filamentous fungus rhizopus oryzae nrrl 395 lactic acid ... · production yield of lactic acid was...
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Filamentous fungus Rhizopus oryzae NRRL 395
lactic acid
Studies on Lactic Acid Production using Filamentous Fungus
Rhizopus oryzae NRRL 395
Filamentous fungus Rhizopus oryzae NRRL 395
lactic acid
Studies on Lactic Acid Production using Filamentous Fungus
Rhizopus oryzae NRRL 395
Lactic acid lactic acid bacteria Lactobacillus
. Lactobacillus L-(+)-lactic acid 95-98%
. filamentous fungus Rhizopus oryzae 100%
L-(+)-lactic acid .
Lactobacillus . lactic acid
.
, lab-scale 2.5 L jar fermenter Rhizopus oryzae pH
buffer , CaCO3 NaOH . NaOH
pH buffer CaCO3 lactic acid
. Fermenter NaOH pH buffer
CaCO3 , lactic acid 13%
. CaCO3 pH buffer lactic acid
.
Rhizopus oryzae L-(+)-lactic acid CaCO3
. CaCO3 2% 24
, lactic acid 0.67g lactic acid/g
glucose .
cellobiose lactic acid
, .
cellulose solka floc
SSF(simultaneous saccharification and fermentation, )
.
Abstract
Lactic acid and its salt are being widely used in food, chemical, and pharmaceutical
industries. Recently, there has been an increasing interest in lactic acid because it is one
of the raw materials for the production of environmentally benign polymers. Polylactic
acid (PLA) is used in the manufacture of new biodegradable plastics, and will play an
important role in solving a world-wide environmental problem, abandoning of waste
plastic.
Generally, lactic acid is produced by lactic acid bacteria such as Lactobacillus
rhamnosus, Lactobacillus lactis, and its species. However, lactic acid bacteria produces
not only L-(+)-lactic but also 5% D-(-)-lactic acid. Unlike most bacteria, lactic
acid-producing Rhizopus oryzae generate only L-(+)-lactic acid as a fermentation product.
There are fewer organic acids in fermentation broth of Rhizopus oryzae than
Lactobacillus rhamnosus. Therefore, Rhizopus oryzae may be better than Lactobacillus
rhamnosus in the viewpoint of purification.
The calcium carbonate has an effect on pH control and increases lactic acid
production in Rhizopus oryzae fermentation. The optimal concentration and addition time
of calcium carbonate is 2% and at culture time of 24 hr, respectively. The maximum
production yield of lactic acid was 0.67 g lactic acid/g glucose.
Cellobiose, as a carbon source, decreased lactic acid production but increased the cell
growth rate. The enzyme hydrolysis and fermentation process and the Simultaneous
saccharification and fermentation process were performed in Rhizopus oryzae with solka
floc as a carbon source.
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Abstract ��������������������������������������������������������������������������������������������������������������������������
List of Tables ������������������������������������������������������������������������������������������������������������������
List of Figures ����������������������������������������������������������������������������������������������������������������
1. ���������������������������������������������������������������������������������������������������������������������������1
1-1. �������������������������������������������������������������������������������������������������������������������1
1-2. Lactic acid �����������������������������������������������������������������������������������������������2
1-3. Rhizopus oryzae ��������������������������������������������������������������������������������������2
1-4. �����������������������������������������������������������������������������������������������������4
1-5. Simultaneous Saccharification and Fermentation �������������������������������������5
2. ���������������������������������������������������������������������������������������������������������������9
2-1. Rhizopus oryzae ��������������������������������������������������������������������������������������9
2-1-1. ��������������������������������������������������������������������������9
2-1-2. ����������������������������������������������������������������������������������������������������9
2-1-3. ��������������������������������������������������������������������������������������������������10
