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̣ m cơ bả n
Chương 4. Thiết kế bể phản ứng theo mẻ, bể phản ứng theo mẻ có bổ sung cơ chất, bể phản ứng liên tục 4.1. Khá i niê 4.2. Cá c dạ ng thiế t bị phả n ứ ng sinh họ c 4.3. Cá c tho ng só trong cá c thiế t bị phả n ứ ng 4.4. Quy trình thiế t kế bể phả n ứ ng sinh họ c ̣ thó ng bể phả n ứ ng sinh họ c 4.5. Đá nh giá hê
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that add complexity
Bioreactor Design Bioreactors have requirements
compared to simpler chemical reactors Usually three-phase (cells, water, air) Need sterile operation Often need heat removal at ambient conditions
But biological reaction systems have many advantages Some products can only be made by biological routes Large molecules such as proteins can be made Selectivity for desired product can be very high Products are often very valuable (e.g. Active Pharmaceutical
Ingredients: APIs)
Selective conversion of biomass to chemicals Well established for food and beverage processes
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Bioreactor Design Enzyme catalysis Cell growth and metabolism Cleaning and sterilization Stirred tank fermenter design Other bioreactors
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Enzyme catalysis Enzymes are biocatalysts and can sometimes be isolated
from host cells
Low cost enzymes are used once through: amylase, ligninase High cost enzymes are immobilized for re-use
Enzymes are usually proteins
Most are thermally unstable and lose structure above ~60ºC Usually active only in water, often over restricted range of pH,
ionic strength
Enzyme kinetics: Michaelis-Menten equation:
R = reaction rate C = substrate concentration α, β = constants
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Enzyme Catalysis: Immobilization
Enzymes can sometimes be adsorbed onto a solid or encapsulated in a gel without losing structure. They can then be used in a conventional fixed-bed reactor
ultrafiltration
If the enzyme is larger than the product molecule, it can be contained in the reactor or using nanofiltration
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Bioreactor Design Enzyme catalysis Cell growth and metabolism Cleaning and sterilization Stirred tank fermenter design Other bioreactors
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Cell Growth Cell growth rate can be limited by many factors
Availability of primary substrate
Typically glucose, fructose, sucrose or other carbohydrate
Availability of other metabolites
Vitamins, minerals, hormones, enzyme cofactors
Availability of oxygen
Hence mass transfer properties of reaction system Inhibition or poisoning by products or byproducts
E.g. butanol fermentation typically limited to a few % due to toxicity
High temperature caused by inadequate heat removal
Hence heat transfer properties of reaction system
factors are exacerbated at higher cell
All of these concentrations
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I
II
III
IV
V
adapt
n o i t a r t n e c n o c l l e c e v i L
t c u d o r p r a l u
caused
n o i t a r t n e c n o c
through Cell growth goes several phases during a batch I Innoculation: slow growth while to new cells environment II Exponential growth: growth rate proportional to cell mass III Slow growth as substrate or other factors begin to limit rate IV Stationary phase: cell growth rate and death rate are equal V Decline phase: cells die or by often sporulate, product build-up
l l e c a r t n I
Batch time
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I
II
III
IV
V
Intracellular product accumulation is slow at first (not many cells) Product
n o i t a r t n e c n o c l l e c e v i L
t c u d o r p r a l u
accumulation continues even after live cell count falls still cells (dead contain product)
n o i t a r t n e c n o c
l l e c a r t n I
Batch time
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Cell Growth Kinetics
Cell growth rate defined by:
x = concentration of cells, g/l t = time, s μg = growth rate, s-1
s = concentration of substrate, g/l Ks = constant μmax = maximum growth rate, s-1
Cell growth rate usually has similar dependence on substrate concentration to Michaelis-Menten equation: Monod equation:
Substrate
consumption must
allow
for
cell
maintenance as well as growth
mi = rate of consumption of substrate i to maintain cell life, g of substrate/g cells.s Yi = yield of new cells on substrate i, g of cells/g substrate
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Metabolism and Product Formation
Product formation rate in biological processes is often not
closely tied to rate of consumption of substrate Product may be made by cells at relatively
low
concentrations
Cell metabolic processes may not be involved in product
formation
It is usually not straightforward to write a stoichiometric
equation linking product to substrate
Instead, product formation and substrate consumption are linked through dependence of both on live cell mass in reactor:
pi = concentration of product i, g/l ki = rate of production of product I per unit mass of cells
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Exercise: Where Should We Operate?
I
II
III
IV
V
Intracellular
product,
batch process
n o i t a r t n e c n o c l l e c e v i L
Batch operation should continue into Phase V to maximize the product assay (increase reactor productivity)
t c u d o r p r a l u
Probably not economical to go to absolute highest product concentration
n o i t a r t n e c n o c
l l e c a r t n I
Batch time
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Exercise: Where Should We Operate?
I
II
III
IV
V
Intracellular
product,
If
the
continuous process product
n o i t a r t n e c n o c l l e c e v i L
is harvested from the cells then we need a high rate of production of cells operate and would toward the upper end of phase III
t c u d o r p r a l u
n o i t a r t n e c n o c
l l e c a r t n I
Batch time
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Exercise: Where Should We Operate?
