Kenji Sonomoto1, 2 - PowerPoint PPT Presentation

Title: Kenji Sonomoto1, 2


1
Modern application of lactic acid bacteria for
new polymer materials
V International Brazil-Japan Workshop Biofuel,
Environment and New Products from Biomass October
29-November 1, 2007 UNICAMP, Campinas, Brazil

• Kenji Sonomoto1, 2
• 1Faculty of Agriculture, and 2Bio-Architecture
  Center, Kyushu University, Fukuoka, Japan

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Hokkaido
Kyushu University
Tokyo
Gifu
Kyoto
Honshu
Fukuoka
Shikoku
Kyushu
3
Background
Oil-refinery
CO2 SOX,NOX
Bio-refinery
Carbon neutral
Petroleum will be exhausted soon.

• Utilization of carbon source
• from biomass, especially
• non-edible materials
• L-LA and D-LA fermentation
• for polylactic acid (PLA)

CO2
Biodegradable recycling plastic (PLA)
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Plant for PLA
Pilot Plant at Toyota
Plant at Nature Works ( up to 140,000 ton/year)
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RAUM (2003)
Bio-FRP (Kenaf/PLA) Spare Tire Cover
PLA Fiber (Floor Mat)
Toyota Eco Plastics
RAUM was got on sale in 2003. PLA was used in
spare tire cover and floor mat.
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Bio-Plastic Utilization
i-unit
(PLA)
                                                
               
                                                
               
Bio-Plastic involving PLA was utilized as
interior materials.
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Development of Polylactic Acid (PLA)
Polylactic acid
Lactic acid
Poly-L-lactic acid

• Transparent
• thermal-resistance

Low
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Direct lactic acid fermentation from sago starch
and xylooligosaccharide with novel lactic acid
bacteria

• Direct fermentation of L-lactic acid from sago
  starch (and rice starch with amylose mutation)
• 2. Direct fermentation of D-lactic acid from
  xylooligosaccharides

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Direct L-lactic acid fermentation by Enterococcus
faecium No.78
YLA/TS yield of lactic acid to consumed total
sugar PLA lactic acid productivity (Max lactic
acid production/the indicated time)
Shibata et al., Enzyme Microb. Technol., 41, 149
(2007)
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About Sago

• - Starch one of the most abundant plant
  products in the world
• - World production of starch approx. 27.5 million
  tons
• - Starch market dominated by corn, potato and
  tapioca
• - World consumption of sago starch is between
  200,000 to 300,000 tons/yr
• in Malaysia, there are several sources of starch,
  such as
• tapioca
• corn
• yam
• sweet potato
• rice
• Sago has several advantages over other cash crops
  
• it is the highest starch producer among all
  starch crops of the world.
• in a year, one hectare of sago plantation can
  produce 25 t of starch, higher than rice (6 t),
  corn (5.5 t), wheat (5 t) and potato (2.5 t).
• Starch content is 80 by mass
• Low price of sago starch (US0.10/kg)

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Sago Planting, Harvesting Starch Production
- sago matures after 8-10 years - the long sago
trunk is cut into logs using chainsaw
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Sago Planting, Harvesting Starch Production
- each log is approx. 1m long, weighing 120-150kg
- these logs are transported to the sago mill
using river or lorries
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Direct lactic acid fermentation with E. faecium
No.78 and the other amylolytic lactic acid
bacteria (sago starch, pH 6.5)
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Comparison of direct lactic acid fermentation
with E. faecium No.78 and the other amylolytic
lactic acid bacteria
YLA/TS yield of lactic acid to consumed total
sugar PLA lactic acid productivity (Max lactic
acid production/the indicated time) This
experiments were carried out twice and the
average data shown with error.

