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Optimization of Medium Composition for Erythritol Production from Glycerol by Yarrowia lipolytica Using Response Surface Methodology a
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Anita Rywińska , Marta Marcinkiewicz , Edmund Cibis & Waldemar Rymowicz
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Department of Biotechnology and Food Microbiology , Wrocław University of Environmental and Life Sciences , Wrocław , Poland b
Department of Bioprocess Engineering , Wrocław University of Economics , Wrocław , Poland Published online: 11 Nov 2014.
To cite this article: Anita Rywińska , Marta Marcinkiewicz , Edmund Cibis & Waldemar Rymowicz (2015) Optimization of Medium Composition for Erythritol Production from Glycerol by Yarrowia lipolytica Using Response Surface Methodology, Preparative Biochemistry and Biotechnology, 45:6, 515-529, DOI: 10.1080/10826068.2014.940966 To link to this article: http://dx.doi.org/10.1080/10826068.2014.940966
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Preparative Biochemistry & Biotechnology, 45:515–529, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2014.940966
Optimization of Medium Composition for Erythritol Production from Glycerol by Yarrowia lipolytica Using Response Surface Methodology
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Anita Rywin´ska and Marta Marcinkiewicz Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
Edmund Cibis Department of Bioprocess Engineering, Wrocław University of Economics, Wrocław, Poland
Waldemar Rymowicz Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland Several factors affecting erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 strain were examined in batch fermentations. Ammonium sulfate, monopotassium phosphate, and sodium chloride were identified as critical medium components that determine the ratio of polyols produced. The central composite rotatable experimental design was used to optimize medium composition for erythritol production. The concentrations of ammonium sulfate, monopotassium phosphate, and sodium chloride in the optimized medium were 2.25, 0.22, and 26.4 g L1, respectively. The C:N ratio was found as 81:1. In the optimized medium with 100 g L1 of glycerol the Wratislavia K1 strain produced 46.9 g L1 of erythritol, which corresponded to a 0.47 g g1 yield and a productivity of 0.85 g L1 hr1. In the fed-batch mode and medium with the total concentration of glycerol at 300 g L1 and C:N ratio at 81:1, 132 g L1 of erythritol was produced with 0.44 g g1 yield and a productivity of 1.01 g L1 hr1. Keywords erythritol, glycerol, optimization, response surface methodology model, Yarrowia lipolytica
INTRODUCTION Erythritol, a four-carbon sugar alcohol, occurs widely in nature, especially in several seaweeds, mushrooms, algae, fruits, and fermented foods. Animal toxicological studies and clinical surveys have consistently demonstrated the safety of erythritol, even when consumed in high amounts on Address correspondence to Anita Rywin´ska, Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Chełmon´skiego Str. 37=41, 51-630 Wrocław, Poland. E-mail: Anita. [email protected]
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a daily basis.[1] More than 90% of ingested erythritol is not metabolized by the human body and is excreted intact with urine without changing blood glucose nor insulin levels. Therefore, erythritol could be used in special foods for individuals suffering from diabetes and obesity.[2] As an all-natural bulk sweetener it has unique sensorial and functional properties: Its sweetness level is 60–80% that of sucrose, and it is noncaloric (1.26 kJ g1), noncariogenic, nonglycemic, and does not have an aftertaste. This sugar alcohol has positive enthalpy of solubilization (23.3 kJ mol1), thus providing a strong cooling effect in ingestion.[3] Erythritol is used as a food ingredient, as a sugar replacer, and as an intermediate for the production of pharmaceuticals and industrial chemicals. This sugar alcohol can be synthesized from starch by a high-temperature chemical reaction in the presence of a nickel catalyst.[1] This process has not been industrialized because of its low efficiency. Erythritol can also be produced microbiologically using osmophilic yeasts like Pichia sp.,[4] Candida magnolia,[5] Torula sp.,[6,7] Trigonopsis variabilis,[8] Moniliella tomentosa var. pollinis,[9,10] Trichosporon sp.,[11] fungus Aureobasidium sp.,[12] Pseudozyma tsukubaensis,[13] and some lactic acid bacteria under anaerobic conditions.[14] It has as well been produced commercially using a mutant of Trichosporonoides megachiliensis SN-G42 cultivated on glucose media.[12] Glucose from chemically and enzymatically hydrolyzed corn or wheat starches is used as major carbon source for erythritol production on the industrial scale. A high initial concentration of glucose and the presence of salts, such as sodium chloride or potassium chloride, in the production medium facilitates erythritol production by osmophilic microorganisms. Generally, an increased concentration of sugar and osmotic pressure enhances erythritol production rate and yield in the batch processes.[6,8] The enzymatic pathway involved in the formation of erythritol in osmophilic yeasts and the regulation of erythritol production from glucose has been reported by many investigators.[1,15–17] These results suggest that erythritol may be produced mainly through the pentose phosphate pathway, with erythrose reductase[15,16] and transketolase[17] being the key enzymes for high erythritol production in osmophilic yeast cells. Erythritol biosynthesis in synthetic media containing glucose or fructose has been extensively studied in various aspects.[1] Effects of vitamins and related compounds such as nitrogen and phosphorus sources, the presence of inositol, phytic acid (myoinositol hexaphosphate),[18] and cuprum and manganese ions,[19] as well as the presence of fumarate[15] and 1,8-dihydroxynaphtalene-melanin,[16] on erythritol production have been studied also. Only a few reports showed the production of this polyol by Yarrowia lipolytica yeast on media containing glycerol as carbon and energy sources.[20–22] Y. lipolytica is one of the most extensively studied ‘‘nonconventional’’ yeasts. According to Holzschu et al.,[23] this yeast is considered nonpathogenic, and several processes based on Y. lipolytica were classified as generally regarded as safe (GRAS) by the Food and Drug Administration (FDA). Y. lipolytica yeasts produce relatively large quantities of organic acids such as citric acid, pyruvic acid, and aketoglutaric acid.[24] Currently, Y. lipolytica is used as a model for the study of protein secretion, degradation of hydrophobic substrates, and several new fields of application.[24–26] The ability of this yeast to produce a high amount of erythritol from glycerol has not been thoroughly studied so far. In our recent preliminary study we have found that Y. lipolytica yeasts are suitable to produce high amounts of erythritol and citric acid in media containing glycerol at pH 5.5, deemed optimal for citric acid biosynthesis by Y. lipolytica.[21] At low pH, 3.0, and under the conditions of carbon excess and nitrogen deficiency in the cultivation medium, the Wratislavia K1 strain of Y. lipolytica produced a high amount of erythritol without citric acid.[21,22] The effect of culture
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medium constituents on erythritol production from glycerol by Y. lipolytica yeast has not been studied yet. Among different statistical methods, the response surface methodology model has been successfully used for media optimization.[27] Based on the preceding information, the overall goal of this study was to improve the production of erythritol on glycerol media with Y. lipolytica Wratislavia K1 strain. The specific objectives of this research were (i) to investigate the effect of nitrogen and phosphorus sources and sodium chloride concentration on the efficient production of erythritol and (ii) to design an optimal medium composition using response surface methodology.
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MATERIAL AND METHODS Materials All medium components were laboratory grade or better. Pure glycerol (99.5% wt wt1), yeast extract, bacto-peptone, malt extract, and other chemicals were purchased from Sigma-Aldrich. Unpurified crude glycerol was derived from methyl ester production and contained 76% wt wt1 of glycerol and 7.3% wt wt1 of NaCl. Distilled water was used for the preparation of culture media. Microorganism Yarrowia lipolytica Wratislavia K1 strain was employed for erythritol production with glycerol as a source of carbon. The strain was originally obtained from the yeast culture collection belonging to the Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences, in Poland. The yeast strain was maintained at 4 C on agar slants. Media and Culture Conditions The medium for agar slants preparation contained: 10 g L1 of glucose, 3 g L1 of yeast extract, 3 g L1 of malt extract, 5 g L1 of Bacto Peptone, 20 g L1 of agar. The growth medium for seed culture preparation contained: 50 g L1 of glycerol, 3 g L1 of yeast extract, 3 g L1 of malt extract and 5 g L1 of bacto-peptone. A seed culture was carried out in a 300-mL flask containing 50 mL of growth medium on an Elpan (Poland) rotary shaker at 30 C for 3 days. An inoculum of 100 mL was introduced into a bioreactor containing 1.3 L of the production medium. Batch and fed-batch cultivations were carried out in 5-L stirred-tank reactors (Biostat B Plus, Sartorius, Germany) at 30 C with a working volume of 1.4 and 2 L, respectively. The aeration rate was fixed at 0.7 L min1. The stirrer speed was adjusted to 800 rpm and the dissolved oxygen concentration was maintained at 35 5% saturation. pH was maintained automatically at 3.0 by the addition of 10% (w v1) of NaOH solution. All cultures were carried out in three replications. All the cultures were cultivated until the complete consumption of glycerol. Effect of Nitrogen Source and Phosphorus and Sodium Chloride Concentration The effect of nitrogen sources and phosphorus and sodium chloride concentration on erythritol production was evaluated in submerged cultivations in a stirred tank reactor as described earlier. In the
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production medium various nitrogen sources—NH4Cl, (NH4)2SO4, (NH4)2HPO4, NH4NO3, urea, yeast extract, and corn steep liquor—were applied. Nitrogen content of each nitrogen source was equal to that of 2.44 g L1 ammonium sulfate (nitrogen concentration 0.52 g L1). Monopotassium phosphate (potassium dihydrogen phosphate) as a source of phosphorus, from 0 to 2 g L1, and varying concentrations of sodium chloride from 0 to 90 g L1 were used.
