PENGANTAR BIOPROSES

(STK-1210) / Introduction to Bioprocess

MEDAN, 15th March 2022

6th Course

Bode Haryanto, S.T., M.T., Ph.D

Rivaldi Sidabutar, S.T., M.T.
Nisaul Fadilah Dalimunthe, S.T., M.Eng
Capaian Pembelajaran MK
CP-1 : Mahasiswa mampu menjelaskan hubungan protista dengan proses fermentasi dan
produk fermentasi

CP-2 : Mahasiswa mampu memahami dan menggambar kurva pertumbuhan

mikroorganisme
 - Memahami bagian-bagian susunan kimia, struktur, dan fungsi sel bakteri
- Memahami arti pertumbuhan dan perbanyakan mikroba, serta kurva
pertumbuhan bakteri
- Memahami jenis dan fungsi nutrisi bagi pertumbuhan mikroba serta
mekanisme metabolisme mikroba
- Memahami faktor-faktor lingkungan yang mempengaruhi kehidupan mikroba

CP-3 : Mahasiswa mampu menentukan jumlah generasi dan waktu penggandaan

Rujukan
1. Ketchum, P., Microbiology: Concepts and Application, John Wiley & Sons, New York,
1988.
2. D. Dwijosaputro. Dasar-dasar Mikrobiologi. Penerbit Djambatan 1990.
3. Krueger, et al., Introduction to Microbiology, Mc Millan, 1979.
4. Frosbisher, et al., Fundamental of Microbiology, Saunders (Toppan), 1974
5. Bailey dan Ollis. Biochemical Engineering Fundamentals, Mc Graw Hill, 1987
6. Aiba, et al., Biochemical Engineering, Tokyo University Press, 1973
7. Cooney, Fermentation and Enzyme Technology, John Wiley, 1973
8. Wang, D.C., Cooney, C.L., Dunnill, P., Humphrey, A.E., & Lilly. M.D. Fermentation and
Enzyme Technology. John Wiley & Sons, Singapore, 1979.
Jadwal dan Penilaian
Jadwal Perkuliahan

Kelas A : Senin, 10.00 – 11.40 WIB

Kelas B : Selasa, 15.10 – 16.50 WIB
Kelas C : Selasa, 13.30 – 15.10 WIB

Jika ada hal yang perlu dibicarakan dengan para pengajar diluar waktu kuliah, mahasiswa
dapat bertemu setelah kuliah berakhir

Pertemuan pada waktu-waktu yang lain dapat diadakan bila diperlukan atau dengan
menghubungi komting

Penilaian

Penilaian dilakukan terhadap masing-masing capaian pembelajaran dengan rincian

- Tugas (orang/kelompok)
- Ujian Pengukuran CP
 Bioprocess study aspects

Utilization by technology and

industry

1 3

Biological agents (microbes, Produced products and

enzymes) services
The scope of BIOPROCESS

Laboratory scale: microbial selection steps or

enzyme performance description: 1-5 liter
fermenter

Pilot-plan scale: optimization of bioprocess

conditions/variables: fermenter 5 – 500 Liter

Industrial scale: considering the economic

calculation: fermenter 500 – 5000 liter
 BIOPROCESS PERFORMANCE

1st Identification of products,

substrates and intermediates 3st Bioprocess rate kinetics

2st Stoichiometry of process 4st Reactor design/modelling

 Bioprocess needs research assistance

01 02 03 04

Microbiology: Biochemistry: Genetics: the

Physiology: cell
understanding of chemical reactions genetic material
processes and
microbes (types, and processes in in cells
activities of an
structures and organism cells
components of cells)
 Microbes in bioprocess

The size is small, so the simple genetic material

ratio of area to cell
volume is high

relatively does not

produce toxic waste (safe
for the environment)
Can grow on a variety of
media

Reproduction is rapid
What is a BIOPROCESS
ENGINEER??
 Technically trained to understand,
design, and efficiently handle
bioreactors.
 Ensures that a favorable sustainable
state or predictable outcome of a
bioprocess is achieved
 Differentiating from other engineers,
training in biological sciences,
especially quantitative and analytical
biological sciences, and green
chemistry
Bioprocess applications and products

01 Medical field:

antibiotics, vaccines, vitamins, steroids, hormones,

antibodies, interferons etc.

