CHE 4171 Biochemical Engineering

Revision for final exam

Sterilization

Steps to avoid contamination

Use pure inoculum
Media sterilization

Fermenter sterilization
Sterilize all raw materials
Maintain aseptic conditions during process

Sterilization agents
Thermal

preferred for economical large-scale sterilizations of liquids

and equipment.

Chemical (preferred for heat-sensitive equipment)

ethylene oxide (gas) for equipment
70% ethanol-water (pH=2) for equipment/surfaces
3% sodium hypochlorite for equipment

Radiation

UV for surfaces, x-rays for liquids (costly/safety)

Filtration

membrane filters having uniform micropores

depth filters of glass wool

Question
A fermentation medium has a concentration of
spore c = 103 spores /L. It is known that specific
death constant of spore is 0.98 min-1 at 1210C,
and medium is sterilized at 121C. The
destruction of microorganisms by steam may be
described as a first order reaction.

Calculate the sterilization time needed for 1 L

and 10,000 L tank to ensure only 1 out of 1000
fermentation is unsuccessful.

For 1L
N0= 103
Probability of unsuccessful, 1-P= 0.001
From chart:
kt = 14
Hence, t = 14/0.98 = 14.3 min

For 10, 000L

No= 107
1-P= 0.001,
From chart:
kt = 23
Hence, t = 23/0.98 = 23.5 min

Question
Based on your calculation above,
Compare the difference in sterilization time
required for small and large bioreactors.
What is the consequence of this difference?

Solution

Sterilization time is longer for larger bioreactor.

Energy consumption will be higher; quality of
medium (nutrients) may change depending on
the thermal stability.

Question
Describe one alternative way of sterilization to
avoid the undesirable consequence identified in
step (ii)

Solution
To avoid thermal instability, we could sterilize by
passing through filter.

Del factor

Del factor ( ) = ln ( No/Nt )

Del factor ( )

ln t = E/RT + ln (/A)

ln t

1/T

Del factor ( ) = degree of sterilization

Principles of Fermentation Technology,- Peter F. Stanbury,
Allen Whitaker, Stephen J. Hall, Second Edition.

12

Heat Sterilization
Two mode of heat sterilization processes:
Batch
Continuous
ADVANTAGES OF CONTINUOUS:

Continuous mode

1. Shorter time
Reduction of sterilization cycle time
2. Higher temperature
Ease of scale-up
Superior maintenance of medium quality (less destruction)
Reduced surge capacity for steam (more efficient plant use)

ADVANTAGES OF BATCH:

Lower capital cost (bioreactor used as autoclave)

Lower contamination risk (less transfers of liquids)
Presence of solids (particles) less of a problem

Batch mode
1. Longer heat-up/cool down time
2. Incomplete mixing
13

Flow conditions
to meet cell
reduction
requirements

Pe = uL / Dz
Ratio of (cell death rate) / (cell flow rate)
Bioprocess Engineering, Doran
Academic Press, 1997.

Flow in pipes
Plug flow uniform velocity
profile little mixing or variation
in fluid velocity.
Turbulent flow: Re > 2x104
Laminar flow: Re < 2100
parabolic velocity profile
Plug flow
Turbulent flow
Laminar flow

: Dz = 0
: Dz = moderate
: Dz = large

For sterilization processes, we want plug flow!

Operating Consideration for

Bioreactor

Question
Cell immobilization system is another commonly
used bioreactor systems for microbial, animal and
plant cells culture.
i) Identify two commonly used active immobilization
methods. Give one example for each method.

1) Entrapment in a porous matrix.

E.g. Polymeric beads, Encapsulation, hollow

fiber membrane

2) Cell Binding to Inert Supports.

Eg: Micro-porous support.

ii) Name 4 advantages and 2 disadvantages of

cell immobilised system.
Advantages:
High cell concentration; cell reuse without cell
recovery facility; eliminates cell washout at a high
dilution rates; high volumetric productivities; provide
favourable microenvironment; genetic stability;
protection from shear damage.

Disadvantages:
Mechanical disruption of the immobilizing matrix;
mass transfer limitation; limited to extracellular protein
production.

Synthetic Biology

Synthetic Biology the goal

modifying living organisms for beneficial purposes such as
production of renewable energy, pharmaceuticals and
sensors
a straightforward process:
characterise gene sequences that perform needed functions
(parts)
combine the parts into devices to achieve more complex
functions
insert the devices into cells or chassis

develop a synthetic biology toolbox of reusable genetic

components as biological versions of transistors and
switches
For more information: Kwok R (2010) Nature 463: 288

Synbio jargon
A part is a DNA sequence encoding some part of the
genetic machinery, including but not limited to:

Promoters
Ribosome binding sites (RBS)
Protein coding regions
Terminators

A device is a group of parts that work together for specific

functions, such as:

Protein production
Sensing/reporting
Measurement
Signal inversion
Cell signaling
Cell motility

A chassis:

Host cell for the parts/devices. Mainly E. coli, some B. subtilis and
yeast plus new emerging chassis

Question
Synthetic biology is increasingly being used to
improve or invent new biological production
processes for fuel, chemical and nutraceutical
products.

