BIOREACTOR SYSTEM

ERT314

Dr Huzairy Hassan
School of Bioprocess Engineering, UniMAP
Semester 2, 2017/2018
STERILIZATION

 Commercial fermentations require thousands of liters of liquid

medium and millions of liters of air  must be free from
contaminating organisms.

 Methods of sterilization: chemical treatment, exposure to

ultraviolet, gamma, and X-ray radiation, sonication,
filtration, and heating.
STERILIZATION

1) Batch Heat Sterilization of Liquids

2) Continuous Heat Sterilization of Liquids

3) Filter Sterilization of Liquids

4) Sterilization of Air
STERILIZATION
 STERILIZATION

1) Batch Heat Sterilization of Liquids

• The liquid is heated to the sterilization

temperature by introducing steam into the
coils or jacket of the vessel.
• Alternatively:
• - steam is bubbled directly into the medium
• - vessel is heated electrically

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https://www.indiamart.com/proddetail/jacketed-process-vessels-8011609433.html
 STERILIZATION

1) Batch Heat Sterilization of Liquids

• Holding time, 𝒕𝒉𝒅 - determined to achieve desired level of cell

destruction + destroying contaminating organisms.
• Heat sterilization is also capable of destroying nutrients in the
medium.
• To minimize this loss  𝑡ℎ𝑑 at the highest sterilization T should be
kept as short as possible.
• Cell death occurs at all times during batch sterilization, including
within the heating-up and cooling-down periods.
 STERILIZATION
1) Batch Heat Sterilization of Liquids
 STERILIZATION

1) Batch Heat Sterilization of Liquids

First, we determine the number of cell 𝑵𝟏 𝒂𝒏𝒅 𝑵𝟐 in order to determine 𝒕𝒉𝒅  by
considering the kinetics of cell death:

𝑑𝑁
= −𝑘𝑑 𝑁 , where N = number of viable cells; 𝑘𝑑 = specific death constant.
𝑑𝑡

𝑵
𝒍𝒏 𝑵𝟏
𝑁
- Integrating during T constant (holding period)  ln 𝑁1 = 𝑘𝑑 𝑡ℎ𝑑 or 𝒕𝒉𝒅 = 𝟐
2 𝒌𝒅
 STERILIZATION

1) Batch Heat Sterilization of Liquids

𝑵𝟏 = number of viable cells at the start of holding
𝑵𝟐 = number of viable cells at the end of holding

𝑵𝟏 𝒌𝒅  evaluated as a function of T using Arrhenius

𝒍𝒏 equation:
𝑵𝟐
𝒕𝒉𝒅 = 𝑘𝑑 = 𝐴 𝑒 −𝐸𝑑 /𝑅𝑇
𝒌𝒅
A = Arrhenius constant / frequency factor
𝑬𝒅 = activation energy for thermal cell death
 STERILIZATION

1) Batch Heat Sterilization of Liquids

−𝑬𝒅
𝑑𝑁 𝒅𝑵
Combining = −𝑘𝑑 𝑁 & 𝑘𝑑 = 𝐴 𝑒 −𝐸𝑑/𝑅𝑇  = −𝑨 𝒆 𝑹𝑻 𝑵
𝑑𝑡 𝒅𝒕

Integrating

𝑵𝟎 𝒕 𝑵𝟐 𝒕
𝒍𝒏 = ‫𝒆𝑨 𝟏 𝟎׬‬−𝑬𝒅/𝑹𝑻 𝒅𝒕 𝒍𝒏 = ‫𝒆𝑨 𝒇 𝒕׬‬−𝑬𝒅/𝑹𝑻 𝒅𝒕
𝑵𝟏 𝑵𝒇 𝟐

Heating period Cooling period

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STERILIZATION

What about ---

 liquid contains solid particles (cell flocs & pellets)

