lecture notes(process biotechnology) 1

7/28/2019 Lecture Notes(Process Biotechnology) 1

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Bio-Process Kinetics

Process Biotechnology

Anondho WIJANARKO

University of Indonesia

Simplified Lecture Notes


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To Imanti

andAdam, Musa, Isa and Muhammad


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CONTENTS

Microbial Growth

Microbial Cell Growth

Classical & Empirical Growth Kinetics Environmental Alteration Studies

Light I llumination Effect

Temperature Effect

Microbial Kinetic Studies

Non Elementer Reaction

Microbial Growth Reaction Kinetics


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Microbial Growth


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Microbial Cell Growth

Mode of GrowthSelective assimilation of nutrients and

convert into and also include Chemical

rearrangement of protoplasmic material

characteristic of the particular organism

Production of an increased amount ofnuclear substance and cell division


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Growth Phase


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Growth Phase

Induction Phase (Lag Phase)

Transient Phase (Acceleration Phase)

Exponential Phase

Stationary Phase (Declining Phase)

Death Phase


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Question Sheet

Why microbial growth have an lag phase?

Why death phase could be occurred in

microbial growth?

What is essential nutrient for growth of

organism especiallyprokaryotes?

What is important factor for cell division?


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Answer Sheet

Keywords

Environmental Adaptation

Saturated microbial population and rare of

nutrient

Generally : Organic materials/CO2,

Phosphate, Nitrate/NH3,Sulfate, Mg2+, K+

Mg2+


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Growth Approximation

G th C t t


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Growth ConstantsExponentialStationary Growth PhaseTotal Biomass Production(G)

Incident growth rate, Incident mean division rate (mf)

Specific growth rate, Beginning mean division rate (m)

Doubling time of population (tD); exponential growthphase

dt

dX

Xtt

X

X

1

12

ln1

2

mf

dt

dX

Xt 1

lim0m

)( im XXG

mt

2ln

D


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Problem solving


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Microbial population growth A new microorganism has been discovered which at each cell

division yields three daughters. From the growth rate data below

calculate the mean time between successive cell divisions?

t [h] X[g/dm3]

0.0 0.100.5 0.15

1.0 0.23

1.5 0.34

2.0 0.51


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Answer Sheet

1

815.0

815.0

100.0 3

h

eXdm

gt

mf

mf4ln~

Dt

htD

70.1

815.0

4ln~


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Classical Growth KineticsEmpirical Approximation

MonodGrowth Kinetic

TessierGrowth Kinetic

MoserGrowth Kinetic

Contois Growth

Kinetic

s

s

s

11 mmf sKs

sKse/

1 mmf

sXB

s

mmf


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Empirical Growth KineticsMedium constituent Inhibition

Andrews Growth

Kinetic

Aiba Growth Kinetic

i2Ks

sK

s

s

mmf

pKsKKs

ps

p

mmf


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Growth KineticsMultiple essential nutrient

Bailey Growth Kinetic

...33

3

22

2

11

1

sK

ssK

ssK

s

sss

mmf


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Home Work

Which kinetic approximation do you choose in caseof microbial growth of Hepatotoxin producedOscilatoria Agardhii NIVA CYA 97 in low

temperature? Which empirical equation that you choose of

inoculation of microorganism in case of multiplecontent limitation of nutrients, such as Mg2+,

phosphate, Nitrate and organic compound?

Which kinetic approximation do you choose ofcultivation photosynthetic microorganism that didnot grew up in pH above 7.8?


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Answer Sheet

Keywords

Aiba Equation

Hepatoxin was produced by all photosyntheticheterocystis microbes inoculation in case of coldenvironmental condition

Bailey Equation

Multiple limitation of nutrient contents

Andrew Equation

Generally, Increasing pH was increase culture [HCO3-]

that was known as essential nutrient for growthmechanism in cytoplasm and pH around 7.8 tend

optimum [HCO3-] in case of inoculation of commonhotos nthetic microbe.


