1. 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

2. Akselerostaat-tehnoloogia laboratoorsed rakendused
Tööstuslike protsesside optimeerimine
Mikroobide iseloomustamine
Söötmete väljatöötamine
Mikroobifüsioloogia uurimine
Mikroobide selektsioon
Metaboolika

3. Cultivation methods Batch Chemostat Turbidostat/auxostat Fed-batch
Accelerostat (A-stat)
D-stat
Turbidostat/auxostat
Auxo-accelerostat
Fed-batch
m-stat (substrate limited fed-batch)
The classical cultivation techniques
Batch, chemostat, turbidostat and Fed-batch
Have been known already for long time
And during past 50 years
Only little attention has been payd to develop
The new, more efficient techniques
Are the old methods already so perfect

4. Fed-batch ehk juurdevoolukultuur
Kasvatamismeetodid
Fed-batch ehk juurdevoolukultuur Si
Läbivoolukultuur
sööde
raku-kultuur
rakud
Batch

5. Batch culture lnX YXS=dX/dS tana=m=dX/dt/X
Is very convinient for determination of the maximum specific growth rate
Using semilogarithmic plot the maximum for current environmental conditions growth is equal to slope tanA
It is not always that simple, in many cases the organsims will have not long enough the phase of exponentioal
Or balanced growth.
tana=m=dX/dt/X
TIME (h)

6. Batch culture of S. cerevisiae, pH=3.6; T=30oC
In case of Saccharomyces cerevisisae, constant equal to maximum growth can be observed during few generations, at higher biomass concentations the growth rate starts to decrease.

7. 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

8. 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
m=D; YXS= (SFEED - S)/X
Chemostat is considered to be the most precise method of culture characterisation
KING of the cultivation methods
Because the culture characteristics can be vey simply
And precisely measured
Often all you need to know is feeding rate, culture volume and limiting substrate concentration in the feeding

9. 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 .
Keeping dilution rate constant steady state will be obtained in the chemostat
To prove that steady-state is obtained, it is usually sufficient that biomass concentration remains constant during time span of the observation.

10. Chemostat culture of E. coli glucose (10 g L-1), T=30 oC, pH=6.6
1.0
2.0
3.0
4.0
5.0 X
g/l
glc
ace
0.10
0.20
0.30
0.40
0.50
1/h
Dilution rate D = m
All the points in this curve are the steady-state point, which steady-state was ensured passing 3-5 culture volumes throug the fermentor.
The maximum specific growth rate, equal to dilution rate in chemostat was h-1
At higher dilution rates the wash-out of the culture was observed.
mmax=0.39 h-1

11. 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
Thats not all, shift up of the dilution rate can
Cause oscillations in case of yeast culture

12. Oscillations of S. cerevisiae at D=0.1 h-1
All culture parameter including
CO2 production rate
Oxygen consumption rate
Titrant consumption rate
As well as
Optical tensity oscillate in
Time span of observation.

13. Smooth change of D instead of step-wise
To prevent the negative effects descibed before We
Used the step-wize shange instead of step-wize
After obtaining stedy state in chemostat, we started to increase the dilution rate smootly at constant acceleration rate
In result the time and media consumption of the experiment decreased at least two-fold
and

14. A-stat cultivation of E. coli
The wash-out of the culture at much higher dilution rate than in chemostat was obtained.
Due to the dilution rate was increased smootly at constant acceleration rate a
The culture behaviour demonstrated that at certain dilution rate the capacity of respiratory chain in E. coli becomse exhausted and overflow metabolites like acetate start to accumulate.
Also maximum specific growth rate 0.59 h-1 was calculated..

15. Calculation of culture characteristics in A-stat
In calculations of specific growth rate beside dilution rate also change of biomass concentration should be taken into account.

16. 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.
To carry out the A-stat or accelerostat experiments conviniently we developed the software FermExpert
Which was the first Microsoft windows based cultivation control software in the word
And is sold until now by Applikon.

17. A-stat cultivation of Saccharomyces cerevisiae
Hier one more accelrostat experiment with Saccharomyces cerevisiae.
In RED the behaviour of growth rate is shown.
The growth rate remains lower than dilution rate, however the maximum growth rate 0.45 h-1, the same that in batch was observed

18. Chemostat based methods
Accelerostat (A-stat)
D-stat (a=0)
T, Si, pH, pO2
Fed-batch with changing culture volume (quasi-steady-state culture)
There are some more chemostat based methods we developed,
the D-stat in which dilution rate is kept constant
And one of the environmental or cultivation parameters is changed smoothly

19. D-stat with increase of temperature
Hier you see one experiments represented yesterday on the poster session
The dilution rate was kept constant and
Temperature was increased at constant rate
In result the intracellular threhalose concentration increased and then at 42 oC started to decrease,
In the same time the specific growth rate obtained a zero value.

