免费文献传递   相关文献

Acetone-butanol-ethanol fermentation from cane molasses by Clostridium beijerinckii DSM 6422

一株拜氏梭菌利用甘蔗废糖蜜发酵生产丙酮丁醇



全 文 :!10
"!

#
2012
$

%
M
 

 
Ê
 
“
 
Ë
 
Ì
ChineseJournalofBioprocessEngineering
Vol.10No.3
May2012
doi:10.3969/j.issn.1672-3678.2012.03.002
OPQI
:2011-03-21
KCR!

ÍÎûÄÅÆÇ8ÉÚÛ
(863
ÚÛ

ÓÔÕÖ
(2006AA02030109)
ST.U

k
 
l
(1985—),
m

%nYoã

påÆÇM

ÆÇæç

M¶¶ªo

q¾r

‚êã
),
ëì
,Email:weiyunw@126.com
¹Šº»¼`5½¾¿ÀÁÂdyeÃÄfÅ
B
 
C
1,
DEF
1,
G
 
H
2,

 
F
2,
I
 
J
2,
BKL

(1
MNO89: ;PQ::A

RS
230036;2
,59: ;<7@:A
78;,^
_
214122)
7
 
l

ÛÜÝÞÅßàáâã

äå
ClostridiumbeijerinckiDSM6422
£¤æ綷©è¥¦¬é껼

ëìÀ
#

íå
H2SO4îïðÅß,éÅñ¸òó60g/L,(NH4)2SO42g/L,CaCO310g/L,ôó30℃,pH55~70,õö
¸
6%(
÷¹Öø
),
ù
5L
¥¦úû¥¦üý
96h,
þÿ!¨¸á
1617g/L,
 û©èñ¸òóá
1007g/L,
þÿ
!¨Òá
302%,
ÅäåÒá
893%。
mno
:Clostridiumbeijerincki;
ÜÝÞÅß

¶·©è
pqrst
:TQ923    
6uvBw
:A    
6x;t
:1672-3678(2012)03-0006-06
Acetonebutanolethanolfermentationfrom
canemolassesbyClostridiumbeijerinckiDSM6422
SUNShan1,WANGWeiyun1,NIYe2,WANGYun2,SONGGang2,SUNZhihao2
(1SchoolofLifeScience,AnhuiAgriculturalUniversity,Hefei230036,China;
2KeyLaboratoryofIndustrialBiotechnologyoftheMinistryofEducation,SchoolofBiotechnology,
JiangnanUniversity,Wuxi214122,China)
Abstract:AcetonebutanolethanolfermentationusingsugarcanemolassesbyClostridiumbeijerinckiDSM
6422wasstudied.ThemolasseswerepretreatedwithH2SO4andthefermentationconditionsincluding
carbonsource,nitrogensource,temperatureandpH,etc.,wereoptimizedTheresultsshowedthatthe
optimalfermentationconditionswasasfolows:initialsugarconcentration60g/L,(NH4)2SO42g/L,
CaCO310g/L,inoculationamount6%(V/V),pH5570,fermentedat30℃ for96h.Underthese
conditions,thetotalsolvent,butanolratio,solventproductivity,andsugarutilizationinthe5Lbioreac
torwere1617g/L,1007g/L,302%,and893%,respectively.
Keywords:Clostridiumbeijerincki;sugarcanemolasses;acetonebutanolethanol
  
($)

ËÌstÓu>vw~$%xD>y
X

†óÀAZM¶zl>;P{ãcÜ)Ü©
>×|

ý·q>sX‰Sþ

M¶89‰M:N
O;PàQ}~¯û

0$%Öø€©ŠØ

,
^8Ɂ#Ö
[1-2]。
  
KLíbK“”ËÌ:M>ï:ù

¼Q1˜
>ò89K

çIK
),
,Íí‚Ö>89al

ƒ~
20
„…
30
$Z

†QKL89M:NO;P
>‡F*ˆ
[3]。
͉;P89ÆÇ¥¦1©óK
—al

ûÆÇ>_·4BíCDE4
(Clostridim
beijenncki)[4-11]。
Ͳ($îQAŠKL89M:
NO;P>ÆÇ*ˆ
[12-14]。
‹ÐFGKLalM
:NO;PŒQA£>ÆÇ)Ù

