BIO2013LN

CBB 20104

Chapter 2: Bioprocess
Reaction

CONTENT
MAJOR METABOLIC PATHWAYS
INTRODUCTION TO METABOLISM
GLUCOSE METABOLISM
GLYCOLYSIS, KREBS CYCLE, RESPIRATION
BIOSYSTHESIS

FERMENTATION
BIOTRANSFORMATION
BIOCONVERSION
OPERATING CONSIDERATIONS FOR BIOREACTORS

FOR SUSPENSION AND IMMOBILIZED CULTURES

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MAJOR METABOLIC PATHWAYS

INTRODUCTION
Metabolism is the collection of enzyme -catalyzed

reactions that convert substrates that are external

to the cell into various internal products

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CHARACTERISTICS OF METABOLISM
Varies from organisms to organism
Many common characteristics

Affected by environmental conditions

O2 availability: Saccharomyces cerevisiae
Aerobic growth on glucose more yeast cells
Anaerobic growth on glucose ethanol
Control of metabolism is important in bioprocesses

TYPES OF METABOLISM
Catabolism
Metabolic reactions in the cell that degrade a substrate

into smaller / simpler products.

Glucose CO2 + H20
Produces energy for the cell
Anabolism
Metabolic reactions that result in the synthesis of
larger /more complex molecules
Glucose to glycogen
Requires energy

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BIOENERGETICS
The source of energy to fuel cellular metabolsim is

reduced forms of carbon (sugars, hydrocarbons, etc.)

The Sun is the ultimate source via the process of

Photosynthesis in plants
CO2 + H2O + hv CH2O + O2

Figure 5.1: Classes of Reactions

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ATP - Adenosine Triphosphate

Catabolism of carbon-containing substrates
generates high energy biomolecules

ATP - Reactions

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ATP: Energy Currency of the Cell (Fig.

5.2)

NAD+ and NADP + (Fig. 5.3)

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GLUCOSE METABOLISM:
Catabolic Pathways of Primary Importance
1.
2.
3.

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Glycolysis: from glucose to pyruvate.

Krebs or tricarboxylic acid (TCA) cycle for
conversion of pyruvate to CO2.
Respiration or electron transport chain for
formation of ATP by transferring electrons from
NADH to an electron acceptor (O2 under aerobic
conditions).

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Glycolysis: in Eucaryotes

Krebs or TCA Cycle

In Mitochondria of eucaryotes
provides e- (NADH) and ultimately energy (ATP) for

biosynthesis
provides intermediates for amino acid synthesis
generates energy (GTP)

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Krebs or TCA Cycle

Krebs or TCA Cycle

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Complete Oxidation of Glucose

Energetics of Glucose Oxidation

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ATP Yields

Respiration

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Respiration
Goals of Respiration
1. Regenerate NAD+
2. Generate ATP

Oxidative
Phosphorylation

BIOSYNTHESIS
The EMP pathway and TCA cycle are critical

catabolic pathways and also provide important

precursors for the biosnythesis of amino acids,
nucleic acids, lipids and polysaccharides.
The Hexose - Monophosphate pathway (HMP)

is used for biosynthesis

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HMP
Pathway

Amino Acids by Various Pathways

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Fermentation:
No TCA Cycle or Respiration

Products from
fermentation

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Metabolic Engineering (ME)

Repeated mutations were necessary to create strains of the mold

Penicillium chrysogenum which produce high titers of penicillin; that became
the foundation of a commercial process and changed human health
care.
Radiation and chemical agents were employed
by investigators to induce mutations in the
microorganism.

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Scope of metabolic engineering

Modify host cells, host multicellular organisms, or

product
Improved production, in selectivity or in quantity, of
chemicals already produced by the host organism
Extended substrate range for growth and product
formation
Addition of new catabolic activities for degradation of
toxic chemicals
Production of chemicals new to the host organism
Modification of cell properties

General methodology of metabolic

engineering
1. Identify the target phenotype or trait
2. Increase the frequency of occurrence of gene(s) that may confer the
phenotype
Increase the mutation frequency in producing cells by Mutagen
treatment (UV, X-ray, chemical mutagen) (Classical method)
Introduce additional gene(s) (that may already exist or absent in the host
cell) known to give cells the desired properties (Genetic Engineering)
Introduce genetic element to inactivate or activate the gene by random
insertion of extra sequence
3. Identify the mutants (clones) that have the desired trait.
Two general means
Screening
Selection

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Strategies of improving production

Typical objectives of metabolic engineering for chemical

production formation
Modify the pathway
Amplify the rate limiting enzyme
Redirect the flux at the divergent branch (or node) of the pathway
Remodel the regulatory element of the protein by protein engineering
or using a heterologous enzyme
Replace an enzyme(s) that is energetically or kinetically more
favorable.
Amplify the first enzyme in the pathways, then identify the potential
rate limiting steps

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Strategies of improving production

Enhancing the precursor and energetic supply by

engineering central metabolism

Engineering the transport system
Engineering the substrate and precursor uptake
Increase the rate
Change the specificity to use new substrate of new precursor
Engineering the product secretion
Engineering the tolerance to its own product or high
substrate concentration
Decouple the growth and production
Need biomass for product formation
Too much biomass diverts the sources if the objective is to
produce the product.

