Module 1. Basic elements of the design and modeling of bioreactors.

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Upstream Downstream

“Midstream”

Bioreactor

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 What is a bioreactor?

Control for optimum

processes

Control for an Control for quick

economical process processes

A bioreactor is a vessel system that maintains a

biologically active system, thus it must provide a
controlled environment that maximizes the production
of a biotechnological product.
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 Characteristics of a bioreactor process

Uniform distribution of cells Allowing for the difussion of

within the vessel. gasses.

Keeping a pure (axenic) and

Homogeneous temperature.
aseptic culture.

Preventing sedimentation. Optimizing production.

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 Which products can be obtained through a bioprocess?

Recombinant
Exopolysaccharides
proteins

Polymers Insecticides

Antibiotics Saponins

Alkaloids

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 Basic parts of a bioreactor

Heating element/jacket
Inoculum feeding port

Jar/vessel
Air filters (Whatman 0.2 μm) Air and sample outlets

Acid/base/antifoam feed
Impellers

Sterile gas inlet (O2, N2, CO2)

pH, O2, temperature sensors

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Parts of a bioreactor

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 Acid/base/antifoam feed

O2 sensor

Air feed

Rotor 0.2 μm filter

pH meter

Inoculation port Impeller

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 Axial

Rushton 2p

Rushton 3p
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 Temperature sensor

Air oulet/disposal port

Air outlet

Butterfly screw
Sampling port

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Culture medium

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 RPM O2 Control

Temperature

Vessel pressure

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Calculations: Mass balance

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 What is a mass balance?

It is the process of counting the components of a system.

Law of conservation of mass.

General equation:

I+𝐺−𝑂−𝐶 =𝐴

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 Reactionless systems

𝐼+𝐺−𝑂−𝐶 =𝐴 Reaction, steady state

𝐼−𝑂 =𝐴 𝐼+𝐺−𝑂−𝐶 =𝐴
No reaction, steady state
𝐼+𝐺 =𝑂+𝐶
𝐼−𝑂 =𝐴
𝐼=𝑂
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 Differential mass balance

The composition in a system at a given

timeframe is known. This composition is
represented in mass and time units (ex. kg/h)

Fed-batch and continuous systems

Integral mass balance

The reaction happens in a closed-off system,

the compositions of interest are, in general,
only the composition at t= 0 and t=n, where n
is the final process time.

Discontinuous (batch) systems

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 Differential mass balance (no reaction)

A feed (1000 kg/h) containing 10% EtOH, 20% sucrose and the balance of water is
mixed with a second feed (2000 kg/h) containing 25% EtOH, 50% sucrose and the
balance of water. Calculate the final composition of the resulting stream.

L1
1000 kg/h
10% EtOH L3
20% Sucrose ?? kg/h
Mixing tank ?? EtOH
?? Sucrose
L2 ?? Agua
2000 kg/h
25% EtOH
50% Sucrose
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 Differential mass balance (no reaction)

For the production of a bacterial metabolite, a culture medium is prepared by

feeding 100 kg/h molasses (75% fructose, 20% solids) into a tank. Another
required feed contains 55 kg/h banana peels (60% fructose, 30% solids).
Aditionally, 1% reagent grade fructose is added, and the process has a total of
80% water loss.

• Calculate the total resulting mass.

• Calculate the final composition of the mixture.
• If only 40% water was lost, how much water would the mixture contain?
• Which substitution(s) could be done to make a cheaper substrate?

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 Differential mass balance (reaction)

You’re planning on using a strain of Saccharomyces cerevisiae to produce ethanol.

You know that the strain has a 92% yield when using glucose (8% is turned into
water). If your proposal requires treating sugarcane bagasse to obtain 500 g of
glucose per 1 ton of bagasse:
• How much bagasse is required to produce 3 L of ethanol??
• How much water would be produced?
• If Zymomonas mobilis (99% yield) was used, how much more ethanol would be
produced?

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 Differential mass balance (reaction)

Xanthan gum is produced by Xanthomonas campestris in batch processees. The reaction

stoichiometry for its metabolism is as follows:

1 g glucose+ 0.23 g O2 + 0.01g NH3 → 0.75 g gum + 0.09 g biomass + 0.40 g CO2
• How much substrate is required to produce 200 g of xanthan gum?
• If a 15 L vessel containing 5% (w/v) glucose is innoculated with X. campestris, how
much gum could be produced (hypothetically)?
• Would this be actually possible?