.9àc1Ž
89M:>Z“”‘’

  
QRFG“Ï”ÍüM¶4s°
(DSMZ)
>ABCDE4
ClostridiumbeijerinckiDSM
6422
ÈæHIKL89M:NO;P>½–ÆÇ

ÿ#óFGJKL89:NO;P>“pXM:
“”>v/~ŠX‘—rs˜™

1 
~%€
11 
`‚ƃ„K
  ClostridiumbeijerinckiDSM6422,
“Ï”ÍüM
¶4s°
(DSMZ)。
  
®Xwx)

¥}{
5g,
\}{
5g,
9y
š
10g,
tuK
5g,
›œq
1mg,
ƒÂ
40mL,
€
h=ƒ=ƒ
05g,
žŸÂ£ •
1L。
  
î-wx)

9yš
3g,
¬\š
10g,
}{
10g,
򡯢z
1g,
tuK
5g,
€h=ƒ=ƒ
05g,
dX£
3g,
¤=£
3g,
›œq
3mg,
žŸÂ
£ •
1L,pH67。
  
89wx)

KL
30~80g,CaCO310g,
(NH4)2SO42g,K2HPO405g,žŸÂ£ •1L,
pH55~70。
12 
ƒ„€
  
4î®Xwx

S¥¦§‡4®Xwx
)s
,¨
Œ
37℃
CD©ªwx
36~48h,
«³‘®
Xwx)Н®X
2~3


‘’î-wx)¨Œ
37℃
©ªwx
12~18h,
¬î-Ȓ0ìM—#Þ
‘’89wx)Èæ89

  
­®wx

‡®XÖ>4îÿ
6%(
–?iì

>‘î*“˜ó
150mL
>
250mL
KT®
s
,¨
Œ
30℃
©ªwx
96h,
¬89¯ޣ£

  
89”wx

‡
3L
89wx)ʒ
5L
89
”s
,121℃
"°~ž±²4
15min,
³´Þÿ
6%
>‘’Œ®XÖ>4î
,30℃
©ª89
wx

13 
r…%¨’
  C.beijerinckiDSM6422
89:Mµ¶NO

;
P

[P

[=ý;=øò¡¯Z[:¶

·G¸S
¹º‰È梣
[15]。
  
¹º‘’
:¸
S¹º»
(VARIAN3900),
¹º
¼ó
PEG 20M,
õ½¾-X¢£¹
(FID),
¼Œ
Ì@`Œ

¿¸ó
N2,ÀÁ™10mL/min,õ¸Â
™
30mL/min,
À¸Â™
300mL/min。
ȨÌU
180℃,FID
ŒU
220℃,
Ȩ˜
10μL,iÂþ
90∶1。
ÿB;P󲯐È棘iž

  
K¼˜££

«K…U££·G<Ä)ÂÅ=
(DNS)
‰
[10],
££<}GtuKó¯°¶

  
4–M¶˜>££

‡4ÆǏÈY>…
U

GÀwx)†óÀ0`

·G
752
iÉÉU
Ú~
660nm
©££E
OD660ð。
  
«¡}>:š£±ó¹óí
1g
«KµF:M
>«¡}»ì

ÿÊiìzè

KFGš£±óó
íKË̒K>—˜Êiþ

0*KL

Kÿ«K

)。
2 
†‡%ˆ)
21 DSM6422
dyeÁÂiÇÈ
211 
ŒU089>?@
  
89ŒU0Ÿ M—~¡}:˜ÇQÍ1?
@

,^}~9;ŒU‘’Èæwx

ÂÃÄY
1。
/Y

òÅ
:C.beijerinckiDSM6422
>ÎÈ89Œ

30℃,
û*΋*
30℃
Ï0NO;P>89
M:QA£>Ј?@

È89ŒUû*
30℃
<

¡}:˜ËÌ89ŒU>`ûÍ"#

òFí89
ŒUËû

4–M—™šËÑ

y84–˃ϡ

:¶YZ‘„Ò

:˜"ú

89ŒUˋ

E4
Ÿ Z["Ó

89Ô#՗

Ÿ M:þUîS
‡ú‹

q
1 
ɝDSM6422
deÊËijk
Fig.1 Efectsofdiferenttemperatureduringthe
fermentationbyDSM6422
212 pH
089>?@
  
i†ÿ9;>wx)½¾
pH
)VW0­®w
x>89:¡}>?@

ÂÃÄY
2。
/Y

òÅ

w
x)½¾
pH
‹*
55
Îû*
70
<

¡}:˜êÖ
7 
!

