595-761

Advanced Pharmaceutical Biotechnology I

Microbial Culture

Teerapat Nualnoi, PhD

Dept of Pharmaceutical Technology
[email protected]
Introduction
• Cellular organisms
Prokaryotes Eukaryotes
3 domains: Bacteria Archaea Eukaryotes

5 kingdoms: Monera Protista Fungi Animals Plants

• Viruses
• Prion (transmissible spongiform encephalopathy: TSE)

SAR-COV-2 (NIH) Escherichia coli (NIH) Saccharomyces cerevisiae (wiki)

Phylogeny
the study of relationships among different groups of organisms
and their evolutionary development.

Molecular Phylogeny
the branch of phylogeny that analyzes genetic, hereditary
molecular differences to gain information on an organism's
evolutionary relationships.
16S rRNA gene is by far one of the most common genes for
studying bacterial phylogeny.
Phylogenetic Tree
a visual representation of the relationship between different
organisms, showing the path through evolutionary time from a
common ancestor to different descendants.
Prokaryotes Eukaryotes

Three domains of life

Prokaryotes vs Eukaryotes
Eubacteria and
Archaebacterial

Archaea?
Bacteria, Archaea, Eukarya
Structure features Bacteria Archaea Eukarya
Chromosome number 1 1 >1
Nuclear membrane present present absent
Membrane lipid present present absent
Peptidoglycan present absent absent
Lipid membrane
Glycerol esters

lipid bilayer membrane

Diether lipid

lipid “monolayer” membrane Tetraether lipid

Bacterial peptidoglycan

Peptidoglycan is not
present in archaea
Bacteria, Archaea, Eukarya
Gene structure Bacteria Archaea Eukarya
Intron in genes Rare Rare Common
Transcription coupled
Yes Maybe No
with translation
Polygenic mRNA Yes Yes No
Poly A tail absent present present
Ribosome 30S, 50S 30S, 50S 40S, 60S
Initiator amino acid f-Met methionine methionine
Bacteria, Archaea, Eukarya
Metabolic process Bacteria Archaea Eukarya
Oxidative Membrane Membrane In
phosphorylation dependent dependent Mitochondria

Membrane Membrane In
Photosynthesis
dependent dependent chloroplast
Nitrogen fixation Yes Yes No

Modes of metabolism Pr Eu
Heterotrophs Autotrophs Chemoautotrophs ✓ -
Chemoheterotrophs ✓ ✓
Chemotrophs Phototrophs
Photoautotrophs ✓ ✓
Lithotrophs Photoheterotrophs ✓ -
Microorganisms Acidophiles
Some bacteria and archaea
Virus
(and fungi) are extremophiles
Bacteria
Protein adaptations
Archaea
Sources of enzymes/proteins that
Fungi function in hostile environments Yellowstone NP, WY
Live without O2 and produce
Methanogens methane

Halophiles Thermophiles Barophiles

Great Salt Lake, UT Yellowstone NP, WY Deep sea

Bacteria
Shapes Spirochaete
Vibrios
Bacillus (rod) (curved rod)
Spirillum

Cocci

Pair of
cocci
Chain of
Chain of Group of bacilus
cocci cocci
Examples
Escherichia coli
• a Gram-negative, facultative
anaerobic, rod-shaped, non-spore
forming, coliform bacterium
• Normal microbiota (harmless)
strains benefit host by producing
vitamin K
• Pathogenic strains: food poisoning
• Chemoheterotrophs: culture medium must include a
carbon source
• Most widely studied prokaryotic organism
• Host organism for producing recombinant proteins
Examples
Staphylococcus aureus
• a Gram-positive, facultative
anaerobic, round-shaped
bacterium
• Normal microbiota: upper
respiratory tract and skin
• May become an opportunistic pathogen causing skin
infection (abscesses), sinusitis, etc.
Examples
Pseudomonas aeroginosa
• a Gram-negative, rod-shaped,
encapsulated bacterium
• Commonly found in the
environment such as soil and
water
• Opportunistic pathogen that causes severe acute and
chronic infections at different sites within the body such as
urinary tract, skin (burn or surgical wounds), and the
respiratory tract

Capsule is located immediately exterior to peptidoglycan layer

of gram-positive bacteria and the outer membrane
(Lipopolysaccharide layer) of gram-negative bacteria
Examples
Capsule is located immediately exterior to peptidoglycan layer
of gram-positive bacteria and the outer membrane
(Lipopolysaccharide layer) of gram-negative bacteria

