I HC QUC GIA THNH PH H CH MINH

TRNG I HC KHOA HC T NHIN

K THUT LN MEN
CNG NGHIP
(Bn tho)














TS. HONG VN QUC CHNG













Thnh ph H Ch Minh - 2011

1
MC LC

CHNG 1. KHI NIM LN MEN V NG DNG ................................... 3
1. Khi nim ln men ................................................................................................. 3
2. ng dng ca cc qu trnh ln men ..................................................................... 3
2.1. Sn xut sinh khi vi sinh vt .......................................................................... 3
2.2. Sn xut enzyme vi sinh vt .............................................................................. 4
2.3. Sn xut cc cht trao i vi sinh vt .............................................................. 4
2.4. Sn xut cc sn phm ti t hp .................................................................... 5
2.5. H tr cc qu trnh bin np .......................................................................... 8
3. Qu trnh pht trin ca cng nghip ln men ....................................................... 8
CHNG 2. NG HC TNG TRNG CA VI SINH VT
TRONG QU TRNH LN MEN 9
1. Phn loi phng php ln men ................................................................................ 9
2. Phng php ln men m.......................................................................................... 9
3. Phng php ln men lin tc................................................................................. 12
4. Phng php ln men m-b sung (fed-batch) ....................................................... 14
5. Ln men mt cao ................................................................................................ 16
CHNG 3. S LU TRNH H THNG LN MEN CNG
NGHIP 18
1. Gii thiu ................................................................................................................ 18
2. Cc cng on ca qui trnh ln men ...................................................................... 19
3. H thng thit b lin quan n qui trnh ln men .................................................. 22
CHNG 4. MI TRNG NUI CY TRONG LN MEN CNG
NGHIP 27
1. Gii thiu ................................................................................................................ 27
2. Thit k mi trng ln men .................................................................................. 27
3. Cc thnh phn ca mi trng ln men ................................................................ 30
4. nh gi mi trng ............................................................................................... 43
5. Phng php ng ha to dung dch glucose t tinh bt ................................... 43
6. Phng php x l mt r ng to dung dch ng phc v cho ln men ........ 45
CHNG 5. KH TRNG TRONG LN MEN CNG NGHIP .................. 46
1. Mc ch kh trng trong ln men ......................................................................... 46
2. Yu cu khi kh trng ............................................................................................ 47
3. Phng php kh trng ........................................................................................... 47
4. ng hc s cht ca vi sinh vt bi nhit ............................................................. 48
5. Kh trng mi trng ............................................................................................. 51
5.1. Kh trng theo phng php lin tc .............................................................. 52
5.2. Kh trng theo phng php m ..................................................................... 55
6. Kh trng ni ln men ............................................................................................ 55
7. Lc khng kh ......................................................................................................... 57
CHNG 6. KIM SOT NUI CY ............................................................ 58
1. Kim sot tng trng t bo .................................................................................. 58
2. Kim sot pH .......................................................................................................... 60
3. Kim sot nhit .................................................................................................. 61
4. Kim sot hm lng oxygen ha tan trong mi trng ln men .......................... 64
5. Kim sot tc b sung mi trng dinh dng trong qu trnh ln men ........... 67
6. Ph bt trong qu trnh ln men .............................................................................. 70
6. 1. To bt v cc nhn t gy to bt ................................................................. 70
2
6.2. Nhng tr ngi gy nn bi bt ....................................................................... 70
6.3. Kim sot bt ................................................................................................... 70
7. Theo di v tnh ton kt qu ln men .................................................................... 73
1. Nng sn phm .............................................................................................. 73
2. Sn lng ............................................................................................................ 73
3. Hiu sut ............................................................................................................. 74
CHNG 7. TI U HO H THNG LN MEN ...................................... 78
1. Ci tin ging ......................................................................................................... 78
2. Ci tin thit b ...................................................................................................... 79
3. Tng cng cc iu kin thch hp trong qu trnh nui cy ......................... 79
CHNG 8. NHIM TP KHUN V NGUYN TC PHNG
CHNG TP NHIM ............................................................................................. 84
1. Hu qu ca s nhim tp khun ............................................................................ 84
2. Pht hin tp nhim ................................................................................................. 84
3. Nguyn nhn tp nhim .......................................................................................... 85
4. Nguyn tc phng chng nhim trong ln men ...................................................... 85
5. Cch pht hin - nh gi ngun gc tp nhim ..................................................... 86
6. Mt s im cn lu khi truy tm ngun gc gy tp nhim ............................... 88
CHNG 9. THU HI V TINH CH SN PHM LN MEN .................. 89
1. Gii thiu ............................................................................................................. 89
2. Cc phng php x l thu hi sn phm ln men ................................................. 92
CHNG 10. X L CHT THI CA QUI TRNH LN MEN ................ 97
1. Gii thiu v x l cht thi v cc phng php x l .................................... 97
2. Tip cn x l cht thi ca qui trnh ln men cng nghip ........................... 102
CHNG 11. BI TP P DNG ................................................................... 104
1. Bi tp .................................................................................................................. 104
2. Bi gii ................................................................................................................. 106
TI LIU THAM KHO ..................................................................................... 110
PHN PHC LC ................................................................................................. 110



3
CHNG 1. KHI NIM LN MEN V NG DNG

1. Khi nim ln men
Thut ng ln men bt ngun t ng t fervere trong ting Latin, c ngha
l lm si bt (ni bt), nhm m t hin tng hot ng ca nm men trong dch
chit tri cy hoc dch chit i mch. Hin tng si bt ny l do CO
2
sinh ra bi
s phn gii hiu kh ca cc loi ng hin din trong dch chit. Tuy nhin, i vi
cc nh sinh ho v cc nh sinh vt hc th thut ng ln men cn c ngha khc.
V phng din sinh ha th ln men c ngha lin quan n s sinh nng lng bi
qu trnh phn gii cc hp cht hu c, trong khi , v phng din vi sinh vt hc
trong cng nghip th ngha ca ln men rng hn.
S phn gii ng l mt qu trnh oxy ho dn n vic to thnh cc phn t
pyridine nucleotides dng kh v sau cc phn t ny li tip tc b oxy ho cho
cc qu trnh tip theo. Trong iu kin hiu kh, s oxy ha ca cc phn t pyridine
nucleotides din ra bng s chuyn in t thng qua h thng cytochrome m trong
oxygen ng vai tr l cht nhn in t cui cng. Tuy nhin, trong iu kin k
kh, s oxy ho cc pyridine nucleotides dng kh din ra cng vi s kh ca mt
hp cht hu c m thng l sn phm trung gian ca mt qu trnh phn gii. i
vi trng hp v s hot ng ca nm men trong dch nc tri cy hoc dch chit
ng cc, NADH c hnh thnh nh s kh ca acid pyruvic thnh ethanol. Cc
chng vi sinh vt khc nhau c kh nng kh pyruvate thnh cc sn phm khc nhau
v rt a dng. Do vy, thut ng ln men c s dng ng theo ngha sinh ha l
mt qu trnh sinh nng lng trong cc cht hu c ng vai tr va l cht cho
va l cht nhn in t.
Qu trnh sinh tng hp alcohol nh tc ng ca nm men trong dch chit i
mch hoc dch chit tri cy c thc hin vi qui m ln t rt lu v tr
thnh cht trao i ca vi sinh vt c sn xut cng nghip u tin. Do vy, cc
nh vi sinh vt hc cng nghip m rng khi nim ln men m t bt k qui
trnh sn xut no m sn phm c sinh ra nh nui cy vi sinh vt. Vic sn xut
bia hoc cc dung mi hu c c xem nh l qu trnh ln men theo c ngha v
sinh ho hc v vi sinh vt hc. Trong ti liu ny, thut ng ln men c s dng
theo ngha rng bao hm c hai
Cn phn bit cc khi nim:

Cht cho in t Cht nhn in t cui cng
Ln men (sinh ho)
H hp hiu kh
H hp k kh
Cht hu c
Cht hu c
Cht hu c
Hp cht hu c
Oxygen
Hp cht v c (nitrate, sulphate)

Thut ng ln men m rng bao gm c ba khi nim trn.

2. ng dng ca cc qu trnh ln men
2.1. Sn xut sinh khi vi sinh vt
Cng nghip sn xut sinh khi vi sinh vt c th c chia thnh hai qui trnh chnh.
(1) sn xut nm men dng trong cng nghip bnh m, (2) sn xut t bo vi sinh vt
dng trong thc phm cho con ngi v cho ng vt (protein n bo). Nm men
4
bnh m c sn xut theo qui m cng nghip t nhng nm 1900 v nm men c
sn xut dng lm thc phm cho con ngi c trong i th chin th I. Tuy
nhin mi n nhng nm 1960 th sn xut sinh khi vi sinh vt mi c khai thc
mnh m. V do vy t nhng nm 1970, cc qui trnh lin tc theo qui m ln
c thit lp v a vo sn xut sinh khi vi sinh vt phc v cho ng vt.

2.2. Sn xut enzyme vi sinh vt
Enzyme thng mi c sn xut t thc vt, ng vt v vi sinh vt. Tuy nhin,
enzyme vi sinh vt c nhiu u th vt tri l do c th sn xut vi lng rt ln
bng k thut ln men. Hn na rt d dng lm tng sn lng ca h thng vi sinh
so vi cc qui trnh sn xut t ngun thc vt hay ng vt. Bn cnh , s tin b
ca k thut DNA ti t hp gip c th sn xut cc enzyme c ngun gc ng
vt nh cc chng vi sinh vt. Cc enzyme ny c dng ch yu trong cng nghip
thc phm v cc ngnh cng nghip c lin quan khc (Bng 1. 1). Qu trnh nui
cy vi sinh vt sn xut enzymes c kim sot rt cht ch. Ngy nay, ngi ta
s dng mt s k thut sinh hc phn t tng cng nng sut sn xut ca cc
chng nh b sung cht cm ng, t bin genes, hay k thut ti t hp genes.

2.3. Sn xut cc cht trao i vi sinh vt
Cng vi cc giai on tng trng, s thay i trong qu trnh nui cy mt
chng vi sinh vt c th c m t thng qua sn phm m chng sinh ra trong cc
phase khc nhau ca ng cong tng trng. log phase, sn phm c sinh ra ch
yu l phc v cho tng trng ca t bo, bao gm cc amino acids, nucleotides,
lipids, carbohydrates Nhng sn phm ny c xem l sn phm s cp ca s
bin dng, v phase ny c gi l trophophase.
Rt nhiu sn phm s cp c gi tr kinh t cao v ngy nay c sn xut bng
phng php ln men (Bng 1. 2).
S tng hp cc cht trao i s cp ca cc chng vi sinh vt hoang di ch cho
nhu cu trong t bo ca chng. Do vy nhim v ca cc nh vi sinh vt hc cng
nghip l phi lm sao ci bin cc chng ny v cung cp cc iu kin nui cy cn
thit ci thin nng sut sinh tng hp cc cht ny.
Trong phase n nh, mt s chng tng hp ra cc hp cht m chng khng tng
hp c trong log phase, cc cht ny khng c vai tr r rng i vi qu trnh bin
dng ca t bo v chng c xem l cht trao i th cp, v phase ny gi l
idiophase. Vi sinh vt tng trng trong mi trng t nhin tc thp, iu ny
khin ngi ta cho rng, trong t nhin th idophase chim u th hn trophophase.
Khng phi ton b vi sinh vt u c bin dng th cp, v d nh i vi trng
hp ca vi khun Enterobacteriaceae th khng tm thy c sn phm th cp. i khi
rt kh phn bit mt sn phm l s cp hay th cp v ng hc sinh tng hp
ca nhng hp cht nht nh c th thay i ty thuc vo cc iu kin nui cy.
Vai tr sinh l ca bin dng th cp trong cc t bo vi sinh vt c tranh lun
nhiu, nhng ngi ta quan tm n vai tr quan trng ca cc cht trao i trung gian
nhiu hn. C rt nhiu cht trao i th cp c hot tnh khng khun, l cc enzyme
c ch, mt s cht thc y tng trng v rt nhiu cht c dc tnh. Do vy, sn
phm ca bin dng th cp hnh thnh nn c s ca nhiu qui trnh ln men
khc nhau. Cng ging nh trng hp i vi cc cht trao i s cp, cc chng vi
5
sinh vt hoang di c khuynh hng tng hp cc cht trao i th cp vi nng
thp v s tng hp ny c kim sot bi s cm ng, s c ch d ha v h thng
phn hi.

2.4. Sn xut cc sn phm ti t hp
S pht trin ca cng ngh DNA ti t hp m rng tim nng ng dng ca cng
ngh ln men, to ra cc sn phm ln men khc nhau, phc v cho cuc sng ca con
ngi. Cc genes ca cc sinh vt bc cao c th s c a vo t bo vi sinh vt v
cc chng ny c kh nng tng hp ra cc protein ngoi. C nhiu chng vi sinh vt
khc nhau c dng lm t bo ch cho nhng h thng biu hin bao gm E.coli,
Sac. cerevisiae v cc nm si. Cc sn phm c sinh ra t cc chng c bin i
di truyn nh th ny gm: interferon, insulin, albumin ca huyt thanh ngi, cc
nhn t tng trng biu b, chymosin ca b, v somatostatin ca b. Khi thit k cc
h thng biu hin cc sn phm ti t hp cn ch n mt s vn : s tit ca
sn phm, gim thiu s phn hy sn phm v s kim sot sinh tng hp trong ton
b tin trnh ln men, cng nh ti u ha biu hin ca genes ngoi.
6
Bng 1. 1. Cc ng dng ca enzyme trong thng mi
Cng nghip ng dng Enzyme Ngun gc
Cng nghip bnh m v
xay bt

Cng nghip bia


Cng nghip ng cc
Trong ch bin socola
Coffee
Trong xay caf


Siro bp



Ch bin sa




Trong chin-xo
Nc p tri cy

Git i
Thuc da
Tht
Gim nht trong qu trnh nho bt, tng cng mm
mi, v duy tr ti ca bt
Ci thin cu trc, gim thi gian trn, tng th tch ca bnh
m
Tng cng nghin nh
Chng li nh hng ca kh lnh
Ci thin lc tinh
X l cc thc phm n nhanh
Sn xut cc dch si-r
Ln men ht c-f
Lm ch phm c-f c c
Sn xut cc lai bnh ko
Sn xut cc lai siro c hm lng maltose cao
Sn xut cc loi siro c ch s DE thp
Sn xut glucose t siro bp
Sn xut siro fructose
Sn xut dch thy phn protein
On nh sa bay hi
Sn xut sa c, kem, v cc thc n trng ming dng ng
lnh
Sn xut sa th
Tch lai glucose
Lm trong cc loi dch
Loi oxygen
S dng lm cht ty
Chng mc lng
Lm mm mi
Amylase

Protease
Amylase
Protease
b-glucanase
Amylase
Pectinase
Pectinase
Pectinase, hemicellulase
Invertase, pectinase

Amylase
Amylase
Amyloglycosidase
Glucoseisomerase
Protease
Protease
Lactase

Protease
Glucose oxidase
Pectinase
Glucose oxidase
Protease, lipase
Protease
Protease
Nm

Nm/vi khun
Nm/vi khun
Nm/vi khun
Nm/vi khun
Nm
Nm/vi khun
Nm
Nm
Nm/vi khun

Nm
Vi khun
Nm
Vi khun
Nm/vi khun
Nm
Nm men

Nm/vi khun
Nm
Nm
Nm
Vi khun
Nm/vi khun
Nm
7
Trong y dc


Trng nh
Dch thy phn protein
Ch bin thc ung
Cng nghip dt
Rau qu
H tr tiu ha
Chng ng mu
Trong cc th nghim lm sng
Thu hi bc t film s dng
Trong sn xut dch thy phn
To s n nh
Chng s xp xp theo kch thc ca cc si
Cc ch phm dng soup hoc nghin nh
Amylase, protease
Steptokinase
Numerous
Protease
Proteases
Glucose oxidase, catalase
Amylase
Pectinase, amylase,
cellulase
Nm
Vi khun
Nm/vi khun
Vi khun
Nm/vi khun
Nm
Vi khun
Nm

Bng 1. 2. Cc sn phm s cp ca bin dng vi sinh vt v ng dng trong thng mi
Cht trao i s cp ngha thng mi
Ethanol
Citric acid
Glutamic acid
Lysine
Nucleotides
Phenylalanine
Polysaccharides
Vitamins
Thnh phn hot tnh trong cc thc ung c cn, s dng lm nhin liu thay th xng du.
C nhiu ng dng trong cng nghip thc phm
Cht tng v
Cht h tr thc phm
Cht tng v
Cht tin thn ca aspartame, cht gy ngt
ng dng trong cng nghip thc phm, tng cng kh nng thu hi du bo
Cht h tr thc phm

8
2.5. H tr cc qu trnh bin np
Cc t bo vi sinh vt c th c s dng chuyn mt hp cht vo mt cu
trc lin quan c gi tr hn v chng c tc dng nh cc cht xc tc vi tnh c trng
v tr v lp th cao. Cc qu trnh vi sinh vt c trng hn cc qu trnh ha hc v cho
php c th thm, loi b hoc thay i tng nhm chc nng nhng v tr c trng
nht nh trn cc phn t phc tp m khng cn dng n cc ho cht. Cc phn ng
c xc tc bi enzyme bao gm phn ng kh hydrogen, phn ng oxy ho, phn ng
hydroxyl ho, phn ng kh nc, phn ng kh carboxyl, amin ho, kh amin, phn
ng chuyn v. Cc qu trnh vi sinh vt cn c thm mt thun li na l thc hin
nhit v p sut phn ng thp, khng i hi cc cht xc tc nh cc kim loi nng.

3. Qu trnh pht trin ca cng nghip ln men
Qu trnh pht trin ca cng nghip ln men din ra qua nm giai on c m t
trong Bng 1. 3. Qu trnh pht trin ngnh cng nghip ln men trc giai on 1900
c m t l giai on 1, cc sn phm trong giai on ny ch n thun gii hn
dng alcolhol ung c, hoc dm n.
Bng 1. 3. Qu trnh pht trin ca ngnh cng nghip ln men.


Giai on Sn phm chnh Loi bn Kim sot qu trnh Phng php nui cy
Kim sot
cht lng Cc thit b pilot Chn lc ging
Alcohol
- Bng g, t n 1500
barrels
- Bng ng thau
S dng thit b o
nhit , bo mc,
trao i nhit
Dng m
Hu nh khng
kim sot
Khng c
Nm men
thun nht
s dng
Carlberg
(1886)
Dm
Bng cc thng phuy, khay
hay trong cc lp lc chy
dng
Dng m
Hu nh khng
kim sot
Khng c
Chn lc
chng
ln men dm
tt
2. 1900-1940
Nm men bnh m,
glycerol, citric acid,
lactic acid,
acetone/butanol
Bn bng kim loi 200m3 cho
sn xut acetone/butanol
S dng h thng phn phi
Khuy trn c s dng
S dng u d
pH,
u d kim sot
nhit
Dng m v h thng
fed- batch
Hu nh khng
kim sot
Hu nh khng
c
S dng ging
thun nht
3. 1940 n nay
Pennicillin,
Streptomicin
cc khng sinh
khc
gibberelin,
amino acid,
nucleotides,
cc cht chuyn
ho, enzyme
Cc bn ln men c trang b h
thng sc kh, vn hnh trong
iu kin v trng- ng ngha
bn ln men
S dng u d
pH,
DO c th thanh
trng c. S
dng cc thit b
kim sot m sau
ny c th kim
sot trn my tnh
- Thng thng l
dng m v fed-batch.
- Pht trin nui cy
lin tc cho ln men
bia v mt vi cht
trao i s cp
Rt quan trng Tr nn thng dn
S dng cc
chng
trnht bin v
chn lc c
bn
4. 1964- nay
Sinh khi n bo,
protein,
hydrocarbon, cc
cht phc v chn
nui
Cc bn ln men chu p lc,
c thit k gii quyt vn
trao i kh, nhit
S dng my tnh
kim
sot cc thng s
vn hnh
Nui cy lin tc
vi vic hi lu mi
trng nui cy
Rt quan trng Rt quan trng
S dng k
thut di truyn
5. 1979- nay
Sn xut cc
protein
khc dng
(heterologous) bi
vi sinh vt v t
bo ng vt, Sn
xut khng th n
dng bi t bo
ng vt
S dng ni ln men c pht
trin trong giai on 3, 4. V
pht trin thm ni nui cy t
bo ng vt
Pht trin cc iu
kin
kim sot v u
d nh trong giai
on 3, 4
Dng m, fed-batch,
hoc lin tc.
Pht trin nui cy lin
tc dng chy trn
nui cy t bo ng
vt
Rt quan trng Rt quan trng
S dng k
thut ti t hp
1. Trc 1900
9
CHNG 2. NG HC TNG TRNG CA VI SINH VT
TRONG QU TRNH LN MEN

1. Phn loi phng php ln men

Tu theo tng c im c th m vic phn loi ln men c khc nhau. C th phan loi
ln men nh sau:
1. Theo tnh cht mi trng: Lng; Rn; Bn rn
2. Theo c tnh s dng oxygen ca chng vi sinh vt: Ln men hiu kh; Ln
men k kh
3. Theo trng thi: Trng thi tnh (khng khuy trn/sc kh); Trng thi ng
(c khuy trn/sc kh)
4. Theo mc kim sot ca qui trnh: C kim sot ( tp nhim, c cht,
pH,); Khng kim sot (t nhin)
5. Theo qui trnh ln men: Ln men theo m (batch); Ln men theo dng lin tc
(continuous); Ln men theo dng bn lin tc (fed-batch, dng m-b sung)
Nhm nh gi din tin c th ca mt chu k ln men, phng php ln men
theo m c chn lm kiu mu nh gi, trn c s s m rng nh gi i vi
cc phng php ln men cn li.

2. Phng php ln men m
y l mt h thng nui cy khp kn, gii hn v dinh dng, ngha l c cht
dinh dng khng c b sung trong sut qu trnh ln men, tri qua bn phase khc
nhau.
Trong giai on u (lag phase, (a)) ca qu trnh nui cy, hu nh khng c s
tng trng ng k no xy ra, ch yu l giai on thch nghi ca t bo trong mi
trng mi. Trong thc t sn xut cn phi rt ngn thi gian ca phase ny cng nhiu
cng tt a h thng ln men nhanh chng bc vo giai on tng trng v sinh
tng hp sn phm quan tm. Tip theo, vi sinh vt dn dn thch nghi, tng trng
nhanh dn v t n tc ti a - log phase (b).

Hnh 2. 1. H thng ln men theo phng php m.
10
S tng trng trong phase ny c th c biu din theo phng trnh ton hc
sau:
dx/dt = x (2.1)
Trong x sinh khi vi sinh ti thi im nui cy c t gi, l tc tng
trng c trng (hr
-1
), tc ny khc nhau ty theo cc giai on trong chu k tng
trng. T phng trnh trn ta c:
dx/x = dt

} }
=
t
to
t
to
dt x dx /
(ln x
t
- ln x
0
) = (t-t
0
) (2.2)
x
0
: lng sinh khi ban u (t
0
), x
t
: lng sinh khi sau thi gian nui cy t (gi).


Hnh 1. 1. S ng hc tng trng ca vi sinh vt khi nui cy dng m. (a) lag- pha,
(b) log- pha, (c) pha n nh, (d) pha suy vong.
Trong log phase, giu dinh dng, s t
max
. Mi chng vi sinh vt trong
nhng iu kin ti thch nht nh s c
max
khc nhau. Tc tng trng ph thuc
rt ln vo ngun dinh dng ca mi trng nui cy v thnh phn cc sn phm do
chnh hot ng ca vi sinh vt to ra trong mi trng. Nu vn duy tr vic b sung
dinh dng vo mi trng nui cy th tng trng vn gi tc cao.
Bng 2. 1. Mt s gi tr
max
ca mt s chng vi sinh vt trong iu kin nui cy c
trng
Chng vi sinh vt
max
(gi
-1
) Tham kho
Vibrio natriegents
Methylomonas methanolytica
Aspergillus nidulans
Penicilium chrysogenum
Fusarium graminearum Schwabe
4,24
0,53
0,36
0,12
0,28
Eagon (1961)
Dostalek et al (1972)
Trinci (1969)
Trinci (1969)
Trinci (1992)
0
0.2
0.4
0.6
0.8
0 4 8 12 16 20 24 28 32 36
Th i gian nuo i ca y (gi )
L
n
(
n
o
n
g

o

t
e

b
a
o
)
(a) (b)
(c)
(d)
11
Nui cy t bo thc vt
Nui cy t bo ng vt
0,01-0,046
0,01-0,05
Petersen & Alfermann (1993)
Lavery (1990)

S tng s lng t bo din ra trong log phase theo cp s nhn vi c s 2 theo
phng trnh:
N = N
0
.2
n
(2.3)
trong , N
0
v N l s lng t bo ban u v ti thi im kho st, n l s th h.
lg(N/N
0
) = n.lg2
n = 3,32.lg(N/N
0
) (2.4)
Thi gian th h t
g

t
g
= t/n (2.5)
vi t l thi gian m vi sinh vt nhn i qua n th h. i vi vi khun t
g
thng khong
20-60 pht, i vi nm men t
g
thng khong 1-3 gi.
Hng s tc phn chia k l s ln phn chia sau 1 gi nui cy
k =1/g = n/t (2.6)
Trong giai on n nh (stationary phase, c), lng sinh khi to thnh c th m
t bng phng trnh:
x = Y(s
i
-s
r
) (2.7)
trong , x l hm lng sinh khi, Y l hiu sut tng hp sinh khi, s
i
l hm lng c
cht ban u, s
r
l hm lng c cht cn li. Tng trng ca vi sinh vt s dng li khi
s
r
= 0 hoc khi m trong mi trng c s tch ly cc cht c th gy c hi cho chng.
Gi tr Y nhm gip nh gi hiu sut chuyn i mt c cht thnh sinh khi vi sinh
vt. Do vy c th dng Y tnh ton lng cn thit ca mt c cht khi mun tng
hp mt lng sinh khi nht nh. Hiu sut Y khng phi l mt hng s n nh m
thay i ty thuc vo iu kin nui cy nh nhit , pH, hm lng oxygen ho
tan, s gii hn ca cc c cht,... S suy gim tng trng ph thuc vo c cht gii
hn khi cn kit trong mi trng c th c m t theo phng trnh (theo Monod,
1942):
=
max
s
r
/(K
s
+s
r
) (2.8)
Trong K
s
l hng s s dng c cht,
Hnh 2. 2. i vi mt c cht nht nh th mi chng vi sinh vt c K
s
khc nhau
(Bng 2. 2).
Khi s
r
= K
s
th = 1/2
max
. Khi = 0, bc vo giai on phase n nh. y l
giai on chnh ca vic sn xut cc cht trao i.






s
i
K
s
s
r
N
o
n
g

o

c

c
h
a
t

g
i

i

h
a
n
Th i gian nuo i ca y
12
Hnh 2. 2. S biu th tr s K
s
.
Bng 2. 2. Mt s gi tr K
s
ca mt s chng thng thng
Chng vi sinh vt C cht K
s
(mg/dm
3
) Tham kho
E.coli
Sac. cerevisiae
Pseudomonas sp.
Glucose
Glucose
Methanol
6,8.10
-2

25,0
0,7
Shehata and Marr (1971)
Pirt and Kurowski (1970)
Harison (1973)

S thay i hm lng cc cht trao i c th biu din:
dp/dt = x (2.9)
trong , p l nng sn phm, l tc sinh tng hp c trng. Mt khc, s to
thnh sn phm ty thuc vo hot tnh ca chng (hiu sut trn mt n v t bo):
dp/dx = Y
p/x
(2.10)
vi Y
p/x
l hiu sut to sn phm trn mt n v sinh khi (nng sut sn xut).
T hai phng trnh trn ta c:
dx/dt = x/Y
p/x

m dx/dt = x
Do vy ta c: = .Y
p/x
(2.11)


Nh vy tc sn xut c trng ph thuc vo , v t gi tr cao nht khi =
max
.

3. Phng php ln men lin tc
Khc vi cc giai on trong nui cy theo m (gm 4 pha ring bit lag phase,
log phase, phase n nh v phase suy vong), i vi nui cy lin tc, log phase c
duy tr bng cch b sung nguyn liu vo ni ln men v qu trnh ny c lp i lp
li cho n khi ni ln men y. Trong nhiu trng hp, ngi ta lp t thm h thng
thng lu nhm gi mc (th tch) ca dch ln men trong ni lun mc n nh cao
nht, v khi tc np liu v tc chit dch c cn bng, nng t bo trong
ni ln men ph thuc vo tc np liu.
Ta c:
D = F/V (2.12)
Trong , F: tc np liu (KL/hr), V l th tch dch ln men trong ni (KL), D l
pha long (gi
-1
).
S thay i mt t bo trong dch ln men c th biu din:
dx/dt = growth output
dx/dt = x Dx (2.13)
Trong iu kin n nh, nng t bo trong dch khng i, dx/dt = 0.
Lc : = D (2.14)
Tc tng trng c trng ph thuc vo pha long v nh vy trong nui
cy lin tc th s
max
.
13


Hnh 2. 3: S h thng ln men lin tc.
(A): H thng ln men lin tc n tng, khng hi lu sinh khi;
(B): H thng ln men lin tc n tng, hi lu sinh khi;
(C): H thng ln men lin tc a tng.
Trong mt s trng hp, ngi ta dng D kim sot . Tng trng ca vi
sinh vt trong nui cy lin tc c iu khin bi cc cht trong thnh phn ca mi
trng nui cy. Ln men theo cch ny cn c gi l chemostat. Theo Monod ta c:
=
max
s /(K
s
+s ) (2.15)
trng thi cn bng, D = , nn D =
max
s
r
/(K
s
+s
r
), s
r
: nng c cht trng
thi cn bng.
s = K
s
D/(
max
- D) (2.16)
Nh vy, nng c cht cn li ph thuc vo pha long. Trong thc t, iu
ny xy ra do s tng trng ca t bo lm cn kit ngun dinh dng khi m ngun
cung cp cho tng trng ngang bng vi pha long. Nu tc s dng c cht ca
vi sinh vt nh hn tc np liu cho s tng trng t bo th mt s c im sau s
xy ra: Tc tng trng ca t bo nh hn tc pha long, v do vy t bo s b
chit ra khi ni ln men vi tc cao hn tc m chng c sinh ra. iu ny gy
14
nn suy gim mt t bo trong ni ln men. Nng c cht trong ni ln men s tng
dn v trong ni ln men cn t t bo hn s dng. Khi nng c cht cao hn, tc
tng trng ca vi sinh vt tng ln v cao hn tc pha long v khi , sinh khi
tng dn ln v trong trng hp ny th trng thi n nh mi c thit lp.
Do vy, ngi ta cn gi chemostat l h thng nui cy t cn bng theo gii hn
dinh dng, v c th duy tr trong mt gii hn rng ca tc tng trng c trng
ph (sub-
max
).
Nng t bo trong nui cy lin tc giai on cn bng ( x ) c th c biu
din:
x = Y(s
i
+ s
f
- s
r
) (2.17)
Phng trnh to sn phm:
S thay i nng sn phm = (tng sn phm lng sn phm chit);
dp/dt = .x - Dp (2.18)
giai on n nh, dp/dt = 0. Khi :
p = . x /D (2.19)
trong p l nng sn phm giai on n nh. Trong nui cy lin tc, ngi ta
cn chia ra nhiu kiu nui cy khc nhau:
1. Nui cy dng a h thng: Dch chit ra t h thng ln men ny c a
ngay vo mt h thng ln men khc. u im ca phng php ny l c th thay i
c ngun nguyn liu cc giai on khc nhau trong qu trnh ln men.
2. Phng php hi lu t bo vi khun trong qu trnh ln men: Dch chit ra
trong qu trnh ln men ngoi sn phm cn cha mt lng rt ln t bo vi khun. Nu
lng t bo ny c thu hi v ti b sung vo h thng ln men th s tng cng
c sn lng ca h thng. C th dng phng php lc hay ly tm tch t bo ri
cho hi lu vo h thng. Tuy nhin, rt kh thc hin vic hi lu ton b t bo theo
dch chit v vn chng tp nhim i vi h thng theo kiu ny cng l mt tr ngi
ln.