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Design of Experiment The range of erythritol concentration was related to three factors: ammonium sulfate concentration (X1), potassium dihydrogen phosphate concentration (X2), and sodium chloride concentration (X3). The conditions allowing the maximal extent of erythritol concentration were determined by the response surface method and the rotatable design of the experiment, that is, the design of a spherical distribution of variance. An attempt was made to use such an experimental design that is based on a full factorial design of second order (where the experimental data are approximated by a second-degree polynomial). Then, if the rotatability requirement is to be fulfilled, the star arm size at three independent variables should be 1.682. Choosing an appropriate number of replicates at the center point of the rotatable design, we obtain an almost constant variance of the approximated values of the second-degree polynomial for the points that are distant from the design center by no more than 1. For the design being used in this study, the required number of replicates is 6. In total, 20 experiments were performed; more specifically, 8 experiments were according to the two-level full factorial design of type 23, 6 experiments at the star points (1.682, 0, 0), (0, 1.682, 0) and (0, 0, 1.682), and 6 experiments at the center point of the design. The values (both coded and real) of the variables X1, X2, and X3 used in the experiments, as well as the experimental values of erythritol concentration, are summarized in Table 1. Batch shake-flask cultivations were carried out in a 300-mL Erlenmayer flask with 30 mL of the production medium at 30 C for 7 days, using a rotary shaker (G10 New Brunswick) with agitation of 160 rpm. pH was maintained at 3.0 by the addition of 10% (w v1) of NaOH solution. One milliliter of seed culture (see subsection on Media and Culture Conditions) was transferred into the each production medium. Analytical Methods To determine concentrations of dry biomass, substrate, and products, 10 mL of the fermentation broth (2 times a day) was taken from the bioreactor. After centrifugation (at 5000 rev min1 for 5 min), cells were harvested by filtration on 0.45-mm pore-size membranes, washed twice with distilled water, and dried to a constant weight at 105 C. The supernatant was determined for contents of erythritol, mannitol, glycerol, and citric acid. Concentrations of glycerol, erythritol, mannitol, and citric acid were determined by highperformance liquid chromatography (HPLC; UltiMate 3000, Dionex, USA) on a carbohydrate column (HyperREZ XP, Thermo Scientific, USA) coupled to an ultraviolet (UV; k ¼ 210 nm) and refractive index (RI) detector. The column was eluted with 25 mM of trifluoroacetic acid at 65 C at a flow rate of 0.6 mL min1. Polyols and citric acid were identified and quantified with reference to authentic standards. The treatment diluted the sample by a factor of 10. The
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TABLE 1 Central Composite Rotatable Design for the Optimization of Three Nutrients (Each of Five Levels) for Erythritol Concentration Coded values and real values
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Experiment number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1
(NH4)2SO4 (g L ), X1 1 1 1 1 1 1 1 1 1.682 þ1.682 0 0 0 0 0 0 0 0 0 0
(1.61) (3.39) (1.61) (3.39) (1.61) (3.39) (1.61) (3.39) (1.0) (4.0) (2.5) (2.5) (2.5) (2.5) (2.5) (2.5) (2.5) (2.5) (2.5) (2.5)
1
KH2PO4 (g L ), X2 1 1 1 1 1 1 1 1 0 0 1.682 1.682 0 0 0 0 0 0 0 0
(0.20) (0.20) (0.80) (0.80) (0.20) (0.20) (0.80) (0.80) (0.5) (0.5) (0) (1.0) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5)
Experimental values 1
NaCl (g L ), X3 1 1 1 1 1 1 1 1 0 0 0 0 1.682 1.682 0 0 0 0 0 0
(16.2) (16.2) (16.2) (16.2) (63.8) (63.8) (63.8) (63.8) (40) (40) (40) (40) (0) (80) (40) (40) (40) (40) (40) (40)
erythritol (g L1), Y 29.1 25.9 30.9 39.7 17.9 36.1 16.0 17.7 18.6 18.9 25.4 17.8 10.4 13.3 38.9 22.4 22.4 23.2 23.0 32.0
sample was also filtered before HPLC analysis. The retention times for mannitol, erythritol, glycerol, and citric acid were 9.8, 11.3, 12.9, and 7.9 min, respectively. The osmotic pressure was measured by an automatic osmometer (MARCELL, Poland). Calculation of Fermentation Parameters The yield of erythritol production from glycerol, expressed in grams per gram, was calculated from erythritol concentration divided by total amount of glycerol consumed. The volumetric erythritol production rate, expressed in grams per liter per hour, was calculated as erythritol concentration divided by duration of the fermentation process. RESULTS AND DISCUSSION Effect of Nitrogen Sources Inorganic and organic nitrogen sources served to stimulate the cell growth and erythritol production.[28] The ability of the selected Wratislavia K1 strain of Y. lipolytica to produce erythritol was examined in the medium containing 100 g L1 of glycerol and various nitrogen sources: inorganic nitrogen sources such as ammonium chloride, ammonium sulfate, ammonium
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TABLE 2 Effect of Various Nitrogen Sources on the Cell Growth and Erythritol Production From Glycerol by Y. lipolytica Wratislavia K1 Strain
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Nitrogen source
Parameter
NH4Cl
(NH4)2SO4
(NH4)2HPO4
NH4NO3
Urea
Yeast extract
Corn steep liquor
Time of cultivation (hr) Dry cell weight (g L1) Erythritol (g L1) Mannitol (g L1) Citric acid (g L1) Erythritol yield (g L1) Erythritol productivity (g L1 hr1)
75 13 0.5 25 1.5 21 1.1 1 0.2 0.25 0.33
71 13 0.8 26 1.8 24 1.3 1 0.3 0.26 0.37
91 12 0.4 13 1.0 18 1.3 1 0.2 0.13 0.14
91 13 0.7 18 1.3 21 1.5 nd 0.17 0.20
99 13 0.6 14 1.2 23 1.7 2 0.4 0.14 0.14
70 15 0.5 25 0.9 13 1.5 3 0.5 0.24 0.36
93 13 0.5 20 0.8 25 1.4 2 0.3 0.20 0.22
Note. Results are means SD from triple experiments. Culture conditions: 100 g L1 of pure glycerol; 0.25 g L1 of KH2PO4; amount of nitrogen, 0.52 g L1, corresponding to the addition of appropriate nitrogen source; nd, not detected.
hydrogen phosphate, and ammonium nitrate, and organic nitrogen compounds such as corn steep liquor, urea, and yeast extract. The total nitrogen concentration in each medium was similar, about 0.52 g L1. Relevant data are summarized in Table 2. As can be seen in Table 2, ammonium chloride, ammonium sulfate, and yeast extract were the best nitrogen sources for erythritol production by Wratislavia K1 strain. In the presence of these alone, the production of erythritol reached 25 and 26 g L1, respectively. A lower concentration of erythritol was obtained in the media containing urea and ammonium hydrogen phosphate, that is, 14 and 13 g L1, respectively. The source of nitrogen was also found to affect mannitol production by this strain. Its concentration was in the range from 13 to 25 g L1. The highest erythritol yield (0.26 g g1) and productivity (0.37 g L1 hr1) were achieved with ammonium sulfate. Inexpensive inorganic nitrogen sources were the most suitable for erythritol production by Wratislavia K1 strain. This is a good feature of this yeast strain, because organic nitrogen sources (yeast extract and casein hydrolysate) commonly used in glucose fermentation are very expensive and therefore cannot be necessarily advantageous to industrial production. Various nitrogen sources such as inorganic salts (ammonium chloride and ammonium sulfate) and organic salts (yeast extract, corn step liquor, urea, soybean flour, potato protein, peptone, malt extract, beef extract, tryptone, and soytone) were tested for maximizing the production of erythritol and mannitol by different osmophilic yeasts.[13,28] Particularly, yeast extract and corn steep powder gave the most effective production of erythritol and mannitol by osmophilic yeasts.[29–31] In the medium containing 10 g L1 of yeast extract, C. zeylanoides produced 43.2 g L1 of erythritol, which was about twofold higher than in the culture with ammonium chloride.[30] On the other hand, the addition of a yeast extract or a meat extract resulted in an increased production of another polyol, that is, mannitol, concomitant with erythritol. According to Lee et al.,[19] the medium for erythritol production by Torula sp. contained up to 20 g L1 of the yeast extract. Sawada et al. [17] reported that thiamine was an effective compound for the enhancement of erythritol production by T. megachiliensis SN-G42 strain. Supplementation of the culture medium with 4 mg L1 of thiamine resulted in erythritol concentration increase from 87.9 (without thiamine) to 205.1 g L1. It
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should be noted that the yeast extract contains a high amount of this vitamin. In other investigation, corn steep powder was proved to be the best nitrogen source for erythritol production by P. tsukubaensis KCCM 10356. This strain produced 149 g L1 of erythritol, which corresponded to 49.7% yield and 1.65 g L1 hr1 productivity.[13] Additionally, when corn steep liquor was used as the main nitrogen source, erythritol produced may be brown colored or contain a large quantity of salt. For these reasons the purification process is difficult, which is disadvantageous to economical production. Moreover, urea as a source of organic nitrogen is not admitted as a food additive and therefore it cannot be used for the production of erythritol to be used in foodstuffs. In further investigations, ammonium sulfate will be used as a nitrogen source for Wratislavia K1 strain.
