REDUCTION OF BY PRODUCTS IN ERYTHRITOL BIOSYNTHESIS
FROM GLYCEROL BY Y. LIPOLYTICA
Ludwika Tomaszewska, Anita Rywińska, Waldemar Rymowicz
Wrocław University of Environment al and Life Sciences; Department of Biotechnology and Food Microbiology; Chełmońskiego Str. 37/41, 51-630 Wrocław, Poland
INTRODUCTION
In recent years, because of increased interest in environmental protection, especially much attention was devoted to utilization of the industrial and agro-industrial by-products by its
application in biosynthesis of valuable compounds. A great example of such efforts is application of glycerol – one of the major by-products of biodiesel production – in erythritol biosynthesis by
Yarrowia lipolytica yeast. Erythritol is a compound that naturally occurs in human diet (e.g. present in fruits, honey, mushrooms, seaweed). This sugar alcohol has 60-80% of sweetness in
comparison to sucrose and very low caloric value (0 – 0.2 kcal/g). Moreover, it is safe for consumption, non-cariogenic, its intake did not affect blood insulin level, and it does not cause any
gastric side-effects.
The aim of the study was to improve the parameters and purity of erythritol production from glycerol by Y. lipolytica Wratislavia K1 strain.
MATERIALS AND METHODS
Microorganism. Y. lipolytica Wratislavia K1 used in this study originated from the yeast culture collection of the Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences in
Poland.
Substrate. In the study the carbon and energy source were: pure glycerol – 98% wt/wt (POCH Gliwice; Poland) and crude glycerol derived from biodiesel production - 78% wt/wt (LOTOS; Poland).
Media. Growth medium contained of: 50 g of pure glycerol, 3 g of yeast extract (YE), 3 g of malt extract and 5 g of bactopepton in 1 liter of distilled water.
Bioreactor production medium consisted of: glycerol - 150 g (batch culture) or 250 g (fed-batch mode), 1g of MgSO4•7H2O, 0.2 g of KH2PO4, 5 or 10 g of YE, 25 g of NaCl in 1 liter of tap water.
Culture conditions. The growth cultures were carried out in 0.3-L flasks containing 0.1 L of growth medium on rotary-shaker at 29.5 °C and 140 rpm for 3 days. In order to inoculate media for the bioreactor experiments,
0.2 L of growth culture was used. Bioreactor cultures were carried out in a 5-L stirred-tank reactor (Biostat B Plus, Sartorius; Germany) with a working volume of 2 L at 30°C, the aeration rate fixed at 0.6 v/v/min and the
stirrer speed adjusted to 800 rpm. The pH was maintained automatically at 3.0 by the addition of a 20% (w/v) NaOH solution.
Analytical methods. In the samples biomass was determined gravimetrically after drying at 105°C. The concentrations of glycerol, erythritol, mannitol, arabitol, citric acid, α-ketoglutaric acid were determined by HPLC
method on an HyperRez XP Carbohydrate H+ column (Thermo Scientific, USA) coupled to an UV detector (λ=210 nm) and an RI detector (Shodex, Japan). The column was eluted with 25 mM of trifluoroacetic acid at 65°C
and a flow rate of 0.6 mL/min. The protein content of the biomass was analyzed by Kiejdahl method. Minerals content was analyzed by atomic absorption spectrophotometry (AAS) [Miśta et al. 2012]
RESULTS
0
20
GLY
X
40
60
Time (h)
ERY MAN ARA
80
CA
B
180
160
140
120
100
80
60
40
20
0
9
8
7
6
5
4
3
2
1
0
0
100
KA
20
GLY
40
X
ERY
60
80
Time (h)
MAN ARA
CA
Tab. 1. Comparison of erythritol biosynthesis processes by Y. lipolytica Wratislavia K1 strain in dependance
on the amount of YE in the medium.
MAN, ARA, CA, KA (g/L)
9
8
7
6
5
4
3
2
1
0
X, GLY, ERY (g/L)
180
160
140
120
100
80
60
40
20
0
MAN, ARA, CA, KA (g/L)
A
X, GLY, ERY (g/L)
In the batch culture with pure glycerol media (150 g/L) the increase of YE concentration stimulated biomass growth. In the culture with 10 g/L of YE biomass concentration reached 28 g/L
and was doubled, in comparison to the culture with 5 g/L of the nitrogen source. In the process with lower dose of YE erythritol concentration was higher and reached 78 g/L, corresponding to
0.51 g/g production yield and productivity of 1.01 g/Lh. Lower erythritol concentration (62.1 g/L) and its production yield (0.40 g/g) in the process with 10 g/L of YE may be explained by
enhanced biomass growth in this culture. The protein concentration of the biomass derived after the cultures was in the range of 16.7 – 19.8%.
