Bioreactors and Fermentation

Outline
• Terminology
– Bioprocess, bioprocessing, biochemistry,
biotechnology, bioengineering, biomedical engineering,
biophysics
– Biopharmaceutical, biofuel, biodiesel, bioethanol, E10,
E85, B20, biomass, substrate, reaction kinetics,
 catalyst, enzyme
• Yeast
• Fermentation
• Bioreactor
• Analysis equipment
– Hemacytometer, Spectrophotometer, Cellometer,
HPLC system
Terminology
• Bioprocess
– Any process that uses living cells or their components
(e.g., bacteria, enzymes, chloroplasts) to obtain desired
products
• Bioprocessing
 – R&D and manufacturing of products prepared from or used by
biological systems (food, feed, biopharmaceuticals, and
cosmetics)
• Biochemistry
– Study of chemistry and biological processes of living
organisms and the molecular basis for the changes occurring in
living cells
• Biotechnology
– Use of biological systems to make/modify
products/processes (plant regeneration, gene
manipulation/transfer). In the past, producers used cross-
hybridization to alter a plant's genetic makeup. With
biotechnology, DNA can be altered directly.
 Terminology (contd.)
• Bioengineering
– Application of engineering in biological sciences. Bioprocess
engin. (biocatalysis, bioseparation, bioinformatics, bioenergy),
genetic engin. (sub-set of biotechnology), cellular engin.
(tissue culture), and biomedical engin. are sub-sets of
bioengineering.
• Biomedical engineering
– It combines chemical, electrical, and mechanical engineering
to improve medical diagnosis (ultrasound, MRI), monitoring,
and therapy (artificial hips, knees and other joints). Sometimes,
biomedical engineering is also referred to as bioengineering.
• Biophysics
– Use of physics to study biological systems. Overlaps with
biochem., nanotechnology, bioengin., agrophysics. May
involve molecular level issues (DNA, RNA, microscopy),
structural biology, enzyme kinetics etc. Biomedical physics is a
 Terminology (contd.)
sub-set.
• Biopharmaceutical
– Medical drugs produced using biotechnology. They are proteins
(including antibodies), nucleic acids (DNA, RNA or antisense
oligonucleotides), recombinant vaccines, and monoclonal
antibodies used for therapeutic or in vivo diagnostic purposes,
and are produced by means other than direct extraction from a
native (non-engineered) biological source.
• Biofuel
– Solid, liquid or gaseous fuel derived from organic (relatively
recently dead biological matter) material and is distinguished
from fossil fuels, which are derived from long dead biological
material. They can be produced from any (biological) carbon
source, with the most common source being photosynthetic
plants.
• Biodiesel (Fatty acid alkyl ester)
– Biofuel produced from vegetable oils (corn, cotton, rapeseed,
soybean, palm, mustard), restaurant waste oils, animal fats or
algae
– A non-petroleum-based diesel fuel consisting of long chain
alkyl (methyl, propyl or ethyl) esters, made by
transesterification of vegetable oil or animal fat (tallow), which
can be used (alone, or blended with conventional petrodiesel) in
unmodified diesel- engine vehicles (resulting in better emissions
in the form of decreased particulates, CO, and hydrocarbons)
– 100 lbs of oil + 10 lbs of methanol –> 100 lbs of biodiesel + 10 lbs of glycerol
• Bioethanol
– An alcohol made by fermenting sugars from biological
materials (starch crops -- corn) or cellulosic biomass (trees,
grasses)
– Can be used as vehicle fuel in its pure form or as a gasoline
additive to increase octane & improve vehicle emissions (CO2)
• E10
– Fuel that contains 10% ethanol, mixed with 90% gasoline
• E85
– A mixture of 85% ethanol and 15% gasoline. This
will not work in most cars.
• B20
– A blend of 20% biodiesel and 80% petroleum diesel
• Biomass
– Biological material (C, H, O, N, alkali, alkaline earth, heavy
metals) derived from living, or recently living organisms (plant
or animal based)
• Substrate
– The surface a plant or animal lives upon (serves as food source
for cells). It can be biotic or abiotic (non-living) materials.
• Reaction (chemical) kinetics
– Mechanism and factors influencing the speed of chemical reactions
• Catalyst
– A substance that increases the rate of a chemical reaction (by
providing a lower activation energy reaction mechanism to occur)
without being consumed or produced by the reaction. Proteins
that act as catalysts in biochemical reactions are called enzymes.
• Enzyme
– A specialized chemical that helps living organisms perform a task.
In bioreactors, enzymes created by microorganisms attack the
plant cell wall and break it up to get the glucose out.
 Yeast
• Unicellular fungus (more than 1000 species identified)
– Commonly used to leaven bread and ferment alcoholic beverages
– Most yeasts belong to the division Ascomycota
– A few yeasts (eg., Candida albicans) can cause infection in humans
– Saccharomyces cerevisiae (most commonly used yeast), was
domesticated for wine, bread, and beer production 2000+ yrs
ago
• Yeast physiology can be either obligately aerobic
or facultatively anaerobic (fermentative)
– There is no known obligately anaerobic yeast
• In absence of O2, fermentative yeasts produce energy by
converting sugars into carbon dioxide and ethanol
(alcohol)
• In brewing, ethanol is the desired product, while in
baking, carbon dioxide raises the bread and the ethanol
evaporates
 Yeast (contd.)

