AUKSOSTAAT-AKSELEROSTAAT-TEHNOLOOGIA

Klassikalised fermentatsiooniprotsessid toidutööstuses on perioodilised.

Pidevprotsesside rakendamine on olnud seni väheedukas, kuna need ei taganud vajalikku

kvaliteeti ja olid saastumistundlikud. Auksostaattehnoloogia võimaldab aga protsesside mikrobioloogilise saastuse

probleemid edukalt lahendada, paranda toidu kvaliteeti, luua uusi tooteid ja optimeerida

protsesse

Akselerostaat-tehnoloogia laboratoorsed rakendused

Tööstuslike protsesside optimeerimine

Mikroobide iseloomustamine Söötmete väljatöötamine Mikroobifüsioloogia uurimine Mikroobide selektsioon Metaboolika

Cultivation methods Batch Chemostat

Accelerostat (A-stat) D-stat

Turbidostat/auxostat Auxo-accelerostat

Fed-batch -stat (substrate limited fed-batch)

Kasvatamismeetodid

Fed-batch ehk juurdevoolukultuur

Si

Läbivoolukultuur

sööde sööderaku-kultuur

rakud

Batch

Batch culture

lnX

TIME (h)

tan=dX/dt/X

YXS=dX/dS

Batch culture of S. cerevisiae, pH=3.6; T=30oC

0

2

4

6

8

10

X g/l

0

5

10

15

20

25

eth g/l

0

0.1

0.2

0.3

0.4

0.5

1/h

0

5

10

15

20

25

YATP

g/mol

0

10

20

30

40

50

QATP

mmol/g/h

2 4 6 8 10 12

Time (h)

X eth

YATP

QATP

Läbivoolukultuur Kontrollitakse lahjenduskiirust (D)

Kemostaat, hoitakse D-d ja keskkonna tingimusi konstantsena

A-staat, muudetakse lahjenduskiirust sujuvalt D-staat, muudetakse keskkonna tingimusi sujuvalt

Kontrollitakse biomassi kontsentratsiooni Turbidostaat (kostantne hägusus) pH-auksostaat (konstantne pH) CO2-auksostaat pO2-auksostaat

Kui kasutatakse sujuvat kasvutingimuste muutmisest nimetatakse meetodit aukso-akselerostaadiks

Chemostat

The most precise method of culture characterization

The steady-state can be obtained keeping D and T, , air, SFEED etc. constant

D=feeding rate/culture volume (1/h) The culture characteristics

=D; YXS= (SFEED - S)/X

Steady-state the culture conditions in which X, Si, Pi,

YXS, pH, pO2, T, V, biomass composition etc. are constant

the biomass concentration, not changing in time span of observation shows, usually that steady-state is achieved in chemostat

.

Chemostat culture of E. coli glucose (10 g L-1), T=30 oC, pH=6.6

0

1.0

2.0

3.0

4.0

5.0

Xg/l

0

1.0

2.0

3.0

4.0

5.0

glcg/l

0

1.0

2.0

3.0

4.0

5.0

aceg/l

0 0.10 0.20 0.30 0.40 0.50

1/h

Dilution rate D =

Xglc

ace

max=0.39 h-

1

The reasons for development of accelerostat

Significantly slower growth rate in chemostat than in batch culture

Long time and big amounts of media are required to obtain the chemostat curves

Oscillations after the step-wize change of D can occure in chemostat

Development of computer controlled cultivation systems

Oscillations of S. cerevisiae at D=0.1 h-1

19.0

19.5

20.0

20.5

21.0

21.5

O2%

0

0.5

1.0

1.5

2.0

2.5

CO2%

0

2

4

6

8

10

ODU

0

0.15

0.30

0.45

0.60

0.75

Ialk

8 10 12 14 16 18

hours

time

O2

CO2

OD

Ialk

Smooth change of D instead of step-wise

0

0.4

0.8

1.2

1.6

2.0

0 25 50 75 100 125

hours

time

a=0.01 h-2

a=0.005 h-1

a=0.02 h-2

D

D (h-1)

D0=0.1 h-1

Vst=3V

A-stat cultivation of E. coli

0

3

5

8

10

13

OD U

0

0.2

0.4

0.6

0.8

1.0

size µ³

0

30

60

90

120

150

glc C-mM

0

30

60

90

120

150

ace C-mM

5

7

9

11

13

15

QO2 mmol/g/h

0.20 0.30 0.40 0.50 0.60 0.70

1/h

D = Do + at

A

OD

size

glc

ace

QO2

Calculation of culture characteristics in A-stat

ODdt

ODdD

)(

ODdt

ETHd

OD

DETHQETH

*

)(

FermExpert BioXpert First Microsoft Windows based cultivation

soft-ware for fermentation control (1992) The program enabled

to program the behavior of cultivation parameters and

on-line calculation of the culture parameters using differential equations

Possibility to change the dilution rate smoothly.