 scaling up to larger volumes

 STERILIZATION

2) Continuous Heat Sterilization of Liquids

 High-temperature, short-exposure-time

 Reduce thermal damage to the medium significantly compared to batch

sterilization, while achieving high levels of cell destruction
 Improved steam economy and more reliable scale-up (amount of steam needed is 20
– 25% of that used in batch)
 Time required is significantly reduced because heating and cooling are almost
instantaneous.
Figure 14.38 Continuous sterilizing equipment:
(a) Continuous steam injection with flash cooling
(b) Heat transfer using heat exchangers
(a) CONTINUOUS STEAM INJECTION WITH
FLASH COOLING: Descriptions
1. Raw medium entering the system is first pre-heated by hot,
sterile medium in a heat exchanger. This economises on
steam requirements for heating and cools the sterile
medium.
2. Steam is then injected directly into the medium as it flows
through a pipe; the liquid temperature rises almost
instantaneously to the desired sterilisation temperature.
The time of exposure to this temperature depends on the
length of pipe in the holding section of the steriliser.
3. After sterilisation, the medium is cooled instantly by
passing it through an expansion valve into a vacuum
chamber (flash cooler)
4. Further cooling takes place in the heat exchanger where
residual heat is used to pre-heat incoming medium.
(b) HEAT TRANSFER USING HEAT EXCHANGERS:
Descriptions

• Raw medium is pre-heated with hot, sterile medium in a

heat exchanger
• then brought to the sterilisation temperature by further
heat exchange with steam.
• The sterilisation temperature is maintained in the
holding section
• Temperature is reduced to the fermentation temperature
by heat exchange with incoming medium.
STERILIZATION
STERILIZATION

Disadvantages
• Heat-exchange systems :
• are more expensive to construct than injection devices;
• fouling of the internal surfaces also reduces the efficiency of heat transfer
between cleanings.

• Steam injection:
• dilution of the medium by condensate
• foaming from direct steam injection can also cause problems with operation
of the flash cooler.
STERILIZATION

(a)Plug Flow: Fluid velocity is the same across

the diameter of the pipe.

(b)Turbulent Flow: The velocity distribution

approaches plug flow, however, there is some
reduction of flow speed at the walls.

(c)Laminar Flow: Fluid velocity is lowest at the

walls of the pipe and highest along the central
axis of the tube
 STERILIZATION

2) Continuous Heat Sterilization of Liquids

 Deviation from plug flow behavior is characterized by axial dispersion 
the degree to which mixing occurs along the length of axis of the pipe.
 Axial dispersion is a critical factor affecting the design of continuous
sterilizers.
 If axial dispersion is high, the performance of the sterilizer is reduced.
 STERILIZATION

2) Continuous Heat Sterilization of Liquids

 The relative importance of axial dispersion & bulk flow in the transfer of
material through pipes is represented by a dimensionless variable, Peclet
number, Pe
𝒖𝑳
𝑷𝒆 = Perfect plug flow  D𝑧 = 0
D𝒛 Pe is infinitely large
u = average linear fluid velocity
Typical Pe value: 3 - 600
L = pipe length
D𝑧 = axial dispersion coefficient
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STERILIZATION

𝑁1 = number of viable cells

entering the sterilizer
𝑁2 = number of cells leaving the
sterilizer
𝑘 𝐿
𝐷𝑎 = 𝑑 = Damköhler number
𝑢

𝑁2
 The lower the value 𝑁1
, the greater is cell
destruction
 At a given sterilization T, 𝑘𝑑 , 𝐷𝑎, the
performance of sterilizer declines significantly
as the Pe decreases.
STERILIZATION

What about ---

 liquid contains solid particles (cell flocs & pellets)

 compared to batch?
Example 14.8

Liquid medium at a flow rate of 2 m3 h-1 is to be sterilized by heat

exchange with steam in a continuous sterilizer. The medium contains
bacterial spores at a concentration of 5 X 1012 m-3. Values of the activation
energy and Arrhenius constant for thermal destruction of these
contaminants are 283 kJ gmol-1 and 5.7 X 1039 h-1, respectively. A
contamination risk of one organism surviving every 60 days of operation is
considered acceptable. The sterilizer pipe has an inner diameter of 0.1 m
and the length of the holding section is 24 m. The density of the medium is
1000 kg m-3 and the viscosity is 3.6 kg m-1 h-1. Determine the required
sterilizing temperature.
 STERILIZATION
3) Filter Sterilization of Liquids