YE
(g /L)
Time
(h)
Selectivity
(%)
Y
(g /g-)
Q
(g /Lh)
Protein
(%)
1.01
1.24
16.7
19.8
0.61
12.4
20.3
16.3
28.7
Batch cultures (150 g/L of pure glycerol)
77
12.5
78.0
90,6
0.51
50
28.1
62.1
93,8
0.40
Fed-batch cultures (250 g/L of crude glycerol)
238
16.7
145.5
90.0
0.59
285
25.4
165.5
97.9
92
27.3
134.2
92.7
0.54
138
31.5
141.2
99.1
5
10
5*
10*
100
Biomass Erythritol
(g /L)
(g /L)
1.46
*The results in the second line present the changes obtained as a consequence of last fed, made
after complete glycerol exhaustion
Y – erythritol production yield, Q – erythritol volumetric productivity
KA
Fig. 1. Effect of yeast extract (YE) on biomass and erythritol production from 150 g /L of pure glycerol in bioreactor batch cultures. Culture conditions: 5 g /L of YE (A), 10 g /L of YE (B).
1
3
0
50
100
GLY
X
ERY
150
Time (h)
MAN
200
ARA
250
CA
180
160
140
120
100
80
60
40
20
0
1
3
0
GLY
KA
Fig. 3. The time course of erythritol fed-batch biosynthesis from 250 g /L of crude glycerol by Y. lipolytica Wratislavia K1 in medium with 5 g /L
of YE. The arrows indicate periodical fed of glycerol (1 and 2) to the total concentration of 250 g /L and fed of 10 g of (NH4)2SO4 (3).
2
30
60
ERY
90
120
X
150
Time (h)
180
MAN
210
ARA
240
270
CA
9
8
7
6
5
4
3
2
1
0
300
MAN, ARA, CA KA (g /L)
2
9
8
7
6
5
4
3
2
1
0
300
X, GLY, ERY (g /L)
180
160
140
120
100
80
60
40
20
0
MAN, ARA, CA, KA (g./L)
X, GLY, ERY (g./L)
To maximize the final product concentration and improve its purity the fed-batch mode and the medium with 250 g/L of crude glycerol and 5 or 10 g/L of YE was applied. The processes
were launched as batch cultures in which the initial glycerol concentration was about 125 g/L. Next, two pulsed additions of glycerol (about 75 g/L) were made as it is indicated by the arrows.
After glycerol utilization 10 or 20 g of (NH4)2SO4 was added to the culture and the process was maintained for further 48 h.
KA
Fig. 4. The time course of erythritol fed-batch biosynthesis from 250 g /L of crude glycerol by Y. lipolytica Wratislavia K1 in medium with 10 g /L
of YE. The arrows indicate periodical fed of glycerol (1 and 2) to the total concentration of 250 g /L and fed of 20 g of (NH4)2SO4 (3).
CONCLUSIONS
Application of fed-batch mode allowed to obtain 145.5 g/L and 132 g/L of erythritol in the processes with 5 and 10 g/L, respectively. Extension of the culture after glycerol exhaustion resulted in
utilization of by products by yeast and the increase of erythritol concentration up to 165.5 g/L. The selectivity of erythritol biosynthesis was increased up to 99.1% by the addition of nitrogen
source after glycerol depletion. Simultaneously, the protein content in the yeast biomass increased up to 28.7%.
REFERENCES
Miśta D, Rząsa A, Szmańko T, Zawadzki W, Styczyńska M, Pintal A, Króliczewska B. The effect of humic-fatty acid preparation on production parameters and meat quality of growing rabbits. J Ann Anim Sci 2012;12:117–126.
Acknowledgments. This work was co-sponsored by grant No. N N312 256640 from the National Science Centre (Poland) and by the Ministry of Science and Higher Education of Poland and European Union under Project No. POIG 01.01.02-00-074/09.