4. Phng php ln men m-b sung (fed-batch)
Ln men m-b sung l phng php nui cy m nguyn liu c b sung vo
ni ln men lin tc hoc gin on, dch ln men c th c chit ra trong qu trnh
nui cy. Ban u nui cy theo m, sau nguyn liu c np vo ni ln men. Cc
c cht khi c b sung vo mi trng ang nui cy c th l (1) ging nh thnh
phn mi trng ban u hoc ch mt s c cht gii hn. i vi c cht gii hn khi
b sung vo c th (2) nng tng ng vi mi trng ban u, hoc (3) cao hn
hoc (4) rt m c. H thng m-b sung theo cch (1), (2) gi l m-b sung thay i
th tch; theo cch (3), (4) gi l h thng m-b sung c nh th tch.

15

Hnh 2. 4. S t thng ln men m-b sung.
(A): Ln men m-b sung gii hn th tch
(B): Ln men m-b sung khng gii hn th tch

4.1. Ln men m-b sung thay i th tch
ng hc ca h thng ny c m t ln u bi Pirt (1975), c xem xt nh
h thng nui cy m nhng b gii hn bi nng ca mt c cht. Sinh khi ti bt k
thi im no trong qu trnh nui cy c trnh by theo phng trnh:
x
t
= x
o
+ Y(s
i
- s
r
) (2.20)
trong x
o
l nng ging np vo h thng.
Khi s
r
= 0 v x
t
= x
max
; do x
0
<< x
max

x
max
= Y.s
i
(2.21)
Nu ti thi im s
r
= 0, mi trng c b sung vo vi D <
max
th ton b
mi trng va mi c b sung s c s dng ngay (ds/dt = 0), v ta c:
F.s
i
= .(X/Y) (2.22) vi X = x.V.
Theo cch ny, tng sinh khi s tng dn trong khi nng t bo hu nh
khng tng (dx/dt = 0), do vy = D. y c xem nh l trng thi cn bng. Khi th
tch dch ln men trong ni tng, t l pha long gim xung, v nh vy:
D = F/(V
0
+ F.t) (2.23)
Theo phng trnh Monod, Khi s
r
gim th D s gim, do vy s x tng ln. Tuy
nhin trong h thng nui cy m-b sung th bt k gi tr no ca th s
i
>> K
s
nn
trong thc t s
r
thay i rt nh. Do vy, trng thi c coi nh l cn bng n nh th
D <
max
v K
s
<< s
i
.
S thay i nng sn phm trong h thng ny tng t nh trong nui cy lin
tc c biu din theo cn bng:
dp/dt = x Dp (2.24)
trng thi cn bng tc l lng sn phm sinh ra cn bng vi pha long do
np dch b sung vo th nng sn phm trong h thng s khng thay i, dp/dt = 0,
khi :
16
p = .x/D (2.25)

4.2. Ln men m-b sung khng thay i th tch
Theo Pirt (1979), c s ca h thng ny c xem nh trong trng hp nui cy
m trong do s tng trng ca chng gy nn s cn kit c cht gii hn t n
mt mc nht nh. Sau c cht ny c b sung vi nng cao, khng lm thay
i n th tch dch ln men. ng hc trong h thng ny c m t nh sau:
dx/dt = GY (2.26)
trong G l tc np c cht.
Do dx/dt = x, nn:
GY = x
= GY/x (2.27)
Nu GY/x <
max
th khi b sung c cht vo chng s c s dng ngay, lc
ds/dt = 0. Tuy nhin do dx/dt > 0 v x v X tng theo thi gian nn:
x
t
= x
0
+ GYt (2.28)
Trong x
0
, x
t
l nng t bo khi bt u v sau khi vn hnh h thng m-b sung
c t gi nui cy.
Khi x tng ln, gim dn. Nng ca cht gii hn hu nh khng thay i,
sinh khi tng dn v nng cc cht khng gii hn trong mi trng gim dn.
S cn bng sn phm trong h thng ny c biu din nh sau:
dp/dt = x
t
(2.29)
dp/dt = (x
0
+ GYt) (2.30)

5. Ln men mt cao
Nh cp trong phng php ln men m, tng lng sinh khi sinh ra trong
qu trnh ln men l t ngun dinh dng nht nh trong mi trng chun b ban u.
Nng cc thnh phn mi trng c thit k bo m tc tng trng l tt
nht. Do vy, tng mt t bo cn phi b sung dinh dng trong qu trnh nui cy
v nh vy, ln men mt cao ch c th t c trong trng hp nui cy m-b
sung v thnh phn b sung vo phi c nng cao th tch dch ln men hoc khng
thay i hoc thay i t.
Trong ln men sn xut cng nghip, cho d sn phm sinh ra l ni bo hay ngoi
bo, vi mt iu kin thch hp th chng nui cy s c mt gi tr v tc sn xut
c trng () nht nh:
= (P/X)/t (2.31)
P = .X.t (2.32)
trong P l tng lng sn phm thu c sau thi gian nui cy t, X l tng lng sinh
khi tham gia sn xut. T phng trnh trn ta thy, thu c P nhiu, cn phi tng
X.
X = x.V (2.32)
17
vi V l th tch dch ln men, x l nng t bo trong dch ln men. Do th tch dch
ln men b gii hn bi dung tch ni ln men nn t X cao cn phi tng nng t
bo x trong qu trnh ln men.
Vn t ra l lm th no tng nng t bo v nng t bo ny c th
tng c ti mc no? Chng ta s cp vn ny trong nhng chuyn sau.

18
CHNG 3. S LU TRNH H THNG LN MEN CNG
NGHIP

1. Gii thiu
Trong phn ny, chng ta tp trung phn tch qui trnh ln men cng nghip theo
phng php chm, hiu kh, c kim sot v y l qui trnh bao gm y cc thnh t
ca mt qui trnh ln men tiu chun. Qui trnh ny thng bao gm cc cng on sau:
- Gi ging
- Hot ha ging
- Nhn ging (s cp, th cp)
- Ln men sn xut
- Thu hi sn phm ln men
- X l cc cht thi sinh ra trong qu trnh sn xut sn phm ln men
Ty theo chng vi sinh vt, yu cu ca sn phm mc tiu v h thng cng ngh
thit b m iu kin ca tng bc trong qui trnh ln men c th khc nhau. Tuy nhin,
m bo c hiu qu sn xut tt v n nh nht th ton b cc hot ng c lin
quan n ln men c mt c im chung l cn c thc hin trong iu kin c kim
sot v trng.
Nu khng cp n cc yu t v kiu ni ln men, mt h thng ln men cn
bn bao gm su thnh t sau:
1. Cng thc mi trng s dng trong qui trnh nui cy vi sinh vt t giai on
nhn ging cho n giai on ln men sn xut.
2. iu kin v trng ca mi trng nui cy, ni ln men v cc thit b lin
quan trong h thng.
3. Bo m nhn ging s lng vi thun nht v cht lng cao trc khi
np vo ni ln men chnh.
4. S tng trng ca chng vi sinh vt trong sn xut di iu kin ti u
to thnh cc sn phm mong mun.
5. Chit tch, tinh sch v kt tinh sn phm.
6. X l cht thi ca qu trnh sn xut.

Cc yu t ny lin quan cht ch vi nhau theo mt qui trnh c m t trong
Hnh 3. 1. Tng yu t c lp c th c nghin cu ci tin ph hp cui cng
nng cao hiu qu sn xut ca ton h thng.
Trc khi tin hnh xy dng h thng ln men cn phi chn lc chng ging vi
sinh vt bo m sinh tng hp ng sn phm mong mun vi hm lng cao. Vic
chng lc chng ging thng c thc hin bi cc phng nghin cu c nng lc v
kinh nghim trong lnh vc pht trin chng ging, nh gi hiu qu v tnh n nh ca
chng. Vic tuyn chn chng ging phc v sn xut cng nghip da trn mt s tiu
ch sau:
- c tnh dinh dng ca chng (dng c cht thng dng, r tin)
19
- Nhit ti thch ca chng (chn chng c nhit ti thch cao, gim chi ph
h thng gii nhit trong ln men cng nghip)
- Tnh n nh ca chng trong qu trnh s dng
- Nng sut sinh tng hp sn phm cao
Da vo loi sn phm ln men v cc yu cu v tinh sch ca sn phm, h
thng thu hi chit tch v tinh sch sn phm c thit k bo m hiu sut thu hi
cao, t tiu chun nh mong mun. Trong qu trnh vn hnh sn xut, c ba yu t
chnh: chng ging, qui trnh ln men, qui trnh thu hi cn phi c nghin cu ci tin
lin tc nhm thu nhn c nhiu sn phm vi cht lng cao.

2. Cc cng on ca qui trnh ln men
2.1. Gi ging
y l bc rt quan trng trong hot ng vn hnh mt h thng ln men nhm
bo m c ngun cung cp ging n nh cho sn xut. Qu trnh gi ging cn phi
bo m mt s yu cu sau:
- Duy tr hot tnh ging trong thi gian di (khng xy ra hin tng thoi ha,
gim kh nng tng trng hoc/v nng lc sn xut ca ging)
- Khng b tp nhim
Cc phng php gi ging khc nhau trong cng nghip bao gm:
a. Gi ging nhit thp
- Gi trn mt thch nghing 20 ~ 5
0
C, khong 6 thng cy chuyn mt ln. Nu
ging c gi trn b mt thch nghing c ph mt lp du bo v th thi gian gi
ging c th ko di n 1 nm.
- Gi trong nit lng (ng su). Khi gi ging nhit thp (-150 ~ -190
0
C),
hot ng bin dng ca vi sinh vt gim hn. y l phng php gi ging kh an
ton cho nhiu loi vi sinh vt khc nhau (vi khun, nm mc, nm men, virus). Thng
thng ngi ta cho chng vi sinh vt cn gi pht trin n phase n nh, b sung cht
bo v (thng l glycerol 10%), sau lm lnh v gi.
b. Gi trong iu kin kh nc
- Nui cy kh. Phng php ny thng c p dng i vi bo t nm si.
Cc bc tin hnh bao gm t v trng vi bo t nm khong vi ngy cho pht
trin, lm kh nhit phng khong 2 tun, sau ct gi iu kin khng kh kh
hoc t lnh.
- Phng php ng kh. Lm ng mt dung dch t bo trong iu kin chn
khng, ko theo s thng hoa ca cc phn t nc. Dch nui cy phi t n giai on
phase n nh, sau to huyn ph t bo trong mt mi trng bo v nh sa, serum,
hoc glutamate natri. Dch ny c lm ng kh sau gi trong t lnh v c th bo
qun n hn 10 nm.
20
Noi len
san xuat
(> 1000L)
Kh vao
M
Noi nuoi
cay mam
(10-100L)
M
Nuoi cay lac
(200-1000mL)
Nuoi cay
giong goc
(5-10mL)
Nguyen lieu
Moi trng
Thanh trung
Ly tam/loc
Dch noi Sinh khoi
X ly nc thai
Chiet xuat san pham
Tinh che san pham
ong goi
D

c
h

s
a
u

n
u
o
i

c
a
y
Thanh pham

Hnh 3. 1. S lu trnh h thng ln men trong cng nghip.
21
2. 2. Hot ha ging
Hot ho ging l bc rt quan trng thng c thc hin bng phng php
cy chuyn nhm mc ch khi phc li hot tnh ca ging sau thi gian lu gi. Cy
chuyn thng c thc hin trn mi trng thch nghing trong ng nghim, trong
t m nhit thch hp n khi vi sinh vt pht trin thnh mt lp trn mt thch.
Mi trng nui cy trong giai on ny phi bo m cc yu t dinh dng cn thit,
iu kin nui cy nh nhit , pH, thong kh phi t gi tr ti u cho chng.
Cc im cn lu trong cng on ny bao gm:
- B mt thch cn phi m bo m nht nh, khng c qu kh v nh th
s gy nh hng n s pht trin ca chng sau khi cy ria ln mt thch.
- Trong qu trnh cy ria, cn phi bo m ria u (theo ng zic-zc) trn mt
thch, khng ria qu gn mp thch nghing v y lp thch mng, kh v t dng
cht, khng cho sinh khi t bo khi khun lc pht trin ln.
- Cn phi kim sot tt thi gian t c lng sinh khi vi hot tnh ti
u v n nh. Trnh trng hp thi gian qu di, sinh khi t bo pht trin qu nhiu
(over growth), lp t bo trn cng ca khun lc khng c tip xc trc tip vi dinh
dng ca mi trng thch trong thi gian di s nh hng n cht lng ca ging
sau ny.

2.3. Nhn ging
Mc ch ca nhn ging l to mt lng sinh khi ln a vo qui trnh
sn xut. Qu trnh nhn ging thng dng li giai on cui ca log phase, khi
hot tnh ca ging l mnh nht. Qu trnh nhn ging thng chia thnh hai giai on:
a. Nhn ging s cp
Cng on nhn ging s cp thng c thc hin trong phng nhn ging
(phng clean room) qui m 100-500 ml vi thit b s dng c th l:
1. My lc vi bnh tam gic c th tch phi gp ti thiu 4 ln th tch dch nui
cy bo m khng gian lc trn dch nui cy v cung cp lng oxy cn
thit. Tc lc l mt yu t quan trng nh hng n tng trng ca chng trong
qu trnh nhn ging. Nu lc tc thp s khng bo m c ha tan oxy tt
trong dch ln men, chng s pht trin chm. Nu lc qu nhanh, dch nui cy s tip
xc vi phn nt bng (cotton plug) lm m t nt bng d gy ra hin tng tp nhim
v s cn tr vic trao i khng kh gia bn trong v bn ngoi ca bnh tam gic. Mt
khc, khi lc tc qu cao, t bo vi sinh vt s b nh hng bi nhng tc ng c
hc.
2. Ni ln men nhn ging vi kch thc nh. H thng ny c y cc yu t
bo m cho chng pht trin tt nht nh khuy trn, h thng cung cp khng kh,
h thng hiu chnh pH, nhit . Tuy nhin do c nhiu h thng lin quan phc tp
tc ng vo trong qu trnh nui cy nn nguy c xy ra tp nhim trong trng hp ny
l rt cao.
Khi qu trnh nhn ging kt thc cn phi tin hnh kim tra, nh gi cc thng
s quan trng trc khi a vo sn xut nh OD (DCW- dried cell weight), hiu sut
sinh khi, pH, so vi tiu chun. Mt trong nhng vn quan trng ca cng on
22
nhn ging s cp l phi bo m ging mm khng b tp nhim. t c iu ny
cn phi bo m mi trng v thit b s dng c thanh trng trit , qu trnh thao
tc vn hnh phi c chun ha, p ng c cc tiu ch v trng. kim tra tp
nhim ca ging mm thng thng s dng phng php tri trn a pettri. Trong mt
s trng hp do mt tp nhim qu nh, khng th pht hin c, ngi ta thng
nui cy tng cng (enrichment culture) nhm tng s lng t bo tp nhim (nu c)
trong mu th kim tra. Phng php thc hin nh sau: dng khong 10-50 ml dch
nhn ging sau khi kt thc nui cy np vo bnh tam gic cha mi trng tng t
nh mi trng nhn ging v tip tc nui cy cng iu kin trong thi gian 24 gi.
Sau ly mu dch tri ln a pettri kim tra tp nhim. Nu tp nhim xy ra th
kh nng ging mm ban u b tp nhim rt ln. bo m kt qu nh gi chnh
xc, qu trnh nui cy tng cng cn phi c thc hin trong iu kin tuyt i v
trng.
b. Nhn ging th cp
Ty theo qui trnh m h thng nhn ging th cp c thc hin qua mt hoc
nhiu bc. Qu trnh nhn ging thc cp c thc hin trong ni nui cy mm vi th
tch np ging tng dn, khong 5-10% cho mi bc. Cn phi bo m cc iu kin nh
thnh phn dinh dng trong mi trng nui cy, pH, nhit ti u chng tng
trng tt nht. Mc ch ca nhn ging th cp l to c lng sinh khi ln, hot
tnh tt phc v cho ln men sn xut.

2.4. Ln men sn xut
y l cng on chnh ca qui trnh, do vy cn phi kim sot cht ch cc iu
kin nui cy nh lng t bo cn thit cho sn xut, nhit , pH, hm lng oxygen
ha tan, nng cc c cht gii hn nhm t c hiu qu ln men cao nht. Trong
qu trnh nui cy cn phi rt ngn lag phase, tng cng tc tng trng c trng
t gi tr ti a,
max
v trong pha n nh phi c duy tr tt. Mt s thnh phn
dinh dng s b cn kit theo thi gian do vy cn phi b sung bo m duy tr c
hot tnh sn xut ca chng (m- b sung). Theo thi gian, cn phi phn tch, nh gi
hiu qu ln men xc nh thi gian nui cy thch hp, mang li hiu qu sn xut
cao nht.

3. H thng thit b lin quan n qui trnh ln men

Mt h thng ln men hiu kh c kim sot bao gm cc thit b lin quan nh
sau (Hnh 3. 2):



H
cng ty K
(1
(2
Hnh 3.3. H
Khoa hc v
1) Bn ln m
2) T iu k
ho tan
(1)
Hnh 3. 2.
H thng thi
v K thut
men th tch
khin: Kim
(2
. M hnh h
it b ln m
Vnh An (V
h 50 L (2 b
m sot cc t
)
h thng ln

men qui m
Vit Nam).
n)
thng s: t
(5)
n men kim
pilot c
c khuy
(4)
sot t n
c thit k v
y trn, pH
(3)
ng.

v gia cng
H, nhit
23

g bi
, oxy

(3
(4
(5

3.1. N
-
1000 KL
-
ni v t
-
nhit lm
nui cy
nhit
tower). C
(jacket)
gn bn











-
qua nh
men, tn
thng
men khi
trong dc
-
hiu kh
thng cy
hnh th
ln men
qua dch
do tc
a tr l
3) My lm
4) My nn
5) L hi
men v k
i ln men
Thn ni: T
L.
H thng k
ng cng
Thit b gi
m cho nhi
y th cn ph
thp 5-25
C 3 dng
bao bn ng
trong ni l
H thng p
ng l c k
ng din tch
c lp
i i t y
ch nui cy
H thng th
, khng kh
yclone c
nh v v t
n. Phn ln
h ln men,
ng c hc
li ni ln m
lnh nc
kh cung c
in nhm c
kh trng m
Ty theo qu
khuy trn:
ha tan c
i nhit: Do
t ca d
hi gii nhi
o
C, c c
thit b gi
goi ni ln
n men.
phn phi
ch thc n
h tip xc c
t gn y
bn n m
y.
hot kh v
h c sc
c gn nh
trng ca
bt kh ny
i vo h th
, phn kh t
men qua
gii nhi
p khng kh
cung cp h
mi trng tr
ui m sn x
: Nhm m
ca oxygen
o qu trnh
ch ln men
t cho ni
ung cp b
i nhit th
n men; dng
kh (air spa
nh, to i
a bt khn
y bn ln m
mt dch l
hi lu dc
c vo t
h bn. Tron
bt nh nn
y s thot r
hng thu h
trong bt s
ng ng h
it sinh hc
h cho qu
hi nc n
rc khi nu
xut m th
c ch to
vo dch l
ln men si
n tng ln.
ln men. T
i h thng
ng c s
g ng vng
arger): Nh
u kin cho
ng kh vi d
men, bo m
di nht
ch bt tan (c
y ni ln m
ng qu trnh
n ton b b
ra khi ni
i dch (cycl
thot ra ng
i lu gn
trong qu
trnh ln m
ng phc v
ui cy.
tch bn l
s ng nh
n men.
inh ra nng
kim so
Tc nhn gi
g my lm
s dng cho
xon (coil)
m phn ch
o khng kh
dch ln me
m hnh trn
tng tnh
cyclone):
men, sau
h ln men do
bt s ni l
ln men c
lone). tro
goi v ph
y cyclon
trnh ln m
en
v cho than
n men s
ht ca dch
g lng sinh
ot nhit
i nhit th
mt nc (
o ni ln m
) v dng b
hia khng k
h tip xc
en. H thn
nh ca bt k
hiu qu h
i vi tr
s thot r
o c rt nhi
n b m
ng vi kh
ong cyclone
n dch (m
ne.
en
nh trng n
khc nhau
h ln men t
h hc di
thch hp
ng l n
(chiller, coo
men: (1) dn
ng (tube
kh sc vo
tt vi dch
ng phn ph
kh trong n
ha tan ca
ng hp ln
ra ngoi qu
iu bt kh
t dch trong
ng kh sau
e, bt s b v
ng bt) s
24
i ln
t 1-
trong
dng
p cho
c c
oling
ng o
e set)
o bn
h ln
i kh
i ln
a oxy
n men
ua h
c
g ni
u khi
v ra
c

-
ho tan (
lm thay
xuyn
cm bin
d cm b
ng m
trnh nu
ni ln m
nhn v
gi tr c
hiu ch
cho pH t
din v t
nh vy
men.
-
ng ny
dch np

3.2. H
-
ln men
cho h th
gii nhi

-
tch n
H thng c
(DO
2
), ph
y i cc
c duy tr
n ghi nhn
bin gn tr
kim
i cy. V d
men c
gi gi tr n
i t v sau
nh pH OH
-
tng dn ln
thng tin s
y, pH nui c
Cc ng
u c gn
p vo hoc c
thng cun
My nn k
. Lu l n
hng thit b
t thng d
Hn
H thng t
c ngng (c
cc u d
bt t ng
c im l-
gi tr p
v hiu ch
c tip vo
sot lng
d trong tr
ci t l 7
ny v thit
u s gi
-
s c cu
n v khi v
c x l
cy ca dc
g ng: np l
n van nhm
chit ra khi
ng cp oxyg
kh: ht khn
nhit pht r
b nn v kh
ng l nc
nh 3. 3. My
tch nc n
cyclone). D
cm bin
g . Trong
-ha ca d
ph hp v
nh kp th
ni ln men
tc nhn h
ng hp k
7,5. Khi pH
t b iu kh
tn hiu n
ung cp v
t qua kh
l, van t
h ln men
iu, np gi
m c lp cc
cn thit.
gen (khng
ng kh v n
ra trong qu
hng kh n
c lm m
y nn khng
ngng trong
Do khng k
(sensor)
g qu trnh
ch ln men
i nhu cu c
i. Mt h th
n, thit b g
hiu chnh
kim sot pH
H gim xu
hin trung t
n van t
o ni ln m
i ngng c
ng s ng
c kim
ng, ly m
c h thng h
kh nn ho
nn li p
trnh nn
n trc kh
t (chilled w
g kh s dn
g kh nn, b
kh sau khi
iu khin
ln men, do
n. bo
ca vi sinh
hng iu k
ghi nhn v
cung cp v
H t ng.
ng di 7,
m. y s
ng. Van t
men. Khi OH
i t 7,5 th
g li, ngng
sot t n
u, ng x
h tr vi n
oc ngun o
sut 1-3 kg
khng kh
hi cung cp
water/ cooli
ng trong c
bao gm c
c nn l
n pH, nhit
o hot ng
m cc yu
h vt, cn ph
kin t n
x l tn hi
vo ni ln
Gi tr pH
,5 u d c
s phn tch
ng s m
H
-
vo ni
h u d c
g cung cp
ng trong su
x dch T
ni ln men
oxygen lng
gf/cm
2
cu
do vy cn
vo ni ln
ing tower w

ng nghip.
c thit b h
li nhit
t , ox
g ca vi sinh
u t ny th
hi c h th
ng bao gm
iu, van t
n men trong
vn hnh t
cm bin s
h v so snh
m ra v tc
ln men s
m bin s
ngun OH
ut qu trnh
Tt c cc
n v iu
g)
ung cp cho
n phi gii n
n men. Tc
water).
h nhit, thi
s tng ca
25
xygen
h vt
ng
hng
: u
ng
g qu
trong
ghi
h vi
nhn
lm
nhn
H
-
. C
h ln
ng
u tit
o ni
nhit
nhn
it b
ao v

trong kh
ngng v
gim lu
-
ch s d
-
3.3. H
-
-
Hnh 3.

3.4. H
-
-
-

3.5. L
C
ni ln m
lng (LP
boiler).

hng kh c
v h nhit
u lng kh
Thit b lc
dng.
Cc thit b
thng n
Thp gii n
Bm cp n
4. S h
thng chu
Bn cha c
H thng th
Thp lu
hi
Cung cp h
men, mi tr
PG), kh n

m ln
. Hm l
qua cc lc
c kh. Kch
iu khin
c gii nhi
nhit, my l
ngun nc
thng my
un b v th
cc thnh ph
hit b trao
i nc nn
ng nui c
n thin nhi

nn trc k
ng m cao
c khi v c
thc l l
n p sut, l
t
lm lnh (ch
nhit th
y lm lnh n
anh trng
hn nguyn
i nhit li
ng vi p s
cy. C t
n (CNG)

khi s dng
o trong kh n
th gy tp
c c th th
u lng.
hiller)
hp cho ni
nc cung c
mi trng
n liu, bm,
n tc
sut >1,5 k
th dng c
t l ho
g cho ln m
nn s lm
p nhim.
hay i t 0
ln men

cp cho h
g lin tc
, thit b ph
kgf/cm
2
ph
c nhin liu
oc dng i
men cn ph
tng th tc
0,01 - 0,5 m
thng ln m
h tr
c v cho v
u nh du D
in tr t
i tch loi n
ch dch ln
mm ty theo
men.
vic thanh t
DO, FO, kh
nng (elect
26
nc
men,
mc
trng
h ha
trical
27
CHNG 4. MI TRNG NUI CY TRONG LN MEN
CNG NGHIP

1. Gii thiu
Mi trng nui cy c thit k ty theo yu cu ca chng vi sinh vt, phng
php ln men v sn phm mc tiu. Trc khi thit k mi trng ln men cn kho st
chi tit v nhu cu dinh dng ca chng, ngun c cht trc tip to ra sn phm v h
thng thit b-cng ngh ln men. Tuy nhin cc mi trng ln men u c chung mt
s yu cu cn bn. Ton b cc chng vi sinh vt trong qu trnh sinh trng, pht trin
v to sn phm trao i cht u cn nc, ngun nng lng, ngun carbon, ngun
nitrogen, ngun khong vi lng, vitamin v oxygen (i vi trng hp hiu kh). i
vi qui m phng th nghim th vic chun b mi trng tng i n gin v thng
dng cc hp cht tinh khit. Tuy nhin vi qui m sn xut cng nghip th cng on
ny kh phc tp.
Trong cng nghip ngi ta dng cc ngun dinh dng khc nhau chun b
mi trng vi yu cu l cng tha mn cc iu kin sau y cng tt.
- Tng cng ti a hiu sut to sinh khi hoc/v sn phm
- Tng cng ti a nng sinh khi hoc sn phm
- Cho php to c tc sn xut ti a
- Hn ch n mc ti thiu hiu sut tng hp cc sn phm khng mong mun.
- n nh cht lng v sn sng cho s dng quanh nm
- Hn ch n mc ti thiu vic xy ra cc vn trong qu trnh chun b v
thanh trng mi trng.
- Hn ch n mc ti thiu cc vn ny sinh nh gy tr ny cho vic khuy
trn, chit tch, tinh sch v x l cht thi.
Cn lu l vic chn mi trng c lin quan mt thit n vic thit k ni ln
men, nh hng n qu trnh thu hi sn phm v qu trnh x l cht thi sau ln men
v cc yu t lin quan n php lut, vn ha, tn gio,
2. Thit k mi trng ln men
Thit k mi trng ln men l bc rt quan trng quyt nh s thnh cng
trong nui cy vi sinh. Thnh phn mi trng nui cy phi bo m lng phc
v cho vic tng sinh khi, to cc cht trao i v cung cp nng lng cho vic duy
tr mt t bo v s sinh tng hp. Phng trnh cn bng vt cht trong qu trnh ln
men c m t nh sau:
[C v E] + [N] + O
2
+ Cc yu t khc Sinh khi + Sn phm + CO
2
+ H
2
O + Nhit
Thnh phn mi trng bao gm cc yu t a lng, trung lng v vi lng.
- Yu t a lng bao gm: Ngun nc, ngun carbon, ngun nit.
28
- Yu t vi lng bao gm: Ngun mui khong, ngun vitamin, cc cht h tr
tng trng, cht kim sot
Da vo thnh phn v hm lng ca cc cht trong t bo vi sinh vt thit k
thnh phn v lng cn thit nhm thu nhn c sinh khi theo yu cu, bo m
sn xut. Bng 4.1 m t mt s thnh phn cn bn ca sinh khi cc loi vi sinh vt
khc nhau, cho thy carbon chim khong 50% trng lng kh t bo, v cc nguyn t
C, O, N, H chim tng cng khong 92%.
Bng 4. 1. Thnh phn v hm lng cc nguyn t trong t bo vi sinh vt

n v: % theo trng lng kh ca t bo.

