Microbial bioreactors/bioreactions: tools for basic knowledge on

workkinkajouBiotechnology

Dec 5, 2012 (4 years and 10 months ago)

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KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Saccharomyces cerevisiae

:

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Evoutions in Bio Sciences


Ecology


Quantitative ecology


Physiology,


Quantitative biology


Systemic Biology


Holistic Biology

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Yeast as cell factory

Yeast

Aerobiosis

Semi Anaerobiosis

Anaerobiosis

Baker yeast

Yeast extract

Flavouring agents

Metabolites, ex food

additives

Waste water treatment

Yeast as co product

animal feed

Recombinant
yeast



enzyme
pharmacentical

Ethanol (ETBE)

Ethanol solvant

chemistry

alcoolic beverages


KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

MICROBIAL/BIO REACTOR
ENGINEERING:A BASIC TOOL FOR
KNOWLEDGE IN HOLISTIC BIOLOGY

G.Goma,S Guillouet,C Jouve,J L Uribellarea

Laboratoire d ingenierie des systémes biologique
et des procédés

UMR CNRS,INRA,INSA


KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse


Intersections
on technology
and common
fields

White
biotechs

Red biotechs


Agro
-
food
biotechs

Green
biotechs


Basic knowledges



Focused on




life sciences …




敮杩g敥物e 獣楥s捥c




扩潭慴桥a慴楣a




灨祳楣p



Economy, sociology, ...

Generic technology



卹S瑨整楣猬i灡瑨睡祳



B楯捡瑡汹獩猠敮杩g敥物湧



B楯灲潣敳獩湧

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Microbial Engineering:a part of
biotechnologies

Find and improve the microorganisms for bio
processing

Find the conditions of bio processing where the
microrganism is economicaly performant


A multidisciplinary approach

A contraint : find the bottlenecks,eliminate them

An obligation:need of handling a complete tool
box:from genes to bioproducts and bioprocess


KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

What kind of technological
strategy?


Low tech ?


High tech ?


Right tech for the goal


What are the criteria of production ?


Production of «

active agents

»


Cost ?


Invisible technology


Relatively safe technology


Reproducible protocols simplest as possible


Semi speciality



«

de novo

» technology?


use of existing tools of production?

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

The IB Value Chain

Biofuels

H2

Ethanol

Sugars

Agricultural

(by)products

Biochemicals

Food Ingredients

Pharmaceuticals

Fine Chemicals

Biomaterials

Polylactic acid

1,3 propane diol

PHAs

Physical treatment

and/or enzymes

(Micro
-
)organisms

biocatalysis

Bulk

Fine

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

The steps


Factory and his environment


The reactors ,biorector:biocatalist,srategy


Raw materials and biocatalist,bioreaction
engineering


The biocatalist


Global implementation ;find the differents
bottleneks and solve the problems


Need a tool box,and combining experimentals
datas(strategy?) and simulations


KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Si


X


products:j

X

Feeds,

Substrat(s), air,
regulations and
controls

Take down,
culture medium,

gaz out,
biomass,
products,,,,,,,,,,

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Industrial (White) Biotechnology

Sugars

Biofuels

Biomaterials

Biochemicals


Cell factories

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Measures

Régulations

Correction pH,
Antifoam

Tank,mixing,
température control

Gaz out:analyse

Gaz in

control

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Dual use of fermentors


RPM


Qair


Pressure


C
O
2
,

O
2
?


Gas balance


OD?


Ph (controlled)


Temperature

For this 2 controlled parameters,
the analysis of the «

work

» of the
control regulator gives
informations


Starters


milk, silage, …


Baker yeast


bread


Alcoholic beverages


Lactic acid/organic acids
(citric)


Antibiotics


Vaccines


Monoclonal antibodies


Recombinant proteins (or toxin
?)