A-stat cultivation of Saccharomyces cerevisiae

0

10

20

30

40

50

glc

0

30

60

90

120

150

eth

0

0.10

0.20

0.30

0.40

0.50

D

0

2

4

6

8

10

X

0

0.10

0.20

0.30

0.40

0.50

0

2

4

6

8

10

QO2

0 8 16 24 32 40

hours

time

Saccharomyces cerevisiae Alko743 growth on glucose in aerobic conditions

glc

eth

D

X

QO2

Chemostat based methods

Accelerostat (A-stat) D-stat (a=0)

T, Si, pH, pO2

Fed-batch with changing culture volume (quasi-steady-state culture)

D-stat with increase of temperature

50

60

70

80

90

100

pO2%

25

30

35

40

45

50

T°C

0

0.05

0.10

0.15

0.20

0.25

trehg g-1 dwt

0

0.10

0.20

0.30

0.40

0.50

μh-1

0

0.10

0.20

0.30

0.40

0.50

Dh-1

0 3 5 8 10 13

time [h]

D=0.08 h-1

Dμ

pO2

Ttreh

D-stat with increase of temperature

0

0.2

0.4

0.6

0.8 1.0

eth g L-1

0

0.04

0.08

0.12

0.16 0.20

suc g L-1

0

0.4

0.8

1.2

1.6 2.0

ace g L-1

0

0.02

0.04

0.06

0.08 0.10

D h-1

30 34 38 42 46 50

C

T - temperature

ethanol

sucrose

acetate

Dilution rate

D-stat with changing culture volume

0

0.4

0.8

1.2

1.6

2.0

VL

0

0.02

0.04

0.06

0.08

0.10

D1/h

0

0.02

0.04

0.06

0.08

0.10

myNc1/h

0 70 140 210 280 350

hours

time

A

V

DmyNc

Effect of growth rate on

titrant X

CO2

X CO2

titrant

Neutral range of biomass

for max

Auxostat (turbidostat) The biomass concentration can be kept

constant by feed-back control of Optical density OD pH Dissolved oxygen concentration pO2

Oxygen concentration in exhaust gas O2

CO2 concentration in exhaust gas etc.

by dilution rate D. For steady-state culture there may be no

difference as set-point can be adjusted to desired biomass concentration X

Feed-back control in auxostat

X, pH, pO2

V

IF Z>ZsTHEN PUMP1=HIGHELSE PUMP1=LOW

IF V>VsetTHENPUMP2=ONELSEPUMP2=OUT

PUMP1

PUMP2

Auxostat

Chemostat PUMP1 = constant

Obtaining steady-state in pH-auxostat

0

5

10

15

20

25

pmp

20

30

40

50

60

70

T

0

0.15

0.30

0.45

0.60

0.75

D

1/h

0 4 8 12 16 20

hours

time

pmp

T

D

Experimental strategy of auxo-accelerostat

1. The steady-state is obtained by keeping cultivation conditions Y {Tset, pHset, Vset, feeding medium composition etc.} controlling biomass concentration X = * Z at desired level

2. One of the culture parameters (T, S, I etc.) is changed at constant rate

pH-auxo-accelerostat of S. cerevisiae with increase of biomass concentration

0

1.0

2.0

3.0

4.0

5.0

X g/l

0

0.10

0.20

0.30

0.40

0.50

1/h

0

4

8

12

16

20

YATP g/mol

0

15

30

45

60

75

QATP mmol/g/h

19 21 23 25 27 29 Time (h)

X

YATP

QATP

Batch culture of S. cerevisiae

0

2

4

6

8

10

X g/l

0

5

10

15

20

25

eth g/l

0

0.1

0.2

0.3

0.4

0.5

1/h

0

5

10

15

20

25

YATP

g/mol

0

10

20

30

40

50

QATP

mmol/g/h

2 4 6 8 10 12

Time (h)

X eth

YATP

QATP

Effect of biomass concentration

Usually no direct effect Indirect effect

Growth promoting compounds Growth inhibiting compounds

Primary metabolites Secondary metabolites Toxins

Auxostat methods

Turbidostat pH-auxostat pO2-auxostat CO2-auxostat T-auxostat Ethanol-auxostat

pH-auxoaccelerostat Advantages

Very sensitive to change of biomass concentration,

Well proportional to biomass concentration Technically simple and reliable

Disadvantages Affected by change of pH in the feeding Complicated in studies of pH effect