20. D-stat with increase of temperature
Also the accumulation of acetate started at that temperature

21. D-stat with changing culture volume
D-stat can be carried out using changing culture volume. Those results were presented yesterday on poster session by dr Monika Drews
Keeping dilution rate constant in fed-batch culture you can save lot of culture media to achieve the steady state culture
Later, you can take out big omounts of culture media for you experiments without disturbing steady state.

22. Effect of growth rate on
Sorry for long introduction, however it is time now to go to the auxostat.
At near maximum growth rates in chemostat small change in m=D caused the big change in biomass concentration, pH, CO2 production etc.
Thefore it is impossible to keep the culture in this range using chemostat based methods
Biomass can be however to be kept constant using the feed-back loops of biomass control.

23. 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
The methods in which biomass concentration is kept constant be feedback control of dilution rate are called auxostat methods.
For feed-back contro any parameter which is proportional to biomass concentration can be used.

24. Feed-back control in auxostat
PUMP1
PUMP2
IF Z>Zs
THEN PUMP1=HIGH
ELSE PUMP1=LOW
IF V>Vset
THEN
PUMP2=ON
ELSE
PUMP2=OUT
X, pH, pO2 V
Auxostat needs to pumps to be controlled
First is used to keep the control parameter Z constant
The second one is for culture volume (NB you cannot to collect the culture from surface, it affects the results!!)
For example if the biomass concentration is higher than desired the pump A is swithced on at high speed
And if it lower than desired value it will be switched on at low speed.
Chemostat PUMP1 = constant

25. Obtaining steady-state in pH-auxostat5 10 15 20 25
pmp 30 40 50 60 70 T
0.15
0.30
0.45
0.60
0.75 D
1/h 4 8 12 16
hours
time
Instead of biomass concentration (turbitity) pH can used as well as control parameter.
To obtain the new steady-stat charactarised in case of auxostat with constant dilution rate in time span of observation
Up to 10 culture volumes may be required to pass through culture medium

26. Experimental strategy of auxo-accelerostat
The steady-state is obtained by keeping cultivation conditions Y {Tset, pHset, Vset, feeding medium composition etc.} controlling biomass concentration X = g * Z at desired level
One of the culture parameters (T, S, I etc.) is changed at constant rate
To study the effect of environmental conditions on culture characteristics in auxostat the environmental conditions are usually changed step-wise.
We like in case of accelerostat used the smooth, gradual change instead and called the method
AUXOACCELEROSTAT

27. pH-auxo-accelerostat of S
pH-auxo-accelerostat of S. cerevisiae with increase of biomass concentration
In this slaid a pH-auxoaccelerostat experiment is presented in which we increased the biomass concentrartion by increasing the pH of the feeding medium smoothly.
The experiment demonstrated that different physiological states of the culture can correspond to the same growth rate and even biomass concentration in axostat culture,
The behaviour of YXS and QATP suggest that at maximum growth rate the growth of yeast cells is limited by anabolic (biosynthetic capacity) not by capacity of ATP production.
With increase of biomass concentration over 0.7 g l-1 the growth rate decreases

28. Batch culture of S. cerevisiae
The same was observed also in batch culture of Saccaromyces cerevisisae.
The cross of YATP and QATP was however not observed, like in parallel auxoaccelerostat experiment with longer stabilisation time

29. Effect of biomass concentration
Usually no direct effect
Indirect effect
Growth promoting compounds
Growth inhibiting compounds
Primary metabolites
Secondary metabolites
Toxins
So for auxoaccelrostat experimets the biomass concentration should be carefully chosen
and kept in neutral range not affecting the culture characteristics.

