FE 463 Food Biotechnology

(Upstream Processing)

Md. Mostafa Kamal

Department of Food Engineering
Gopalganj Science and Technology University
Gopalganj-8100
The upstream processing in a fermentation process includes:

✓ preparation of the fermentation medium

✓ sterilization of air and fermentation medium

✓ inoculation of the fermentor

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Fermentation Medium
A medium is defined as the substance surrounding the cells, which enables the
microorganisms to grow and form products.

Medium used for industrial fermentations should have the following criteria:
✓ Maximum yield and concentration of product or biomass per unit mass of substrate
✓ Minimum production of undesirable metabolites
✓ Consistent quality of product
✓ Minimal problems during sterilization and fermentation

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The medium used for fermentation may be classified as: defined, complex and technical

medium.

Defined medium:

✓ Consists only of precisely chemically defined substrates

✓ At laboratory and pilot plant level where physical, chemical, and physiological

parameters need to be standardized first

✓ Only defined media should be used for studies.

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Complex medium

✓ Composed of substrates with undefined composition, such as extracts or

hydrolysates from waste products, which are cheap substrates commonly used in

industrial production.

✓ Relatively expensive substrates, such as yeast extract, brain heart infusion, peptone,

and tryptone are often used for complex medium.

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Technical media
✓ Used on an industrial scale and are cheaper
✓ Normally, but not necessarily, complex substrates are the main components
✓ Examples are soy meal, whey, fishmeal, malt extract, and sulfite waste liquor
✓ Waste water from monosodium glutamate production, which contains high levels of
chemical oxygen demand (COD), sulphate, and ammoniacal nitrogen at a low pH,
has been used as the nitrogen and water source, with sugar beet pulp as the carbon
source, for the production of pectinase by solid-state fermentation

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Optimization of Fermentation Medium
✓ Done by using an experimental design considering the variables, mostly medium
ingredients
✓ Experimental design for the fermentation process involves the determination of the
optimum medium composition
✓ Design of experiments (DOE) is a systematic approach to problem solving which is
applied to data collection and analysis to obtain information rich data
✓ The primary goal of designing an experiment is to obtain valid results using a
minimum of effort, time, and resources

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Components of Industrial Fermentation Medium
✓ In industrial processes, the cheapest substrates are used to keep the production costs
low.
✓ Some of the cheap substrates available include barley, barley malt, blood meal, cane
molasses, corn gluten meal, corn meal, corn steep liquor, cotton seed meal, dried
distillers’ soluble, fish soluble, fish meal, linseed meal, meat meal, bone meal, oat
flour, peanut meal, rice bran, rice flour, soybean meal, wheat flour, whey powder,
and yeast hydrolysate.

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Nutritional sources used in fermentation medium

Nitrogen source
Inorganic Medium
Carbon source Organic Inorganic elements Vitamins additives
Carbohydrates, Urea, Nitrates, Phosphorus, Thiamine, Growth
Amino Nitrites, Sulphur, Riboflavin, factors,
Alcohols, acids, Ammonia, Magnesium, Pyridoxin, Precursors,
Carboxylic Purines, Molecular Potassium, Biotin, Detergents,
Pyrimidines nitrogen. Calcium, Pantotheni Anti-
acids, Chlorine, c acid, foaming
Fats, Complex Cobalt, Niacin, agents,
sources Copper, Iron, Inositol, Anti-
Hydrocarbons, such as Manganese, Choline. microbial
Gaseous dried Molybdenum agents.
yeasts, Zinc.
substrates. protein
hydrolisates
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Sterilization

✓ Sterilization is a process by which microorganisms are either killed or removed

from the material or equipment.

✓ Sterilization is necessary to ensure that only the desired microorganism is present to

carry out the fermentation

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Medium Sterilization Techniques
The sterilization techniques applicable for fermentation media sterilization include:
✓ Sterilization by high temperature achieved by direct or indirect steam or electric
heating.
✓ Sterilization of the medium for animal cell culture by membrane filtration with
absolute rating of 0.1–0.04 μm.
✓ Microwave irradiation, which is used commonly in food industries and has been
reported to cause cell death.
✓ High voltage pulses and photo semiconductor powders, which involve the rupture of
the cell membrane by increasing the trans-membrane electric field.