2-2. Rhizopus oryzae optical purity ������������������������������������������������������������12
2-2-1. optical purity ����������������������������������������������������������������������������������������������12
2-2-2. ��������������������������������������������������������������������������������������������������12
2-3. Enzymatic hydrolysis and fermentation �����������������������������������������������������������12
2-4. Simultaneous Saccharification and Fermentation ���������������������������������������������13
3. �������������������������������������������������������������������������������������������������������������14
3-1. Rhizopus oryzae optical purity ������������������������������������������������������������14
3-2. Rhizopus oryzae CaCO3 �������������������������������������������������16
3-3. Lab-scale 2.5 L fermenter Rhizopus oryzae ������������������������22
3-3-1. Fermenter Rhizopus oryzae ������������������������������������������22
3-3-2. NaOH pH Rhizopus oryzae ���������������������26
3-3-3. Fermenter CaCO3 ����������������������29
3-4. cellobiose �����������������������������������������������������33
3-5. �������������������������������������������������������������������������������������������������������38
3-6. Rhizopus oryzae ����������������������������������42
3-3-1. Enzymatic hydrolysis and fermentation ����������������������������������������42
3-3-2. Simultaneous Saccharification and Fermentation ���������������������������43
4. �����������������������������������������������������������������������������������������������������������������������49
������������������������������������������������������������������������������������������������������������������������51
������������������������������������������������������������������������������������������������������������������������54
List of Tables
Table 1. Compositions of medium for Lactobacillus rhamnosus and Rhizopus
oryzae
Table 2. Organic acids in fermentation broth of Lactobacillus rhamnosus and
Rhizopus oryzae
Table 3. Compositions of media for filamentous fungus Rhizopus oryzae NRRL
395 culture
List of Figures
Fig. 1 Model of glucose metabolism of the filamentous fungus Rhizopus oryzae.
Ext-: extracellular, G-6-P: glucose-6-phosphate, F-6-P: fructose-6-phosphate,
F-1,6-bP: fructose-1,6-bisphosphate.
Fig. 2. HPLC chromatograms of fermentation broth of Rhizopus oryzae and
Lactobacillus rhamnosus. A: Rhizopus oryzae, B: Lactobacillus
rhamnosus.
Fig. 3. HPLC chromatograms of fermentation broth of Lactobacillus rhamnous and
Rhizopus oryzae NRRL 395. A : Lactobacillus rhamnosus, B : Rhizopus
oryzae NRRL 395.
Fig. 4. Time course of glucose concentration with varying CaCO3 addition time.
: addition at 0 hr, : addition at 12 hr, : addition at 24 hr, :
control.
Fig. 5. Time course of lactic acid concentration with varying CaCO3 addition
time. : addition at 0 hr, : addition at 12 hr, : addition at 24 hr,
: control.
Fig. 6. Dry cell weight with varying CaCO3 addition time.
Fig. 7. Time course of glucose concentration with varying initial CaCO3
concentration. : 1% CaCO3, : 2% CaCO3, : 3% CaCO3.
Fig. 8. Time course of lactic acid concentration with varying initial CaCO3
concentration. : 1% CaCO3, : 2% CaCO3, : 3% CaCO3.
Fig. 9. Time course of glucose concentration at pH 5 and pH 6. : pH 5, :
pH 6.
Fig. 10. Time course of lactic acid concentration at pH 5 and pH 6. : pH 5, :
pH 6.
Fig. 11. Time course of pH of fermentation broth. : pH 5, : pH 6.
Fig. 12. Time course of glucose concentration using NaOH solution as pH buffer.
: pH 5, : pH 6.
Fig. 13. Time course of lactic acid concentration using NaOH solution as pH
buffer. : pH 5, : pH 6.
Fig. 14. Time course of glucose concentration with additional CaCO3 in medium.
: control, : 1% CaCO3.
Fig. 15. Time course of lactic acid concentration with additional CaCO3 in medium.
: control, : 1% CaCO3.
Fig. 16. Dry cell weight with additional CaCO3 in medium.
Fig. 17. Time course of substrate concentration with varying carbon source. :
cellobiose, : glucose after cellobiose, : glucose, : addition of
glucose medium.
Fig. 18. Time course of lactic acid concentration with varying carbon source. :
cellobiose, : glucose after cellobiose, : glucose.
Fig. 19. Dry cell weight with varying carbon source. : after 34 hr, : after
69 hr.
