Biochemical Engineering
CELL CUTIVATION
Engr. A. Corpuz 09/2022
Chemical Engineering
Cagayan State University – Carig Campus, Tuguegarao City
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1. MICROBIAL CELL CULTIVATIONS
1.1 Microbial Cells
1.2 Bacteria
1.3 Fungi
1.4 Culture Media
2. ANIMAL CELL CULTIVATIONS
2.1 Animal Cells
2.2 Growth Media
2.3 Monoclonal Antibodies
3. PLANT CELL CULTIVATIONS
3.1 Plant Cells
3.2 Secondary Metabolite Production
3.3 Types of Plant Tissue Culture
3.4 Culture Media
4. CELL GROWTH MEASUREMENT
4.1 Bacterial Cell Growth
4.2 Measurement of Cell Number
4.3 Measurement of Cell Mass
4.4 Indirect Methods
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Microbial Cell Cultivation
Traditional applications
• preparation of food, alcoholic beverages, milk products, textiles, etc
resultant fungal cellulase was subsequently
utilised in textile waste hydrolysis for
recovery of glucose and polyester
https://doi.org/10.1016/j.psep.2018.06.038
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Microbial Cell Cultivation
Emerging applications
• production of pharmaceuticals (antibiotics, therapeutic proteins, enzymes and insulin), industrial
chemicals, enzymes, agricultural chemicals, wastewater treatments, mineral leaching, and
recombinant DNA technologies
Penicillin fermentation
Penicillium
chrysogenum
The main role of anaerobic bacteria in sewage treatment
is to reduce the volume of sludge and produce methane
gas from it.
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Microbial Cell Cultivation
Emerging applications
• production of pharmaceuticals (antibiotics, therapeutic proteins, enzymes and insulin), industrial
chemicals, enzymes, agricultural chemicals, wastewater treatments, mineral leaching, and
recombinant DNA technologies
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Classification of Living Organisms
o Plasmodium
o slime moulds
o euglena
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Prokaryotes
• cell wall – thicker than cell membrane,
protects the cell from external
influences
• cell / cytoplasmic membrane – a
selective barrier between the interior
and exterior of the cell
the inner side of the cell wall
• cytoplasm – holds the ribosomes
• ribosomes – composed of protein and
ribonucleic acid (RNA), site of
important biochemical reactions for
protein synthesis
• nuclear region – contains
deoxyribonucleic acid (DNA), which
contains genetic information that
determines the production of proteins
and other cellular substances and
structures
(bacteria) Genus species:
Prokaryotes are organisms made up of cells that lack a cell
nucleus or any membrane-encased organelles.
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Bacillus subtilis
B. Albus
B. coagulans
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Eukaryotes
• Cell membrane – protective membrane
• Cytoplasm – contains large numbers of
granules called ribosome
• Ribosome – involved in continuous reactions
to synthesize cell materials
• Endoplasmic reticulum – irregular network of
interconnected membrane-delimited
channels, holds ribosomes on surface
• Mitochondria – contain the electron transport
enzymes that utilize oxygen in the process of
energy generation
• Vacuole and lysosome – organelles that serve
to isolate various chemical reactions in a cell
• Nuclear membrane – double membrane with
pores (40 - 70 mμ wide) surrounding nucleus
• Nucleus – contains chromosomes, controls
hereditary properties and all vital cell
activities
• Chromosomes – long and threadlike bodies
found in the nuclei of cells, contain the genes
arranged in linear sequence in nucleoproteins
Eukaryotes are organisms made up of cells that possess a
membrane-bound nucleus that holds genetic material as well
as membrane-bound organelles.