• It is considered a virulent factor

• Escherichia coli (some strains)
Neisseria meningitides
Cell capsule Pseudomonas aeruginosa
Salmonella
• Streptococcus pyogenes
Streptococcus pneumonia
Bacillus anthracis

CPS: capsular polysaccharide

LPS: lipopolysaccharide (endotoxin)
Examples
Bacillus subtilis
• A spore forming, motile, rod-
shaped, Gram-positive,
facultative aerobe
• Production of enzymes and
others
Endospore
• Produced by Bacillus spp., Clostridium spp.
• Resistance to heat, disinfectants, desiccation and
radiation than vegetative cells
• Single cell differentiates to single spore
• Germinate in suitable condition
• Enable the cell to survive periods of hardship
Terminology
Prokaryotes Chemotrophs
Eukaryotes Lithotrophs
Archaea Phototrophs
Archaebacteria
Extremophiles
Eubacteria Halophiles
Phylogeny Thermophiles
Molecular phylogeny Acidophiles
Phylogenetic tree Thermoaciophiles
16S rRNA Barophiles
Psychrophiles
Heterotrophs
Autotrophs
Terminology
Cocci Microbiota
Bacilli Bacterial capsule
Curved bacilli Endospore
Spirilla Vegetative cells
Spirochetes Capsular polysaccharide
Facultative Lipopolysaccharide
Anaerobes Saprophytic
Obligate Zoonotic
Aerobes
Fastidious
Other clinically significant bacteria
Rickettsiae
Obligately intracellular Gram-negative bacteria

Chlamydiae
Obligately intracellular Gram-negative bacteria
No peptidoglycan

Mycoplasma
Very small, lack of cell wall, but can grow in cell-free medium
Saprophytic and pathogenic

Actinomycetes
Gram-positive, produce branching filamentous, reproductive spores
Some Norcardia spp. are pathogenic to humans
Streptomyces spp. produce a wide range of antibiotics
Other clinically significant bacteria

https://www.youtube.com/watch?v=FZPSfMpgo0o

https://www.youtube.com/watch?v=H-dEmghGIwI
Applied Microbiology in Industry
Agricultures
Biofertilizers
Biopesticides

Environmental
Bioremediation
Biomining
Applied Microbiology in Industry
Food industry
Fermented foods: milk products, meat preparations, etc.
Fermented beverages: wine, beer, whiskey, Kombucha
Applied Microbiology in Industry
Commodity chemicals Fine chemicals
Ethanol Antibiotics
Acrylamide Vitamins
Citric acid Drugs
etc. Amino acids

Enzyme productions
Therapeutic enzymes
Other enzymes

Vaccines and other

biologics
Pharmaceutical Microbiology
Pharmaceutical Microbiology
Produce Pharmaceuticals
Production of recombinant proteins

Fermentation and fermenter (bioreactor)

Pharmaceutical Microbiology
Control of products and manufacturing environments

Sterility test
Bacterial endotoxin test
Antimicrobial effectiveness test
Microbial limit test
Mycoplasma test
Tests for adventitious agents
Microorganisms

SAR-COV-2 (NIH) Escherichia coli (NIH) Saccharomyces cerevisiae (wiki)

Influenza virus Mycoplasma Cryptococcus neoformans

Which ones can grow in culture medium?

Microbial growth curve
Microbial growth curve
Typical phase of microbial growth
1. Lag phase:
The time taken by the cells to adapt to the new environment
2. Log (Exponential) phase:
Cell number is increased exponentially
Primary metabolites are essential to grow
amino acids, vitamins, nucleotides, etc.
ethanol, acetone, butanol (by-product of metabolism)
Specific growth rate
number of division per cell per unit time
affected by growth condition
Generation time: time required for the population to double
Example of primary metabolites
Product Application
A-Arginine Alleviation of hyperammonemia and liver
disorders by stimulating arginase
L-Citrulline Similar to arginine
L-Histidine B6-deficiency and pregnancy
L-Homoserine Deficiency diseases
L-Glutamine As histidine and treatment of gastric ulcers
L-Isoleucine Deficiency diseases
L-Leucine Deficiency diseases
L-Ornithine Similar to arginines
L-Phenylalanine Deficiency diseases
L-Proline Deficiency diseases
L-Threonine Dietary uses
L-Valine Deficiency disease
Citric acid Blood coagulant
Calcium gluconate Calcium deficiency
Vitamin B12 Pernicious anemia, dietary additive
Microbial growth curve
Typical phase of microbial growth
3. Stationary phase:
Equilibrium between number of cells dividing and dying
Often due to a presence of growth-limiting factor
depletion of one or more essential nutrients
accumulation of inhibitory or toxic by-products e.g. acids
Products synthesized in this phase do not have any role in
cell metabolism – production of secondary metabolites

