MEMBRANE SEPARATION & BIOPROCESS

TECHNOLOGY

Presenter: Dr. T. Mamvura

Lecturer, Chemical, Materials & Metallurgical
Engineering
 BIOPROCESS TECHNOLOGY

• Bioprocess technology is a technique that produces a

biological material
• Objectives of bioprocess technology are:
✓ Biomass production
✓ Enzyme production
✓ Metabolite production
✓ Recombinant protein production
✓ Metabolic biotransformation
Milestones of Bioprocess Development
 HISTORY OF BIOPROCESS TECHNOLOGY

 10,000-7,000 BC Wine making developed in Eastern

Mediterranean
 7,000-5,000 BC Beer developed in Egypt and Babylon
 5.000 BC Cheese making & some medicinal plants were
developed
 4,000 BC Vinegar was referenced in old testament
 500 AD Algae was cultivated for food by Aztecs
 500 AD Yogurt, sauces & fermented meats were
developed
 1600 AD The name fermentation was used
 1680 AD Van Leeuwenhoek observed yeast cells
 1781 AD Pressed Baker’s yeast produced by Dutch
(this was the first improvement process
in handling Baker’s yeast).
 1789 AD Jenner demonstrated vaccination against
smallpox infection (First immunization)
 HISTORY OF BIOPROCESS TECHNOLOGY

 1837 AD Cagniard-Latour, Schwann and Kutzing independently

hypothesized that yeast is a living thing. (First
knowledge on cell biology).
 1847 AD Blondeau studied fermentation of lactic acid, butyric
acid, acetic acid and urea. He hypothesized that
different fermentations carried out by different
organisms (fungi?)
 1857 AD Pasteur demonstrated that living yeast cells ferment
sugar into ethanol and carbon dioxide. Pasteur noted
cylindrical organisms produce butyric acid only in
absence of oxygen. (First knowledge on anaerobic
fermentation).
 1859 AD Darwin published the Origin of Species.
 1877 AD Pasteur noted relationship between microbes/
infectious disease. (First knowledge on pathogenic
organisms).
 HISTORY OF BIOPROCESS TECHNOLOGY

• 1881 AD Koch developed methods for handling microbial

cultures (First knowledge on microbial technique
and maintenance).
• 1881 AD First commercial production of lactic acid by
anaerobic fermentation.
• 1894 AD Takamine patented a process to isolate diastase
enzymes from molds that can breakdown starch.
(First knowledge on enzymes it’s applications).
• 1916 AD Germany produces baker’s yeast grown on
molasses as protein supplement. Also, produced
glycerol by yeast fermentation.
 1918 AD Great Britain produced acetone and butanol by
anaerobic fermentation process.
 1923 AD Commercial production of citric acid by surface
cultures.
 HISTORY OF BIOPROCESS TECHNOLOGY

 1929 AD Fleming demonstrated that mold contaminant in a

petri-dish causes bacterial death. (Fist discovery of
microbial antibiotics)
 1934 AD Gautheret successfully cultured plant cells. (First
knowledge on plant tissue culture).
 1940 AD Florey and Chain isolated penicillin, elucidated its
structure and demonstrated its bacterial properties on
G(+) bacteria.
 1940s Waxman discovered streptomycin's. and its properties
on G(-) bacteria (first microbial screening method for
new antibiotics discovery) Waxman also discovered
vitamin B12.
 1950s The production of Cortisone at the cost of $200/g (now
it’s cost is $16/g). The production of polio and peruses
vaccines.
 1960s The production of Xanthus gum. The production of
alkine protease for detergents industry.
 HISTORY OF BIOPROCESS TECHNOLOGY

 1970s The discovery of glucose isomerase and the production of

high fructose corn syrup (HFCS ) as a sweetener.
 1970s Human growth hormone was synthesized. The
development of recombinant human insulin.
 1980s Kohler and Milstein developed monoclonal antibodies.
The production of poly (hydroxybutyrate).
US Supreme Court approved the principle of
patenting organisms.
First recombinant DNA vaccine for livestock.
FDA approved Insulin drug.
Interferon the first anticancer drug.
The first pest resistant corn (Bt corn).
FDA approve the first hepatitis C vaccine.
The first transgenic animals
 HISTORY OF BIOPROCESS TECHNOLOGY

 1990s The production of amino acids (lysine, threonine and

isoleucine).
The production of antibodies.
The discovery of first breast cancer gene.
The discovery of gene associated with Parkinson's disease.
A sheep named Dolly become the first animal cloned from
an adult cell.
FDA approved bovine (BST) for dairy cows.
The commercialization of first genetically engineered crops
The first gene therapy for immune disorder.
 HISTORY OF BIOPROCESS TECHNOLOGY

 2000s The production 1,2-propandiol.

