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Optimization of PHAs production by Bacillus sp. PB-3 from waste activated sludge

废弃活性污泥中产聚羟基脂肪酸酯菌株Bacillus sp. PB-3的发酵条件优化



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ChineseJournalofBioprocessEngineering
Vol.10No.5
Sep.2012
doi:10.3969/j.issn.1672-3678.2012.05.006
NOPH
:2011-12-08
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(31100088);
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),
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,Email:lq_ujn@126.com
01ÖÞ23eûúJ45÷6t“
Bacilussp.PB 3^
]•– ¡
–/—
1,2,
‰
 
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2,
y
 
p
3,
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2,
›
 
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“pž0F05

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NOÌ%zÍÎ5ÖêϙÛÐÐÑ

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òšãÄ ÆØٗÚ
(PHAs)
Óñò
BacilusspPB 3,
ÛܯÎÝ5Þa“ñò½ßã2!Äβ Æ¯—Ú(PHB)。ÏÄÅÆ
‡ì¡ÑÒ()=>Y

àkþgÄÅáâ
:12g/L
Ó£¤/!

ã
,2g/L
Óäåæ!

ã

%&
pH75,
ÄÅÆç‘Ü
80mL,
¬ô!
200r/min,37℃
ÄÅ
48h,PHB
cÜìídÿñÎècÜÓ
3209%,
6=>
lïš
30%。
bcd

Ä ÆØٗÚ

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:A    
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:1672-3678(2012)05-0028-06
OptimizationofPHAsproductionbyBacilusspPB3from
wasteactivatedsludge
HAODake1,2,LIQiang2,HANSheng3,WANGJiajia3,LUOYan2,GONGKai2
(1.ShandongAcademyofMedicalSciences,Jinan250062,China;2.SchoolofMedicineandLifeSciences,
UniversityofJinanandShandongAcademyofMedicalSciences,Jinan250022,China;
3.SchoolofChemistryandChemicalEngineering,UniversityofJinan,Jinan250022,China)
Abstract:AhighyieldstrainofPHAs,Bacilussp.PB3wasisolatedfromwasteactivatedsludgeby
Nileredstainingandfluorescencemicroscopyscreening.Gaschromatographyanalysisindicatedthatthe
intracelularproductionofthestrainwaspolyhydroxybutyrate(PHB).Furthermore,mediumcomposition
andfermentationfactorswereinvestigatedandoptimized.Theoptimalcarbonandnitrogensourceswere
glucose(12g/L)andbeefpaste(2g/L),respectively.TheoptimalcultivationconditionswerepH75,
37℃ for48h,and80mLmediumatanagitationrateof200r/min.Underthefermentationconditions,
theyieldofPHBwasraisedto3209% (w/w),30% higherthanthatbeforeoptimized.
Keywords:PHAs;PHB;screen;fermentation
  