4. Death (decline) phase:

Cell death rate is higher than cell division rate
Lack of nutrients and increase of waste products
Examples of secondary metabolites

Secondary Metabolite Pharmaceutical Relevance

Penicillin Antibiotic
Cephalosporin Antibiotic
Tetracycline Antibiotic
Streptomycin Antibiotic
Griseofulvin Antibiotic (antifungal)
Actinomycin Antitumor
Pepstatin Treatment of ulcers
Cyclosporin A Immunosuppressant
Krestin Cancer treatment
Bestatin Cancer treatment
Fermentation
Use to produce:
• Microbial cells or biomass
• Microbial metabolites
• Microbial enzymes
• Recombinant proteins
• Biotransformations i.e. modification of compounds
Factors influencing fermentation process
• Temperature
• pH
• Medium composition Modes of operation
• Dissolved oxygen
• Precursors
Media for Industrial Fermentation
Nutritional requirements of microorganisms are varied not only
at the broader sense i.e. fungi, bacteria, yeast, etc, but at
species and strain levels.

Besides, the culture media must be formulated to suit the

stages of the fermentation process i.e. inoculum propagation,
main production (production of primary metabolites, production
of secondary metabolites)
Media for Industrial Fermentation
TWO TYPES OF CULTURE MEDIA
1. Chemically defined or synthetic medium
• pure compound in precisely defined concentration are
used
• Preferred for research purposes to establish specific
nutrition requirement for growth or product formation
• Reproducible
• Low tendency to foam
• Easy for product recovery and purification
• Unsuitable for industrial fermentation because they are
expensive
Media for Industrial Fermentation
TWO TYPES OF CULTURE MEDIA
2. Natural or complex media
• Corn steep liquor, Molasses, Yeast Extract, etc.
• Not completely chemically defined

Example:
• Corn steep liquor (CSL) 50 g/L
• Glucose/Sucrose 30 g/L
• (NH4)2SO4 10 g/L
• KH2PO4 2 g/L
• CaCl2.2H2O 0.06 g/L
Media for Industrial Fermentation
Example of chemically defined medium:
• Sucrose 20 g/L
• Lactose 10 g/L
• Peptone 5 g/L
• (NH4)2SO4 13 g/L
• KH2PO4 3 g/L
• Na2SO4 0.5 g/L
• EDTA 0.25 g/L
• MgSO4.7H2O 0.05 g/L
• CaCl2.2H2O 0.25 g/L
• FeSO4.7H2O 0.02 g/L
• MnSO4.4H2O 0.02 g/L
• ZnSO4.7H2O 0.01 g/L
• Na2MoO4.2H2O 0.01 g/L
• CuSO4.5H2O 0.005 g/L
Media for Industrial Fermentation
Nutrition requirement and medium compositions
• Carbon
• Nitrogen
• Inorganic salts (sulfate, phosphate)
• Trace elements (Zn, Mg, Fe, K, Ca, Cu, Mn, etc.)
• Vitamins