Fig. 20. Time course of glucose concentration at 37 and 42 . : 37 , :
42 .
Fig. 21. Time course of lactic acid concentration at 37 and 42 . : 37 , :
42 .
Fig. 22. Dry cell weight at 37 and 42 .
Fig. 23. Glucose concentration in the enzyme hydrolysis and fermentation process.
: cellulase, : cellulase + -glucosidase, : inoculation of Rhizopus
oryzae.
Fig. 24. Lactic acid concentration in the enzyme hydrolysis and fermentation
process. : cellulase, : cellulase + -glucosidase.
Fig. 25. Dry cell weight in the enzyme hydrolysis and fermentation process.
Fig. 26. Glucose concentration in the simultaneous saccharification and ferrmentation
process. : cellulase, : cellulase + -glucosidase.
Fig. 27. Lactic acid concentration in the simultaneous saccharification and fermentation
process. : cellulase, : cellulase + -glucosidase.
1.
1-1.
Lactic acid 3 (2-Hydroxypropanoic acid,
COOH-HCOH-CH3) . Lactic acid
55%, 40%, 5%
, .
polylactic acid .
polylactic acid
.
lactic acid
(acetic acid, butyric acid, lactic acid, propionic acid)
.
.
succinic acid
.
.
Lactic acid
, . lactic
acid lactic acid
Cargill ,
Argonne , Purdue G. Tsao
.
lactic acid
. lactic acid
.
1-2. Lactic acid
lactic acid L-form D-form 50%
racemic mixture . lactic
acid racemic mixture lactic acid
racemic mixture .
lactic acid lactic acid bacteria
Lactobacillus filamentous fungus Rhizopus oryzae
.
1-3. Rhizopus oryzae
Rhizopus oryzae lactic acid filamentous fungus
. , lactic acid, ethanol,
fumaric acid, pyruvic acid [Fig. 1].
Fig. 1 Model of glucose metabolism of the filamentous fungus Rhizopus oryzae.
Ext-: extracellular, G-6-P: glucose-6-phosphate, F-6-P: fructose-6-phosphate,
F-1,6-bP: fructose-1,6-bisphosphate.
Rhizopus oryzae fungi
. lactic acid Lactobacillus
.
. , Lactobacillus 3 - 5% D-(-)-lactic acid
Rhizopus oryzae 100% L-(+)-lactic acid
PLA .
Lactobacillus yeast
extract ammonium sulfate . scale-up
(Table. 1). Rhizopus oryzae
lactic acid Lactobacillus
(Fig. 1, Table 2). lactic acid
.
1-4.
Rhizopus oryzae glucose .
glucose glycolysis 2 pyruvate . pyruvate
pathway citric acid cycle ATP
. , glucose glucose citric
acid cycle substrate inhibition . glycolysis
pyruvate pathway
. Fig. 1 Rhizopus oryzae glucose metabolism model
.
cellobiose glucose , Rhizopus oryzae
cellobiose (Enock Y. Park, 2000).
cellobiose Rhizopus oryzae glycolysis citric acid cycle
ATP ,
.
1-5. Simultaneous Saccharification and Fermentation
SSF enzymatic hydrolysis fermentation
. cellobiose glucose
(Enari, 1983). SSF
single cell protein, SCP
(Spindler and Wyman, 1989). scp enzymatic hydrolysis
50 , fermentation 30-35 . SSF
.
SSF lactic acid
(Takagi, 1984; Abe and Takagi, 1991). , Lactobacillus
rhamnosus SSF , enzymatic hydrolysis
50 fermentation 37
42 (Lee, S. M, Y. M. Koo, 2001).
SSF , scale-up
. ,
.
Table. 1. Compositions of medium for Lactobacillus rhamnosus and Rhizopus
oryzae.