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Simple Bio-Production Kinetic

Cellular growth rate

Monod approximation

Yield factor

Substrate Utilization

Product Formation(Beginning ofStationary Phase)

s

s

s

dtdsdt

dX

sXs

XY

/

X

dt

Xd f

dtdXdt

dP

XP X

PY

/

sK

Xs

dt

Xd

s

sK

Xs

Y

dt

sd

ssX

/

sK

XsY

dt

Pd

s

XP

m/


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EnvironmentalAlteration Studies


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Microbial Growth KineticEnviromental Condition

Direct Effects

Light Illumination (Energy Source)

Temperature

Essential nutrients content

Indirect Effects

Gas inlet volumetric rate

Gas inlet content

Liquid circulation rate

Non essential nutrients content


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Light Illumination Effect Oscillatoria agardhi Gomont

(Post AF, R de Witt, LC Mur, J. Plank. Res., 7 (1985) 487-495)


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Temperature effect

ModifiedArhenius Model

R

dH

RdS

a

T

RTE

e

TeA

1

/m


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Classification of

Microorganism


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Question Sheet

What is happen if microorganism is at 90oC? Why?

In case of decreasing of temperature about 20oCfrom optimum temperature, what is happen in caseof microbial growth rate?

In case of ethanol production that was S. sake haveethanol tolerance around 10%, what do you do tomake an whisky industry?

Why a shade microbe does not grew well in highlight illumination and commonly have not hightemperature resistance?


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Answer Sheet

Death, thermophile did not survived at temperature 85oC up.

Refer to Arhenius approximation, microbial growth was

decreased to quarter.

Ethanol production was set below 7-8% and purified to 40%

Photo-bleaching (chlorolysis), shade microbe commonly

psychrophile, that have a limitedness to growth at both of

high temperature and light illumination.


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Problem solving


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Temperature variation of Growth Johnson, EyringandPolisaarrepresent growth ofE. coli between

18oC and 46oC by the following equation for the specific growth

rate m:

Plot this function as log m versus 1/T

Show that this equation can be represented as the product of two function

whose form is suggested by the plot in above part, and what explanation

rationalizes these two individual functions and the value of aboveparameters?

In this interpretation of m as function of T, what implicit assumption are

made with regard to irreversible deactivation?

T

TT

75200

239exp1

7520exp1096.9 9

m


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Answer Sheet

T

7770exp1084.6 12m

0.0034364 17.494 18.000

0.0034247 19.178 19.000

0.0034130 21.011 20.000

0.0034014 23.005 21.000

0.0033898 25.174 22.000

0.0033784 27.530 23.000

0.0033670 30.089 24.000

0.0033557 32.867 25.0000.0033445 35.880 26.000

0.0033333 39.147 27.000

0.0033223 42.687 28.000

0.0033113 46.520 29.000

0.0033003 50.667 30.000

0.0032895 55.151 31.000

0.0032787 59.990 32.000

0.0032680 65.199 33.000

0.0032573 70.773 34.000

0.0032468 76.667 35.000

0.0032362 82.737 36.000

0.0032258 88.620 37.000

0.0032154 93.488 38.000

0.0032051 95.692 39.000

0.0031949 92.579 40.000

0.0031847 81.570 41.000

0.0031746 63.206 42.000

0.0031646 42.683 43.000

0.0031546 25.748 44.000

0.0031447 14.433 45.000

0.0031348 7.7740 46.000

T53000exp1096.5 72m


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Answer Sheet Deactivation line

This decreasing line presented a rapid decrease in growth rate as the temperature

approaches the upper limit for survival of the microorganism tend that the mostthermally sensitive essential protein denatures and this hypothesis also has beenconfirmed in several instance by genetic studies in which mutation of a single genehas caused a large change in the maximum tolerable temperature formicroorganism

One physical mechanism for this phenomenon is obvious as the temperatureincreases, the atoms in the enzyme molecule have greater energies and a greater

tendency to move. Eventually, they acquire sufficient energy to overcome theweak interactions holding the globular protein structure together, and deactivationfolows.

Activation line This increasing line clarified at low temperature, apparently, the metabolic activity

of cell increase with increasing temperature as the activities of its enzyme rises.Notice in this activation line, that was also commonly called to Arrhenius plot,

that classical Arrhenius behavior appears at low temperature, exactly, below ofthe maximum tolerable temperature for microorganism.

