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UEMK3233 Topic 1 8

1) Production of microbial cell (biomass)

Two main applications:-

a) as a source of protein Example: SCP

b) as a commercial inoculum (starter culture)

a) SCP production refer to microbial biomass used as food and feed

additives

either the isolated cell protein or the total cell materialmay be called SCP

great nutritional value high protein, vitamin, and lipid

content and the almost complete range of all essential

amino acids

Single Cell Protein

Microbial biomass or protein extracted there from

processes in which bacteria, yeast, fungi or algae are

cultivated in large quantitiesAs human or animal protein supplement in animal feed

or in human nutrition

As protein supplement (for infant as nutritional food

protein)

UEMK3233 Topic 1 10

Sample of the single cell protein

biosolids after the drum dryerUEMK3233 Topic 1 11

Component Alkane

yeast

Methanol

bacterium

Alga Soya

meal

Milk

powde

r

Raw protein 60 80 72.6 42 34

Fat 9 9.5 7.3 4 1

Mineral salts 6 9.5 4.7 6.5 8

moisture 4.5 2.8 3.6 10 5

Table 1: Composition (%) of SCP compared with soya meal

and milk powder

Protein content in bacteria 60 to 65%

Selected fungi and yeast 33 to 45%

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UEMK3233 Topic 1 12

Some psychological barriers:

microorganism involved must be safe & acceptable;

non-pathogenic; non-toxic forming (for example,

aflatoxins are produced by some fungi); geneticallystable

Toxic or carcinogenic substances used during the

production of microbial e.g., hydrocarbon, polycyclic

aromatic compounds .

may cause indigestion and allergic reactions

UEMK3233 Topic 1 13

b) as a commercial inoculants (or known as

starter cultures)

Major applications (as food starter cultures),

such as, in baking and dairy industries

Functions of food starter cultures:-

Texture modifications

Preservation

Flavor development

Nutritional improvement

UEMK3233 Topic 1 14

Examples:-

i. Bakers yeast bread making

ii. Cheese-starter cultures Streptococcus

cremoris, Streptococcus lactis

iii. Yoghurt manufacturing Stretococcusthermophilus, Lactobacillus bulgaricus, L.

acidophilus

Topic 1Topic 1 --ProkaryotesProkaryotes 1515

1 m 2 m 5 m

(a) Spherical (cocci) (b) Rod-shaped (bacilli) (c) Spiral

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UEMK3233 Topic 1 20

Examples of commercial applications of microbial enzymes

UEMK3233 Topic 1 21

c) Production of microbial metabolites

Growth of microbial divided into few stages:-

i. Lag phase

ii. Log orexponential

phase

iii. Stationary phase

iv. Death phase

Growth curve of a bacterial culture in

batch conditions

UEMK3233 Topic 1 22

Phases Processes

Lag Growth does not occur;

adaptation period

Log/exponential

Cells grow gradually at constantrate until reach maximum

growth rate

stationary Growth ceases and growth rate

reduced

death Viable cell number declinesUEMK3233 Topic 1 23

Product formation during microbial growth:-

a) Primary metabolites

referred asprimary products of metabolism

are essential for life and reproduction of cells

produced during log phase (trophophase)

e.g., amino acids, proteins, carbohydrates,lipids.

examples (refer Table 3 and 4)

also known as ????

produced by wild type OR genetically modifiedmicroorganism ???

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UEMK3233 Topic 1 24

some may be produced in concentrations far

higher than those required by the producing

strains

Table 3: Excessive production of certain primary metabolites

UEMK3233 Topic 1 25

Primary metabolite Commercial applications

Ethanol Active ingredient in alcoholic

beverages;

Citric acid Food industry

Acetone & butanol Solvents

Glutamic acid Flavour enhancer

Vitamins Feed supplements

Polysaccharides Food industry; oil industry

Table 4: Examples of primary product from microbial

metabolism and their commercial application

UEMK3233 Topic 1 26

UEMK3233 Topic 1 27

b) Secondary metabolites secondary products of metabolism produced during

stationary phase (idiophase) (Figure A)

are seemingly not essential for growth and reproduction

produced from the intermediates and products of primarymetabolism (refer Figure B) and their formation is extremely

dependent on environmental conditions every secondary metabolite is formed by only a few

organisms

Importance of secondary metabolites in fermentation

industries

a) Antimicrobial activity

b) Growth promoters

c) Pharmacological properties

Wild-type microbial ??? Genetically modified microbial??

also known as ?????