• pH, O2
• Others (precursor, inducer, inhibitor)
• Antifoam
Media for Industrial Fermentation
CARBON
• One of the vital nutrients
• Biomass is typically 50% carbon on dry weight basis
• Building blocks for major biomolecules e.g. proteins,
carbohydrates, lipids and nucleic acids
• Metabolic energy source (ATP production)
• A source of biosynthesis precursors e.g. antibiotics
• Carbon sources:
• Simple sugars e.g. glucose, fructose
• Disaccharides e.g. maltose, lactose, sucrose
• Sugar polymers e.g. starch, dextrin, cellulose
• Fat and oils e.g. soybean oil
• Polyols e.g. glycerol, mannitol
• Alcohol e.g. methanol (utilized by methylotrophic yeast)
Media for Industrial Fermentation
CARBON
• Supplied as “pure” or in “feed-stock”
• Cheese whey (by-product of dairy industry)
• an excellent source of lactose
• Malt extract (source of monosaccharide and disaccharide)
• Molasses (result of sugar refining, derived from sugarcane
or sugar beets
• an excellent source of Sucrose
• Most widely used carbon sources in fermentation industry
Media for Industrial Fermentation
Component of beet molasses
• Water 19.2 %
• Sucrose 48.9 %
• Glucose + fructose 0.5 %
• Raffinose 1.3 %
• Organic nonsugars 18.0 %
• Ash:
(K2O 6.4%, CaO 0.21%, MgO 0.12%, P2O5 0.03%, Na2O 1.6%,
Fe2O3 0.03%, S 0.74%, Cl 0.8%)
• Vitamins (mg/100 mL)
• Thiamine (B1) 0.01
• Riboflavin (B2) 1.1
• Nicotinic acid (B3) 8.0
• Ca pantothenate (B5) 0.7
• Folic acid (B9) 0.025
Media for Industrial Fermentation
NITROGEN
• Required for the synthesis of N-containing compounds in
the cells e.g. amino acids, proteins or nucleic acids
• May be supplied in inorganic or organic nitrogen sources
• Inorganic N sources
• e.g. ammonium salts, nitrates or nitrites
• Can be used by algae and fungi
• Bacteria and yeasts find difficulty utilizing N in this form
• Organic N sources
• Amino acids or urea
• Complex sources e.g. Corn steep liquor (CSL), Yeast extract
soybean meal, Peptone
Media for Industrial Fermentation
Corn Steep Liquor (CSL)
• By-product during starch extraction (wet corn milling) from
maize
• One of the most preferable N sources in industry
fermentation
• It contains approximately 4% w/v of N (various amino acids)
• Other compounds present in CSL include vitamins, minerals
and lactose
• The composition may vary depending on the quality of maize
and processing condition.
Media for Industrial Fermentation
Yeast Extract
• An autolysate of Baker’s yeast, Saccharomyces cerevisiae
• Typical composition of yeast extract
Total N content 8 – 12 % (50 – 75 % protein)
Amino acid N content 3 – 5.2 %
Total carbohydrate content 4 – 13%
Minerals and vitamins are also present

Peptone
• Prepared by acid or enzyme hydrolysis of high protein
materials such as meat, casein, gelatin, soybean, etc.
Media for Industrial Fermentation
Soybean meal
• Finely ground defatted soybean seeds
• By-product of soybean oil extraction
• Consist of approximately 8% w/w nitrogen
• It has been used for the commercial production of
streptomycin
Media for Industrial Fermentation
INORGANIC SALTS AND TRACE ELEMENTS
Component Range (%)
KH2PO4* 1.0 – 4.0
MgSO4.7H2O 0.25 – 3.0
KCl 0.5 – 12.0
CaCO3 5.0 – 17.0
FeSO4.4H2O 0.01 – 0.1
ZnSO4.8H2O 0.1 – 1.0
MnSO4. H2O 0.01 – 0.1
CuSO4.5 H2O 0.003 – 0.01
Na2MoO4.2H2O 0.01 – 0.1

* also acts as a buffering agent

Media for Industrial Fermentation
PRECURSORS
substances added prior to or simultaneously with the
fermentation which are incorporated without any major change
into the molecule of the fermentation product and which
generally serve to increase the yield or improve the quality of
the product
Media for Industrial Fermentation
Phenylacetic acid as a precursor for penicillin production

Phenylacetic acid
Penicillin G
(precursor)
Media for Industrial Fermentation
D-threonine
A precursor for L-isoleucine biosynthesis by the bacterium Serratia
macescens

Anthranilic acid
A precursor for tryptophan biosynthesis by the yeast Pichia (Hansenula)
anomala

tryptophan
biosynthesis pathway
Media for Industrial Fermentation
INDUCERS
substrates or substrate analogs incorporated into culture
medium or added at a specific point during fermentation to
trigger or induce production of products (e.g. enzymes,
secondary metabolites, recombinant proteins)

Starch or dextrin
Induce the production of amylase

Pectin
Induce the production of pectinase
Media for Industrial Fermentation
IPTG inducer

lactose

IPTG

Isopropyl β-D-1-
thiogalactopyranoside
(lactose analog)
Media for Industrial Fermentation
IPTG inducer

Gene of Interest

IPTG
Media for Industrial Fermentation
INHIBITORS
They are used to redirect the metabolism toward target product
Inhibitor
Pathway I Pathway I
Product B Product B