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Sterilization by Heat
When a medium containing microorganisms is heated above a certain temperature limit,
the microorganisms are unable to survive.

Batch sterilization:
Sterilization of medium in the fermentor can be carried out in batch mode by passing
steam through the available heat transfer area (jacket or limpet coil) or through direct
steam sparging or with electrical heaters. The highest temperature to be operated for batch
sterilization of medium is 121 °C.

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The batch heat sterilization is described by a first order kinetics resulting in the equation:

𝑁𝑡
ൗ𝑁𝑜 = 𝑒 −𝑘𝑡 ……… (1)

where Nt is the number of surviving microorganisms at t, No is the initial number of

microorganisms, k is the specific death rate, and t is time.
Equation (1) can be rearranged as:

𝑁𝑜
ln ൗ𝑁𝑡 = 𝑘𝑡 ………. (2)

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The reaction or sterilization rate increases with increase in temperature due to an increase
in the reaction rate constant or specific death rate (k). The relationship between
temperature (T) and specific death rate (k) can be expressed as:

𝑘 = 𝐴 𝑒 −𝐸/𝑅𝑇 …….. (3)

where A is the Arrhenius constant, E is the activation energy, R is the gas constant and T is
the absolute temperature. Substituting k from Equation (3) into Equation (2):

𝑁𝑜
ln ൗ𝑁𝑡 = 𝐴 𝑒 −𝐸/𝑅𝑇 . 𝑡 ……….. (4)

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The term “ln (No/Nt)” has been used as a design criterion for sterilization and is
represented as the Del factor (∇), which is a measure of the fractional reduction in viable
cell count produced in a certain temperature and time regime.
Thus, we have:
𝛻 = 𝐴 𝑒 −𝐸/𝑅𝑇 . 𝑡 ……… (5)
where t is the time required to achieve a certain ∇ value.
Rearranging Equation (5):
ln (t) = E/RT + ln (∇/A) ……… (6)
From a plot of ln (t) vs 1/T, the thermal death characteristics, namely activation energy (E)
and Arrhenius constant (A), can be determined.

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The batch sterilization cycle consists of heating, holding, and cooling cycles. The overall
Del factor for destruction of cells during the sterilization period is represented as:
𝛻𝑜𝑣𝑒𝑟𝑎𝑙𝑙 = 𝛻ℎ𝑒𝑎𝑡𝑖𝑛𝑔 + 𝛻ℎ𝑜𝑙𝑑𝑖𝑛𝑔 + 𝛻𝑐𝑜𝑜𝑙𝑖𝑛𝑔 ……… (7)

The advantages of batch sterilization are low capital equipment costs, low risk of
contamination, easier manual control, and suitability for media containing a high
proportion of solids.

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Continuous sterilization:
Continuous sterilization has the advantages of relatively straight forward energy
recovery and lower nutrient degradation of heat labile substances due to shorter holding
time.
However, a disadvantage is that the presence of solids may give rise to inefficient
sterilization, requiring a proper design.

Continuous heat sterilization essentially consists of a heating section using heat

exchangers (typically spiral-type plates on an industrial scale), and a holding section where
the temperature of the medium is maintained constant at the sterilization temperature.

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Sterilization of Air
✓ Aerobic fermentation processes require sterile air.
✓ The sterilization of air in the strict bacteriological sense means the complete elimination
of all viable microorganisms.
✓ Depth filters consisting of granular carbon or fibrous media has been almost universally
adopted.
✓ In most fermentation systems, a pre-filter usually made of cellulose, viscose, or glass
wool is installed prior to the absolute filter to remove dust, oil droplets, and moisture
from the process air.
✓ The absolute filters have a rating of 0.2–0.01 μm (for total removal of bacteria, viruses,
bacteriophage or spherical particles of that size) and can be sterilized using steam.
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Inoculation
✓ The process of inoculation is the transfer of seed material or inoculum into the
fermentor.
✓ Inoculation of a laboratory fermentor is generally done using pasteurized tubing
and a peristaltic pump.
✓ However, on a larger scale, inoculum transfer is done by applying a positive
pressure on the inoculum fermentor and connecting it aseptically to the production
fermentor.
✓ Inoculum transfer is commonly done with vegetatively growing biomass
determined by parameters such as turbidity, packed cell volume, dry weight, wet
weight, or morphological characteristics
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Fermentation Process/ Processing
✓ Actual growth of the microorganism
✓ Formation of the product under agitation and aeration
✓ provide uniform environment and adequate oxygen to the cell for growth, survival,
and product formation