# k
 


ABCDE4FGHIJKL89M:NO;P
ú‹

~½¾
pH
ó
55~70
<

:¡}Âðþ͘
£

wx)½¾
pH
ó
70
<

¡}:˜Îû

q
2 pH
DSM6422
deÊËijk
Fig.2 EfectofdiferentpHduringthefermentation
byDSM6422
213 
î×089>?@
  
‡ˆ‰~tŒ">4îØ-ِ‘îî-
wx)Þ

~
37℃
¨ŒwxÚsi†wx
16、18、
20、22
~
24h,
££9;î×î->M¶˜

«Û
6%
‘•
150mL
89wx)s

©ª*
30℃
¨ŒwxÚwx
96h,
££«¡}:˜

ÂÃçY

µè

/Y

òÅ

~
22h
<

î-M—ÜÝ

®n
û

³‘89Þ>¡}:˜î{Îû

ÑòFí
,órsˆ•>4îíÿÞߟ ÎRØ-ó_

Èî-wx
22h
<

î-®n{Îû

ÍËÌ
î-wx<Ê>՗

4–*+àX

89¡}:
˜J¾"ú

,^

}~
22h
óî-wx<Ê

q
3 
‚ÌDSM6422
deÊËijk
Fig.3 Efectsofdiferentseedagesduringthe
fermentationbyDSM6422
214 
‘î˜089>?@
  
~
250mL
KT®s“’
150mL
89wx)

‘’9;–?>î×ó
22h
>î-
,30℃
©ªw
x
96h
Þ£«¡}>:˜

ÂÃçY

µè

/Y

òÅ

O4ÍÈá>‘î˜ó
4%~8%,
ȑî˜
ó
6%
<

«¡}:˜Îû

,^

}~
6%
>‘î
˜†ó89>Îȑî˜

q
4 
͂DSM6422
deÊËijk
Fig.4 Efectofdiferentinculumratioduringthe
fermentationbyDSM6422
215 
KL⩪æ‰089>?@
  
aKLs¼Q1˜>ã–

Ùäåi

Òæ
-øM—

ъ¶—òF=?@4B>M—~8
9

!ûKLs>Ùäåi=?@4–M¶˜>
££

KL¡>çUÍ1<

î=?@Ÿ >e
Y89
[16]。
,^

rsG詪>aKL89†0
`

VW9;aKL⩪æ‰0NO;P89>
?@

à–©ªæ‰ç"

1)
°©ª
 
‡aKLÆÇA£…UÞ

12000r/min
°
15min
hé¢

2)H2SO4©ª ‡aKLÆÇÞ,G1mol/L>
H2SO4¡.pH•30,60℃"©ª©ª3hޏ
°’‹®

«G
5mol/L
>
NaOH
.
pH
•
65。
3)
®¯ê©ª
 
‡
5g
åi®¯êʒ
100mL
ÆÇÞ>KLs
,50℃
"©ª©ª
20min
ÞËëªh®¯ê

G
5mol/L
>
NaOH
.
pH
•
65。
/z

òÅ

aKLìË⩪Þ89:˜þ
詪>KL89:˜QÞê>‘û

Esÿ·G
H2SO4⩪aKL89íÃÍÖ。~KLsʒ
H2SO4=XÞ,òÿHAŠò¡¯>îi(ç)
=ƒ~øiQR=ƒ

Zó9¡¯>
CaSO4Ď
¢

òÕÖתhKLs>M—

!ïðøiã–

,^·Gñò†>
H2SO4=X⩪>æ‰)©
ªKL

216 
½¾«K…U089>?@
  
i†ÿ9;…U>½¾KLób>89wx
)Èærs

ÂÃÄY
5。

M
 

 
Ê
 
“
 
Ë
 
Ì
  
!
10
"
 