3

54

1

a

2

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60

, h

-1

3

5

c

4

1

2

0

5

10

15

20

25

0 10 20 30 40 50 60T, oC

YA

TP,

g d

wt m

ol A

TP

-1

35

4

1b

2

0

30

60

90

120

150

0 10 20 30 40 50 60

QLA

C, m

mol

g d

wt-1

h-1

Determination of culture characteristics of different LAB in pH-auxo-accelerostat

Strain characterization

Determinations of growth characteristics of Saccharomyces cerevisiae Effect of biomass Effect of ethanol Effect of propanol Effect of temperature Effect of oxygen Effect of yeast extract Effect of pH Effect of salt

pH-auxoaccelerostat, ethanol

0

0.15

0.30

0.45

0.60

0.75

1/h

0

0.10

0.20

0.30

0.40

0.50

X g/l

0 10 20 30 40 50

Ethanol (g/l)

X

pH-auxo-accelerostat, NaCl

0

0.11

0.22

0.33

0.44

0.55

1/h

0

0.15

0.30

0.45

0.60

0.75

YGE

mol/mol

0

4

8

12

16

20

YATP

g/mol

0

15

30

45

60

75

QATP

mmol/g/h

0 8 16 24 32 40

NaCl (g/l)

YGE

YATP

QATP

pH-auxoaccelerostat, pH

0

0.10

0.20

0.30

0.40 0.50

1/h

0

0.10

0.20

0.30

0.40 0.50

YGE

mol/mol

0

5

10

15

20 25

YATP

g/mol

0

10

20

30

40 50

QATP

mmol/g/h

3.7 3.4 3.1 2.8 2.5 2.2

pH

YGE

YATP

QATP

pH-auxoaccelerostat, T

0

0.15

0.30

0.45

0.60 0.75

1/h

0

0.05

0.10

0.15

0.20 0.25

YGE

mol/mol

0

10

20

30

40 50

YATP

g/mol

0

11

22

33

44 55

QATP

mmol/g/h

25 29 33 37 41 45

T (C)

YG/E

YATP

QATP

YGE

pH-auxosccelerostat, yeast extract, T=37oC

0

0.15

0.30

0.45

0.60 0.75

1/h

0

0.10

0.20

0.30

0.40 0.50

YGE

mol/mol

0

3

6

9

12 15

YATP

g/mol

0

20

40

60

80 100

QATP

mmol/g/h

0 0.8 1.6 2.4 3.2 4.0

Yeast extract (g/l)

max=0.69 h-1

YGE

YATP

QATP

pH-auxoaccelerostat, pO2

0

0.10

0.20

0.30

0.40 0.50

1/h

0

5

10

15

20 25

YATP

g/mol

0

10

20

30

40 50

QATP

mmol/g/h

0

4

8

12

16 20

pO2

3.0 4.0 5.0 6.0 7.0 8.0

Time (h)

YATP

QATP

pO2

CO2-auxo-accelerostat

CO2 concentration is proportional to X and growth rate

Advantages Not affected by pH of the feeding medium Good sensitivity and precision

Disadvantages Solubility of CO2 affects the concentration Delay in measurements Significant change in biomass concentration

complicates interpretation of results

CO2-auxo-accelerostat

0

1.5

3.0

4.5

6.0

7.5

CO2 %

1.5

2.0

2.5

3.0

3.5

4.0

pH

0

5

10

15

20

25

pO2

0

0.5

1.0

1.5

2.0

2.5

OD

0

1.0

2.0

3.0

4.0

5.0

C5 mM

0

0.10

0.20

0.30

0.40

0.50

X 1/h

14.0 15.0 16.0 17.0 18.0 19.0

hours

time

CO2

pH

pO2

OD C5

X

pO2-auxostat

Allows to keep biomass concentration at desired level determined by the stirrer speed, aeration rate etc.

Difficult to use in case of low oxygen consumption

pO2-auxo-accelerostat

0

10

20

30

40

50

pO2

0

200

400

600

800

1000

balance g

0

0.05

0.10

0.15

0.20

0.25

OD

0

0.2

0.4

0.6

0.8

1.0

1/h

0

0.5

1.0

1.5

2.0

2.5

Ptry

0 2 4 6 8 10

hours

time

pO2

balance

OD

Trypsine feeding

T-auxostat

Very perspective in industrial scale Heat production is proportional to

biomass production Both T of the fermentation medium

and T of cooling water can be used as set-point

Auxostat with change of culture volume

Biomass with maximum activity is required repeatedly for carrying out Infection experiments Physiologic studies Food fermentations