Modes of Operation
✓ The choice of the fermentation mode is dependent on the relation of consumption of
substrate to biomass and products.
✓ Usually operated in: batch, fed batch, or continuous fermentation.

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Batch fermentation
✓ A sequence of operations
✓ Growth rate is normally uncontrolled and is highest at the start
✓ The productivity is calculated by the final concentration of biomass divided by the
complete time of batch
Continuous Fermentation
✓ An open system
✓ Continuously adding fresh medium and removing the culture medium at the same
rate.
✓ Two modes of operation for continuous fermentation are chemostats and auxostats.
✓ The commonly used auxostats include turbidostats, the pH auxostat, and the nutristat
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Chemostat:
✓ The chemostat is the most widely used apparatus for studying microorganisms
under constant environmental conditions.
✓ It is a continuous fermentation.
✓ Performed in a Continuous Stirred Tank Reactor (CSTR).
✓ Maintaining a steady growth rate through continuous deeding a growth limiting
nutrient and withdrawing part of medium at the same rate.

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Auxostat:
✓ Continuous culture technique
✓ Dilution rate is regulated based on metabolic activity of the culture
✓ A chemostat is essentially used for operation at moderate to low dilution rates
but an auxostat is used at high dilution rates.
✓ Practical applications include high-rate propagation, destruction of wastes with
control at a concentration for maximum rate, open culturing because potential
contaminating organisms cannot adapt before washing out, and operation of
processes that benefit from careful balance of the ratios of nutrient concentrations.

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Turbidostat:
✓ Controls the feed rate depending on the optical density of the fermentation broth,
which is proportional to the biomass concentration.
✓ The culture cannot washout as in a chemostat.
✓ This mode of operation is ideal only when operated near maximum growth.

Nutristat:
✓ The nutristat involves regulation of the feed rate to maintain the residual
substrate concentration during fermentation.
✓ Ion selective electrodes (NH4) have been used for control in the nutristat.
✓ The lack of reliable and accurate sensors is a drawback to nutristat operation. 24
Fed Batch Fermentation
✓ It is a technique in between batch and continuous fermentation
✓ Fed batch fermentation involves two phases: growth phase and production phase
✓ After the initial growth phase, one or more of the nutrients are supplied to the
fermentor
✓ Fed batch fermentation is well suited for producing product or cells when:
o Substrate is inhibitory and there is a need to maintain low substrate
concentration (e.g., citric acid, amylase)
o Product or biomass yields at low substrate concentrations are high (e.g., baker’s
yeast, antibiotic production).

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 Shaft power
# Torsion dynamometer
# Strain gauge measurement
Air inlet Filter Exit gas analyser
Oxygen - paramagnetic
Carbon dioxide - IR
Mass spectrometer

Pressure gauge
# Diaphragm type

Agitator speed
# Tachometer
Air compressor
Foam detection
# Capacitance/ conductance
Airflow Controller
pressure

pH (Glass electrode)
Control
DO (Polarographic) system
Temp (Thermocouple)

Computer
data
station
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Agitation
✓ Mixing and dispersion of air in the fermentation broth is achieved
✓ Impellers brings about uniform mixing of microorganisms and nutrients, and
dispersion of air in the nutrient solution, resulting in efficient mass and heat transfer
✓ The types of impellers used in bioreactors are broadly classified based on flow pattern
as radial flow (e.g., turbine) and axial flow (e.g., high efficiency impeller).
✓ Multiple impeller bioreactors are becoming popular due to:
Efficient gas distribution, higher gas phase residence time, increased gas hold up, superior
liquid flow characteristics, and lower power consumption per impeller as compared to
single impeller systems

