Industrial Microbiology – Introduction and Overview

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12 Δεκ 2012 (πριν από 4 χρόνια και 4 μήνες)

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Industrial Microbiology


Introduction and Overview




Dr. Gerard Fleming

ger.fleming@nuigalway.ie


ext. 3562

The Scope:


This course seeks to introduce students to
those aspects of applied microbiology which
they are likely to encounter in the
Fermentation/Medicare sector. Knowledge of
the techniques for growing microorganisms
together with sterilization practices
contributes to Good Manufacturing Practice

Learning outcome


Demonstrate a knowledge and understanding of
Industrial Bioprocesses by successfully
attempting an examination question and
accruing marks for the same at the end of
semester 1.


Take elements from the course that you might
apply to your 4
th

year project next year.

Ger: 6 lectures

Research, development and scale
-
up:


Typical objectives
-

qualitative and quantitative
(titre, yield and volumetric productivity) and
restraints.


Primary and secondary screening
-

the use of
shake flasks, lab fermenters and pilot plant.
New approaches to screening.

Organisms:

Choice and storage.

Process improvement by strain selection
-
avoiding induction, repression and inhibition
-
use
of auxotrophs


Media and Process manipulation

Economic considerations
-

crude v defined
-

carbon sources
-
nitrogen sources
-

vitamins and
growth factors
-

minerals
-

inducers
-
precursors
-

inhibitors.

The Process
….continued…



What is a bioprocessor (fermenter)
-

pH,
temperature, foam/antifoams and
agitation/aeration.



Industrial batch cultures
-

inoculation
development and fermentation build up
-

when
to harvest
-

fed batch cultures.

Continuous cultures with and without recycling.

Dr. Paul McCay: (4 lectures)


Sterility and Asepsis
-

Definitions and
reasons:

Lecture 8 and 9 Basic heat treatments and
large (industrial) scale heat sterilisation


Recommended Text:
Principles of
Fermentation Technology by P.F. Stanbury, A

Whitaker and S.J. Hall (2nd ed.) Pergamon
Press, 1995.


What’s it all about?

Substrate

Organism

What’s it all about?

Substrate

Organism

Process

What’s it all about?

Substrate

Organism

Process

Product

What’s it all about?

Substrate

Organism

Process

Product

MONEY

Learning About Industrial
Microbiology


Come to Lectures


Dip in and out of:

Principles of Fermentation Technology;
PFT

(Stanbury Whittaker and Hall)… if you get
stuck



My door is always open….do not hesitate
to drop down


Today


Large and small scale processes


Improving process economics


The large
-
scale process


Biomass, enzymes, primary and
secondary metabolites


Need for growth of the organism?


Large and Small Scale
Processes

Large Scale Process


Example:


300,000L (63,000
gal) Bioprocessors


30m high


Producing MSG

Corneybacterium used
for production of

200,000 tons MSG
(Glutamine) and

65,000 Tons Lysine

Large Scale Processes

Volume

10,000L to 100,000L+

Product value

Low (Low value added)

Product types

Biomass, Bulk chemicals,
Antibiotics, Most enzymes

R & D
development

Fermentation
Technology/process
engineering, strain and
medium manipulation etc. to
improve process economics

R & D Cost

Low

How can we improve process
economics?


Better Product Yields



Higher Product Titres



Improved Volumetric Productivity

Product Yield


The amount of product we get for a
given amount (or in practice, cost) of
substrate (raw material).



Important when substrates are a major
proportion of product costs.

Product Titre


The concentration of product when we
harvest the bioprocess



Important when purification costs are a
major proportion of product costs

Volumetric Productivity


The amount of product produced per unit
volume of production bioprocessor per unit
time. (or, in crude terms “how fast does the
process go”)



NOTE: “Time” includes down time, turn
-
round
time etc.



High Volumetric Productivity minimises the
contribution of fixed costs to the cost of the
product.

How can we improve process
economics?


Better Product Yields


Higher Product Titres


Improved Volumetric Productivity


IMPORTANT:

Bear these in mind when we
discuss Organisms. Media and Processes.


We try to OPTIMISE the above.

Small Scale Processes

Volume

100L to 1,000L

Product value

High (High value added)

Product types

Therapeutics, Diagnostics,
Products from recombinant
micro
-
organisms & cell cultures.

R & D Thrust

Initial product development,
validation and approval. Genetic
Engineering

R & D Cost

High

Small Scale Processes


150 L System



NOTE: Containment
is a concern when
working with
recombinant micro
-
organisms

Traditional Processes

Some makers of :

Alcoholic Beverages

Cheese, Yoghurt etc.

Vinegar

May take advantage of
scientific knowledge, but
do not operate modern
“industrial fermentations”



Traditional Processes


It is difficult to
quantify what makes
a good product


There is no
substitute for a
craftsman


If it isn’t broke don’t
fix it!