The implicit assumption are made regard to irreversible deactivation :irreversibility alteration of whole active forms of enzyme to inactive forms incase of a large change in the maximum tolerable temperature for eachmicroorganism


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Temperature EffectCellular Consideration

Psychrophile Obligate

Protococcus Agardh SS 100-3

Oscillatoria redekei Van Goor

Oscillatoria sp. SS 100-5

Facultative Anabena cylindrica Lemmerman

Oscillatoria Agardhi Gomont

Nostoc commune Antartica

Mesophile

Synechococcus leopoliensis

Anabaena variabilis IAM M3

Microcystis Aeruginosa IAM M228

Thermophile Mastigocladus laminosus HTF

Synechococcus lividus OH75S

Synechcocus elongatus It 7S


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Home Work Why optimum specific growth rate values of

psychrophile factually, lower than

thermophile? Why GC content of microbial DNA is

important for classification of organism in

terms of growth rate dependence ontemperature?

What is DBI?


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Answer Sheet

Keywords

Arhenius Limitation

GuaninCytosin of DNA have a very strong of 3 pairs

hydrogen bound, that was responsible in high temperature

tolerance characteristic of microbe.

DBI was value that defined content of double bond of

cellular membrane fatty acid in the each strains plasma-

membrane.


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Problem solving

Pl l 3 b f k f 90

O. agardhi A. nidulans S. lividus


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Please solve 3 numbers for mark of 90

or 4 numbers for mark of 100

Number b and e must be done

a. Calculate mf of each strain

at S=0.2 g/dm3?b. Calculate A of each strain!!!

Calculate m at 285 K and 310 K

of each strain!

Think deeply and carefully before

answer these question!!!

c. Define DBI and GC Content!

d. Write DBI equation!

e. For each type of strain

Calculate the DBI?

Calculate GC contens (GCDNA)?

What type? Why?

O. agardhi A. nidulans S. lividus

Fatty acids contents 14:0 0 0.011 0

14:1 0 0.012 0

16:0 0.292 0.477 0.54

16:1 0.217 0.385 0.1

16:2 0.033 0 0

18:0 0.004 0.037 0.22 18:1 0.073 0.074 0.14

18:2 0.146 0 0

18:3 0.235 0 0

Growth Characteristic Tm 355 K 361 K 371K

T 295 K 301 K 328 K

Ki 0.001 g/dm3 10 g/dm3 1 g/dm3

Km 10 g/dm3 10 g/dm3 100 g/dm3

m 0.012 h1 0.014 h1 0.048 h1

Ea 46.5 Kj/mole 128 Kj/mole 240 Kj/mole

DNAGCC

mT o 0.413.69)(

Kmole

JR

314.8

RT

Ea

eA

m

i

m

K

s

s

K 1

1f

A Sh t


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Answer Sheetf mf[h-1] A [h-1]

Oscillatoria agardhi Gomont 0.003984 0.0000478 2.06*106

Anacystis nidulans 0.01960 0.0010584 2.29*10

20

Synechococcus lividus 0.001995 0.0000958 8.00*1036

m[h-1](285K) (310K) DBI GCDNA

Oscillatoria agardhi Gomont 0.00618 0.0 1.353 0.310 psychrophile

Anacystis nidulans 0.00306 0.0618 0.471 0.456 mesophile

Synechococcus lividus 0.0 0.0479 0.240 0.700 thermophile

DBI was value that defined content of double bond of cellular membrane fatty acid in the each strains

plasma-membrane.

Percentage of Guanin & Cytosin in whole of DNA

FA

nNUFAnDBI

n

:0


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Microbial Kinetic Studies


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Non Elementer Reaction

Common reaction rate

N = integer Elementer

N = non integer Non Elementer

Non Elementer Example :

nsk

dt

ds

HBrBrH 222 22

22

3

212

'][

'

BrkHBr

HBrk

dt

Brd

COCHCHOCH 43

2333 CHOCHkdtCHOCHd

R ti M h i


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Reaction Mechanism

2222

1

2 OHNHNO

k

022

dt

OHd22

OH

OHNHNO 222 222

OHHOHk

2222 22

0222221 HOHkHNOk 2

2

122 NO

k

kOH

2

212

2

2

122222

2 HNOkHNO

k

kkHOHk

dt

OHd

R i M h i


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Reaction Mechanism

22

1

1

2 ONNOk

k

2222 / OHON

022

dt

ONd

OHOHH

k

2222 23

0

22

dt

OHd

OHNHNO 222 222

222222

2

OHNHON

k

022232222 HOHkHONk

2232

22ON

k

kOH

2

22

21

212

2

2

21

122222

2

11 H

HNO

kk

kkH

H

NO

kk

kkHONk

dt

Nd

022222212

1 HONkONkNOk

2

2

21

122

1 H

NO

kk

kON


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Question Sheet

What is mechanism path?

