Chapter Two

1
Introduction

✓ In biochemical engineering processes, measurement of dissolved oxygen

(DO) is essential.

✓ The production of SCP may reach a steady-state condition by keeping the

DO level constant, while the viable protein is continuously harvested.

2
Introduction Cont.

✓ The concentration of protein is proportional to oxygen uptake rate. Control of

DO would lead us to achieve steady SCP production.

✓ Variation of DO may affect retention time and other process variables such as
substrate and product concentrations, retention time, dilution rate and
aeration rate. Microbial activities are monitored by the oxygen uptake rate
from the supplied air or oxygen.

3
Introduction Cont.

✓ Microbial cells in the aerobic condition take up oxygen from the gas and
then liquid phases.

✓ The rate of oxygen transfer from the gas phase to liquid phase is important.
At high cell densities, the cell growth is limited by the availability of oxygen in
the medium.

4
Introduction Cont.

✓ The growth of aerobic bacteria in the fermenter is then controlled by the

availability of oxygen substrate, energy sources and enzymes. Air has to be
supplied for aerobic process in order to enhance the cell growth.

✓ Oxygen limitation may cause a reduction in the growth rate. The supplied
oxygen from the gas phase has to penetrate into the microorganism. Several
steps are required in order to let such a phenomenon take place.

5
Measurement of dissolved oxygen concentrations

✓ The concentration of dissolved oxygen in a fermenter is normally measured

with a dissolved oxygen electrode, known as a DO probe. There are two
types in common use:

✓ Galvanic electrodes

✓ Polarographic electrodes

6
Measurement of dissolved oxygen cont.

✓ In both probes, there are membranes that are permeable to oxygen. Oxygen
diffuses through the membrane and reaches to cathode, where it reacts to
produce a current between anode and cathode proportional to the oxygen
partial pressure in the fermentation broth.

✓ The electrolyte solutions in the electrode take part in the reactions and must
be located in the bulk of liquid medium.

7
Measurement of dissolved oxygen cont.

I. Oxygen transfer rate (OTR)

✓ Once batch mode studies are completed and the required data are collected,
without dismantling the bioreactor, liquid media is prepared with 33 g 𝑘𝐻2 𝑃𝑂4
and 𝑁𝑎2 𝐻𝑃𝑂4 , 10 g yeast extract and 500 g glucose in 10 liters of distilled
water.

✓ Then liquid media is sterilized with autoclave at 121℃/15 min. Then, it is

cooled down to room temperature with air flow rate of 100 𝑚𝑙 𝑚𝑖𝑛−1 .

8
Measurement of dissolved oxygen cont.

✓ The fluid residence time of 10 hours is expected to give maximum cell optical
density. Otherwise, the effect of media flow rate has to be carried out
separately. This is the basic assumption made in this experiment.

✓ The aim of this set of experiments is to determine a suitable air flow rate with
variation from 0.025 to 1 vvm.

9
Measurement of dissolved oxygen cont.

✓ Backer yeast production using continuous mode of operation for 3.5 days
using isolated strains from the waste stream of food processing plant is
given below.

10
Measurement of dissolved oxygen cont.

II. Respiration quotient (RQ)

✓ Measurements of inlet and outlet gas compositions of a culture vessel have
been considered as an indicator for cell activities in the fermentation broth.

✓ The continuous monitoring of gas analysis would lead us to understand the

oxygen consumption rate and carbon dioxide production, which originate from
catabolism of carbon sources.

11
Measurement of dissolved oxygen cont.

✓ Respiration is a sequence of biochemical reactions resulting in electrons

from substances that are then transferred to an exogenous electron accepting
terminal.

✓ Respiration in a cell is an energy-delivery process in which electrons are

generated from oxidation of substrate and transferred through a series of
oxidation–reduction reactions to electron acceptor terminals.

12
Measurement of dissolved oxygen cont.

✓ In biosynthesis, the end products result from a respiration process.

✓ Since oxidation of carbonaceous substrate ends with carbon dioxide and

water molecules, the molar ratio of carbon dioxide generated from oxidation–
reduction to oxygen supplied is known as the respiration quotient:

𝒅𝑪𝑪𝑶𝟐
ൗ 𝒅𝑪𝑪𝑶𝟐
𝑹𝑸 = 𝒅𝒕 =
𝒅𝑪𝑶𝟐ൗ 𝒅𝑪𝑶𝟐
𝒅𝒕
13
Aeration and agitation

✓ In the biochemical engineering profession, there are various bioprocesses

actively involved in the synthesis and production of biological products.

✓ Understanding of all the processes may require basic knowledge of biology,

biochemistry, biotechnology, and real knowledge of engineering processes.

✓ Transfer of oxygen is a major concern in many bioprocesses that require air

for microbial growth such as single cell protein and production of antibiotics.

14
Aeration and agitation cont.

✓ Agitation in a fermentation unit is directly related to oxygen transported from

the gas phase to liquid phase followed by oxygen uptake by the individual
microbial cell.

✓ The activities of microorganisms are monitored by the utilization of oxygen

from the supplied air and the respiration quotient.

✓ Aeration and agitation are implemented in most fermentation processes.