 1 
ÁÂÎÏЀDSM6422
deÊËijk
Table1 EfectsofpretreatmentmethodsonthesolventproductionfrommolassesbyDSM6422
g·L-1
©ªæ‰ ρ(NO) ρ([P) ρ(;P) ρ(«¡})
詪
065±01 035±03 309±01 439±03
°©ª
053±04 044±01 351±01 484±05
H2SO4©ª 130±05 053±03 578±02 761±10
®¯ê©ª
133±07 057±02 489±01 679±10
 
q
5 
ŽÁÂÑÁҐœDSM6422
deÊËijk
Fig.5 Efectsofinitialtotalsugarconcentration
duringthefermentationbyDSM6422
  
/Y

òÅ

ËÌ89wx)½¾KL«K—
˜…U>9GPÊ

«¡}>:˜ô‹`õö

È
89wx)½K—˜…Uó
60g/L
<

«¡}:˜
{Îû

Ë̽K—˜…U>Ϝ‘û

«¡}
:˜Ë½K—˜…U>PÊÍ"#

!û÷K—˜
…UêÖPÊ

øÞȽK—˜…Uû*
60g/L
<

û—˜…U>K9F*‘û¡}:˜î9F*
ùúM:Ze

,^}~
60g/L
ó89>½¾«K
—˜…U

217 
‰Ê

u089>?@
  
KLalsª1¼Q
40%~50%
>«K

_·Z
iíIK

tuK~ÃK

‰
,`
¼Q#˜>

u

Ë~89wx)sÆÊ9;>AR

u~QR

u

ÿÆÊ«>¼

˜Ú~
2g/L
ãä
),
VW9;‰
Ê

u0O4FGKL89M:NO;P>?@

Â
ÃÄY
6。
/Y

òÅ

O4FGÍÖ×FGAR

uÈæ¡}>89M:

û™z

9;AR

u>ü
ý

S89>ìþ¯Vÿ

}~
(NH4)2SO4†ó89
wx)>

u

q
6 


ÓDSM6422
deÊËijk
Fig.6 Efectsofdiferentaddednitrogensources
duringthefermentationbyDSM6422
 
2 
ÔÕ

ÓÖחØ
Table2 Pricesofdiferentinorganicnitrogensources
AR

u
(NH4)2SO4 !=" ¤=" NH4Cl #¿ (NH4)2HPO4 NH4HCO3
üý
/(
$
·
%
-1) 950 4500 6200 1500 2000 5300 1600
218 CaCO3Æʘ089>?@
  
ÈüM¶~M—ËÌs9G:=<

ʒ>
CaCO3&9GסÃ!ý=ÈæЇ,ÿ CO2>
(Ç)å)

s~1wx)>*=¯

,^~NO
;P89ËÌsàQ#Ö>Ó+
pH
>†G

VW
CaCO3>Æʘ0O489:¡}>?@,ÂÃç
Y

µè

  
/Y

òÅ
:CaCO3>ÆÊ0O4FGKL8
9:«¡}QêÖ>?@

ËÌ
CaCO3Æʘ>


«¡}>:˜îËÉP—

È
CaCO3—˜…
U{
10g/L
Þ

ϜÆÊ
CaCO3089>?@
91

µÿ·G
10g/L
ó
CaCO3>ÎÈÆʘ。
9 
!

# k
 


ABCDE4FGHIJKL89M:NO;P
q7 CaCO3š›\DSM6422deÊËijk
Fig.7 EfectsofdiferentCaCO3concentration
duringthefermentationbyDSM6422
219 
ŠX‘’"­®89rs
  
~ÿ‹ŠX‘’"Èæ­®rs

½¾«K—
˜…Uó
60g/L,
89ŒUó
30℃,
½¾
pH70,
CaCO3>Æʘó10g/L,ÂÃÄz3。/z3ò
Å
:96h
Þ8 9 ¡  s > ; P — ˜ … U ó
1032g/L,
«¡}:˜ó
1627g/L,
;P~«¡}
:˜i†Ïþ0`rs

KL½K—˜…U
50g/L,
½¾
pH65)
‘û1

é

÷KS
3882g/L
ú•
682g/L,
K>FGš‘û1
533%。
 
3 
ÙÚÛܧÊËe—Ø
Table3 Comparisonofsolventproductionunderinitial
andoptimizedfermentationconditions
g·L-1
¡} ρ(NO)ρ([P)ρ(;P)ρ(÷K)ρ(«¡})
ŠX
0.65 0.35 3.09 38.82 4.39
ŠXÞ
4.09 1.86 10.32 6.82 16.27
22 5L
dÝpi
DSM6422
dÞß
  