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Aeration
✓ Cheapest mode of supplying oxygen to the fermentation media is air.
✓ It comprises an air compressor, an air filter (prefilter and sterile filter) and a sparger.
✓ Compressor depends upon the discharge pressure, type of drive, capacity, and cost.
✓ OTR is the rate can be expressed as:
OTR = kLa (c* - cL)
where kLa is the volumetric mass transfer coefficient, c* is the dissolved oxygen
concentration in equilibrium with the gas phase, and cL is the dissolved oxygen
concentration in the liquid

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✓ The oxygen mass transfer coefficient, kLa, is directly related to power input by aeration
and aeration rate by:
kLa = K (Pg/ V)a (vs)b
where Pg/ V is the gas power input by aeration per unit volume and vs is the
superficial gas velocity.
✓ A high kLa (volumetric mass transfer coefficient) indicates a high oxygen transfer rate
(OTR) in the fermentation process.
✓ OTR varies with the nature and the scale of the fermentation.
✓ For a 1m3 vessel, at an OTR between 250 and 300 mmol/L/h

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Process Monitoring and Control
✓ Monitoring and control to ensure optimum process performance.
✓ The process control variables broadly classified as physical, chemical, and biological.
✓ The physical variables include temperature, impeller speed, aeration rate, and
pressure.
✓ Measured by inline sensors
✓ The chemical variables include pH, dissolved oxygen, dissolved carbon dioxide, and
redox potential.
✓ Measured by online sensors.
✓ The biological variables include biomass, oxygen uptake rate, carbon dioxide
production rate, and respiration quotient.
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STEADY-STATE CONTINUOUS CULTIVATION THEORY

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Mass balance in a series of vessels
To provide a general model, a series of equivalent vessels, n in total number, will be
considered.

F X1 Xn-1 Xn
Xo (=0) P1 Pn-1 Pn
Po (=0) S1 Sn-1 Sn
So

n vessels in series, F= flow rate of medium, X= cell mass concentration, P= product

concentration, S= limiting substrate concentration
Script 1, 2, …. n = 1st, 2nd, …. n-th vessels whereas the subscript 0 is for the incoming medium.
If a fresh medium is charged into the 1st vessel, Xo = 0 and Po = 0 32
In the above figure, the rate of medium flow through the series of vessels as F l/hr and the
volume of each vessel as V l, the mass balance equations regarding concentration of cell
mass X, product P and limiting substrate S can be expressed respectively as follows:

General equation: Accumulation = Input – Output + Generation

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For X: 𝑑𝑋𝑛 𝑑𝑋𝑛
𝑉 = 𝐹𝑋𝑛−1 − 𝐹𝑋𝑛 + 𝑉 … … … … … (1)
𝑑𝑡 𝑑𝑡 𝐺𝑟𝑜𝑤𝑡ℎ 𝑖𝑛 𝑛−𝑡ℎ 𝑣𝑒𝑠𝑠𝑒𝑙

𝑑𝑋𝑛 1 𝑑𝑋𝑛
𝑉 = 𝐹(𝑋𝑛−1 −𝑋𝑛 ) + 𝑉 ∙ ∙ 𝑋𝑛
𝑑𝑡 𝑋𝑛 𝑑𝑡

𝑑𝑋𝑛
= 𝐷(𝑋𝑛−1 −𝑋𝑛 ) + 𝜇𝑛 𝑋𝑛 … … … … … (2)
𝑑𝑡

Where,
D= F/V= dilution rate hr-1

1 𝑑𝑋𝑛
𝜇𝑛 = = Specific growth rate of cells in n-th vessel.
𝑋𝑛 𝑑𝑡

Subscripts n, (n-1) = n-th, (n-1)-th vessel respectively

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Similarly,
𝑑𝑃𝑛 𝐹 𝑑𝑃𝑛
For P: = (𝑃𝑛−1 − 𝑃𝑛 ) + ( ) ..… (3)
𝑑𝑡 𝑉 𝑑𝑡 𝑃𝑟𝑜𝑑𝑢𝑐𝑡 𝑖𝑛 𝑛−𝑡ℎ 𝑣𝑒𝑠𝑠𝑒𝑙