UdIefXg
(PHAs)
ÝÑTL{coT
Â[\t}çw2é(P

{¯˜Lp

ÅÓ0
Gsé“ATL3ÆJK

Ýã;”³2´Y`é
2}~ª°kà
[1-2]。
éÁ"©ª
PHAs
#ß6ã
T>ªÄµm2ö÷

ž+µž‰ï2GHIã;
‡ß2±´

  
_`abÝ]böœc¹º}ª2ÑTL„
;†L2U©,

sc—;Ÿ(2
PHAs
©ª
S
[3-4],
PJ¤aT>
PHAs。
(abcqnN2©
ª
PHAs
2Sh

P²abçèGHI

²•Gab
2¾{9ˤ
[5-6],
*Ù²J)/T>ªÄ

ä[/a‡

ã;‡ß2ÅÓP•b5¤ïA
[7-8]。
  
6xy
[9]
(³ý´]^2_`abc

@
µoµ™‡Ó

²ÏqnN

h
PHAs
>TS
HG B 1,
hû»è¶·š¸HGS˜

01
GHÇ
PHAs
2>(è
234%。
¹Ã(y
[10]

º»x¨Ä7

(abcqž

h>U β dI.
Xg
(PHB)
Sh
AE13,
hû»è¦¦S˜


8901[\žý
PHB
>(è
085g/L。
2˜Î
y
[7]
-Nl¼GS2
phaG
Ižd6m

(
A/O
aböœYZ2{|º=2_`abc

q
nµž
PHAMCL©ªS,0GQSéJNO<è C
{

ŽTÂ2|I©ª
PHAMCL,>%öŽá
p(2
3137%。
  
Š‹(]^_`abc²Ïqný

h>
PHAs
2Sh
BacilusspPB 3,
-s©ª2
PHAs
¼Ùd/»`P»(²³

Ļs#ߪ
²è
PHB。
ÄKcŠ‹ÈˤabçèGHI#
ߪ²

-
PB 3
>
PHB
GH[\d89

#d
è01T>
PHB
çN;ùŽõ

1 
op%rs
11 
tu
  
›Ï“ÄŸaböœ´2_`abqnN2
m>
PHAs
Sh

12 
vwJ
qnGHI
(g/L):
‰½^
5,
”“¡
5,NaCl
2,
¢e
20;pH75(¯
°)
50℃
`a
,´
l
50
mg/L
º»™‡©Jqn
PHA
©ªS
)。
  
z*GHI
(g/L):
NO<
10,
‰½^
2,CaCl2
01,MgSO402,NH4Cl008,NaCl1,KH2PO4005,
Na2HPO4·12H2O006;pH75。
  
01GHI
(g/L):
NO<
10,
‰½^
2,CaCl2
01,MgSO402,NH4Cl008,NaCl1,KH2PO4005,
Na2HPO4·12H2O006;pH75。
13 
t“^78
131 
Sz2²Ï
  
²_`abçڐ½L

«¬Àqn2GHI



30℃
IÍÆ
30min,
VϮGH
3d。
132 
Sz2ƒ9
  
¤±zÛ¾­GS¿

±zý€52Ÿ,GH
Ic§¯

R9j$Z)žýŸÏ

}~:Þ2S
¿



žýƒGH2ShÇ

ÆÀ
4℃
°±

[11]。
133 
Œq
  
@µº»™‡Ó

©¨ÄñÑllÖ


PHAs
Sh2Œq

134 
9q
  
²Œqµž2Shdލƒ9Ç

dž³0
1GH

@槯
PHAs
2—(



h>
PHAs
éå±Ü2N0Sh

14 
t“^9Ù
141 
Sh2}~1N
  
Ÿ,GHI¯GHÇ

1NS¿}~r©,
01TÂ|†

@µ)ld1N

142 
º»Sh2TœT9R5
  
@µµC9HFTª

ð›©9

‚bK

À
ÁTª

©ªXsˤ

ÂXsDŒ
、H2S}ªrVP
¼–y

º»sTœT9R5

143 
Sh
16SrDNA
2¿º»
[12-13]
  DNA
,­→PCR6mR5→)Ã1N→PCR
>Lƒ9r
16SrDNA
¿º»

15 
ë^ÙÞg¨
  
ˤ
GC
Öº->Ld»`²³
[14]。
16 PHAs^

  
S,.d
:100mL
01©
,6000r/min
ÏÐ
20
min,
S,¤KÏ*b“c

ÏÐ

‡9

w
,¯
¯á
âÇ

vp(¤

  
²¬³2S,ÆÀ
80%
p/c
,50℃
Ä,
90min,
öœÇ2S,c´lÅÆPp/R©©

,ç°
2∶1),80℃
tÄ,
5h,
µ·

·©Çw¸
KÅÆ
,´
lüb¥/³N
PHAs,¯
¯ÏÐ
20
min(5000r/min、10℃)
Ç

‡9

w

³N2 
¡
60℃
e á

ž ý È “ Ó É Ò ™
PHAs,
v
p(
[15]。
17 
vwJ&g®]•–^ ¡
  
GHIª²289£¤Ig01GHI

9:
s¬ª²{„

²Ý´l{|2

{
、C
{

  
GH[(


ή
pH,
Úµ
,N、C
{´µ

´©(

289£¤Ig01GHI

Èލ89
J¯Þ892[\èIg

92 
!