1000 – 10,000x larger and more complex than prokaryotic cells
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Bacteria
Bacteria – microscopic, 1500 known species,
0.5 to 1 μm dia
Asexual reproduction
Multiply by geometric progression…
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Bacteria
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Bacterial doubling time
The relationship between the number of bacteria in a population at a given time (Nt), the original number of
bacterial cells in the population (No), and the number of divisions (n) those bacteria have undergone during that
time can be expressed by the following equation:
Also, doubling time td or generation time is defined as:
𝑡
=𝑛
𝑡𝑑
Example. Escherichia coli, under optimum conditions, has a generation time of 20 minutes. If one started with only
10 E. coli (No = 10) and allowed them to grow for 12 hours.
𝑛=
The number of generations is computed as:
12 ℎ × 60 𝑚𝑖𝑛ൗℎ
20 𝑚𝑖𝑛
= 36
The number of bacteria after 12 hours (Nt) would be
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Bacterial doubling time: Practice exercises
1. A culture is inoculated at noon with 103 cells/ml. At 10 pm the population is determined to be 109 cells/ml. What
is the number of generations? What is the doubling time?
2. An experiment was performed in a lab flask growing cells on 0.1% salicylate and starting with 2.2 x 104 cells. As
the experiment below shows, at the end there were 3.8 x 109 cells. How many doublings or generations occurred?
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Bacterial Growth: Nutritional Requirements
Inputs:
Carbon source
Autotrophs – inorganic (CO2, carbonates)
Heterotrophs – minerals, organic
substances( glucose, amino acids)
Outputs:
Nitrogen source
Atmospheric N2, inorganic N
compounds, derived nitrogen cpds
Other nutrients
Cell
S, P (elementary, inorganic or organic sulfur),
metallic elements (Na, K, Ca, Mg, Mn, Fe,
Zn, Cu, Co), vitamins.
Metabolites (CO2, H2O, etc)
Products
Energy source
Phototrophs – radiant energy
Chemotrophs – chemical reactions
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Newly
generated
biomass
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Bacterial Growth: Physical Conditions
▪ Temperature response:
HW 3A. Identify at
least 3 bacteria per
category.
Due 1 Oct
▪ Oxygen response:
▪ pH response:
Optimum pH 6.5 - 7.5 (most species)
Feasible limits pH 4 - 9 (most species)
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Bacteria
Spore formation
•
•
Spore formation (sporulation) occurs when
nutrients are depleted.
Spores are more resistant than normal cells to
heat, drying, radiation, and chemicals.
Cell wall structure
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Fungi
o plants devoid of chlorophyll hence cannot
synthesize own food
o range in size and shape from single-celled yeasts to
multicellular mushrooms
Yeast (Saccharomyces cerevisiae)
•
•
•
shape is spherical to ovoid
size is 1-5 µm in width and from 5-30 µm in length
found in fruits, grains, in the soil, air, on the skin and in
the intestines of animals
budding
Mold (Aspergillus and Penicillium)
•
•
filamentous fungi
A single reproductive cell or spore is germinated to
form a long thread, hyphae, which branches repeatedly
as it elongates to form a vegetative structure called a
mycelium.
branching
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Bacterial Cell Cultivation
The growth of microbial population in artificial environments.
pure culture - contains only 1 kind of microorganism
mixed culture - contains more than 1 kind of microorganism
Natural media - complex media containing peptones, beef extract, or yeast extract
- nutrient broth (liquid), nutrient agar (solid)
Synthetic media - consist of dilute, reproducible solutions of chemically pure, known
inorganic and/or organic compounds; for research purposes
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Animal Cell Cultivation
Tissue culture methodology has given researchers the opportunity to study cancer cells, to
classify malignant tumors, to determine tissue compatibility in transplantation, and to study
specific cells and their interactions.
The mammalian cell culture technique can be employed to produce clinically important
biochemicals such as human growth hormones, interferon, plasminogen activator, viral vaccines,
and monoclonal antibodies.
Has the capability of the post-translational modifications, which involve proteolytic cleavage,
subunit association, or a variety of additional reactions such as glycosylation, methylation,
phosphorylation, or acylation, that are important for proper biological activity of a product.
HW 4B. Cite 6 reasons why it is difficult to cultivate large quantities of
mammalian cells. Due 1 Oct
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Animal Cells
They are bound together by intercellular material to form tissue (epithelium, connective
tissue, muscle, and nerve).