Describe one biological production process that

has been developed using synthetic biology
methods.

Solution
Artemisinin new gene pathway into yeast
Lycopene improved gene pathway in E
coli/bacteria
Isobutaldehyde new fuel made from CO2 in
algae
Biosensor/quorum oscillator engineering light
production in bacteria
Synthetic life/new organism producing a new
genome outside the cell then inserting it into anucleate cell

Artemisinin pathway engineering in yeast

The complete biosynthetic pathway that provides an efficient biosynthetic route to artemisinic
acid, with fermentation titres of 25 g L-1 of artemisinic acid Saccharomyces cerevisiae.
Paddon CJ et al (2013) High-level semi-synthetic production of the potent
antimalarial artemisinin. Nature, 10th April online

Direct photosynthetic recycling of carbon dioxide to

fuels/chemicals
Atsumi S et al (2009) Nature Biotech 27:1177

New pathway engineered into Synechococcus

to enable light + CO2 isobutaldehyde
directly

Low vapour
pressure

Synechococcus elongatus
PCC7942

A synchronized quorum of genetic clocks

Danino T. et al (2010) Nature 463: 326

A gene network engineered with global intercellular

coupling to generate synchronised oscillations in a growing
cell population
Investigated collective synchronisation properties along
with spatiotemporal waves occurring at mm scales
Used computational modelling to measure the observed
dependence of the period and amplitude of the bulk
oscillations on the flow rate

Macroscopic biosensor with an oscillatory output

Question
List major issues that a synthetic biology project
is likely to encounter.

Synthetic Biology current status

Many of the biological parts are undefined
Poor quality control of parts
Inherent variability from random insertion events during transformation
into genomes
Needs: new improved curated collections of parts; directed insertion of genes

The circuitry is unpredictable

The parts may not work as expected when put together and much trialand-error is still required
Needs: greater use of computer modelling and directed evolution strategies should
speed this process

The complexity is unwieldy

Gene discovery, pathway assembly and optimisation the 12-gene route
for artemisinin (more later) synthesis by microbes took around 150
person-years of work
Needs: more automated processes, dedicated assembly cells for gene cloning
For more information: Kwok R (2010) Nature 463: 288

Synthetic Biology current status

Many parts are incompatible
Promoters from one organism may not work in another or can be
blocked/shut down/toxic/mixed response
Need: separate production systems; physical separation of
production from homeostatic processes

Variability crashes the system

Inherent variability in gene expression, protein production and
modification among homogeneous cell populations
Needs: careful control of growth conditions and cell-cell
communication can reduce variability; embrace variability;
improving DNA replication and mutation rate
For more information: Kwok R (2010) Nature 463: 288

Solution
Complexity; genes do not work as expected;
faulty genes; variable expression; bottlenecks;
unpredictable; non-exchangeable genes; large
pathways etc

Plant Tissue Culture

INITIATION OF A PLANT CELL SUSPENSION

1)

Remove
explant from
whole plant.

FRAGMENTS OF
PLANT MATERIAL
2) Surface sterilise explant. Use bleach,
bleach + surfactant, or mercury chloride.

PLANT
3) Place explant on solid
medium for callus induction.

5) Place callus in liquid

medium and shake to form a
plant cell suspension
culture. This takes 34 weeks
under good conditions.

4) Under appropriate
conditions, callus
forms in 36 weeks.

CALLUS TISSUE
SUSPENDED PLANT CELLS

Question
Anticancer drug, paclitaxel was originally extracted
from bark of the Pacific yew. However, it is now
produced using plant cell culture technology.
What are the advantages of using plant cell culture
to produce paclitaxel in comparison to the
extraction from the barks of Pacific yew trees?

Anticancer drug Paclitaxel (taxol) (I)

Taxol displayed some promise as an anticancer agent in 1971.

Paclitaxel was found to cause regression of mammary tumors in 1978.

Mechanism: An antimicrotubule agent, blocking cell growth by stopping
mitosis (cell division).
In 1992, the FDA approved the drug. Bristol-Myers Squib sold it at a
wholesale price of just under $1000. Most expensive drug by then.

http://www.paclitaxel.org/background.html

Anticancer drug Paclitaxel (taxol) (II)

Supply problems:
Each tree could supply about 2 kilograms of bark.
12 kilograms of bark was needed to produce one half gram of Taxol.
The Yew tree dies after stripping the bark.
In 2011 the International Union for Conservation of Nature designated a

species of Asian Yew Tree used to harvest paclitaxel as "endangered"

http://www.paclitaxel.org/background.html

http://en.wikipedia.org/wiki/Paclitaxel

Advantages of production of chemicals

from plant cell culture
Control of supply of product independent of the availability of plant itself.
Cultivation under controlled and optimized conditions.
Strain improvements with programs analogous to those used for
microbial systems.
Novel compounds not present in nature can be synthesized by feeding
of compounds analogous to natural substrates.
No need to use harmful herbicides and pesticides.
Not affected by climate, geographical location, insect infestation, plant
disease, etc.