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Effect of Ammonium Sulfate For in-depth analysis of the effect of ammonium sulfate concentration on erythritol production, experiments were conducted in a medium culture containing various amounts of ammonium sulfate (from 0.62 to 9.8 g L1). The effect of ammonium sulfate concentration on erythritol production by Wratislavia K1 strain is shown in Table 3. An increase in ammonium sulfate concentration up to 9.8 g L1 increased biomass level from 8 to 20 g L1. The highest concentration of erythritol, 26 g L1, was achieved in the medium containing 2.44 g L1 of ammonium sulfate. In this medium, the C:N ratio was at 75:1. The best yield of erythritol production, 0.25–0.26 g g1, was achieved when the C:N ratio ranged from 150:1 to 75:1, whereas the highest erythritol productivity, 0.37 and 0.33 g L1 hr1, was obtained when the C:N ratio was 75:1 and 100:1, respectively. According to Burscha¨pers et al.,[10] nitrogen limitation was the prerequisite for erythritol formation by M. tomentosa var. pollinis. On the other hand, nitrogen limitation caused also considerable foam formation, which was very difficult to suppress. Therefore, careful control over the concentration of a nitrogen source is necessary to obtain high erythritol productivity without foam formation. This can be best realized by a fed-batch operation. Nitrogen deficiency not only induces erythritol biosynthesis but also stimulates the production of citric acid by yeasts. TABLE 3 Effect of Ammonium Sulfate on the Cell Growth and Erythritol Production From Glycerol by Y. lipolytica Wratislavia K1 Strain (NH4)2SO4 (g L1) 0.62
1.22
1.86
2.44
4.88
9.8
C:N ratio Parameter 1
Dry cell weight (g L ) Erythritol (g L1) Mannitol (g L1) Citric acid (g L1) Erythritol yield (g g1) Erythritol productivity (g L1 hr1)
300:1
150:1
100:1
75:1
38:1
19:1
8 1.1 21 1.6 18 1.4 6 0.8 0.22 0.26
10 1.4 23 2.6 19 1.3 3 0.6 0.25 0.31
12 1.6 25 1.8 20 1.6 2 0.4 0.25 0.33
13 0.8 26 1.8 24 1.3 1 0.3 0.26 0.37
18 0.6 19 1.4 13 0.6 1 0.2 0.2 0.32
20 1.7 18 1.5 10 0.9 1 0.2 0.19 0.26
Note. Results are means SD from triple experiments. Culture conditions: 100 g L1 of pure glycerol; 0.25 g L1 of KH2PO4.
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Effect of Monopotassium Phosphate Phosphate is one of the most important medium components; it additionally affects cell physiology and metabolism in the culture of microorganisms. It was shown that an excessive amount of phosphate supplied in a glucose or a glycerol medium had a negative effect on polyols production.[7] Culture media require phosphate in the range of 0.3–300 mM for the growth of microorganisms, and a phosphate concentration much lower than this may inhibit the production of many metabolites. Therefore, the effect of phosphate was examined during glycerol fermentation with the Wratislavia K1 strain. In order to select the optimal amount of phosphate for erythritol production, monopotassium phosphate was tested in the range from 0 to 2 g L1. As summarized in Table 4, the concentration of a phosphate source had no effect on cell growth as it resulted in similar amounts of biomass produced (13–14.5 g L1). However, it played a significant role in the production of erythritol and mannitol. The maximal concentration of erythritol (26 g L1), corresponding to 0.26 g g1 yield and 0.37 g L1 hr1 productivity, was achieved in the medium containing 0.25 g L1 of potassium dihydrogen phosphate. According to other investigators, phytic acid (monoinositol hexaphosphate) was the best phosphate source for erythritol biosynthesis by C. magnoliae[18] and Torula sp.[7] In our investigation we have found that the higher phosphate supplementation (0.5–2 g L1) stimulated mannitol production. As a result, 24–27 g L1 of mannitol was produced as shown in Table 4. According to Hattori and Suzuki,[29] the polyol production was apparently differentiated by the level of phosphate concentration in the culture medium. Mannitol production occurred at a high concentration of phosphate and ceased when its concentration was reduced below 0.04 m g mL1 as monopotassium phosphate, while erythritol production was initiated at this level of phosphate concentration.[29] The production of mannitol from glycerol by resting cells of C. magnoliae under aerobic conditions was investigated by Khan et al.,[31] who observed that the addition of a high amount of potassium dihydrogen phosphate (5 g L1) decreased mannitol concentration from 51 to 3 g L1 and that a low amount of erythritol was also produced, which was otherwise not observed. Effect of Sodium Chloride Erythritol-producing strains can be characterized as osmophilic yeasts. It has been reported that sugar- and salt-tolerant yeasts produced and intracellularly accumulated polyols TABLE 4 Effect of KH2PO4 on the Cell Growth and Erythritol Production From Glycerol by Y. lipolytica Wratislavia K1 KH2PO4 (g L1) Parameter 1
Dry cell weight (g L ) Erythritol (g L1) Mannitol (g L1) Citric acid (g L1) Erythritol yield (g g1) Erythritol productivity (g L1 hr1)
0
0.05
0.25
0.50
1
2
13 1.2 19 1.5 14.5 1.5 3 1.6 0.19 0.21
14 1.4 23 1.5 16 1.6 3 1.2 0.23 0.32
13 0.8 26 1.8 24 1.3 1 0.3 0.26 0.37
14 1.5 17 0.6 26 2 1 0.4 0.17 0.18
14.5 1.5 14 0.7 24 1.5 1 0.2 0.16 0.15
13.5 1.3 16 1.1 27 2.1 1 0.3 0.14 0.16
Note. Results are means SD from triple experiments. Culture conditions: 100 g L1 of pure glycerol; 2.44 g L1 of (NH4)2SO4.
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TABLE 5 Effect of NaCl Concentration on the Cell Growth and Erythritol Production From Glycerol by Y. lipolytica Wratislavia K1 NaCl (g L1)
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Parameter Osmotic pressure (mOsm kg1) Dry cell weight (g L1) Erythritol (g L1) Mannitol (g L1) Citric acid (g L1) Erythritol yield (g g1) Erythritol productivity (g L1 hr1)
0
7.5
15
30
60
90
1500 13 0.8 26 1.5 24 1.4 1 0.3 0.26 0.37
1600 14 0.7 29 1.6 23 1.5 1 0.5 0.29 0.41
1900 13 0.8 32 2.0 20 1.0 3 0.8 0.32 0.47
2400 14 0.5 37 2.1 2 1.1 2 0.6 0.38 0.6
3700 11 0.6 37 2.4 1 0.6 2 0.8 0.37 0.57
3800 4 0.4 36 1.5 2 0.5 2 1.0 0.34 0.12
Note. Results are means SD from triple experiments. Culture conditions: 100 g L1 of pure glycerol; 0.25 g L1of KH2PO4; 2.44 g L1 of (NH4)2SO4.
(erythritol, mannitol, glycerol and arabitol) in the presence of high concentrations of sugars or when exposed to a high concentration of sodium or potassium chloride.[32] To evaluate the effect of sodium chloride on erythritol production by the Wratislavia K1 strain, its initial concentration varied from 0 to 90 g L1(Table 5). Sodium chloride supplementation increased the osmotic pressure from 1500 (without salt) to 3800 mOsm kg1 (90 g L1 of sodium chloride). The effect of sodium chloride addition on cell growth and production of erythritol and mannitol was dependent on its concentration, as shown in Table 5. The maximal production of erythritol (37 g L1), corresponding to 0.38 g g1 yield and 0.55 g L1 hr1 productivity, occurred when the fermentation medium contained 30 g L1 of sodium chloride. A similar effect was shown by Hattori and Suzuki.[30] In their investigations, C. zeylanoides produced a higher amount of erythritol from n-alkane in the medium containing sodium chloride or potassium chloride. In our study we have found that an increase in sodium chloride concentration up to 90 g L1 decreased the amount of mannitol and biomass from 24 to 1 g L1 and from 14 to 4 g L1, respectively. On the other hand, Hajny et al.[28] demonstrated that sodium chloride had no effect on erythritol fermentation from glucose by yeast belonging to the genus Torula, whereas potassium chloride addition to the medium stimulated the formation of glycerol as a by-product. Finally, Lin et al.[9] reported that yeast strains of Moniliella produced less erythritol in the presence of sodium chloride or potassium chloride than in the media without these salts. The differences in these two studies could be due to different yeast strain and substrate in both fermentations.
Optimization of Medium Composition Ammonium sulfate, potassium dihydrogen phosphate, and sodium chloride were identified as critical medium components that determine erythritol, mannitol, and citric acid production with the Wratislavia K1 strain on media containing glycerol. Therefore, the next step of this study involved the optimization of culture media using response surface methodology.