• Xiaodong, W. et al. Biotechnol Lett 1997 19
  841-843
• Sanni AI. et al. Int J Food Microbiol 2003 80
  77-87

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Schematic diagram of continuous culture
systemwith high cell density
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Continuous L-lactic acid fermentation with high
cell density
Shibata et al., Enzyme Microb. Technol., 41, 149
(2007)
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Continuous L-lactic acid fermentation with high
cell density and amylase recycling

• Strain Enterococcus faecium No.78
• Medium feed solution MRS medium
• (carbon source
  sago starch)
• Conditions pH 6.5, 30oC, Working volume 400 mL
• Cell concentration recycle unit
• Hollow-fiber microfiltration (MF) module
• (Microza PMP-102)
• Enzyme recycle unit
• Hollow-fiber ultrafiltration (UF) module
• (Minimate TFF Capsul)
• MWCO 100,000

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Schematic diagram of continuous culture system
with high cell density and amylase recycle
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Continuous culture with high cell density
amylase recycle system
VCC Viable cell count, Av average YLA/TS yield
of lactic acid to consumed total sugar, PLA
Lactic acid productivity
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Rice Mutant Library Preserved in Institute of
Genetic Resources of Kyushu University
CM Line 6000Lines
Mutants for morphological
properties induced by MNU
cv. Kinmaze
3000 Lines

cv,Taichung 65

3000 Lines


Mutations for All of genes in Rice Genome
(about 40, 000 genes)
High Quality of Gene Pool for Scientific Use
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CM Line ?Morphological Physiological Mutant
Lines ?
CM Line consists of the mutant strains screened
for morphological and physiological traits in
rice by MNU treatment of fertilized egg cells in
rice.
Various kinds of mutants for the traits of leaf,
panicle, spikelets, plant height or heading date
have been stocked and they are used as materials
for the identification of responsible gene and
the gene action.
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Enzymes for starch biosynthesis
Amylose
Amylopectin
wx
BE
BEI BEIIa BEIIb
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Mutations on amylose content
wx
du1
du2
du3
du4
du5
KINMAZE (WT)
I2KI
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Direct lactate production from rice starch with
amylose mutation KINMAZE (Wild type?amylose
15.58)?wx (Mutant? amylose 0.76) Strain
E. faecium No.78, Lb. manihotivorans JCM12514
LA 13.46 g/L
LA 14.81 g/L
LA 19.43 g/L
LA 10.13 g/L
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Direct lactic acid fermentation from sago starch
and xylooligosaccharide with novel lactic acid
bacteria

• Direct fermentation of L-lactic acid from sago
  starch (and rice starch with amylose mutation)
• 2. Direct fermentation of D-lactic acid from
  xylooligosaccharides

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In our laboratory
Leuconostoc lactis SHO-47 (SHO-47)
D Leuconostoc lactis SHO-54 (SHO-54)
D Lactococcus lactis subsp. lactis IO-1 (IO-1)
L
Xylose-fermenting Lactic acid bacteria
SHO-47, SHO-54 PK pathway (theoretical YLA 1
mol-LA/mol-xylose) IO-1
PK pathway PP pathway (theoretical YLA
1.67)
Xylose Xylooligo
Lactate (pure L- or D-)
Few reports on xylooligosaccharide-fermenting
lactic acid bacteria
Aim
Xylooligosaccharide fermentation by these strains
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Xylan-hydrolysate fermentation
SHO-54
SHO-47
IO-1
2.0
0.8
xylose
xylotetraose
xylobiose
xylopentaose
1.6
0.6
xylotriose
xylohexose
1.2
Xyose,xylobiose, xylotriose(g/l)
Xyotetraose,xylopentaose, xylohexaose(g/l)
0.4
0.8
0.2
0.4
0.0
0.0
0
6
12
18
24
0
24
6
12
18
24
0
6
12
18
Time (h)
LAmaxl22.97 mM Ylac1.09 mol/mol-xylose Plac0.27
g/l/h
LAmax 18.37 mM Ylac0.81 mol/mol-xylose Plac0.25
g/l/h
LAmax13.67 mM Ylac0.53 mol/mol-xylose Plac0.07
g/l/h
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Conclusion

• SHO-47, SHO-54 and IO-1 could not ferment xylan
  but xylooligosaccharides (monohexaose).
• SHO-47 and SHO-54 had higher b-xylosidase
  activities than IO-1.
• b-Xylosidase was localized in cytoplasm.
• Xylooligosaccharides were uptaken and then
  degraded to xylose. Surplus xylose might be
  transported outside cell and then uptaken to be
  fermented.