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UEMK3233 Topic 1 28

Figure A: Production of antibiotics

(e.g.Penicillium spp. & Streptomycetes)

UEMK3233 Topic 1 29

d) Recombinant products

advent of recombinant DNA technology extend the range

of potential fermentation products

genes from higher organisms may be introduce into

microbial cells recipients are capable of synthesizingforeign proteins

hosts E. coli, S. cerevisiae and filamentous fungi

products from GMO interferon, insulin, epidermal growth

factor, chymosin etc

important factors to consider

i. secretion of the product

ii. minimization of the degradation of the product

iii. maximizing the expression of the foreign gene

UEMK3233 Topic 1 30

e) Microbial transformation / Bioconversions

to chemically modify a wide variety of organic

compound to convert a compound into a

structurally related, financially more valuable

compound

examples:

1. production of vinegar conversion of ethanol to acetic

acid

2. production of steroids, antibiotics and prostaglandins

reaction involved:- oxidation, dehydrogenation,

dehydration, condensation, amination,

isomerization,

UEMK3233 Topic 1 31

advantageous over the use of chemical

reagents:-

operate at low temperature

without requirement of any potential heavy metalpollution

disadvantages:-

require large amount of biomass

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UEMK3233 Topic 1 32

Oxidation of ethanol to acetic acid

UEMK3233 Topic 1 34

UEMK3233 Topic 1 35

Many words have been used to describe

engineering working with biotechnology:-

Bioengineering is a broad title include work

on medical and agricultural systems itspractitioners include agricultural, electrical,

mechanical, industrial, environmental

Biological engineering is similar but emphasizes

applications to plants and animals

UEMK3233 Topic 1 36

Biochemical engineering

extension of chemical engineering principles to systems using a

biological catalyst to bring about desired chemicaltransformations

often subdivided into bioreaction engineering and bioseparation

engineering

Biomedical engineering

the application of engineering techniques to the understanding ofbiological systems and to the development of therapeutictechnologies and devices.

kidney dialysis, synthetic skin, artificial joints.. etc are some

products of biomedical engineering.

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UEMK3233 Topic 1 39

Approach to research

biologists vs engineers

fundamental training distinctly different

For biologist,mathematical theories and quantitative methods play 2nd

role

very strong with respect to laboratory tools (interpretation oflaboratory data from complex systems) BUT they are often

have incomplete backgrounds in mathematics

usually better at the formation of testable hypotheses,

experimental design, and data interpretation from complex

systems results are qualitative and descriptive

UEMK3233 Topic 1 40

Engineerusually possess a very good background in the physical and

mathematical sciences

theory formulate mathematical equation (predictive model)

and then the validity of the theory is tested by comparingpredicted responses to those in experiments

typically unfamiliar with the experimental techniques andstrategies used by life sciences

UEMK3233 Topic 1 41

The skills of engineer and life sciences are

complementary integration of skills

To become a bioprocess engineer:

needs a solid understanding of biology (need to take further

courses in microbiology, biochemistry, cell biology.)