𝑑𝑃𝑛
= D (𝑃𝑛−1 − 𝑃𝑛 ) + 𝑌𝑝/𝑥 𝜇𝑛 𝑋𝑛 ………… (4)
𝑑𝑡

Where,
𝑌𝑝/𝑥 = ∆𝑃Τ = yield of product based on cell mass
∆𝑋

For S: 𝑑𝑆𝑛 𝐹 𝑑𝑆𝑛

= 𝑆𝑛−1 − 𝑆𝑛 + ( ) 𝐶𝑜𝑛𝑠𝑢𝑚𝑝𝑡𝑖𝑜𝑛 𝑖𝑛 𝑛−𝑡ℎ 𝑣𝑒𝑠𝑠𝑒𝑙 …….. (5)
𝑑𝑡 𝑉 𝑑𝑡

𝑑𝑆𝑛 1
= 𝐷 𝑆𝑛−1 − 𝑆𝑛 − 𝜇𝑛 𝑋𝑛 ……………… (6)
𝑑𝑡 𝑌𝑋/𝑆

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 𝑑𝑆𝑛 𝑌𝑃/𝑋
= 𝐷 𝑆𝑛−1 − 𝑆𝑛 − 𝜇𝑛 𝑋𝑛 ……………… (6)
𝑑𝑡 𝑌𝑃/𝑆

Where,
𝑌𝑋/𝑆 = − ∆𝑋Τ∆𝑆 = yield of cell growth based on limiting substrate
𝑌𝑃/𝑆 = − ∆𝑃Τ = yield of product based on limiting substrate
∆𝑆

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At steady-state condition, the left-hand side of all equations from (1) to (6) is zero. Then
from equation (2),
𝐷(𝑋𝑛−1 −𝑋𝑛 ) + 𝜇𝑛 𝑋𝑛 = 0

D𝑋𝑛−1 − 𝐷𝑋𝑛 + 𝜇𝑛 𝑋𝑛 = 0

𝐷𝑋𝑛−1
𝑋𝑛 = (n ≠ 1) ……… (7)
𝐷−𝜇𝑛

𝐷𝑃𝑛−1 + 𝑌𝑃/𝑋 𝜇𝑛 𝑋𝑛
The steady-state equation for P is given by: 𝑃𝑛 = ……….. (8)
𝐷

1 1
Lastly, the steady-state for S is given by: 𝑆𝑛 = 𝑆𝑛−1 − ∙ 𝜇𝑛 𝑋𝑛 …………. (9)
𝐷 𝑌𝑋/𝑆
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MASS BALANCE IN SINGLE VESSELS WITH RECYCLING

F Fa, X Fe, Xe

V
X
Fex, Xx
ωF, Xx

F = flow rate of fresh medium, Fa = flow rate of cell suspension from reactor
Fe = flow rate of effluent from separator, Fex = flow rate of concentrate cell suspension from
separator, ω = recycle ratio, ωF = flow rate of cell suspension recycled, X = cell mass
concentration in reactor
Xe = cell mass concentration in effluent from separator
Xx = cell mass concentration in recycled suspension
V = reactor (working) volume 38
 From figure,
Fa = F + ωF = F (1+ω) -------------- (1)
F = Fe + Fex -------------- (2)

For complete mixing in the reactor, the mass balance by general equation:

𝑑𝑋 𝑑𝑋
𝑉 = 𝑋𝑥 𝜔𝐹 − 𝐹𝑎 𝑋 + 𝑉
𝑑𝑡 𝑑𝑡 𝑔𝑟𝑜𝑤𝑡ℎ

𝑑𝑋 1 1 𝑑𝑋
= 𝑋 𝜔𝐹 − 𝐹𝑎 𝑋 +
𝑑𝑡 𝑉 𝑥 𝑉 𝑑𝑡 𝑔𝑟𝑜𝑤𝑡ℎ

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For steady-state condition 1 1 𝑑𝑋
𝑋𝑥 𝜔𝐹 − 𝐹𝑎 𝑋 + =0
𝑉 𝑉 𝑑𝑡 𝑔𝑟𝑜𝑤𝑡ℎ