Major Groups of Large Scale
Processes

1.
Biomass

2.
Enzymes

3.
Metabolites


Primary Products of
Catabolism e.g. Citric acid


Intermediates


e.g. glycine in Nitrogen
metabolism


Secondary products

e.g.
penicillin

4.
Biotransformations

Growth =
production

No Growth
Needed

Biomass


Bakers Yeast (
Saccharomyces
cerevisiae
)



Bacterial Insecticides (
Bacillus
thuringensis
)



Nitrogen Fixing Inoculants (bacteria:
e.g.
Rhizobium
)

Biomass


Single cell protein:


For Animal feed


Upgrading low value agricultural
products:


Cellulose


Starch


Use yeasts or fungi


Profit margins very small


competitive
market


For Human consumption


Fungi (eg Quorn)
Fusarium venenatum


Enzymes (see table 1.1 PFT)



Often depolymerases (eg. Amylases,
Proteases)


Large range of uses (and purities):


Food


Pharmaceuticals


Detergents


Industrial Microbiology (Medium
Preparation)


Leather Preparation


Enzymes (see table 1.1 PFT)


Organisms used for production:


Bacteria (especially
Bacillus
)


Yeasts (eg
Saccharomyces
)


Fungi (eg
Mucor
)


Problems caused the cell’s control systems
(induction, repression) may need to be
overcome:


Mutate/engineer organism


Medium formulation


Process manipulation (substrate supply)

Primary Metabolites


Products of Catabolism


By
-
products of the cell’s energy yielding
processes


“Normal” cells produce significant
quantities (but we can improve on this!)


Examples:



Ethanol


Alcoholic Beverages (

0.07/l)


Fuel (and industrial) Alcohol (

0.9/l)

Ethanol:


C
3
H
6
O
3

Converts to C
2
H
5
OH+ CO
2


Beverages


Organism: Yeast (
Saccharomyces

cervisiae

or
uvarum
)


Some substrates immediately available:


Grape juice (Wine, Brandy)


Sugar Cane (Rum)


Some substrates need pre
-
treatment to
depolymerise starch and protein:


Malt (Beer, Whisky)


Cereals, potatoes etc. plus malt , enzymes etc
(vodka, other spirits, some beers etc.)


Post
-
fermentation treatment may include
distillation (spirits) and/or maturation.

Ethanol


Fuel/Industrial Alcohol


Organisms:


Yeasts


Bacteria (
Zymomonas
): fast but sensitive to
product.


Substrates: Cheap Agricultural products:


Sucrose (Sugar Cane)


Starch type products (Depolymerise with
enzymes etc. or obtain organism with amylase
activity)


Very low value added/Competitive market (but
Government support?).


Conventional distillation step can make the
process uneconomical:


Use vacuum (low temperature) distillation
during fermentation.

Primary Metabolites

Metabolic Intermediates


Intermediates in metabolic pathways
(TCA cycle, pathways leading to protein
and nucleic acid production etc.).


Levels of intermediate pools generally
low in healthy “wild type” organisms


Need to develop industrial strains:


Overcome feedback inhibition/repression.

Citric Acid Cycle


Primary Metabolites

Metabolic Intermediates


Examples:


Citric Acid (Soft Drinks, Foods etc.)


Lysine (Essential AA, Calcium absorption,
Building blocks for protein)


Glutamic acid (Monosodium Glutamate
precursor)


Phenylalanine (Aspartame precursor)



Organisms Yeasts. Fungi, Bacteria:


Corynebacterium

for amino acid production

Secondary Metabolites


Not part of the “central” metabolic pathways
(see Fig 1.2 of the book)


Producers:


Actinomycetes (eg
Streptomyces
)


Fungi (eg
Penicillium
)


Sporeforming bacteria (
Bacillus
)



Produced as growth slows/stops in batch
cultures


Antibiotics

are of major industrial importance

Secondary Metabolite
production in Batch Culture


1. Trophophase


Culture is
nutrient sufficient


Exponential
Growth


No Product
Formation

Secondary Metabolite
production in Batch Culture


2 Idiophase


Carbon limitation


Growth slowing or
stopped


Product formation


HARVEST AT THE
END OF THIS
PHASE

Secondary Metabolite
production in Batch Culture


3 Senescence


Product formation
ceases.


Degeneration/lysis of
mycelium (Fungi,
Actinomycetes)


Product
degraded/used by
culture.

Biotransformation


Use cells as “catalysts” to perform one or two
step transformation of substrate.


Use cells several times:


Fungal/Actinomycete mycelium


Immobilised bacteria or yeast cells packed
into a column


Examples:


Transformations
of plant sterols by
Mycobacterium fortuitum
”.



Ethanol to Acetic acid (immobilised
Acetobacter
)

Growth


A necessary Evil?


When a culture grows more cells are
produced. Unless our product is biomass this
seems a waste of materials and time.

BUT


Cells are the agents responsible for product
formation. We must have enough for this to
take place rapidly and efficiently.

Growth


A necessary Evil?


A major challenge is to balance growth and
product formation:


The two process separate naturally for
secondary metabolites (batch culture)


We may manipulate the process to
separate them e.g. temperature
-
sensitive
promoters


The growth phase is then optimised for
growth and the production phase for
product formation.