What definition of intermediate species?

What was become determining factor ofreaction rate?


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Answer Sheet

Mechanism path is microscopic description of a

chemical reaction that was composed in term of

elementer reactions

Intermediate species is an imaginary reactant that

was proposed in mechanism path and was predicted

have a share in deciding of reaction rate

Slowest elementer reaction of the proposedmechanism path

Microbial Gro th


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Microbial GrowthEnzymatic Reaction/Kinetic consideration

Michaelis-Menten Kinetics Reaction mechanism

Kinetic equation

Substrate Actvation and Inhibition Reaction mechanism

Kinetic equation

Product Activation and Inhibition Reaction mechanism

Kinetic equation

sK

s

m

mmf

ESSEk

k

1

1

EPESk

2

ESSEk

k

1

1

2

2

2

ESSESk

k

EPESk

3

im KssK

s

/2

mmf

ESSEk

k

1

1

EPESk

2

ESPPESk

k

3

3

pKsKKs

ps

p

mmf


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Michaelis-Menten Kinetics

Reaction mechanism

Kinetic derivation

ESSEk

k

1

1

EPESk

2

ESEE 0

ESESkk

kES

kk

kES

ESkkESkES

ES

dtddt

d

0

21

1

21

1

211

0

00 ES

m

dtd

dtd

KS

SP

k

kk

S

SEkESkP

k

kkS

SEES

max

1

21

022

1

21

0


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Home Work

Please exhibit kinetic derivation of substrate

activation and inhibition?

Please exhibit kinetic derivation of productactivation and inhibition?

What do you think about reaction kinetic if

Km is high that was indicated inbioremediation of toluene by C. nivalis?


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Answer Sheet

Substrate Activation & Inhibition

Reaction mechanism

Kinetic equation

Equation constant

im

KssK

s

/2

mmf

ESSEk

k

1

12

2

2

ESSESk

k

EPES

k

3

1

31

k

kkKm

2

2

k

kKi

03 Ek m

Answer Sheet


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Answer Sheet Product Activation & Inhibition

Reaction mechanism

Kinetic equation

Equation constant

Reaction kinetic become First order

XCX

s

KX

sK

sX

dt

dX

mm

1

mmmf

ESSEk

k

1

1

EPESk

2

ESPPES

k

k3

3

pKsKKs

ps

p

mmf

3

3

1

21

k

k

k

kkKs

3

3

k

kKp

02 Ek m


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Problems

Solve problems in Biochemical Engineering

Fundamental (JE Bailey and DF Ollis) of

page 446 to 447 number 7.2 to 7.3 !!!


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Problem solving


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Cellulose Hydrolysis Kinetics

The enzymes which degrade cellulose, producing the dimer cellobiose, a

simplified reaction network can be written :

Where G1, G2 are insoluble cellulose and soluble ccllubiose and E1 is

indicative of the enzyme involved in the slowest step leading to cellobiose.

Derive a reaction rate of this hydrolysis studies?

1212

1211

1111

3

3

2

1

EGEG

EGEG

EGEG

k

k

k

k


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Answer Sheet

Fast reaction

Slow reaction

Inhibition step

Intermediate species

1212

1211

1111

3

3

2

1

EGEG

EGEG

EGEG

k

k

k

k

1112 & EGEG

121110

EGEGEE

0

0

12

11

dt

EGd

dt

EGd 0

0

123123

112111

EGkEGk

EGkEGk

123

312

11

2

111

EGk

kEG

EGk

kEG

210

1

3

3

2

1

1 GG

EE

k

k

k

k


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Rate of cellulose hydrolysis Reaction

210

23112

123112

123112

1

2

2

1231122

33

211 GG

EGkGk

dt

Gd

EGkGkdt

Gd

EGkEGk

k

kdt

Gd

EGkEGkdt

Gd

kk

kk

2121

022

3

3

2

1

2

3

2

1

1 GG

GGEk

dt

Gd

k

k

k

k

k

k

k

k

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