The production of xylitol
The production of hydroxpropanoic acid.
FDA approved the first gene target for patient
with chronic myeloid leukemia.
FDA approved the first transgenic rootworm
resistant corn.
Sequencing human genome.
FDA approved the first anti-angiogenic drug for
cancer.
FDA approved the recombinant HPV vaccine.
FDA approved the first H5N1 avian flue vaccine.
 HISTORY OF BIOPROCESS TECHNOLOGY

 2000s FDA approved the first transgenic animal for

the production of human recombinant anti-
thrombine.
USDA granted the first regulatory for a plant
made vaccine.
Global biotechnology crops reaches over 330
million acres.
 2010s The creation of first synthetic cell.
Advances in stem cell technology.
Advances in 3-D printing technology leading to
skin printing.
BIOPROCESS TECHNOLOGY
 BIOPROCESS TECHNOLOGY

• Tools of Industrial bioprocessing:

❑Genetic engineering
❑Protein engineering
❑Metabolic engineering
❑New developments in Synthetic biology
• Challenges of Industrial bioprocessing:
❖Agricultural raw materials are increasing costly
❖Low-cost materials like cellulose cannot be readily used for
microbial process
❖Bioprocess is still not effective as chemical processing
❖Chemical industry is also emerging as competitive
❖Large amount of funding is needed for Bioprocess
 BIOPROCESS TECHNOLOGY

• Impact of Bioprocessing:
✓ Make agriculture more competitive and sustainable
✓ Improve quality of life of people while reducing impact by
development of innovative and affordable products
✓ Helps to increase industrial economy and environmental
efficiency and sustainability
BIOPROCESS TECHNOLOGY: BIOREACTOR
 Two Commercial Sustainable Biofuels

Ethanol Biogas
• 90% of World´s • Local waste to fuel
biofuel • Good for local fleets
• Local & global fuel • Can be combined with
Compressed Natural Gas (CNG)
• Sustainable volume
• Expensive infrastructure
production
• CO2 reduction >95 %
• Stable fuel
• CO2 reduction 71%
 Biogas

? What is biogas
A: A mixture of methane and carbon dioxide
 How is it made?

• Biogas is produced by the breakdown of organic waste by bacteria

in the absence of O2 (anaerobic digestion or fermentation)

Leftover food from houses,

shops, restaurants and factories

What types of organic waste could

be turned in biogas? Leftover straw
Cow, sheep and
Sewage and crops from
chicken manure
farming

Leftover meat and blood

from abattoirs
 How is it made? Anaerobic digestion

Two Types of Bacteria

Each Relying On The Other

First Stage Second Stage

“TWO-STAGE” Continuous Process

 Anaerobic Digestion Process: 1st Stage

Organic Material hydrolysis (broken down) and Changed

By Acid Forming Bacteria

To Simple Organic Material

Organic Organic
Matter + Bacteria Acids

First Stage
 Anaerobic Digestion Process: 2nd Stage

Methane-Forming Bacteria
Use Organic Acids

Produce Carbon Dioxide and Methane

CH4
Organic
Acids + Bacteria +
CO2

Second Stage
 Anaerobic Digestion Process

Food Sources: Soluble organic materials (not inorganics)

Cell Acids
Membrane

Enzymes

(Absorption)
Adsorbed
Typical Acid Forming Bacteria Particle
 Anaerobic Digestion Process

Acid Forming Methane Forming

CH4
Organic Organic
Bacteria +
Matter Acids
CO2

First Stage Second Stage

Volatile
Acid Phase Acids

Acids Used at Rate Produced

 Anaerobic Digestion Process

Acids Used at Rate Produced

If Not Used  Drop in pH
Start-up
Upset
“Sour”
“Stuck”