Waste water treatment


Bioleaching

Instrumentation of a
fermentor

Use of fermentors

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Réalisation


Mixing

FLUIDIC Mixing

Jets


KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

AERATION : TECHNOLOGIES d’AERATION

ICI, Ltd. factory, Billingham, UK,
(Chem. Eng. News, 18
-
Sep
-
78)


FERMENTEUR type air
-
lift

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Metabolic descriptor


Mass conservation


Elemental biologicals reactions


Macroscopic kinetics


Matrix of reactions combining kinetics and
stoechiometry of elemental reaction of metabolic
pathways


Combining kinetics observed by on line
measurements by robusts sensors evaluation of
metabolics fluxes «

on line

» and nutritionals
needs


Identification of some bottlenecks

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse



Phenomenogical models



Behavioural models



Structured models and stoechiometric/metabolic descriptors

Experimental strategies

S

P

X

O
2

CO
2

O
2

CO
2

Q
e

Q
s

PEP

ATP

NADH,H

+

Glucose

ATP

NADH,H

+

ATP

IsoCitrate

Suc
-
CoA

Malate

HS
-
CoA

HS
-
CoA

GTP

Fumarate

Succinate

ATP

ATP

Citrate

a

Kglu

NADH,H

+

CO

2

CO

2

CO

2

CO

2

+

NADPH,H

NADH,H

+

+

NADPH,H

CO

2

FadH

2

NADH,H

+

CO

2

ANABOLISME

ATP

CO

2

NADH,H

+

+

NADPH,H

ATP

+

NADPH,H

2

CO

2

Acétate

Pyruvate

Glucose6
-
P

Fructose
-
P

TrioseP

Glycerate3P

Pentose P

Sedoheptulose7 P

Erythrose4P

OAA

NAD

2

2

+

H 0 + 4 H

2

1/2 O

2

+

FAD

FADH

2

H 0 + 2 H

3 H

+

ATP

1/2 O

NADH,H

+

GlycerolP

glycerol

ATP

SH
-
CoA

SH
-
CoA

Acetyl CoA

Qresp

%pO
2

pH

Temps

Predictive modelisation and
implementation of microbial
processes


KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Système

métabolique


Système

protéique

Système

génomique

Système:

d’adaptation et de

défense

Interface de la cellule et échanges

Système

d’échanges

1

2

3

Plate
-
forme métabolomique, fluxomique


Vers une
biologie des systèmes

par la réconciliation
des niveaux métaboliques, génétiques et moléculaires

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Prerequisite to “Systemic Biology”

Analytical methods

Kinetics

Flux, Stocks

Technology

in situ

continue

on line

in parallel

micro samples

Metabolome

Data base

(x2 every 18 months
)

Metabolic pathways

coupled kinetics

relaxation time,regulations,

«

OMICS

»

Sequences



genes

Profiles



proteins

Definition

of

functions ,


networks


KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Top Down strategy


Fit the macroscopic environnment,bioreactor


Find reproducible conditions:signature recognition


Biokinetics


Quantitative physiologie


Metabolic pathways


Proteomic


transcriptomics

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

-

How

osmotic

conditions

affect

response

to

ethanol?


-

G
enes

and

mechanisms

i
nvolved

?





Analysis of first fermentation



Comparison with another fermentation with
better performances



> sequencial feeding glucose

> Titer 50 h = 147 g/L,


viability = 30%

> Viability = 80% at 120g/L

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

2



Normally, we have sensors only for the


environmental variables.




Physiological states are tracked through


offline measurements and analysis, with an


implied delay.




The physiological state can be identified by


the fusion of environmental measurements.

The physiological state recognition

The cell population expresses stable characteristics within
every physiological state, thus an invariant control strategy
can be effectively applied in each state.

Motivation

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Identification and Classification
of Physiological States


A bottlenek for «

the omics

»studies,for
control strategies and «

quality

»


Morphometry


Kinetics and stoechiometrics «

parameters

»


Differentiation of biologicals and
environmental effects

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Yeast :Axenic culture gives a population
production linked to some mechanims

G
1
, G
2
, G
3
,G
4
,…


Cycle

The family growth by
budding

S1 sugar

S2 oxygene
yes/no

S3 ethanol

My job is
bioconversi
on

I have a
limitation

I do
nothing

I am stressed



I became a
filament

My job is to
produce
cell
biomass

I work

I work

I am ill

I am injuried

Finish : End ; cryptic
growth !!! I am a substrate

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

The Tool box

Bio: the “omics”

+

Traditional technologies

+

mathematical tools


“the rule of innovation”



KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Biocatalysis strategy

Diversity

Natural

Eco
-
systems

Natural

Diversity of

Eco
-
systems





Screening



















Screening







Engineering

metabolic*





Building strains




DNA shuffling



Global analysis “Omics

and engineering”