Suspension cells – cells from connective tissues with liquid matrices (blood or lymph fluids), or nonanchorage dependent
when grown in culture
Anchorage-Dependent Cells - require a (wettable) surface for attachment and growth like plastic and glass
Anchorage: Petri dishes and roller bottles to spongy polymers, a ceramic matrix, hollow fibers, microcarriers
(capsules, beads)
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Animal Cell Cultivation
Growth media: Unlike microorganisms, animals do not metabolize inorganic nitrogen.
Therefore, many amino acids and vitamins should be provided.
• contains amino acids, vitamins, hormones, growth factors, mineral salts, and glucose
and supplemented with 2-20% v/v mammalian blood serum
• Serum-free media formulations contain purified hormones and growth factors as
substitute for costly serum supplements which may be contaminated with virus,
mycoplasma or proteins.
Polypeptide
chain
Disulfide
bond
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ANTIBODIES (IgG, A, M, D, E)
- produced by B-lymphocytes but which
cannot be maintained in a culture medium
- Research/clinical application: detect the presence
and level of drugs, bacterial & viral products,
hormones, and other antigens in blood samples
- have binding sites that recognize the shape of
particular determinants on the surface of the
foreign substance, or antigens
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Animal Cell Fusion
A fused cell, hybrid-myeloma (or hybridoma) cell is a hybridized B-Iymphocytes from the
mouse spleen with mouse myeloma cells.
Monoclonal antibody (MAb) – coming from 1 line of
B-lymphocytes, recognizes only 1 chemical
structure
Hybridoma cell – can be cultured,
Acquires the characteristics of the both cell lines:
• the production of specific antibodies
• immortality
.
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Plant Cell Cultivation
•
•
•
•
In order to produce secondary metabolites from a plant, plant cells are cultivated as a suspension culture.
By synthetic organic chemistry, either too difficult or too costly
Secondary metabolites - chemical compounds such as pharmaceuticals, flavors, pigments, fragrances,
agrochemicals produced in trace quantities in plants
only high value-added, low market-volume products are economically feasible to produce due to more
sophisticated handling of plant cell culture
.
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Plant Cell
20-40 μm dia
100-200 μm long
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distinctive features: rigid wall, a large vacuole, presence of
chloroplasts
cell wall: outer layer (for support)- middle lamella cont. heavy
layer of pectin (a polygalacturonan) that serves as glue to hold 1
plant cell to another;
inner layer (for migration of substances) - cell membrane cont.
protein and lipid
vacuole - a receptacle for waste metabolic products or secondary
plant substances, covered by plasma membrane
cytosol - liquid around floating structures bet. nucleus and cell
membrane
chloroplast - site of photosynthesis, contains chlorophyll that is
responsible for trapping the light
nucleus - cell's control center, contains DNA (prot. translation,
transcription)
Golgi apparatus – proteins synthesized are sorted and packaged
into its vesicles
Endoplasmic reticulum – network of tiny tubes that interconnect
the different
parts of the cells
.
Ribosome – actively involved in the synthesis of proteins
Mitochondrion – cont. genetic material and enzymes for cell
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metabolism
Plant Cell Cultivation
Unorganized growth - occurs frequently when pieces
of whole plants are cultured in vitro
Organized growth - occurs when organized plant parts
are transferred to culture medium where they may
continue to grow with their structure preserved
Callus cultures - small organs or tissue sections
Root cultures
Suspension (or cell) cultures - cells and cell
aggregates growing dispersed in liquid medium
Embryo cultures
Protoplast cultures – cells without cell walls
CULTURE MEDIA:
• Major nutrients (N, K, Ca, P, Mg, S salts)
• Plant growth regulators (hormones: auxins, cytokinins)
• Minor nutrients (Fe, Mn, Zn, B, Cu, Mo, Co salts)
• . Carbohydrate source (sucrose)
• Organic supplements (vitamins, amino acids, others) • Solidifying agent for semisolid (agar)
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Plant Cell Cultivation
Most
used
.