Substrate Substrate
A A

Product C Product C
Pathway II Pathway II

Sodium bisulfite
Shifting the metabolic pathway toward glycerol production in
Saccharomyces cerevisiae
Media for Industrial Fermentation
ANTIFOAM
• Foam formation during fermentation may lead to blocking of
filters or outlets, causing contamination and over-pressure
• Complex media tend to produce more foam as compared to
chemically defined media
• Chemical antifoaming agents:
• Silicone oil, alkylated glycol, polyalcohol
• Natural antifoaming agents:
• Soybean oil, sunflower oil, canola oil
• Mechanical foam breaker or ultrasound
Modes of Fermentation
• BATCH FERMENTATION

• CONTINUOUS FERMENTATION

• FED-BATCH FERMENTATION
Modes of Fermentation
BATCH FERMENTATION

Working
seed Shake Scale-up (50 L) Production
flask 2 – 10 % of production vol. bioreactor
(1000 L)
Seed formation trail

BIOREACTOR
Clean and prepare for the Harvest
next batch
Downstream
processing Batch Time
Modes of Fermentation
BATCH FERMENTATION
• The culture is maintained under optimal fermentation condition for
a defined duration (batch time) for the maximum product formation
• No media adding during the process
• Product is harvested only at the defined batch time
• There is a downtime between two batches
Modes of Fermentation
CONTINUOUS FERMENTATION

Fresh medium in
Pump
Air in Air out

Sterile air filter

Overflow weir
Nutrient
reservoir

Medium outflow
Fermenter

Harvest
reservoir
Modes of Fermentation
CONTINUOUS FERMENTATION
• Medium in the bioreactor is replaced by fresh nutrient medium
while the fermentation process in ongoing i.e. in exponential
growth phase
• Steady-state is achieved and the fermentation continues non-stop
• Steady-state formation of new biomass in the bioreactor is
equivalent to the loss of cells from the bioreactor
• Specific growth rate at this steady-state is a function of dilution
rate, which is controllable
• Thus, the primary objective of continuous culture is to control cell
growth at an optimum level of productivity
Modes of Fermentation
CONTINUOUS FERMENTATION
CHEMOSTAT MODE
• Steady cell growth is maintained by a continuous inflow of fresh
medium nutrients (phosphorous, nitrogen, glucose) at a
concentration that the growth is controlled
• Increase or decrease concentration of growth limiting factor in the
adding medium is corresponding to Increase or decrease of the
growth rate of cells
Modes of Fermentation
CONTINUOUS FERMENTATION
TURBIDOSTAT MODE
• Turbidity  Cell density
• The specific growth rate is controlled by controlling the turbidity
inside the bioreactor
• If the turbidity tends to increase, the feed rate is increased to
dilute turbidity back to its set point
• When the turbidity tends to fall, the feed rate is lowered so that
growth can restore the turbidity to its set point
Modes of Fermentation
FED-BATCH FERMENTATION
• Intermediate between batch and continuous fermentation
• Culture medium is either periodically or continuously added to the
inoculated fermentation batch
• Penicillin production (two-stage fermentation) achieved by fed-
batch fermentation
• Initial growth phases
• Production phase: the cells in maintained at relatively low growth
rate by feeding glucose at a low dilution rate
phenylacetic acid precursor which is toxic to Penicillium
chrysogenum above threshold concentration is also added slowly
along with the medium
Modes of Fermentation
FED-BATCH FERMENTATION
FIXED-VOLUME FED-BATCH
• The volume in bioreactor is fixed so that the culture is not diluted
by the feed solution added
• A very concentrated feed solution or a powder form of nutrient is
supplied to the bioreactor
• Cyclic fed-batch culture (an extended version)
• A portion of culture is harvested periodically and replaced by
sterile water or medium containing feed substrate
• As a result of dilution, the biomass concentration is decreased
and results in an increase in specific growth rate
Modes of Fermentation
FED-BATCH FERMENTATION
VARIABLE-VOLUME FED-BATCH
• The volume changes with the fermentation time due to substrate
feed
• The medium added to the bioreactor can be one of the following
I. Same medium as used in batch mode
II. Solution of the limiting substrate of the same concentration
as in the initial medium
III. Concentrated solution of limiting substrate (adding at the rate
slower than I and II)
Modes of Fermentation
FED-BATCH FERMENTATION
VARIABLE-VOLUME FED-BATCH
• Single fed-batch (SFB)
• No culture is removed until the end of the batch
• The batch time is limited by the fermenter volume
• Not full fermenter volume is utilized until the end of the batch
• Repeated fed-batch (RFB)
• When the fermentation reach a certain stage (no longer
effective for the production), a quantity of culture is removed
and refilled by fresh medium
Components of Bioreactors