#
  
­ŸPy

]^_`abc>UdIefXgSh
Bacilussp.PB 3
201[\89
2 
xy%z)
21 
t“^g:q9Ù
  
(_`abcqž

hm>
PHAs
2Sh

Ü
Ýè
PB 3,
èÊËÖv`S

S¿ó•“Ó

Ì
}

{gð

Z?
021~046cm,
2À„GHI²
Ï

S,ó¨Ÿ2ÍÌ}¡™

ÊTμ

ã;
º`

  PB 3
ShébK‚

±ÏFPÐE


VP
R5

ÂXsDŒR5óv`

„NO<ç¤>
X{>«

›*Ñá;ðñÒÓ

Pí³œË¤<
P;†X

ÖÛJ¯|†P}~|†

"ÛKL
[16],
ŽS
PB 3
AŒû»èš¡S˜
(Bacilussp)
Sh

  
Sh2
16SrDNAPCR
)ÁyUÞ



¤
BLAST
Ê\„
Genbank
cg"Ô2
16SrDNA
¿
d|{`°±žà

„
PB 3
|{`ý
95%
J¯2Sz

œ¦š¡S
(Baciluslicheniformis)、
:;š¡S
(Bacilussubtilis)、
K‚š¡S
(Bacilusamyloliquefaciens)。
›+ÕñQS˜š
¡S˜

1—Maker;2,3—
Sh
PCR
y
f
1 16SrDNAPCR
V;xy
Fig.1 Resultsof16SrDNAPCRelectrophoresis
22 
ë^ÙÞg¨
  
²>L
PHAs
ˤ«€ÓÔ
(GC)
²³

yU
Þ



›Þ




t=35min`
aNG2ã
„
PHB
QñRã2NGIʀ|

ØðSh>T2
Lpè
PHB。
!

™ÓÇ$èQñÙ#$

•$è¼Ù#$
f
2 
t“^
GC
õØxy
Fig.2 ResultsofGCanalysis
23 
vwJ^ ¡
231 C
{-S,TÂr
PHB
çw234
  C
{-S,TÂr
PHB
çw234ÚÞ
3。
f
3 C
íZtY„…®
PHB
<=^_`
Fig.3 Efectsofdiferentcarbonsourcesoncel
growthandPHBaccumulation
  
›Þ



‚

:l°±;ËÀS,2T
Â

;PHB
2æ¾

{

;PHB
2æ¾

{

éÝ
PHB

S,cTö2°*±)

ž+

nýNO<çèæ
¢

{

232 N
{-S,TÂr
PHB
çw234
  N
{-S,TÂr
PHB
çw234ÚÞ
4。
›
Þ





ÝS,TÂP©ª
PHB
±èœ?
2

{

scJ‰½^èæ¢

{

{î;ËÀS
,TÂ

|ID;ËÀ
PHB
2çw
,PHB
p(´µ

099g/L。
ü†

{c2Â~

áéAS,
ˤ

TžŽ2áp(±m

-sÝ
NaNO3,S,
ÂS”³

Žáp(Pý
281g/L,
驪2
PHB
{“œ?

{ËÀ
PHB
2çw


PB 3
S
ˤ;†

{2éå8Àü†

{

›À
PHB
Ä
Ö{—f

‰½^çèÞz:x

{érQS,
03
T
 
L
 
´
 
Y
 
µ
 

  
!
10
"
 
f4 N
íZtY„…®
PHB
<=^_`
Fig.4 Efectsofdiferentnitrogensourcesoncel
growthandPHBaccumulation
{|TÂI#-

{2]ß
,^
s´µ-
PHB

ª34{Ÿ

ž+Ç:J
2g/L
‰½^çè

{

24 
vw•–^ ¡
241 
GHIÊ-S,TÂr
PHB
çw234
  
GHIÊ-S,TÂr
PHB
çw234Ú
Þ
5。
f
5 
vwŽ#ZtY„…®
PHB
<=^_`
Fig.5 Efectsoftrainingtimeoncelgrowth
andPHBaccumulation
  
