STERILIZATION

notions: STERILITÁS -- STERILITY

ASZEPTIKUSSÁG -- ASEPTICITY

ELSZIGETELÉS, IZOLÁLÁS -- CONTAINMENT

Killing microbes Protecting the environment from microbes

Protect the system from the microbesaseptic operation=maintaining sterility

PatogenesVírusesGMO-s

rDNS production problems

OECD 1986 – Recombinant DNASafety Considerations

EC 1990 Council Directive on the Contained use od GMOs

vakcinatermelés

GMO I.: small risk

II.: others

STERILIZATION

CONTAMINATIONDECREASING YIELDALTERATION IN PROCESS BEHAVIOUR (KINETICS)PLUS STERILIZATION NEEDWHOLE CHARGE GOES WRONG

(SCALE DEPENDENT DEMAGE)PROBLEM AT DOWN-STREAM

Extra work, money

METHODS for removal and killing of microbial cellsmechanical methods: filtration ,

centrifugation,flotation,

electromagnetic irradiation: UV, X ,chemical methods: dezinfection,heat.

STERILIZATION

Thermal death of microorganisms

Temperature ranges of the growth of microbes

µ

STERILIZATION

Vegetative bacterium- and yeast cells

Conidiospores of phylamentous fungi

Rel

ativ

e

h

eat

resi

sten

cy Vegetative cells

conidiospores

Viruses and bacteriophages

Bacterium spores

Rel

ativ

e

h

eat

resi

sten

cy

endospores

- heat sensitivity depends upon (given species): life history of the cell, age of the cell

(e.g.: cells from the exponencial growth phase are more sensitive than cells from the stacioner phase)

- cells are more sensitive against moist heat than against dry heat

STERILIZATION

- heat sensitivity ( thermal death) increases with incr. temperature

- heat sensitivity depends on media pH, viscosity, osmotic pressure,presence of defending colloids,

KINETICS OF THERMAL DEATH AT CONSTANT TEMP.

dNdt

kN= −

N number of living cells [pc/cm3]k thermal death rate (decay) constant [min-1].

ktNln

−=

STERILIZATION

kte0NN

N

0N

N

0N

t

0kdtdlnN =

NdN

kt0N

ln

−=

→∫ ∫ ∫−=

−=

METHOD FOR THE DETRMINATION OF k

what k depends on?microbe ....sort, „form”mediumtemperature

Empirical constant

Apparent activation energy of thermal death (KJ/mol)Determination method

Microbe T[oC ] k[min-1] Ea [KJ/mol]

Bacillus subtilis(vegetative) 110 27 310

Bacillus subtilis(spores) 121,1 3 -

Bacillus stearothermophilus 104 0,051 283

(spores) 125 6,06 283130 17,52 283

STERILIZATION

130 17,52 283 Clostridium botulinum

(spores) 104 0,42 344Hemoglobin(heatdenaturation) 68 6,3·10 -3 312

Medium components heat decay apparent activation energies [[[[kJ/mol]]]]

Reaction between carbohydrates and proteins 130,6B1 vitamin decay 87,9 B2 vitamin decay 98,8

tk

1 = Mean life span

=k

3,2decimal reduction timeDDDD

N

Nlg 0

STERILIZATION

1000

100

10

1

DDDD

t

tk

1 = Mean life span

Probabilistic approach of thermal death

Kinetic description is good if No>>>>>>>>1 !Thermal death is also a stochastic process

Definition: the life span of one cell (spore)is the length time duringwhich the cell (spore) will just remain viable.

STERILIZATION

mean life span of the population

N0 no of all the sporesNi no of the spores with life span of ti

∑∞

==

−

1iitiN

0N1t

Life span

1

tk=Mean thermal decay constant

!

CONDITIONSIf temp is the same everywhere in the vessel, No growth, (!!!!)Behaviour of the individual spores is independent of the others.

Probability of the event that at time t the no of the survivors isexactly N ( N= 0,1,2,...No), follows a binomal distribution:

( ) ( )[ ] ( )[ ]( )NNN0N

0tp1tp N

N tP −−

=

STERILIZATION

( ) ( )[ ] ( )[ ]N tp1tp N

tP −

=

probab. that one sporeis survivor at time ( )p t e kt= −

( ) ( ) ( ) ( )( )P t

N !

N N !N!e 1 eN

0

0

ktN

ktN N0

=−

−− − −

What is the prob. that all the spores had already died by the time t ?

( ) ( ) ( ) ( )( )P t

N !