Agriculture:
02
biopesticides, animal feed, xylase enzymes, compost and
fertilizers, Nitrogen fixing bacteria etc..

03 Chemical Industry

Ethanol, acetone, butanol, organic acids, biopolymers,

surfactants, perfumes etc.

04 Agroindustry :

Alcoholic beverages, fermented dairy products, PST,

organic acids, enzymes, antioxidants, sweetening agents,
dyes, aromas, etc.
General Bioprocess

Feedstock Bioprocessing Product

GAS Cell culture

Biocatalyst Bioreactor Recovery PRODUCT

LIQUID product
LINES
SOLID Enzymatic

Feedstock Bioprocessing Product

 Gas  Immobilized Enzymes  Separation  Pharmaceuticals
− Syn. Gas −Ambient to Extreme − In situ  Fine chemicals
− CO2  Fermentation − Secondary  Specialty Chemicals
− Organic vapor − Immobilized  Media  Feedstock
 Liquid − Free cell − Gaseous
 Bulk chemicals
− Organic −Ambient to Extreme −Aqueous
− Sugar solution  Bioreactors − Organic
 Solid − Continuous Systems
− Biomass − Membrane
− Consumer Waste − Batch or Fed-batch
Microbial
Growth
Objectives
• Define microbial growth
• Distinguish between binary fission and budding
• Explain growth cycle of Population
• Classify microbes into five groups on the basis of preferred temperature range
• Explain the importance of osmotic pressure to microbial growth
• Explain how microbes are classified on the basis of O2 needs
• Identify ways in which aerobes avoid damage by toxic forms of O2
• Explain microbial growth at low and high pH
• Provide a use for each of the four elements (C, N, S, P) needed in large amounts for
microbial growth
• Distinguish between chemically defined and complex media
• Justify the use of each of the following: anaerobic techniques, living host cells,
candle jars, selective, differential, and enrichment media
• Review some direct and indirect methods of measuring bacterial cell growth
Definition

Microbial Growth Defined:

1. Mother or parent cell doubles in size

2. Divides into two daughter cells

• Microbial growth is defined as the increase in the number of

cells, which occurs by cell division
• Binary fission (equal cell division): A cell duplicates
its components and divides into two cells

• Budding (unequal cell division): A small, new cell

develops from surface of exisiting cell and subsequently
separates from parent cell

• Septum: A partition that grows between two

daughter cells and they separate at this location
Binary Fission
Thin section of the bacterium Staphylococcus,
undergoing binary fission
Budding
 Phases of Growth
• Consider a population of organisms introduced into a
fresh, nutrient medium
• Such organisms display four major phases of growth

1. The lag phase

2. The logarithmic phase
3. The stationary phase
4. The death phase
 Bacterial Growth Curve
Foundation
Illustrates the dynamics of growth Fig 6.15
Phases of growth
• Lag phase
• Exponential or
logarithmic (log) phase
• Stationary phase
• Death phase (decline
phase)
Compare growth in liquid
Lag Phase
• Organisms do not increase significantly in number
• They are metabolically active
• Grow in size, synthesize enzymes, and incorporate
molecules from medium
• Produce large quantities of energy in the form of
ATP
Log Phase
• Organisms have adapted to a growth medium
• Growth occurs at an exponential (log) rate
• The organisms divide at their most rapid rat
• a regular, genetically determined interval (generation
time)
• The longest and most productive phase
• Stationary Phase:
1. Cell division decreases to a point that new cells are
produced at same rate as old cell die.
2. The number of live cells stays constant.
• Decline (Death) Phase:
1. Condition in the medium become less and less
supportive of cell division
2. Cell lose their ability to divide and thus die
3. Number of live cells decreases at a logarithmic
rate
Microbial Growth
Microbial growth: Increase in cell number, not cell size!
Physical Requirements for Growth: Temperature (cardinal
temperature)
• Minimum growth temperature
• Optimum growth temperature
• Maximum growth temperature
Five groups based on optimum growth temperature
1. Psychrophiles
2. Psychrotrophs
3. Mesophiles
4. Thermophiles
5. Hyperthermophiles