1 𝑑𝑋 𝐹𝑎 𝜔𝐹 𝑋𝑥
= − ∙ -------------- (3)
𝑋 𝑑𝑡 𝑔𝑟𝑜𝑤𝑡ℎ 𝑉 𝑉 𝑋

𝐹(1+𝜔) 𝜔𝐹 𝑋𝑥
𝜇= − ∙
𝑉 𝑉 𝑋
𝑋𝑥
𝜇 = 𝐷 1 + 𝜔 − 𝐷𝜔 ∙
𝑋

𝑋𝑥
𝜇 = 𝐷 1 + 𝜔(1 − ) -------------- (4)
𝑋

Here,
µ = specific growth rate
D = dilution rate l-1
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Another mass balance of cells regarding the separator (right side of the figure) in steady-
state gives:
𝐹𝑎 𝑋 = 𝐹𝑒 𝑋𝑒 + 𝐹𝑒𝑥 𝑋𝑥 + 𝜔𝐹𝑋𝑥

𝑋𝑒 𝑋𝑥
𝐹𝑎 = 𝐹𝑒 + (𝐹𝑒𝑥 +𝜔𝐹)
𝑋 𝑋
𝑋𝑒
𝑋𝑥 𝐹𝑎 − 𝐹𝑒 𝑋)
(
=
𝑋 𝐹𝑒𝑥 + 𝜔𝐹
𝑋𝑒 𝐹𝑒 𝑋𝑒
𝑋𝑥 𝐹(1 + 𝜔) − 𝐹 (
𝑒 𝑋 ) 𝐹 (1 + 𝜔) − ( )
= 𝐹 𝑋
=
𝑋 𝐹 − 𝐹𝑒 + 𝜔𝐹 𝐹
𝐹(1 − 𝑒 + 𝜔)
𝐹

𝐹 𝑋
𝑋𝑥 (1+𝜔)− 𝐹𝑒 ( 𝑋𝑒 )
= 𝐹 ------------- (5)
𝑋 1− 𝐹𝑒 +𝜔
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From equation (4)

1+𝜔 −𝐹𝐹𝑒 ∙ 𝑋𝑋𝑒

𝜇=𝐷 1 + 𝜔(1 − ) -------------- (6)
1− 𝐹𝐹𝑒 + 𝜔

𝑋𝑒 1+ 𝜔
If ൗ𝑋 = 0, then 𝜇 = 𝐷{1 + 𝜔(1 − 𝐹 )}
1+ 𝜔 − 𝑒
𝐹

𝜇 1+ 𝜔
𝐷
= {1 + 𝜔(1 − 𝐹𝑒 )} ------------ (7)
1+ 𝜔 − 𝐹

𝐹𝑒 𝜇
If = 0, then Τ𝐷 = 1 𝑜𝑟 𝜇 = 𝐷
𝐹
𝐹𝑒 𝜇
If = 1, Τ𝐷 = 0
𝐹
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Exercise:
Activated sludge process accomplishes essentially two operations. It supplies O2 to the
microbial population and separates the activated sludge flock from the mix liquor. Usually,
;part of the sludge is fed back to the aeration unit. Data recorded (at 27 oC) in steady state
operation at a sewage treatment plant are as follows:

Flow rate of raw sewage = 310,000 m3/day

Flow rate of excess sludge = 6,700 m3/day
Working volume of aeration unit = 68,000 m3
Recycle ratio = 0.197
Concentration of a specific species of microbe in aeration basin = 21,000 /ml
Concentration of the microbe in effluent from separator = 51 ml
Assuming complete mixing in the aeration unit and that no growth of the sludge occurs in
the separator, estimate the volume of specific growth rate of the sludge.

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Solution
Flow rate of effluent from separator: Fe = F – Fex = 310,000 – 6,700
= 303,300 m3/day
In addition,
Xe/X = 52/ 21,000 = 2.476 x 10-3
From equation (6)

52 303,300
310,000 1 + 0.197 − ∙
21,000 310,000
𝜇= 1 + 0.197(1 − )
68,000 303,300
1 + 0.197 −
310,000
= 0.549 day-1

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