Methane Formers Must Be Active

 Anaerobic Digestion Process

Methane Formers:
Slow Growers Loading
Very Sensitive to Changes pH
Temperature

• Digester Operation Depends On Maintaining

Proper Environment for METHANE FORMERS

A BALANCE IS NEEDED
 Anaerobic Digestion Process

• Products of Digestion Process:

1. Gases
Methane (CH4)
Carbon Dioxide (CO2)
2. Scum
Lighter Solids
3. Supernatant
Liquid Removed
4. Digested Sludge
“Stabilized”
 Ethanol

? What is ethanol
A: A mixture of ethanol & water. Ethanol is a natural byproduct of plant
fermentation by yeast & also can be produced through the hydration of
ethylene

• How is ethanol made?

A: Ethanol is primarily produced from the fermentation of starch in
corn/maize grain or sugar molasses or any feedstock with sugar in it
• In the fuel industry, biorefineries use state-of-the-art technologies to
convert grains, beverage & food waste, cellulosic biomass & other
feedstocks collectively known as BIOMASS into high-octane ethanol
• Is ethanol & alcohol the same?
A: Ethanol aka alcohol or ethyl alcohol is a clear, colourless liquid & the
principle ingredient in alcoholic beverages like beer, wine or brandy
Ethanol Production
Ethanol
Typical Ethanol Producing Plant
 Fermentation of Sugars

• Certain species of yeast (e.g., Saccharomyces cerevisiae)

metabolize (breakdown) sugar to produce ethanol & CO2
• Chemical equations below summarize the conversion:
• Glucose ---------> Ethanol + Carbon Dioxide
𝑌𝑒𝑎𝑠𝑡
• 𝐶6 𝐻12 𝑂6 2𝐶𝐻3 𝐶𝐻2 𝑂𝐻 + 2𝐶𝑂2
• Sucrose + Water -----> Ethanol + Carbon Dioxide
𝑌𝑒𝑎𝑠𝑡
• 𝐶12 𝐻22 𝑂11 + 𝐻2 𝑂 4𝐶𝐻3 𝐶𝐻2 𝑂𝐻 + 4𝐶𝑂2
• Alternatively:
𝐼𝑛𝑣𝑒𝑟𝑡𝑎𝑠𝑒
• 𝐶12 𝐻22 𝑂11 + 𝐻2 𝑂 2𝐶6 𝐻12 𝑂6
• Enzyme invertase breakdown sucrose to glucose & yeast
then ferments the glucose
 Fermentation of Sugars

• Fermentation is the process of culturing yeast under

favourable thermal conditions (35–40oC) to produce
alcohol
• Toxicity of ethanol to yeast limits the ethanol
concentration obtainable by brewing
• Higher concentrations are obtained by fractional
distillation
• The most ethanol-tolerant yeast strains can survive up to
18% ethanol by volume
Distillation of Ethanol-Water Mixture
 Fermentation of Sugars

• To produce ethanol from starchy materials such as cereals, starch

must first be converted into sugars

• In beer brewing this has traditionally been accomplished by

allowing the grain to germinate or malt, which produces the
enzyme amylase
• When the malted grain is mashed, the amylase converts the
remaining starches into sugars
 Fermentation of Sugars

• Batch fermentation usually employed

• Fermenter volume – 600 m3
• 3% inoculum (3 x 106 yeast cell/ml)
• Within 12 days yeast produces 10% ethanol by volume
• If process is carried out at 35 – 38oC & pH 4 – 4.5;
maximum production is 1.9 g/hr
• 80% cells removed in separator & recycled into fermenter
• When high quality molasses is used, max yield is 95%
• For continuous fermentation, centrifuge used to recycle
yeast cells
• For continuous fermentation more than one tank is used
Beer Production Process - KBL
Beer Production Process - KBL
CELLS AND CELL STRUCTURE
 DEFINITION OF CELLS

A cell is chemical system that is able to maintain its structure and

reproduce or in simpler terms, “the basic unit of life”
 DISCOVERY OF CELLS

In 1665, Robert Hooke used a microscope to examine a thin

slice of cock (dead plant cell walls). He saw “row of empty
boxes”. He named them “CELLS” because they looked like the
small rooms that the monks lived in called Cells.
 CELL THEORY

The cell theory proposed by Theodor Schwann in 1839,

states that;
– Cells are the fundamental units of life
– All living organisms are composed of cells
– All cells come from pre-existing cells
– Evolution through natural selection explains the diversity of
modern cells.
CELL SIZE
 CELL SIZE Cont.