Lactobacillus rhamnosus Rhizopus oryzae
Gucose
Yeast extract
K2HPO4
KH2PO4
MgSO4 7H2O
MnSO4 H2O
FeSO4 7H2O
Sodium acetate
Glucose
Ammonium sulfate
KH2PO4
MgSO4 7H2O
ZnSO4 7H2O
A
Lactic acid
BA
Lactic acid
B
Fig. 2. HPLC chromatograms of fermentation broth of Rhizopus oryzae and
Lactobacillus rhamnosus. A: Rhizopus oryzae, B: Lactobacillus
rhamnosus.
Table. 2. Organic acids in fermentation broth of Lactobacillus rhamnosus and
Rhizopus oryzae.
Lactobacillus rhamnosus Rhizopus oryzae
Citric acid
Pyruvic acid
Malic acid
Succinic acid
Lactic acid
Formic acid
Fumaric acid
Acetic acid
Propionic acid
Citric acid
Pyruvic acid
Malic acid
Lactic acid
Fumaric acid
2.
2-1. Rhizopus oryzae
2-1-1.
Lactic acid filamentous fungus Rhizopus oryzae NRRL
395(Northern Regional Research Center, USDA, Peoria, Illinois) ATCC
. Potato dextrose agar(Difco Lab.)
slant 30 7 , 4
.
(Table 3).
lactic acid glucose
carbon source .
2-1-2.
250 ml flask 50 ml Shaking incubator
(KMC-8480SF, VISION SCIENTIFIC CO.) 37 , pH 6, 200 rpm
18 .
shaking incubator lab-scale 2.5 L fermenter(Korea Fermenter Co.)
18 . shaking
incubator pH
24 CaCO3 . Lab-scale 2.5L fermenter
0.5 vvm aeration
pH 5 N sodium hydroxide .
2-1-3.
glucose concentration, cellobiose concentration, lactic acid
concentration . filter paper(Whatman No. 1)
,
DCW(dry cell weight) .
Glucose concentration glucose analyzer(YSI 2700 SELECT, Yellow Springs
Intrument Co.) . Lactic acid LC-10AD
pump(Shimadzu Co.) Waters 486 UV detector(Waters Co.) HPLC
system Aminex HPX-87H column(7.8 × 300 mm, Bio-Rad, USA)
, 0.008N H2SO4 0.5 mL/min
. Cellobiose concentration OROM pump(OROM Tech Co.) 500
ELSD(Evaporated Light Scattering Detecter, Alltech Co.) HPLC system
carbohydrate column(Waters Co.) , 75% acetonitile
1.2 mL/min .
Table. 3. Compositions of media for filamentous fungus Rhizopus oryzae NRRL
395 culture.
Contents Seed culture medium lactic acid production medium
glucose
(NH4)2SO4
KH2PO4
MgSO4 7H2O
ZnSO4 7H2O
30 g/L
3 g/L
0.2 g/L
0.2 g/L
0.08 g/L
various carbon source
3 g/L
0.2 g/L
0.2 g/L
0.08 g/L
2-2. Rhizopus oryzae optical purity
2-2-1. optical purity
Rhizopus oryzae fermentation broth Lactobacillus rhamnosus
fermentation broth 100 320 HPLC
.
2-2-2.
L-(+)-Lactic acid D-(-)-lactic acid LC-10AD
pump(Shimadzu Co.) Waters 486 UV detector(Waters Co.) HPLC
system CHIRALPAK MA(+) column(Daicel. Co.) ,
2 mM CuSO4 0.5 mL/min .
2-3. Enzymatic hydrolysis and fermentation
cellulose solka floc glucose enzyme
hydrolysis enzyme Trichoderma reesei Rut C-30
crude cellulase -glucosidase Novozym 188(Novozymes Co.)
. 30 g/L solka floc 5 U/mL crude cellulase
2 U/mL -glucosidase shaking incubator 42 , 200 rpm
enzyme hydrolysis . 48 Rhizopus oryzae
.
2-4. Simultaneous Saccharification and Fermentation
SSF(Simultaneous Saccharificaton and fermentation) cellulose
glucose glucose lactic acid enzymatic
hydrolysis fermentation . Rhizopus oryzae
solka floc , 5 U/mL crude cellulase
2 U/mL -glucosidase , Rhizopus oryzae
shaking incubator 42 , 200 rpm . 24 pH
CaCO3 .