›Þ



Sh2T¾5#è
12h,12h
ÇdlP£TÂ#
,48h
Çdl‚×#

ìíT
L(2m´

>L
PHB
—(ùúm´

›+Pà

PHB
2çwPm´Ý›TL(2m´åˆ2

!
ð
PHB
2çwéå„TL(ªÍ°

ìÇ2
24~36h
Ý
PHB
çw2_Ø#
,36h
Ç
PHB

wótJRS


48h
IýæmA

W
48h
è
æ¢GHIÊ

242 
ή
pH
-S,TÂr
PHB
çw234
  
›Àž³01µ¶c2
pH
øJF¬

ïé
F¬01©2ŒÆ
pH。
ž+

ļ–Ù¬/æ¢
GHIÇ

ž³01GH[\tGH
48h,
M
NGHIή
pH
-S,TÂr
PHB
çw234

yUÞ



f
6 
vwJ>?
pH
ZtY
PHB
<=^_`
Fig.6 EfectsofinitialpHcelgrowth
andPHBaccumulation
  
›Þ



QShã;±2Xë¾5âã


pH
è
6~8
2âã}:é01GHIc”³


TL(P
PHB
>(„9{Ÿ


pH
è
6~
75
2âã}
,pH
-
PHB
,}©ª234±Ï


pH
è
75~80
ÊI

jºS,TL(J
)

é
PHB
2©ª;mWRS

ž+PJeº
PHB
ë`ôõ›`p±Y»

áXÝ!ë`ôõ-À
S,
PHB
2çw34{Ÿ

;ÅÓ0G
[17],pH≥8
I;ËÀ¤©ª]bˆ92_`abT>
PHB,
å
pH

7~8
âã}I

ˆ9º=ab©ª
PHB

å{ü
pH
234

*á!ð_`abPJY»©
ª
PHB。

pH
è

P

I
,PHB
©ªüý
pH
2
34±Ÿ

ž+T>
PHB
IP@µF¬
pH

75
è梌Æ
pH,
(^,m
PHB
>(

243 
NO<-S,TÂr
PHB
çw234
  
NO<-S,TÂr
PHB
çw234ÚÞ
7。
f
7 
ñò¤¼€½‹Z
PHB
<=^_`
Fig.7 Efectsofglucoseconcentrationoncelgrowth
andPHBaccumulation
  
›Þ



ìíNO
Öº
ý2TL(„
PHB
—(óÞZ¯ó2RS
,^ PHB
—(NO12g/L
Iýæm

ìí
13 
!

#
  
­ŸPy

]^_`abc>UdIefXgSh
Bacilussp.PB 3
201[\89
NO14g/L
I
,PHB
—(ä;T
tJ

WNO<2p(´µè
12g/L
æ¢

ÑTL
PJƔ²sˤ

^£¤mÀ
12g/L
2NO<
p(´µd01

01©c¹
sŒžPéÝNO<12AÑTLˤdl7=
ÚôÇ


PHB
©ª>?}ª
PHB;^
ìíN
O
m´µ2NO<{VËÀS
,çw
PHB,
ŒžÝ…Û2ôõê;ËÀSh
PHB
2çw

*ÞG܄KL
[18]
2Sh-ŠL2]
³(€5

244 
GHÚµ-S,TÂr
PHB
çw234
  
GHÚµ-S,TÂr
PHB
çw234UÞ



f
8 
vw!‹ZtY
PHB
<=^_`
Fig.8 Efectsofculturetemperaturesoncel
growthandPHBaccumulation
  
›Þ



S,TÂìÚµ2ómðñóI
€Ým^ǂ[2RS

Úµè
35℃
I

Öºý
PHB
>L—(PTL(æm

ÝS,TÂP
PHB
©
ª2æ¾TÂÚµ

åÚµmÀ
35℃
I

S,áp
(

WTL(

„
PHB
 ¡:ótJRS

245 
GHI´©(-S,TÂr
PHB
çw2
34
  
GHI´©(-S,TÂr
PHB
çw234
ÚÞ
9。
›Þ



GHI´©((-S,Ž
áp(234{Ÿ

éÝ-
PHB
çw234íŸ

å±z(PGúÞ»I

ìí01GHI,ç2m
´

Žáp(€[,PHB
—(áðñtJ

åGHI´(è
30mL
I
,PHB
—(æŸ

…ð
PB 3
S201Ý]C~01

@µƒ„´©(a
EM@«(
,´
©(ÏF@«(Ÿ

GHI}&C
bcám

¾©S,TÂ

|I;ËÀ
PHB
2çw

MÞý01µ¶cGH©2w0JrS,7=
P>(yžE234

£¤
50mL
2GHI´©(

f
9 
vwJ@€ZtY<=
PHB
^_`
Fig.9 Efectsofculturemediumvolumeoncel
growthandPHBaccumulation
  
@µ¯µ-GHIª²JrGH[\289

(^;ž89Ç2
PHB
—(PJý
3209%,
±
89",m/
30%。
3 
x
 
)
  