need to have improvements in experimental tools

learn more advanced work in biochemical engineering

Biochemical engineering

extension of chemical engineering principles to systems using a

biological catalyst to bring about desired chemical transformations

often subdivided into bioreaction engineering and bioseparation

engineering

UEMK3233 Topic 1 42

How biologists and engineers

work together

Figure 1: Steps in development of a complete

bioprocess for commercial manufacture of a new

recombinant-DNA-derived product

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UEMK3233 Topic 1 43

6 Plasmids

2 Animal tissue

3 Part of animalchromosome

4 Gene cut fromchromosome

5 Microoganism such

as E. coli

1 biochemicals

7 Cut plasmid

8 Recombinantplasmid

9 Insertion int omicroorganism

10 Plasmid

multiplication

and gene

expression

11 Celldivision

12 Small scale culture

13 Bench-top bioreactor

14 Pilot scale bioreactor

15 Industrial scale bioreacto r16 Product recovery

17 Packaging and marketingUEMK3233 Topic 1 44

Steps in bioprocess development

The interdisciplinary nature of bioprocessing is

evident if we look at the stages of development

required for a complete industrial process

as shown in Figure 1, several steps are required to

convert the idea of the product into commercially

reality

these stages involve different types of scientific

expertise

UEMK3233 Topic 1 45

6 Plasmids

2 Animal tissue

3 Part of animalchromosome

4 Gene cut from

chromosome

5 Microoganism suchas E. coli

1 biochemicals

7 Cut plasmid

8 Recombinantplasmid

9 Insertion int o

microorganism

10 Plasmidmultiplication

and gene

expression

11 Celldivision

12 Small scale culture

13 Bench-top bioreactor

14 Pilot scale bioreactor

15 Industrial scale bioreacto r16 Product recovery

17 Packaging and marketingUEMK3233 Topic 1 46

Step 1 to 11

concerned with genetic manipulation of host organism a

gene from animal DNA is cloned into Escherichia coli

genetic engineering

done in laboratories on a small scale by scientists trained in

molecular biology and biochemistry experiment tools: petri dishes, micropipettes,

microcentrifuges, nano- or microgram quantities of

restriction enzymes, electrophoresis gels for DNA, protein

fractionation

Parameters of major importance stability of the

constructed strains and level of expression of the desired

productbioprocess development

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UEMK3233 Topic 1 47

Step 12 Small-scale cultivation (lab-scale) is mostly carried out

using shake flasks (250-mL to 1-L) to study the growth

and production characteristics of cells

culture environment (medium composition, pH,temperature..and other environmental condition) allow

optimal growth of microorganism and productivity of

product

practical skills microbiology, fermentation

calculated parameters cell growth rate, specific

productivity and product yield used to describe

performance of the organism

UEMK3233 Topic 1 48

Step 13 to 14 (scaling up)

once the culture conditions for production are known, scale-

up of the process starts

Bench-top bioreactor (cultures can be more closely

monitored in bioreactor than in shake flasks better

control over the process)

Lab scale 1- or 2-litre

Equipped with instruments for measuring and adjusting temperature,

pH, DO, stirrer speed, and many other process variables

Information collected: oxygen requirements of the cells, shear

sensitivity, foaming characteristics .

Identify the limitation imposed by the reactor on the activity of the

organismwhether or not the reactor can provide conditions for

optimal activity of the cells is of prime concern!!

UEMK3233 Topic 1 49

Step 14 Pilot-scale bioreactor

Engineers trained in bioprocessing are normally involved in

pilot-scale operations a vessel of capacity 100 to 1000 L

is built according to specifications determined from the

bench-scale prototype

Aim of pilot-scale studies to examine the response of

cells to scale-up

Changing the size of the equipment seems relatively trivial; however, loss or

variation of performance often occurs

Even though the geometry of the reactor, method of aeration and mixing,

impeller design and other features may be similar in small and large

fermenters, the effect on activity of cells can be great!

Loss of productivity following scale-up may or may not be recovered

Economic projections often need to be re-assessed as a result of pilot-scale

findings

UEMK3233 Topic 1 50

Step 15 (Industrial-scale operation)

This part of process development is clearly in the

territory of bioprocess engineering

As well as the reactor itself, all of the auxiliary

service facilities (air supply and sterilisation

equipment; steam generator; supply lines; medium

preparation; sterilisation facilities; cooling-water

supply. etc) must be designed and tested

Need to ensure the fermentation to be carried out

aseptically

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UEMK3233 Topic 1 51

Step 16 (Product recovery or downstreamprocessing)

After the production process, raw broth is treated in a seriesof steps to obtain pure product

Product recovery is often difficult and expensive

accounts for 60% or 80 90% of the total processing cost Actual procedures used depend on the nature of the product

and the broth physical, chemical or biological methods

Techniques applied industrially for downstream processingare first developed and tested using small-scale apparatus many operations which are standard in the laboratorybecome uneconomic or impractical on an industrial scale

Scientists trained in chemistry, biochemistry, chemicalengineering and industrial chemistry play important rolesin designing product recovery and purification systems

UEMK3233 Topic 1 52

Step 17 (Packaging and marketing)

After the product has been purified in sufficient purity, it

can be packaged and marketed

New pharmaceuticals products require medical and

clinical trials to test the efficacy of the product

Bioprocess engineers with a detailed knowledge of the

production process are often involved in documenting

manufacturing procedure for submission to regulatory

authorities manufacturing standards must be met,

particularly in the case for recombinant products where a

greater number of safety and precautionary measures is

required!

UEMK3233 Topic 1 53

As shown in this example, a broad range of

disciplines is involved in bioprocessing

Scientists working in this area are constantly

confronted with biological, chemical,

physical, engineering and sometimes

medical questions!!