3.
3-1. Rhizopus oryzae optical purity
Rhizopus oryzae L-(+)-lactic acid , Lactobacillus
rhamnosus L-(+)-lactic acid D-(-)-lactic acid .
L-form D-form Rhizopus oryzae Lactobacillus
rhamnosus fermentation broth 100 , 300 , HPLC
Daicel chiral column CHIRALPAK MA+
.
Optical purity Fig. 3 . Fig. 3 Rhizopus oryzae
Lactobacillus rhamnosus fermentation broth L-(+)-lactic acid
D-(-)-lactic acid , Rhizopus oryzae 100%
L-(+)-lactic acid , Lactobacillus rhamnosus 98%
L-(+)-lactic acid 2% D-(-)-lactic acid .
D-(-)-Lactic acid
L-(+)-Lactic acid
A B
D-(-)-Lactic acid
L-(+)-Lactic acid
A B
Fig. 3. HPLC chromatograms of fermentation broth of Lactobacillus rhamnous and
Rhizopus oryzae NRRL 395. A : Lactobacillus rhamnosus, B : Rhizopus
oryzae NRRL 395.
3-2. Rhizopus oryzae CaCO3
Rhizopus oryzae CaCO3 pH
. CaCO3 pH
buffer . , Rhizopus oryzae pH
lactic acid
. CaCO3
.
CaCO3 0 , 12 , 24
, CaCO3 .
Fig. 4-6 . 0 12 CaCO3
glucose lactic acid .
24
. , lactic acid 24
(Fig. 4-5). 24
(Fig. 6). CaCO3
glucose lactic acid
.
CaCO3 1%, 2%, 3% , 24
. Fig. 7 8 . 2% 3% CaCO3
lactic acid . 1% CaCO3 2%
3% lactic acid . lactic acid
0.67 g lactic acid/g glucose (Fig. 8).
Time (hr)0 20 40 60 80
Glu
cose
(g
/L)
0
10
20
30
40
50
60
70
Fig. 4. Time course of glucose concentration with varying CaCO3 addition time.
: addition at 0 hr, : addition at 12 hr, : addition at 24 hr, :
control.
Time (hr)
0 20 40 60 80
Lac
tic
acid
(g
/L)
0
5
10
15
20
25
30
Fig. 5. Time course of lactic acid concentration with varying CaCO3 addition
time. : addition at 0 hr, : addition at 12 hr, : addition at 24 hr,
: control.
CaCO3 addition time
0 hr 12 hr 24 hr control
Dry
cel
l w
eig
ht
(g/L
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Fig. 6. Dry cell weight with varying CaCO3 addition time.
Time (hr)
0 20 40 60
Glu
cose
(g
/L)
0
10
20
30
40
50
60
Fig. 7. Time course of glucose concentration with varying initial CaCO3
concentration. : 1% CaCO3, : 2% CaCO3, : 3% CaCO3.
Time (hr)
0 20 40 60
Lac
tic
acid
(g
/L)
0
10
20
30
40
50
Fig. 8. Time course of lactic acid concentration with varying initial CaCO3
concentration. : 1% CaCO3, : 2% CaCO3, : 3% CaCO3.
3-3. Lab-scale 2.5 L fermenter Rhizopus oryzae
3-3-1. Fermenter Rhizopus oryzae
scale-up . lab-scale
pilot-scale plant-scale scale-up
. lab-scale fermenter
.
lab-scale 2.5 L jar fermenter Rhizopus oryzae
. 500 mL flask 100 mL
, 1% shaking incubator 36 , 200 rpm 24
. 2 2.5 L fermenter 1 L ,
24 . fermenter pH 5
pH 6 , pH CaCO3 . CaCO3
, on-line pH
. 36 ,
200 rpm 0.5 vvm aeration .
Fermenter Rhizopus oryzae Fig. 9-11
. pH factor . On-line
pH pH
. pH ,
CaCO3 pH buffering (Fig. 11),
buffer solution(CaCO3 solution) total volume
lactic acid . lactic acid
pH 6 0.5 g lactic acid/g glucose (Fig. 10-11).
