fermentation technology chapter vvi

8/10/2019 Fermentation Technology Chapter Vvi

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1

Chapter V

Black box growth


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2

Growth without non-catabolic product

Single substrate limited growth


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Hyperbolic kinetic equation for specific

substrate uptake rate (qS)

-qS

0.5 qSmax

qSmax

0 KS CS

SS

S

SS CK

Cqq

max)(

KS: substrate affinity [kg substrate/m3]

qSmax: maximal uptake rate [kg S consumed/ kg X present-h]


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4

Substrate uptake for maintenance

Some part of substrate is used for producing energy needed for

cells maintenance. This part is catabolized with a rate mS.

Hence, mS= kg substrate catabolized for maintenance / kg

biomass presenthour.


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5

Herbert-Pirt relation for substrate

)(Y

)( maxSX

SS mq

It means the substrate taken into cells are used for growth and

maintenance only. Both mSand YSXmaxare model parameters.

-qS

0

(-mS)

max

1

SXY

Herbert-Pirt linear plot for

substrate, non-catabolic

product is absent


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6

Combination of the qSequation with Herbert-

Pirt relation

maxmaxSq-

SXSSS

S YmCK

C

CS= 0 , = mSYSXmax= -kd

CS>>>, = [-qSmax-(mS)]YSX

max= max

= 0, (-qS) = (-mS), which occurs at CS= CSmin.

The specific growth rate equation based on single substrate is

then:

SS

SS

CK

CC

minmax


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The plot of - CS

max

-kd CSmin

CS

For mS= 0, (Monod equation)SS

S

CK

C

max


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Using Herbert-Pirt relation to calculate other

rates

Example:

The case of aerobic growth on glucose, N-source is ammonia

SupposeqS= 0.3125 + 0.0015

Growth reaction:

-0.3125 C6H12O6+ aNH4++ bO2+ cH

++ dH2O +

C1H1.8O0.5N0.2+ eCO2

Use the yield and elemental balance to obtain a, b, c, d, e

The catabolic reaction is: -C6H12O6- 6O2+ 6CO2 + 6H2O

What are (-qNH4+), (-qO2), qCO2, qH+, qH2O?


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How to measure the kinetic parameters?

Obtaining

max

and YSXmax

.Using batch fermentation at constant volume where CS>>

Hence = max(constant, during the exponential phase)

qS= qSmax

(constant),YSX= YSX

max= (CX-CXo)/(CSo-CS)

Obtaining KSand mS.

Using chemostats, is the variable to get -qS. Apply Herbert-

Pirt relation and plotqSvs. to get YSXmaxand mS.

PlotqSvs. CSto obtain KSand qSmax.


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Further reading

The Herbert-Pirt relation including non catabolic productformation

Extended Herbert-Pirt relation

Different qP- functionEffect of temperature and pH.


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11

Chapter VI

Growth and product formation in

bioreactors


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12

Fermentation processes

substratefermenter

Downstream

processing

wastes

products

The upstream processing costs about 2050 %, whereas thedownstream processing costs about 50-80 %


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The type of reactors and processes

The reactors systems:

Aerated stirred tank reactor 0.11000 m3with cooling

equipment

Bubble column 0.110,000 m

3

with airlift and coolingequipment

Bioreactors for immobilized cells/biocatalysts (packed bed,

fluidized bed, trickle bed)

The types of processes used are batch, continuous, and fedbatch systems. Mostly, batch and fed batch reactors are used in

industry whereas continuous reactors (chemostats) are used in

laboratories.


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Fermenter utilities (contd)

Air filtration, using membrane / bed filter with dry air about 60

m3air/m3broth-h.

Compressor, 2 bar, 150oC.

Heat removal

Cooling jacket

Coil

With cool water temperature at 10oC and capacity is about

50,000 kJ/m3reactor-h.


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Packed bed fermenter


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What do we want?

High yield of biomass

High max

High temperature tolerant

Grow on low cost substrate

High qPat low

Extra cellular products

Low viscous biomass

Generally regarded as safe (GRAS)

Mineral medium

Thus, it is important to

analyze the kinetics of

growth and product

formation, designing the

optimal feed profile, role

of transport processes,

maybe use bigger

reactors.

