM2 and C:N ratio of 80:1