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As already mentioned in the Design of Experiment section, attempts were made to approximate the experimental data for the extent of erythritol production by a second-order polynomial. The polynomial takes the form of the following relation: Y¼
3 X
aij Xi Xj þ
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i;j¼1 ij
3 X
ai Xi þ a0
ð1Þ
i¼1
where a0, ai, and aij are parameters of the polynomial, Y is the extent of erythritol production, and X1, X2, and X3 are as indicated in Table 1. It is essential to note, however, that during approximation, values of these variables are coded. The estimated parameter values of the polynomial (1) that approximate the values of the extent of erythritol concentration, as well as the determination coefficient obtained, are compiled in Table 6. Six replications at the center point of the design made it possible to assess the adequacy of the model (polynomial (1)) in terms of test statistics with Snedecor’s F-distribution of (5, 5) degrees of freedom. Table 6 also includes the value of the statistics and the value of the corresponding limit significance level (a). The limit value of a indicates that the polynomial (1) proposed is adequate if the assumed significance-level values are lower than 0.3622. The values in Table 6 were calculated using Microsoft Office Excel 2003 software. Because of the spherical distribution of information in the rotatable design, the values of the variables X1, X2, and X3 at which these functions reach their maxima were sought in the set defined by the inequality of Eq. (2): X21 þ X22 þ X23 3
ð2Þ
that is, on the inside and on the border of a sphere with a center lying in the design center, and with a radius that equals the distance from the center point to one of the points of the 23 full-factorial design. Using the mathematical conditions for the maximum of a multivariable function it was confirmed that the optimal point lay inside the set defined by the inequality TABLE 6 Estimated Parameters of the Polynomial Approximating the Extent of Erythritol Concentration, and the Statistical Parameters of Estimation Parameter a11 a22 a33 a12 a13 a23 a1 a2 a3 a0 Coefficient of determination Value of F-Snedecor statistics p Value for F-Snedecor statistics
Value 3.3155 2.3081 5.7544 0.6750 1.4750 1.5000 0.4170 3.2788 4.7680 27.0451 0.6612 1.3938 .3622
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TABLE 7 Optimal Conditions for Erythritol Production
Type of value
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Coded Real
(NH4)2SO4 (g L1),
KH2PO4 (g L1),
NaCl (g L1),
Erythritol (g L1),
X1
X2
X3
Y
0.286 2.245
0.938 0.221
0.573 26.37
30.26
of Eq. (2). Hence, it was possible to evaluate analytically the optimal values of X1, X2, and X3 and the corresponding maximal Y. These four values are shown in Table 7. Figure 1 (made by using the MATLAB software, version 4.2c.1) relates the extent of erythritol concentration to each pair of independent variables when the third variable takes the optimal value.
FIGURE 1 Effect of medium components on erythritol production (in g L1): (A) influence of (NH4)2SO4 and KH2PO4 concentration at the optimal value of NaCl concentration; (B) influence of (NH4)2SO4 and NaCl at the optimal value of KH2PO4 concentration; (C) influence of KH2PO4 and NaCl at the optimal value of (NH4)2SO4 concentration. Dotted lines indicate the contours of the range of independent variables (X21 þ X22 þ X23 3) for encoded values of X1, X2, and X3.
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TABLE 8 Results of Erythritol Production From Glycerol in Optimal Medium Substrate Parameter
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Dry cell weight (g L1) Erythritol (g L1) Mannitol (g L1) Citric acid (g L1) Erythritol yield (g g1) Erythritol productivity (g L1 hr1)
Pure glycerol
Crude glycerol
15.5 46.9 0.8 0.4 0.47 0.82
18.1 39.3 2.5 0 0.40 0.66
Thus, the optimized medium containing 2.25 g L1 of ammonium sulfate, 0.22 g L1 of monopotassium phosphate and 26.4 g L1 of sodium chloride was applied in bioreactor batch cultures with 100 g L1 of pure and crude glycerol. In such a medium the C:N ratio was found at 81:1. As it can be seen in Table 8, 46.9 and 39.3 g L1 of erythritol was produced with the yield of 0.47 and 0.40 g g1 from pure and crude substrate, respectively. In the optimized medium the amount of by-products does not exceed 2.5 g L1. These values of erythritol concentration were significantly higher in comparison to the result achieved via mathematical modeling. This phenomenon can be explained by the oxygenation conditions, because the experiments for the purpose of mathematical modeling were carried out in a shake flask, where oxygenation is lower than in a bioreactor. Due to the worse oxygenation conditions, during the shake-flask culture not the whole amount of glycerol was utilized (data not shown) and therefore the calculated optimal erythritol concentration was lower than that obtained in the bioreactor culture conducted in the optimal medium.
Production of Erythritol in Fed-Batch Process It is known that effective erythritol production is conducted in batch and fed-batch cultures with osmophilic yeast from glucose in the concentration of 200–400 g L1.[7,13,19,33–35] To date, little is known about erythritol production by Y. lipolytica from glycerol. Recently, a novel two-stage osmotic pressure control fed-batch strategy was developed for erythritol production from this substrate by Y. lipolytica and a high concentration of erythritol was obtained, 194.3 g L1, with a productivity of 0.95 g L1 hr1. However, as a by-product mannitol was formed at a high level, 36.8 g L1.[36] In the next step of the presented study, the medium with total glycerol of 300 g L 1 and C:N ratio of 81:1 was used in the fed-batch culture. The fed-batch fermentation profile of erythritol production from pure glycerol by Wratislavia K1 was shown in Figure 2. The process was launched as batch culture in medium with glycerol concentration of 100 g L1 (at initial volume of bioreactor of about 1.8 L); pulsed additions of glycerol (about 100 g L1) were made after 2 and 4 days. The Wratislavia K1 strain produced 132 g L1 of erythritol with a productivity of 1.0 g L1h1. The concentration of by-products, mannitol and citric acid, did not exceed 5 g L1.
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FIGURE 2 Time course of biomass (.), glycerol (~), erythritol (&), mannitol (&), and citric acid () during fed-batch fermentation by Y. lipolytica Wratislavia K1 strain. The arrow indicates periodic feeding of glycerol to total concentration of 300 g L1.
CONCLUSIONS Currently, most crude glycerol is produced from a biodiesel production process and its price is determined by the demand and production of biodiesel. The price of crude glycerol varied from $40 to $330=ton over the last decade.[37] Glycerol could be utilized as a carbon source in microbial bioconversions for the production of a range of chemicals to be used as end products in various industries. One of them is erythritol, which could be produced by Y. lipolytica yeast. The price of erythritol obtained from glucose by Moniliella pollinis ranged from $3700 to $3900=ton over recent years. In our opinion, the erythritol price obtained from crude glycerol will be correlated with the glycerol global market price and the total cost of erythritol production by microbial method, including cost of fermentation, purification, and crystallization processes. Therefore, the price of crystalline erythritol (more than 99.5% pure) obtained from crude glycerol by a selected strain of Y. lipolytica may in the future be attractive as a new alternative technology for its cheap production at commercial scale. Based on the data presented in this study, it appears that the production of erythritol by Y. lipolytica Wratislavia K1 from glycerol could potentially compete with the more traditional processes run with sugars and other yeasts. The results obtained suggest that the concentrations of glycerol, ammonium sulfate, monopotassium phosphate, and sodium chloride affect the production of erythritol, keeping the by-products at minimal possible levels in this fermentation process. Further optimization of the culture conditions and cultivation systems will contribute to improving the production of erythritol using biological processes. FUNDING The authors are thankful to the Polish Ministry of Science and Higher Education and the European Union for financial support under research project PO IG 01.01.02-00-074=09.
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25. Lazar, Z.; Walczak, E.; Robak, M. Simultaneous Production of Citric Acid and Invertase by Yarrowia lipolytica SUCþ Transformants. Bioresource Technol. 2011, 102(13), 6982–6989. 26. Madzak, C.; Gaillardin, C.; Beckerich, J.M. Heterologous Protein Expression and Secretion in the Non-Conventional Yeast Yarrowia lipolytica: A Review. J. Biotechnol. 2004, 109, 63–81. 27. Laxman, S.; Ramchandra, V.G.; Bhalchandra, K.V.; Karthik, N. Strain Improvement and Statistical Media Optimization for Enhanced Erythritol Production With Minimal By-Products From Candida magnoliae Mutant R23. Biochem. Eng. J. 2011, 55, 92–100. 28. Hajny, G.J.; Smith, J.H.; Garver, J.C. Erythritol Production by Yeast Like Fungus. Appl. Microbiol. 1964, 12, 240–246. 29. Hattori, K.; Suzuki, T. Large Scale Production of Erythritol and Its Conversion to D-Mannitol Production by n-Alkane Grown Candida zeylanoides. Agric. Biol. Chem. 1974, 38, 1203–1208. 30. Hattori, K.; Suzuki, T. Production of Erythritol by n-Alkane-Grown Yeasts. Agric. Biol. Chem. 1974, 38, 581–586. 31. Khan, A.; Bhide, A.; Gadre, R. Mannitol Production From Glycerol by Resting Cells of Candida magnolia. Bioresource Technol. 2009, 100(20), 4911–4913. 32. Park, Y.C.; Lee, D.Y.; Lee, D.H.; Kim, H.J.; Ryu, Y.W.; Seo, J.H. Proteomics and Physiology of Erythritol-Producing Strains. J. Chromatogr. B 2005, 815, 251–260. 33. Lin, S.J.; Wen, C.Y.; Liau, J.C.; Chu, W.S. Screening and Production of Erythritol by Newly Isolated Osmophilic Yeast-Like Fungi. Process Biochem. 2001, 36, 1249–1258. 34. Ryu, Y.W.; Park, C.Y.; Park, J.B.; Kim, S.Y.; Seo, J.H. Optimization of Erythritol Production by Candida magnoliae in Fed-Batch Culture. J. Ind. Microbiol. Biotechnol. 2000, 25, 100–103. 35. Savergave, L.S.; Gadre, R.V.; Vaidya, B.K.; Narayanan, K. Strain Improvement and Statistical Media Optimization for Enhanced Erythritol Production With Minimal By-Products From Candida magnoliae Mutant R23. Biochem. Eng. J. 2011, 55, 92–100. 36. Yang, L.B.; Zhan, X.B.; Zheng, Z.Y.; Wu, J.R.; Gao, M.J.; Lin, C.C. A Novel Osmotic Pressure Control Fed-Batch Fermentation Strategy for Improvement of Erythritol Production by Yarrowia lipolyica From Glycerol. Bioresource Technol. 2014, 151, 120–127. 37. Ayoub, M.; Abdullah, A.Z. Critical Review on the Current Scenario and Significance of Crude Glycerol Resulting From Biodiesel Industry Towards More Sustainable Renewable Energy Industry. Renew. Sustain. Energy Rev. 2012, 16, 2671–2686.