·GŠXÞ>­®‘’Èæ,1wx

VW
C.beijerinckiDSM6422
FGKLalÈæNO;P
89>=¯
。5L
89”
(BIOF6005A)
s“˜ó
3L,
89½¾KL«K—˜…Uó
60g/L,
½¾
pH
ó
70,CaCO3>Æʘó10g/L,‘î˜ó6%,8
9ŒU
30℃,
¸ó
100% CO2,CD8996h,§
sÂÃÄY
8。
  
/Y

òÅ
:C.beijerinckiDSM6422
~89È
æ•
22h
ãä

4–M—-™

4–M—>ËÌs
.ËÌQR=>?@
、CO2~H2øsÊ:¶>:M。
:=6789
pH
-™"ú

¿Q#˜>=³X
ó¡}

È
pH
ú•Î‹<

89>`a¯/çP
q
8 DSM6422
ràdyeÊË
Fig.8 Timecoursesofcelgrowthandproductionof
solventduringbatchfermentationbyDSM6422
þ

89s1˜?@>=&`aZNO;Pø

pH
J¾0`

89Ȓ:¡}67

È¡}:˜
{A£…UÞ

,;PøQR¡}04–Qá1†
G

4–J¾2àÎÏ¡

89õ*¯

/Y
8`
òÅ

O4FGKL89>½¾«K—˜…Uó
60g/L,96h
<«¡}—˜…Uó
1617g/L,
Es
;P—˜…Uó
1007g/L,
«¡}:šó
302%,
KFGšó
893%。
3 
†
 
)
  
Ë0­®wx‘’ÈæŠX

؏
Clostridi
umbeijerinckiDSM6422
FGKL89M:NO;
P>Îȑ’

½¾«K—˜…U
60g/L,CaCO3
10g/L,
89ŒU
30℃,
½¾
pH55~70,
!ûO
4FÍÖ×FG3ü>AR

u
(NH4)2SO4。·G
ŠX>­®89‘’~
5L
89”sÈæ)1§
s


96h
<«¡}{
1617g/L,
Es;P—
˜…U
1007g/L,
«¡}:š
302%,
KFG
š
893%。
M©6u

[1] NiYe,SunZhihao.Recentprogressonindustrialfermentative
productionofacetonebutanolethanolbyClostridiumacetobutyli
cum inChina[J].ApplMicrobiolBiotechnol,2009,83:
415423.
[2] 
kN4

M¶;P:p>8É5öýxD6L
[M]∥2009“
pM¶ÄÅ8É*7

8j

Ñoå9:
,2009:335344.
[3] AroyoR.Artofproducingbutanolandacetonebyfermentationof
molasses:US,2113471[P].19380405.
[4] KeisS,ShaheenR,JonesDT.EmendeddescriptionsofClostridi
umacetobutylicumandClostridiumbeijerincki,anddescriptions
ofClostridiumsaccharoperbutylacetonicumsp.novandClostridi
umsaccharobutylicumsp.nov[J].IntJSystEvolMicrobiol,
01
M
 

 
Ê
 
“
 
Ë
 
Ì
  
!
10
"
 
2001,51(6):20952103.
[5] QureshiN,SahaBC,DienB,etal.Productionofbutanol(abio
fuel)fromagriculturalresidues:Ⅰ.useofbarleystrawhydroly
sate[J].BiomassandBioenergy,2010,34:559565.
[6] QureshiN,SahaBC,HectorRE,etal.Productionofbutanol(a
biofuel)fromagriculturalresidues:I.useofcornstoverand
switchgrasshydrolysates[J].BiomassandBioenergy,2010,34:
566571.
[7] EzejiT,MilneC,PriceND,etal.Achievementsandperspec
tivestoovercomethepoorsolventresistanceinacetoneandbuta
nolproducingmicroorganisms[J].ApplMicrobiolBiotechnol,
2010,85(6):16971712.
[8] QureshiN,SahaBC,HectoraRE.Butanolproductionfrom
wheatstrawbysimultaneoussaccharificationandfermentationu
singClostridimbeijenncki:Ⅰ.batchfermentation[J].Biomass
andBioenergy,2008,32:168175.
[9] EzejiTC,QureshiN,BlaschekHP.Bioproductionofbutanol
frombiomass:fromgenestobioreactors[J].CurOpinBiotechn
ol,2007,18:220227.
[10] QureshiN,LolasA,BlaschekHP.Soymolassesasfermentation
substrateforproductionofbutanolusingClostridiumbeijerincki
BA101[J].JIndMicrobiolBiotechnol,2001,26:290295.
[11] ParekhM,FormanekJ,BlaschekHP.Pilotscaleproductionof
butanolbyClostridiumbeijerinckiBA101usingalowcostfer
mentationmediumbasedoncornsteepwater[J].ApplMicrobiol
Biotechnol,1999,51:152157.
[12] 
;vÀ