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CELL GROWTH MEASUREMENT
• Balanced growth is defined as growth during which a doubling of the biomass is accompanied by a
doubling of all other measurable properties of the population such as protein, DNA, RNA, and intracellular water.
Measurement of Cell Number
▪ Microscopic counts
▪ Viable plate count
▪ Coulter counter - conductivity method
.
Measures changes in electrical resistance
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CELL GROWTH
There are distinct growth phases in the growth curve of a microbial culture. A typical growth curve
includes the following phases 1) Lag phase; 2) Growth or Log Phase (accelerating, exponential, and
decelerating growth phase); 3) Stationary phase and 4) Death Phase
.
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CELL GROWTH
Lag phase - a period of adaptation of cells to a new environment (e.g., change in pH, increase in nutrient
supply, reduced growth inhibitors etc.) The cells increase in size and weight rather than increase in
numbers.; considered a non-productive period of a fermentative process thus often minimized or
controlled.
Log phase - reproduction at maximum rate (shortest generation time); exponential growth 1>2>4>8>16..
- At the late lag period, the cells have adjusted to the new environment and begin to grow and
multiply (accelerating growth phase) and eventually enter the exponential (logarithmic phase)
growth phase, where the cells grow and divide rapidly, at a relatively constant rate, as the
exponential functions of the time.
Stationary phase - no net increase, balance between cell division, cell "death", maintenance, growth rate
is equal to death rate; when a required nutrient is exhausted, or when inhibitory end products are
accumulated or when physical conditions changed.
Death phase - Death phase is the result of the inability of the bacteria to carry out further reproduction
as condition in the medium becomes less and less supportive of cell division. Eventually, the number of
. rate.
viable bacterial cells begins to decline at an exponential
• Industrial fermentation is usually interrupted at the end of the exponential growth phase or before the
death phase begins.
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Exponential growth model
Exponential growth can be described by below with k as exponential growth
rate:
Nt= N0 𝑒 𝑘𝑡
Exercise. A bacterial culture starting with 200 bacteria grows at a rate proportional to its size. After 3 hours there will
be 900 bacteria.
1.
2.
3.
4.
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What is exponential growth rate?
Express the population after t hours as a function of t?
What will be the population after 6 hours?
When will the population reach 5000?
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Ans: k=
Ans: Nt= 200 𝑒 0.501359𝑡
Ans: 4050 bacteria
Ans: t = 6.42 h
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CELL GROWTH MEASUREMENT
Balanced growth is defined as growth during which a doubling of the biomass is accompanied by a
doubling of all other measurable properties of the population such as protein, DNA, RNA, and intracellular water.
Measurement of Cell Mass
▪ Cell Dry Weight – for dense cell suspensions
▪ Turbidity – determine amount of light scattered
I
I0
.
Centrifugal settling then drying
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Spectrophotometer
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CELL GROWTH MEASUREMENT
Indirect methods are based on the overall stoichiometry for growth and product formation. The change of the cell
mass can be monitored indirectly by measuring nutrient consumption, product formation, cell components, heat
evolution, or other physical properties of broth.
▪
▪
▪
▪
▪
Nutrient Consumption - Phosphate, sulfate,
or magnesium (nutrient not likely to be used
to synthesize a metabolic product)
Product Formation - hydrogen ion, CO2
(growth associated)
Cell Components - macro-molecular cell
components (protein, RNA, DNA)
Heat Evolution – need to complete energy
balance of a fermentation system, typically 5
kcal/g
Viscosity - apparent viscosity measured at a
fixed shear rate can be used to estimate cell
or product concentration
.
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Reference
• Dutta, R. (2008). Fundamentals of Biochemical Engineering.
Springer.
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Requirements
• HW3/1: Sept29/Oct1
• HW4: Oct1
• Short test 1: Oct 8
• Coverage: Introduction/Enzyme Kinetics/Cell Cultivation
• Short test 2: Before Midterms
• Coverage: Stoichiometry of Microbial Growth and Product Formation/
Kinetics of substrate utilization, etc
HW 5: Stoich of microbial growth and product formation
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