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
Optimization of Medium Composition for Erythritol Production from Glycerol by Yarrowia lipolytica Using Response Surface Methodology a
a
b
Anita Rywińska , Marta Marcinkiewicz , Edmund Cibis & Waldemar Rymowicz
a
a
Department of Biotechnology and Food Microbiology , Wrocław University of Environmental and Life Sciences , Wrocław , Poland b
Department of Bioprocess Engineering , Wrocław University of Economics , Wrocław , Poland Published online: 11 Nov 2014.
To cite this article: Anita Rywińska , Marta Marcinkiewicz , Edmund Cibis & Waldemar Rymowicz (2015) Optimization of Medium Composition for Erythritol Production from Glycerol by Yarrowia lipolytica Using Response Surface Methodology, Preparative Biochemistry and Biotechnology, 45:6, 515-529, DOI: 10.1080/10826068.2014.940966 To link to this article: http://dx.doi.org/10.1080/10826068.2014.940966
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Preparative Biochemistry & Biotechnology, 45:515–529, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2014.940966
Optimization of Medium Composition for Erythritol Production from Glycerol by Yarrowia lipolytica Using Response Surface Methodology
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Anita Rywin´ska and Marta Marcinkiewicz Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
Edmund Cibis Department of Bioprocess Engineering, Wrocław University of Economics, Wrocław, Poland
Waldemar Rymowicz Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland Several factors affecting erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 strain were examined in batch fermentations. Ammonium sulfate, monopotassium phosphate, and sodium chloride were identified as critical medium components that determine the ratio of polyols produced. The central composite rotatable experimental design was used to optimize medium composition for erythritol production. The concentrations of ammonium sulfate, monopotassium phosphate, and sodium chloride in the optimized medium were 2.25, 0.22, and 26.4 g L1, respectively. The C:N ratio was found as 81:1. In the optimized medium with 100 g L1 of glycerol the Wratislavia K1 strain produced 46.9 g L1 of erythritol, which corresponded to a 0.47 g g1 yield and a productivity of 0.85 g L1 hr1. In the fed-batch mode and medium with the total concentration of glycerol at 300 g L1 and C:N ratio at 81:1, 132 g L1 of erythritol was produced with 0.44 g g1 yield and a productivity of 1.01 g L1 hr1. Keywords erythritol, glycerol, optimization, response surface methodology model, Yarrowia lipolytica
INTRODUCTION Erythritol, a four-carbon sugar alcohol, occurs widely in nature, especially in several seaweeds, mushrooms, algae, fruits, and fermented foods. Animal toxicological studies and clinical surveys have consistently demonstrated the safety of erythritol, even when consumed in high amounts on Address correspondence to Anita Rywin´ska, Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Chełmon´skiego Str. 37=41, 51-630 Wrocław, Poland. E-mail: Anita. [email protected]
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a daily basis.[1] More than 90% of ingested erythritol is not metabolized by the human body and is excreted intact with urine without changing blood glucose nor insulin levels. Therefore, erythritol could be used in special foods for individuals suffering from diabetes and obesity.[2] As an all-natural bulk sweetener it has unique sensorial and functional properties: Its sweetness level is 60–80% that of sucrose, and it is noncaloric (1.26 kJ g1), noncariogenic, nonglycemic, and does not have an aftertaste. This sugar alcohol has positive enthalpy of solubilization (23.3 kJ mol1), thus providing a strong cooling effect in ingestion.[3] Erythritol is used as a food ingredient, as a sugar replacer, and as an intermediate for the production of pharmaceuticals and industrial chemicals. This sugar alcohol can be synthesized from starch by a high-temperature chemical reaction in the presence of a nickel catalyst.[1] This process has not been industrialized because of its low efficiency. Erythritol can also be produced microbiologically using osmophilic yeasts like Pichia sp.,[4] Candida magnolia,[5] Torula sp.,[6,7] Trigonopsis variabilis,[8] Moniliella tomentosa var. pollinis,[9,10] Trichosporon sp.,[11] fungus Aureobasidium sp.,[12] Pseudozyma tsukubaensis,[13] and some lactic acid bacteria under anaerobic conditions.[14] It has as well been produced commercially using a mutant of Trichosporonoides megachiliensis SN-G42 cultivated on glucose media.[12] Glucose from chemically and enzymatically hydrolyzed corn or wheat starches is used as major carbon source for erythritol production on the industrial scale. A high initial concentration of glucose and the presence of salts, such as sodium chloride or potassium chloride, in the production medium facilitates erythritol production by osmophilic microorganisms. Generally, an increased concentration of sugar and osmotic pressure enhances erythritol production rate and yield in the batch processes.[6,8] The enzymatic pathway involved in the formation of erythritol in osmophilic yeasts and the regulation of erythritol production from glucose has been reported by many investigators.[1,15–17] These results suggest that erythritol may be produced mainly through the pentose phosphate pathway, with erythrose reductase[15,16] and transketolase[17] being the key enzymes for high erythritol production in osmophilic yeast cells. Erythritol biosynthesis in synthetic media containing glucose or fructose has been extensively studied in various aspects.[1] Effects of vitamins and related compounds such as nitrogen and phosphorus sources, the presence of inositol, phytic acid (myoinositol hexaphosphate),[18] and cuprum and manganese ions,[19] as well as the presence of fumarate[15] and 1,8-dihydroxynaphtalene-melanin,[16] on erythritol production have been studied also. Only a few reports showed the production of this polyol by Yarrowia lipolytica yeast on media containing glycerol as carbon and energy sources.[20–22] Y. lipolytica is one of the most extensively studied ‘‘nonconventional’’ yeasts. According to Holzschu et al.,[23] this yeast is considered nonpathogenic, and several processes based on Y. lipolytica were classified as generally regarded as safe (GRAS) by the Food and Drug Administration (FDA). Y. lipolytica yeasts produce relatively large quantities of organic acids such as citric acid, pyruvic acid, and aketoglutaric acid.[24] Currently, Y. lipolytica is used as a model for the study of protein secretion, degradation of hydrophobic substrates, and several new fields of application.[24–26] The ability of this yeast to produce a high amount of erythritol from glycerol has not been thoroughly studied so far. In our recent preliminary study we have found that Y. lipolytica yeasts are suitable to produce high amounts of erythritol and citric acid in media containing glycerol at pH 5.5, deemed optimal for citric acid biosynthesis by Y. lipolytica.[21] At low pH, 3.0, and under the conditions of carbon excess and nitrogen deficiency in the cultivation medium, the Wratislavia K1 strain of Y. lipolytica produced a high amount of erythritol without citric acid.[21,22] The effect of culture
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medium constituents on erythritol production from glycerol by Y. lipolytica yeast has not been studied yet. Among different statistical methods, the response surface methodology model has been successfully used for media optimization.[27] Based on the preceding information, the overall goal of this study was to improve the production of erythritol on glycerol media with Y. lipolytica Wratislavia K1 strain. The specific objectives of this research were (i) to investigate the effect of nitrogen and phosphorus sources and sodium chloride concentration on the efficient production of erythritol and (ii) to design an optimal medium composition using response surface methodology.