15
Aeration and agitation cont.

✓ The word ‘aerobe’ refers to the kind of microorganism that needs molecular
oxygen for growth and metabolism.

✓ ‘Aerobic’ is the condition of living organisms surviving only in the presence of

molecular oxygen.

✓ Aerobic bacteria require oxygen for growth and can be incubated to be grown
in atmospheric air.

16
Aeration and agitation cont.

✓ Oxygen is a strong oxidizing agent which has the ability to accept electrons
for yielding energy, a process known as respiration.

✓ A bioreactor is a reaction vessel in which an organism is cultivated in a

controlled manner to produce cell bodies and/or product.

✓ Initially the term ‘fermenter’ was used to describe these vessels, but in strict
terms, fermentation is an anaerobic process whereas the major proportion of
fermenters use aerobic processes.

17
Aeration and agitation cont.

✓ Thus, in general terms, ‘bioreactor’ means a vessel in which organisms are

grown under aerobic or anaerobic conditions. If a bioreactor or a reaction vessel
operates under aerating conditions, the system is called an aerobic bioreactor.

✓ Sterile air is supplied as a source of intake for respiration of microorganisms.

✓ The oxygen is dissolved in the liquid phase. The microorganisms consume

the oxygen that is dissolved in the liquid media.

18
Aeration and agitation cont.

✓ Growth of the aerobic bacteria in the fermenter is controlled by the availability

of substrate, energy and enzymes.

✓ Microbial cultures are always known as heterogeneous systems, as cells are

solid and nutrients are in the liquid phase.

✓ If the process is aerobic, air has to be supplied to enhance cell growth,

otherwise the limited dissolved oxygen is used up and then oxygen limitation
may cause a decrease in the growth rate.

19
Aeration and agitation cont.
✓ The rate of reaction depends on substrate concentration and product
presence.

✓ High concentrations of substrate and product may cause growth inhibition, as

the microorganisms are intoxicated at high levels of substrate or product; such
phenomena may easily happen in batch culture.

✓ The aerobic activity depends upon the local bulk oxygen concentration, the
oxygen diffusion coefficient and the respiration rate of microbes in the aerobic
region.
20
Aeration and agitation cont.
✓ The transfer of oxygen from the gas to the microorganism takes place in
several steps.

✓ The oxygen must first travel through the gas–liquid interface, then the bulk of
liquid and finally into the microbial cell.

21
Effect of agitation on dissolved oxygen
✓ Aerobic bacteria are easily grown at a small scale in tubes and flasks by
incubating the media under normal atmospheric conditions.

✓ In large-scale operations, the media has to be exposed to air, and sufficient

air must be present for respiration of all living microorganisms.

✓ The indication of availability of oxygen in the liquid phase is to measure the

amount of dissolved oxygen.

22
Effect of agitation on dissolved oxygen cont.
✓ DO probes are available on the market, and most fermenters are equipped
with a DO meter. For aerobic fermentation, the bioreactor must be equipped
with a DO meter.

✓ The level of DO in the media is a function of temperature. Higher operating

temperatures decrease the level of DO.

✓ To have sufficient oxygen, an air sparger is required to purge compressed air

or pressured air to be bubbled into the media. The availability of oxygen is a
major parameter to be considered for effective microbial cell growth rate.
23
Air sparger
✓ Air under pressure is supplied through a tube end consists of an ‘O’ ring with
very fine holes or orifices.

✓ The size of bubbles depends on the size of hole and type of sparger. For very
fine bubbles with effective gas dispersion, a micro-sparger is used in the
fermenter.

✓ A micro-sparger is in fact a highly porous ceramic material and is used

instead of a gas sparger.

24
Air sparger cont.
✓ The size of bubbles affects the mass transfer process. Smaller bubble size
provides more surface area for gas exposure, so a better oxygen transfer rate is
obtained.

✓ The size of gas bubbles and their dispersion throughout the tank are critical to
bioreactor performance.

25
Oxygen transfer rate in a fermenter
✓ The molar flux of oxygen is generalized in a simple equation, with the
concentration gradient as the major driving force in the transfer of oxygen from
gas and liquid interface to the bulk of liquid.

✓ The rate of oxygen transfer in a fermenter broth is influenced by several

physical and chemical parameters that change either 𝑘𝐿 , or the value of
interfacial area of bubbles (a) or the concentration gradient known as the driving
force for the mass transfer.

26
Oxygen transfer rate in a fermenter cont.
✓ At low concentrations of the soluble gas, the molar flux of oxygen transported to the
fermentation media is:

𝑁𝐴 = 𝑘𝐿 𝑎 𝐶 ∗𝐴𝐿 − 𝐶𝐴𝐿

Where;

NA = oxygen flux in kmol 𝑚−2 𝑠 −1 , 𝑘𝐿 = liquid side mass transfer coefficient in 𝑚𝑠 −1 , 𝐶 ∗𝐴𝐿 = oxygen
𝑘𝑚𝑜𝑙
concentration in equilibrium with the liquid phase at the interface in , 𝐶𝐴𝐿 = oxygen concentration
𝑚3

𝑘𝑚𝑜𝑙
in the bulk of liquid in , 𝑎 = interfacial area in surface area of bubbles per unit volume of
𝑚3

broth(𝑚2 𝑚−3 ).

27