Filamentous fungus Rhizopus oryzae NRRL 395 lactic acid ... · production yield of lactic acid was 0.67 g lactic acid/g glucose. Cellobiose, as a carbon source, decreased lactic acid

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.

��

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


2-1-1. ��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


3-3-1. 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


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.


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


Fig. 27. Lactic acid concentration in the simultaneous saccharification and fermentation


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.


2-1-1.

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


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


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.


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.


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 .


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


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


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


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

,

.

1. Jianxin Du, Ningjun Cao, Cheng S. Gong, and George T. Tsao, Production of

L-lactic acid by Rhizopus oryzae in a bubble column fermenter, Applied

Biochemistry and Biotechnology, 70-72: 323-329.

2. C. W. Yang, Zhongjing Lu, and George T. Tsao, Lactic acid production by

pellet-form Rhizopus oryzae in a submerged system, Applied Biochemistry and

Biotechnology, 51/52: 57-71.

3. Ying Zhou, Jose M. Dominguez, Ningjun Cao, Jianxin Du, and George T.

Tsao, Optimization of L-lactic acid production from glucose by Rhizopus

oryzae ATCC 52311, Applied Biochemistry and Biotechnology, 77-79: 401-407.

4. Peimin Yin, Naoki Nishina, Yuuko Kosakai, Kazutoyo Yahiro, Yongsoo Park

and Mitsuyasu Okabe, Enhanced production of L(+)-lactic acid from corn

starch in a culture of Rhizopus oryzae using an air-lift bioreactor, Journal of

Fermentation and Bioengineering, 84(3): 249-253.

5. Barbara E. Wright, Angelika Longacre and Jacqueline Reimers, Models of

metabolism in Rhizopus oryzae, J. theor. Biol. 182: 453-457.

6. Adriana Bramorski, Pierre Christen, Martha Ramirez, Carlos R. Soccol and

Sergio Revah, Production of volatile compounds by the edible fungus Rhizopus

oryzae during solid state cultivation on tropical agro-industrial substrates,

Biotechnology Letters, 20(4): 359-362.

7. Adenise L. Woiciechowski, Carlos R. Soccol, Luis P. Ramos, Ashok Pandey,

Experimental design to enhance the production of L-(+)-lactic acid from steam-

exploded wood hydrolysate using Rhizopus oryzae in a mixed-acid

fermentation, Process Biochemistry, 34: 949-955.

8. Peimin Yin, Kazutoyo Yahiro, Tooru Ishigaki, Yongsoo Park, and Mitsuyasu

Okabe, L(+)-Lactic acid production by repeated batch culture of Rhizopus

oryzae in air-lift bioreactor, Journal of Fermentation and Bioengineering, 85(1):

96-100.

9. Yan Sun, Y.-L. Li, S. Bai, Modeling of continuous L(+)-lactic acid production

with immobilized R. oryzae in an airlift bioreactor, Biochemical Engineering

Journal, 3: 87-90.

10. Yuji Oda, Katsuichi Saito, Hiroaki Yamauchi, Motoyuki Mori, Lactic acid

fermentation of potato pulp by the fungus Rhizopus oryzae, Current

Microbiology, 45: 1-4.

11. Enock Y. Park, Yuuko Kosakai, and Mitsuyasu Okabe, Efficient production of

L-(+)-lactic acid using mycelial cotton-like flocs of Rhizopus oryzae in an

air-lift bioreactor, Biotechnol. Prog., 14: 699-704.

12. Yuuko Kosakai, Yong Soo Park, Mitsuyasu Okabe, Enhancement of L(+)-lactic

acid production using mycelial flocs of Rhizopus oryzae, Biotechnology and

Bioengineering, 55(3): 461-470.

13. Lee, S. M., Production of cellulase and optimization of deinking process for

waste paper recycling in the mid-scale, M. S. thesis, Dept. of Biological

Engineering, Inha University, Inchon.

14. Vichien Kitpreechavanich, Sunee Chotineeranat, Klanarong Sriroth, Busaba

Yongsmith, Yoshihito Shirai, and Yusaku Fujio, L(+)-Lactic acid production

from tapioca starch by Rhizopus oryzae, Biotechnology for Sustainable

Utilization of Biological Resources in Tropics, 14: 217-225.

15. Christopher D. Skory, Freer SN, Bothast RJ, Production of L-lactic acid by

Rhizopus oryzae under oxygen limiting conditions, Biotechnology Letters,

20(2): 191-194.

16. L. Xuemei, L. Jianping, L. Mo'e, Peilin, L-lactic acid production using

immobilized Rhizopus oryzae in a three-phase fluidized-bed with simultaneous

product separation by electrodialysis, Bioprocess Engineering, 20: 231-237.

17. Y. Sun, Y.-L. Li, S. Bai, H. Yang, Z.-D. Hu, Stability of immobilized R.

oryzae in repetitive batch productions of L(+)-lactic acid: effect of inorganic

salts, Bioprocess Engineering, 19: 155-157.

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

Documents

Filamentous fungus Rhizopus oryzae NRRL 395 lactic acid ... · production yield of lactic acid was 0.67 g lactic acid/g glucose. Cellobiose, as a carbon source, decreased lactic acid