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MATERIAL AND METHODS Materials All medium components were laboratory grade or better. Pure glycerol (99.5% wt wt1), yeast extract, bacto-peptone, malt extract, and other chemicals were purchased from Sigma-Aldrich. Unpurified crude glycerol was derived from methyl ester production and contained 76% wt wt1 of glycerol and 7.3% wt wt1 of NaCl. Distilled water was used for the preparation of culture media. Microorganism Yarrowia lipolytica Wratislavia K1 strain was employed for erythritol production with glycerol as a source of carbon. The strain was originally obtained from the yeast culture collection belonging to the Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences, in Poland. The yeast strain was maintained at 4 C on agar slants. Media and Culture Conditions The medium for agar slants preparation contained: 10 g L1 of glucose, 3 g L1 of yeast extract, 3 g L1 of malt extract, 5 g L1 of Bacto Peptone, 20 g L1 of agar. The growth medium for seed culture preparation contained: 50 g L1 of glycerol, 3 g L1 of yeast extract, 3 g L1 of malt extract and 5 g L1 of bacto-peptone. A seed culture was carried out in a 300-mL flask containing 50 mL of growth medium on an Elpan (Poland) rotary shaker at 30 C for 3 days. An inoculum of 100 mL was introduced into a bioreactor containing 1.3 L of the production medium. Batch and fed-batch cultivations were carried out in 5-L stirred-tank reactors (Biostat B Plus, Sartorius, Germany) at 30 C with a working volume of 1.4 and 2 L, respectively. The aeration rate was fixed at 0.7 L min1. The stirrer speed was adjusted to 800 rpm and the dissolved oxygen concentration was maintained at 35 5% saturation. pH was maintained automatically at 3.0 by the addition of 10% (w v1) of NaOH solution. All cultures were carried out in three replications. All the cultures were cultivated until the complete consumption of glycerol. Effect of Nitrogen Source and Phosphorus and Sodium Chloride Concentration The effect of nitrogen sources and phosphorus and sodium chloride concentration on erythritol production was evaluated in submerged cultivations in a stirred tank reactor as described earlier. In the
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production medium various nitrogen sources—NH4Cl, (NH4)2SO4, (NH4)2HPO4, NH4NO3, urea, yeast extract, and corn steep liquor—were applied. Nitrogen content of each nitrogen source was equal to that of 2.44 g L1 ammonium sulfate (nitrogen concentration 0.52 g L1). Monopotassium phosphate (potassium dihydrogen phosphate) as a source of phosphorus, from 0 to 2 g L1, and varying concentrations of sodium chloride from 0 to 90 g L1 were used.
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Design of Experiment The range of erythritol concentration was related to three factors: ammonium sulfate concentration (X1), potassium dihydrogen phosphate concentration (X2), and sodium chloride concentration (X3). The conditions allowing the maximal extent of erythritol concentration were determined by the response surface method and the rotatable design of the experiment, that is, the design of a spherical distribution of variance. An attempt was made to use such an experimental design that is based on a full factorial design of second order (where the experimental data are approximated by a second-degree polynomial). Then, if the rotatability requirement is to be fulfilled, the star arm size at three independent variables should be 1.682. Choosing an appropriate number of replicates at the center point of the rotatable design, we obtain an almost constant variance of the approximated values of the second-degree polynomial for the points that are distant from the design center by no more than 1. For the design being used in this study, the required number of replicates is 6. In total, 20 experiments were performed; more specifically, 8 experiments were according to the two-level full factorial design of type 23, 6 experiments at the star points (1.682, 0, 0), (0, 1.682, 0) and (0, 0, 1.682), and 6 experiments at the center point of the design. The values (both coded and real) of the variables X1, X2, and X3 used in the experiments, as well as the experimental values of erythritol concentration, are summarized in Table 1. Batch shake-flask cultivations were carried out in a 300-mL Erlenmayer flask with 30 mL of the production medium at 30 C for 7 days, using a rotary shaker (G10 New Brunswick) with agitation of 160 rpm. pH was maintained at 3.0 by the addition of 10% (w v1) of NaOH solution. One milliliter of seed culture (see subsection on Media and Culture Conditions) was transferred into the each production medium. Analytical Methods To determine concentrations of dry biomass, substrate, and products, 10 mL of the fermentation broth (2 times a day) was taken from the bioreactor. After centrifugation (at 5000 rev min1 for 5 min), cells were harvested by filtration on 0.45-mm pore-size membranes, washed twice with distilled water, and dried to a constant weight at 105 C. The supernatant was determined for contents of erythritol, mannitol, glycerol, and citric acid. Concentrations of glycerol, erythritol, mannitol, and citric acid were determined by highperformance liquid chromatography (HPLC; UltiMate 3000, Dionex, USA) on a carbohydrate column (HyperREZ XP, Thermo Scientific, USA) coupled to an ultraviolet (UV; k ¼ 210 nm) and refractive index (RI) detector. The column was eluted with 25 mM of trifluoroacetic acid at 65 C at a flow rate of 0.6 mL min1. Polyols and citric acid were identified and quantified with reference to authentic standards. The treatment diluted the sample by a factor of 10. The
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TABLE 1 Central Composite Rotatable Design for the Optimization of Three Nutrients (Each of Five Levels) for Erythritol Concentration Coded values and real values
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Experiment number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1
(NH4)2SO4 (g L ), X1 1 1 1 1 1 1 1 1 1.682 þ1.682 0 0 0 0 0 0 0 0 0 0
(1.61) (3.39) (1.61) (3.39) (1.61) (3.39) (1.61) (3.39) (1.0) (4.0) (2.5) (2.5) (2.5) (2.5) (2.5) (2.5) (2.5) (2.5) (2.5) (2.5)
1
KH2PO4 (g L ), X2 1 1 1 1 1 1 1 1 0 0 1.682 1.682 0 0 0 0 0 0 0 0
(0.20) (0.20) (0.80) (0.80) (0.20) (0.20) (0.80) (0.80) (0.5) (0.5) (0) (1.0) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5) (0.5)
Experimental values 1
NaCl (g L ), X3 1 1 1 1 1 1 1 1 0 0 0 0 1.682 1.682 0 0 0 0 0 0
(16.2) (16.2) (16.2) (16.2) (63.8) (63.8) (63.8) (63.8) (40) (40) (40) (40) (0) (80) (40) (40) (40) (40) (40) (40)
erythritol (g L1), Y 29.1 25.9 30.9 39.7 17.9 36.1 16.0 17.7 18.6 18.9 25.4 17.8 10.4 13.3 38.9 22.4 22.4 23.2 23.0 32.0
sample was also filtered before HPLC analysis. The retention times for mannitol, erythritol, glycerol, and citric acid were 9.8, 11.3, 12.9, and 7.9 min, respectively. The osmotic pressure was measured by an automatic osmometer (MARCELL, Poland). Calculation of Fermentation Parameters The yield of erythritol production from glycerol, expressed in grams per gram, was calculated from erythritol concentration divided by total amount of glycerol consumed. The volumetric erythritol production rate, expressed in grams per liter per hour, was calculated as erythritol concentration divided by duration of the fermentation process. RESULTS AND DISCUSSION Effect of Nitrogen Sources Inorganic and organic nitrogen sources served to stimulate the cell growth and erythritol production.[28] The ability of the selected Wratislavia K1 strain of Y. lipolytica to produce erythritol was examined in the medium containing 100 g L1 of glycerol and various nitrogen sources: inorganic nitrogen sources such as ammonium chloride, ammonium sulfate, ammonium
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TABLE 2 Effect of Various Nitrogen Sources on the Cell Growth and Erythritol Production From Glycerol by Y. lipolytica Wratislavia K1 Strain
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Nitrogen source
Parameter
NH4Cl
(NH4)2SO4
(NH4)2HPO4
NH4NO3
Urea
Yeast extract
Corn steep liquor
Time of cultivation (hr) Dry cell weight (g L1) Erythritol (g L1) Mannitol (g L1) Citric acid (g L1) Erythritol yield (g L1) Erythritol productivity (g L1 hr1)
75 13 0.5 25 1.5 21 1.1 1 0.2 0.25 0.33
71 13 0.8 26 1.8 24 1.3 1 0.3 0.26 0.37
91 12 0.4 13 1.0 18 1.3 1 0.2 0.13 0.14
91 13 0.7 18 1.3 21 1.5 nd 0.17 0.20
99 13 0.6 14 1.2 23 1.7 2 0.4 0.14 0.14
70 15 0.5 25 0.9 13 1.5 3 0.5 0.24 0.36
93 13 0.5 20 0.8 25 1.4 2 0.3 0.20 0.22
Note. Results are means SD from triple experiments. Culture conditions: 100 g L1 of pure glycerol; 0.25 g L1 of KH2PO4; amount of nitrogen, 0.52 g L1, corresponding to the addition of appropriate nitrogen source; nd, not detected.