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 Production analysis

Kinetic parameters Productivity

Yields

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 Production yields and volumetric productivity

These parameters allow us to know how an

organism uses its substrates as a source for
growth or metabolite production.
Biomass/substrate Volumetric
yield productivity
𝑔
Δ𝑥 Product/substrate Product/biomass 𝑄𝑝 =
𝑀𝑎𝑥. ℎ𝑎𝑟𝑣𝑒𝑠𝑡 ൗ𝐿
𝑦= yield yield
𝑇𝑖𝑚𝑒 ℎ
Δ𝑠
Δ𝑝 Δ𝑝
𝑦= 𝑦=
Δ𝑠 Δ𝑥

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Hra PHB (g/L) DE Biomasa (g/L) DE pH DE Azucares reductores (g/L) DE
0 0.000 0.000 1.240 0.042 6.05 0.087 14.94 0.10
6 0.239 0.030 2.385 0.134 4.82 0.025 7.33 0.15
12 0.714 0.148 3.340 0.297 5.19 0.040 5.92 0.28
24 1.514 0.028 3.980 0.467 6.99 0.091 3.88 0.25
36 1.705 0.093 4.228 0.078 7.14 0.04 2.18 0.07
48 2.211 0.198 4.995 0.064 7.22 0.026 1.78 0.15
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 Hra PHB (g/L) DE Biomasa (g/L) DE pH DE Azucares reductores (g/L) DE
0 0.000 0.000 1.240 0.042 6.05 0.087 14.94 0.10
6 0.239 0.030 2.385 0.134 4.82 0.025 7.33 0.15
12 0.714 0.148 3.340 0.297 5.19 0.040 5.92 0.28
24 1.514 0.028 3.980 0.467 6.99 0.091 3.88 0.25
36 1.705 0.093 4.228 0.078 7.14 0.04 2.18 0.07
48 2.211 0.198 4.995 0.064 7.22 0.026 1.78 0.15

Biomass/substrate yield Product/substrate yield

Δ𝑥 (4.99 − 1.24) Δ𝑝 (2.21 − 0.00)

𝑦= 𝑦= 𝑦= 𝑦=
Δ𝑠 (14.94 − 1.78) Δ𝑠 (14.94 − 1.78)
3.75 𝑔/𝐿 2.21 𝑔/𝐿
𝑦= 𝑦 = 0.28 𝑔/𝑔 𝑦= 𝑦 = 0.17 𝑔/𝑔
13.16 𝑔/𝐿 13.16 𝑔/𝐿

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 Hra PHB (g/L) DE Biomasa (g/L) DE pH DE Azucares reductores (g/L) DE
0 0.000 0.000 1.240 0.042 6.05 0.087 14.94 0.10
6 0.239 0.030 2.385 0.134 4.82 0.025 7.33 0.15
12 0.714 0.148 3.340 0.297 5.19 0.040 5.92 0.28
24 1.514 0.028 3.980 0.467 6.99 0.091 3.88 0.25
36 1.705 0.093 4.228 0.078 7.14 0.04 2.18 0.07
48 2.211 0.198 4.995 0.064 7.22 0.026 1.78 0.15

Product/biomass yield Volumetric productivity

𝑔
Δ𝑝 2.21 𝑔/𝐿 𝑃𝑟𝑜𝑑𝑢 ൗ𝐿
𝑦= 𝑦= 𝑄𝑝 =
Δ𝑥 4.99 𝑔/𝐿 𝑇𝑖𝑚𝑒 ℎ
2.21 𝑔/𝐿
𝑄𝑝 =
𝑦 = 0.44 𝑔/𝑔 = 44% 48 ℎ
aka production efficiency 𝑄𝑝 = 0.046 𝑔/𝐿/ℎ
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Bioreactor operation

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 Discontinuous (batch)
Continuous system
systems

The total process charge is The feed and drain line are
Fed-batch systems
loaded and left to ferment for both active, maintaining a
a determined amount of time specific growth condition
(final processing time). within the vessel.
The vessel is fed at certain
intervals to increase biomass
and/or product yield.

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 Class activity: Types of systems for ApoAI
and SAA

As a team, look up 2-3 scientific articles on the production of the proteins of

interest for the challenge (at least one per type of protein of interest) and
determine the type of bioreactor operation used and the reasoning behind its
selection (between batch, fed-batch, continuous, perfusion, bleed, etc.). (45 min)

Prepare 1 slide per article to present. (5 mins per team)

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