N N !N!e 1 eN

0

0

ktN

ktN N0

=−

−− − −

( ) ( )P t 1 e 10kt

N0

= − − <

Always higher than 0:

STERILIZATION

( ) ( )1- P t 1 e 00kt

N0= − − −1 >

( ).eN=Nin which

e1tP1tk

0

N0

−

−−≅−

At a common sterilization process N0 >>1

tk0

eN eNe1tk

0 −− ≈−=−

e-x ~ 1-x+...according to a Taylor serie

Sterility criterion? Pl .:

99,9% P0(t)=0,999 1-P0(t)=0,001=10-3

The probability that the sterilzitation had failed, i.e. there remained at least one survivor: 10-3

The probability that the sterilization succeeded,

STERILIZATION

The probability that the sterilization succeeded, No remaining living cells : 0,999

At every 1000th of sterilizations is allowed to occur one unsuccesful one.It is probable that one in thousand went wrong.

After the sterilization process the number of survivors (Cell in the whole system)

steam

Electic heating

water

Steam out

Door

Steam in

Steam

Steam

cooling

cooling water

Batch sterilization of culture media

Steam cooling water

water

cooling water

condensate condensate

Heat penetration curve

120-130 oC

Commeasurable sections!

STERILIZATION

Temperature of fermentation

20-25 oC

t1 t2 tvScale dependent

STERILEZÉS

Thermal death during heating period:

1t

0kdt

N0N

ln ∫ ∇== heating

Thermal death during holding period: ( ) . holding1t2tholdingk

2N1N

n ∇=−=l

Thermal death during cooling period:

∫ ∇==vt

t coolingkdtvN2N

nl

STERILIZATION

cooling period:2t

coolingvN

v

2

2

1

1

0

v

2

2

1

1

0

v

0

coolholdheat

N

Nln

N

Nln

N

Nln

N

N

N

N

N

Nln

N

Nln ++=

=

∇+∇+∇=∇

0,20 0,75 0,05e.g.:

factor=

N0

Nln Scale sensitive!

10-3 N0 = 105 / ml

32,2 1010

10.10liter 100 13

3

55

=∇=−

STERILIZATION

39,2 1010

10.10.10 m 100

36,8 1010

10.10.10 m 10

10

163

3553

153

3453

3

=∇=

=∇=

−

−

−

10x: increases with 2,3

Continuous sterilization of culture media

Fermentor size limit productivity: (kg product/fermentor.m3.year). Advantages of the cont. Sterilization process:

-at higher temp.(130-140oC) with shorter process time

increased safetyless thermal decay of culture medium components

STERILIZATION

-the continuous process more reproducible,

-stable quality of the sterile mediathis may increase the fermentation yield

-cont. Ster. Equipment and the process easily controllable,automatization possible.

steam

Media

vacuum

Steam injection

Holding section

Expansion valve

Sterile media

time

STERILEZÉS

Plate and frame heat exchanger

steam

Holding sectionsterilemadium

cooling

precooling

heating

medium

time

oC

preheating

20s

20s2-3 min

STERILIZATION

SPIRAL HEAT EXCH.

STERILIZATION

STERILIZATION

STERILIZATION

STERILIZATION

CONTINUOUS MEDIUM STERILIZATION PROCESS OUTLINE

CULT.MEDIUM

BYPASS

STERILEMEDIUM

HOLDING SECTION30 s 140 o C

CLEANING CIRCUIT

gõz

COOLING WATER

COOLER

HEATER

REGENERATINGHEATER-COOLER

WATERDETERGENTS

REGENERATION BYPASS

MEASURMENT POINTS

STERILIZATION

Continuous sterilizer (design) calculation

lnN

Nk t k

Lw

q

0

v

= =∆For holding section:

BUT!!!plug flow

u u= max

L – length of holding section tube (m)w –volumetric rate (m3/min)q – tube cross sectional area (m2)

Turbulent stream

Laminar stream

max

u u= 05, * max

maxu*82,0u =

STERILIZATIONBIM 2 SB2002

HOT POINTS

BOTTOM DRIVE

MECHANICAL SEAL

ELEKTRODES

STERILEZÉS

AIR OUT

MIX.SHAFTMECHANICAL STEAL

AEROSOL

AIR FILTER

ACID,BASE,HBG

INOKULUM

HOT POINTS

TOP DRIVE

EMPTYING

SAMPLING

STERILIZATION

MIXING SHAFTAIR IN, OUTINGREDIENTS:

PIPESVALVES

INOKULUM LINESENSORSPUMPS

DOWN-STREAM: STERILENONSTERILE

STERILIZATION

SLIPPING SURFACE

DOUBLE MECHANICAL SEAL

STERILE WATER –LUBRICATION

STERILIZATION

STERILE INOCULATION

STERILIZATION

STERILE INOCULATION 2

STERILIZATION

THERMODYNAMIC STEAM TRAP

STERILIZATION

MEMBRANE VALVES

VISSZACSPÓSZELEP

STERILIZATION

AIR FILTRATION

PALL

Absolute filter

EMFLONR

STERILIZATION

AIR FILTRATION DEPTH FILTER

STERILIZATION

CIP(CLEANING IN PLACE)