Fig. 6 .3
Temperature
 Physical Requirements for Growth:
pH and Osmotic Pressure
Most bacteria grow best between pH 6.5 and 7.5: Neutrophils
Some bacteria are very tolerant of acidity or thrive in it: Acidophiles
(preferred pH range 1 to 5)
Alkaliphiles grow between pH range 9 to 11
Molds and yeasts grow best between pH 5 and 6
Hypertonic environments (increased salt or
sugar) cause plasmolysis
Obligate halophiles vs.
facultative halophiles

Fig 6.4
pH
Chemical Requirements for Growth: Oxygen

O2 requirements vary greatly

Table 6.1: The Effects of Oxygen on the Growth of Various Types of Bacteria
Chemical Requirements for Growth: Carbon, N,
S, P, etc.
• Carbon
•  Half of dry weight
• Chemoheterotrophs use organic carbon sources

• Nitrogen, Sulfur, Phosphorus

• Needed for ?
• Found in amino acids and proteins (most bacteria decompose proteins)
• S in thiamine and biotin
• Phosphate ions (PO43–) Vit B1

• Also needed K, Mg, Ca, trace elements (as cofactors), and organic growth
factors
Vit B7
Culture Media
• Culture medium: Nutrients prepared for microbial growth
• Have to be sterile (not contain living microbes)
• Inoculum: Microbes introduced into medium
• Culture: Microbes growing in/on culture medium
• Chemically defined media: Exact chemical compo-
sition is known (for research purposes only)
• Complex media: Extracts and digests of yeasts,
meat, or plants, e.g.:
• Nutrient broth
• Nutrient agar
• Blood agar
Agar
• Complex polysaccharide
• Used as solidifying agent for
culture media in Petri plates,
slants, and deeps
• Generally not metabo-
lized by microbes
• Liquefies at 100°C
• Solidifies ~40°C
Anaerobic Culture Methods
• Use reducing media, containing chemicals (e.g.:
thioglycollate) that combine with O2

• Are heated shortly before use to drive off O2

• Use anaerobic jar

• Novel method in clinical labs:

Add oxyrase to growth media
 OxyPlate (no need for
anaerobic jar)

Fig 6.5 22
Capnophiles: Aerobic Bacteria Requiring High
CO2
• Low oxygen, high CO2 conditions
resemble those found in Candle jar
• intestinal tract Fig 6.7
• respiratory tract and
• other body tissues where
pathogens grow
• E.g: Campylobacter jejuni

• Use candle jar, CO2-generator packets,

or CO2 incubators
Selective Media and Differential Media
Selective medium: Additives
suppress unwanted and
encourage desired microbes –
e.g. EMB, mannitol salt agar
etc.

Differential medium: changed

in recognizable manner by
some bacteria  Make it easy
to distinguish colonies of
different microbes – e.g.  and
 hemolysis on blood agar;
MacConkey agar, EMB, mannitol
salt agar etc.
Enrichment Media/Culture
• Encourages growth of desired microbe
• Example: Assume soil sample contains a few phenol-
degrading bacteria and thousands of other bacteria
• Inoculate phenol-containing culture medium with the
soil and incubate
• Transfer 1 ml to another flask of the phenol medium
and incubate
• Transfer 1 ml to another flask of the phenol medium
and incubate
• Only phenol-metabolizing bacteria will be growing
Pure Cultures
Contain only one species or strain.
Most patient specimens and
environmental samples contain
several different kinds of bacteria
Streak-plate method is commonly
used
Colony formation: A population of cells arising from a
single cell or spore or from a group of attached cells
(also referred to as CFU).
Only ~1% of all bacteria can be successfully cultured
 Streak Plate Method

3 or 4
quadrant
method
 Preserving Bacterial Cultures
• Deep-freezing: Rapid cooling of pure culture in suspension liquid to –50°to –
95°C. Good for several years.
• Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a
vacuum. Good for many years.
The Growth of Bacterial Cultures
Binary fission – exponential growth

Budding

Generation time – time required for cell to divide (also

known as doubling time)
Ranges from 20 min (E. coli) to > 24h (M. tuberculosis)