Time (hr)
0 10 20 30 40 50 60 70
Glu
co
se
(g
/L)
0
20
40
60
80
100
120
Fig. 9. Time course of glucose concentration at pH 5 and pH 6. : pH 5, :
pH 6.
Time (hr)
0 10 20 30 40 50 60 70
La
cti
c a
cid
(g
/L)
0
10
20
30
40
50
60
Fig. 10. Time course of lactic acid concentration at pH 5 and pH 6. : pH 5, :
pH 6.
Time (hr)
0 10 20 30 40 50 60 70
pH
1
2
3
4
5
6
7
8
9
10
Fig. 11. Time course of pH of fermentation broth. : pH 5, : pH 6.
3-3-2. NaOH pH Rhizopus oryzae
Rhizopus oryzae factor pH .
CaCO3 on-line pH
scale-up .
on-line pH NaOH solution
.
2 fermenter 1 L , 24
. 36 , 200 rpm 0.5 vvm aeration
, fermenter pH 5 pH 6 . pH
5 N NaOH solution .
NaOH Rhizopus oryzae Fig. 12 13
. Rhizopus oryzae NaOH pH , pH
6 pH 5 glucose
lactic acid . pH 6 glucose lactic acid
0.53 g lactic acid/g glucose (Fig. 13). NaOH pH
CaCO3 CaCO3 on-line
pH NaOH
.
Time (hr)
0 10 20 30 40 50 60
Glu
cose
(g
/L)
0
20
40
60
80
100
Fig. 12. Time course of glucose concentration using NaOH solution as pH buffer.
: pH 5, : pH 6.
Time (hr)
0 10 20 30 40 50 60
Lac
tic
acid
(g
/L)
0
10
20
30
40
50
60
Fig. 13. Time course of lactic acid concentration using NaOH solution as pH
buffer. : pH 5, : pH 6.
3-3-3. Fermenter CaCO3
CaCO3 pH buffer , aggregation
. Rhizopus oryzae
CaCO3 pH
morphology lactic acid
. CaCO3
.
Rhizopus oryzae 2 2.5 L fermenter .
fermenter 1 L 1% CaCO3 ,
fermenter 1 L . 24
36 , 200 rpm, 0.5 vvm aeration . pH
5 N NaOH solution .
Fig. 14-16 . 1%
CaCO3 glucose lactic acid
, lag phase
. lactic acid 0.63 g lactic acid/g glucose ,
50% 13% (Fig. 14
Fig. 15). CaCO3 pH buffer ,
lactic acid .
1% CaCO3 (Fig.
16).
Time (hr)
0 10 20 30 40 50 60 70
Glu
cose
(g
/L)
0
20
40
60
80
100
120
Fig. 14. Time course of glucose concentration with additional CaCO3 in medium.
: control, : 1% CaCO3.
Time (hr)
0 10 20 30 40 50 60 70
Lac
tic
acid
(g
/L)
0
10
20
30
40
50
60
70
Fig. 15. Time course of lactic acid concentration with additional CaCO3 in medium.
: control, : 1% CaCO3.
Dry
cel
l wei
gh
t (g
/L)
0
2
4
6
8
No CaCO3 CaCO3
Dry
cel
l wei
gh
t (g
/L)
0
2
4
6
8
No CaCO3 CaCO3
Fig. 16. Dry cell weight with additional CaCO3 in medium.
3-4. cellobiose
Rhizopus oryzae
cellobiose ,
. Rhizopus oryzae cellobiose ,
.
glucose glucose ,
glycolysis citric acid cycle pathway .
citric acid cycle glycolysis 2 pyruvate
. , cellobiose
glycolysis citric acid cycle
, ATP .
. Cellobiose
, cellobiose 34 glucose
glucose .
34 dry cell weight .
30 g/L .
Rhizopus oryzae Fig. 17-19 .
34 cellobiose
, (Fig.
19). Lactic acid cellobiose 34 glucose
21 g/L , glucose
17 g/L , cellobiose 13 g/L
(Fig. 18).
