, &

,
,

2460

, .

, 2011

UNIVERSITY OF PATRAS
DEPARTMENT OF CHEMISTRY
SECTION OF APPLIED, ANALYTICAL & ENVIRONMENTAL CHEMISTRY

DISSERTATION

USE OF FREEZE DRIED YEAST, FREE AND IMMOBILIZED ON DELIGNIFIED CELLULOSIC

MATERIALS, FOR GLUCOSE FERMENTATION AT DOMESTIC SCALE

KOKKALI ELENI
2460

Supervisor
Dr. Bekatorou Argyro, Assistant Professor

PATRAS, 2011



(
). ,


( ). ,
Saccharomyces cerevisiae AXAZ-1
().
,
,
0-10 C.


.

, , ,
(HPLC)

Sacharomyces cerevisiae -1,

Abstract
The aim of this thesis was the research on glucose fermentation efficiency of a freeze
dried immobilized biocatalyst (yeast cells immobilized on delignified cellulosic materials).
Additionally, another aim of this thesis was to study the effect of temperature on the
rate of fermentation to evaluate the possibility for a domestic scale application
(fermentation in the consumers refrigerator). Specifically, the psychrophilic and alcohol
resistant yeast strain Saccharomyces cerevisiae AXAZ-1 was immobilized on delignified
cellulosic material (sawdust). The immobilized biocatalyst, as well as the free yeast cells,
were freeze dried and used for glucose fermentations at 0-10oC. The fermented liquids
were analyzed by Gas Chromatography and High Pressure Liquid Chromatography for
alcohol and residual sugar, and the results showed potential for commercial application.
Key words
Alcoholic Fermentation, Freeze-drying, immobilization, Sacharomyces cerevisiae 1,High Pressure Liquid Chromatography (HPLC).

Table of Contents
.................................................................................................7
1. ................................................................................................7
1.1 .............................................................................................. 7
1.1.1

1.1.2

1.1.3

13

1.1.4

13

1.1.5

13

1.1.6

14

1.2 .................................................................................. 14
1.2.1

14

1.2.2

15

1.2.3

15

1.2.4

16

1.2.5

16

1.2.6

17

1.2.7 18
1.2.8

18

1.3 ...................................................................... 19
1.3.1

19

1.4 ...................................................................................................... 19
1.4.1

19

1.4.2

19

1.4.3

21

1.4.4

21

1.4.5

22
1.4.6

23

1.4.7

24

2. .......................................................................... 25
2.1 ............................................................................. 25
2.1.1

25
4

2.1.2

25

2.1.3

26

2.1.4

26

2.1.5

27

2.2 -1 ............................................................... 27
2.3 ........................................................ 27
2.3.1

28

2.3.2

28

2.3.3 28
2.4 .......................................................................................... 28
2.4.1

28

2.4.2

28

2.4.3

29

2.4 ........................................................................................................... 30
2.4.1

30

3. - ................................................................. 31
3.1 ...................................................................................... 42

4. ...................................................................... 47
5. ..................................................................................... 48
5.1 ....................................................................................... 48
5.2 ..................................................................................... 50
5.3 .................................................................................... 50


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C6H12O6 2 C2H5OH + 2 CO 2 + 40 kcal
, , P (14,8 kcal)
ADH. TP

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C6H12O6 + 2 ADP + 2 Pi + 2 NAD+ 2 CH3COCOO + 2 ATP + 2 NADH + 2 H2O + 2H+

C6H12O6 + 2C2H5OH + 2CO2

:
(http://en.wikipedia.org/wiki/)

10

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cerevisiae. .

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(Topt) (Tmax) .
Topt 20-30 C.

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Iconomopoulou,2002)
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14

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, 500 mg/L,
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400 mg/L
.

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S. cerevisiae ,
pH.

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(,2007, , 2011, Kourkoutas et al.,2004, Norton & D' amore,1994).
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(, 2007).

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21

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25

1.4.3
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(101.3 kPa = 1 atm).
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(
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27

1.4.5


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(Miyamoto-Shinohara et al. , 2000, Palmfeldt et al., 2003, Carvahlo et al., 2004).
1.
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(Gerke rt
al.,1992). S. cerevisiae,
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(1991).
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28

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Abadias et al. (2000) Cerrutti et al. (2000)

Candida sake , .

Hua et al. (2009) pH

Oenococcus oeni.

(2007),
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Iconomopoulou et al. (2000) (2001)

30

 AXAZ-1
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(Iconomopoulou et al.,2002,2003).
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(,2001).

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S. cerevisiae Gram-
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31

2.
2.1
2.1.1

:

2% w/v

0.1% w/v (4)2SO4

0.1% w/v KH2PO4

0.5% w/v MgSO47 H2O

0.4% w/v (yeast extract)

4% w/v
12% w/v .
- 2% w/v .
p 5.5 - 5.7
,
. pH
2SO4, NaOH.
130 C 1,5 - 2 atm.