Bng 4. 2. Phn tch thnh phn ca mt s chng vi sinh vt.
Chng vi sinh
vt
Ngun
carbon
Tc
tng
trng
Thnh phn
Cng thc c
nh
Trng
lng
phn t
C H N O
Klibsiella
aerogenes
Glycerol 0,1 50,6 7,3 13,0 29,0 CH
1,74
O
0,43
N
0,22
23,7
Aerobacter
aerogenes
Complex 48,7 7,3 13,9 21,1 CH
1,78
O
0,33
N
0,24
22,5
Aerobacter
aerogenes
Complex 0,9 50,1 7,3 14,0 28,7 CH
1,73
O
0,24
N
0,43
24,0
Sacchromyces
cerevisiae
47,0 6,5 7,5 31,0 CH
1,66
O
0,49
N
0,13
23,5
Cc nguyn t E.coli Vi khun Nm men Nm mc
C 50 50-53 45-50 40-63
O 20
H 8 7 7
N 14 12-15 7,5-11 7-10
P 3 2-3 0,8-2,6 0,4-4,5
S 1 0,2-1,0 0,01-0,24 0,1-0,5
K 1 1-4,5 1,0-4,0 0,2-2,5
Na 1 0,5-1,0 0,01-0,1 0,02-2,5
Ca 0,5 0,01-1,1 0,1-0,3 0,1-1,4
Mg 0,5 0,1-0,5 0,1-0,5 0,1-0,5
Cl 0,5 0,5 - -
Fe 0,2 0,02-0,2 0,01-0,5 0,1-0,2
Khc 0,3
29
Sacchromyces
cerevisiae
50,3 7,4 8,8 33,5 CH
1,75
O
0,15
N
0,50
23,9
Candida utilis Glucose 0,45 46,9 7,2 10,9 35,0 CH
1,84
O
0,56
N
0,20
25,6
Candida utilis Ethanol 0,43 47,2 7,3 11,0 34,6 CH
1,84
O
0,55
N
0,20
25,5

T cc kt qu phn tch thnh phn t bo Bng 4.2 trn y cho thy, hm
lng carbon thay i t 46-50%, H t 6-7%, N t 8-14% v O t 29-35%. S thay i
ny ty thuc vo c cht v iu kin tng trng. Trong tnh ton k thut, ngi ta s
dng cng thc chung ca vi sinh vt l: CH
1,8
O
0,5
N
0,2
. Da trn cng thc ny, trng
lng phn t t bo c xc nh l: 24,6.
V d: Mun sn xut thu 10 g t bo khi s dng glucose lm ngun carbon th
lng glucose ti thiu cn thit trong mi trng phi l bao nhiu?
Cng thc c nh ca t bo: CH
1,8
O
0,5
N
0,2
Glucose: C
6
H
12
O
6
: MW 180.
S phn t mol t bo cn l: 10/24,6
S mole glucose cn 1/610/24,6
Lng glucose cn: 1/610/24,6180 = 12,2 g.
Mt s mi trng thng dng:
1. Mi trng sinh tng hp Amylase (Underkofler, 1966)
Dch thy phn u nnh 1,85%
Dch chit nm men thy phn 1,5%
Dch thy phn casein 0,65%
Lactose 4,75%
MgSO
4
.7H
2
O 0,04%
Cht ph bt 0,05%
2. Mi trng sn xut glutamic acid (Gore et al., 1968)
Dextrose 270g/L
NH
4
H
2
PO
4
2g/L
(NH
4
)
2
HPO
4
2g/L
K
2
SO
4
2g/L
MgSO
4
.7H
2
O 0,5g/L
MnSO
4
.H
2
O 0,04g/L
FeSO
4
.7H
2
O 0,02g/L
Polyglycol 2000 0,3g/L
Biotin 12mg/L
Penicillin 11mg/L
30
3. Mi trng sn xut Pennicillin (Perlan, 1970)
Glucose hoc r ng (feed lin tc) 10%
Dch chit bp 4-5%
Phenylacetic acid 0,5- 0,8 %
Lard oil (hoc du thc vt) dng lm cht ph bt 0,5%
pH 6.5-7.5 (acid hoc kim)
4. Mi trng sn xut Endotoxin t Baccillus thuringiensis (Holmberg et al., 1980)
R ng 0-4%
Bt u nnh 2-6%
KH
2
PO
4
0,5%
K
2
HPO
4
0,5%
MgSO
4
0,0005%
MnSO
4
0,003%
FeSO
4
0,001%
CaCl
2
0,005%
Na(NH
4
)
2
PO
4
.4H
2
O 0,15%


3. Cc thnh phn ca mi trng ln men
3.1. Nc
T bo vi sinh vt cha 70% l nc, do vy nc l yu t quan trng trong tt c
cc mi trng ln men. Ngoi ra nc rt cn thit cho cc hot ng khc trong cng
ngh ln men nh l tc nhn gia nhit, gii nhit hoc phc v cho vic v sinh thit b.
Ngun nc cung cp phi n nh, sch bo m cc yu t nh pH, cc mui
khong ha tan v mc tp khun trong nc.
Hm lng khong trong nc rt quan trng i vi cng nghip ln men sn
xut bia. V d nc cng c hm lng CaSO
4
cao rt tt cho bia ng Bourton ca
Anh, nc vi hm lng carbonate cao th tt cho cc loi bia c mu sm hn nh bia
nu. Ngy nay, trc khi dng cho qui trnh ln men nc thng c x l bng
phng php kh ion, hay cc k thut khc nh b sung mui khong, hiu chnh pH,
lc b tp khun,
Trong thc t, tit kim, gp phn gim thiu gi thnh sn xut, ngi ta
thng nghin cu ti s dng ngun nc thu hi t cc qui trnh khc. Trong mt s
qui trnh sau ln men, giai on thu hi hoc kt tinh, mt lng ln nc thot ra t
qui trnh ny do hot ng c c. Lng nc ny khi thot ra trong qu trnh bay hi
ko theo mt s hp cht khc trong dch nh NH
3
(cn li trong dch ln men ca m
trc), cc hp cht hu c d bay hi, sau c a vo ti s dng cho ln men,
gp phn lm gim ti cho qui trnh x l nc thi ca h thng.
3.2. Ngun nng lng
31
Nng lng cho s tng trng ca vi sinh vt bt ngun hoc l t s oxy ha cc
thnh phn dinh dng trong mi trng hoc l t nh sng. Hu ht trong cng nghip
vi sinh, cc chng thng dng l ha dng. Do vy, ngun nng lng thng dng
nht c dng l t ngun carbon nh cc carbohydrate, lipid v protein. Mt vi chng
cn dng c c hydrocarbon hay methanol lm ngun C v nng lng cho qu trnh
tng trng v trao i cht. Chu trnh TCA va cung cp cc acid hu c lm khung C
hnh thnh cc cht quan trng khc cung cp cho t bo mt khc cn cung cp cc
cht mang nng lng ln nh NADH, FADH, cc cht ny sau di vo chui chuyn
in t tng hp ATP cung cp ngun nng lng ln cho hot ng ca t bo.

32
Acetyl-CoA
CoA
Citrate
3-
Isocitrate
3-
Aconitate
3-
Succinyl-
CoA
o-Ketoglutarate
2-
Succinate
2-
Oxaloacetate
2-
Malate
2-
Fumarate
2-
Pyruvate-(C3)
NADH
CO
2
CO
2
CO
2
CoA
NAD(P)H
CoA+NAD
+
NADH
GTP
GDP+Pi
FADH
FAD
NADH
NAD
+
NADP
+
Overall reaction: Pyruvate-+4NAD++FAD 3CO
2
+4NADH+FADH
(1)Substrate level: GDP+Pi
phosphorylation GTP+ADP
(2)Electron transport 4NADH = 12ATP
phosphorylation FAD = 2ATP
GTP
GDP+ATP
(3)Sum: CAC plus Glycolysis
38 ATP per glucose
15 ATP

Hnh 4. 1. Chu trnh TCA v cn bng nng lng.

3.3. Ngu
N
cc sn
men cn
-
-
-
C
m. N
C. Ngu
bo, ole
glucose.
vi mt
K
nit hu
ng Mai
tng tr
-
-
ketosam
-
cht c m

un carbon
Ngun carbo
phm trao
n da trn c
Sn phm c
Gi thnh s
tinh kh
Cc ngun C
Ngoi ra, m
n carbon t
eic, linoleic
Ngun hyd
s qui trnh
Khi thanh tr
u c v ch
illards, cara
ng ca t b
Phn t glu
Sau hn
mine
Ketosamin
mu, gy c
n
on c s
i s cp
c yu t sa
chnh ca q
sn phm
it ca nguy
C thng d
t r ma
du (du
, linolenic,
docarbon v
h ln men n
rng mi tr
ng d dng
amen ha) t
bo. Phn
ucose kt h
nh thnh im
ne b dehyd
c ch tng t
dng th
p v th cp
au:
qu trnh
yn liu (c
dng l car
ng hoc m
olive, du b
), c th
v cc dn x
ngi ta cn
ng ln m
g phn ng
to nn hp
ng Maillar
p vi amin
mmonium io
drate ha v
trng ca v
ng c nh h
p. Khi chn
c ion kim l
rbohydate:
mt r c c
bp, du co
cung cp
xut: n-alka
n dng cellu
men, nn tc
g vi NH
2
-
p cht cha
ds xy ra th
no acid h
on v din r
kt hp v
vi sinh vt.
hng n
n la ngun
loi)
bt ng, b
i ng c
otton, du
nng lng
ane, methan
ulose lm ng
ch ring ng
trong cu t
a nitrogen c
heo 3 giai
hnh thnh N
ra qu trnh
vi reduton

s tng h
n carbon ch
t ng cc
ng c d
u phng
g cao hn
ne, methano
gun cung
gun ng
trc ca am
c mu en
on:
N glucoside
h isomer h
nes hnh

p sinh kh
ho qui trnh
, khoai ty
dng lm n
) m (cc
nhiu so v
ol. Ngoi ra
cp C.
g kh vi n
mino acid (
gy c ch
e
a hnh t
h thnh cc


33
i hay
h ln
y, bt
gun
c acid
i t
a, i
gun
(phn
cho
thnh
c hp

3.4. Ngu
H
ngun n
men, dc
cnc
mi tr
1990). C
vi sinh v
dng th
nn pH
khi ngu
lm ngu
chng G
ng ho
ca vi si
amino a
hn nu
amino a
sung m
acid ny
N hu c
sn xut
nnh
C
u nnh
un nitroge
Hu ht cc
nitrogen l v
ch chit tht
c mm, ).
ng nui c
Cc mui c
vt s dng
gy nn s
acid khi NH
n NH
4
+
b
un N. Tuy
Gibberella f
NH
4
+
ca
inh vt chuy
acids, protei
chng ta cu
acid nht
t lng nh
y. Tuy nhin
phc tp,
t lysine, ng
b sung v
Cc hp ch
h, u xanh
en
chng vi s
v c (amm
t, dch chit
. NH
3
c
y Sac. cer
a ammoni
g NH
4
+
v g
s thay i
H
4
+
c s
cn kit, c
nhin c m
fujikuroi (B
a vi sinh vt
yn dn qua
in, urea. Tr
ung cp ngu
nh cho tn
t nh ami
n, cc amin
, thng kh
i ta s d
o mi trn
t N dng l
h, t ht co
inh vt s d
monia, nitrat
t cao bp, d
dng va
rivisiae sn
um nh am
ii phng g
dn sang
dng v s
s chuyn
mt ngoi l
Borrow et al
t. khi pH t
a NH
4
+
. Ng
rong nhiu
un N l h
ng trng. T
no acid i
no acid dn
hng ng n
ng methion
ng nui cy
lm ngun
oton, cao n
dng trong
te, nitrite,) v
dch thy ph
hiu chn
xut cng
mmonium su
gc acid t
iu kin k
s gy c c
n dn sang
cho kiu
l., 1961, 19
ng dn n
gun N hu
trng hp
u c, v m
Trong ln m
i vi cc ch
ng b sun
nht, r tin
nine v thre
y thay th ch
cung cp a
m men. Ng
cng nghi
v hu c (
hn u nn
nh pH va
nghip dc
ulphate, th
do. Mt kh
kim. Ban
ch qu trn
iu kin k
trao i ch
964). pH
n gi tr thc
u c c s
p, s tng tr
mt s chng
men amino
hng t bi
ng vo mi
n, n nh. T
eonine thu n
ho loi tinh
amino acid
goi ra cc
p c th d
(amino acid
nh, peptone,
l ngun cu
ch albumin
ng gy n
hc, khi gc
u, ammon
nh ng ha
kim khi ni
ht ngun N
2,8 -3,0, n
ch hp, s
dng c th
rng ca
g vi sinh v
acid cng
n khuyt d
i trng nu
Trong cng
nhn t dch
h khit, t t
l dch chi
dch ny c
ng c c
d, urea, cao
, dch thy
ung cp N t
ngi (Co
nn tnh acid
c nitrate
nium nitrat
a nitrate. Sa
trate c
N ny l
nitrate c ch
ng ho ni
h t cc n
chng s n
t i hi m
nghip, c
ng cc am
ui cy l n
nghip ln
h thy phn
tin.
t cao bp,
cn b sung
34

c hai
nm
phn
trong
llins,
d khi
c s
e to
au
dng
i vi
h s
itrate
gun
nhanh
mt s
n b
mino
gun
n men
n u
dch
g cc

yu t k
ca chn
v thi g


T
chun
cn quan
-
mt enz
hin din
ngun N
cho thy
khc nh vi
ng c th b
gian lu gi
Trong sn xu
n b mi tr
n tm n c
C ch ki
zyme lin q
n ca amm
N c s d
y rng NH
4
tamin, kho
b nh hn
.
Hnh 4.
ut vaccine
ng, khng
cc yu t sa
m sot vi
quan n vi
onia. Nguy
dng thch h
4
+
c ch s
ng. Trong
ng bi tc
. 2. Cc con
es cho ng
g nn dng
au:
c s dng
c chuyn h
yn nhn c
hp hn. N
s hp th
qu trnh c
ng ca y
n ng ch
i, cn phi
g amino acid
ngun nitr
ho nitrate
a qu trnh
Ngi ta
cc amino
ct gi cc
u t mi tr
uyn ha N
s dng ng
d t protein
rogen tn t
thnh ion
h ny l do a
c cc th
acids ni
sn phm
rng nh
Nit.
gun amino
n. Khi chn
i bi enzy
NH
4
+
, b
ammonia h
nghim kh
chung v c
ny, cht l
m, nhi
o acid tinh k
n la ngun
yme kh nit
c ch khi c
hoc ion NH
ho st trn
cc amino a
35
ng
t

khit
n nit
trate,
c s
H
4
+
l
nm
acids
36
mng ni ring. i vi Aspergillus nidulans, ammonia iu ho s sn xut cc protease
kim tnh v trung tnh. V th, trong mt hn hp ngun N, tng thnh phn ca N c
nh hng n s iu ha trao i cht v do vy c s chn la ngun N thch hp
s dng trc cho n khi ngun ny cn kit.
- Ngi ta chng minh rng vic sn xut khng sinh bi nhiu chng vi sinh
vt khc nhau chu nh hng bi loi v nng nitrogen trong mi trng nui cy.
Ngun N d ng ha nh NH
4
+
, NO
3
-
, cc amino acid c th c ch s sinh tng hp
cc cht khng sinh. S sn xut khng sinh ch bt u tng trong mi trng nui cy
sau khi ton b ngun N c s dng ht.
Trong th nghim nui cy lc trong bnh tam gic, mui ca cc acid yu (ammonium
succinate) c b sung vo lm ngun N v hn ch s thay i pH, c th dng
mui ammonium ca cc acid mnh nh Cl
-
hoc SO
4
2-
. Theo cch ny, ngi ta c th
dng mui phosphate c nng thp hn trong mi trng lm h m pH, nhng
nu nng mui phosphate cao c th gy c ch vic sn xut nhiu cht trao i th
cp.
- Trong ln men sn xut gibberellin, ngun N nh hng ln vic nh hng sn
xut cc loi gibberellin v t l tng quan gia cc loi ny (Jefferys, 1970).
Nm 1963, Rhodes chng minh rng nng ti thch ca ngun N cho sn
xut griseofulvin c s bin thin ty thuc vo kiu cy ging v loi ni ln men ang
s dng. Nh vy, cc yu t ny cn phi c cp khi gii thch cc kt qu cc
chng trnh nghin cu pht trin cc loi mi trng nui cy vi sinh vt. Mt s
nguyn liu c cha ngun N phc tp, vi sinh vt khng th s dng c v gy tr
ngi vi cc qui trnh thu hi v qui trnh x l cht thi.
3.5. Cc nguyn t khong trung-vi lng
Hu ht cc chng vi sinh vt i hi mt s nguyn t trung-vi lng nht nh
cho s tng trng v trao i cht. Trong nhiu mi trng nui cy, Mg, P, K, S, Ca,
Chlorine l cc thnh t rt cn thit. Ty tng loi mi trng m nng yu cu cng
khc nhau v do vy chng thng c b sung vo mi trng t cc thnh phn ring
bit. Cc nguyn t khc nh Co, Cu, Mn, Mb v Zn cng l nhng nguyn t ng vai
tr quan trng thng c sn trong cc thnh phn nguyn liu nhng ch vi hm lng
rt nh nn cn phi phn tch mi trng nui cy nu cn thit th phi b sung thm
nhm lun bo m rng cc nguyn t vi lng t hm lng theo nhu cu ca vi sinh
vt trong mi trng nui cy. Trong thc t, cht lng ca ngun nguyn liu khng

n nh
Bng 4.
Bng 4.
Tng hm
Hm ln
Tng ln
Tng ln
Tng ln
Alanin
Argini
Glutam
Leucin
Proline
Isoleuc
Threon
Valine
Phenyl
Methio
Cystin
Hm l

C

(v ngun
3 cho thy
3: Thnh p
m lng cht r
ng acid qui v
ng ng kh
ng ng kh
ng nitrogen:
ne
ne
mic acid
ne
e
cine
nine
e
lalanine
onine
ne
ng tro
Cc nguyn
Hnh 4. 3.
gc t nhi
thnh phn
phn dch th
rn
lactic acid
t do
t do sau th
t vi lng
M hnh en
in) nn h
n cc cht tr
hu phn ca
y phn
5
15
5.
6.

4%
25
8
8
6
5
3.
3.
3.
2.
1.
1.
1.
g ny ng v
nzyme hydro
m lng c
rong dch th
ao bp
1%w/v
5%w/v
.6%w/v
.8%w/v
%w/v
5
.5
.5
.5
.0
.0
.0
.25%w/v
vai tr rt q
olase vi ng
a cc ngu
hy phn ca
Potassium
Phosphoru
Sodium
Magnesium
Iron
Copper
Calcium
Zinc
Lead
Silver
Chromium
Vitamine
Aneurine
Biotin
Ca-pantoth
Folic acid
Nicotinam
Riboflavin
quan trng t
guyn t Zn
uyn t ny
ao bp.
m
us
m
m
thenate
d
mide
ne
20%
1-5
0,3
0,0
0,0
0,0

0,0

0,0

41-
0,3
14,
0,2
30-
3,9
trong cu tr

n tm ho

y cng thay
%
5%
3-1%
003-0,3%
01-0,3%
01-0,03%
003-0,08%
001-0,003%
-49 mg/g
34-0,38 mg/g
,5-21,5 mg/g
26-0,6 mg/g
-40 mg/g
9-4,7 mg/g
rc ca enzy
t ng.
37
y i.
yme.
38
Bng 4. 4. Vai tr ca cc nguyn t vi lng
Nguyn t kim loi Vai tr
Coban (Co) Cn to Vitamin B12, transcarboxylase ca vi khun ln
men propionic.
Molypden (Mo) C trong cu trc ca molybdoflayvoprotein tham gia kh
nitrate v trong nitrogenase kh N
2
.
ng (Cu) C trong mt s enzyme tham gia h hp nh cytochrome C;
c trong enzyme tham gia quang hp nh plastocyanin v mt
s superoxide dismutase.
Mangan (Mn) Lm hot ha nhiu enzyme, c trong mt s superoxide
dismutase, l nhng enzyme rt quan trng kh c dng
c O
2
.
Niken (Ni) C trong cc enzyme hydrogenase c chc nng nhn hoc
phng thch H
2
.
Tungsten v Selen C vai tr trong nh dng cu trc enzyme dehydrogenase
St (Fe) C trong nhiu enzyme trong t bo c bit l cc enzyme h
hp.
Km (Zn) ng vai tr trong cu trc nhiu enzyme: carbonic anhydrase,
alcol dehydrogenase, DNA polymerase, RNA polymerase.
Thng ng vai tr ni cc tiu n v protein trong s sp
xp cu hnh c bit cho hot ng ca enzyme.

Khi dng mi trng tng hp nui cy vi sinh vt, cn phi b sung mt lng
chnh xc cc nguyn t vi lng. Cc loi mui v nng thng dng trong chun b
mi trng nui cy c tm tt theo Bng 4.5.
Bng 4. 5. Khong nng thch hp ca mt s thnh phn khong
Thnh phn Khong nng
thch hp (g/L)
KH
2
PO
4

MgSO
4
.7H
2
O
KCL
CaCO
3

FeSO
4
.4H
2
O
ZnSO
4
.8H
2
O
1,0-4,0
0,25-3,0
0,5-12,0
5,0-17,0
0,01-0,1
0,1-1,0
39
MnSO
4
.H
2
O
CuSO
4
.5H
2
O
NaMoO
4
.2H
2
O
0,01-0,1
0,003-0,01
0,01-0,1

Nng phosphate cc mi trng nui cy trong th nghim thng cao hn
nng cc khong khc. Mt phn ca phosphate ny c dng ng vai tr l cht
m, gim thiu s thay i pH khi khng s dng h thng hiu chnh pH t bn ngoi
trong qu trnh nui cy.
Trong mt s qui trnh c trng, nng ca mt s cht khong nht nh rt
quan trng. Cc qu trnh sinh tng hp cht trao i th cp c gii hn chp nhn i
vi phosphate v c thp hn so vi qu trnh tng trng. Ngun phosphate ny thp
cho vic ng ho trc khi kt thc trophosphase. Garner v cng s (1950) cho rng
chc nng quan trng ca mui calcium trong mi trng ln men l lng t cc
phospate v c d tha v tng cng hiu sut ca streptomycine. Nng phosphate
v c trong mi trng cng nh hng n s sn xut ca bacitracins, citric acid (trong
nui cy b mt), ergot, monomycin, novobiocin, oxytetraclin, polyenes, ristomycin,
rifamycin Y, streptomycin, vancomycin v viomycin. Tuy nhin, trong mi trng nui
cy sinh tng hp pyrronitrin, thiopeptin v methylenomycin th cn phosphate vi nng
cao. Hai khng sinh monomycin c sn xut chn lc bi chng streptomyces
jamaicensis khi nng phosphate l 0,1 mM hoc 0,4 mM. S iu ha phosphate cng
c tho lun bi Weinberg (1974), Aharonowitz v Demain (1977), v nhiu tc gi
khc.
Trong bo co tng kt ln men sinh tng hp khng sinh, Liras v cng s (1990)
nhn din c enzyme ch b (a) c ch bi phosphate, (b) ngn chn bi phosphate,
(c) c ch sinh tng hp mt enzyme nhng khng r rng. Th t cc bc kim sot
ca phosphate cng c xc lp v xc nh cc c tnh t promotor iu ho
phosphate kim sot s sinh tng hp candicidin.
Weiberg (1970) tng kt 9 nguyn t vi lng c nh hng sinh hc. Trong s
9 nguyn t ny, nng ca Mn v Zn l nhy cm quan trng nht trong c ch trao
i cht th cp. Trong tt c cc h thng trao i cht bc 2 m trong , ngi ta
thu thp y d kin cho thy rng hiu sut sn phm bin thin tuyn tnh vi nng
nng (theo hm logarit) ca mt kim loi quan trng (cha kho). Mi tng quan
tuyn tnh ny khng c xc lp khi nng ca kim loi vi lng ny khng hoc
c c tnh i vi tng trng ca t bo. Hiu sut sinh tng hp mt s sn phm sinh
hc bc mt v bc hai chu s nh hng bi nng ca cc kim loi vi lng hn
40
nng i hi cho tng trng ti a. Theo Foster (1949), Chlorine khng ng vai tr
nh l mt thnh phn dinh dng trong qu trnh trao i cht ca nm mc.
3.6. Chelator (Cht km)
i vi nhiu loi mi trng ln men thng c tnh trng l ngay sau khi pha
ch hoc sau khi thanh trng c s hnh thnh cht lng cn dng phc hp ca
phosphate kim loi khng tan. Gaunt v cng s (1984) chng minh rng khi thanh
trng mi trng Mandels v Weber (1969), mt cht lng cn mu trng c hnh
thnh c cha ton b Fe v hu ht cc kim loi nh Ca, Mn, Zn hin din trong mi
trng. Hin tng hnh thnh lng cn ny va lm gim nng cc cht cn thit cho
s pht trin v bin dng ca vi sinh vt va gy cn tr cho qu trnh thu hi sn
phm v v sinh thit b.
gii quyt vn v s hnh thnh cc phosphate kim loi khng tan trong mi
trng ln men, ngi ta b sung cc cht chelator nng thp nh ethylene diamine
tetraacetic acid (EDTA), citric acid, polyphosphates vo mi trng nui cy. Cc
cht ny to cc phc vi kim loi trong mi trng v c s dng dn trong qu trnh
nui cy. Ngi ta nhn thy rng khi thm vo mi trng Mandels v Weber lng
EDTA c nng 25 mg/dm
3
th khng thy c s xut hin ca cht lng cn sau thanh
trng. Lu vic chn la cc chelator khng gy c ch cc chng vi sinh vt trc
khi a vo s dng. i vi mi trng dng qui m cng nghip, khng cn phi b
sung chelator, do cc thnh phn phc nh dch chit nm men, pepton, s to phc vi
kim loi v bo m rng kim loi c gii phng dn trong qu trnh tng trng.
3.7. Nhn t tng trng
Mt s chng vi sinh vt khng th tng hp ton b cc thnh t cu to nn t
bo, do vy chng i hi cn c mt s tin cht nh cc vitamin, mt s amino acid
c trng, acid bo hoc sterol, nhng cht ny gi l nhn t tng trng. Cc nhn t
tng trng ny thng c trong cc ngun nguyn liu t nhin cung cp ngun C v N
dng trong cc cng thc mi trng nui cy. Tuy nhin trong mt s trng hp th
cc nhn t tng trng ny trong mi trng khng p ng nhu cu ca vi sinh
vt nn cn thit phi b sung vo. V d ngi ta b sung calcium pentothenate trong
cng thc mi trng sn xut dm, hay trong qui trnh ln men sn xut glutamic
acid, biotin cng c b sung vi mt nng nht nh.
3.8. Cc cht m pH
41
Mun thu nhn c sinh khi vi nng sut ti a, pH mi trng phi c
kim sot gi tr ti u. Mt s hp cht khi b sung vo mi trng nui cy ngoi vai
tr l thnh phn dinh dng, cn ng vai tr c trng l cht m nhm iu chnh pH
mt gi tr n nh. Nhiu mi trng c kim sot pH 7.0 nh s kt hp ca m
CaCO
3
. Khi pH gim, carbonate b phn hy hiu chnh pH n nh. Ngoi vai tr
quan trng i vi dinh dng t bo phosphate cn ng vai tr l cht m pH. Tuy
nhin, nu nng phosphate cao s gy c ch sinh tng hp cc cht trao i th cp.
S cn bng v t l ngun C v N trong mi trng cng hnh thnh nn mt c
s cho vic kim sot pH nh vai tr ca mt h m. Ngun C v N ny thng c
cung cp bi protein, peptides, amino acids nh trong dch chit bp. pH ca mi
trng nui cy c th c iu chnh bng cc cht nh NH
3
, NaOH, H
2
SO
4
ty theo
yu cu.
3.9. Cht tin thn
Mt s ha cht khi c b sung vo mt s loi mi trng ln men s h tr
trc tip cho vic tng hp mt sn phm mong mun. V d khi b sung cao bp (corn-
steep) vo mi trng ln men sn xut penicilin lm tng s tch lu sn phm t 20 n
v/cm
3
ln 100 n v/cm
3
dch mi trng. Trong cao bp c cha phenylalanine lm
tng cng sn xut benzyl penicillin.
3.10. Cht km hm
Khi b sung cc cht km hm vo mi trng ln men, mt sn phm c trng
nht nh s c tng hp nhiu hn, hoc mt cht trao i trung gian theo con ng
thng thng s c tch ly dn. S sinh tng hp glycerol bng vi sinh vt l mt v
d tiu biu. S sn xut glycerol ph thuc vo bin i ca ethanol khi loi b
acetaldehyde. Khi thm sodium bisulphite vo mi trng s dn n s hnh thnh
acetaldehyde bisulphite. Do vy khng cn acetaldehide ti oxy ho NADH
2
, ng vai
tr l cht nhn hydrogen ca n b mt i bi dihydroacetone phosphate, sinh ra trong
qu trnh glycolysis. Sn phm ca phn ng ny l glyceral 3-phosphate, sau chuyn
thnh glycerol. ng dng ca cc cht km hm c trng trong ln men c ghi nhn
trong Bng 4. 6.
Bng 4. 6. Cc cht km hm dng trong ln men
Sn phm Cht km hm nh hng chnh Chng vi sinh vt
-Glycerol Sodium bisulphite c ch sn xut acetaldehyde Sac. cerevisiae
-Tetracycline

Bromide

c ch s hnh thnh
Chlortetracycline
Strep.aureofaciens

42
-Glutamic acid Penicilline Tnh thm ca mng t bo Mic. glutamicus
-Citric acid Cht kim metal/
phosphate, pH<2.0
c ch Oxalic acid

Asp. niger

-Valine

Nhiu cht c ch
khc nhau
Nhiu nh hng vi cc cht c
ch khc nhau
Bre. roseum
-Rifamycine -B Di-ethyl barbiturate

Km hm cc Rifamycine khc

Nocardia
mediterranei
-7-chloro-6 demethyl-
tetracycline
Ethione nh hng ln cc phn ng
chuyn 1 carbon
Strep.aureofaciens

3.11. Cht cm ng
a s cc enzyme c sn xut trong cng nghip u l enzyme cm ng. Cc
enzyme ny ch c sinh tng hp khi c s p ng vi s hin din ca cht cm ng
trong mi trng nui cy. Cc cht cm ng thng l cc c cht bt hoc ng i
vi amylase; maltose i vi pullunase v pectin i vi pectinase. Mt s cht c ch c
nh hng rt mnh nh isovaleronitrile cm ng sinh tng hp nitralase. Cc c cht
tng ng khng b phn hy bi cc enzyme cng c th ng vai tr l cht cm ng.
Hu ht cc cht cm ng l cc c cht tng ng nhng cng c khi l cht trao i
trung gian hoc sn phm lm cht cm ng. V d maltodextrine cm ng sinh amylase,
acid bo cm ng sinh lipase. Tuy nhin, do gi thnh cao nn trong cng nghip ngi
ta khng dng chng lm cht cm ng. Mt ng dng ly nm men lm cht cm ng
trong sn xut streptomycin. Trong qu trnh ln men, streptomycin v
mannosidostreptomycin c sinh tng hp. Bi v mannosidostreptomycin ch c hot
tnh khong 20% so vi streptomycin, do vy n l sn phm khng mong mun. Chng
Strep.griseus c th c cm ng bi nm men mannan sinh |-mannosidase, enzyme
ny s chuyn mannosidostreptomycin thnh streptomycin.
Hin nay ngi ta c th sn xut mt lng ln protein c ngun gc khc nhau (t
virus, ngi, ng vt, thc vt v vi sinh vt) trong nm men, nm si v vi khun. Tuy
nhin cc protein ny i khi li c c tnh i vi t bo ch v c nh hng ln ln
tnh n nh ca s biu hin protein. Cng ging nh s c ch ln tng trng ca
chng, tnh c ca cc protein ny s gy nh hng n cc iu kin chn lc cho cc
t bo phn ly m khng th tng hp protein vi hm lng cao. Do vy, iu kin
chng tng trng ti thch th s khng ph hp ng thi sinh tng hp protein
ngoi c, v iu ny ch thc hin c khi gy cm ng sau khi to lng sinh
khi trong bn ln men. V d trng hp ca Sac. cerevisiae c promoter Gal1 l mt
43
phn ca h thng biu hin, s hnh thnh sn phm ch thc hin c khi b sung
galactose vo mi trng nui cy c cha glycerol hoc glucose nng thp.
3.12. Yu cu v oxygen
Mc d oxygen khng c b sung vo mi trng nui cy vi sinh vt nh l
mt thnh phn tuy nhin oxygen ng vai tr rt quan trng i vi s tng trng v s
sinh tng hp cc sn phm trao i cht ca vi sinh vt. Tnh cht ca mi trng nh
hng ln hm lng oxygen theo cc cch nh sau:
- C ch bin dng nhanh: Nu nng ng trong mi trng nui cy mc
ph hp cho tng trng, hm lng oxygen nhanh chng gim xung do nhu cu s
dng oxygen xy ra ng thi khi ng ha ng.
- Tnh lu bin ca mi trng: Cc thnh phn ca mi trng s gp phn to
nn nht ca mi trng. Khi nht ca mi trng cao, tnh lu bin ca mi
trng thp v do vy s nh hng trc tip ln s sc kh kh nng khuy trn, hn ch
s khuch tn ca oxygen khng kh trong mi trng trong qu trnh ln men.
- Cht ph bt: Cht ph bt ng vai tr l cht hot ng b mt v lm gim tc
khuch tn ca oxygen. Khi nng cht ph bt cao, ha tan ca oxygen trong
mi trng s gim ng k, gy nh hng n s tng trng v trao i cht ca vi
sinh vt.
4. nh gi mi trng
Mc ch ca nh gi mi trng
Bo m v mt k thut: ph hp, hiu qu cao
Yu t kinh t: gi thnh sn xut thp, tnh n nh
Yu t an ton cho sn phm
Da trn cc tiu ch:
1. Thnh phn
2. Lng
3. Cht lng
4. Gi c
5. Tnh n nh ca ngun nguyn liu