. glucose
(cellulose complex) ,
complex , cellulose chain cellobiose
glucose endo- -1,4-glucanase(CMCase) cellulose chain
cellobiose exo- -1,4-glucanase(1,4-D-glucan
cellobiohydrolase) cellobiose glucose -glucosidase
. cellobiose
-glucosidase
.
Time (hr)
0 20 40 60 80
Su
bs
tra
te (
g/L
)
0
5
10
15
20
25
30
35
Time (hr)
0 20 40 60 80
Su
bs
tra
te (
g/L
)
0
5
10
15
20
25
30
35
Fig. 17. Time course of substrate concentration with varying carbon source. :
cellobiose, : glucose after cellobiose, : glucose, : addition of
glucose medium.
Time (hr)
0 20 40 60 80
Lac
tic
acid
(g
/L)
0
5
10
15
20
25
Fig. 18. Time course of lactic acid concentration with varying carbon source. :
cellobiose, : glucose after cellobiose, : glucose.
Cellobiose Cellobiose + Glucose Glucose
Dry
cel
l w
eig
ht
(g/L
)
0
1
2
3
4
Fig. 19. Dry cell weight with varying carbon source. : after 34 hr, : after
69 hr.
3-5.
SSF(simultaneous saccharification and fermentation) glucose
enzymatic hydrolysis glucose lactic acid
fermentation one-step . SSF
Lactobacillus rhamnosus (Lee, S. M. and Y. M. Koo,
2001). . Rhizopus
oryzae SSF
. SSF
42 Rhizopus oryzae
. 37 .
Rhizopus oryzae Fig. 20-22
. 37 glucose lactic acid
, 42 cell growth rate lag
phase . Lactic acid 37 42
(Fig. 20 21). , 42
(Fig. 22).
Time (hr)
0 20 40 60 80
Glu
cose
(g
/L)
0
10
20
30
40
Fig. 20. Time course of glucose concentration at 37 and 42 . : 37 , :
42 .
Time (hr)0 20 40 60 80
Lac
tic
acid
(g
/L)
0
5
10
15
Fig. 21. Time course of lactic acid concentration at 37 and 42 . : 37 , :
42 .
Dry
cel
l wei
gh
t (g
/L)
0
1
2
3
4
5
37 oC 42 oC
Fig. 22. Dry cell weight at 37 and 42 .
3-6. Rhizopus oryzae
row material
.
.
Rhizopus oryzae .
glucose .
(cellulose
complex) .
, , enzymatic
hydrolysis fermentation enzymatic hydrolysis
fermentation one-step SSF(Simultaneous Saccharification and
Fermentation) .
3-6-1. Enzymatic hydrolysis and fermentation
enzymatic hydrolysis ,
Rhizopus oryzae . Fig. 23-25 . Lactic
acid crude cellulase -glucosidase crude
cellulase (Fig. 24). Rhizopus oryzae
cellobiose -glucosidase lactic acid
. Crude cellulase -glucosidase lactic
acid ,
(Fig. 25).
3-6-2. Simultaneous Saccharification and Fermentation
SSF enzymatic hydrolysis fermentation one-step
. , ,
.
Rhizopus oryzae SSF ,
Fig. 26 27 . SSF enzymatic hydrolysis
fermentation lactic acid ,
Rhizopus oryzae .
Time (hr)
0 20 40 60 80 100 120 140
Glu
co
se
(g
/L)
0
5
10
15
20
25
30
35
Fig. 23. Glucose concentration in the enzyme hydrolysis and fermentation process.
: cellulase, : cellulase + -glucosidase, : inoculation of Rhizopus
oryzae.
Time (hr)
0 20 40 60 80
La
cti
c a
cid
(g
/L)
0
2
4
6
8
10
12
14
16
Fig. 24. Lactic acid concentration in the enzyme hydrolysis and fermentation
process. : cellulase, : cellulase + -glucosidase.