Leuconostoc lactis SHO-47 and SHO-54 were found
to produce D-lactic acid from xylooligosaccharide
efficiently. Ohara et al., J. Biosci. Bioeng.,
101, 415 (2006)
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Bio-architecture of lactic acid bacterium
Xyl
ose isomerase

Xylulokinase

PP pathway
PK pathway
Transketolase

Phosphoketolase
Transaldolase

ADP

Acetate kinase
ATP

Transketolase

Acetic acid

CoA
-
SH

Fructose
-
bisphosphate aldolase

Phosphotransacetylase

Pi

Pathway for xylose metabolism in L. lactis IO-1
Pyruvate
-
formate lyase
Triosephosphate isomerase

Glyceraldehyde
-
3
-
phosphate dehydrogenase

Tanaka et al., Appl. Microbiol. Biotechnol., 60,
160 (2002) Ohara et al., J. Biosci. Bioeng., 103,
92 (2007)
Lactate dehydrogenase
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Cascade utilization of biomass resource with
lactic acid fermentation
Lactic acid bacteria
Lactic acid fermentation
Utilization of lactate etc as second
fermentation material
Optically active lactic acids (D- or L-lactic
acid) Organic acid (DL-lactic acid, acetic acid,
etc.)
Polymerization
H2-producing bacteria
PHA-producing bacteria
Acetone -butanol- producing bacteria
Bio-hydrogen
Stereocomplex poly-lactic acid
Polyhydroxyalkanoate (PHA)
Acetone, Butanol
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Anaerobic fermentation with biomass (in my lab)

• Lactate fermentation
• (Lactate?Bacteriocin?Functional food
  materials)
• Acetone-Butanol-Ethanol(ABE)Fermentation
• Ethanol-anaerobic fermentation

Biomass studied

• Sago starch(waste)
• Hemicellulose
• Shochu distilled waste
• Garbage
• Waste sludge
• Oil sludge
• Waste of vegetables, fluits etc..

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(by Dr. Dulce M. Flores)
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Direct L-lactic acid fermentation with various
carbonsources by Enterococcus faecium No.78
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Comparison of direct lactic acid fermentation by
amylolytic lactic acid bacteria
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Conclusions
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Thermal gelatinization of starch by amylose
mutation
( ) amylose content ()
Amylose content modifies the termination
temperature of thermo-gelatinization for starch
and the swelling power
38
Preparation of xylan-hydrolysate by xylanase
treatment

60?, 20 min incubation
10 min boiling
centrifugation

• Xylooligosaccharides from xylanase-treated xylan
  (8.25 g/l)

Culture same as xylooligosaccharide
fermentation All sugar phenol-sulfate methods
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Halolactibacillus miurensis

• Halolactibacillus, salt (loving) lactic acid
  rodlet.Isolated from living and decaying marine
  organisms collected
• from Oura beach, Miura Peninsula, Kanagawa
  Prefecture,
• Japan, in July 1998.

• Cells are Gram-positive, non-sporulating,
  straight rods,
• occurring singly, in pairs, or in short chains
  and elongated. Motile with peritrichous flagella.
• It is slightly halophilic, highly halotolerant
  and alkaliphilic.

?Aim? Direct lactate fermentation from xylan by
Halolactobacillus miurensis
1) Ishikawa et al., Int. J. Syst. Evol. Microb.,
55, 2427 (2005)
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Characteristics of Halolactibacillus miurensis
Lactate yield(g/g)
Initial pH 8.5? 37oC?Cultivation for 7 days at
initial pH 8.5 37oC

• M23-4 strain was able to produce lactate from
  various xylan and showed high lactate yield.