Consider reproductive potential of E. coli

Fig 6.13

Figure 6.1
g=t/n
Serial Dilution and Standard Plate Counts

• Standard plate count: One method of measuring

bacterial growth

• Agar plate: A petri dish containing a nutrient

medium solidified with agar

• Serial dilutions are used to dilute the original

bacterial culture before you transfer known volume
of culture onto agar plate
Serial Dilution
 Calculation of the number of bacteria per
milliliter of culture using serial dilution
• Pour plate: made by
first adding 1.0ml of
diluted culture to 9ml
of molten agar
• Spread plate: made
by adding 0.1ml of
diluted culture to
surface of solid
medium
Direct Measurements of Microbial Growth

Viable cell counts: Plate counts: Serial dilutions

put on plates CFUs form colonies

Fig 6.16
Fig 6.17

Figure 6.15, step 1

Additional Direct Measurements
1. Filtration method of choice for low counts
2. Direct microscopic count: Counting chambers
(slides) for microscope

Fig 6.20
 Direct Microscopic Counts
• Another way to measure bacterial growth

• Petroff-Hausser counting chamber

• Bacterial suspension is introduced onto chamber with a calibrated pipette

• Microorganisms are counted in specific calibrated areas

• Number per unit volume is calculated using an appropriate formula

The Petroff-Hausser Counting Chamber
Most Probable Number (MPN)
• Method to estimate number of cells

• Used when samples contain too few organisms to give reliable

measures of population size by standard plate count

• Series of progressively greater dilutions

• Typical MPN test consists of five tubes of each of three

volumes (e.g.
10, 1, and 0.1ml)
A MPN test: those tubes in which gas bubbles are
visible (labeled +) contain organisms
Positive carbohydrate fermentation test

+ Gas/+Acid + Acid - No Acid or Gas

Turbidity, or a cloudy appearance, is an indicator of
bacterial growth in urine in the tube on the left
Estimating Bacterial Numbers by Indirect
Methods

Spectrophotometry to measure turbidity

OD is function of cell number
Bacterial colonies viewed through the magnifying glass
against a colony-counting grid
A Spectrophotometer: This instrument can be used to
measure bacterial growth by determining the degree of light
transmission through the culture
Measuring Microbial Growth - Overview

Direct Methods Indirect Methods

• Plate counts • Turbidity
• Filtration • Metabolic activity
• MPN • Dry weight
• Direct microscopic count
Culturing Bacteria
• Culturing of bacteria in the laboratory presents two problems:

1. A pure culture of a single species is needed to study an organism’s

characteristics
2. A medium must be found that will support growth of the desired
organism

• Pure culture: a culture that contains only a single species of

organism
•Chemostat
Continuous culture, the
experimenter can control growth
rate and population density
independently of each other
• Batch culture
Closed system culture, fixed
volume
The Streak Plate Method uses agar plates to
prepare pure cultures
A Streak Plate of Serratia marcescens. Note the greatly
reduced numbers of growth /colonies in each successive region
Types of Culture Media
• Natural Media: In nature, many species of microorganisms grow together in
oceans, lakes, and soil and on living or dead organic matter

• Synthetic medium: A medium prepared in the laboratory from material of

precise or reasonably well-defined composition

• Complex medium: contains reasonably familiar material but varies slightly in

chemical composition from batch to batch (e.g. peptone, a product of enzyme
digestion of proteins)
Commonly Used Media
• Yeast Extract

• Casein Hydrolysate

• Serum

• Blood agar

• Chocolate agar
Selective, Differential, and Enrichment
Media
• Selective medium: encourages growth of
some organisms but suppresses growth of
others
(e.g. antibiotics)

• Differential medium: contains a constituent that

causes an observable change (e.g. MacConkey agar)

• Enrichment medium: contains special nutrients that

allow growth of a particular organism that might not
otherwise be present in sufficient numbers to allow it
to be isolated and identified
Preserved Cultures
• To avoid risk of contamination and to reduce mutation rate, stock culture
organisms should be kept in a preserved culture, a culture in which organisms
are maintained in a dormant state
1. Lyophilization
2. Frozen at -70oC
3. Refrigeration

• Reference culture (type culture): a preserved culture that maintains the

organisms with characteristics as originally defined