To obtain the steady-state culture using minimum amount of culture media

CO2-auxostat with volume change

0

2

4

6

8

10 pmp

6.0

6.2

6.4

6.6

6.8

7.0 pH

0

1.0

2.0

3.0

4.0

5.0 V

0

0.005

0.010

0.015

0.020

0.025 ECO2

0

0.4

0.8

1.2

1.6

2.0 Nv

60 70 80 90 100 110

hours

time

pmp

pH

V

ECO2

Nv

Determination of growth rate in auxo-accelerostat

= (dVOUT/dt + dV/dt)/V + d(X*V)/dt)/(X*V)

Balance

VOUT,XVFEED

vIN vOUT

Stirrer control

Z LV, X, pH,

pO2

Dilution rate

Application of the new methods in food technology

Auxostat – can be used to improve the performance of food fermentation processes

D-stat and accelerostat – optimization on fermentation conditions for biomass production

Auxo-accelerostat – culture characterization

Principle possibilities of application of auxostat in food fermentations

Two tank processes1. Continuous auxostat culture2. Batch maturation

One tank processes1. Auxostat with changing culture

volume (tank filling)2. Batch maturation

Two tank auxostat process

pH, pO2, T, V, strSi, Pi

Värske toore

KONT-ROLLER

pump

BIOXPERT pump=low p

H

Järel-valmimine

pump=high

ARVUTI

pHSET

One tank auxostat process

Toore

juuretis

pH

Applications of auxostat technology

Piiritusetööstus Piimatööstus

Hapendatud piimatooted Jogurt Kohupiim, juust

Toiduäädikas Õlletööstus

Piirituse pidevtootmine MOE piiritusetehases on seadistatud

momendil mitmeastmeline kemostaattehnolooia, mis on väga tundlik protsessi saastumisele võõrmikroflooraga.

Auksostaat tehnoloogia võimaldab alandada kultiveerimise pH-d väärtuste 3-3,6 juurde, mille juures kasvul maksimaalse kiiruse 0,3-0,4 h-1 juures on praktiliselt võõrmikrofloora ülekasv välistatud ja

tootlikus võrreldes kemostaadiga on fermentatsiooni esimeses astmes 4 kordne.

topsi-jogurti pidevtootmine

pH-auksostaadis (pHset=5.8) on võimalik hoida jogurtibakterite Streptococcus thermophilicuse ja Lactobacillus bulgaricuse kooslust kasvukiiruse 1 h-1 juures

Villides selle kultuuri topsidesse valmib topsijogurt.

orgaaniliste hapete tootmine

Auxostat-tehnoloogia on sobilik veiniäädika tootmiseks

Suure puhtusastmega orgaaniliste hapete stereoisomeeride tootmisel, näiteks piimhappe bakterite abil

Vadaku vääristamine

Hapustamine pH-auksostaadis Kontsentreerimine pöördosmoosil Kontsentraadi (laktaatsiirupi)

kasutamine pärmi substraadina või piimhappe tootmiseks

Juuretised

Auksostaat-tehnoloogia võimaldab saada pidevalt optimaalsete füsioloogiliste omadustega juuretisi

Vähendab starterjuuretise kulu Lülitada tootesse optimaalses

füsioloogilises seisundis probiootilisi juuretisi

Probiootiliste jookide automaadid.

Naturaalkalja tehnoloogia

Piimhappe bakterid + pärm kooskasvatamine kalja ekstraktil auksostaadis

Mikrofiltratsioon Villimine pudelisse + mikroobid

kaasa

Toksilisuse pidev monitooring

Biopuhastusseadmete korral on väga oluline vältida reaktori toksilist šokki.

Kuna auksostaat reageerib momentaalselt toksilisuse kasvule on võimalik meetodi abil monitorida heitvete toksilisust ja võtta selle alusel kasutusele asjakohaseid meetmeid

Rekombinantide tootmine

Maksimaalse kasvukiirusega kasvavad rakud on kõige paremini infekteeritavad viiruste ja faagide poolt

Rekombinantide tootmine bakuloviirussüsteemis

Kokkuvõte Akselerostaattehnoloogia on efektiivne

uudsete toidu- ja biotehnoloogiliste protsesside disaini meetod

Auxostaattehnoloogia on perspektiivne fermentatsiooni meetod toiduainete tehnoloogias Vähendab juuretiste kulu Võimaldab paremini kontrollida protsesse Võimaldab luua põhimõtteliselt uusi tooteid

Auksostaatprotsessid tagavad

suure tootlikkuse Toidukvaliteedi, kuna toimub

täpselt defineeritud tingimustes Toiduohutuse, kuna

fermentatsiooni tingimused on kogu kasvu vältel on mittesobivad patogeensete mikroorganismide kasvuks