30. Auxostat methods Turbidostat pH-auxostat pO2-auxostat CO2-auxostat
T-auxostat
Ethanol-auxostat
We used different auxo-accelerostat methods

31. pH-auxoaccelerostat Advantages Disadvantages
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
pH is very sensitive to …

32. Strain characterization
Determination of culture characteristics of different LAB in pH-auxo-accelerostat
We used the pH auxostat for characterisation of technological properties of lactic acid bacteria and Saccharomyces cerevisisae.
Result show that the technological parameters of different species of LAB are very different

33. 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
In case of Saccharomyces cerevisisae we studied …

34. pH-auxoaccelerostat, ethanol
You see the yeast has optimal in respect of growth rate concentration of ethanol (about 1%)

35. pH-auxo-accelerostat, NaCl
The NaCl concentration up to 10 g l-1 do not affect the growth reate

36. pH-auxoaccelerostat, pH
The pH down to 2.8 does not ffect the growth rate

37. pH-auxoaccelerostat, T
The growth rate optimum of Saccharomyces cerevisiae is
at 37oC
However YATP decreases with increase of temperature

38. pH-auxosccelerostat, yeast extract, T=37oC
Adding yeast extract maximum growth rate of 0.69 h-1can optained at optimal temperature T=37 oC

39. pH-auxoaccelerostat, pO2
At pO2 concentration lower than 0.4 % of air saturaation the growth rate and YATP of yeast starts to decrease.

40. 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
In some cases if the change of pH is very small
Like in the cultures of insect cells the or
If effect weak acids is studied the pH-auxo-accelrostat can not be conviniently used.

41. CO2-auxo-accelerostat
In CO2 auxoaccelerostat experiment with smooth increase of pentanoic acid concentration the grwoth rate starts to decrease almost imminiently after pentanoic acid is added

42. 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
PH auxostat in turn ….

43. pO2-auxo-accelerostat
This is an example in which tryptone concentration in the feeding was increased

44. T-auxostat Very perspective in industrial scale
Heat production is proportional to biomass production
Both T of the fermentation medium and DT of cooling water can be used as set-point

45. 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
To derive biomass with maximum biosynthetic activity …

46. CO2-auxostat with volume change
Using CO2 auxostat is that after decreasing the culture volume the release of specific CO2 realeas incrreases at expence of accumulatd in the culture media CO2.

47. Determination of growth rate in auxo-accelerostat
vIN
vOUT
VFEED
VOUT,X Z L
Balance
V, X, pH, pO2
Stirrer control
Determination of growth rate in axo-accelerostat has many sources of errors
The dilution rate fluctuates in accordience of contro parameter control
If the biomass concentration changes, it also increases error of measurements.
Dilution rate
m = (dVOUT/dt + dV/dt)/V + d(X*V)/dt)/(X*V)

48. 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

49. Principle possibilities of application of auxostat in food fermentations
Two tank processes
Continuous auxostat culture
Batch maturation
One tank processes
Auxostat with changing culture volume (tank filling)

50. Two tank auxostat process
pH, pO2, T, V, str
Si, Pi
Värske toore
KONT- ROLLER
pump
BIOXPERT
pump=low pH
Järel-valmimine
pump=high
ARVUTI
pHSET

51. One tank auxostat process
Toore
juuretis pH

52. Applications of auxostat technology
Piiritusetööstus
Piimatööstus
Hapendatud piimatooted
Jogurt
Kohupiim, juust
Toiduäädikas
Õlletööstus

53. 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.

54. 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.

55. orgaaniliste hapete tootmine
Auxostat-tehnoloogia on sobilik veiniäädika tootmiseks
Suure puhtusastmega orgaaniliste hapete stereoisomeeride tootmisel, näiteks piimhappe bakterite abil

56. Vadaku vääristamine Hapustamine pH-auksostaadis
Kontsentreerimine pöördosmoosil
Kontsentraadi (laktaatsiirupi) kasutamine pärmi substraadina või piimhappe tootmiseks

57. Juuretised
Auksostaat-tehnoloogia võimaldab saada pidevalt optimaalsete füsioloogiliste omadustega juuretisi
Vähendab starterjuuretise kulu
Lülitada tootesse optimaalses füsioloogilises seisundis probiootilisi juuretisi
Probiootiliste jookide automaadid.

58. Naturaalkalja tehnoloogia
Piimhappe bakterid + pärm kooskasvatamine kalja ekstraktil auksostaadis
Mikrofiltratsioon
Villimine pudelisse + mikroobid kaasa

59. 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

60. Rekombinantide tootmine
Maksimaalse kasvukiirusega kasvavad rakud on kõige paremini infekteeritavad viiruste ja faagide poolt
Rekombinantide tootmine bakuloviirussüsteemis

61. 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

62. Auksostaatprotsessid tagavad
suure tootlikkuse
Toidukvaliteedi, kuna toimub täpselt defineeritud tingimustes
Toiduohutuse, kuna fermentatsiooni tingimused on kogu kasvu vältel on mittesobivad patogeensete mikroorganismide kasvuks