hydrogen phosphate, and ammonium nitrate, and organic nitrogen compounds such as corn steep liquor, urea, and yeast extract. The total nitrogen concentration in each medium was similar, about 0.52 g L1. Relevant data are summarized in Table 2. As can be seen in Table 2, ammonium chloride, ammonium sulfate, and yeast extract were the best nitrogen sources for erythritol production by Wratislavia K1 strain. In the presence of these alone, the production of erythritol reached 25 and 26 g L1, respectively. A lower concentration of erythritol was obtained in the media containing urea and ammonium hydrogen phosphate, that is, 14 and 13 g L1, respectively. The source of nitrogen was also found to affect mannitol production by this strain. Its concentration was in the range from 13 to 25 g L1. The highest erythritol yield (0.26 g g1) and productivity (0.37 g L1 hr1) were achieved with ammonium sulfate. Inexpensive inorganic nitrogen sources were the most suitable for erythritol production by Wratislavia K1 strain. This is a good feature of this yeast strain, because organic nitrogen sources (yeast extract and casein hydrolysate) commonly used in glucose fermentation are very expensive and therefore cannot be necessarily advantageous to industrial production. Various nitrogen sources such as inorganic salts (ammonium chloride and ammonium sulfate) and organic salts (yeast extract, corn step liquor, urea, soybean flour, potato protein, peptone, malt extract, beef extract, tryptone, and soytone) were tested for maximizing the production of erythritol and mannitol by different osmophilic yeasts.[13,28] Particularly, yeast extract and corn steep powder gave the most effective production of erythritol and mannitol by osmophilic yeasts.[29–31] In the medium containing 10 g L1 of yeast extract, C. zeylanoides produced 43.2 g L1 of erythritol, which was about twofold higher than in the culture with ammonium chloride.[30] On the other hand, the addition of a yeast extract or a meat extract resulted in an increased production of another polyol, that is, mannitol, concomitant with erythritol. According to Lee et al.,[19] the medium for erythritol production by Torula sp. contained up to 20 g L1 of the yeast extract. Sawada et al. [17] reported that thiamine was an effective compound for the enhancement of erythritol production by T. megachiliensis SN-G42 strain. Supplementation of the culture medium with 4 mg L1 of thiamine resulted in erythritol concentration increase from 87.9 (without thiamine) to 205.1 g L1. It
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should be noted that the yeast extract contains a high amount of this vitamin. In other investigation, corn steep powder was proved to be the best nitrogen source for erythritol production by P. tsukubaensis KCCM 10356. This strain produced 149 g L1 of erythritol, which corresponded to 49.7% yield and 1.65 g L1 hr1 productivity.[13] Additionally, when corn steep liquor was used as the main nitrogen source, erythritol produced may be brown colored or contain a large quantity of salt. For these reasons the purification process is difficult, which is disadvantageous to economical production. Moreover, urea as a source of organic nitrogen is not admitted as a food additive and therefore it cannot be used for the production of erythritol to be used in foodstuffs. In further investigations, ammonium sulfate will be used as a nitrogen source for Wratislavia K1 strain.
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Effect of Ammonium Sulfate For in-depth analysis of the effect of ammonium sulfate concentration on erythritol production, experiments were conducted in a medium culture containing various amounts of ammonium sulfate (from 0.62 to 9.8 g L1). The effect of ammonium sulfate concentration on erythritol production by Wratislavia K1 strain is shown in Table 3. An increase in ammonium sulfate concentration up to 9.8 g L1 increased biomass level from 8 to 20 g L1. The highest concentration of erythritol, 26 g L1, was achieved in the medium containing 2.44 g L1 of ammonium sulfate. In this medium, the C:N ratio was at 75:1. The best yield of erythritol production, 0.25–0.26 g g1, was achieved when the C:N ratio ranged from 150:1 to 75:1, whereas the highest erythritol productivity, 0.37 and 0.33 g L1 hr1, was obtained when the C:N ratio was 75:1 and 100:1, respectively. According to Burscha¨pers et al.,[10] nitrogen limitation was the prerequisite for erythritol formation by M. tomentosa var. pollinis. On the other hand, nitrogen limitation caused also considerable foam formation, which was very difficult to suppress. Therefore, careful control over the concentration of a nitrogen source is necessary to obtain high erythritol productivity without foam formation. This can be best realized by a fed-batch operation. Nitrogen deficiency not only induces erythritol biosynthesis but also stimulates the production of citric acid by yeasts. TABLE 3 Effect of Ammonium Sulfate on the Cell Growth and Erythritol Production From Glycerol by Y. lipolytica Wratislavia K1 Strain (NH4)2SO4 (g L1) 0.62
1.22
1.86
2.44
4.88
9.8
C:N ratio Parameter 1
Dry cell weight (g L ) Erythritol (g L1) Mannitol (g L1) Citric acid (g L1) Erythritol yield (g g1) Erythritol productivity (g L1 hr1)
300:1
150:1
100:1
75:1
38:1
19:1
8 1.1 21 1.6 18 1.4 6 0.8 0.22 0.26
10 1.4 23 2.6 19 1.3 3 0.6 0.25 0.31
12 1.6 25 1.8 20 1.6 2 0.4 0.25 0.33
13 0.8 26 1.8 24 1.3 1 0.3 0.26 0.37
18 0.6 19 1.4 13 0.6 1 0.2 0.2 0.32
20 1.7 18 1.5 10 0.9 1 0.2 0.19 0.26
Note. Results are means SD from triple experiments. Culture conditions: 100 g L1 of pure glycerol; 0.25 g L1 of KH2PO4.
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Effect of Monopotassium Phosphate Phosphate is one of the most important medium components; it additionally affects cell physiology and metabolism in the culture of microorganisms. It was shown that an excessive amount of phosphate supplied in a glucose or a glycerol medium had a negative effect on polyols production.[7] Culture media require phosphate in the range of 0.3–300 mM for the growth of microorganisms, and a phosphate concentration much lower than this may inhibit the production of many metabolites. Therefore, the effect of phosphate was examined during glycerol fermentation with the Wratislavia K1 strain. In order to select the optimal amount of phosphate for erythritol production, monopotassium phosphate was tested in the range from 0 to 2 g L1. As summarized in Table 4, the concentration of a phosphate source had no effect on cell growth as it resulted in similar amounts of biomass produced (13–14.5 g L1). However, it played a significant role in the production of erythritol and mannitol. The maximal concentration of erythritol (26 g L1), corresponding to 0.26 g g1 yield and 0.37 g L1 hr1 productivity, was achieved in the medium containing 0.25 g L1 of potassium dihydrogen phosphate. According to other investigators, phytic acid (monoinositol hexaphosphate) was the best phosphate source for erythritol biosynthesis by C. magnoliae[18] and Torula sp.[7] In our investigation we have found that the higher phosphate supplementation (0.5–2 g L1) stimulated mannitol production. As a result, 24–27 g L1 of mannitol was produced as shown in Table 4. According to Hattori and Suzuki,[29] the polyol production was apparently differentiated by the level of phosphate concentration in the culture medium. Mannitol production occurred at a high concentration of phosphate and ceased when its concentration was reduced below 0.04 m g mL1 as monopotassium phosphate, while erythritol production was initiated at this level of phosphate concentration.[29] The production of mannitol from glycerol by resting cells of C. magnoliae under aerobic conditions was investigated by Khan et al.,[31] who observed that the addition of a high amount of potassium dihydrogen phosphate (5 g L1) decreased mannitol concentration from 51 to 3 g L1 and that a low amount of erythritol was also produced, which was otherwise not observed. Effect of Sodium Chloride Erythritol-producing strains can be characterized as osmophilic yeasts. It has been reported that sugar- and salt-tolerant yeasts produced and intracellularly accumulated polyols TABLE 4 Effect of KH2PO4 on the Cell Growth and Erythritol Production From Glycerol by Y. lipolytica Wratislavia K1 KH2PO4 (g L1) Parameter 1
Dry cell weight (g L ) Erythritol (g L1) Mannitol (g L1) Citric acid (g L1) Erythritol yield (g g1) Erythritol productivity (g L1 hr1)
0
0.05
0.25
0.50
1
2
13 1.2 19 1.5 14.5 1.5 3 1.6 0.19 0.21
14 1.4 23 1.5 16 1.6 3 1.2 0.23 0.32
13 0.8 26 1.8 24 1.3 1 0.3 0.26 0.37
14 1.5 17 0.6 26 2 1 0.4 0.17 0.18
14.5 1.5 14 0.7 24 1.5 1 0.2 0.16 0.15
13.5 1.3 16 1.1 27 2.1 1 0.3 0.14 0.16
Note. Results are means SD from triple experiments. Culture conditions: 100 g L1 of pure glycerol; 2.44 g L1 of (NH4)2SO4.
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TABLE 5 Effect of NaCl Concentration on the Cell Growth and Erythritol Production From Glycerol by Y. lipolytica Wratislavia K1 NaCl (g L1)
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Parameter Osmotic pressure (mOsm kg1) Dry cell weight (g L1) Erythritol (g L1) Mannitol (g L1) Citric acid (g L1) Erythritol yield (g g1) Erythritol productivity (g L1 hr1)
0
7.5
15
30
60
90
1500 13 0.8 26 1.5 24 1.4 1 0.3 0.26 0.37
1600 14 0.7 29 1.6 23 1.5 1 0.5 0.29 0.41
1900 13 0.8 32 2.0 20 1.0 3 0.8 0.32 0.47
2400 14 0.5 37 2.1 2 1.1 2 0.6 0.38 0.6
3700 11 0.6 37 2.4 1 0.6 2 0.8 0.37 0.57
3800 4 0.4 36 1.5 2 0.5 2 1.0 0.34 0.12
Note. Results are means SD from triple experiments. Culture conditions: 100 g L1 of pure glycerol; 0.25 g L1of KH2PO4; 2.44 g L1 of (NH4)2SO4.