Remember!

qS or completelydetermines the microbial

behavior. It must be

controlled at an optimal

value


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Batch, continuous, and fed batch processes

In batch reactor: and qSare at the maximum value and notcontrolled.

In chemostat: is controlled at optimum

In fed batch reactor: qSis controlled.

batch fed

batchchemostat


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Constant volume batch reactor

The steps:1. Add sterile growth medium solution (substrate consist of

C, N, P source, K+, Mg+, salts, vitamin, trace elements)

and choose the right T and pH.

2. Add electron acceptors (O2, NO3-, etc)

3. Inoculate microorganism at initial time with concentration

CXo.

4. Run the fermentation and harvest the broth, proceed to thedownstream processing.


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Constant volume batch reactor

What is the plot CSand CXwith time?

Derive from the mass balance, one can obtain that the growth

is exponential, the slope of the curve CXvs. time is rX.

Parameters State

variables

reactor operator micro

organism

V CS0

CX0

qSmax

maxYSX

max

mS

KS

CS

CX


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Substrate concentration profile

At constant volume:

For batch reactor:

t = 0, CS= Cso ;

Hence, by putting CS= 0, t end of batch is obtained

The slope in CSvs. t plot is rS.

XSSS Cqr

dt

dC

t

XoSXS

S eCqCqdt

dC maxmaxmax

1)exp(

max

max

max

tCq

CC XoS

SSo


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Conclusion for the batch reactor

, YSX, and qSare constant at the maximum values

Microbial model parameters are determined by combining the

exponential equation with CSand CXdata versus time.

, the most important rate, can not be controlled

Try to sketch the curve CS, CX, , -qS, YSX, rX, -rSversus

time for batch reactor!


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Chemostat

Parameters State

variables

reactor operator micro

organism

V

L,out

CS,in

L,in

qSmax

YSXmaxmS

KS

CS

CX

Steady state: constant volume, constant in and out flow rates.Constant CSand CX.


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Steps in operating continuous culture Sterilized reactor is filled with sterile growth medium

solution containing substrate at CS,in

Air sparging starts to provide electron acceptor

Add a small amount of microorganism (inoculum)

Start the medium in flow (often L,inand L,outare nearly

equal, not always)

Wait until steady state is achieved

The main property of chemostat:

1. CSand CXare independently manipulated by the user bymanipulating transport (to and from the reactor) of substrate

and biomass.

2. Excellent experimental tool to study microbial kinetics,

stoichiometry, under controlled conditions.


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Biomass mass balance in chemostat

Inout + gen = acc

The is chosen by manipulating exit flow rate

Hence, all other qi-values are set (stoichiometric coupling)

Does CSdepend on CS,in? Use (CS) to prove it.

DV

VCC

VrC

outL

XoutLX

XoutLX

,

,

,

00


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Critical dilution rate

The maximal value for D (=) attainable in the chemostat, is

achieved when the maximal CSin achieved. At critical dilution

rate, CSCS,in.

Note: CS,innormally in order 10000 mg/l, CS,minorder 1 mg/l, KS

order 10 mg/l.

Wash out

If D > Dcrit, the cell will be washed out

Then, sketch the graph CSvs. D.

max

,

min,,max

inSS

SinS

crit CK

CCD


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Substrate mass balance

outL

inL

SS,inS

SoutSoutLinSinL

DCCr

VrCC

,

,

,,,,

0

This equation together with Herbert-Pirt relation provide rSand

(-qS) from measured concentration, flows, and volume.

What about CX?

SinSSXS

SX

SinS

S

SX CCY

mY

D

CCD

q

rC

,

max

,

)(


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The application of chemostat

1. Kinetic studies, at different = D one can varies qS, CStoobtain YSX

max, mS, qSmax, KS.

2. Physiological and genomic micro array study studies of

microorganisms under defined steady state, I.e. substrate,

electron acceptor, N-source, type of limitation for growth.