5. Phng php ng ha to dung dch glucose t tinh bt
Tinh bt (starch) l ngun nguyn liu c ngun gc nng nghip rt ph bin,
c s dng sn xut ng glucose, phc v cho nhiu mc ch khc nhau trong
lnh vc thc phm, ln men.
44
Phng trnh phn ng thy phn tinh bt thnh ng glucose:
(C
6
H
10
O
5
)nH
2
O + (n-1)H
2
O nC
6
H
12
O
6

MW: (162n+18) 180.n
(n 1)
Hiu sut ng ha = 180.n/(162.n+18) vi n l s n phn glucose trong tinh
bt. Ty theo loi tinh bt m n c khong gi tr khc nhau. Do vy, i vi mi loi
tinh bt th hiu sut ng ha cc i khc nhau. Hiu sut ng ha ln nht khi
ton b cc n phn glucose trong phn t tinh bt c ct ri hon ton, khi s
lng n phn trong dung dch t cc i.
Hiu sut ng ha ti a =

+
n
n n im ) 18 162 /( 180 ( 100 = 111,11%
Tc nhn thy phn tinh bt thnh ng glucose c th l H
2
SO
4
hoc enzyme.
Vic s dng H
2
SO
4
thy phn tinh bt trong cng nghip gp phi nhiu nhc im
nh hiu sut thy phn khng cao, km n nh, thit b s dng t tin, km an ton
cho cng nhn vn hnh, dung dch glucose c mu cao, cha nhiu d lng ca
SO
4
2-
gy nh hng xu khi s dng cho mi trng ln men. Do vy trong sn xut
cng nghip sn xut ng glucose t tinh bt hin nay ngi ta thng dng cc
enzyme nh amyloase, glucose amylase thy phn.
S dng phng php enzyme thy phn tinh bt thnh ng cn thng qua
hai bc:
- Qu trnh dch ha (liquefaction):
S dng enzyme a-amylase thy phn tinh bt thnh cc on oligosacchride
mch ngn. Phn ng c thc hin pH 5-6,5, nhit 70-105
o
C trong thi gian
khong 90-120 pht vi t l enzyme s dng l 0,8-1,3 L/t DS. Nng tinh bt trong
dch bt thch hp cho phn ng l 30-35%. Sn phm ca qu trnh dch ha l dung
dch cha cc oligosacchride vi DE (dextrose equivent) khong 12-20%. Vi thnh
phn ny th s rt thun li cho giai on phn ct tip theo.
- Qu trnh ng ha (saccharification):
Phn ng thy phn c thc hin bi enzyme glucose amylase, c c im l
thy phn cc oligosaccharide thnh cc n phn glucose.
45
Phn ng din ra pH 4,0-4,5, nhit ti u khong 60-62
o
C. Vi t l enzyme s
dng 0,35-0,5 L/t DBS (dried base starch) th sau khong thi gian 60-80 gi s thu c
dung dch ng glucose vi hiu sut ng ha t khong 110%.
6. Phng php x l mt r ng to dung dch ng phc v cho ln men

1. Thnh phn ca mt r ng Vit Nam
Bng 4. 7. Thnh phn ca mt r ng
Thnh phn %
Hm lng ng tng s 46-60
Hm lng Ca
2+
0,3-1,2
Hm lng K
+
1,8-5,0
Hm lng Mg
2+

Mu l
400nm
(*250) 0,2-1,3
T trng 1,4-1,7

2. Phng php x l mt r ng trc khi dng cho qui trnh ln men
Thng thng H
2
SO
4
c s dng thy phn ng sucrose thnh ng
glucose v fructose ng thi SO
4
2-
s phn ng vi Ca
2+
to mui CaSO
4
dng t tan
v c th loi khi dung dch ng bng cch ly tm.
Sucrose + H
+
Glucose + Fructose
Ca
2+
+ SO
4
2-
CaSO
4
(t tan)







46
CHNG 5. KH TRNG TRONG LN MEN CNG NGHIP

1. Mc ch kh trng trong ln men
Mt sn phm ln men c to ra bng mt chng hoc mt nhm chng vi sinh
vt nht nh trong mi trng dinh dng. Nu qu trnh ln men b xm nhim bi vi
sinh vt ngoi th thng xy ra cc hu qu sau y:
- Mi trng nui cy thng giu dinh dng gip cho s tng trng tt khng
ch cho chng vi sinh vt sn xut m cn cho c chng tp nhim, do vy thng dn
n vic tht thot sn lng.
- Trong trng hp ln men lin tc, chng tp nhim c th tng trng vt
qu chng sn xut v thay th chng sn xut trong trong qu trnh ln men.
- Chng tp nhim c th gy nhim ln sn phm cui, v d trong trng hp
sn xut protein n bo th t bo chng tp nhim s ln trong sn phm cui.
- Chng tp nhim c th sinh tng hp cc hp cht nh hng kh khn cho
qui trnh chit tch sn phm cui.
- Chng tp nhim c th phn hu sn phm mong mun. y l trng hp
thng xy ra i vi ln men sn xut khng sinh khi m cc chng tp nhim khng li
nhng nh hng c ch ca cht khng sinh v vic phn hu cht khng sinh l mt
trong nhng c ch khng thng thng. V d trong trng hp phn hu khng sinh |-
lactam bi chng sn xut |- lactamase.
- S tp nhim ca phage i vi chng ln men sn xut thng dn n vic
phn hu t bo trong dch nui cy.

Mt trong nhng vn quan trng ca ln men c kim sot l phi kh trng
nhm loi b cc yu t gy tp nhim sinh hc (nh nm mc, nm men, vi khun,
phage). Ty theo vo loi sn phm v qui trnh ln men m mc kim sot v trng
trong qu trnh ln men c khc nhau. Trong trng hp ln men sn xut cc ch phm
sinh hc dng lm thuc tr bnh th cn phi kim sot tp nhim cht ch, do vy mc
kh trng l rt nghim ngt. i vi trng hp ln men sn xut cc sn phm dng
trong thc phm th mc kim sot tp nhim km nghim ngt hn. iu kin v
trng trong nui cy c kim sot t c khi:
- S dng ging khng b tp nhim
- Thanh trng mi trng trc khi s dng
- Thanh trng ni ln men
- Thanh trng ton b cc nguyn liu b sung vo ni ln men trong qu trnh
nui cy
- Duy tr iu kin v trng trong qu trnh ln men
47
2. Yu cu khi kh trng
- Loi b mt phn hoc ton b tc nhn gy nhim sinh hc nh hng ln qu
trnh ln men. Tc nhn c th bao gm c t bo sinh dng hoc bo t.
- Qu trnh kh trng khng gy nn nhng bin i ng k v cht lng ca
mi trng. Khng to ra cc cht c hi c th gy km hm hay c ch s pht trin,
hoc lm suy gim hot tnh sinh tng hp ca chng nui cy sau ny.
- Tc nhn kh trng khng c hi i vi ngi vn hnh, mi trng, thao tc
n gin, r tin, d p dng cho qui m sn xut ln, d t ng ha.

3. Phng php kh trng
3.1. S dng cc tc nhn vt l
- Phng php lc
Phng php lc c s dng lc khng kh hoc mi trng. Trong trng
hp thnh phn mi trng c cc cht km bn vi cc tc nhn nh nhit hoc chiu
x, hoc mi trng c cha cc thnh phn d bay hi th s dng phng php lc l
mt gii php rt hu hiu.
C cc dng lc thng dng nh lc mng, lc ti, lc cartridge
- Khng kh nng (hot air)
S dng khng kh nng lm kh cc thit b ln men.
- Phng php dng hi nc nng (steam)
Hi nc nng l tc nhn thanh trng hiu qu, d p dng cho qui m sn xut
cng nghip v c s dng rng ri thanh trng mi trng nui cy vi sinh vt v
thanh trng thit b ln men.
- Phng x (tia UV, tia X, tia g), siu m

3.2. Tc nhn ha hc
Cc ha cht thng dng l phenol, alkohol, formol, hologen, H
2
O
2
hoc cc
dung dch ha cht chuyn dng nh Germicide, Stabilon BP, Triquart
- Phenol. c dng dng dung dch st trng cc dng c, thit b. Tu theo
nng m dung dch phenol c tc dng dit khun hay c ch vi khun. Hot tnh ca
phenol b gim trong mi trng kim v trong mi trng c mt cht hu c. Ngc li
trong mi trng c mui th hot tnh ny tng ln. Phenol v cc dn xut ca n tc
ng ch yu ln cc lp mng ca t bo, ph hoi tnh bn thm ca mng t bo cht
v lm bin tnh protein. Chng thng c dng kt hp vi x phng lm dung
dch st trng da.
- Cc alcohol nh ethanol, methanol c tc dng st khun rt mnh, thng gy
nn s ng t protein mng. Khi nng cao, cc alcohol c tnh kh nc mnh do
48
rt nc khi t bo, cn tr s xm nhp ca alcohol vo ni bo nn ch c tc dng
c ch vi khun. Ethanol 70% c tnh st khun tt hn loi 90%.
- Cc halogen c tnh c vi vi sinh vt. Tc dng dit khun ca c hologen l
do vic hnh thnh acid pecloric. Acid ny b phn hu tip hnh thnh acid clohydric
v oxy. Oxy trng thi ny l mt cht oxy ho mnh c tc dng ph hu cc thnh
phn ca t bo.
+ Cl
2
v cc hp cht ca clo nh NH
2
Cl (cloramin), NHCl
2
(cloran) u c
tc dng dit khun tt. Cu trc ca t bo c th b ph hu do tc dng trc tip
ca Cl2. C ch tc dng ca clo c m t nh sau:
- Clo c th thay th hydro trong nhm amin ca protein to thnh cloramin
lm cho cu trc ca protein b thay i, do cu trc ca t bo cng b ph
hu.
R-CO-NH-R- + Cl
2
R-CO-NCl-R + HCl
- Clo cn tc dng vi nc to thnh oxi nguyn t
Cl
2
+ HOH HOCl + HCl
HClO HCl + O
- Clo trong hp cht vi Ca
2+
to thnh clorua vi Ca(OCl)
2
cng c kh nng
dit khun tt.
Ca(OCl) + 2H
2
O Ca(OH)
2
+ HClO
HClO HCl + O
- Clo tc dng vi NaOH to thnh natrihypoclorit cng l mt cht dit khun
rt tt.
Cl
2
+ 2NaOH NaOCl + NaCl + H
2
O
+ Iod l mt cht dit khun khc thuc nhm halogen. Iod d ho tan trong
alcohol v trong dung dch v trong cc dung dch kim Na, K. Iod ch c tc dng
dit khun trn b mt nn thng ch c dng kh trng b mt, khng kh.
4. ng hc s cht ca vi sinh vt bi nhit
ng hc v s hy dit t bo vi sinh vt do hi nc nng c m t theo
phng trnh sau:
-dN/dt = kN (5.1)
Trong , N l s t bo sng hin din, t l thi gian thanh trng, k l tc cht
c trng ca t bo vi sinh vt (pht
-1
). Cn lu thm rng k ch t hng s di iu
kin nhit thanh trng khng thay i.
T phng trnh (5.1) trn ta c:
49
-dN/N = k.dt
) / (
}
N dN =
}
dt k.
Ln(N
t
/N
o
) = -kt (5.2)
N
t
/N
o
= e
-kt
(5.3)
N
o
v N
t
l s t bo vi sinh vt ti thi im bt u x l thanh trng v sau khi
thanh trng c thi gian t. Gi tr k khng ch ph thuc vo chng vi sinh vt m cn
ph thuc vo dng trng thi sinh l: v d khi dng bo t th kh nng chu nhit cao
hn rt nhiu so vi dng t bo sinh dng. Do vy khi kh trng phi chn la iu
kin thch hp c th thanh trng ht ton b cc nhn t gy nhim.
iu kin (100
o
C, 40 pht), hu ht cc chng vi sinh vt (k c bo t) u b
dit ngoi tr bo t ca Streptomyces sp ngay c vi iu kin nhit nh trn trong
vng 3 gi. cc iu kin (115
o
C, 60 pht), (120
o
C, 30 pht), (130
o
C, 5 pht) th hu
nh ton b vi sinh vt b cht, ngay c i vi trng hp ca bo t cc chng chu
nhit (thermophile). Trong iu kin (160
o
C, 30 pht) bo t kh ca cc chng chu
nhit cng b dit.
T phng trnh (5.3) v th trn Hnh 5.1 cho thy:
1. Thi gian thanh trng c kh nng khng cn t bo vi sinh vt tn ti l v
cng (N
t
= 0)
2. Sau mt thi gian thanh trng nht nh th lun lun tn ti kh nng c di
mt t bo ang sng. Trong thc t, ngi ta quan nim rng vi N
t
< 1 th lun tn ti
mt t bo sng sau khi x l kh trng nhit.
iu ny c ngha l cho d thanh trng nhit no, trong thi gian bao lu th
nguy c b tp nhim cng vn cn! Do vy, vn l cn phi hn ch s lng vi sinh
vt tp nhim trc khi thanh trng nh v sinh thit b, s dng nguyn liu c tinh
sch cao, n nh ch khng nht thit l phi tng cng cc iu kin kh trng.
Gi s D l thi gian kh trng gim s lng t bo vi sinh vt sng xung 10
ln ta c: N
t
= 1/10.N
o

Ln(1/10) = - kD
Hay D = 2,303/k
50


0,1
1,0
10
100
10 20 30 40 50
70
o
C 60
o
C
50
o
C
Th i gian te ba o suy
gia m 10 la n (D)
T
y

l
e

t
e

b
a
o

s
o
n
g

s
o
t
(
t
h
e
o

t
y

x

c
h

l
o
g
)
Thi gian (phut)

Hnh 5. 1: nh hng ca nhit ln s bin d ca cc chng vi khun a nhit trung
bnh. Thi gian suy gim t bo 10 ln (D) c c ca cng mt chng vi sinh vt cc
nhit khc nhau. D l thi gian m ch 10% s t bo vi sinh vt cn li mt nhit
nht nh. i vi nhit 70
o
C, D = 3 pht; 60
o
C, D = 12 pht; 50
o
C, D= 41 pht.
Bng5. 1: ng hc nhit dit theo thi gian
t kt
e
kt
N
t
0 - 1 10,000,000
10 1.5 4 2,231,302
20 3.0 20 497,871
30 4.5 90 111,090
40 6.0 403 24,788
50 7.5 1808 5,531
60 9.0 8103 1,234
70 10.5 36316 275
80 12.0 162755 61
90 13.5 729416 14
100 15.0 3269017 3.06
110 16.5 14650719 0.68
120 18.0 65659969 0.15
130 19.5 294267566 0.03
140 21.0 1318815734 0.0076
150 22.5 5910522063 0.0017
No = 10,000,000 t bo
k = 0.15 /pht Hnh 5. 1: ng hc nhit dit theo thi gian kh trng

1,000,000
2,000,000
3,000,000
4,000,000
5,000,000
6,000,000
7,000,000
8,000,000
9,000,000
10,000,000
0 50 100 150
S

n
g

s
a
u

Thigianxlnhit,pht
51



Hnh 5. 2: Mi quan h gia nhit v tc cht c biu th bi thi gian t bo
suy gim 10 ln ca hai chng vi sinh vt khc nhau. i vi chng (a), nhit
110
o
C, D< 20 giy. Trong khi i vi chng (b)(chu nhit cao) D = 10 pht.
5. Kh trng mi trng
Kh trng mi trng trong nui cy vi sinh vt c th c thc hin bng
phng php dng hi nc hay phng php lc. Yu cu ca phng php lc l mi
trng lng, ng nht, cn b th trong mi trng t. i vi cch kh trng dng hi
nc th c nhiu thun li, d p dng. Dng hi nc nng kh trng c hai cch:
(1) gia nhit gin tip: nh trong trng hp ca Autoclave, ca kh trng lin tc, (2)
gia nhit trc tip nh trong trng hp ca kh trng theo m.
Khi tng nghim ngt kh trng th s gy bin i thnh phn mi trng do
s phn ng gia cc thnh phn ny hoc lm phn hy cc cht khng bn nhit, d
bay hi T lm cho kt qu ln men gim ng k (theo bng sau).
Cn lu rng khi pH ca mi trng thp, hiu qu thanh trng tng ln, v nu
nng cc cht trong mi trng (ng, protein, cht bo) cao s lm gim hiu qu
thanh trng ca mi trng.
Bng 5. 1: nh hng thi gian thanh trng ln nng glucose v tc tch ly sn
phm trong ln men to khng sinh (Corbett, 1985)
Thi gian thanh trng
121
0
C (pht)
Nng
glucose cn li (%)
Tc tch ly
sn phm tng i


C
thc t s
m th th

5.1. Kh
Mt s
1
2
3
4
5
6
Hnh 5.
phng
thanh tr
trng t
li, mc
60
40
30
C hai dng
sn xut cn
hanh trng l
trng the
u im ch
. Duy tr t
. D dng n
. D dng k
. Gim s t
. Lng ti
. Gim thi
3: M hnh
php m. T
ng l rt n
trong trn
c tng l
g thanh trn
ng nghip:
lin tc c n
o phng p
nh ca h t
t cht lng
nng cp qu
kim sot t
tng th tch
u th ca h
u s n m
thay i nh
Trong tr
ngn (~5 ph
ng hp ny
n t nh
ng thng
Thanh trn
nhiu u i
php lin t
thng kh t
g mi trn
ui m sn xu
ng
h mi trn
hi nc nn
n ni ln m
hit tron
ng hp thn
ht) v
l vng S1
hit than
35
46
64
c s d
ng lin tc v
im v do v
c
trng lin t
ng sau thanh
ut
ng do hi n
ng n nh
men

ng thanh tr
nah trng li
c lm ngu
. Trng h
nh trng di
ng than
v thanh tr
vy thng
c:
h trng
c ngng t
trong sut
ng theo ph
in tc (a),
i nhanh; t
p thang tr
n ra chm,
9
9
10
nh trng m
ng m. So
c dng
t
qu trnh

hng php
thi gian
ng tc n
rng theo m
, v qu tr
0
2
00
mi trng t
vi thanh t
nhiu hn.
lin tc v
t nhi
ng nhit ca
m (b) th ng
nh h nhi
52
trong
trng

theo
t
a mi
gc
t sau
53
thanh trng cng chm, do vy, tng tc ng nhit ca mi trng (S1+S2+S3) trong
trng hp ny di hn (50 pht).

Hnh 5. 4: M hnh h thng thanh trng mi trng lin tc trong ln men cng nghip.
H thng thanh trng lin tc bao gm:
(1) Bn chun b mi trng;
(2) Bm;
(3) Thit b hiu chnh lu tc truyn dch
(4) Thit b gia nhit s cp
(5) Thit b gia nhit
(6) Thp lu
(7) Thit b kim sot nhit thanh trng.

Nguyn l hot ng ca h thng bao gm hai giai on l (1) thanh trng thit b
ca h thng v (2) thanh trng mi trng.
1. Thanh trng thit b ca h thng
H thng trc khi s dng cho thanh trng mi trng th bn thn n cn phi
c thanh trng loi b cc tc nhn gy nhim. Tc nhn thanh trng l nc nng
t bn cha c bm (2) vo h thng qua thit b gia nhit (5). y, nhit ca
nc c nng ln 120
o
C bng hi nc nng c cung cp trc tip vo thit b gia
nhit. Nc nng sau i vo thp lu v c hi lu v bn cha sau khi i qua thit
b gia nhit s cp. Thi gian tun hon nc thanh trng trong h thng khong t 1- 2
gi th bo m c tnh v trng.

H
cha n
ny
vo thit
N
bng h
sau khi q
vo thi
bin nhi
thm h
t ng
T
Hnh 5.
pha dch


Hnh 5.
Dng t
T
thng th
mt thi
T
theo cn

tr
trng n
V
tc b
2. Th
H thng thi
c nng sa
c bm a
t b gia nhi
Nhit tha
thng t
qua thit b
t b gia nhi
it s nhn
i nc nn
ny s ng
Thit b gia
5 a) c
h trao i nh

5. Thit b t
m; (b) dng
Thi gian th
hanh trng.
i gian nht
Thi gian th
ng thc:

rong , t l
np vo h t
V d: Vi t
m dch m
hanh trng
it b sau kh
ang hng b
vo h th
t (5).
anh trng
ng gm
gia nhit v
it. Khi nhi
din v tru
g cho h th
g li, hn ch
nhit c th
lp ghp li
hit cho nha
(a)
trao i nh
g xon c, s
hanh trng
Thp lu
nh ty thu
hanh trng
t = V/F
thi gian
thng.
th tch th
i trng v
mi trng
hi c than
bn chun b
ng thanh tr
c ci t
u cm bi
v mt van t
it thanh
uyn tn hi
hng. Ng
h hi nc
h l dng
in k nhau
au hoc dn
it s dng
spiral; (c) D
c kim
c thit k
uc vo lu
c kim s
(5.5)
thanh trng
p lu V=10
o h thng
g
nh trng xo
b mi tr
ng qua thi
gi tr th
t nhit gn
t ng kim
h trng ch
u n b
c li, khi nh
c nng i v
cc tm tr
u to nn c
ng xon c
(b)
g trong h th
Dng cun l
sot bng l
nhm lu
u tc truyn
sot thng q
g, V l th t
0 KL, th
g thanh trn
ong, chuyn
ng. Khi
it b gia nh
ch hp (v
trn ng
m sot ln
a t theo g
iu van t
hit v
o thit b g
trao i nhi
c khoang h
(spiral, coil

hng thanh
l xo, coil.
u lng d
gi dch m
n dch.
qua lu l
tch thp l
hi gian tha
ng F = 10/15
n van 3 chi
mi trn
hit s cp
d 120
o
C)
g ng dn d
ng hi nc
gi tr ci
ng m
t qu gi tr
gia nhit.
it (heat e
h bn tron
l) (Hnh 5.
(c)
trng mi t
dch mi tr
mi trng s
ng dch b
u, F l lu
anh trng t
560 = 40 K
u t hng
ng trong b
(4) v sau
v t kim
dch mi tr
c nng cung
t, thit b
ra cung
r ci t th
exchange pl
ng cho php
5 b, c).
trng. (a)
ng np v
sau khi gia n
m vo h th
lng dch
t t 15 ph
KL/hr.
54
g bn
n (1)
i
m sot
ng
g cp
cm
g cp
van
lates,
p hai
o h
nhit
hng
h mi
t th
55
Lu tc dch np vo h thng c kim sot t ng bng mt thit b o lu
lng (3) trn ng ng v van t ng. Khi lu lng dch khc bit vi gi tr ci t
th van t ng s t ng/m iu chnh lu lng ng theo gi tr ci t.
5.2. Kh trng theo phng php m
Ton b cc thnh phn mi trng c chun b trong ni ln men, sau gia
nhit trc tip vo bn n khi t nhit nht nh cn cho thanh trng (thng thng
121-125
o
C). Tnh thi gian thanh trng k t khi nhit mi trng t gi tr ci t.
Mi trng sau thanh trng c h nhit bng cch cp nc lnh vo h thng gii
nhit ca ni (thng l dng ng xon vng, coil hoc dng thp ng, tube). Trong qu
trnh thanh trng cn lu l phi khuy trn dch mi trng nhm bo m gia nhit
ng nht cho ton b mi trng. u im ca phng php ny l thao tc n gin,
d thc hin, d kim sot qu trnh thanh trng v hiu qu thanh trng tt. Tuy nhin,
phng php ny tn ti mt s nhc im nh thi gian gia nhit di, lm ngui mi
trng chm do vy tng thi gian chu nhit ca mi trng cao, gy nh hng ln n
cht lng ca mi trng sau khi thanh trng. Do phi gia nhit trc tip nn mt lng
ln nc ngng t hi nc nng lm cho th tch mi trng sau thanh trng tng ln
rt ng k, lm gim nng cc cht trong mi trng. khc phc tnh trng ny,
ngi ta thng gim th tch nc pha long khi chun b mi trng. Mt nhc im
na ca phng php ny l tnh t ng ha km.
xc nh iu kin thanh trng cho mt loi mi trng no chng ta cn
thc hin hai lot th nghim thay i nhit v thi gian thanh trng, sau nh gi
s tn ti ca vi sinh vt tp nhim (bng cch tri cc mi trng sau thanh trng ln a
pettri ri hoc thc hin nui cy lc) v kt qu ln men khi nui cy chng sn
xut.
6. Kh trng ni ln men
Ty mc nghim ngt trong kim sot tp nhim ca qui trnh ln men m
phng php v iu kin kh trng ni c khc nhau. V d i vi cc trng hp mc
kim sot khng cao nh trong ln men ru, ln men dm th khng cn phi dng
hi nc kh trng m c th dng alkohol tit trng trc khi s dng. Trong
trng ln men amino acids, ln men khng sinh th cn phi dng hi nc nng
thanh trng vi p sut cao (thng 1,5 kgf/cm
2
t nhit thanh trng ~125
o
C)
trong thi gian di (2 gi). Trong qu trnh thanh trng cn phi bo m l hi nc
nng phi c lun chuyn trong bt k khng gian no ca thit b, nc ngng t li
y ni phi c lin tc x ra nhm trnh mt s v tr ca ni khng bo m nhit
thanh trng. iu cn lu l sau khi kh trng xong phi lun lun gi ni ln men
56
p sut dng bng khng kh c lc sch, trnh khng kh t bn ngoi xm nhp
vo ni mt khi p m. Qui trnh thao tc thanh trng ni ln men (Hnh 5. 6) nh sau:


Hnh 5. 6. S bn ln men v cc ng ng lin quan.

1. Cc van x, van thot kh phi trng thi m trc khi bt u thanh trng
(V1, V2, V4, V9, V12).
2. ng van kh nn (V5), van cp mi trng (V7), van np ging (V11) v m
cc van V3, V8, V10.
3. Cp hi nc nng vo ni, m V6.
4. Khi nhit t 100
o
C, m nh cc van x (V1, V2, V4, V9, V12).
5. Khi nhit t yu cu (125
o
C) bt u tnh thi gian thanh trng
6. Khi thi gian thanh trng t yu cu, dng qu trnh thanh trng bng cch
ng van cp hi nc nng (V6) v ngay lp tc m van cp khng kh nn
(V5).
7. Khi ton b nc ngng (condensate) trong ni ln men, trong ng ng u
c thot ra ngoi, ng cc van x (V1, V2, V9, V12).
8. Duy tr p sut khng kh trong bn v sn sng nhn mi trng chun b
cho nui cy.

57
7. Lc khng kh
Khng kh trc khi sc vo ni ln men cn phi c lc b cc tc nhn gy
tp nhim. Phng php v trng khng kh thng l s dng thit b lc. Thit b ca
h thng lc bao gm:
+ Lc th: nhm loi b cc ht t c kch thc ln. Kch thc lc 5-30 m,
thng l dng mng lc lm bng si thy tinh.
+ Lc tinh: nhm lc b cc t bo vi sinh vt (v c bo t). Kch thc mng lc
thng 0,1-0,3 m, thng l dng module (cartridge) lm bng cc lp si PE
(polyethylene).