• Cell size is limited by the surface area-to-volume ratio

• As an object grows larger, its volume increases more rapidly than its
surface area
• Cells must maintain a large surface area-to-volume ratio in order to
function
Input and Output
Cultivation Media
Microorganisms
 NUMBER OF CELLS

Although all living things are made of cells, organisms may be:
• Unicellular – composed of one cell
• Multicellular- composed of many cells that may organize into
tissues, etc.
 MICROBIAL DIVERSITY

• Living cells can be found almost anywhere where water is in the

liquid state. The right temperature, pH, and moisture levels vary
from one organism to another
• Some cells can grow at -20°C (in brine to prevent freezing), while
others can grow at 120°C (where water is under high enough
pressure to prevent boiling)
• Cells that grow best at low temperatures (below 20°C) are usually
called psychrophiles, while those with temperature optima in the
range of 20° to 50°C are mesophiles. Organisms that grow best at
temperatures greater than 50°C are thermophiles.
• Many organisms have pH optima far from neutrality; some prefer
pH values down to 1 or 2, while others may grow well at pH 9.
Some organisms can grow at low pH values and high
temperatures
 MICROBIAL DIVERSITY Cont.

• Although most organisms can grow only where water activity is

high, others can grow on barely moist solid surfaces or in
solutions with high salt concentrations
• Some cells require oxygen for growth and metabolism. Such
organisms can be termed aerobic. Other organisms are
inhibited by the presence of oxygen and grow only
anaerobically. Some organisms can switch the metabolic
pathways to allow them to grow under either circumstance.
Such organisms are facultative
• Some bacteria are photosynthetic and can convert CO2 from
the atmosphere into the organic compounds necessary for life.
They can also convert N2 into NH3 for use in making the
essential building blocks of life. Such organisms are important
in colonizing nutrient-deficient environments
 CELL TYPES

There are two primary cell types:

–Procaryotic cells
–Eucaryotic cells
PROKARYOTIC CELL
 PROKARYOTIC CELLS

• Procaryotes are a kingdom of organism including bacteria and

cyanobacteria
• Procaryotes have a simple structure with a single chromosome
• Procaryotic cells have no nuclear membrane and no
organelles, such as the mitochondria and endoplasmic
reticulum
• Procaryotic cells grow rapidly, with typical doubling times of
one-half hour to several hours
• The sizes of most procaryotes vary from 0.5 to 3 micrometres
(µm) in equivalent radius
EUKARYOTIC CELL
 EUCARYOTIC CELL Cont.

• Fungi (yeasts and molds), algae, protozoa, and animal and plant
cells constitute the eukaryotes
• Eucaryotes have a more complex internal structure, with more
than one chromosome (DNA molecule) in the nucleus
• Eucaryotes are five to ten times larger than procaryotes in
diameter (e.g., yeast about 5 mm, animal cells about 10 mm, and
plants about 20 mm)
• Eucaryotic cells have a true nuclear membrane and contain
mitochondria, endoplasmic reticulum, golgi apparatus and a
variety of specialized organelles
• Golgi apparatus (aka golgi body, golgi complex) - made up of
membrane sacs and is used to process and bundle macromolecules
like proteins and lipids as they are synthesized within the cell
Cell Structure and Functions
 CELL MEMBRANE (PLASMA MEMBRANE)

• Each cell has a limiting boundary, the cell membrane, plasma membrane
or plasmalemma. It is a living membrane, outermost in animal cells but
next to cell wall in plant cells.
• The plasma membrane encloses the cell contents.
• It provides cell shape (in animal cells) e.g. the characteristic shape of red
blood cells, nerve cells, bone cells, etc
• It allows transport of certain substances into and out of the cell but not
all substance, so it is termed selectively permeable
 MEMBRANE PROTEINS