ã¥<

=4

ø

FGKL89M:;P4B>i
>}–A£
[J].
?@ÑÄ
,2010(5):3235.
[13] 
Aeù

lBC

D¥E

ø

FGFGKLkl89M:;
P
[J].
M¶Ê“ËÌ
,2010,8(6):69.
[14] 
$ÉH

+IM

JK

KL89M:NO;P>ÆÇ
[J].
“
püM¶
,1999,29(2):2832.
[15] 
ÜLM

N¥O

PQR

SŸ§¸S¹º‰ižNO~;P
89:¶
[J].
0ùý89“p
,2007,33(11):115117.
[16] LiuYupeng,ZhengPu,SunZhihao,etal.Economicalsuccinic
acidproductionfromcanemolassesbyActinobacilussuccinogenes
[J].BioresTechnol,2008,99(6):
櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒櫒
17361742.
,-./
p,«¬áây#$ã&ä½V¼`i勌 ¡æç
sÍÑo4üM¶Æǵ+TÆÇV~UÕðïÆÇVVy>ÆÇòEJ8åAWÈG*NO;P
E4>ûíxyz{êq

Oêq&fXó
pGusA22tetO1,
íÿdr¿ö)³YV),>òZ-ó)Ù

Ë[’)Ï
Tn10
³\->
tet
./z{$’uv>MYxyÁz{

Oêq>_·=Øí
:1)
ûí
 
~ÆÊμg/L]>3Âô$r¿‘’",xyz{þU¸ò‘û2]ì
˜]
;2)
ò.
 
z{þ*ò{xy¶3Âô$r¿…U>./
;3)^
_
 
E^_¯òÿ`›AŠá¯}
67),>[’
;4)
ñ*HG
 
~¼Kwx)sÆÊ3Âô$r¿¸òrùxy

ÍA¶abwx)

·
GÑWxyÁz{êq

òÿ0NO;PE4s>NOc),ÈæJ

Nò†

ŠX),>z{˜

ÿ–8
ÉÀÁ>ûí<·ò†êq

ódmYZM¶oef~NO;PE4s>‡GX£)Ù

SNÆÇL¥
2012
$8z~
《MetabolicEngineering》
!



èhéêëìíîïpðñ
Íghijo#

klm

n>ÆÇor

kpl=òQíú‹qrsst

0Aqrsuvw>
ã[

íÃÕóÞê

/81j4xyø

gszÑoÎ{Z>ÆÇêè

kpl=FHqrs8Mstú‹
18%。
ËÌ¡
âÔ#՗

8Mqrs>st"ú

IÌ{

$ÿ‹<

stò"ú
29%。
AuvQw>ã[kpl=>
íÃÖ*QuvQwR

Rqrs8Mstò"ú
25%,
zÞkpl=>A]â|íÃÎÖ

}G~ÂÃsl=¼˜~

QÔ*ú‹;Á€h=Âð

ÐÍ

Sâ|qrs>TUVÿ

€
0‘—>l=˜`9G

¶·‚’A£˜>l=b}Îl=þX0ù



l=>kp!9탩ƒÖ

=†í„G1}˜l==…†¾M¿
B12
>w‡

>Z9òJ>ˆìêq‰Š

€‰

ç쭋R„
G1˜l=

QŒÈ¬­M—>òF

µÿ

~Q¶·kp<

Î֍ŽA"jM


Ú[%

11 
!

# k
 


ABCDE4FGHIJKL89M:NO;P