41
Direct lactate production from xylan and xylan
hydrolase production from H. miurensis M23-4
As xylan hydrolase activity of supernatant
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
Time (Day)
Time (Day)
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Effect of pH on enzyme reaction of xylan
hydrolysis (H. miurensis M23-4)
pH 8.6
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Conclusions

• Induction of expression of xylan hydrolase

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Laboratory of Microbial Technology, Division of
Microbial Science and Technology, Department of
Bioscience and Biotechnology, Faculty of
Agriculture, Graduate School, Kyushu University
6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581,
Japan http//www.agr.kyushu-u.ac.jp/biosci-biotech
/biseibutu/
Kenji SONOMOTO Prof., Dr. sonomoto_at_agr.kyushu-u.a
c.jp
Takeshi ZENDO Assis. Prof., Dr. zendo_at_agr.kyushu-
u.ac.jp
Jiro NAKAYAMA Assoc. Prof., Dr. nakayama_at_agr.kyus
hu-u.ac.jp
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Research Topics in Laboratory of Microbial
Technology

• Diversified microbial utilization of renewable
  resources (Biodegradable plastic materials,
  Biodiesel fuel)
• Bioprocess development and control of anaerobic
  fermentation (Lactic acid fermentation,
  Acetone-ethanol fermentation)
• Biochemistry, genetics and fermentation of
  bacteriocins of lactic acid bacteria
• Functional analysis and development of molecular
  chaperons from halophilic lactic acid bacteria
• Characterization of microbial diversity in
  fermented foods and human intestine by molecular
  approaches (Nukadoko, correlation between
  microflora and allergy)
• Characterization of chemical signals controlling
  microbial behavior (bacterial pheromone)

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Lab. of Microbial Technology Fac. of
Agriculture Kyushu University
Kumagawa River, Kumamoto RAFTING !! September 4,
2006
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Amylose content of starch
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Xylose fermentation
IO-1
SHO-54
SHO-47
7
4
12
OD562nm
3
5
8
pH
Concentration (g/L)
OD562nm
pH
2
Xylose
3
4
Lactate
1
1
Acetate
0
0
0
0
6
12
18
24
6
12
18
24
0
0
6
12
18
24
Time (h)
Lactate 0.97 mol/mol Acetate 1.05 mol/mol
Lactate 0.97 mol/mol Acetate 0.99 mol/mol
Phosphoketolase pathway
Glyceraldehyde- 3-phosphate
Lactate
Xylulose- 5-phosphate
Xylose
PK
Acetate
Acetyl phosphate
53
Xylose fermentation by SHO-47, SHO-54 and IO-1
Ylac, Yace, Yield of lactate or acetate to
xylose Plac, Lactate production rate
54
Xylooligosaccharide fermentation (xylooligosacchar
ide mixture of xylobiose and xylotriose)
SHO-47
IO-1
SHO-54
7
4
7
OD562nm
Xylose
pH
3
5
5
Xylobiose
Lactate
pH
2
Concentration (g/L)
3
3
OD562nm
Acetate
1
1
1
Xylotriose
0
0
0
0
6
12
18
24
0
6
12
18
24
0
6
12
18
24
Time (h)
55
Xylooligosaccharide fermentation by SHO-47,
SHO-54 and IO-1
Ylac, Yace, Yield of lactate or acetate to total
sugar
Plac , Lactate production rate
56
Xylooligosaccharide fermentation
YLa, Yield of lactate to total sugar-consumed PLA-
max, Maximum lactate production rate
57
Conclusion

• Leu. lactis SHO-54 and Leu. lactis SHO-47
  metabolized xylose to lactate and acetate through
  phosphoketolase pathway.

• They fermented xylooligosaccharide.

• They showed higher values of lactate yield,
  lactate
• production rate and xylooligosaccaride
  degradation
• rate than those of IO-1 strain.

• They had high b-xylosidase activities, which were
  
• induced by xylose or xylooligosaccaride.

• The b-xylosidases degraded xylotetraose,
• xylopentaose and xylohexaose exo-geneously.

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