(_`abc²Ïqný

hm>
PHA
2S
h

hTœT9R5r
16SrDNA
¿°-

û»Q
S蚏¡S
(Bacilussp)PB 3,
ˤ
GC
-}
PHA
d²³

Øðs#ߪ²è
PHB。
  
@µGžE¼–-sGHIª²r01[d/89

Ä»æ¢


12g/L
NO<

æ¢


2g/L
‰½^

æ¢01[\èGHIŒÆ
pH75,
GHÚµ
35℃,
GHIÊ
48h,
GHI´©
(
50mL。
  
89Ç
PHB
—(ý
3209%,
°89",m
/
30%,
|fÅÓc>(±m

èßÇˤab
01T>
PHAs
,Ž/œ+Ã

|I;xBK/
6¡9¼©ª°àPŸ(abÍMT.,2&‡
a‡ö÷

èT>PJK2ô9°à,Ž/‰ï2
Έ

ßÇ2•–cPMÞ;¤]bçèGH
I

¾(o´¥Xs

J#µž©ª
PHBV(
Ud
I.XàXMUg

201[,
è]b2¾{9
ˤ¶N;ùŽõ

L6j

[1] MadisonL,HuismanGW.Metabolicengineeringofpoly(3
hydroxyalkanoates):fromDNAtoplastic[J].MicrobiolMolBiol,
1999,63(1):2153.
[2] RandriamahefaS,RenardE,GuérinP,etal.Fouriertransformin
fraredspectroscopyforscreeningandquantifyingproductionof
PHAsbyPseudomonsgrownonsodiumoctanate[J].Biomacro
mol,2003,4(4):10921097.
[3] SatohH,IwamotoY,MinoT,etal.Activatedsludgeasapossible
23
T
 
L
 
´
 
Y
 
µ
 

  
!
10
"
 
sourceofbiodegradableplastic[J].WaterSciTechnol,1998,
38(2):103109.
[4] LawKH,LeungYC,LawfordH,etal.Productionofpoly
hydroxybutyratebyBacilusspeciesisolatedfrommunicipalacti
vatedsludge[J].ApplBiochemBiotechnol,2001,91/92/93:
515524.
[5] 
Ü



âãä

y

[Ö_`ab3ƾ{9ˤÑ
TLdª®¯
[J].
ÑTL¼@i
,2011,38(4):481486.
[6] 
!åó

¶æ-

[Ö_`aböœ®¯r0hRS
[J].
ç
—îl9Y
,2009,37(1):7174.
[7] 
2˜Î

èñÿ

éêë

y

_`abc
PHASMCL©ªS2
qnr|`ÅÓ
[J].
‹Æôõ»¼¼i
,2006,25(2):
517521.
[8] DiasJML,LemosPC,SerafimLS,etal.Recentadvancesin
polyhydroxyalkanoateproductionbymixedaerobiccultures:from
thesubstratetothefinalproduct[J].MacromolBiosci,2006,6
(11):885906.
[9] 
6x

ìIí

îï‰

y

>UdIefXgŽS2qn
„û»
[J].
ÑTL¼i
,2010,50(11):14881493.
[10] 
¹Ã(

ðñx

òó

>U β dI.XgSh2qnr
01[\289
[J].
TL®¯@i
,2010(1):103106.
[11] CharterisWP,KelyPM.Antibioticsusceptibilityofpotenticaly
probioticactobacilusspecies[J].JFoodProtect,1998,61(12):
16361643.
[12] JohnG,NoelR,PeterH,etal.Bergey′smanualofdeterminative
bacteriology[M].Baltimore:Wiliam&Wilkins,1992:768770.
[13] 
Î3ô

õö»

…ÚŽSÚ¡û»Ÿ÷
[M].
¿À

»¼N
Á•
,2001:346348.
[14] ChaiXS,DongCX,DengYL.Insitudeterminationofbacterial
growthbymultipleheadspaceextractiongaschromatography[J].
AnalChem,2008,20:78207825.
[15] WendlandtKD,GeyerW,MirschelG,etal.Possibilitiesforcon
trolingaPHBaccumulationprocessusingvariousanalytical
methods[J].JBiotechnol,2005,117(1):119129.
[16] BuchananR,GibonnsNE.
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[M].8
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