3. Waste water treatment

4. Industrial fermentation, which is not widely applied


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Chemostat optimization

Normally, the maximal rX

andrS

are needed as the economic

parameter

Try to find the optimum D where drS/dD and drX/dD = 0.

Note that rShas a maximum at higher D.

Chemostat wash out dynamic

What happen when D is close to Dcrit?

What happen to CXwhen D = D>Dcrit?

tDCC

CDCdt

dC

ssXX

XXX

'exp

'

max,


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Pro and cons of chemostat

(+)

Excellent experimental tool because is defined

(-)

Low biomass and product concentration

Loss of biomass in outflow

Relatively prone to be contaminated compare to batch or fed

batch reactors

Microbial selection for non-producing mutants


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Fed batch fermentation

In batch reactor, CS

and CX

are high. No transport of S or X and

no control on .

In chemostat, CSand CXare low. Transport of S or X and

control on .

In fed batch reactor. Substrate transport in, not out. No biomasstransport.

Why fed batch?

1. Low CS

no toxicity / osmotic problem2. High CXhigh CPeasier downstream processing

3. Control of ?


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Fed batch fermentation

Batch phase

time

CSO

CS

Start feeding

Feeding phase under substrate

limited conditions

CS= 150 mg/l.

CSO5000

20000 mg/l

In substrate limited feeding phase, CS is very low. Thus, one

can use the pseudo steady state condition for substrate mass

balance


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Substrate mass balance in fed batch reactor

V

Cr

VrCdt

VCd

inSinSS

SinSinS

S

,,

,, 00

Hence, in a fed batch reactor the substrate conversion (-rS

) is

controlled by the operator.

Through controlled rS, is controlled.

It is obvious that the reactor volume changes with time.

However, since the change is very small, for simplicity we canassume constant volume, constant density.


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Rates in fed batch reactor

We start with an assumption that volume is a variable

Biomass mass balance

Pseudo steady state substrate mass balance

Product mass balance

X

X

XXX

VCdt

VCd

VCVrdt

VCd

1

X

insins

X

SS

VC

C

C

rq

,,

X

PP

XPP

VCdt

VCdq

VCqdt

VCd

1

P ibl b f di i i f d


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Possible substrate feeding strategies in a fed

batch fermentation

1. Substrate input (S,inCS,in) is constant

2. is maintained at optimum that gives maximal qPor

maximal YSP.

3. Substrate input is determined by other known reactorlimitations (oxygen, heat, etc)

Apply Herbert-Pirt equation and biomass mass balance to

calculate rXand CX.


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Biomass mass balance in fed batch reactor

Assume constant volume (DV


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Biomass mass balance in fed batch reactor

XSSXinSinS

SXX CmY

V

CY

dt

dC

max,,max

The first explains growth, the later is about maintenance.

InitiallyCXis low in a fed batch reactor. By neglecting

maintenance:

CXincreases linearly with time

Later, CX increases, more and more substrate needed for

maintenance

dCX/dt decreases.End, all substrate needed for maintenance, dCX/dt = 0

constant

,,max

V

C

Ydt

dC inSinS

SX

X

SinSinS

Xm

VCC

/,,max


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Fed batch reactor with constant feeding rate

The analytical solution for CX(t) in feeding phase.

t = time after start feeding

CXf= biomass concentration at start feeding

Note:

For t >>> 0,For t


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Fed batch reactor with constant feeding rate

rXdecreases

sharply decreases

qSdecreasesCSdecreases slowly

)(max,,max tCmYVCY

dtdC

XSSXinSinS

SXX

tC

trt

X

X

tC

rtq

X

SS-


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Fed batch reactor with constant optimum

= opt

= constant qi, Yijare all constant.

qSconstant at qSoptCSconstant.

Biomass concentration increases exponentially

All rates increases exponentially

Substrate feeding rate increases exponentially

tf= time when feeding starts

XoptXX Crdt

dC

tCqtr Xoptii

ttCVrtC optfinSinSSinSinS exp,,,,

fermentation technology chapter vvi

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