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BIOPROCESS REACTIONS

BIOPROCESS REACTION
Fermentation
Biotransformation
Bioconversion
Bioremediation

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FERMENTATION
Definition: microbial process in which enzymatically

controlled transformations of organic compounds occur

Fermentation has been practiced for years and has

resulted in foods such as bread, wine, and beer

Some important fermentation products

Product
Ethanol
Glycerol
Lactic acid
Acetone and butanol
-amylase

Organism
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Lactobacillus
bulgaricus
Clostridium
acetobutylicum
Bacillus subtilis

Use
Industrial solvents,
beverages
Production of
explosives
Food and
pharmaceutical
Solvents
Starch hydrolysis

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The Range Of Fermentation Processes

There are four major groups of commercially important

fermentations :
Those that produce microbial cells (or biomass) as the
product (Bakers yeast and food)
Those that produce microbial enzymes(Amylase)
Those that produce microbial metabolites (Ethanol,
Citric acid)
Those that modify a compound which is added to the
fermentation the transformation processes (ethanol to
acetic acid)

The fermentation plant

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The fermentation plant cont..

The component parts of a fermentation process

The formulation of medium

Sterilizing the medium
Seed fermenter
Production fermenter
Extraction and purification
Disposal effluent

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Media for Industrial Fermentations

The media is the feed solution
It must contain the essential nutrients needed for the microbe
to grow

Factors of consideration when choosing media

Quality consistence and availability
Ensure there are no problems with Media Prep or other aspects
of production process

Ex. Cane molasses, beet molasses, cereal grains

Sterilization
Sterilizing the feed solution is essential because the media
cannot contain foreign microbes because this could severely
hinder the growth of the production microbe

Most popular method is heat sterilization of the feed solution

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The Development of Inocula for

Industrial Fermentations
The inoculum is the starter culture that is injected into
the fermenter
It must be of sufficient size for optimal growth kinetics

Since the production fermenter in industrial

fermentations is so large, the inoculum volume has to be
quite large
A seed fermenter is usually required to produce the
inoculum volume
The seed fermenters purpose is not to produce product but
to prepare inoculum

Schematic diagram

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BIOTRANSFORMATION
Definition- biological process whereby an organic

compound is modified into a recoverable product by

simple, chemically defined reactions catalyzed by enzymes
contained in the cells.
Differ from fermentation results from complex
biosynthetic machinery primary and secondary metabolites
Mechanism-substrate added to microbes to transform.
Examples: production of steroids, conversion of antibiotics
and prostaglandins.

The essential difference between fermentation and

biotransformation is that there are several catalytic

steps between the substrate and the product in
fermentation while there is only one or two in a
biotransformation.
The distinction is also in the fact that the chemical

structures of the substrate and the product resemble

one another in a biotransformation, but not
necessarily in fermentation

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Example of biotransformation

Acetobacter

Chemical
D- glucose

D- sorbitol

L- sorbitol

Chemical

Ascorbic acid

The world of biotransformation

Chemical modification (or modifications) made by an organism on

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a chemical compound.
If this modification ends in mineral compounds like CO2, NH3+
or H2O, the biotransformation is called mineralisation.
Biotransformation means chemical alteration of chemicals such as
(but not limited to) nutrients, amino acids, toxins, or drugs in the
body. It is also needed to render nonpolar compounds polar so
that they are not reabsorbed in renal tubules and are excreted.
Biological process obey law of chemistry
Important when high specificity required
Process condition: low temperature, low pressure and aqueous
Dilution is main disadvantage

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BIOCONVERSION
Using microorganism to biocatalyze specific chemical reaction

beyond the capabilities of organic chemistry

Involves growth in fermentors with specific condition
After process, desired product extract and purified.
Glucose to fructose by glucose isomerase

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Diagram

BIOREMEDIATION
A process that uses naturally occurring or genetically

engineered microorganisms such as yeast, fungi and

bacteria to transform harmful substances into less toxic
or nontoxic compounds
Degrade contaminants as a source of carbon and energy
sources
Application-decomposting waste landfills

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Advantages and disadvantages

Advantages : Bioremediation is ecologically sound, natural

process; it destroys target chemicals at the contamination

site, less expensive
Disadvantages : using bioremediation often takes longer than

other remedial method such as excavation or incineration

OPERATING CONSIDERATIONS FOR

BIOREACTORS FOR SUSPENSION
AND IMMOBILIZED CULTURES
Cultivation method
Batch and continuous reactors
Immobilized cell systems
Solid state fermentations

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CHOOSING THE CULTIVATION METHOD

BATCH
CONTINUOUS

Most of commercial bioprocesses are batch systems

Why ?
Productivity
Many secondary products are not made by growing cells;
growth represses product formation.
Under such circumstances, product is made only at low dilution
rates
For secondary products, the productivity in a batch reactor may
significantly exceed that in a simple chemostat

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Genetic instability
Biocatalyst has undergone extensive selection
These highly bred organisms often grow less well than the parental

strain
Back mutation from the productive specialized strain to one similar
to the less productive parental strain is always present for chemostat
In the chemostat, the less productive variant will become dominant,
decreasing productivity