Membrane Protein Function

Channels or Transporters Move molecules in one direction

Receptors Recognize certain chemicals

Glycoproteins Identifies cell type
Enzymes Catalyze production of substances
 TRANSPORT MECHANISM – SMALL MOLECULES

Small molecules can be transported across the plasma membrane by any

one of the following three methods:
• Diffusion: molecules of substances move from their region of higher
concentration to their region of lower concentration. This does not
require energy. E.G. absorption of glucose in a cell
• Osmosis: movement of water molecules from the region of their
higher concentration to the region of their lower concentration
through a semipermeable membrane. There is no expenditure of
energy in osmosis. This kind of movement is along concentration
gradient
• Active Transport: When the direction of movement of a certain
molecules is opposite that of diffusion i.e. from region of their lower
concentration towards the region of their higher concentration, it
would require an “active effort” by the cell for which energy is needed.
This energy is provided by Adenosine Triphosphate (ATP). The active
transport may also be through a carrier molecule
 TRANSPORT MECHANISM – LARGE MOLECULES

During bulk transport the membrane changes its form and shape. It
occurs in two ways:
(i) endocytosis (taking the substance in)
(ii) exocytosis (passing the substance out)

Endocytosis is of two types :

 THE FLUID MOSAIC MODEL

• Plasma membrane is composed of a lipid bilayer of phospholipid

molecules into which a variety of globular proteins are
embedded
• Each phospholipid molecule has two ends, an outer head
hydrophilic i.e. water attracting, and the inner tail pointing
centrally hydrophobic, i.e. water repelling
• Protein molecules are arranged in two different ways:
(i) Peripheral proteins or extrinsic proteins: these proteins are present on the
outer and inner surfaces of lipid bilayer
(ii) Integral proteins or intrinsic proteins: These proteins penetrate lipid
bilayer partially or wholly
 THE CELL WALL

• The cell wall of eucaryotic cells shows considerable variations.

Some eucaryotes have a peptidoglycan layer in their cell wall;
some have polysaccharides and cellulose (e.g., algae)
• The plant cell wall is composed of cellulose fibres embedded
in pectin aggregates, which impart strength to the cell wall
• Animal cells do not have a cell wall but only a cytoplasmic
membrane. For this reason, animal cells are very shear-
sensitive and fragile. This factor significantly complicates the
design of large-scale bioreactors for animal cells
 THE CELL WALL

• It is fully permeable

• Surrounds the plasma membrane

• It is 5 to 10 nm thick

• It gives shape, support and

protection to the cell
 THE CYTOPLASM

cytoplasm

• Jelly-like substance enclosed by cell

membrane

• It provides a medium for chemical

reactions to take place

• Components of cytoplasm include

fluid (cytosol), organelles (excluding
nucleus, storage substances and,
interconnected filaments and fibres
 THE CYTOSKELETON

• Made up of filaments and

fibres

• Made of 3 fibre types

– Microfilaments
– Microtubules
– Intermediate filaments

• 3 functions:
– mechanical support
– anchor organelles
– help move substances
 FLAGELLA AND CILLIA

• Made of protein tubes called microtubules

• Microtubules are in a 9 + 2 arrangement, i.e.
two central microtubules and nine set
surrounding them
• They are responsible for moving cells, fluids and
small particles across the cell surface

• Flagella
– Longer and fewer in cells
– Found on sperms

• Cilia
– Shorter and more numerals in cells
– Protoplamic projection and membrane
bound
 FLAGELLA AND CILLIA Cont.