(erythritol, mannitol, glycerol and arabitol) in the presence of high concentrations of sugars or when exposed to a high concentration of sodium or potassium chloride.[32] To evaluate the effect of sodium chloride on erythritol production by the Wratislavia K1 strain, its initial concentration varied from 0 to 90 g L1(Table 5). Sodium chloride supplementation increased the osmotic pressure from 1500 (without salt) to 3800 mOsm kg1 (90 g L1 of sodium chloride). The effect of sodium chloride addition on cell growth and production of erythritol and mannitol was dependent on its concentration, as shown in Table 5. The maximal production of erythritol (37 g L1), corresponding to 0.38 g g1 yield and 0.55 g L1 hr1 productivity, occurred when the fermentation medium contained 30 g L1 of sodium chloride. A similar effect was shown by Hattori and Suzuki.[30] In their investigations, C. zeylanoides produced a higher amount of erythritol from n-alkane in the medium containing sodium chloride or potassium chloride. In our study we have found that an increase in sodium chloride concentration up to 90 g L1 decreased the amount of mannitol and biomass from 24 to 1 g L1 and from 14 to 4 g L1, respectively. On the other hand, Hajny et al.[28] demonstrated that sodium chloride had no effect on erythritol fermentation from glucose by yeast belonging to the genus Torula, whereas potassium chloride addition to the medium stimulated the formation of glycerol as a by-product. Finally, Lin et al.[9] reported that yeast strains of Moniliella produced less erythritol in the presence of sodium chloride or potassium chloride than in the media without these salts. The differences in these two studies could be due to different yeast strain and substrate in both fermentations.
Optimization of Medium Composition Ammonium sulfate, potassium dihydrogen phosphate, and sodium chloride were identified as critical medium components that determine erythritol, mannitol, and citric acid production with the Wratislavia K1 strain on media containing glycerol. Therefore, the next step of this study involved the optimization of culture media using response surface methodology.
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As already mentioned in the Design of Experiment section, attempts were made to approximate the experimental data for the extent of erythritol production by a second-order polynomial. The polynomial takes the form of the following relation: Y¼
3 X
aij Xi Xj þ
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i;j¼1 ij
3 X
ai Xi þ a0
ð1Þ
i¼1
where a0, ai, and aij are parameters of the polynomial, Y is the extent of erythritol production, and X1, X2, and X3 are as indicated in Table 1. It is essential to note, however, that during approximation, values of these variables are coded. The estimated parameter values of the polynomial (1) that approximate the values of the extent of erythritol concentration, as well as the determination coefficient obtained, are compiled in Table 6. Six replications at the center point of the design made it possible to assess the adequacy of the model (polynomial (1)) in terms of test statistics with Snedecor’s F-distribution of (5, 5) degrees of freedom. Table 6 also includes the value of the statistics and the value of the corresponding limit significance level (a). The limit value of a indicates that the polynomial (1) proposed is adequate if the assumed significance-level values are lower than 0.3622. The values in Table 6 were calculated using Microsoft Office Excel 2003 software. Because of the spherical distribution of information in the rotatable design, the values of the variables X1, X2, and X3 at which these functions reach their maxima were sought in the set defined by the inequality of Eq. (2): X21 þ X22 þ X23 3
ð2Þ
that is, on the inside and on the border of a sphere with a center lying in the design center, and with a radius that equals the distance from the center point to one of the points of the 23 full-factorial design. Using the mathematical conditions for the maximum of a multivariable function it was confirmed that the optimal point lay inside the set defined by the inequality TABLE 6 Estimated Parameters of the Polynomial Approximating the Extent of Erythritol Concentration, and the Statistical Parameters of Estimation Parameter a11 a22 a33 a12 a13 a23 a1 a2 a3 a0 Coefficient of determination Value of F-Snedecor statistics p Value for F-Snedecor statistics
Value 3.3155 2.3081 5.7544 0.6750 1.4750 1.5000 0.4170 3.2788 4.7680 27.0451 0.6612 1.3938 .3622
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TABLE 7 Optimal Conditions for Erythritol Production
Type of value
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Coded Real
(NH4)2SO4 (g L1),
KH2PO4 (g L1),
NaCl (g L1),
Erythritol (g L1),
X1
X2
X3
Y
0.286 2.245
0.938 0.221
0.573 26.37
30.26
of Eq. (2). Hence, it was possible to evaluate analytically the optimal values of X1, X2, and X3 and the corresponding maximal Y. These four values are shown in Table 7. Figure 1 (made by using the MATLAB software, version 4.2c.1) relates the extent of erythritol concentration to each pair of independent variables when the third variable takes the optimal value.
FIGURE 1 Effect of medium components on erythritol production (in g L1): (A) influence of (NH4)2SO4 and KH2PO4 concentration at the optimal value of NaCl concentration; (B) influence of (NH4)2SO4 and NaCl at the optimal value of KH2PO4 concentration; (C) influence of KH2PO4 and NaCl at the optimal value of (NH4)2SO4 concentration. Dotted lines indicate the contours of the range of independent variables (X21 þ X22 þ X23 3) for encoded values of X1, X2, and X3.
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TABLE 8 Results of Erythritol Production From Glycerol in Optimal Medium Substrate Parameter
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Dry cell weight (g L1) Erythritol (g L1) Mannitol (g L1) Citric acid (g L1) Erythritol yield (g g1) Erythritol productivity (g L1 hr1)
Pure glycerol
Crude glycerol
15.5 46.9 0.8 0.4 0.47 0.82
18.1 39.3 2.5 0 0.40 0.66
Thus, the optimized medium containing 2.25 g L1 of ammonium sulfate, 0.22 g L1 of monopotassium phosphate and 26.4 g L1 of sodium chloride was applied in bioreactor batch cultures with 100 g L1 of pure and crude glycerol. In such a medium the C:N ratio was found at 81:1. As it can be seen in Table 8, 46.9 and 39.3 g L1 of erythritol was produced with the yield of 0.47 and 0.40 g g1 from pure and crude substrate, respectively. In the optimized medium the amount of by-products does not exceed 2.5 g L1. These values of erythritol concentration were significantly higher in comparison to the result achieved via mathematical modeling. This phenomenon can be explained by the oxygenation conditions, because the experiments for the purpose of mathematical modeling were carried out in a shake flask, where oxygenation is lower than in a bioreactor. Due to the worse oxygenation conditions, during the shake-flask culture not the whole amount of glycerol was utilized (data not shown) and therefore the calculated optimal erythritol concentration was lower than that obtained in the bioreactor culture conducted in the optimal medium.
Production of Erythritol in Fed-Batch Process It is known that effective erythritol production is conducted in batch and fed-batch cultures with osmophilic yeast from glucose in the concentration of 200–400 g L1.[7,13,19,33–35] To date, little is known about erythritol production by Y. lipolytica from glycerol. Recently, a novel two-stage osmotic pressure control fed-batch strategy was developed for erythritol production from this substrate by Y. lipolytica and a high concentration of erythritol was obtained, 194.3 g L1, with a productivity of 0.95 g L1 hr1. However, as a by-product mannitol was formed at a high level, 36.8 g L1.[36] In the next step of the presented study, the medium with total glycerol of 300 g L 1 and C:N ratio of 81:1 was used in the fed-batch culture. The fed-batch fermentation profile of erythritol production from pure glycerol by Wratislavia K1 was shown in Figure 2. The process was launched as batch culture in medium with glycerol concentration of 100 g L1 (at initial volume of bioreactor of about 1.8 L); pulsed additions of glycerol (about 100 g L1) were made after 2 and 4 days. The Wratislavia K1 strain produced 132 g L1 of erythritol with a productivity of 1.0 g L1h1. The concentration of by-products, mannitol and citric acid, did not exceed 5 g L1.
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FIGURE 2 Time course of biomass (.), glycerol (~), erythritol (&), mannitol (&), and citric acid () during fed-batch fermentation by Y. lipolytica Wratislavia K1 strain. The arrow indicates periodic feeding of glycerol to total concentration of 300 g L1.
CONCLUSIONS Currently, most crude glycerol is produced from a biodiesel production process and its price is determined by the demand and production of biodiesel. The price of crude glycerol varied from $40 to $330=ton over the last decade.[37] Glycerol could be utilized as a carbon source in microbial bioconversions for the production of a range of chemicals to be used as end products in various industries. One of them is erythritol, which could be produced by Y. lipolytica yeast. The price of erythritol obtained from glucose by Moniliella pollinis ranged from $3700 to $3900=ton over recent years. In our opinion, the erythritol price obtained from crude glycerol will be correlated with the glycerol global market price and the total cost of erythritol production by microbial method, including cost of fermentation, purification, and crystallization processes. Therefore, the price of crystalline erythritol (more than 99.5% pure) obtained from crude glycerol by a selected strain of Y. lipolytica may in the future be attractive as a new alternative technology for its cheap production at commercial scale. Based on the data presented in this study, it appears that the production of erythritol by Y. lipolytica Wratislavia K1 from glycerol could potentially compete with the more traditional processes run with sugars and other yeasts. The results obtained suggest that the concentrations of glycerol, ammonium sulfate, monopotassium phosphate, and sodium chloride affect the production of erythritol, keeping the by-products at minimal possible levels in this fermentation process. Further optimization of the culture conditions and cultivation systems will contribute to improving the production of erythritol using biological processes. FUNDING The authors are thankful to the Polish Ministry of Science and Higher Education and the European Union for financial support under research project PO IG 01.01.02-00-074=09.
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