Hnh 5. 7. M hnh h thng cc thit b cung cp v x l khng kh nn cung cp cho h
thng ln men. H thng bao gm:
(1) My nn kh
(2) Thit b gii nhit
(3) Thit b ly tm (cyclone) tch nc
(4) Thit b gia nhit
(5) Thit b lc th
(6) Thit b lc tinh
Khng kh c nn li vi p sut t 1-3kgf/cm2 bng my nn kh cng nghip.
Tu theo nhu cu s dng ca h thng ln men m cng sut ca my nn kh c la
chn khc nhau. Khng kh sau khi c nn nhanh p sut cao thng sinh nhit.
Thit b gii nhit gip h nhit (thng t 15- 20
o
C) ca kh nn t ngt lm cho hi
m trong kh nn ngng t li v c loi b qua thit b tch m (cyclone). Sau ,
khng nn ny c sy gia nhit tr li (thng l khong 38-40
o
C) lm cho phn
nc ngng (nu cn) trong kh nn khuych tn ng nht tr li, trnh tnh trng cc
git nc ngng ny (nu cn) gy m trn mng lc v lm gim tnh hiu qu ca
thit b lc. Phn kh nn ny sau i qua thit b lc th nhm loi b nhng phn t
bi c kch thc ln sau qua thit b lc tinh, loi b cc tch nhn gy nhim sinh
hc. Tu theo mc nghim ngt ca h thng m kch thc lc ca thit b lc tinh
khc nhau.
58
CHNG 6. KIM SOT NUI CY

1. Kim sot tng trng t bo
Kim sot tng trng l hot ng nhm hn ch s tng trng ca chng trong
nhng giai on cn thit ca qu trnh ln men. kim sot c s tng trng ca
chng trong qu trnh ln men cn phi nh lng (trc tip hoc gin tip) c lng
sinh khi hin din trong tng thi im ca qu trnh nui cy. Cc phng php thng
c s dng theo di tng trng bao gm:
1. Phng php m trc tip
- Thng p dng i vi cc vi sinh vt n bo c kch thc ln nh nm men,
to
- m trc tip di knh hin vi (s dng bung m hng cu hoc bung m
Breed). Mu dch ln men c pha long v c m trc tip bng phng m di
knh hin vi. Phng php ny c u im l c th xc nh nhanh c s lng t bo
tng thi im nhng c nhc im ln chnh xc khng cao v khng phn bit
c t bo sng/cht.
- C th m trc tip bng knh hin vi hunh quang. Nhum mu cha vi khun
bng cc cht nhum c kh nng pht hunh quang nh acridin cam (AODC), 4,6-
dianidino-2-phenyl-indol (DAPI), fluorescein isothiocyanate (FITC). Theo phng php
ny, s lng vi sinh vt trong mu c xc nh tng i chnh xc, tng ng t l
vi lng sinh khi v do vy thng c dng c lng sinh khi vi sinh vt c
trong mu.
2. Phng php m khun lc
Phng php ny c thc hin thng qua vic nui cy mu trn mi trng c
trng. Mu dch ln men c pha long (theo dy thp phn) t t l thch hp v tri
trn mi trng thch c trng sau c nhit ti tch chng pht trin.
m s khun lc hin din trn mt thch sau thi gian nui cy thch hp, s khun lc
tng ng vi s t bo vi sinh vt sng ti thi im ly mu (cfu-colony forming unit).
u im ca phng php ny l xc nh c chnh xc s lng t bo sng trong
mi trng ln men ng thi c nhy cao v thng c dng xc nh s lng
t bo sng ngay c khi trng hp mt thp. Tuy nhin phng php ny c nhc
im l thi gian nh gi rt lu.
3. Thng qua my o mt quang (OD, optical density).
y l phng php gin tip rt thng dng nh gi nhanh s bin ng nng
t bo trong qu trnh nui cy.
Nguyn tc ca phng php ny l khi pha lng c cha nhiu phn t khng tan
th s hnh thnh mt h huyn ph to c do cc phn t ny cn nh sng, lm phn
tn chm nh sng ti qua dch. T bo vi sinh vt c xem nh l mt thc th c th
cn nh sng to c khi hin din trong dch huyn ph. c ca dch huyn ph
t l vi mt t bo vi sinh vt trong dch. Trong mt khong gii hn nht nh, c
th xc lp c quan h tuyn tnh gia c v mt t bo. Do vy c th s dng
my o c vi bc sng nh sng thch hp (thng l t 550 610 nm) xc
nh gin tip mt t bo hin din trong mu.
59
Nhc im ca phng php ny l kt qu o thng b nh hng bi cc phn
t to mu ca mi trng do vy chnh xc khng cao. khc phc tnh trng ny
thng thng ny ta s dng chnh mi trng ln men lm dch huyn ph hiu chnh
my so mu v v tr 0 trc khi tin hnh o mu.
4. Phn tch trng lng t bo kh (DCW, Dried Cell Weight). Thu mt th tch
mu dch ln men nht nh (V, ml), ly tm thu sinh khi v sy chn khng khong 60
o
C
n khi trng lng khng i (m, g). T s m/V (g/ml) chnh l nng t bo kh
trong dch ln men ti thi im thu mu. Cn ch l trong trng hp mi trng ln
men c cha nhiu phn t khng tan, khi ly tm, cc phn t ny cng lng vi sinh
khi nn kt qu s khng chnh xc. khc phc tnh trng ny thng thng ngi ta
s dng lc c kch thc thch hp loi b v ra li sinh khi nhiu ln vi
acid/base.
5. Thng qua cc thng s ch cng trao i cht.
Bng cch xc nh lng O
2
hp th hoc lng CO
2
sinh ra trong qu trnh ln
men, ngi ta c th gin tip bit c lng sinh khi tham gia ln men trong tng thi
im nht nh. Nhc im ca phng php ny l i khi lng sinh khi nhiu
nhng do iu kin ln men khng bo m lm cho qu trnh trao i cht din ra chm.

Ti sao phi kim sot tng trng?
Bt k mt h thng ln men no th cng c s gii hn v cng sut thit k.
Trong qui trnh thit b ln men, s gii hn l th tch ni ln men, h thng sc
kh, h thng nc gii nhit Do vy, lun bo m cung cp ngun dinh dng,
ngun oxygen cho vi sinh vt trong qu trnh sn xut, ngi ta phi gii hn lng t
bo tham gia sn xut mt mc nht nh, ph hp vi cng sut thit k. Nu lng
sinh khi t bo tham gia sn xut nh hn cng sut thit k th sn lng thu c
khng t yu cu lm tng chi ph c nh (nh ph) ca sn phm. Ngc li nu s
lng t bo pht trin qu ln, vt qua gii hn thit k th kh nng p ng ca qui
trnh lin quan khng lm cho hiu qu ln men km, tng chi ph bin ng (bin ph)
v nh vy s lm tng chi ph sn xut. Do vy cn phi kim sot s tng trng t bo
mt mc nht nh, ph hp vi cng sut ca cc h thng thit b lin quan. Cng cn
lu thm rng, trong qu trnh nui cy, vic theo di mt t bo theo thi gian cn
c ngha nhn din tnh trng bnh thng hay bt thng ca qu trnh ln men.
Bn cnh vic theo di tng trng cn gip nhn din nu c hin tng tp
nhim xy ra. V d khi nhim phage, mt t bo t ngt gim do mt lng ln t
bo vi sinh vt b v ra khi b phage tn cng, cn nu b nhim tp khun th mt t
bo tng ln bt thng.

60

Hnh 6. 1. S thay i OD trong qu trnh nui cy. (1): Trng hp bnh thng, kim
sot c, (2): Trng hp OD tng lin tc, khng kim sot c, c th do b tp
nhim hoc cc yu t kim sot khng ph hp. (3): OD gim t ngt c th l do
nhim phage.

Kim sot tng trng ca chng nui cy bng cch no?

- B sung cc cht c ch tng trng (Tween 40, 60, penicilin)
- Thay i yu t sinh l (nhit , pH ca mi trng ln men)
- Gii hn hm lng c cht gii hn

2. Kim sot pH
Kim sot pH nhm bo m pH sinh l ca t bo trong qu trnh pht trin v
sinh tng hp cc cht. Vi sinh vt rt nhy cm vi s thay i pH ca mi trng. Cn
lu l pH ti thch ca mi trng l pH ti thch cho h enzyme ca chng nui cy.
Tc nhn dng iu chnh pH trong qu trnh ln men l acid (H
2
SO
4
, HCl ) hoc
kim (NaOH, KOH, NH
3
, urea ). Yu cu chung ca tc nhn hiu chnh pH trong qui
trnh ln men sn xut qui m cng nghip l r tin, d hp th, khng lm thay i ng
k th tch dch ln men, khng lm tng ng k p sut thm thu trong mi trng, d
t ng ha. Trong ln men fed-batch sn xut amino acid ngoi vic b sung ngun
carbon, ngi ta cn phi b sung thm ngun N bo m cung cp N cho s tng
trng ca vi sinh vt v cho vic tng hp amino acid. Urea hoc NH
3
thng c
chn lm ngun cung cp N v c th lm tc nhn hiu chnh pH trong trng hp pH
mi trng gim xung do s tch ly sn phm amino acid. Trong qui trnh ln men
cng nghip, ngi ta thng dng h thng kim sot pH t ng. H thng ny bao
gm (Hnh 6. 2):
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0 2 4 6 8 10 12 14 16 18 20
O
D
Thi gian nui cy (gi)
-(1)
-(2)
-(3)
61

Hnh 6. 2. S h thng kim sot pH t ng ca ni ln men.

1. in cc o pH (electrode) gn trc tip vo bn ln men trong qu trnh nui
cy.
2. H thng trung chuyn v khuych i tn hiu (transmiter)
3. H thng iu khin (controller): cho php ci t gi tr pH cn kim sot,
hin th gi tr pH hin ti trong bn ln men, so snh s chnh lch ny v
a tn hiu iu khin m hay ng van np tc nhn (H
+
, OH
-
) hiu chnh
pH vo.
4. Van t ng (control valve): cho php np hay ngt tc nhn chnh pH (acid
hay base) vo bn ln men.

3. Kim sot nhit
Vi sinh vt c th c phn loi da vo nhit thp nht m bt u din
ra s tng trng. Mi nhm vi sinh vt c khong nhit thch hp cho tng trng
khc nhau. i vi nhm a nhit thp (psychrophiles) th nhit ti thch cho tng
trng di 20
o
C. i vi nhm chu nhit trung bnh (mesophiles) th nhit ti thch
cho tng trng l t 20-45
o
C. Trong khi , nhit ti thch cho nhm chu nhit
(thermophiles) l 45-60
o
C.


H
N
T
Arrheun
L
T
N
T
tng gia
vi qui t
1
tr ch y
Hnh 6
Hnh 6. 4. N
Nhit nu
Tc ca
nius:
Logv =
H A
Trong ,
Nh vy Log
Trong nui c
ai on ca
trnh c khu
. Hot n
yu trong qu
6. 3. Nhit
Nhit ti
i cy nh h
phn ng
RT
H
303 , 2
+
v: tc p
H A : nng
R: hng s
T: nhit
gv ph thu
cy vi sinh
qu trnh n
uy trn) sin
ng ca vi sin
u trnh ln
ti thch
thch ca v
hng n t
thay i p
+ constant
phn ng,
g lng ho
kh,
tuyt i
c vo 1/T.
vt, cn b
nui cy, nh
nh ra l do:
nh vt. y
n men.
ca cc nh
vi khun sin
tng trng
ph thuc v
(6.1
t ha,
.
o m n
hit c th

y l nhit l
m vi sinh v
nh lactic aci
g nh th n
vo nhit
1)
ng nhit
th thay i
ng c ngu

vt khc nh

id v ca n
o?
theo ph
ti thch c
i. Nhit tron
un gc sin
hau.
m men.
hng trnh
a chng. T
ng ln men
nh hc, n
62
h ca
Trong
n (i
g vai
63
[C] + O
2
+ [N] [P] + CO
2
+ E (6.2)

Mt s v d:
C
6
H
12
O
6
+ C
3
H
6
O
3
(lactate) + 32,4 Kcal (6.3)
C
6
H
12
O
6
+ C
2
H
5
OH (ethanol) + 41,9 Kcal (6.4)
C
6
H
12
O
6
+ C
5
H
9
NO
4
(glutamate) + 193,4 Kcal (6.5)

2. Ngun gc vt l. Nhit lng sinh ra do cc tc ng l hc nh do khuy
trn, do nhit t khng kh sc vo, Lng nhit ny thng khng ln.
Tu tng giai on c th ca qu trnh ln men v tu vo nhit mi trng
m hot ng kim sot nhit nui cy c khc nhau. Trong giai on u, nhit ln
men nh, nu nhit mi trng thp, cn gia nhit (dng hi nc nng). Trong giai
on tng trng mnh, nhit ln men nhiu, cn gii nhit (dng nc nhit thp).
Trong thc t, ngi ta thng dng nc nhit thp (5~27
o
C) gii nhit. Thit b
trao i nhit c th l dng ng (coil, package) t trong ni ln men hoc dng o
(jacket) t bn ngoi ni ln men. Kh nng gii nhit ph thuc vo lu lng, nhit
ca nc gii nhit v din tch tip xc gia hai loi dch. Mt h thng iu khin nhit
bao gm:
- Thit b trao i nhit (heat exchanger), lm bng vt liu c h s truyn nhit
cao.
- H thng lm lnh nc gii nhit (cooling system) hoc h thng gia nhit
(thng dng l in tr t nng-heater hoc hi nc nng-steam)
- Bm cung cp nc gii nhit
- B cm bin nhit t ng (thermo sensor), iu khin h thng van nhm cho
php cc van ny ng hay m kim sot nhit nui cy theo gi tr ci t.

Cch tnh s gii nhit ca h thng ln men
Gi s c h thng gii nhit cho ni ln men dng jacket (Hnh 6.1)

T1: Nhit u vo ca nc gii nhit
T2: Nhit u ra ca nc sau khi gii nhit
T3: Nhit ca dch ln men
F: Lu tc nc gii nhit (KL/hr)
A: Din tch trao i nhit (m
2
)
ATm =
) 2 3 (
) 1 3 (
) 2 3 ( ) 1 3 (
T T
T T
Ln
T T T T

(6.6)
Nhit lng trao i Q, Mcal/hr, Q = ( ) F T T 1 2 (6.7)
Hiu sut trao i nhit U ca thit b, Mcal/hr.m
2
.
0
C, U =
Tm A
Q
A
(6.8)

64

Hnh 6. 5: S h thng gii nhit ni ln men dng jacket.

V d:
Trong trng hp gii nhit ni ln men vi nhit ln men theo yu cu l T3,
din tch trao i nhit A, bng h thng gii nhit vi nhit cung cp l T1, lu lng
dng cp l F. Nhit nc sau khi qua ni ln men l T2. Hy tnh nhit lng sinh ra
t ni ln men v hiu sut trao i nhit ca thit b.

T1 = 16
o
C, T2 = 22
o
C, T3 = 32
o
C, F=75 KL/gi, A=25,6 m
2
.
Ta c: ATm=12.5
o
C;
Q = 450 Kcal;
U=1.41 Mcal/hr.m
2
.
o
C.

4. Kim sot hm lng oxygen ha tan trong mi trng ln men
Ty theo chng vi sinh vt v giai on pht trin m nhu cu s dng oxygen
cng khc nhau. i vi cc chng k kh th khng cn cung cp oxygen trong qu trnh
ln men. i vi cc chng hiu kh khng bt buc th trong tng giai on c th ca
qu trnh, nhu cu s dng oxygen c khc nhau. i vi cc chng hiu kh bt buc,
cn cung cp oxygen trong sut qu trnh nui cy. V d i vi trng hp ca nm
men, trong iu kin hiu kh th nm men sinh sn tng s lng, nhng trong iu k
kh th chng s chuyn ho glucose thnh ru ethanol. c tnh ny c khai thc
trong qui trnh cng ngh sn xut bia ru v nc gii kht t qui trnh ln men.
Oxygen t khng kh i vo t bo vi sinh vt trong qu trnh nui cy thng qua 3
giai on:
- T khng kh vo dch ln men
- T dch ln men vo t bo
- S s dng oxygen bi t bo vi sinh vt
Trong mi trng nc, hm lng oxygen ho tan ti a l 8,4 mg/L 25
o
C.
Nhu cu s dng oxygen ca vi sinh vt hiu kh trong qu trnh nui cy din ra lin tc
do vy cn phi cung cp oxygen lin tc.
Vai tr ca oxygen c xc nh l to ATP v ti to NAD(P) t NAD(P)H
2

65
V d trong ln men sn xut glutamic acid, vi sinh vt s dng mt phn t
glucose cn 2,33 phn t oxygen. Khi thiu oxygen, cc sn phm ph (nh lactic acid)
c hnh thnh nhanh chng, do vy mt phn ngun C tiu tn cho cc sn phm ph
ny dn n hiu sut ln men thp. Trong mt s trng hp, chnh cc sn phm ph
ny c tc ng ngc li gy c ch qu trnh tng trng v sinh tng hp cc sn
phm mong mun.
Hm lng oxygen ha tan trong mi trng ln men ph thuc:
- Kh nng cung cp khng kh hoc oxygen dng kh
- khuy trn dch men
- p sut kh trong trong ni ln men
Tc ha tan oxygen t khng kh vo dch ln men c th m t qua phng
trnh sau:
dC
L
/dt = K
L
a.(C* - C
L
) (6.9)
C
L
: nng oxygen ha tan trong dch ln men ti thi im nh gi
(mmoles/dm
3
), C*: nng oxygen ha tan bo ha, K
L
h s dch chuyn ha tan
(cm/hr), a l din tch b mt tip xc gia phase kh v phase lng. Trong thc t, ngi
ta thng nh gi kh nng ha tan oxygen trong ni ln men thng qua ch s K
L
a.
Hm lng oxygen ha tan trong ni ln men phn nh s cn bng gia lng cung cp
v lng s dng oxygen bi vi sinh vt.
OTR v OAR l hai ch s c dng trong vic nh gi kh nng cung cp
oxygen cho ni ln men v lng oxygen c s dng bi vi sinh vt.
OTR: Kh nng cung cp oxygen ti a ca h thng, b gii hn bi cng sut
thit k ca h thng. OAR(R-ab): tc s dng oxygen ca chng nui cy, thng
gi l B-rab. Trong trng hp nui cy khng nn p, cch tnh B-rab nh sau:
B-rab =
1000
1
.
4 , 22
1
.
100
1
. ) .(
) ( 100
) ( 100
.
) 273 (
273
.
)
`

+
+
+
xo xi
yi xi
yo xo
T V
Q
(mmol/min/ml) (6.10)
Trong , Q: lu lng khng kh sc vo ni ln men
V: th tch dch mi trng ln men
T: nhit nui cy
x
o
v x
i
: nng oxygen ca khng kh (%) trc khi sc vo v sau khi
thot ra t ni ln men
y
o
v y
i
: nng CO
2
ca khng kh (%) trc khi sc vo v sau khi thot
ra t ni ln men.
S nh hng ca oxygen ln tng trng ca vi khun
i vi ln men thu nhn amino acid, cn phi tng cng ti a t l s lng t
bo /lng glucose (hiu sut sinh khi) s dng trong phase tng trng v t l gia
lng amino acid /lng glucose (hiu sut chuyn i c cht thnh sn phm) s dng
trong phase sn xut. Trong iu kin thiu oxygen (pL < pLcrit) din ra trong phase tng
trng, cc sn phm ph l cc acid hu c nh lactic acid s tch ly dn, gy nh
66
hng n hiu sut ln men. Do vy cn phi sc kh - khuy trn trong giai on ny
bo m cung cung oxygen theo yu cu cho s h hp ca vi khun.
Bng 6.1. S ph thuc ca hm lng Arg sn xut vo p sut ring phn ca Oxygen
trong dch ln men cc pha khc nhau.
p sut O
2
trong
phase tng trng, atm
p sut O
2
trong
phase sn xut, atm
Nng Arg,
mg/ml
0,01-0,05 0,01-0,05 30,3
0,01-0,05 0,32-0,42 28,4
0,32-0,42 0,01-0,05 19,5
0,32-0,42 0,32-0,42 22,8

Tuy vy, ngi ta cng nhn thy rng trong iu kin ngun oxygen cung cp
qu cao th vic tng hp cc amino acid ca vi khun cng khng hiu qu. Hin tng
ny lin quan cht ch vi p sut vn hnh bn trong ni ln men trong qu trnh nui
cy. Trong trng hp ln men di p sut khng kh, nui cy bt u vi p sut l
0,21 atm ca pL. Trong khi nu p sut nui cy l 1,5 kg/cm
2
th khi bt u nui cy
p sut ring phn l 0,315 atm pL v vi iu kin p sut 2,0 kg/cm
2
th p sut ring
phn s l 0,42 atm pL trong. Qu trnh ln men sn xut Arg b nh tr khi p sut nui
cy cao. Do vy trong thc t sn xut, p sut vn hnh cng gn p sut kh quyn th
cng tt. Nu vi sinh vt tng trng vi pL t 0,01-0,05 atm, nn dng kh vi hm
lng oxygen 5% trong iu p sut khng kh.
Nh cp trn y, iu kin cung cp oxygen ti u c kho st thng
qua s thay i mc pL trong phase tng trng v phase sn xut cc amino acids,
vi kt qu ghi nhn c ty thuc vo iu kin ti u ca oxygen.
- Amino acids ca nhm Glu nh l Gln, Glu, Pro v Arg c sinh ra ti a
trong iu kin h hp thch hp cho vi khun v gim mnh trong iu kin sn xut
thiu oxygen.
- Nhm Leu, Phe, v Val c tng hp ti a trong iu kin thiu oxygen.
- Cc amino acids ca nhm Asp nh l Lys, Ile v Thr c tng hp ti a
trong iu kin h hp thch hp cho vi khun v gim khng ng k trong iu kin
thiu oxygen.
S cn thit ca oxygen trong ln men amino acid
Trong ln men cc amino acids i hi phi cung cp ngun oxygen do hai
nguyn nhn chnh sau:
67
1) Vi khun cn c ngun ATP cho tng trng ca chng v s sinh tng hp
amino acids bng c ch bin dng nng lng hiu kh.
2) NAD c ti to bi oxygen t NADH
2
, m NADH
2
uc sinh ra t qu trnh
sinh tng hp cc amino acids.
Do vy, hiu qu ca s khuy trn rt c ngha trong ln men sn xut amino
acids. Trn c s ca s lng mol ATP c yu cu cho s sinh tng hp ca nhiu
loi amino acids khc nhau v s lng mols NAD(P)H
2
to ra trong qu trnh c gi
l qu trnh sinh tng hp. Bng 6.2 tng hp t l cn bng ca ATP v NAD(P)H
2

trong phn ng ln men cc amino acid t c cht l glucose.
Bng 6. 2. Bng tnh ton lng NADH
2
v ATP sinh ra trong qu trnh tng hp cc
amino acid thng qua chu trnh Glyoxylate v con ng PEP carboxylation
Amino acid
Theo con ng
Glyoxylate
Theo con ng
c nh CO
2

NADH
2
ATP NADH
2
ATP
Glu
Gln
Pro
Arg
+5 1/3
+5 1/3
+4 2/3
+4
+2
+1 1/3
+1 1/3
-2/3
+3
+3
+2
+1
+1
0
0
-3
Leu
Phe
Val
+2
0
0
+2
-1 2/5
+2

Lys
Ile
Thr
+2 2/3
+2
+4
+2/3
+2/3
0
-2
-3
-4
-1
-4
-4

Cc amino acids nh Leu, Phe v Val c sinh tng hp khng thng qua chu
trnh TCA vi hiu sut ti a khi h hp b cn tr bi iu kin thiu oxygen; iu ny
c cho l do cc nguyn nhn sau:
- Khi s quay vng ca chu trnh TCA b ngn tr do thiu oxygen, ngun PEP
hoc pyruvic ngoi bo m c cha cc amino acids c gia tng.
-Nhiu oxygen l khng cn thit cho s ti oxy ha NAD(P)H
2
bi v khng c
NAD(P)H
2
sinh ra do s sn xut ca cc amino acid (Phe v Val) hoc bi v n c
sinh ra t hn so vi trong trng hp ca cc amino acids thuc nhm Glu v Asp.
5. Kim sot tc b sung mi trng dinh dng trong qu trnh ln men
Nh chng ta bit, i vi nui cy vi sinh vt, mt trong nhng vn quan
trng cn kim sot l phi duy tr hm lng dinh dng ca cc cht trong mi trng
68
ln men khng qu tha hoc qu thiu, c bit l i vi mt s c cht gii hn. V d
trong ln men sn xut glutamic acid cng nghip, nu tc np ng glucose vo mi
trng nui cy qu cao, lm nng ng glucose tch ly trong mi trng nhiu, lc
ny vi khun sinh glutamic acid s ng ha glucose vi tc cao cng vi vic s
dng nhiu oxygen. Tuy nhin do tc sc kh v khuy trn khng thay i nn nng
oxygen ha tan trong dch ln men gim xung, lc ny trong mi trng ln men xy
ra tnh trng thiu ht oxygen dn n vic hnh thnh cc sn phm ph m ni bt nht
l lactic acid. Vic hnh thnh cc sn phm ny lm gim hiu sut ln men v chnh cc
sn phm ph l yu t c ch gy nh hng n hot tnh ca chng trong qu trnh ln
men. Ty theo c im ca tng loi ln men m phng php np b sung nguyn liu
v kim sot s np liu khc nhau. Tuy nhin phi lun bo m mt s yu cu cn bn
sau:
- Ch np b sung theo nhu cu s dng ca chng, khng np lng qu d
tha dn n vic thiu oxygen cung cp.
-Khng np b sung thiu lm hn ch tc tng trng v tc sn xut ca
chng.

Mc ch ca vic b sung dung dch trong qu trnh ln men bao gm:
- Tng lng sinh khi tham gia vo qu trnh sinh tng hp sn phm (nui cy
mt t bo cao) v ko di thi gian nui cy v tng nng sn phm.
- hn ch s nh hng ca p sut thm thu ln vi sinh vt, trong mi trng
nui cy ban u nng c cht gii hn thng thng ch mc ph hp, khng qu
cao.
- Mi trng b sung thng c cha c cht gii hn (thng l ngun C) nn
cn phi duy tr nng nng thch hp trong qu trnh nui cy nhm kim sot s
tng trng v s sinh tng hp sn phm mong mun tng cng hiu sut sn xut
v gii hn s hnh thnh cc sn phm ph.
Bng 6. 3. Nng gy c ch ca mt s thnh phn trong mi trng nui cy
Thnh phn Hm lng gy c ch tng trng
Glucose > 50 g/L
NH
3
> 3 g/L
Fe > 1,15 g/L
Mg > 8,7 g/L
P > 10 g/L
Zn > 0,038 g/L

Cc thnh phn mi trng thng phi np b sung vo trong qu trnh nui cy:
- Ngun C: glucose, fructose, sucrose
- Ngun N: NH
3
, Urea
Cc phng php np b sung (thc hin trong iu kin v trng):
69
- B sung lin tc: Dch mi trng b sung c np vo vi tc thch hp
theo nhu cu dinh dng ca vi sinh vt. Tc b sung c th c nh, tng dn theo
tng bc hoc tng dn theo hm s m. C th dng du hiu gin tip nh da trn c
s ca hm lng oxygen ha tan trong mi trng ang nui cy iu chnh tc
b sung dinh dng p ng nhu cu s dng ca vi sinh vt v duy tr tt s cn bng
hm lng c cht trong sut qu trnh ln men. Phng php b sung lin tc kh thc
hin, i hi h thng thit b hin i, nhn vin k thut phi c trnh tay ngh cao.


Hnh 6. 6. ng hc tng trng t bo trong nui cy m- b sung vi tc b
sung cht dinh dng khc nhau. (a) Tc c nh; (b) Tc tng dn tng
bc; (c) Tc tng theo hm m.

- B sung gin on: Phng php ny n gin gin hn so vi phng php b
sung lin tc. Vic np b sung ch thc hin mt s ln nht nh trong qu trnh nui
cy. Lng b sung ln sau thng cao hn ln trc v cao hn nhu cu s dng ca vi
sinh vt, do vy nng c cht thay i t ngt sau mi ln np liu, gy sc cho t
bo.











Hnh 6. 7. S bin ng nng glucose v pH trong qu trnh ln men theo phng
php np mi trng b sung. (a) B sung dinh dng lin tc; (b) B sung dinh dng
gin on.
0
2
4
6
8
10
12
0.0
0.2
0.4
0.6
0.8
1.0
1.2
4 8 12 16 20 24 28 32 36
%

G
l
u
c
o
s
e
O
D
Thi gian nuoi cay (gi)
(a)
OD
Glu.
pH
0
2
4
6
8
10
12
0.0
0.2
0.4
0.6
0.8
1.0
1.2
4 8 12 16 20 24 28 32 36
%

G
l
u
c
o
s
e
O
D
Thi gian nuoi cay (gi)
(b)
70
6. Ph bt trong qu trnh ln men
Hin tng to bt thng xy ra trong nui cy vi sinh vt ngay c qui m phng
th nghim. i vi cc nh my ln men sn xut cng nghip, c bit trong iu kin
nui cy c sc kh - khuy trn nh trong ln men sn xut khng sinh, sn xut cc
amino acid hoc trong trng hp ln men s dng cc c cht c khuynh hng to bt
mnh nh mt r ng, hin tng to bt xy ra rt mnh. Thng thng nguyn nhn
gy bt l do cc phn t protein c ngun gc t nguyn liu th dng pha ch mi
trng nh mt r ng, cao ng, cao u, dch chit nm men. Cc protein ny b
bin tnh v hin din trn b mt tip xc gia hai phase kh - lng, hnh thnh nn mt
lp o kh v. Bt kh bn s duy tr trong sut qu trnh ln men, ban u l do mi
trng nui cy, sau do hot tnh ca vi sinh vt gy nn.

6. 1. To bt v cc nhn t gy to bt
Bt bng khng kh do sc vo ni ln men t h thng phn phi kh hoc kh
CO
2
sinh ra trong qu trnh ni dn ln b mt dch ln men gy nn mt lp mng mng.
Bnh thng, lp mng mng ny d dng b b ra v khuch tn thnh hi m thot ra
khi ni ln men qua ng ng thot kh. Trong trng hp mi trng nui cy c
mt s thnh phn lm cho mng bng kh ny bn vng, khng v c th s to nn
bt. Theo thi gian, lng bt trong ni ln men tng dn ln v n khi vt th tch bn
th s thot ra ngoi theo dng kh qua ng ng thot kh ca ni ln men. Khi nghin
cu hin tng to bt cn phi lu c hai vn l s to bt v tnh bn vng ca
bt. Sc cng b mt, nht v cng ca dch ln men l cc yu t quan trng gy
nn hin tng to bt. Nhng loi dch lng cha nhng phn t d b hp ph trn b
mt c khuynh hng to bt mnh. Cht to bt thng l cc hp cht hu c nh cht
ty ra, protein, Cc cht ny u c cc u phn cc i nc nh SO
4
2-
, COO
-
,
NH
4
+
, amide, ester, OH
-
, -CO, hoc k nc c cha cc chui carbohydrate. Bt s c
hnh thnh nhiu mt khi c s cn bng gia cc u i nc v k nc.

6.2. Nhng tr ngi gy nn bi bt
- Lm tch t bo vi khun ra khi b mt dch lng ln men, dn n hin tng
t phn xy ra mnh, mt sinh khi tham gia sn xut ng thi cng to nn s bn vng
cho bt.
- Mt lng ln t bo trong phase bt khng c bo m cc yu t thch hp
cho sn xut nh nhit , pH, dinh dng gy suy gim hot tnh ging.
- Gim th tch s dng ca ni ln men.
- Bt tro ra ngoi gy tht thot mt lng ln t bo, gim hiu sut v sn
lng ln men; gy nhim mi trng lm vic.