Dry
cel
l wei
gh
t (g
/L)
0
2
4
6
8
10
12
Cellulase Cellulase + β-Glucosidase
Dry
cel
l wei
gh
t (g
/L)
0
2
4
6
8
10
12
Cellulase Cellulase + β-Glucosidase
Fig. 25. Dry cell weight in the enzyme hydrolysis and fermentation process.
Time (hr)
0 20 40 60 80
Glu
co
se
(g
/L)
0
5
10
15
20
25
Fig. 26. Glucose concentration in the simultaneous saccharification and ferrmentation
process. : cellulase, : cellulase + -glucosidase.
Time (hr)
0 20 40 60 80
La
cti
c a
cid
(g
/L)
0
2
4
6
8
10
12
Fig. 27. Lactic acid concentration in the simultaneous saccharification and fermentation
process. : cellulase, : cellulase + -glucosidase.
4.
Rhizopus oryzae lactic acid L-(+)
.
Lactobacillus rhamnosus . L-(+)-lactic
acid
.
Rhizopus oryzae CaCO3 pH lactic acid
, CaCO3 2% , CaCO3
24 . lactic acid 0.67 g lactic acid/g
glucose .
, lab-scale 2.5 L jar fermenter Rhizopus oryzae pH
buffer , CaCO3 NaOH . NaOH
pH buffer CaCO3 lactic acid
. Fermenter NaOH pH buffer
CaCO3 , lactic acid 13%
. CaCO3 pH buffer lactic acid
.
Cellobiose Rhizopus oryzae lactic acid
, .
.
solka floc Enzymatic hydrolysis and
Fermentation Simultaneous Saccharification and Fermentation
. Rhizopus oryzae
glucose
(enzyme complex) . Trichoderma reesei RUT
C-30 crude cellulase -glucosidase Novozym 188
, 5 U/mL 2 U/mL .
Rhizopus oryzae fungus ,
L-(+)-lactic acid . cellobiose
,
.
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대학원에 입학한지가 엊그제 같은데, 벌써 졸업을 맞이했습니다. 대학원에서의 2년은
금방 지나간다며, 그 짧은 기간동안 많은 것을 얻을 수 있도록 부단히 노력하라던 어느 선
배의 말이 지금에서야 마음에 와 닿는 것 같습니다. 먼저 학부 때부터 대학원에 이르기까
지 많은 관심과 애정을 주신 지도교수님인 구윤모 교수님께 감사드립니다. 때론 저에게
따끔한 질책을 아끼시지 않았기에 제가 여기까지 올 수 있었던 것 같습니다. 제 연구의 마
지막에 많은 조언과 관심을 주신 김영준 박사님께도 감사의 말씀을 드리고 싶습니다. 졸
업논문 심사의 주심이시며, 학부 때부터 항상 존경의 대상이셨던 김동일 교수님께 감사드
립니다. 학생들에게 항상 자상한 아버지와 같은 허병기 교수님, 허태련 교수님께도 감사
드립니다. 생물공학과의 발전을 위해 애써주시는 김은기 교수님, 소재성 교수님, 윤현식
교수님, 이철균 교수님, 김응수 교수님께도 감사드립니다. 2년 동안 동거동락하며 지내왔
던 실험실의 우진이형, 상목이형, 진희, 영식, 윤정, 혜진, 현수 모두에게 앞으로 좋은 결
과가 있기를 기대합니다. 아직은 청년 같은 이종우 박사님께도 밝은 미래가 있으시길 기
대합니다. 대학원 2년 동안 항상 저에게 따뜻한 정을 준 상윤 형에게도 감사드리며, 졸업
동기들에게도 좋은 일이 가득하길 기원합니다. 그 외 생물공학과 대학원생 모두에게 항상
행복과 사랑이 충만하길 기원합니다. 10년 동안 끈끈한 우정으로 지켜주는 태선, 정식,
준양, 명철, 혁, 민호, 석민이에게도 감사의 마음을 전합니다. 1년 반 동안 내 곁에서 큰
힘이 되어준 영화에게 진심으로 감사하고, 이 논문이 나올 수 있도록 지금 이 자리까지
있기 해주신 부모님께 이 논문을 바칩니다.
2003년 1월
청주에서