Genes*


et functions

screening

Genes*


and functions

screening

Production
-
formulation

«

Bioprocédés

»

Production
-
formulation

«

Bioprocesses

»

premières

Raw

materials

Bioprocess strategy


* e.biotechnology's and engineering

Biomolécules

Biomolecules


High added value

Needs in size of
market :


animal feed

Strategy on co
-
products

/bio
-
products

plus value

Co
-
products

Biomaterial

on energy

Increase the value

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Microbial engineering is multidisciplinary : need
of quantitative and “system” biology

+ system biology modelling





Microbial

engineering

Molecular


physiological engineering













Microbial


process analysis

and control

engineering

Microbial
processing

Physiological

engineering

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Dual use of fermentors

What is a fermentor ?

Elemental biokinetics

x Biomass

p Product

s Substrats

x

p

s

t Time

t

x

p

s
1

t

ou

x

p

s
1

t

s
2

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Cell and glucose ethanol
concentration vs time (
Fed batch

with nutritional strategy)

0
1000
2000
3000
4000
5000
6000
7000
0
5
10
15
20
25
30
35
40
45
50
Time
(h)
0
50
100
150
200
250
300
350
400
Glucose
Ethanol
Biomass
Viable
Biomass
(g)
(g/L)
(g)
(g)
0
1000
2000
3000
4000
5000
6000
7000
0
5
10
15
20
25
30
35
40
45
50
Time
(h)
0
50
100
150
200
250
300
350
400
Glucose
Ethanol
Biomass
Viable
Biomass
(g)
(g/L)
(g)
(g)
KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

0
20
40
60
80
100
120
140
160
180
200
0,00
10,00
20,00
30,00
40,00
50,00
60,00
70,00
80,00
0,0
0,2
0,4
0,6
0,8
1,0
1,2
Ethanol
glucose
Biomass
viability
I

II

VI

Ethanol
Glucose

(g/L)

Biomass


(g/L)



2 phenomena:


-

D
ecoupling growth
-
production



-

Loss of viability

Viability

2

4

6

8

10

12

14

16

18

20

III

IV


V

Study of
a
reference fermentation

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Measurement of
intracellular


metabolites

Sample quenching
in

-
60
°
C methanol

Measurement of
extracellular


metabolites
-

direct filtration

through adaptated membrane

Fast sampling

:

Sampling for extraction of RNAs
and proteins

Gas balances

(Mass spectr.)

Biomass sensor

X

estim

control

Q, q

H

+

Controlled

environment

rpm

Q

air

in

Q

air

out

Monitoring

µ

T
°

pH

p

O2

q

O

2

,

q

CO

2

,

Q

resp

Measurement

& rates

/ 20 sec

Studying the fast biological
responses ...

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

The hyper yeast


KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Data Acquisition : Measures

p
a
r
a
m
è
t
r
e
s

d
e

f
e
r
m
e
n
t
a
t
i
o
n
t
e
m
p
s

(
h
)
0
2
4
6
8
1
0
1
2
t
e
m
p
.

(
°
C
)
3
6
.
8
3
7
.
0
3
7
.
2
3
7
.
4
2
D

G
r
a
p
h

2
0
2
4
6
8
1
0
1
2
R
P
M
6
0
0
9
0
0
1
2
0
0
1
5
0
0
1
8
0
0
2
D

G
r
a
p
h

3
0
2
4
6
8
1
0
1
2
p
H
6
.
6
6
.
7
6
.
8
6
.
9
7
.
0
P
a
r
a
m
è
t
r
e
s

d
e

f
e
r
m
e
n
t
a
t
i
o
n
0
2
4
6
8
1
0
1
2
P
r
e
s
s
i
o
n

(
m
b
)
5
1
0
5
1
5
5
2
0
Fermentation
Param
e
ters

Time(h)

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Biocatalyse
enzymatique

Biocatalyse
microbienne

«

impact socio
-
économique

»

Le
microorganisme
est un
système
biocatalytique

évoluant dans
un système

«

interactions de systèmes et hiérarchies

»