6.3. Kim sot bt
C hai quan im cn bn trong vic kim sot bt:
- Kim sot trc qu trnh: X l nguyn liu, lm gim kh nng to bt trc
khi a vo qui trnh. (nh bentonite mt r trong sn xut nm men, lc sellaite dch thy
phn u nnh, thay i thi gian thanh trng)
71
- Ngn chn s to bt bng cch loi tr cc yu t gy nn nh thay i pH,
nhit , thnh phn mi trng. V iu quan trng l phi n lc kim sot bt v s
to bt l khng th trnh khi trong qu trnh ln men, c bit l qui trnh c sc kh v
khuy trn.
bo m vic ph bt t hiu qu cao, ngi ta thng kt hp s dng c hai
quan im kim sot bt.
Mt s phng php chng bt c p dng, da trn nguyn tc k thut, c
th chia thnh ba loi nh sau:
- Chng bt theo phng php ha hc
- Chng bt theo phng php nhit
- Chng bt theo phng php c hc

a. Phng php ha hc
S dng cc ha cht ph bt vo mi trng nui cy. Yu cu cn bn ca cc
cht ph bt:
- ha tan thp trong mi trng c bt
- Sc cng b mt km
- Kh nng khuych tn trong mi trng lng cao.
Trong cng nghip ln men, cc cht ph bt cn phi c thm mt s tnh cht
sau:
- Khng c hi i vi vi sinh vt
- Khng c s dng bi vi sinh vt
- Khng gy nh hng ngc n ngun cung cp oxygen
- Bn i vi nhit
- Khng gy tr ngi i vi cc qui trnh sau
- Khng gy nh hng xu i vi sn phm
- Hot ng tt nng thp
- Duy tr hot tnh tt trong thi gian di
Cht ph bt dng trong cng ngh ln men cng nghip c phn loi nh sau:
- Cht bo t nhin
- Cn c mch C cao (8C- kh nng ph bt tng i thp)
- Cc dn xut ca sorbitan (sorbitane monolaurate, tri-oleate, tri-stearate)
- Polyether (trng lng phn t cao hn 2000)
-Du silicon (polydimethyl siloxane) [(CH
3
)
3
SiO(CH
3
)
2
O]Si(CH
3
)
3
. Silicon oil
khng ha tan trong nc, hi c, v khng th ng ha bi vi sinh vt.
C ch ph bt:
Cht ph bt c cha cc tc nhn gy v bt cc b v c ch s to bt bng
cch gy suy gim tnh n nh ca bt. i vi cc cht gy b bt, mt lng nh b
hp ph v ha tan trn b mt, do vy lm gim ng k sc cng b mt v tr c
hp ph. Do cht ph bt thng khng tan trong nc nn vng hp ph cht ph bt b
hp dn bi khu vc ln cn rt mnh v c khuch rng mt khi mng ca khu vc
mang theo nhng lp dch tng thp hn. Mng ca lp cht lng mng di chuyn rt
nhanh v khi t n mt mc nht nh, chng lp tc b v ra do s thay i t ngt

c hc,
silicone
theo m
cng,

(
H
P
T
men nh
-
v mc
thi gian
-
men n
(load) c
np c
-
mt mc
nhn bi
L
mt s n
-
-
-
-

b. Phn
C
thng n
cn t m
-
-
-
nhit hoc
v span-20
ng b mt
nht do
(a)
Hnh 6. 8. S
ph
Phng php
Trong sn x
h thng t
Np theo th
to bt
n v lu l
Np theo t
n mc mc
a khuy t
cht ph bt
Da vo
c c xc
t np ch
Lu l khi
nhc im
Hn ch O
c ch tn
c ch sinh
c tnh
ng php ph
C nhiu th
ngi ta k
mt s tiu
Hao tn n
Cu trc
D v sinh
do rung
0 c hp
gy nn s
o vy rt kh
qu tr
h bt; (b),
p b sung c
xut cng n
t ng. C
hi gian (tim
trong mt
ng cht ph
tn hiu ch
nht nh,
ng ln ko
t.
u cm bi
nh trong
ht ph bt.
i dng cht
m sau:
TR
ng trng
h tng hp
bt c h
hit b c k
t hp vi v
chun sau:
ng lng t
n gin
, d thanh t
ng phn t
dn chn l
khng ph
h to b
nh ph bt
c ch ph
cht ph b
nghip, ng
th s dn
mer). Theo
h thng l
h bt np v
th ca am
lp bt trn
theo s gia
n bt (diffe
g ni ln me
ph bt, b
c
kh dng l
vic dng c
t
trng
t khi khu
lc rt mnh
h hp cho
t bn.
(b)
t. (a), hin t
bt.
t:
i ta thn
ng cc nguy
cch ny, y
n men nh
vo cho thc
mpere k. K
n cng s v
a tng dng
ferential tem
en s chm
n cnh u
m phng
c cht ph
ch tn. Ch
nh vo giao
o iu kin
tng lm v
ng b sung
yn tc cn b
yu cu ph
t nh. Tr
ch hp.
Khi bt
va p vo c
g in ca k
mparature se
m vo u c
im l ho
g tin ph b
bt. Cc p
ht c ch t
din ca h
n in-c c
v bt khi b
g cht ph
bn sau:
i bit trc
n c s
c hnh thn
cnh khuy
khuy. y
ensor). Khi
m bin v
ot ng ph
bt trong l
phng tin
to bt nh
hai phase v
n thit nh

b sung ch
bt vo n
c c tin
, c th c
nh trong n
y ph bt lm
y l du hi
i bt dng l
nh vy c
h bt, cn l
n men. Th
n c hc ph
72
du
ko
h
t
i ln
trnh
i t
i ln
m ti
u tt
ln
th
lu
hng
bt

-
Phng
ngt, to
Hnh 6.
kh
tr
ng

7. Theo
1
T
trnh, vi
sn phm
thng c
nng
cng rt
ng kp
acid, bn
lng la
thiu l
np nguy

2
S
trong
men m
ty thu
Khng cn
tin ph b
o lc ko, n
9. Cyclone,
h ca ni
rong cyclon
goi, phn
di v tnh
. Nng s
Trong qu t
c theo di
m m c p
chn phn
sn phm
quan trng
p thi nhm
n cnh vic
actic acid tr
ng oxygen
yn liu
. Sn lng
Sn lng (P
P=

=
n
i
, Vi l th
i c gi tr
c vo s l
n bo tr nhi
t c kh s
nn p, lc t
, thit b lm
ln men. K
ne v do va
dch s
h ton kt q
sn phm
trnh ln m
nng s
phng ph
ng n n g
mong mu
g, gip nhn
m hn ch k
c xc nh n
rong dch l
n ha tan v
ng np v
g
P) l lng

Ci Vi
1

tch dch l
khc nhau.
n chit dch
u
dng t nh
tc ng n
m v bt c
Khi bt the
a chm c h
c hi lu v
qu ln me
men, n
n phm tro
p phn tch
gin, r tin
n, vic n
n bit c
t qu sn x
nng sn
ln men. N
do vy c
o.
sn phm c
(6.11)
n men v
. i vi nu
h ln men r
t mt trong
h hng.
hc thn
eo kh thot
hc bt s v
v li ni l
en
nh gi tc
ong dch ln
h khc nha
n, d thao t
nh gi nng
tnh trng c
xut km. V
n phm l g
Nu nng
th tng c
c trong dc
Ci l nng
ui cy m,
ra trong qu
g bn c ch

ng c lp
t ra s di c
v ra. Phn
n men.
sn xut
n men l rt
au. Tuy nhi
tc. Bn cn
g cc sn
ca qu trn
V d trong
glutamic aci
lactic aci
ng tc
ch ln men c
sn ph
i = 1. i
trnh nui
h: lm thay
p t trn
chuyn theo
n kh trong
t v chiu
t quan trn
in, trong th
nh vic ph
n phm kh
nh ln men
ln men s
id ngi ta
id cao, ngu
sc kh ho
ca mt m
m. Ty th
vi nui c
i cy. i v
y i p su
ng ng t
o hng ly
g bt s tho
hng ca
g. Ty theo
hc t ng
n tch nh
ng mong m
v c nhn
n xut glut
thng o
uyn nhn l
oc gim t
.
eo loi hnh
y m-b su
vi ln men
73
t t
thot
y tm
t ra
a tin
o loi
i ta
h gi
mun
ng tc
tamic
hm
l do
c
h ln
ung, i
n lin
74
tc th vic xc nh P cn ph thuc vo thi gian k t khi chit dch, tuy nhin trong
trng hp ny th khi nim sn lng khng r rng.
3. Hiu sut
L t l phn trm gia lng sn phm thu c v lng nguyn liu a vo s
dng cho qui trnh. V d hiu sut chuyn ha ng thnh ru trong ln men ethanol:
Y
e/s
(%) =
Sugar
Ethanol
100 (6.12)
Hiu sut t bo l lng sinh khi thu c trn lng ng dng cho m ln
men .
Y
b/s
(%) =
Sugar
Biomass
100 (6.13)
nh gi chnh xc hiu qu ln men ca tng giai on trong tin trnh ln
men v nh gi thi im dng ln men vi chi ph sn xut thch hp nht, ngi ta
phn tch sn lng v hiu sut ln men theo thi gian nui cy. Kt qu nh gi ny
gip tr li c cc cu hi nh giai on no th tc tch ly sn phm hoc/v hiu
sut t gi tr cc i? Nn dng qui trnh ln men thi im no th thch hp, mang
li chi ph sn xut thp nht? Vic nh gi c th thc hin theo tng giai on nht
nh trong qu trnh ln men hoc/v nh gi kt qu tch ly ca qu trnh.
V d mt qu trnh ln men c quan tm 3 thi im l 0 gi, 10 gi v 20
gi. Phn tch kt qu ln men ca giai on t 0 10 gi v t 10 20 gi c gi l
nh gi theo giai on. Nu kt qu c xem xt t 0 10 gi ri t 0 - 20 gi th gi
l nh gi theo s tch ly ca tin trnh. Bn cnh , thng phi tnh tc s dng
ng c trng (TS/DCW/Hr) v tc to sn phm c trng (Pro/DCW/Hr) ca
chng thng qua vic phn tch kt qu lng sinh khi nh gi nng lc tiu th
ng v nng lc sn xut sn phm ca chng.
Sau y l mt v d v phn tch kt qu ln men trong qu trnh nui cy (Bng
6.4). Theo di mt m ln men chm, hiu kh, c b sung c cht gii hn l ng
glucose trong qu trnh nui cy khi ng trong mi trng cn kit v c chit dch vi
thi gian nui cy l 20 gi. Th tch dch ln men ban u l 50 KL, nng ng
trong mi trng ban u l 10 g/dl, nng ng trong dch np b sung l 50 g/dl,
lng ng s dng trong qu trnh nhn ging l 2 t. C mi 2 gi, ly mu v phn
tch nng sn phm, v ghi nhn cc ch s k thut nh th tch dch np vo (V
f
), th
tch dch chit ra (V
out
). Trn c s c th tnh c sn lng, hiu sut ca tng giai
on c th.

Bng 6. 4. Kt qu theo di cc thng s ca qu trnh ln men
75


Ch thch:
CT: Culture time, thi gian nui cy
c
p
: concentration of product: nng sn phm
V
out
: Th tch dch ln men chit ra trong qu trnh nui cy
V
inn
: Th tch dch ln men trong ni ln men
V
f
: Th tch dch c cht (mi trng) np b sung trong qu trnh nui cy
S
f
: Tng lng c cht np b sung
s
r
: nng c cht cn li trong mi trng ln men
S
r
: Tng lng c cht cn li trong mi trng ln men
S
u
: Tng lng c cht c vi sinh vt s dng
P: Lng sn phm
Y: Hiu sut (yield) chuyn i c cht
P
hr
: Lng sn phm tng hp c theo thi gian (gi
-1
)
Y
hr
: Hiu sut sinh tng hp theo thi gian nui cy (gi
-1
)
Trong qu trnh nui cy, nng sn phm, th tch dch mi trng b sung, th
tch dch ln men chit ra c phn tch v ghi nhn bi nhng thit h tr. Cc thng s
cn li c tnh ton trn c s ca th tch v nng phn tch c.
V
inn
= V
o
+ V
f
- V
out
, trong V
o
l th tch dch ln men ti thi im bt u
nui cy.
S
f
= V
f
c
f
, trong c
f
l nng c cht trong dch np b sung.
S
u
= S
o
+ S
f
- S
r
, trong S
o
v S
r
ln lt l nng c cht ban u v cn li ti
thi im ng gi.
P = c
p
V, trong V l th tch dch ln men ti thi im nh gi,
V = V
inn
+ V
out

Y = P/S
u

P
hr
= (P
t2
-P
t1
)/(t
2
-t
1
), trong P
t1
, P
t2
l lng sn phm thi im t
1
v t
2

CT c
p
V
out
V
inn
V
f
s
r
S
f
S
r
S
u
P Y P
hr
Y
hr
[hr] [g/dl] [KL] [KL] [KL] [g/dl] [t] [t] [t] [t] [%] [t/hr] [%/hr]
0 0.5 50 0 10.0 0.0 5.0 2.0 0.25 12.50
2 1.2 50 0 5.0 0.0 2.50 4.5 0.60 13.33 0.18 14.00
4 2.4 55 5 2.0 2.5 1.10 8.4 1.32 15.71 0.36 18.46
6 4.5 60 15 2.0 7.5 1.20 13.3 2.70 20.30 0.69 28.16
8 6.8 68 23 2.0 11.5 1.36 17.1 4.62 26.98 0.96 50.10
10 9.0 5 71 26 0.5 13.0 0.38 19.6 6.84 34.86 1.11 89.35
12 11.5 10 71 31 0.4 15.5 0.32 22.2 9.32 42.00 1.24 96.83
14 13.2 15 70 35 0.3 17.5 0.26 24.2 11.22 46.28 0.95 92.07
16 14.0 21 68 39 0.7 19.5 0.62 25.9 12.46 48.15 0.62 75.98
18 14.3 23 69 42 0.4 21.0 0.37 27.6 13.16 47.61 0.35 39.66
20 14.4 25 71 46 0.1 23.0 0.10 29.9 13.82 46.23 0.33 29.40

Y
nh gi
S

p dng
-
S
S
P
Y
-
P
-
Y

Y
hr
= P
hr
/S
uh
.
S
uhr
= (S
ut2
-S
g vo tnh to
Gi s tnh
S
f
(6) = c
f
V
S
u
(6) = S
o
+
P(6) = c
p
(6)
Y(6) = (P(6)
Lng sn
P
hr
(4-6) = (P
Hiu sut s
Y
hr
(4-6) = P
Hnh
hr
, trong
S
ut1
)/(t
2
-t
1
)
on Bng
h ton ti th
V
f
(6) = 50 g
S
f
(6) S
r
(6
(V
inn
(6) +
)/S
u
(6))100
phm sinh
P(6)-P(4))/(
sinh tng h
(4-6)/S
u
(4-6
h 6. 10. Kt
S
uhr
l l
6.2:
i im nu
g/dl15 KL
6) = (2,0 +
V
out
(6)) = 4
0 =(2,7/13,3
tng hp tr
(6-4) = (2,7
p trong gia
6) = [(2,7-1
qu nh g
ng c cht
i cy sau 6
= 7,5 tn
10 g/dl50K
4,5 g/dl(60
3)100 = 2
rong giai o
7-1,32)/(6-4
ai on nu
1,32)/(13,3-
gi ln men
t s dn
6 gi
KL) + 7,5
0 + 0) = 2,7
20,30%.
on t gi n
4) = 0,69 t/g
i cy t 4
-8,4)]100 =
tch ly the
ng trong 1
1,2 = 13,3
7 t.
nui cy 4
gi
6 gi l:
= 28,16 (%/
eo thi gian
gi giai
3 t.
6 l:
/Hr).

n.
76
on

Nhn x
T
tch lu
Tuy nhi
on ny
T
c sn
im nu
tng rt
P
phase su
H
on cn

Hnh 6.
t:
Trong lag ph
trong dch
n hiu su
y, c th do
Trong giai
n xut vi t
ui cy ting
mnh v
Phase n n
uy vong.
Hiu qu ln
n phi xc
11. Kt qu
hase t thi
ln men nh
t sinh tng
o t bo tng
on tip th
tc kh c
g th 12. T
t gi tr cao
nh rt ngn
n men gim
nh dng q
nh gi h
i gian nui
hng khng
hp sn ph
g trng m
heo, qu tr
cao, tng d
rong giai
o nht 95%
, ch khon
m mnh tro
qui trnh ln
hiu qu ln
cy ting t
g nhiu. L
hm khng
nh, to sin
nh ln me
n t 0,7 t/H
on ny, hi
.
ng t 2-4 gi
ong giai o
n men th
n men theo
th 0-4, m
ng sn ph
tng ng
nh khi.
en i vo lo
Hr v t gi
iu sut sin
v qu tr
on t 14 g
i im thch
tng giai
c d nng
hm theo
k (Hnh 6
og phase, l
i tr cc
nh tng hp
nh ln men
gi -18 gi.
h hp nht.

on.
sn phm
cng tng
.11). Trong
ng sn p
i 1,2 t/Hr
sn phm
n bt u
V y l
.
77
m c
dn.
g giai
phm
thi
cng
i vo
giai
78
CHNG 7. TI U HO H THNG LN MEN

Vic ti u ha mt qui trnh ln men l rt quan trng i vi thc t sn xut
cng nghip. Mc ch cui cng ca cng vic ny l lm sao sn xut t hiu qu
cao nht. nh gi hiu qu sn xut, cn phi xem xt ba yu t c bn: nng sn
phm trong dch ln men, tc sn xut sn phm v hiu sut chuyn i c cht (v
d glucose) thnh sn phm mong mun.
Nng sn phm tch ly trong dch ln men c ngha quan trng trong vic
thu hi sn phm. Nng cng cao, hiu qu thu hi cng tt v do vy, chi ph sn xut
s thp. Trong qu trnh ln men sn phm mc tiu c tch ly nhiu ng ngha vi
vic lm gia tng p sut thm thu trong dch nui cy v y l yu t bt li i vi
qu trnh trao i cht v sinh tng hp sn phm ln men chng vi sinh.
Tc sn xut l lng sn phm sinh tng hp c (trong dch ln men) trong
mt n v thi gian. Nu tc sn xut cao, sn lng ln men ln v nh th, cng
sut sn xut ca h thng tng ln, lm gim chi ph u t, gim chi ph c nh.
Hiu sut chuyn i c cht thnh sn phm mc tiu c ngha quan trng trong
c cu gi thnh sn xut, c bit l i vi cc c cht mc tin. Khi hiu sut ln men
cao, nh mc tiu hao nguyn liu gim v nh vy s gp phn lm gim chi ph sn
xut.
Kt qu ca qu trnh ln men chu s chi phi ca nhiu yu t bao gm (1) kh
nng sn xut ca chng ging s dng; (2) h thng thit b ca qui trnh ln men c
bit l cc h thng lin quan trc tip n kim sot vn hnh cc iu kin ln men v
(3) kim sot cc iu kin thch hp cho qu trnh ln men. Do vy, ti u ha hiu
qu ln men, cn thc hin ng b cc yu t nu trn.

1. Ci tin ging
Cng vic ci tin bao gm vic thay i mt s c tnh ca chng nh: tng
cng kh nng sinh tng hp mt sn phm mong mun, hn ch kh nng tng hp cc
sn phm ph, tng cng tnh n nh ca chng trong qu trnh sn xut, tng tnh
chng chu vi cc yu t bt li ca mi trng nh nhit , nng c cht hoc sn
phm, hm lng oxygen thp, tnh khng nhim tt, tng cng hiu sut chuyn i c
cht, (xem thm v cc c ch iu ha biu hin gen). Vic ci tin ging c thc
hin trong cc phng th nghim nghin cu ln c y phng tin thit b, c s vt
cht do vy chng ging khi a vo s dng qui m sn xut thng mi thng l
nhng chng c nh gi tnh n nh v bo m hiu qu sn xut cao.

Bi thc hnh:
Hy nu phng php tin hnh nh gi n nh ca chng trc khi a vo
sn xut. Tnh hung: khi nhn c mt ng ging gc vi yu cu: hy nh gi n
nh ca chng.
Cc bc hoch nh nh gi:
- Cn thng tin khong thi gian d tnh s dng cho sn xut. V d d nh s
dng ng ging ny cho sn xut trong 3 nm.
79
- nh gi n nh theo chiu ngang. T cng mt th h ging, tnh n nh
ca kt qu ln men nh th no.
- nh gi s n nh theo chiu dc: qua nhiu th h nhn ging, kt qu sn
xut c cn c n nh hay khng.

2. Ci tin thit b
- Tng cng oxygen ha tan trong dch ln men
+ Tng lng khng kh sc vo bn
+ Ci tin h thng phn phi kh (air sparger) theo hng chia nh dng
kh tip xc vi dch ln men
+ Tng p sut vn hnh ca ni ln men
- Tng cng ng nht ca dch ln men trong sut qu trnh nui cy
+ Tng tc khuy trn
+ Tng chiu di cnh khuy
- Tng cng tnh chnh xc ca cc thit b cm bin o cc ch s sinh l kim
sot qu trnh ln men nh pH, nhit nhm lm gim bin dao ng so vi
gi tr ci t theo yu cu.
- Tng t ng ha ca thit b nhm gim thiu nhng sai st k thut do vn
hnh.

3. Tng cng cc iu kin thch hp trong qu trnh nui cy
Xt mt v d v kt qu theo di qu trnh ln men trong bn trng hp trn
Hnh 7. 1. Trong trng hp ln men bnh thng, tng trng ca vi sinh vt trong ni
ln men tri qua cc 4 giai on nh m t chng 2. Trong lag phase tng i
di v trong pha log tc tng trng thp ( thp). Pha n nh rt ngn, sau qu
trnh ln men i vo pha suy vong (Trng hp 1).
Trong chng 2, chng ta cp n s ph thuc ca tc sn xut c
trng () vo tc tng trng c trng () v hiu sut chuyn i c cht ca chng
vi sinh vt nui cy.
= .Y
p/x
M =
t X
P
.

Trong , P l lng sn phm, X l sinh khi, t l thi gian nui cy.
Nh vy, P = .X.Y
p/x
.t
Mt khc: X = Y
x/s
.(S
o
- S
r
)
Do vy: P = .Y
x/s
.Y
p/x
.(S
o
- S
r
).t
Nh vy, P ph thuc vo (X), S
r
v thi gian nui cy. C th biu din s ph
thuc ny bng phng trnh:
P =
}
dx.ds
r
.dt
Sn lng P l phn din tch gii hn bi 3 hm s: ng cong tng trng, hm
lng c cht gii hn (ng) cn li v thi gian nui cy (phn mu vng Hnh 7.1).


Hnh 7.

T
v log p
sm hn
(P + P1)
T
trnh ln
T
dch ma
nhng t
men, sn
thc hi
thnh
trng h
T
men tron
1
2
3
trnh nu
chuyn c
1. ng h
Trong trn
ha ( dn
n Trng h
), tt hn tro
Trong trn
n men (pha
Trng hp
au t n
ng thi gia
n lng thu
n phng
nn khng
hp 3.
T trng h
ng 1 m sn
. Tng
. Duy tr ho
. B sung c
ui cy (lm
cc sn ph
c tng tr
g hp c s
n
max
, Tr
p 1. Lc n
ong Trng
g 3, c s t
n nh
4: Lng
gi tr cn
an nui cy
u c trong
n ny gp m
g c p d
hp 2 v 3 t
n xut, chn

max

ot tnh t b
cc cht tc
m tng c
m ny ra kh
ng ca vi s
tc ng l
rng hp
ny, sn l
g hp 1.
tc ng lm
c ko di),
ging np
thit cho s
y ca m kh
g trng h
mt s kh
ng nhiu.
trong v d
ng ta cn qu
bo trong th
c ng cho t
ng kh nn
hi mng t
inh vt tron
lm cho ch
2), qu trn
ng thu
m cho nng
, lc , sn
vo ni l
sn xut, n
hng thay
p ny l (P
khn v h
Chng ta t
trn cho th
uan tm m
i gian di,
tng mc
ng sinh tn
bo)
ng cc iu
ng tng tr
nh ln men
c khi kt th
g t bo
n lng ln
n men tng
ngha l th
i, kt qu
P + P1 + P2
thng nh
p trung qu
hy tng
t s vn cn
, duy tr
ch tng
ng hp sn
u kin ln m
ng tt h
t nng
hc qu trn
c duy tr
n men s l (
g ln, mt
i lng s
l tng
2 + Pa). Tro
n ging nh
uan tm v t
g c ln
n quan tm
c
giai on c
phm cn

en khc nha
n trong lag
t bo c
nh ln men
r trong su
(P+P1+P2)
t bo t
n xut tng
c sn ln
ong thc t,
h cng sut
trng hp
ng sn phm
:
c th trong
ng nh vic
80
au.
g pha
c i
s l
t qu
.
trong
g ln
g ln
, vic
t, gi
2 v
m ln
g qu
c vn
81
4. Gim thiu s suy gim s lng t bo do chit dch ln men trong qu trnh
nui cy.

1. Tng
max

tng cng tc tng trng c trng ca chng trong qu trnh nui cy
ngi ta thng thc hin mt s bin php sau:
- Bo m cc iu kin sinh pH, nhit mi trng ti u trc khi np ging
bt u nui cy. Trong qu trnh nui cy phi duy tr cc yu ny v phi cung cp
lng oxy ha tan cn thit, c bit l gi tr pH. Trong qu trnh ln men, pH ca mi
trng thay i do s suy gim nng cc cht dinh dng ng thi c s hnh thnh
cc sn phm trao i cht. Do vy cn phi thng xuyn hiu chnh pH mi trng
bng cch b sung cc cht H
+
/OH
-
. Tuy nhin ch nn b sung lng nh trnh s
thay i pH qu ln ngay sau khi b sung.
- Bo m khng tn ti cc cht gy c ch tng trng trong mi trng. V d
do hin tng qu nhit trong qu trnh thanh trng mi trng nn lm xut cc thnh
phn gy c ch nhng sn phm ca qu trnh caramen ha.
- Hm lng cc khong vi lng (C, N, P, Mn, Mg, Fe, Vit) phi c cung
cp y cho vic tng hp t bo v duy tr hot tnh ca h enzyme. Cc thnh phn
khong vi lng ny thng tn ti t nhin trong cc loi nguyn liu nh mt r ng,
cao ng Tuy nhin cc thnh phn ny c nhiu bin ng ty theo tng vng/ma
canh tc, tng qui trnh ch bin hay tng m sn xut xut. Do vy, cn phi tnh ton b
sung mt lng khong thch hp di dng tinh khit bo m cung cp y theo
nhu cu vi sinh vt.
- Hn ch p sut thm thu trong mi trng nui cy bng cch gim thiu nng
cc cht gy p sut thm thu cao nh mui khong, ng
- B sung cc ngun c cht cn thit cho vic tng hp t bo m chng ang
nui cy hoc khng th hoc tng hp vi tc rt chm. V d nh ngun amino
acids khng thay th, cc cht tin thn nh PABA, folic acid

2. Duy tr hot tnh ca chng qu trnh nui cy
- Bo m tnh n nh ca cc yu t sinh l trong sut qu trnh nui cy (nhit
, pH khng dao ng nhiu so vi gi tr ti u)
- Hn ch vic tng hp cc sn phm gy c ch tnh n nh ca chng (v d
khi thiu oxygen, hm lng lactic acid tng cao gy nh hng n hot tnh ca vi sinh
vt)
- B sung thm cc cht dinh dng b thiu ht
- Gim p sut thm thu ca mi trng nui cy

3. B sung cc cht tc ng
- Gip tng cng kh nng tng trng (trng hp tng
max
)
- Gip tng cng kh nng sn xut, tng hiu sut. V d b sung biotin trong
qu trnh nui cy cc chng sinh tng hp glutamic acid.
82
- Gip tng cng s bi tit sn phm sinh tng hp ra mi trng nui cy. V
d b sung betaine lm yu cu trc mng, tng cng tnh thm ca mng, gim p sut
thm thu ca mi trng nui cy.

4. Gim thiu s suy gim s lng t bo do chit dch ln men trong qu trnh
nui cy.
Trong qu trnh ln men, c bit l i vi ln men lin tc hoc ln men m-b
sung thay i th tch, do phi b sung cht dinh dng lin tc nn th tch dch ln men
trong ni tng ln. n mt gii hn nht nh, mt phn dch ln men c chit ra
ngoi tin hnh thu sn phm. Trong nhiu trng hp, lng sinh khi t bo trong
dch ln men ny khng c hi lu li nn lm tht thot mt lng ln sinh khi.
iu ny lm gim hiu sut ln men do tn ngun dinh dng, c bit l ngun
carbon, tng hp t bo nhng cc t bo ny khng tham gia sn xut ht thi gian
ln men v do vy sn lng men cng b suy gim ng k. ci thin tnh trng ny
cn phi nghin cu cn i t l chit dch thch hp, c bit l phi tng nng mi
trng dinh dng b sung gim thiu t l chit dch, tng nng sn phm trong
dch ln men. Tuy nhin cn lu l phi kim sot c p sut thm thu thch hp
trong mi trng ln men bo m l khng nh hng n hot tnh ca vi sinh vt.

p sut thm thu:
L p lc gy nn bi s chuyn ng dung mi t dung dch c m c thp
n dung dch c m c cao hn qua mng bn thm.


Hnh 7. 2. M hnh s to thnh p sut thm thu.
Cch tnh p sut thm thu ():
= iMRT
Trong , M: nng phn t gam ca cht tan
R: hng s kh, R = 0,08206 L.atm/K.mol
T: nhit tuyt i ( K), (T = t + 273)
i: hng s van 't Hoff, s lng ion phn ly trong dung dch
i vi cht khng in phn, i = 1; trong trng hp l cht in phn
mnh th i = tng s ion sinh ra t s phn ly ton b cc phn t trong dung dch.

V
io
=
th
si
th
p
th
th
s
tr
n
v

V d i v
on sinh ra tr
= (1 - a) + n
h i = (1 - 0,


p su
inh vt. M
hch cho gia
hm thn
hch hp v
h p sut th
gy nn h
rao i cht
nui cy vi s
vt sn xut





i NaCl, i =
rong trng
na. V d tro
,8) + 2.0,8 =
ut thm th
ng t bo
ai on tng
ng l khc
i p sut th
hm thu ca
hin tng t
t bn trong
sinh vt
hiu qu ca

= 2, CaCl
2
, i
g hp phn
ong trng
= 0,2 + 1,6
Hnh 7. 3.
u c vai tr
vi sinh vt
g trng to
nhau. Nhn
hm thu th
ao th tt h
t bo b m
t bo. Do
c xem l m
ao.
i = 3. i v
ly 100% v
hp ca CH
= 1,8.

. Thit b o
r rt quan
t l mt m
o sinh khi
n chung, tro
hp, trong k
n. Tuy nh
t nc, t b
vy, kim s
mt yu t
vi cht in
a l mc
H
3
COOH,

o p sut th
trng nh h
ng bn th
t bo v g
ong giai o
khi , i v
hin khi p
bo co li v
sot p sut
rt quan tr
n phn yu
phn ly t
gi s p
hm thu.
hng n
m. p su
giai on sin
on pht tr
vi giai o
sut thm t
v nh hn
t thm thu
rng nhm b
, nu gi n
thc t, lc
phn ly a =
hot tnh c
t thm th
nh tng hp
rin, vi sinh
n to sn p
thu qu ca
ng n qu
u trong qu
bo m vi
83
l s
, i
80%
a vi
u ti
p sn
h vt
phm
ao th
trnh
trnh
i sinh
84
CHNG 8. NHIM TP KHUN V NGUYN TC PHNG
CHNG TP NHIM

1. Hu qu ca s nhim tp khun
S nhim tp khun l mt trong nhng vn gy nhiu tr ngi i vi nui cy
vi sinh v chi ph x l mi trng sau khi b tp nhim. Vi khun tp nhim hin din
trong ni ln men s s dng cc thnh phn dinh dng ca mi trng pht trin
gy nn s thiu ht ngun dinh dng nui cy chng ang sn xut, dn n lm
suy gim hiu sut v sn lng ln men. Mt khc, chng tp nhim to ra cc sn phm
khng mong mun trong mi trng, gy c ch cho chng ang nui cy hoc gy tr
ngi cho qui trnh thu hi sau ln men, lm tng hm lng tp cht trong sn phm.
Trong trng hp tp nhim thc khun th (phage), ton b t bo ca chng
ang nui cy c th b v ra trong thi gian ngn nn khng th tip tc qu trnh ln
men. Cn phi dng h thng, tin hnh x l trit trc khi bt u hot ng tr li.