Medium containing heat-labile

- Membranes used: cellulose esters or other
components (serums, differentiation
polymers
factors, enzymes, other proteins)
- Pore diameters: 0.2 – 0.45 μm
easily destroyed by heat
- As medium passes through the filter,
bacteria and other particles (dim.> pore
size) are screened out and collected on
- Membranes blocks medium must be the membrane surface.
pre-filtered to remove any large
particles
- High filtration flow rates require
large membrane surface areas.
 STERILIZATION
3) Filter Sterilization of Liquids
- Membranes must be sterilized by
steam or radiation before use
- Alternative: disposable filters &
filter cartridges

May not be as effective/reliable as

heat sterilization because viruses &
mycoplasma able to pass through the
membranes

0.1 μm membrane size  Ex.

used in mammalian cell culture
 STERILIZATION

4) Sterilization of Air
 No. of microbial cells in air ≈ 103 - 104 m-3
 Most common – Filters
  Depth filters – consisting of compacted beds or pads of
fibrous material (glass wool)
- Distances between fibers: 2 – 10 μm or up to 10 X greater
than bacteria/spores to be removed.
- Air penetrated depth filters & trapped cells are collected
by combination of impaction, interception, electrostatic
effects, & diffusion to fibers
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4) Sterilization of Air

http://www.biologydiscussion.com/biot
echnology/bioprocess-
technology/methods-for-sterilization-of-
media-and-air-with-diagram/10102
 STERILIZATION

4) Sterilization of Air
 STERILIZATION

4) Sterilization of Air

http://www.elmarco.com/applic
ation-areas/depth-air-filtration/
 STERILIZATION

4) Sterilization of Air

Limitation of depth filter:

 Do not perform well if there are large fluctuations in air flow rate
or air is wet  liquid condensing in the filter increases  the
pressure drop, causes channeling of the gas flow, and provides a
pathway for organisms collected on the fibers to grow through the
bed.
 STERILIZATION

4) Sterilization of Air

In industries, depth filters are being replaced by membrane cartridge

filters:
- Use steam-sterilizable or disposable polymeric membranes  trapping
contaminants as on a sieve.
- Contain a pleated, hydrophobic filter (pores 0.45 μm or less)
- Hydrophobic nature of filters  minimize problems with filter wetting
- High filtration area (packed cartridge volume)
- Pre-filters / upstream location  reduce fouling
 STERILIZATION

4) Sterilization of Air

Other applications:
- Used to sterilize off-gases leaving fermenters  prevent release of any
organism (entrained in aerosols in the head-space of the reactor) into the
atmosphere  harmful to plant personnel / environment.
ASSIGNMENT

1) A 15-m3 chemostat is operated with dilution rate 0.1 h-1. A continuous

sterilizer with steam injection and flash cooling delivers sterilized medium
to the fermenter. Medium in the holding section of the sterilizer is
maintained at 130°C. The concentration of contaminants in the raw
medium is 105 mL-1; an acceptable contamination risk is one organism every
3 months. The Arrhenius constant and activation energy for thermal death
are estimated as 7.5 X 1039 h-1 and 288.5 kJ gmol-1, respectively. The inner
diameter of the sterilizer pipe is 12 m. At 130°C, the liquid density is 1000
kg m-3 and the viscosity is 4 kg m-1 h-1.

(a) Assuming perfect plug flow, determine the length of the holding section
(b) What length is required if axial dispersion effects are taken into account?
(c) If the sterilizer is constructed with the length determined in (a) and operated
at 130°C as planned, estimate the frequency of fermenter contamination.
ASSIGNMENT

2) Pseudomonas methylotrophus is used to produce single cell protein from

methanol in a 1000-m3 pressure-cycle airlift fermenter. The biomass yield
from substrate is 0.41 g g-1, 𝐾𝑠 is 0.7 mg L-1, and the maximum specific
growth rate is 0.44 h-1. The medium contains 4% (w/v) methanol. A
substrate conversion of 98% is desirable. The reactor may be operated in
either batch or continuous mode. If operated in batch, an inoculum of
0.01% (w/v) is used and the downtime between batches is 20 h. If
continuous operations are used at steady state, a downtime of 25 days is
expected per year.
Neglecting maintenance requirements, compare the annual biomass
production achieved using batch and continuous reactors.
THANK YOU