Système
d’échange
s

Système
métaboliqu
e


système
protéique

Système

génomique

Système:


d’adaptation
et de défense

Le biotope du
système microbien
crée un
environnement;
«

en soi ,


un système

»

Interface de la cellule et échanges

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Allosteric controls

Mass action law

RNA control

Modification of enzymatic
pools

Cell

Environment

Gradients due to
mixing

Continuous culture

Batch, Fed
-
batch

Phenomenological
model

Metabolic model

Virtual cell

Behavioural models

10
-
6

10
-
5

10
-
4

10
-
3

10
-
2

10
-
1

10
0

10
+1

10
+2

10
+3

10
+4

10
+5

10
+6

s
-

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Extracellular
components

Intracellular components

Perspective :

Use of behavioural modelling

Segregation (size,
viability, …)

Analysis of population or «

dynamic

systems

»

Descriptor of
physiological
state A

Descriptor of
physiological
state B


*

*
Relaxation time

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse



Bacteria



Yeast



Fungi



Eucaryotic cells



In every case

The basic law of biokinetics and
stoechiometry are the same

But, every case have rules of
utilisation with typical profile

What kind of micro
organisms

What kind of profile

dt
V
CO
d
dt
V
CO
d
CO
Q
CO
Q
V
r
dt
V
O
d
dt
V
O
d
O
Q
O
Q
V
r
liq
carbo
dis
gaz
gaz
sort
sot
ent
ent
liq
liq
CO
liq
dis
gaz
gaz
sort
sot
ent
ent
liq
liq
O
)
.
(
)
.
(
.
.
.
)
.
(
)
.
(
.
2
2
2
2
2
2
2
2
2
2











Equations
bilan
Temps (h)
r
O2
r
CO2
moles/h
Coefficient
respiratoire
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0.7
0.8
0.9
1
1.1
1.2
1.3
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
dt
V
CO
d
dt
V
CO
d
CO
Q
CO
Q
V
r
dt
V
O
d
dt
V
O
d
O
Q
O
Q
V
r
liq
carbo
dis
gaz
gaz
sort
sot
ent
ent
liq
liq
CO
liq
dis
gaz
gaz
sort
sot
ent
ent
liq
liq
O
)
.
(
)
.
(
.
.
.
)
.
(
)
.
(
.
2
2
2
2
2
2
2
2
2
2











Equations
bilan
Temps (h)
r
O2
r
CO2
moles/h
Coefficient
respiratoire
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0.7
0.8
0.9
1
1.1
1.2
1.3
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

CATEGORISATION des SIGNAUX

Identification de classes de comportement


Mesures pertinentes / Comportements physiologiques

CLASSES

Item : temps

Item : temps

Temps

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse



Phenomenogical models



Behavioural models



Structured models and stoechiometric/metabolic descriptors

Experimental strategies

S

P

X

O
2

CO
2

O
2

CO
2

Q
e

Q
s

PEP

ATP

NADH,H

+

Glucose

ATP

NADH,H

+

ATP

IsoCitrate

Suc
-
CoA

Malate

HS
-
CoA

HS
-
CoA

GTP

Fumarate

Succinate

ATP

ATP

Citrate

a

Kglu

NADH,H

+

CO

2

CO

2

CO

2

CO

2

+

NADPH,H

NADH,H

+

+

NADPH,H

CO

2

FadH

2

NADH,H

+

CO

2

ANABOLISME

ATP

CO

2

NADH,H

+

+

NADPH,H

ATP

+

NADPH,H

2

CO

2

Acétate

Pyruvate

Glucose6
-
P

Fructose
-
P

TrioseP

Glycerate3P

Pentose P

Sedoheptulose7 P

Erythrose4P

OAA

NAD

2

2

+

H 0 + 4 H

2

1/2 O

2

+

FAD

FADH

2

H 0 + 2 H

3 H

+

ATP

1/2 O

NADH,H

+

GlycerolP

glycerol

ATP

SH
-
CoA

SH
-
CoA

Acetyl CoA

Qresp

%pO
2

pH

Temps

Predictive modelisation and
implementation of microbial
processes


KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Study of a fermentation of
reference