.
2.1.2
2.1.2.1
, .
2.1.2.2

8%, 10%, 14% w/v 80, 100,
140 .
130 C 1,5 - 2 atm 15 - 20
32

 .
2.1.3
-1
Saccharomyces cerevisiae.
.
2.1.3.1

. Petri,

.
48 h 30 C
2 - 4 C.
.
2.1.3.2

4% v/v .
5 mL .
48 h 30 C, 50 mL .
,
500 mL , ,
2 L, . 2
L, 20 g .
2.1.4

1. : SHIMADZU GC-8A Gas Chromatograph

2. oBe
3.
4.
5.
6.
7.
33

8. oBe, 100 mL
9. (), 1000 mL
10. : LABCONCO Flasks 1000 mL
11.
12. Bio cool
13. : LABCONCO FREEZE- DRY SYSTEM/ FREEZONE 4.5
14. : EPI-BION SENTRON pH-System 1001
15. ( ,
)
16. (HPLC): SHIMADZU LC-9A
17.
2.1.5

.

(1997) .
2.1.5.1
5 L 300 g 3 L NaOH 1%
3 ,
.
Bchner
. ,
15 min 130 C 1,5 - 2 atm,
.
2.2 -1
-1
. 300 g 3 L 1% w/v NaOH
3 h
. ,
(DCM)
130 C 15 min. H
16 g S. cerevisae
(-1) 800 mL (12% w/v)
34

100 g DCM. 6-8 h

( < 0,9 Be). ,
12% w/v
600 mL Ringer.
2.3

.

.
.

3 C/min. -45 C

Freeze Dry System, Freezone 4.5 (Labconco).
15 - 5.10 - 3 Bar.
-45 C.
2.3.1

. 3

C/min -45 C. 15 - 5.10 - 3 bar

freeze drying system. ,
.
.
2.3.2

80 C
. 5
85% w/w.
2.3.3

.

35

 .
2.4
2.4.1
(GC)
SHIMADZU GC - 8A. 2 L ,
, ,
.

( Shimadzu LC - 9A HPLC),
Porapac - S 2 , 130-170 C
3 C/min. FID (Flame Ionization, )
210 C 2 - 2
0,6 g/cm2 0,2 g/cm2, .
4 g/cm2.,
Shimadzu C R6A Chromatopack.
2.4.2

(GC)
.
50 ppm 3% .
2.4.3

(HPLC) SHIMADZU LC - 9A.
: (i) Shim-pack, SCR-101 N, (ii) :
, (iii) : 0.8 mL/min (iv)
: 60C, (v) : .
1% v/v
0,45 m.
- 1 0,05 % v/v. ,

0 - 20 g/L.

36

2.4
2.4.1

8, 10, 14 g/L
2.1.1
.
300 mL .
,
.
oBe .
,
.
, 3 (3 atch
). 0, 5 10 0C
. 0
(
) . 5 10 0C
.

.

37

3. -

,
DC . ,
(2
3 batch)
(1o batch).
.
.
10 C
5 C.
0 C
.

1. AXAZ-1
(80 g/L) 5C.

38

 2. AXAZ-1
(100 g/L) 5C.

3. AXAZ-1
(140 g/L) 5C.

39

 4. AXAZ-1
(80 g/L) 5C.

5. AXAZ-1
(100 g/L) 5C.

40

 6. AXAZ-1
(140 g/L) 5C.

7. AXAZ-1
(80 g/L) 10C.

41

 8. AXAZ-1
(100 g/L) 10C.

9. AXAZ-1
(140 g/L) 10C.

42

 10. AXAZ-1
(80 g/L) 5C.

11. AXAZ-1
(100 g/L) 10C.

43

 12. AXAZ-1
(140 g/L) 10C.

3.1


, . ,
5 10 C.

44

 II.

5 C.

DCM

Batch

(g/L)

(Be)

(h)

(g/L)

80

5,3

1869

3,21

80

5,3

594

4,87

80

5,3

604

1,98

1
2
3
1
2
3

100
100
100
140
140
140

6,1
6,1
6,1
7,4
7,4
7,4

1896
648
696
2160
1048
1064

2,38
2,42
0,87
9,78
4,34
1,45

80

5,3

816

0,34

2
3
1
2
3
1
2
3

80
80
100
100
100
140
140
140

5,3
5,3
6,1
6,1
6,2
7,3
7,3
7,3

288
288
984
240
240
960
456
360

0,60
1,19
0,13
0,09
0,10
6,32
5,27
3,34

45

(%
v/v)

 III.

10 C.

DCM

Batch

(g/L)

(Be)

(h)

(g/L)

1
2

80
80

5,3
5,3

360
96

1,70
2,10

3
1
2
3
1
2
3

80
100
100
100
140
140
140

5,3
6,3
6,3
6,3
7,1
7,1
7,1

94
416
124
112
560
192
192

0,20
2,60
2,20
1,40
8,36
3,31
2,20

80

5,2

92

1,45

2
3
1
2
3
1
2
3

80
80
100
100
100
140
140
140

5,2
5,3
6,2
6,2
6,2
7,3
7,3
7,4

54
54
124
68
68
156
92
92

0,37
3,80
2,35
4,47
1,20
3,24
1,53
8,24

46

(%
v/v)

*-

4.

,
.

Sacharomyces cerevisiae -1,
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, .
5 10 C
, .
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.

47

5.
5.1
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5.2
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5.3
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2. www.slideshare.net/SevenAM/consumer-behaviour-final
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4. www.biokemi.org
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6. www.amrita.ac.in
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