2. Pht hin tp nhim
Trong iu kin ln men sn xut bnh thng, qu trnh ln men c lp i lp
li v do vy, ng hc tng trng ca chng v cc thng s k thut khc nh nht
ca dch ln men c phn tch nh gi v ghi nhn. Trong trng hp xy ra tp
nhim, do s tn ti v pht trin ca chng tp nhim nn s gy ra nhng bin ng bt
thng trong qu trnh nui cy, chnh nhng thay i ny s l c s cho vic pht hin
tp nhim.
1. Thng qua vic theo di gi tr OD trong qu trnh ln men. Khi c s hin ca
chng tp nhim trong ni ln men, do l chng hoang di nn trong iu kin thch hp,
chng tp nhim ny s pht trin tng s lng t bo rt nhanh. Do vy khi theo di
nui cy nu gi tr OD tng bt thng th c th h thng ln men b nhim tp
khun. Trng hp OD gim bt thng th c th b nhim thc khun th do thc
khun th tn cng lm v t bo ko theo s suy gim s lng t bo rt nhanh.
2. Dng knh hin vi quan st. Nhn din s khc bit v c im hnh thi ca
tp khun: hnh dng, kch thc t bo, kh nng di ng, to bo t
3. Kim tra trn a pettri cha mi trng dinh dng tng hp: xut hin khun
lc l (c im hnh thi). Cn lu l phi nhng nhit khc nhau pht hin
khun lc, thng thng t 33-39
o
C. Trong thc t, ngi ta dng mi trng dinh dng
c trng (khuyt dng) cho chng nui cy khng mc c. Khi c khun lc xut
hin, c th b tp nhim.
4. Trong qu trnh nui cy, ly mu dch nui cy o nht, nu nht tng
ln t ngt, c kh nng b nhim. Trong mt trng hp nu b tp nhim nng, tnh
cht ca dch nui cy c th biu hin bt thng nh mu, mi.
Trong mt s trng hp nht nh, pht hin tp nhim, cn ly mu tch
ring nui cy qui m v cc iu kin khc nhau.
85
3. Nguyn nhn tp nhim
1. Do thit b
- Thit k h thng thit b khng hp l, cu trc thit b khng ph hp, to nn
cc ti lm cho vic v sinh kh khn hoc to iu kin cho vic hnh thnh cc lp
cn-vy bm chc vo thnh thit b gy tr ngi cho vic v sinh-thanh trng.
- Thit b b r r gy nn s tip xc gia cc phn v trng v khng v trng
trong qu trnh ln men (r r ni ln men, ng ng, lc kh, thit b gia-gii nhit ).
- Cc thit b cm bin hin th cc thng s nh nhit thanh trng, thi gian
thanh trng, p sut thanh trng khng chnh xc: gi tr thc t nh hn gi tr hin
th. V d trong trng hp ci t nhit thanh trng hin th l 120
o
C nhng thc t
ch 110
o
C. Do nhit thanh trng khng t yu cu nn trong mt thi gian trng nht
nh s khng dit ht cc chng tp nhim trc khi a vo h thng ln men.
2. Do nguyn liu
Nguyn liu (bao gm ngun nc), c bit l cc nguyn liu th c ngun gc
t nhin cha vi sinh vt tp nhim vi mt rt cao, trong c c cc chng chu
nhit, to bo t hoc t bo c kch thc nh. Vic loi b ton b cc vi sinh vt
ny l rt kh khn. Mt khc, trong nguyn liu c cha nhiu cn b, tp cht lm
ni n nu tt cho vi sinh vt trnh c tc ng ca nhit khi thanh trng.
3. Do vn hnh
- Thanh trng thit b hoc mi trng khng ng theo nguyn tc c xc
nh.
- Khng bo m cc yu t v trng trong qu trnh thao tc vn hnh c bit
cc cng on np ging, ly mu, np liu
- V sinh, thanh trng thit b khng tt dn n vic nhim dy chuyn t m
nui cy trc .

4. Nguyn tc phng chng nhim trong ln men

C 3 nguyn tc cn bn
1. Khng st li (not remaining). V sinh hoc thanh trng hoc lc bo
m rng khng cn t bo vi sinh vt tp nhim no trong thit b, mi trng trc khi
nui cy.
2. Khng xm nhp vo (not entering). Bo m rng trong qu trnh vn hnh
khng c s xm nhim t bn ngoi vo mi trng hay thit b ang nui cy.
3. Khng ly lan (not spreading). Ngn chn khng cho vi sinh vt tp nhim
ly lan trong mi trng thao tc, hoc ngn chn s ly lan mt khi c tp nhim xy ra.

Thc hnh phng chng tp nhim
- Thng xuyn v sinh thit b trc khi thanh trng. Thng thng ngi ta
dng NaOH long 3~5% hoc HNO
3
long 0,5~1% ra, lm sch ton b cn b
(scale) cn li t m nui cy trc trong thit b ln men.
- Thng xuyn kim tra s r r ca thit b (ni ln men, van, ng ng, cc
mi hn ). Cc phng php kim tra nh np kh nn cao p vo thit b ri dng
86
nc x phng xt bn ngoi kim tra, hoc phng php dng ch th mu nh np
kh nn p sut 0,5-4,0 kgf/cm
2
c cha NH
3
vi nng thp vo thit b ri ph giy c
tm phenolphtalein bn ngoi thit b cc mi ni, ng hn nhn din s r r.
Khi c s r r, NH
3
t bn trong thit b s khuch tn ra v c gi li trn giy c tm
phenolphtalein lm giy i sang mu hng. i vi cc thit b trao i nhit b mt,
ngi ta pht hin cc vt nt bng cch dng cht ch th mu; gm 3 cht cn bn (b
kit): 1. Cht ty sch b mt; 2. Cht nn thm vo trong cc vt nt; 3. Cht hin th mu
khi phn ng vi cht th 2. Sau khi phun cht th 2 ln b mt thit b, dng vi kh lau
sch, sau phun cht th 3. v tr no c xut hin mu ch th, b nt hoc r
r.
- Thng xuyn kim tra chnh xc ca cc thit b o c nh thit b o nhit
, p sut, thi gian, lu lng bng cch so snh vi cc dng c o chun.
- Phi bo m vic vn hnh thanh trng ng qui trnh v cc iu kin thanh
trng thch hp.
- Trong mt s trng hp chng tp nhim chu nhit, c kh nng to bo t nn
rt kh x l. Nhm loi b trit cc bo t cn tn ti trong ni ln men cn s dng
phng n thanh trng-nui cy-thanh trng. Nguyn tc ca phng php ny l sau
khi thanh trng ni ln men, chun b mt mi trng tng hp c thnh phn dinh
dng n gin bao gm cc ngun dinh dng C, N, P, Mg, amino acid vi nng
thp, iu chnh pH 7,0-7,5, nhit 30-35
o
C v tin hnh khuy trn, sc kh trong vng
khong 3-4 gi. y l iu kin thch hp cc bo t ca chng tp nhim cn tn ti
trong ni s n (germination) thnh t bo sinh dng. Sau , lp tc x b ton b
dch nui cy ny v tin hnh thanh trng thit b ngay nhm dit b cc t bo tp
khun cn li trong ni ln men. Khi cn thit c th lp li qui trnh ny 2~3 ln.

5. Cch pht hin - nh gi ngun gc tp nhim
Vic pht hin tp nhim c th thng qua nhiu phng php nh trnh by
trn y tuy nhin trong nhiu trng hp cn phn tch phi hp ng thi nhiu
phng php m trong quan trng nht l kim tra tp nhim theo cch s dng a
pettri cha mi trng tng hp. Trong qu trnh nui cy, theo thi gian, cn ly mu v
tin hnh kim tra trn a petrri bng cch ht khong 1ml dch nui cy tri ln mt a
v nhit thch hp (~37-39
0
C), nu c khun lc l xut hin, c th hin tng tp
nhim xy ra. Ghi nhn s lng khun lc l ny v thc hin nh gi qua cc thng
s: hnh thi, s lng t bo, kh nng to bo t, c tnh di ng v thi gian th h.
Trn c s thi gian th h, s lng v loi t bo tp nhim ti thi im nh gi, ta c
th tnh c thi im tp nhim bt u xy ra ti trong trong qu trnh ln men, t
c th phn tch c kh nng ngun gy nhim l t u. Khi phn tch ngun gc tp
nhim cn tin hnh theo cc bc sau y.

1) C th do cn vi khun tp nhim tn ti trong thit b hoc/v mi trng m
nguyn nhn l do hot ng thanh trng khng trit (remaining).
+ Do khng nhit / thi gian thanh trng (thit b cm bin b h
hng, khng bo m chnh xc theo yu cu.
87
+ Do c s tn ti lp vy cn bm trong thnh thit b (scale), lm ni tr
n tt cho cc vi sinh vt tp nhim tn ti.
+ Do trong thit b/ mi trng c s hin din ca cc chng tp nhim
chu nhit nn khi thanh trng vi iu kin nh thng l th kh nguy c t bo
cc chng ny cn li l rt ln.
+ Do thit b thanh trng mi trng lin tc (thit b gia nhit s cp,
Hnh 5. 4) b r r dn n c s trn ln gia mi trng cha c thanh trng
(u vo) v mi trng c thanh trng (u ra).
+ Do mt t bo tp nhim trong cc thnh phn nguyn liu qu ln,
kh nng thanh trng trit gim.

2) C th ging c cha cc t bo tp nhim hoc hot ng thao tc np ging
gy nn tp nhim.
+ Do ngun ging np vo ni ln men b tp nhim bi qu trnh nhn
ging/ chun b ging.
+ Do hot ng np ging vo ni ln men khng ng thao tc gy nn tp
nhim.

3) C th do vi khun tp nhim thm nhp vo trong qu trnh nui cy m
nguyn nhn l do thit b b r r (van, mi hn ), mng lc (h thng lc kh) b h
hng hoc do thao tc vn hnh ca nhn vin k thut trong qu trnh ln men khng
ph hp (entering).
+ Thit b b r r: van, mi hn, roong m b mt cc v tr lin kt thit
b b b hoc b lng ra trong qu trnh vn hnh. c bit l thit b gii nhit b
r r s gy hin tng tp nhim nghim trng do ngun nc gii nhit cha rt
nhiu vi khun tp nhim c th thm nhm vo dch ln.
+ Mng lc khng kh cung cp cho ni ln men b h hng dn n vic
khng kh cha c lc trit c np vo ni ln men trong qu trnh nui
cy.
+ Hot ng vn hnh ca nhn vin k thut khng ng tun t cc thao
tc chun dn n vic dch/ khng kh bn ngoi hoc t cc qui trnh khng bo
m v trng thm nhp vo ni ln men trong qu trnh nui cy.

V d: Trong nui cy mm (nhn ging- seed cultivation) ln men sn xut lysine
vi th tch dch l 10KL, t l np ging l 0,002%. Theo thi gian ngi ta ghi nhn
c ti gi nui cy th 14 v 16 s t bo tp nhim ca cng mt loi xut hin trn
a pettri tng ng l 21 v 95. Hy nh gi thi im bt u xy ra tp nhim v kh
nng ngun gc tp nhim.
Ta c th tnh c t
g
=120/log
2
(95/21) =~55 pht (~0,918 gi).
Nh vy thi im m trong 1 ml dch nui cy c 1 t bo tp nhim l (c th
pht hin c): 14-0,918log
2
(21/1) = ~10 gi.
Ti thi im bt u nui cy, trong ni ln men c 10 10
6
/2
14/0,918
= 258 t bo.
Trn c s ny ta c th nhn xt v ngun tp nhim:
88
1. Tp nhim xut hin ngay t khi bt u nui cy.
2. Ngun gc ca tp nhim
- Khng phi t ngun ngun ging. V th tch ging np vo l 0,002%10 KL
= 200 ml. Do vy, trong dch ging np vo c 258/200 = 1,3 t bo / ml, tc l c hn 1
t bo tp nhim /1 ml dch ging mm. Vi t l ny chc chn s c pht hin trong
qu trnh kim tra khi kt thc giai on chun bi ging.
- Do s tp nhim l ca mt chng duy nht nn kh nng c th l do hot ng
thanh trng khng hiu qu. Khng phi do thit b thanh trng mi trng s cp b r r
v nu thit b ny b r r th c s trn ln ca mi trng thnh trng vi mi trng
cha thanh trng, m trong mi trng cha thanh trng th khng ch c mt chng gy
tp nhim. Khng phi do trong thnh phn nguyn liu c hm lng tp nhim tng s
cao m c th l do c chng chu nhit hin din trong h thng thit b hoc trong thnh
phn nguyn liu d ch vi mt thp. Do vy, cn c tin hnh cc hot ng kim tra
c thit b c lin quan n qu trnh chun b mi trng v qu trnh thanh trng thit b
nh:
+ Kim tra cc thit b cm bin nhit hin th c chnh xc hay khng.
+ Kim tra v v sinh cc lp vy cn bm vo thit b.
+ Kim tra li cc bc thao tc vn hnh ca nhn vin k thut.

6. Mt s im cn lu khi truy tm ngun gc gy tp nhim
Chng tp nhim thng gp trong qu trnh ln men l thuc h Bacillaceae vi
c im l di ng, to bo t, chu nhit, pht trin nhanh. Ngun gc c th t cc
nguyn liu th (raw materials) hoc trong cc thit b do thanh trng khng trit .
Nu chng tp nhim l thuc h Micrococcus th ngun gc c th t h thng
lc kh, h thng nc gii nhit.
Nu trong ni ln men tn ti t hai chng tp nhim tr ln vi s lng nhiu
v thi gian th h trung bnh ca cc chng tp nhim ngn th c th suy on l do
thit b b r r (h thng thanh trng, h thng lc kh, h thng gii nhit )
Cn nghin cu thm ng hc ca s tc ng nhit khi thanh trng i vi vi
sinh vt.
89
CHNG 9. THU HI V TINH CH SN PHM LN MEN

1. Gii thiu

Chit tch v tinh sch sn phm ln men l mt qui trnh rt phc tp, kh thc
hin v chi ph rt cao. Ngi ta c gng thu nhn sn phm c cht lng cao trong thi
gian ngn vi chi ph u t v chi ph vn hnh thp nht. Trong thc t, chi ph thu hi
sn phm ln men chim t 15% -70% tng gi thnh ca sn phm. Do vy, vic chn
la qui trnh cng ngh thu hi l rt quan trong v ty thuc vo c trng ca tng loi
sn phm.
Nu phn tch dch ln men ti thi im thu nhn th c th thy trong dch c
cha: sn phm mc tiu vi nng thp (nu l sn phm ngoi bo), t bo nguyn
vn, t bo b v, cc cht tan v khng tan ca mi trng nui cy cn li v cc sn
phm trao i cht khc. Sn phm cng c th l nm bn trong t bo, nhy cm vi
nhit hoc d dng b ph v cu trc do cc chng vi sinh vt tp nhim. Ton b nhng
yu t ny gp phn lm tng mc kh khn cho qu trnh thu hi. bo m cho
vic thu hi hoc tinh sch sn phm tt th tc vn hnh cng l yu t quan trng do
mt s sn phm c tnh nhy cm cao. Do vy qui trnh thit b phi ph hp bo m
rng vic thu hi sn phm c thc hin trong mt khong thi gian thch hp nht. S
chn la qui trnh thu hi da trn cc tiu ch sau:
1. Xc nh loi mi trng ln men (rn, bn rn hay mi trng lng)
2. Sn phm thu nhn l ni bo hay ngoi bo
3. Nng ca sn phm trong dch ln men
4. c tnh l - ha ca sn phm (gip cho vic chn la phng n tch)
5. Mc ch s dng ca sn phm
6. tinh khit thp nht c th chp nhn c
7. Tm quan trng ca c tnh sinh hc ca sn phm v ca dch ln men.
8. tp cht trong dch ln men
9. Gi c th trng ca sn phm

Mc ch chnh ca giai on u trong qu trnh thu hi sn phm ngoi bo l
loi b cc ht t rn c kch thc ln v t bo vi sinh vt. Thng thng ngi ta s
dng h thng thit b l my ly tm hoc my lc. Cng on tip theo l dch ln men
c phn on hoc c chit tch nh a vo h thng phn on chnh, c th l h
thng lc tinh, h thng o chuyn p sut thm thu, hp ph, trao i ion, lc gel, sc
k, chit tch h dch lng-dch lng, chit tch dch lng hai phase hoc ng t. Sau
cng, cc phn on c cha sn phm mc tiu s c tinh ch bng cch ng t phn
on v t c tinh sch cao th dng k thut sc k v kt tinh thu hi sn
90
phm. Vic thu hi mt s sn phm khc c th bin i mt vi cng on trong qui
trnh chnh ny (Hnh 9.1).
C th thay i mt s c tnh ca dch sau ln men qua c th vn hnh x l
nhanh hn vi mt qui trnh n gin hn nh mt s k thut sau:
1. Chn la chng vi sinh vt khng sinh tng hp cc cht mu hoc cc cht
trao i khng mun.
2. Thay i iu kin ln men nhm gim thiu s sn xut cc sn phm trao i
cht khng mong mun.
3. Xc nh chnh xc thi im thu hi thch hp nht.
4. Kim sot pH sau khi dng ln men
5. X l nhit sau khi dng ln men
6. B sung cc cht gy kt t
7. Dng enzymes ph thnh t bo

Cn phi lu rng ln men v thu hi sn phm l hai giai on lin tc ca mt
qu trnh v c s tc ng tng h. Darbyshire (1981) quan tm n vn ny
trong qui trnh thu nhn enzyme. Cc thng s lin quan bao gm thi gian thu hoch, s
sn xut cc phn t mu, lc ion v thnh phn mi trng nui cy. Lng ln dch ni
c cha enzyme cn phi c x l ngay sau khi thu nhn dch ln men, hiu sut thu
hi enzyme c th khng d on c. iu ny gy kh khn cho vic thit lp k
hoch thu hi. S sn xut ra cc cht mu gy tr ngi ln cho qui trnh thu hi do
chng lin kt vi resin ging nh cc enzyme. Cht ph bt cn li trong dch ln men
gy nh hng n h thng lc tinh hoc nha trao i ion c s dng trong cc giai
on ca qu trnh thu hi v tinh sch. Cn thc hin cc bc th nghim chn ra
loi cht ph bt no thch hp nht cho qu trnh ln men. Hm lng cc ion trong mi
trng sn xut c th qu cao dn n vic cn phi pha long dch ni sau ly tm vi
nc c kh khong trc khi a dch ni ny vo cng on x l. Cng thc
mi trng nui cy c quyt nh bi cc yu cu sn xut, nhng hm lng protein
trong cc mi trng phc tp nn c nh gi di gc thu hi sn phm enzyme
sau ln men. Quan im ny cng c ng tnh bi Topiwala v Khosrovi (1978),
khi ti s dng nc cho qui trnh ln men thu nhn sinh khi.
S thu hi v tinh sch nhiu hp cht khc nhau c th thc hin c bng
nhiu con ng khc nhau. Trong mt iu kin nht nh, vic chn phng n no
lin quan n vic so snh cc yu t c cp sau y:
1. Vn u t
2. Chi ph x l
3. Cc yu cu ca ton b qui trnh
91
4. Hiu sut thu hi
5. Cht lng thnh phm
6. Tnh sn c cc k thut cng ngh
7. S ph hp vi cc yu cu qui nh
8. Nhu cu x l cht thi
9. Qui trnh lin tc hay theo m
10. Tnh t ng
11. V sinh an ton cho nhn vin vn hnh

Vn chnh hin nay ang i mt trong cc qui trnh thu hi sn phm l vic
tinh sch cc phn t c hot tnh sinh hc trong qui m ln.



Hnh 9. 1. S qui trnh thu hi sn phm sau ln men.
92
2. Cc phng php x l thu hi sn phm ln men
2.1. Cc phng php tch t bo
T bo vi sinh v cc nguyn liu khng tan c th c tch ra ngay khi thu nhn
dch ln men bng cch lc hoc ly tm. Do kch thc ca t bo vi sinh rt nh nn c
th dng thm cc h thng h tr nhm tng cng tc lc. C nhiu phng php
khc nhau c s dng tch loi t bo ra khi dch ln men.
a. Phng php to bt
L phng php phn tch da trn s khc nhau v hot ng b mt ca cc
cht. T bo s c li ko theo trn b mt cc bt kh i ra khi pha lng nn s phn
tch vi pha lng gip d dng loi b.


Hnh 9.2. S h thng tch t bo bng phng php to bt.

b. Phng php lng ta
L phng php da trn s kt lng do trng lc ca cht rn trong mi trng
nc. Phng php ny thng c s dng nhiu trong phng php thu nhn protein.
y l mt qu trnh c hc phn tch mt hn hp khng ng nht bng trng lc.
tng cng tc lng ta ngi ta thng dng mt s phng php thc y qu
trnh lng:
93
+ Phng php to keo t
- Thay i pH (nu khng nh hng n cc bc tip theo)
- B sung cc cht c kh nng lm ng t (mui st hoc mui nhm)
- B sung cc loi keo c in tch nghch
+ Gia nhit dung dch ln men
c. Phng php ly tm
Nguyn tc ca phng php ly tm l da vo trng lng ca t bo. Lc ly tm
cng ln, vic tch loi t bo ra khi dch ln men cng d. Lc ly tm ph thuc vo
tc quay ca rotor (rpm) v bn knh thng quay theo phng trnh:
C = m.w
2
/R
Trong m: khi lng, kg;
w: tc thng quay, m/s
R: bn knh thng quay, m
d. Lc
Nguyn tc ca phng php ny l da vo kch thc ca t bo trong dch ln
men. Lc l phng php phn ring hn hp khng ng nht qua lp lc, b c gi
li trn lp lc, dung dch i xuyn qua lc di mt p sut d so vi p sut bn di
vt ngn.
Tc lc (Darcy v Poiseuille):
U = dV/dt = N.A.( DeltaP. Pi. D
4
)/L. 128m,
Trong : U: tc chy, A: din tch ca phim lc, N: s ng mao qun trn mt
n v din tch; Delta P: Hiu s p sut trn phim; : nht ca dch lc; L: chiu su
ca phim lc; V: th tch.
Cc phng php lc thng c s dng bao gm:
+ Lc chn khng thng quay
+ Lc khun bn
+ Lc tip tuyn (crossfilteration)
Dch t bo khi chy ngang qua b mt lc s c mt p lc to thnh dng chy
vung gc gip cho phn dch i qua mng cn cc phn t s tip tc di chuyn ti gim
thiu kh nng b tc nghn, tc lc cao. Tuy vy, s tc nghn vn xy ra v cc
mng lc vn phi ra bng cc tia nc.

men k
mn bn
phin l
cht tr
tn nh.
C
(mng)
ly dn d
C
1. C
C
Hnh 9.
tng c
ko di chu
ng cch lm
c v tng
lc thng
Cch s dn
lc to thn
dn i trong
Cc kiu phi
Cc loi phi
Cc loi ny
Lc ti
2. S th
ng hiu qu
u k vn h
m ng k
xp ca c
c dng
ng: Trn v
nh mt lp
g qu trnh
im lc:
im lc p:
y c th kh
ip tuyn
hit b lc ti
u lc, ng
nh ca lc.
nh tng
cc ht n
g l t infu
o vi cht d
tng i
lc gi c
thng dn
trng bng
ip tuyn s

i ta thn
Chc nng
i ln ca
ng cn trn p
usoa hay Ki
dng lc
mt ngo
cho lp ny
ng cc loi
g nhit c

dng tron
ng b sung
g ca cht t
a cc l xp
phin nh h
ieselgua, am
c, lm cho c
oi tang trn
y c chiu d
bng gang
cao
ng cng ngh
g cht tr l
tr lc l gi
p trong vt
n hp vi
miang tn m
cht tr lc
ng lc chn
dy c nh.
g hoc bng
hip.
c vo dch
i li cc h
t liu dng
cc ht .
mn, than,
c bm trn p
n khng, sa
.
g thp khn
94
h ln
t rt
g lm
. Cc
t st
phim
au
ng r.

2. C
tr
3. C
m
l
4
yu dng
5
chun sa

2.2. Ph
a
T
ph-n
sinh vt
tc ca t
p sut
chamber
bo v r
99%. T
p sut
lu dch


Cc phim l
rong vt liu
Cc phim l
mt. Ngi t
c v tng h
. Cc phu
g lc m
. Phim lc
au:
- T
- C
- C
- C
ng php p
. Phng p
1. p t
Thit b Mic
ng nht cc
nhm thu n
thit b ny
cao, c th
r (phng t
ra c th
y theo loi
l hai thng
h huyn ph
H
c a: gm
u lc nh g
c kim loi
ta chng m
hiu lc lc
lc chn k
i trng l
v trng: C
Tnh cht l
C kh nng
C th kh t
C kh nng
ph mng t
php vt l:
o p lc ca
crofluidizer
loi dung d
nhn dch h
y l dch t
n 40.00
ng tc)
n kch th
i t bo vi s
g s quan t
nhiu ln
Hnh 5.8. M
mt chng
giy amiang
bng nhn
mt s ln
c bng cch
khng hnh
n men.
Cc vt liu
hc cho ph
g lc nhanh
trng c b
g ti s dng
bo

ao
r (M-110EH
dch. Thit b
huyn ph c
bo sau thu
0 psi, sau
c ph b
c 74 micro
sinh m i
trng quyt
tng c
y ph t b
g a lm b
g hoc cellu
ng a mng
a ny vo t
b sung c
trng l nh
u lc bo
hp chu
bng hi n
g
H-30) c
b ny cn
c cha cc
u hoch t
c
ng kim c
ons, vi hiu
u kin vn
t nh hiu
ng hiu qu
o Microflu
ng khung k
ulose
g thp khn
trong mt t
c cht ph
hng thit b
m mc
c p sut k
c hoc h
s dng tr
c ng d
c sn phm
giai on l
a qua mt
ng, to s
u sut ph m
n hnh khc
sut ph m
ph mng

uidizer M-11
kim loi th
ng r, c rn
tr rng
gia thch h
b c lu l
v trng p
kh trng
i nng.
rong cng n
dng ph
m bn trong
ln men
thit b g
va p m
mng t b
c nhau. Tc
mng. Ng
g t bo.
10EH-30.
a mt co
nh nh trn
lm thnh p
p.
ng ln v
phi c cc
ngh lm h
mng t b
t bo. Ng
c bm nn
i l interra
mnh lm ch
o c th
c np dc
i ta thng
95
, bc
n mt
phim
ch
c tiu
huyn
o vi
guyn
n ln
ction
ho t
t n
ch v
g hi
96
2. ng lnh
3. Nghin
4. Siu m: Cung cp sng vi tn s cao ph t bo
c. Phng php ha hc:
1. Dng p sut thm thu
2. Dng cc tc nhn acid hoc base
3. Dng cht ph mng
4. Enzyme (sinh hc)
2.3. Cc phng php tinh sch sn phm
a. Phng php chit lng- lng
b. Phng php thm tch
c. Phng php ta bng mui
d. Sc k
Da vo tnh cht ca phn t cc cht
- Kch thc: Sc k lc gel
- Trng lc:
- Lc ht
1. Lc ion: Sc k trao i ion
2. Lc Val der Waal: Sc k hp ph
3. i lc: Sc k i lc: da vo i lc c hiu ca protein vi cc
ion gn trn ct.






















97
CHNG 10. X L CHT THI CA QUI TRNH LN MEN

1. Gii thiu v x l cht thi v cc phng php x l
1.1. Gii thiu
Tt c cc qui trnh ln men u s dng nguyn liu sn xut nhiu sn phm
mong mun khc nhau ng thi thi ra mt lng ln cht thi. Cht thi bao gm cc
hp cht v c, hp cht hu c, t bo vi sinh vt (chng nui cy) v nhiu cht rn l
lng khc. Ngi ta thng da vo cc thng s sau nh gi cht lng nc thi.
1. BOD (Biological Oxygen Demand)
2. COD (Chemical Oxygen Demand)
3. pH
4. Nhit
5. Hm lng nitrogen tng
6. Hm lng nitrogen ammonia
7. Hm lng cht rn l lng (TSS, total suspension solid)
8. Mi
9. Mu
10. Hm lng cc kim loi nng
11. Mt vt sinh vt gy bnh

Ty theo thnh phn v hm lng cc cht trong cht thi m xy dng nhng
phng n x l khc nhau. Trc y, ngi ta thng x l theo cch t nhin nh
ti tiu cho mt khu vc ln rng trng i vi cht thi lng hoc chn di t i
vi cht thi rn. Do yu cu v mi trng ngy cng nghim ngt v lng cht thi
ngy cng ln nn hin nay ton b cht thi ca qui trnh ln men phi c kim sot
v x l cht ch.
Trc khi xy dng qui trnh x l cht thi cn phi hiu r tiu chun do a
phng qui nh v c tnh ca loi cht thi cn x l t xy dng phng cch x
l khc nhau.
C ba phng php thng c dng x l cht thi ca qui trnh ln men:
phng php vt l, phng php ha hc, phng php sinh hc. c tnh ca cc qui
trnh x l ny nh sau:
1. X l s cp: dng phng php vt l, phng php ho hc- lng, ng t
2. X l th cp: dng Phng php sinh hc (bn hot tnh) sau khi x l s
cp.
3. X l cp 3: dng phng php vt l, ha hc hoc sinh hc (chng hn dng
microstrainers, lc ct v dng ti c) ci thin cht lng ca dch thi t cc
giai on trc.
4. Bn hot tnh v x b; dng phng php vt l, ha hc v sinh hc. Dng
phng php x l hiu kh kim sot bn (lm cho bn thi tr nn d dng b kh
nc).

98

Hnh 10. 1. S qui trnh x l cht thi lng.
1.2. Cc phng php x l cht thi
2. 1. Phng php vt l
Vic tch b cc cht rn l lng bng trong nc thi bng phng php vt l
trc khi x l sinh hc s lm gim ng k gi tr BOD. i vi hu ht cc qui trnh
ln men, t bo vi sinh vt b tch khi dch thi trong qu trnh thu hi sn phm. Trong
mt s trng hp khi tch b t bo, ngi ta kt hp dng cht tr lc do vy c t bo
v cht tr lc u l cht thi, cht thu hi c th c thc hin theo hai cch cn bn
sau:
- Loi b cht thi m khng cn thng qua x l
99
- Lng cht thi c th s gim xung thng qua h thng kh nc nh lc
p, ly tm, lc quay chn khng Cht thi sau khi c p c th c x l bng
phng n chn di t.
Cht thi rn c sinh ra trong mt s qui trnh trc khi cy ging. i vi
trng hp ca nh my sn xut bia, mt lng rt ln cht thi t ng cc, malt sau khi
s dng c loi ra khi qui trnh. Theo tnh ton c 180L bia c sn xut th c 5kg
cht thi rn dng ny c thi ra. Cc qui trnh x l s cp theo phng php vt l
bao gm cc cng on sau:
a. Lc tch b phn t l lng v ni trong dch
b. Nghin nh gim kch thc cc ht
c. Thi qua cc knh vi tc khng i (0,3 m/s) loi b cc ht ct sn
phng nga s gy hi ln cy trng (khi dng cho bn cy) hoc cc qui trnh sau .
d. Dng cc bn lng tch b nhng ht t l lng nh. H thng ny c
thit k vn hnh lin tc vi thi gian lu dch khong t 6-15 gi. H thng cc bn
lng ny c th tch ti 70% cc cht rn l lng v ph thuc vo c tnh ca cht thi,
c th tch n 40% hm lng BOD. Trong mt s trng hp c th dng thm cht
tr lng. Trong giai on x l bc ba, Phng php vt l c dng lc cc cht cn
li, nhm nng cao cht lng cht thi. Mt s loi lc c th c dng l:
microstraines, lc ct chy chm, lc ct ngc dng tc lc thay i t
3m
3
/m
2
/ngy i vi lc ct chm v t n 700m
3
/m
2
/ngy i vi dng lc
microstrainers. Thng qua phng n ny c th tch b ti 50-70% cht l lng v
khong 30-50% BOD.