First results:genes over expressed

% des genes significatif dans la famille considerée
0,0
2,0
4,0
6,0
8,0
10,0
12,0
14,0
16,0
CELL CYCLE
AND DNA
PROCESSING
CELL FATE
CELL RESCUE,
DEFENSE AND
VIRULENCE
CELLULAR
TRANSPORT
AND
CONTROL OF
CELLULAR
ORGANIZATION
ENERGY
METABOLISM
PROTEIN FATE
(folding,
modification,
PROTEIN
SYNTHESIS
REGULATION
OF /
INTERACTION
TRANSCRIPTION
TRANSPORT
FACILITATION
III ="60g/L ethanol"
IV="80g/L ethanol"
V="90g/L ethanol"
VI="100g/L ethanol"
X="120g/L ethanol"
KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Study of a fermentation of
reference

First results
:genes under expressed

% des genes significatif dans la famille considerée
0,0
5,0
10,0
15,0
20,0
25,0
30,0
CELL CYCLE
AND DNA
PROCESSING
CELL FATE
CELL RESCUE,
DEFENSE AND
VIRULENCE
CELLULAR
TRANSPORT
AND
CLASSIFICATION
NOT YET CLEAR-
CUT
ENERGY
METABOLISM
PROTEIN FATE
(folding,
modification,
PROTEIN
SYNTHESIS
REGULATION OF
/ INTERACTION
WITH CELLULAR
TRANSCRIPTION
TRANSPORT
FACILITATION
TRANSPOSABLE
ELEMENTS,
VIRAL AND
60g/L ethanol
80g/L ethanol
90g/L ethanol
100g/L ethanol
120g/L ethanol
KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Plate
-
forme métabolomique, fluxomique


Exploration fonctionnelle des systèmes
métaboliques microbiens



Analyse des réseaux métaboliques

o

Reconstruction métabolique

o

Analyse topologique

o

Modélisation métabolique




Exploration fonctionnelle



Analyse
in situ
: RMN
in vivo

o

Couplages bioréacteurs / RMN

o

Métabolisme énergétique, carboné, etc..




Métabolomique

o

Identification/quantification des métabolites




Fluxomique

o

Quantification des flux métaboliques

o

Approches isotopiques (
13
C)




Biomathématique/ bioinformatique

o

Modélisatio métabolique

o

Calculs de flux

o

Réconciliation de données

Systèmes métaboliques

Métabolisme central E. col
i :

89 métabolites, 110 réactions

Génome

Environnement

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse


Heterogeneities:gradients(flux,stocks,,,)


:microbe population

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Top Down strategy


Fit the macroscopic environnment,bioreactor


Find reproducible conditions:signature recognition


Biokinetics


Quantitative physiologie


Metabolic pathways


Proteomic


transcriptomics

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Synthesis


Engineers Top down strategies


Biologists,bottum up , bioinformatics!!!!!!!!!


Both strategies are necessary

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Basic concepts

Fuzzy logic


Hierarchical classification


Programmed by

inductive logic

Classification machine

Measures

Biological and engineering
knowledge


Biological modelling

Rules


Hypothesis


or


«

Class

»


3 levels of

multiscale analysis


Single cell, “statistic”

Analysis of information quality

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Tackling complexity in
industrial microbiology for
bioprocessing



h

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse

Système:

d’adaptation et de

défense

Le biotope du système

microbien crée un

environnement;


«

en soi,un système

»

Le biotope du système

microbien crée un

environnement;


«

en soi,un système

»

Système

métabolique


Système

protéique

Système

génomique

Système

d’échanges

1

2

3

Biologie intégrative
descendante

Biologie intégrative
ascendante

Biocatalyse/enzymologie

Construction/sélection
enzyme

Modélisation
moléculaire
PB



Fluxome, métabolome



Construction de
souches



Transcriptome,
régulations

PMM



Biochimie/biologie systémique



Génie moléculaire enzymatique



Génie cellulaire des procaryotes et
eucaryotes inférieurs



Aptitude expérimentation/plate
-
formes



Modélisations locales/globales



Stratégies d’expérimentation/modélisations



Psychologie d’application

PGM



Environnement physico
-
mécanique, physico
-
chimique


Réponse microbienne


Modélisation


Classification


Corrélation
environnement/mise en
œuvre, réponse
transcriptome


Dynamique systèmes


Programmation
expériences

KBDiSB/Aix 09
-
07

GG LISBP INSA Toulouse