I
II
III
IV
V
Extracellular
product,
continuous process
n o i t a r t n e c n o c l l e c e v i L
If the product can be recovered continuously or cells can be recycled then we can maintain highest productivity by operating in Phase IV
t c u d o r p r a l u
n o i t a r t n e c n o c
l l e c a r t n I
Batch time
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Bioreactor Design Enzyme catalysis Cell growth and metabolism Cleaning and sterilization Stirred tank fermenter design Other bioreactors
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Cleaning and Sterilization Biological processes must maintain sterile (aseptic)
operation: Prevent infection of desired organism with invasive species Prevent invasion of natural strains that interbreed with desired organism and cause loss
of desired strain properties
Prevent contamination of product with byproducts formed by invasive species Prevent competition for substrate between desired organism and invasive species Ensure quality and safety of food and pharmaceutical grade products
Design must allow for cleaning and sterilization between
batches or runs Production plants are usually designed for cleaning in place (CIP) and sterilization in
place (SIP)
Continuous or fed-batch plants must have sterile feeds
Applies to all feeds that could support life forms, particularly growth media Including air: use high efficiency particulate air (HEPA) filters
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Design for Cleaning and Sterilization
Reactors and tanks are fitted with special spray nozzles
for cleaning. See www.Bete.com for examples
Minimize dead-legs, branches, crevices and other hard-
to-clean areas
Minimize process fluid exposure to shaft seals on pumps, valves, instruments, etc. to prevent contaminant ingress Operate under pressure to prevent air leakage in (unless
biohazard is high)
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Cleaning Policy
Typically multiple steps to cleaning cycle: Wash with high-pressure water jets Drain Wash with alkaline cleaning solution (typically 1M NaOH) Drain Rinse with tap water Drain Wash with acidic cleaning solution (typically 1M phosphoric or
nitric acid)
Drain Rinse with tap water Drain Rinse with deionized water Drain
Each wash step will be timed to ensure vessel is filled well above normal fill line
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Sterilization Policy
Sterilization is also a reaction process: cell death is typically a 0th or 1st order process, but since we require a high likelihood that all cells are killed, it is usually treated probabilistically
Typical treatments: 15 min at 120ºC or 3 min at 135ºC SIP is usually carried out by feeding LP steam and holding for prescribed time. During cool-down only sterile air should be admitted
Feed sterilization can be challenging for thermally sensitive feeds such as vitamins – need to provide some additional feed to allow for degradation
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Continuous Feed Sterilization
Holding coil must have sufficient residence time at high
temperature
Expansion valve shaft is potential contamination source
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Heat Exchange Feed Sterilization
Uses less hot and cold utility Possibility of feed to product contamination in exchanger Mainly used in robust fermentations, e.g. brewing
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Bioreactor Design Enzyme catalysis Cell growth and metabolism Cleaning and sterilization Stirred tank fermenter design Other bioreactors
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Stirred Tank Fermenter
Most common reactor for biological reactions Can be used in batch or continuous mode Available
from pressure vessel manufacturers
in
7.5
30
1.5 400
3 800
5 1500 2000
15 4000
25 7000 8000
standard sizes Vessel size (m3) 0.5 1.0 300 Vessel size (gal) 150
Typically 316L stainless steel, but other metals are
available
Relatively easy to scale up from lab scale fermenters
during process development: high familiarity
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Typical Stirred Tank Fermenter
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Design of Stirred Tank Fermenters 1. Decide operation mode: batch or continuous
Even in continuous mode, several reactors may be needed to allow for periodic cleaning and re-innoculation
2.
Estimate productivity (probably experimentally)
Establish cell concentration, substrate feed rate, product formation rate per unit volume per unit time Hence determine number of standard reactors to achieve desired production rate: assume vessel is 2/3 full
3. Determine run length: batch time or average length of continuous run 4. Determine mass transfer rate and confirm adequate aeration (see Ch15 for
correlations)
5. Determine heat transfer rate and confirm adequate cooling (see Ch19 for
correlations)
6. Determine times for draining, CIP, SIP, cool down, refilling 7.
Recalculate productivity allowing for non-operational time (CIP, SIP, etc.): revisit step 2 if necessary.
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Bioreactor Design Enzyme catalysis Cell growth and metabolism Cleaning and sterilization Stirred tank fermenter design Other bioreactors
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provide agitation shaft seal
liquid agitation ofof liquid potential source
seal asas potential
source ofof
Shaftless Bioreactors •• UseUse gasgas flowflow toto provide •• Eliminates
pump shaft
Eliminates pump contamination contamination Design requires
•• Design
requires careful
careful attention
hydraulics attention toto hydraulics
Gas loop reactor
Baffle tube reactor
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Example: UOP/Paques Thiopaq Reactor
Biological desulfurization of gases with oxidative regeneration of bugs using air Reactor at AMOC in Al Iskandriyah has six 2m diameter downcomers inside shell
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