Solution
Control of supply of product independent of the
availability of plant itself (Asian Yew Tree is
endangered species)
Cultivation under controlled and optimized
conditions.
Strain improvements with programs analogous to
those used for microbial systems.
No need to use harmful herbicides and pesticides.
Not affected by climate, geographical location,
insect infestation, plant disease, etc.

Question
Cell growth and secondary metabolite production
in plant cell culture usually require different nutrient
and culture conditions.
Suggest a suitable mode of operation and explain
how these limitations can be overcome in the
proposed mode of operation.

Technologies for increasing the production

of chemicals by plant tissue cultures
1)Strain selection
2)Fermenter design
3)Two-stage processing and media optimisation

 Cells in the first stage are cultivated to high cell density using
medium optimised for rapid growth.
The culture is harvested and the cells separated from the
liquid under aseptic conditions.
The biomass is then transferred to the second stage where
appropriate medium and culture conditions are provided to
stimulate secondary metabolite synthesis

Question
Root cultures can produce hyoscyamine but cannot
transform it to scopolamine, while shoot cultures are
able to synthesis scopolamine if hyoscyamine is
supplied as a precursor.

Sketch a suitable fermentation system for the

production of scopolamine. Clearly label the key
components of the proposed system.

Other technologies can be developed to

address these issues:

Immobilised cells

Cell aggregates

Elicitors

Organ cultures roots and shoots

Organ co-cultures

4)

Organ co-cultures

Neither root nor shoot cultures are capable of synthesising

scopolamine on their own.
Root cultures make hyoscyamine but cannot transform it to
scopolamine.

Shoot cultures may be capable of making scopolamine but

required the precursor, hyoscyamine.
A solution to this problem is the use of organ co-cultures,
where hairy roots and shooty teratomas are cultured
together in a shared medium.

Rootshoot co-culture using

dual shake flasks

Rootshoot co-culture in
bioreactors

Single bioreactor

Atropa belladonna
hairy roots

Duboisia hybrid
shooty teratomas

hyoscyamine

Root culture

scopalamine

Shoot culture

Animal Cell Culture

Why animal cells?

Post-translational modification
glycosylation, acylation and phosphorylation
essential for full bio-activity of the protein

Viral vaccine and vector

virus are species specific

Artificial organ/tissue
prolong the life of the patient at the critical stage

Ethical issues
the use of animal for toxicological test

Application of animal cell cultures

Manufacture of biopharmaceuticals.
(e.g., mAb, hormone, interferon)
Propagate virus as vaccine and vector for gene
therapy.
Artificial organs.
Toxicological research replacing the need of animal
test.

Question
Animal cells that originated from fibroblast-like
cells are anchorage dependent.
Identify 4 suitable culture vessels that suitable
for the anchorage dependent cells.

Monolayer cultures
T-Flask , multitray
roller bottle

Immobilised cell bioreactor

hollow fibre
air lift bioreactor
wave bioreactor

Culture vessels

Anchorage independent cells

T-Flask

Hollow fibre

Wave bioreactor
Stirrer tank bioreactor/spinner flask

Air-lift bioreactor

Culture vessels
monolayer cultures
(T-Flask , multitray)

Anchorage dependent cells

- hollow fibre

roller bottle
- air lift bioreactor

microcarrier systems

immobilized cell bioreactor

- wave bioreactor

Question
A finite animal cell lines can only divide for a limited
number of generations.

Identify 4 techniques that can be used to establish

continuous cell lines.

 Inherently, originated from cancerous cells [e.g.,

myeloma cell (NS0)]
Infected with a virus (e.g. SV 40, EBV)
Transfected with a vector derived from a
transforming virus.
Fusion with a continuous cell line (e.g.
hybridoma)

Question
Animal cells are generally fragile, slow growing,
low productivity, and low cell density.
Propose a suitable bioreactor type and mode of
operation for animal cell culture. Justify your
selection.

Suitable type of bioreactor:

stirred tank reactor with low shear impeller
(marine propeller)
wave bioreactor (low shear)
hollow fibre reactor (low shear)
Suitable mode of operation:
fed batch culture (higher productivity can be
achieved in fed batch culture)
perfusion culture (higher productivity and cell
density can be achieved in perfusion culture)