1. Topics
Fermentation
Biotechnology Introduction
Microbiology
Metabolism (Metabolic pathways)
Prof. S.T. Yang
Medium formulation; sterilization
Dept. Chemical & Biomolecular Eng.
The Ohio State University Growth and fermentation kinetics
Industrial Biotechnology Industrial Biotechnology
Today Tomorrow
Baby whole carrots; Fresh-for-two-week Seedless mini melon; Sweeter peas and
tomatoes; Insect-protected crops peppers; Colored cotton
High-laurate oil, for soaps and detergents Healthy low-saturated oil;
Bst-stimulated milk; Vaccinate eggs Faster growing salmon; Non-allergenic cats
Antibiotics, Vitamins, … AIDS vaccine, cancer drugs/vaccines
Citric acid, glutamic acid, lysine, … Artificial tissues and organs
Insulin; Hepatitis B Vaccine; tPA, EPO, .. Bioplastics; Biochemicals; Biomaterials
Ethanol, Methane gas Hydrogen, biofuels
1

2. Four Types of Fermentation Products Industrial fermentation products
Production Microorganism Applications
Cells (biomass) (metric tons)
Proteins, enzymes (cell components) Citric acid 1,200,000 A. niger Food
Ethanol 26,000,000 S. cerevisiae Fuel
Metabolites Glutamate 1,000,000 C. glutamicum Flavoring
Lactic acid 400,000 Lactobacillus sp. Food, Plastics
• Primary metabolites Lysine 800,000 C. glutamicum Feed
Penicillin 60,000 P. chrysogenum Drug
• Secondary metabolites Xanthan gum 100,000 X. campestris Food, Oil drilling
Biotransformation (steroids)
Changing the history Changing the history
- naturally-occurring organisms - genetically modified organisms
Product Application Organism
Product Application Organism
Bacitracin Antiobiotics Bacillus strain
Bovine growth hormone Milk production Escherichia coli
Citric acid Food flavoring Aspergillus niger
Cellulase Cellulose hydrolysis Escherichia coli
Invertase Candy Saccharomyces cerevisiae
Human growth hormone Growth deficiencies Escherichia coli
Lactase Digestive aid Escherichia coli
Human insulin Diabetics Escherichia coli
Pectinase Fruit juice Aspergillus niger
Monoclonal antibodies Therapeutics Mammalian cell culture
Penicillin Antibiotics Penicillium notatum
Ice-minus Prevent ice from plants Pseudomonas syringae
Riboflavin Vitamin Ashbya gossypii
Sno-max Make snow Pseudomonas syringae
Subtilisin Laundry detergent Bacillus subtilis
tPA Blood clots Mammalian cell culture
Tetracycline Antibiotics Streptomyces aureofaciens
Tumor necrosis factor Kill/inhibit tumor cells Escherichia coli
Xanthan gum Rheology modifier Xanthomonas campestris
2

3. Classification of microorganisms Prokaryotes
Prokaryotes – Bacteria, Blue-green algae Unicellular: bacteria
Multicellular: cyanobacteria
Eukaryotes – Fungi (molds, yeasts),
Do not contained membrane-contained
algae, Protozoe nucleus
Archaebacteria Can accept a wide variety of nutrients
Rapid growth
Viruses
Versatile biochemical metabolism
Structure of bacterial cell Essential structure
Essential structures Cell wall:
• 20 nm thick
Non-essential • Consists of peptidoglycan
structures • Structural strength and shape
Cell membrane:
• 7-9 nm thick
• Lipid bilayer
• Semi-permeable - controls the transfer of
chemicals and nutrients
3

4. Essential structure (cont’d) Essential structure (cont’d)
Nuclear body: Cytoplasm:
• DNA • Fluid material
• Control center for all operation
• No nuclear membrane Mesosomes:
• No mitotic apparatus during replication • Bacteria do not have mitochondria, but have
Ribosomes: mesosomes, which are extensions of the
• Sites of important biochemical reactions cytoplasmic membrane
• Protein synthesis • ATP
Non-essential structure Non-essential structure (cont’d)
Pili (fimbria): Capsule (slime layer):
• Gram(-) rods • Secreted by cells to increase viscosity and
• Sexual conjugation impede diffusion
• Adhesive to animal and plant cells • Coating to cell wall
• Insert surface Volutin (mitochromatic) granules:
Flagella: • Highly refractile globules
• Motility of bacteria • Sourse of stored food, e.g. PHB
• Can be polar or peritichous • Appearance influenced by age
4