2. 2. Phng php ha hc
Cc phn t l lng kch thc nh c th b lng t khi dng cc cht tr lng
thng dng nh Fe(SO
4
), Fe
2
(SO
4
)
3
, Al
2
(SO
4
)
3
, Ca(OH)
2
, v cc cht in gii polymer.
Chun b cht tr lng vi nng thch hp trong mt bn sau b sung vo qui trnh
x l nhm gy lng t cc ht t kch thc nh ang l lng trong dch cht thi. Bn
lng ny sau c kh nc v a vo giai on x l sau. Do kch thc ca cc ht
t ny nh nn i hi thi gian lng t di hn, vy nn cn phi thit k h thng vi
mt lot cc bn c kch thc ln.

2. 3. Phng php sinh hc
S dng vi sinh vt (vi khun) phn hy sinh ha cc hp cht hu c, bin cc
hp cht c kh nng phn hy thnh nhng cht n nh, sn phm cui cng l CO
2
,
nc v cc cht v c khc. Hu ht cc cht thi hu c u b phn hy sinh hc din
ra trong iu kin hiu kh hoc/v k kh.

1. Phng php x l hiu kh
- Dng h thng mng film sinh hc vi dng chy nh git (trickling filters).
Sinh khi sinh vt trn mng film s dng ngun oxygen t khng kh thi t di ln v
x l cht thi khi i qua b mt mng film t trn xung lm gim BOD. Mc ch ca
h thng ny l tng cng b mt tip xc ca mng biofilm vi khng kh v dng cht

thi. The
khi np t

Hnh 10
-
nh c
khng
nhiu m
bng nhi
nn to
m
2
/m
3
,
50% v
-
php 1).
di ln
v lc n
u t n
-
-
hnh cu
vn hnh
eo cch ny
tc dch
. 2. H thn
-Dng h th
din tch
p ng
mt bng v
iu module
c th
ng thi c
c th gim
H thng l
Phng ph
n to iu ki
y cho php
nhiu bn l
H thng
H thng c
u lm bng
h vi ti BO
y c th lm
thi 0,06 -
ng mng sin
hng thp.
c trng v
c yu cu
cc h lu
a din b
ng thong
c th lm
m n 70% v
c hiu kh
hp ny bao
in cho vi s
p lm gim
ng
a quay tip
cc trng qu
plastic
OD 6 kg/m
2
m gim t 7
0,12 kg BO
nh hc x l
Do phng
khong k
x l ln
u cha. H
ng plastic n
tt (90-98%
gim BOD
vi ti BOD
sinh hc. P
o gm cc b
sinh vt ph
BOD v N
p xc sinh h
uay. Mt h
c kt li v
2
/ngy.
75-95% gi
OD/m
3
/ngy
nc thi
g php dn
khng thn
ng ln cht
thng thp
nh c th g
%) v din t
D tt. Vi t
D 1,5 kg/m
3
Pht trin tr
b cha c
t trin tt.
NH
3
cng v
hc
thng dn
i nhau. D
tr BOD v
y.
bng ph
ng mng film
ng thong n
thi. Khi m
p vi chiu
gii quyt v
tch b mt
ti BOD 3,
3
/ngy.
rn c s c
h thng k
S kt hp
i cht thi
ng trng qua
ng ny t
v 90-95% c
ng php hi
m sinh hc
nh do vy
m rng qu
u cao khon
vn trn.
c trng
2 kg/m
3
/ng
a biofilm r
kh xuyn q
p c qui trn
rn do vy
ay ln do c
ng t nh d
cht rn l
iu kh.
c vi dng
cng sut
ui trnh ph
ng t 6-9m
Do cu tr
t n 100
gy c th
reactor (ph
qua theo dn
h x l hiu
khng cn
c ng hoc
dng a, c
100
lng

chy
thp
i tn
m lm
c m
0-300
gim
ng
ng t
u kh
n phi
c qu
th

-
trn c
cao.
-
khuy tr
cc phn
cc cht
pht tri
trong n
trong H
+
+
+
+
+

Tuy nhi
kh chu

H
H thng b
c mng film
H thng b
rn cht th
n t hu c
t hu c h
n, thng
c thi ch
nh 10. 3 ba
+ Khuy trn
+ Sc kh k
+ Lm trong
+ Tun hon
+ X bn d
u im c
n thnh ph
u, cn phi c
Hnh 10. 3. Q
h
b dch.
m sinh hc
bn hot t
i trong khi
dng ht tr
a tan v c
c gi l
huyn ha c
ao gm:
n nc thi
t hp vi k
g nc v t
n li lng b
v x l b
a phng p
hn rn l l
c bin ph
Qui trnh x
hot tnh.
y l mt ph
vi lng
nh. H th
c s hin
rong dch g
c cht l l
bn hot t
chng thnh
i cn x l v
khuy trn
ch bn ho
bn cn thi
n
php ny l
ng tn ti
p x l thc
l cht th
hng php
sinh khi l
ng ny bao
din ca vi
i l bn ho
ng lm n
tnh. Vi sin
h cc cht
vi bn ho
cung cp
t tnh ra kh
t t y b
c th x
sau qu trn
ch hp.
hi bng ph
p mi. Gi t
n cho php
o gm ngu
i sinh vt x
ot tnh. N
i khu tr c
nh vt hp t
tr. Qui tr
t tnh
p oxy cho qu
hi hn hp
lng t 2
l c 80
nh cao (bn
hng php
th c din
p x l ch
n cung c
l dng
c thi t
a vi khun
th v oxy
rnh hot
u trnh sin
p bng b l
2 vo b hi
0-90% thn
n hot tnh)
hiu kh qu
tch b m
t thi vi t
p khng kh
g huyn ph
b lng c
n, gip vi k
ha cc ch
ng theo s
nh ha
ng 2
u kh
nh phn hu
) c th gy
ua h thng
101
t ln
c
h v
trn
cha
khun
ht c
s
u c.
y mi

g bn
102
2. Phng php x l ym kh
X l ym kh l qu trnh sinh hc phn hy cc cht hu c trong nc thi
trong iu kin ym kh. Qu trnh ny sinh ra kh CH
4
, CO
2
, NH
3
v cc sn phm v c
khc.
X l ym kh c thc hin nhm gii quyt cht thi hu c bao gm mt s l
do sau:
- Tng cng kh nng x l trong giai on hiu kh
- Gim nh mc s dng in nng trn cng mt n v BOD
- Sn phm cui ca qu trnh x l ny l hu ch nh bn tiu hy c
- Cc cht hu c c c nh theo dng n nh
- C s bin i c tnh kt hp nc lm cho bn d b kh nc hn
- Lng vi sinh vt trong bn hot tnh gim do vy d kim sot bn hn.
- Mc pht trin ca vi sinh vt thp do vy khng cn b sung cc cht dinh
dng thiu trong nc thi.
Qu trnh chuyn ha trong giai on x l ym kh bao gm:
+ Qu trnh phn hy (hydrolysis): thy phn nhng cht c phn t lng cao
thnh cc monomer v polymer c phn t lng thp hn nh h enzyme trong t bo vi
sinh vt.
+ Qu trnh acid ha (Acidogenesis): vi sinh vt ln men cc sn phm ca qu
trnh thy phn thnh cc cht d bay hi nh acetic acid, formic acid, propionic acid,
ru, NH
3
, CO
2
, H
2

+ Qu trnh acetate ha (Acetogenesis): chuyn ha cc sn phm ca qu trnh
acid ha thnh acetic acid thun li trong vic methane ha.
+ Qu trnh methane ha (Methanogenesis): phn hy cc cht n gin ca giai
on trc thnh CH
4
v CO
2
.

2. Tip cn x l cht thi ca qui trnh ln men cng nghip
V d trong trng hp qui trnh ln men sn xut amino acid phc v cho lnh
vc thc phm. Cc nguyn liu chnh s dng c ngun gc t nhin nh tinh bt khoai
m ( ng ha thnh glucose), mt r ma ng v nguyn liu ph nh MgSO
4
,
urea, H
3
PO
4
, cc vitamin. iu cn lu l trong qui trnh ln men, c bit l ln men
trong lnh vc thc phm ton b ngun nguyn liu a vo s dng u an ton khng
cha cc cht c hi v t tiu chun thc phm (food grade). Do vy, tt c cc ngun
pht thi (nu c) th trong thnh phn ca cht thi khng tn ti cc cht gy c hi (v
d nh kim loi nng, cyanua).
1. Lit k v phn loi cc cht thi pht sinh trong qu trnh ln men
1. Cht thi rn: Nguyn liu rn ri vi, cc v bao b cha nguyn liu (bao giy,
bao nylon...)
2. Cht thi lng: Cc nguyn liu dng lng b r r, dch mi trng sau ln men
c thu hi sn phm bao gm c sinh khi t bo vi sinh vt, cc loi dch NaOH,
H
2
SO
4
long dng v sinh thit b
2. Xc nh tiu chun cn x l
X l cht thi t theo tiu chun qui nh ca Vit nam
103
3. Chn la phng php x l thch hp
1. i vi cht thi rn:
a. Cc nguyn liu rn ri vi: thu gom v pha long, tn dng
b. Cc bao b: phn loi v bn ph liu ti ch
2. i vi cht thi lng:
a. i vi nguyn liu dng lng b r r: x l r r; thu gom v a v
trm x l nc thi.
b. i vi dch ln men cha sinh khi t bo sau khi thu hi sn phm:
c th tn dng theo hai hng:
b1. Dng lm phn bn dng lng: B sung cc thnh phn dinh
dng nht nh cn thit cho cy trng, iu chnh pH v hm lng N, P, K ph hp ri
s dng bn cho cy trng.
b2. Dng lm ngun m cung cp cho ch bin thc n chn nui
gia sc: Tin hnh tch sinh khi t bo v sy gim m n mt mc nht nh.
Sn phm sinh khi t bo sau khi sau c s dng lm ngun nguyn liu qu cung cp
ch bin lm thc n cho chn nui gia sc hoc cho lnh vc nui trng thy sn.

3. Trit l khng pht thi
Vi khu hiu khng pht thi (Zero emission) trong qui trnh sn xut, hin nay
nhiu cng ty p dng chng trnh zero emmission mt mt nhm gim gnh nng x
l cht thi, khng thn thin vi mi trng, mt mt gip lm gim chi ph sn xut.
Cht thi pht sinh trong qui trnh sn xut suy cho cng l t chnh cc nguyn-
vt liu u vo do s dng khng hiu qu to ra. Lng cht thi pht sinh trong qui
trnh cng nhiu ng ngha vi ngun nguyn vt liu tht thot cng cao. Do , vic
gim thiu ngun pht thi gip tit kim c ngun nguyn vt liu v nh vy gip h
gi thnh sn xut ca sn phm. t c iu ny thng thng phi u t cho vic
ci tin cng ngh tng hiu qu sn xut.
Ngoi ra, trong mt s trng hp, cht thi pht sinh trong qu trnh cn phi tm
cch ti ch/ ti s dng va lm gim chi ph x l va to c gi tr gia tng cho
chnh cc cht thi ny.










104
CHNG 11. BI TP P DNG

1. Bi tp
Bi 1
Trong mt th nghim nui cy nm men thu sinh khi theo phng php m.
Theo yu cu, lng sinh khi thu c phi t 5 tn/m trong thi gian nui cy l 30
gi. Hiu sut tng hp sinh khi l 56%, th tch dch ln men l 100 KL, t l np
ging l 0,1% (t/KL), nng ng glucose cn li l 0,5 g/dl.
a. Lng ng cn thit a vo h thng thu nhn lng sinh khi trn?
b. Tnh trung bnh ca c qu trnh.
c. Vi nng ru o c sau qu trnh ln men l 3% (v/v), hy cho bit tc
sn xut ru c trng ca nm men trong th nghim trn (d EtOH: 0,789
g/ml).
Bi 2
Trong nui cy theo phng php fed-batch thu nhn sn phm l amino acid ca
mt chng vi khun, ngi ta thc hin trong ni ln men vi th tch mi trng ban
u l 50 KL, nng ng ban u trong mi trng 2,0 g/dl. Th tch ni ln men l
100 KL, vi cng sut ti hn l 80%, D trung bnh l 0,0375 hr
-1
. Lc ngng qui trnh,
nng sn phm l 3,6 g/dl, nng sinh khi l c
b
= 1,2 g/dl, hiu sut tng hp t bo
l 20%.
1. Thi gian np liu cho h thng?
2. Tnh nng ng trong dch np vo h thng.
3. Hy tnh hiu sut sinh sn phm?
Bi 3
Kt qu theo di tin trnh ln men thu nhn amino acid ca mt chng vi khun
theo phng php fed-batch c ghi nhn theo bng sau (Bng 11.1). Th tch dch ln
men khi bt u nui cy V
0
=20 KL, nng ng np b sung c
f
= 25 g/dl.
1. Hy nhn xt v tc tng trng c trng ca chng.
2. Nhn xt hiu sut sinh tng hp sn phm ca tin trnh (thc t).
3. Hiu sut t bo theo tng giai on nui cy, C nhn xt g?

Bng 11.1. S liu theo di qu trnh ln men
105


Ghi ch: CT: thi gian nui cy; c
b
: nng sinh khi; S
f
: lng ng np b
sung tch ly; c
p
: nng sn phm; s
r
: nng ng cn li trong mi trng
nui cy.

Bi 4
Trong nui cy sn xut bia vi th tch 30 KL, ngi ta ghi nhn c s tp nhim
xy ra v cc gi nui cy th 22 v 26 s lng t bo tp nhim tng ng l 23 v
481 trong 1 ml dch nui cy.
1. Hy tnh t
g
(thi gian th h) ca chng tp nhim trn
2. thi im no ca qu trnh nui cy, ngi ta bt u c kh nng pht hin
c tp nhim trn a pettri (1 ml).
3. Khi dng nui cy (gi th 30), c bao nhiu t bo tp nhim trong 1 ml dch.

Bi 5
Trong qui trnh ln men sn xut glutamic acid s dng glucose lm ngun C. C
1 mol glucose c s dng chuyn thnh glutamic acid th c 193,4 Kcal nhit lng
c phng thch ra mi trng nui cy, do vy cn dng nc gii nhit kim sot
nhit ca h thng. Bit rng nhit ngun nc gii nhit cp vo l 28
0
C, sau khi
thot ra khi h thng l 31
0
C, thit din trao i nhit l 20m
2
. Lng ng tiu th ti
thi im cao nht l 2 t/hr.
1. Hy tnh lu lng nc gii nhit ti thiu cn thit c th kim sot c
nhit nui cy (35
0
C).
2. Tnh hiu sut trao i nhit ca thit b



CT c
b
S
f
c
p
s
r
[Hr] [g/dl] [t] [g/L] [g/dl]
0 0.20 0.00 1.0 5.00
2 0.35 0.00 6.5 2.50
4 0.50 0.00 11.0 0.90
6 1.10 1.20 22.5 0.40
8 1.33 1.70 30.0 0.30
10 1.39 1.90 38.5 0.35
12 1.45 2.30 45.0 0.20
14 1.44 2.50 50.0 0.32
16 1.43 2.60 55.0 0.40
18 1.42 2.67 58.5 0.30
20 1.40 2.70 60.5 0.10
106
2. Bi gii

Bi 1
Tm tt:
Ln men phng php m; X = 5 t/B, Y
x/s
= 56%, V
inn
= 100 KL;
T l np ging I
s
=0,1 %, thi gian nui cy CT = 30 gi, s
r
= 0,5 g/dl.
1. Lng glucose cn
Do Y
x/s
= X/S
u, ,
trong S
u
l lng ng s dng
S
u
= X/Y
x/s
= 5/0,56 = 8,93 t.
S = S
u
+ S
r
= 8,93 + 0,5/100100 = 9,43 t.
2. Tc tng trng c trng:
Vi t l np ging 0,1 % (t/KL) cho ni ln men c th tch dch l 100
KL th lng sinh khi ging mm X
o
= 0,1/100100 = 0,1 tn.
= (lnX/X
0
)/t = (ln(5/0,1)/30 = 0,130 hr
-1

3. Lng ethanol tng hp c:
P = 3%100 KL0,789 g/ml = 2,367 t.
Y
p/x
= P/X = 2,367t/5t = 47,34%
Ta c tc sn xut ethanol c trng = Y
p/x

= 0,1300,4734 = 0,062t/t/hr.
Bi 2
Tm tt:
Fed-batch, V
o
= 50 KL, s
i
= 2,0 g/dl, V
fer
= 100 KL, f = 0,8; pha long trung
bnh D
aver
= 0,0375 hr
-1
, c
p
= 3,6 g/dl, x = 1,2 g/dl, Y
x/s
= 20%.

1. Thi gian np liu
V
inn
= 1000,8 = 80 KL,
V
f
= 80 50 = 30 KL,
D
aver
= F
aver
/ V
F = DV = 0,037580 = 3 KL/Hr,
Do vy, T
f
= 30/3 = 10 gi.

2. Nng ng feed
Tng ng feed: S
f
= S
u
-S
i
= (1,280/0,2-2,050)/100 = 3,8 t
c
f
= 3,8/30 = 12.67 g/dl

4. P = 3,6 80 = 2,88 t.
Y
p/s
= P/Su = 2,88/4,8 = 60%.

Bi 3
Tm tt:
Ln men theo phng php fed-batch; V
o
= 20 KL; c
f
= 25 g/dl.
V
inn
: Th tch dch trong ni ln men; V
inn
= V
o
+ V
f

V
f
: Th tch dch ng np b sung vo trong qu trnh nui cy; V
f
= S
f
/c
f

107
S
u
: Lng ng c s dng; S
u
= S
o
+ S
f
- S
r

S
o
: Lng ng trong mi trng lc bt u nui cy, S
o
= s
o
V
o
; s
o
= s
r
ti thi im
bt u nui cy.
S
r
: Lng ng cn li trong qu trnh nui cy; S
r
= s
r
V
inn

P: Lng sn phm thu c; P = c
p
V
inn

B: Lng sinh khi thu c; B = c
b
V
inn


Tc tng trng c trng = (lnB
t
- lnB
0
)/(t - 0)
Hiu sut sn phm ca tin trnh Y
p/s
= (P
t
-P
o
)/(Su
t
-Su
0
)*100
Hiu sut sinh khi theo tng giai on Yb/s
Hr
= (B
t
-B
t-2
)/(Su
t
-Su
t-2
)/(t-(t-2))*100

Bng 11. 2. Tnh ton nh gi kt qu ln men



CT c
b
S
f
c
p
s
r
V
inn
S
u
P B Yp/s Yb/s
Hr
[Hr] [g/dl] [t] [g/L] [g/dl] [KL] [t] [t] [t]
[hr
-1
]
[%] [%]
0 0.20 0.00 1.0 5.00 20.0 0.0 0.02 0.04 0.00
2 0.35 0.00 6.5 2.50 20.0 0.5 0.13 0.07 0.28 22.00 6.00
4 0.50 0.00 11.0 0.90 20.0 0.8 0.22 0.10 0.18 24.39 9.38
6 1.10 1.20 22.5 0.40 24.8 2.1 0.56 0.27 0.50 25.61 13.49
8 1.33 1.70 30.0 0.30 26.8 2.6 0.80 0.36 0.13 29.93 16.12
10 1.39 1.90 38.5 0.35 27.6 2.8 1.06 0.38 0.04 37.19 14.80
12 1.45 2.30 45.0 0.20 29.2 3.2 1.31 0.42 0.05 39.92 9.07
14 1.44 2.50 50.0 0.32 30.0 3.4 1.50 0.43 0.01 43.48 5.30
16 1.43 2.60 55.0 0.40 30.4 3.5 1.67 0.43 0.00 47.49 3.66
18 1.42 2.67 58.5 0.30 30.7 3.6 1.79 0.44 0.00 49.60 0.94
20 1.40 2.70 60.5 0.10 30.8 3.7 1.86 0.43 -0.01 50.24 -4.88

N
1
phase),
gi nui
t l t b
ln men
trc ti
2
kt thc
thy c t
3
cc i
sinh kh

Bi 4.
Tm tt:
1
n
2
S
c
Nhn xt:
. Tc t
t cc i
i cy th 16
bo mt i n
n l thu
ng th 16.
. Hiu sut
nui cy h
th nui cy
. Hiu sut
(15%) gi
i bt u gi
: V
o
= 30KL
. Ta c: N
cy 22 v
t gi nu
n =
t
g
= T/n,
nhim sinh r
t
g

. Kh nng
cy c ch
S th h t
y 22gi l
ng trng
gi nui
6 tr i, ch
nn tc t
sinh khi th
sinh tng h
iu sut vn
y di hn
sinh tng h
ai on t
im mnh
L, CT22 = 2
= N
o
2
n
, N
26 gi, n
ui cy th 2
= log
2
(N/N
o
trong tg
ra qua n th
= (26-22)
g pht hin
ha 1 t bo
bo tp nhi
:
c trng
cu th 10
ng khng t
tng trng
h nn dn
hp sn ph
n tip tc t
thu thm
hp t bo
gi th 7-1
u cho n
23 t bo, C
No, N l s
l s th h
22 n 26.
o
) = log
2
(48
g l thi gia
h.
60/4,4 = 55
n c tp n
o.
im tnh t

ca chng
sau gim
ng trng
g c trng
ng m ln m
m tng dn
ng tuy nhi
sn phm.
ban u th
10. Sau 10 g
n kt thc qu
CT26 = 481
t bo tp n
t bo tp
81/23) = 4,4
an th h, T
5 pht.
nhim trn
lc c kh
g tng mnh
m dn n 0
, t l t b
c gi tr
men ny k
n lin tc th
n mc
p sau t
gi nui c
qui trnh ln
t bo.
nhim ghi n
nhim sinh
4
T l thi l
a pettri kh
nng pht h
h trong gia
0 gi nu
o sinh ra (n
m. Nh vy
khong th
heo thi gia
tng chm
ng mnh
y, hiu su
n men.
nhn c
h ra trong k
ng cn thi
khi trong 1m
hin n l

ai on u
i cy th 16
nu c) thp
y, nu mc
i gian nu
an nui cy
m. iu ny
log pha v
t sinh tng
thi im
khong thi
it t bo
ml ca dch
c thi gian
108
u (lag
6. T
p hn
ch
i cy
. Khi
y cho
v t
g hp
nui
gian
o tp
nui
nui
109
N = log
2
(23/1) = 4,5;
Thi lng (T) t lc c 1 t bo tp nhim n gi nui cy thc 22:
T = 4,555/60 = 4 gi.
Nh vy lc 22-4 =18 gi th c kh nng pht hin tp nhim.
3. Khi dng nui cy thi im dng nui cy, gi th 30:
N
30
= N
22
.2
n
,
vi n = T/t
g
= (30-22)(60/55) = 8,73;
N
30
= 23.2
8,73
= 9.766 t bo.
Trong trng hp tnh t CT26: N
30
= 481.2
(30-26)60/55
= 9.905 t bo.
Ti sao c s khc bit ny?
Nguyn nhn c th l (1) sai s do cch ly mu; (2) t
g
ca chng tp nhim
khng phi khi no cng ging nhau trong qu trnh nui cy.

Bi 5.
Tm tt: T
1
= 28
o
C, T
2
= 31
o
C, T
3
= 35, A = 20 m
2
, S
f

max
= 2,0 t/Hr
Ta c phng trnh:
Glucose + [O] + [N] + [P] + CO
2
+ + 193,4 Kcal
MW ca glucose: 180 g

1. Nng lng sinh ra khi ng ha glucose tng hp glutamic acid thi im
tc ng ha ti a l:
Q
1
= 2.0/180193,4 = 2,148 Gcal/hr.
Mt khc ta c:
Q
2
= m.c.delT vi Q
2
l nhit lng cn phi gii phng khi ni ln men duy
tr nhit nui cy, m l khi lng nc gii nhit cn thit gii ht lng nhit Q
2
,
c l nhit dung ring ca nc c = 1 cal.kg.; delT = T
2
-T
1

ton b lng nhit sinh ra u c gii phng ht th Q
1
= Q
2

Khi m.c.DelT = 2,149,
m = 2,149/[1(31-28)] = 716,3 KL/hr.

2. Hiu sut trao i nhit
Ta c:
U =
Tm A
Q
A
(Hiu sut trao i nhit, Mcal/hr.m
2
.
0
C)
Trong ATm =
) 2 3 (
) 1 3 (
) 2 3 ( ) 1 3 (
T T
T T
Ln
T T T T

;
Q = ( ) F T T 1 2 (Nhit lng trao i, Mcal/hr)
Do vy:
U = 2,148/[20*(31-28)/Ln((35-28)/(35-31))] = 0,0187 Mcal/hr.m
2
.
0
C.


110
TI LIU THAM KHO

1. Stanbury, Whitaker and Hall, 1995. Principles of fermentation technology, Second
edition, Pergamon.
2. Mardigan, Martinko, Parker, 2000. Brock Biology of Microorganisms, Ninth edition,
Prentice Hall.
3. Arnold L. Demain, Julian E. Davies, Ronald M. Atlas, Gerald Cohen, Charles L.
Hershberger, Wei-Shou Hu, David H. Sherman, Richard C. Willson, J. H. David Wu,
1999. Manual of Industrial Microbiology and Biotechnology, second edition, ASM
press, Washington DC.
4. Henry C. Vogel, Celeste L. Todaro, Celeste C. Todaro, 1997. Fermentation and
Biochemical Engineering Handbook: Principles, Process Design, and Equipment,
Noyes Publications, USA.
5. Visvanathan, 2004. Physico-Chemical Processes, Environment Engineering &
Management, Asian Institute of Technology.
6. L Xun Phng, 2001, Vi sinh vt cng nghip, Nxb Xy Dng, H Ni.
7. Tarun K. Ghose, Bioprocess computations in biotechnology, Volume 1, Ellis
Horwood.
8. Trn Linh Thc, 2003, Phng php phn tch vi sinh vt trong nc, thc phm v
m phm, Nxb Gio Dc.
9. Tiu chun Vit Nam TCVN 5945: 2005, Nc thi cng nghip Tiu chun cht
thi, xut bn ln 2, H ni 2006.















PHN PHC LC
111

1. Ph lc 1. Hnh nh cc thit b lin quan ca h thng ln men
2. Ph lc 2. Tiu chun v nc thi Vit Nam (TCVN 5945: 2005)


Ph lc 3. Bng mi quan h gia p sut v nhit hi nc
A B C
1 Nhit oC 40 40 45
2 pH - 6-9 5,5-9 5-9
3 Mi - Khng kh chu Khng kh chu -
4 Mu sc, Co-Pt pH=7 20 50 -
5 BOD
5
(20
o
C) mg/L 30 50 100
6 COD mg/L 50 80 400
7 Cht rn l lng mg/L 50 100 200
8 Arsen mg/L 0.05 0.1 0.5
9 Thu ngn mg/L 0.005 0.01 0.01
10 Ch mg/L 0.1 0.5 1
11 Cadimi mg/L 0.005 0.01 0.5
12 Crome (VI) mg/L 0.05 0.1 0.5
13 Crome (III) mg/L 0.2 1 2
14 ng mg/L 2 2 5
15 Km mg/L 3 3 5
16 Niken mg/L 0.2 0.5 2
17 Mangan mg/L 0.5 1 5
18 St mg/L 1 5 10
19 Thic mg/L 0.2 1 5
20 Xianua mg/L 0.07 0.1 0.2
21 Phenol mg/L 0.1 0.5 1
22 Du m khong mg/L 5 5 10
23 Du thc vt mg/L 10 20 30
24 Clo d mg/L 1 2 -
25 PCBs mg/L 0.003 0.01 -
26 Ho cht bo v tc vt: Ln hu c mg/L 0.3 1
27 Ho cht bo v tc vt: Clo hu c mg/L 0.1 0.1 -
28 Sufua mg/L 0.2 0.5 1
29 Florua mg/L 5 10 15
30 Clorua mg/L 500 600 1000
31 Amoni (tnh theo Nit) mg/L 5 10 15
32 Tng nit mg/L 15 30 60
33 Tng phtpho mg/L 4 6 8
34 Coliform MPN/100ml 3000 5000 -
35 Xt nghin sinh hc (Bioassay) -
36 Tng hot ng phng xa o Bq/l 0.1 0.1 -
37 Tng hot ng phng xa | Bq/l 1 1 -
Gi tr gii hn
90% c sng st sau 96 gi
trong 100% nc thi
Thng s n v TT
112

Ghi ch:
PSIG: Pounds per square inch (gauge)
PSI: Pounds per square inch (absolute). Atmospheric pressure (14.7 psi)
F: Shows the F temperature of steam at the pressure indicated.
C: Shows the C temperature of steam at the pressure indicated.

PSIG PSI
o
F
o
C PSIG PSI
o
F
o
C
0 14.7 212.0 100.0 28 42.7 271.4 133.0
1 15.7 215.5 101.9 29 43.7 272.7 133.7
2 16.7 218.5 103.6 30 44.7 274.0 134.4
3 17.7 221.5 105.3 31 45.7 275.4 135.2
4 18.7 224.5 106.9 32 46.7 276.7 135.9
5 19.7 227.4 108.6 33 47.7 278.1 136.7
6 20.7 230.0 110.0 34 48.7 279.4 137.4
7 21.7 232.4 111.3 35 49.7 280.7 138.2
8 22.7 234.8 112.7 36 50.7 281.9 138.8
9 23.7 237.1 113.9 37 51.7 283.2 139.6
10 24.7 239.4 115.2 38 52.7 284.4 140.2
11 25.7 241.6 116.4 39 53.7 285.6 140.8
12 26.7 243.7 117.6 40 54.7 286.7 141.5
13 27.7 245.8 118.8 41 55.7 287.9 142.2
14 28.7 247.9 119.9 42 56.7 289.0 142.8
15 29.7 249.8 121.0 43 57.7 290.2 143.4
16 30.7 251.7 122.1 44 58.7 291.3 144.1
17 31.7 253.6 123.1 45 59.7 292.4 144.7
18 32.7 255.6 124.2 46 60.7 293.5 145.3
19 33.7 257.2 125.1 47 61.7 294.6 145.9
20 34.7 258.8 126.0 48 62.7 295.6 146.4
21 35.7 260.6 127.0 49 63.7 296.7 147.1
22 36.7 262.3 127.9 50 64.7 297.7 147.6
23 37.7 263.8 128.8 51 65.7 298.7 148.2
24 38.7 265.3 129.6 52 66.7 299.7 148.7
25 39.7 266.8 130.4 53 67.7 300.7 149.3
26 40.7 268.3 131.3 54 68.7 301.7 149.8
27 41.7 269.9 132.2 55 69.7 302.7 150.4
p sut Nhit p sut Nhit