• Lab scale: 2 to 200 L

• Industry scale: 100,000 – 250,000 L
Components of Bioreactors
• Vessel
• Port
• Inlet, outlet
• valve
• Probe,
sensor
• Impeller
• Air sparger
Components of Bioreactors
VESSEL
• Glass or steel (lab scale)
• Steel (industrial scale)

BAFFLES
• Metal strip that are attached radially on the wall of the vessel
• Prevent vortex formation during agitation, promote mixing
• May be designed to oscillate so that the microbial growth on the
baffles and wall are minimized
• Cooling coil may be attached to baffles

COOLING JACKET
• Microbial activity and agitation generates heat during fermentation
• To control the temperature of the bioreactor
Components of Bioreactors
IMPELLER (AGITATOR)
• To drive agitation
• Require power
• Agitation is required to achieve a
number of mixing objective
including:
• Suspension of solid particles
• Bulk fluid mixing
• Air dispersion and oxygen
transfer
• Heat transfer
Components of Bioreactors
Components of Bioreactors
SPARGER
• A device for introducing air into a bioreactor (aeration)
• Provide sufficient oxygen to microorganisms in the fermenter
• Generally, fine bubble aerators are used as they facilitate the
oxygen transfer to a greater extend

Porous sparger: made of sintered glass, ceramics or metal. It is

used only in lab scale-non agitated vessel. the holes tend to be
blocked by growth
Components of Bioreactors
SPARGER
Orifice sparger: used in small stirred fermenter. It is a perforated
pipe kept below the impeller.

Nozzle sparger: Mostly used in large scale. It is single open or

partially close pipe. When air is passed through this pipe there is
lower pressure loss and does not get blocked.
Combined sparger agitator: This is air supply via hallow agitator
shaft. The air is emitted through holes in the disc or blades of agitator
Components of Bioreactors
PROBES OR SENSORS
• To monitor conditions inside the bioreactors
• pH probes
• Thermometer
• Dissolve oxygen probe
• Pressure gauge
• Level probe
• Foam probe
• etc.
Components of Bioreactors
PORTS OR INLET/OUTLET
• Feed port – connect to the medium or nutrition reservoir
• Air stream inlet and outlet
• Inoculation port
• Sampling Port
• Anti-foam port
• Acid and alkali port – to control pH
• Water inlet and outlet

• Filter is installed to maintain sterility

• Various types of valve are used to regulate flows
Types of Bioreactors
SUBMERGED FERMENTERS (SmF)
• Stirred Tank Fermenter (STF)
• Airlift Fermenter (ALF)
• Packed Bed Fermenter
• Fluidized Bed Fermenter

SOLID SUBSTRATE FERMENTERS (SSF)

• Group I: Tray fermenter
• Group II: Packed bed fermenter
• Group III: Rotary drum fermenter
• Group IV: Gas-solid fluidized bed fermenter
Types of Bioreactors

Stirred Tank Airlift or pneumatic fermenter

Fermenter (STF) (A) External loop, (B) Internal loop
• No propeller
• Provide low-shear environment
for animal cell cultures
Types of Bioreactors
• Cells are immobilized on the
support materials
• Nutrient broth is sprayed on
the top of the support
material
• Airflow is countercurrent the
liquid flow
• Preferred for
biotransformation

Packed bed
fermenter
Types of Bioreactors
Solid Substrate Fermentation

• Carried out on a solid support

(substrate) with little or no free water
• May be carried out under non-sterile
condition
• Required less energy as compared to
submerged fermentation
• The water is generally present as thin
film and the film is discontinuous

Tray fermenter
Product manufactured by SSF
Product Substrate Microorganism
Citric acid Cassava bagasse Aspergillus niger

Lactic acid Sugarcane bagasse Rhizopus oryzae

Amylase Cassava bagasse Rhizopus arrhizus

Mycophenolic acid Wheat bran Penicillium brevicompactum

Cepthalosporin Sugarcane bagasse Acremonium chrysogenum

Upstream and Downstream process
UPSTREAM DOWNSTREAM
Seed Media Harvesting
preparation formulation • the separation of cells, cell debris or other
particulate matter from fermentation broth
Stock Media • filtration, centrifugation, precipitation, etc
culture preparation
• grinding, homogenization, or leaching may
be required to recover the products
Active Mixing and
culture filtration Product Isolation
• Solvent extraction, adsorption, precipitation
Seed Media ultrafiltration
Propagation Sterilization
Product Purification
• affinity, size exclusion, reversed phase,
ion-exchange chromatography

Formulation, Packaging, QC
FERMENTATION