Operability and reliability

Batch cultures can suffer great variability from one run to

another
Variations in product quality and concentration create problems
in downstream processing and are undesirable
However, long term continuous culture can be problematic;
pumps may break, controllers may fail and so on
Maintenance of sterility can be very difficult to achieve for
periods of month and the consequences of a loss of sterility are
more severe than with batcch culture

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Market economics
Many fermentations product are required in small amounts, and

demand is difficult to project

Batch process provide much greater flexibility
The same reactor can be used for two months to make product
A and the next three for product B and the rest of the year for
product C

Most bioprocesses are based on batch reactors

Continuous systems are used to make single cell protein
(SCP)
Modified forms of continuous culture are used in waste
treatment

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BATCH AND CONTINUOUS

REACTORS
Batch: Media and cells are added to the reactor and it is run until

a predetermined set point (i.e. time, concentration). The

bioreactor has a constant volume (the initial volume).
Fed-Batch:The bioreactor is a batch process in the beginning and after

a certain point a feed input is introduced and the volume of the vessel
increases.
Continuous:The bioreactor starts with an initial volume and media is

constantly introduced and product is constantly taken out. The inputs

and outputs are at the same rate, so the volume always remains the same.

Modifying batch and continuous

reactors
Chemostat with recycle
Multistage chemostat systems
Fed batch operation

FR, XR
F, X2
F, X0

V,
X1

F+FR,
X1

P10
Growth stage

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P2

Product formation stage

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IMMOBILIZED CELL SYSTEM

Restriction of cell mobility within a confined space
Potential Advantages:
Provides high cell concentrations per unit of reactor

volume.
Eliminates the need for costly cell recovery and
recycle.
May allow very high volumetric productivities.
May provide higher product yields, genetic stability,
and shear damage protection.
May provide favorable microenvironments such as
cell-cell contact, nutrient-product gradients, and pH
gradients resulting in higher yields.

IMMOBILIZED CELL SYSTEM

Potential Disadvantages/Problems:
If cells are growing (as opposed to being in stationary

phase) and/or evolve gas (CO2), physical disruption of

immobilization matrix could result.
Products must be excreted from the cell to be
recovered easily.
Mass transfer limitations may occur as in immobilized
enzyme systems.

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METHODS OF IMMOBILIZATION
Active Immobilization:

1. Entrapment in a Porous Matrix:

METHODS OF IMMOBILIZATION

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METHODS OF IMMOBILIZATION

METHODS OF IMMOBILIZATION
Active Immobilization:

2. Cell Binding to Inert Supports::

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METHODS OF IMMOBILIZATION

METHODS OF IMMOBILIZATION
Binding Forces:
Covalent Bonding: (review enzyme covalent bonding)
Support materials: CMC-carbodiimide
support functional groups
-OH, -NH2, -COOH
Binding to proteins on cell surface

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OVERVIEW OF ACTIVE CELL

IMMOBILIZATION METHODS

PASSIVE IMMOBILIZATION: BIOFILMS

The term biofilm refers to the multilayer growth of cells

on solid support surfaces

Biofilms are micro-colonies of microbial cells attached to
a surface and encased in adhesive polysaccharides
secreted by the cells
Biofilms trap nutrients for cell growth, and help prevent
detachment of cells on surfaces in flowing systems
Basic biofilm formation process involves attachment,
colonization and development

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PASSIVE IMMOBILIZATION (BIOFILMS)

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Biofilm formation is common in industrial fermentation

systems, such as biological wastewater treatment and

mold fermentations
In mixed culture microbial films, the presence of some
polymer-producing organisms facilitates biofilm
formation and enhances the stability of the biofilms
Micro-environmental conditions inside a thick biofilm
vary with position, and affect the physiology of the cells

IMMOBILIZED CELL BIOREACTORS

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SOLID STATE FERMENTATION

Solid state fermentations (SSFs) involve solid substrates

at low moisture levels or water activities

The water content of a typical submerged
fermentation is >95%
The water content of a typical solid state fermentation
is typically between 40-80%
usually used for the fermentation of agricultural
products or foods, such as rice, wheat, barley, corn and
soybeans
The low moisture levels of SSFs acts as a powerful
selection pressure for the growth of mycelial organisms

Advantages of SSFs over

conventional submerged fermentations
The small volume of fermentation mash or rector

volume results in lower capital and operating costs

A lower chance of contamination due to low moisture
levels
Ease of product separation
Energy efficiency
Allows the development of fully differentiated
structures, which is critical in some cases to product
formation

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Disadvantage is the heterogeneous nature of the

media, due to poor mixing characteristics
Results in control problems (pH, DO, temperature)

within the fermentation mash

For large fermentation mash volumes, it can be difficult to
provide sufficient mixing to prevent concentration
gradients from forming
At high agitation speeds, mycelial cells may be damaged
Rotary-tray or rotating-drum fermenters are often used
to provide gentle yet adequate agitation in SSFs

Some Traditional Food

Fermentations

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END OF CHAPTER 2

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