5. Non-essential structure (cont’d) Actinomyces spp.
Chromatophores: Beaded appearance of branched filamentous
• photosynthetic rod shaped bacteria
• Counterpart of chloroplasts for plant cells Cells are smaller
Endospores:
No nuclear membrane
• Highly resistant to destructive effects of
chemical and physical agents No lysozyme
• Contain large amounts of dipicolinic acid Extremely important as a source of powerful
(DPA)
antibiotics
Cyanobacteria Classification of bacteria
Gas vesicles
Grain stain
Gas vesicles are aggregates of hollow cylindrical structures
Morphological type – cocci, rod, spiral
composed of rigid proteins. They are impermeable to water,
but permeable to gas. The amount of gas in the vacuole is Spore forming or not
under the control of the microorganism. Metabolism of sugar (carbon) substrates
Gas vesicles are found in Cyanobacteria, which are Growth requirements:
photosynthetic and live in aquatic systems. In these lakes • Oxygen – aerobic, anaerobic or facultative
and oceans, the Cyanonbacteria want to control their • Nutrients
position in the water column to obtain the optimum amount
of light and nutrients.
Genetic composition (GC content)
5

6. Gram stain Eukaryotes
Unicellular: yeasts, algae
Multicellular: molds, algae
Posses a membrane-contained organelles
Larger than prokaryotes
Complex cell structure
Spatial organization and differentiation
Yeast Yeast
Elliptical or spherical cells
Size: 5 -10 microns
Form spores
Vegetative growth is by budding
Aerobic and anaerobic growth
Colony on agar plate similar to bacteria’s
Important in beverage alcohol industry, ethanol,
baker yeast and single cell proteins (SCP)
6

7. Mold (Filamentous Fungus) Mold
Filamentous
Hyphae
Complicated life cycles
Sexual and asexual spores
Aerobic
Mostly pathogens of plants
Important in industrial fermentations:
• Organic acids (citric, gluconic, gibberellic acid)
• Antibiotics (penicillin, griseofulvin)
• Enzymes (cellulase, protease, amylase)
Cause spoilage in paper, fabrics and food
Eukaryotes
Animal and plant cells Plant cells
Either as callus (undifferentiated plant
tissue)
Or as aggregated cells in suspension
Can produce many commercially
important compounds (perfumes, dyes,
medicines and opiates)
Can catalyze highly specific useful
transformations
7

8. Eukaryotes
Animal cells Eukaryote v.s. Prokaryote
Tissue derived cells
Primary cell lines secondary cell lines
established, permanent cell lines
Anchorage dependent cells
Microcarrier culture techniques
Flagella
Important in large scale production of
vaccines and other useful biochemicals
and therapeutics
Prokaryotes v.s. Eukaryotes Prokaryotes v.s. Eukaryotes
Genome Organelles
Characteristics Prokaryotes Eukaryotes
Characteristics Prokaryotes Eukaryotes Mitochondria No More than one
No. of DNA molecules One More than one Endoplasmic reticulum No Yes
DNA in organelles No Yes Golgi apparatus No Yes
DNA observed as chromosomes No Yes Photosynthetic apparatus Single protein Complex structure,
Nuclear membrane No Yes Simple structure with microtubulus
Mitotic/meiotic division of nucleus No Yes Ribosome Smaller, 70s Larger, 80s
Formation of partial diploid Yes No
Spore Endospores - High Endo/Exospores
heat resistance low heat resistance
8

9. Archaebacteria Phylogenetic Tree of Life
A third ancestral type of cells
Absence of peptidoglycan from the cell
wall
Possess unusual lipids
Methanogens, extreme halophiles,
thermoacidophiles
Viruses Bacteriophages
Not cellular Possible contaminants of bacterial
Informational parasites fermentation
“Alive” when inside the host
Vaccine production from animal viruses
Characteristics of living things
Smallest microbes Recombinant DNA techniques
< 0.2 micron in size
Cloning vehicle in genetic engineering
Genetic materials: DNA or RNA
9

10. Protozoa Algae
Small, single- Photosynthetic
celled animal
Some are Prokaryotes and some are
Most live in water
Eukaryotes
(oceans, lakes,
rivers, ponds)
Eat bacteria
Some are
parasites – e.g.,
Malaria
Metabolism Growth Requirement for Microbe
Catabolism Nutrients - Carbon, nitrogen, and energy
• Glycolysis sources; P, S, minerals, vitamins, etc.
• Aerobic (Respiration) Temperature - mesophilic or thermophilic
• TCA (Krebs) cycle pH - neutral, acidic, or basic
• Electron transport chain
• Anaerobic (Fermentation) Water activity - halophilic
Anabolism (Biosynthesis) Oxygen - aerobic or anaerobic
10