Cilia moving away dust particles from the lungs respiratory system
 NUCLEUS

• It is the largest organelle Nucleus

• It is enclosed by a nuclear membrane of
double membranes that is perforated by
nuclear pores
• Controls cell activities
• Storage of genetic information
 INSIDE THE NUCLEUS

Chromosomes
• Contains small amounts of RNA and
basic proteins called histones attached
to the DNA
• Each chromosome contains a single
linear DNA molecule on which the
histones are attached

The Nucleolus
• Cell may have 1 to 3 nucleoli
• Concentrated area of chromatin, RNA
and proteins
• It is the site of ribosome synthesis
 THE DNA

DNA is the hereditary materials

of the cell

Genes that make up the DNA

molecules code for different
proteins
 RIBOSOMES

• Ribosomes are composed of Proteins and rRNA

• Join amino acids to make proteins, i.e. protein
synthesis
 MITOCHONDRIA

• Cylindrical shaped organelle usually 1

µm in diameter and 2 to 3 µm in length
• Outer membrane is smooth while inner
membrane is highly folded
• A mitochondrion has its own DNA and
it can independently synthesize protein
and reproduce
Functions
• Respiration and oxidative
phosphorylation take place within the
mitochondria – Energy production.
• Signalling
• Apoptosis role – Programmed cell death
 ENERGY PRODUCTION

• Electrochemical proton gradient across the inner

mitochondrial membrane is used to drive ATP synthesis

Respiration
•Raw Materials
– Oxygen
– Pyruvate & Fatty Acids
•Products
– Carbon Dioxide
– Adenosine Triphosphate (ATP)
 APOPTOSIS
• Mitochondria in addition to energy production, have a second major function
related to programmed cell death by apoptosis
• Cytochrome C release activates caspases
• Other changes include
– electron transport, loss of mitochondrial transmembrane potential
– altered cellular oxidation-reduction
– Production of Bcl-2 family proteins (pro- and antiapoptotic)
• Vesicular Mitochondria
– begin to appear during the release of cytochrome C which initiates
mitochondrial mediated apoptosis
– transformation from normal morphology
– with an inner boundary membrane connected to lamellar cristae via crista
junctions
– multiple vesicular matrix compartments
– facilitates membrane fission or fragmentation as the matrix is fragmented at
this stage
– fragmentation of the mitochondrion requires only outer membrane fission
 MITOCONDRIA LOCATION

• Generally located where energy consumption is

highest in the cell

• Cells with high energy requirements: Muscle,

sperm tail, flagella
– Packed around initial segment
– Energy for sperm mobility & microtubules
 ENDOPLASMIC RECTICULUM (ER)

• It is a complex, convoluted
membrane system running
throughout the cytoplasm. It
consists of two types; rough ER and
smooth ER

• The rough ER contains ribosomes on

the inner surface and is the site of
protein synthesis and modification

• The smooth ER lack ribosomes and

is concerned with the synthesis and
transport of lipids and steroids
 GOLGI APPARATUS

• Very small particles composed of

membrane aggregates
• Have shipping side (trans face) and
receiving side (cis face)
• Receives proteins made by ER
• Packaging and shipping station
• Involved in sysnthesis of the cell
wall
GOLGI APPARATUS FUNCTION

1. Molecules come in vesicles

2. Vesicles fuse with Golgi membrane

3. Molecules may be modified by Golgi

4. Molecules pinched-off in separate

vesicle

5. Vesicle leaves Golgi apparatus

6. Vesicles may combine with plasma

membrane to secrete contents
 LYSOSOMES

• Very small membrane –bound

particles that contain and release
digestive enzymes

• Digests nutrients and invading

substances

• Lyse (break open) and release

enzymes to breakdown and recycle
cell parts
 CHLOROPLASTS

• Chloroplasts are relatively large,

chlorophyll-containing, green
organelles that are responsible
for photosynthesis in
photosynthetic eucaryotes, such
as algae and plant cells

• Every chloroplast contains an

outer membrane and a large
number of inner membranes
called thylakoids
 CHLOROPLASTS Cont.

• Chlorophyll molecules are associated with thylakoids, which have

a regular membrane structure with lipid bilayers
• Chloroplasts contain their own DNA and can synthesize protein
 VACUOLE

• Membrane-bound storage sacs

• Vacuoles may occupy large
volumes (up to 90% in plant
cells)
• They are more common in plants
than animals
• Vacuoles are responsible for food
digestion, osmotic regulation and
waste-product storage
For more information contact:

Dr. Mamvura
Faculty of Engineering and Technology
Department of Chemical, Materials and
Metallurgical Engineering

Tel: (+267) 493 1827

E-mail: mamvurat@biust.ac.bw