• Many yeasts can be isolated from sugar-rich

environmental samples such as fruits and berries (grapes,
apples, peaches etc.) and exudates from plants (such as
plant saps or cacti)
• The most common mode of vegetative growth in yeast is
the asexual reproduction by budding or fission
– A small bud (daughter cell), is formed on the parent cell
– The nucleus of the parent cell splits into a daughter nucleus
and migrates into the daughter cell
– The bud continues to grow until it separates from the parent
cell, forming a new cell
• In brewing beer, top-fermenting yeasts (float to the top of
the beer) produce higher alcohol concentrations and
prefer higher temperatures (15-25 °C)
– Eg., Saccharomyces cerevisiae (known to brewers as ale yeast)
• They produce fruitier, sweeter type ale beers
• Bottom-fermenting yeasts ferment more sugars, leaving
a crisper taste and work well at low temperatures (5-10
°C)
– Eg., Saccharomyces uvarum (formerly known as
Saccharomyces) carlsbergensis
• They are used in producing lager-type beers
• Brewers of wheat beers often use varieties of
Torulaspora delbrueckii
 Fermentation
• Conversion of carbohydrate (eg. sugar) into acid or
alcohol by yeast or bacteria
• It is used in brewing and wine making for the conversion
of sugars to alcohol (ethanol – CH3CH2OH)
– This process, followed by distillation, can be used to obtain
pure ethanol (bioethanol) for use as a transport biofuel
• It can also be viewed as the energy-yielding anaerobic
metabolic breakdown (respiration) of a nutrient molecule
such as glucose, without net oxidation (eg., in muscle
cells)
• Fermentation typically refers to the fermentation of sugar
to alcohol using yeast, but other fermentation processes
include making of yogurt, souring of milk, rising of dough
 Bioreactor

• An apparatus (usually jacketed cylindrical SS vessel) for

growing organisms such as bacteria, viruses, or yeast that
are used in the production of pharmaceuticals, antibodies,
or vaccines, or for the bioconversion of organic wastes
• Under optimum conditions of gas (air, oxygen, nitrogen,
and carbon dioxide) flow rates, temperature, pH,
dissolved oxygen level, and agitation speed, the
microorganisms or cells will reproduce at a rapid rate
Controls
Temperature
Pressure
pH
Agitation speed
Air flow rate
 Hemacytometer
A device used to count cells A thick glass microscope slide with a
rectangular indentation that creates a
chamber that is engraved with a
laser- etched grid of perpendicular
lines.
The area bounded by the lines and
depth of chamber are known. Thus,
by counting the number of cells in
that volume of fluid, we can calculate
the concentration of cells in the fluid.
 Cellometer
Automated cell counting – no need
for r.
hemacytomete
ing Chambers
Disposable Countlosed chambers
consist of two encntrolled height. 20
with a precisely
co
l is loaded into the chamber and
inserted into the Cellometer. It
utilizes bright field imaging and
pattern-recognition software to
identify and count individual live
& Trypan
dead cells stained with

Green: Live cells

Cellometer
Red: Dead cells
 Spectrophotometer
Measures amount of
light reflected from an
object or amount of light
absorbed by an object
Biomate 3

Sample
 High Performance Liquid Chromatography
(HPLC) System
HPLC: Mass Separate, identify,
transfer involving
quantify components
adsorption Granular mtl.
(silica, polymers;
in a mixture. Pump a
2-50 m) solvent with the
sample through solid
column of adsorbent
material.
Each component
diffuses at diff. rate,
UV absorbance thereby separating.
Fluorescence Detector identifies &
Refractive index quantifies each
Evaporative light scattering component.
 Summary
• Bioprocess – Any process that uses living cells or their
components (e.g., bacteria, enzymes,
chloroplasts) to obtain desired products
• Bioprocessing
– R&D and manufacturing of products
prepared from or used by biological
systems (food, feed,
biopharmaceuticals, and cosmetics)
• Yeast
– Obligate aerobe or facultative anaerobic
(fermentative)
– In absence of O2, fermentative yeasts
produce energy by converting sugars into
CO2 and ethanol (alcohol)
• Fermentation
– Conversion of carbohydrate (eg. sugar) to
acid/alcohol by yeast/bacteria
• Bioreactor
 – Growing
bacteria/v
irus/yeast
by
controllin
g
temperatu
re, pH, air
flow
• Analysis
equipment
– Hemacyt
ometer,
Spectroph
otometer,
Cellomet
er, HPLC
system