7/27/2019 TekFer1(2013)

1/64

7/27/2019 TekFer1(2013)

2/64

7/27/2019 TekFer1(2013)

3/64

What is BioProcess Technology?

Technology for improving the efficiency ofbioprocess by focusing on microbialperformance.

Knowledge of the metabolism of microbial cells isimportant for :

Optimization of bioreactor operation

Selection of the best microorganism Improvement of the existing m.o

Design (metabolic engineering) of m.o.

7/27/2019 TekFer1(2013)

4/64

Upstream (bioprocess)

first step in which molecules are grown inbioreactors (batch or fed-batch) to reachthe desired density.

Downstream (bioprocess)

process of the cell mass from the

upstream to meet purity and qualityrequirements.

7/27/2019 TekFer1(2013)

5/64

7/27/2019 TekFer1(2013)

6/64

Stage of BioProcess

Stock culture

Shake flask

Seed fermenter

Raw materials

Medium formulation

Sterilization

Computer controlProduction fermenter

Air

Recovery

Purification Products

Effluent treatment

Microorganism

cell preparation

Mediumpreparation

7/27/2019 TekFer1(2013)

7/64

Fermentation/Bioprocess

Technology In the context of industrial microbiology the

term fermentation refers to the growth of

large quantities of cells underanaerobic

or aerobic conditions within a vessel

called a fermenter or bioreactor

7/27/2019 TekFer1(2013)

8/64

Scope of Industrial

Biotechnology First generation processes Fermentation based processes using native organisms

Wine- and beer-making

Baking Second generation processes

Use of native or engineered enzymes/genes to enhance first

generation processes

Textile industry

Starch industry

Third generation processes

Direct use of recombinant and engineered systems

Plant, insect and animal cell culture

7/27/2019 TekFer1(2013)

9/64

Diversity of metabolic/fermentation pathway

among microorganism

Glucose

Pyruvate Butanol,Acetone,

CO2

Acetic acid,

Ethyl alcohol,

CO2

Propionic acid,

Acetic acid,

CO2Ethanol,

CO2

Butanoediol,

CO2

Lactic acid,

CO2

1

5 4

3

27

6

1. Glycolisis

2. Butyric fermentation

3. Butandiol fermentation

4. Alcohol fermentation

5. Homolactic acid ferm.

6. Propionic acid ferm.

7. Mixed-acid ferm.

7/27/2019 TekFer1(2013)

10/64

7/27/2019 TekFer1(2013)

11/64

Fermentation products

Food Industry

~ Beer

~ Bread

~ Cheese

~ Wine

~ Yogurt

Pharmaceutical Industry

~ Insulin

~ Vaccine Adjuvants

Energy

~ Fuel Ethanol

7/27/2019 TekFer1(2013)

12/64

7/27/2019 TekFer1(2013)

13/64

7/27/2019 TekFer1(2013)

14/64

5 major groups of important fermentations:

Microbial cells or biomassMicrobial enzymes

Microbial metabolites

Recombinant products

Transformation Process

7/27/2019 TekFer1(2013)

15/64

Microbial biomass :Yeast production (baking industry)Microbial cells production (SCP as human

or animal food)

Microbial enzymes: amylase (fungi) baking industrypectinase (fungi) coffee industryprotease (bacteria) dairy, meat industrylipase (bacteria)detergents industry

Microbial metabolites: ethanol alcoholic beverages, fuelglutamic acid flavor enhanceralanine sweetener

Recombinant products: genetically modified organism insulin, vitamin

Transformation processes: vinegar production (transformation

from ethanol to acetic acid)

7/27/2019 TekFer1(2013)

16/64

7/27/2019 TekFer1(2013)

17/64

7/27/2019 TekFer1(2013)

18/64

2 major challenges fermentations:

The conditions of reaction must berigidly maintained

The reaction result in the formation of

unwanted by-products

7/27/2019 TekFer1(2013)

19/64

Fermentation Systems Most fermentations use liquid media

Some are non stirred, non aerated and non

aseptically operated (beer, wine) while others

are stirred, aerated and aseptic

7/27/2019 TekFer1(2013)

20/64

Operating Systems

Fermentations in liquid media can be carried out underbatch, fed-batch or continuous culture conditions

Stirred batch fermentor: a closed system where all

nutrients are present at start of fermentation within a

fixed volume

Fed-batch fermentor: fresh medium is fed in

throughout the fermentation and volume of batch

increases with time

Continuous culture: fresh medium is fed into the vessel

and spent medium and cells are removed (fixed volume)

In all systems, pH, temperature, aeration etc is

monitored and adjusted

7/27/2019 TekFer1(2013)

21/64

Batch Fermentor

1. Medium added

2. Fermentor sterilised

3. Inoculum added

4. Fermentation followed to completion

5. Culture harvested

Glucose

Cell biomass

Time

Product

7/27/2019 TekFer1(2013)

22/64

Characteristics of a Batch Fermentation System

Simplest fermentor operation

Sterilisation can be performed in the reactor

All nutrients are added before inoculation

Maximum levels of C and N are limited by inhibition ofcell growth

Biomass production limited by C/N load and productionof toxic waste products

Cells are harvested when biomass levels or product

levels start to decline

7/27/2019 TekFer1(2013)

23/64

7/27/2019 TekFer1(2013)

24/64

Characteristics of Fed Batch Fermentors

Initial medium concentration is relatively low (no inhibition of

culture growth) Medium constituents (concentrated C and/or N feeds) are added

continuously or in increments

Controlled feed results in higher biomass and product yields

Fermentation is still limited by accumulation of toxic end

products

Glucose

Biomass

Product

Biomass

Product

Glucose

BATCH FED-BATCH

7/27/2019 TekFer1(2013)

25/64

Continuous Fermentor

Feedstock

vessel

(sterile)

Pump 1

Collection vessel

Pump 2

Flow rate1 = Flow rate2

7/27/2019 TekFer1(2013)

26/64

Characteristics of Continuous (Chemostat) Fermentors

Input rate = output rate (volume = const.)

Flow rate is selected to give steady state growth (growthrate = dilution rate) Dilution rate > Growth rate culture washes out

Dilution rate < Growth rate culture overgrows Dilution rate = Growth rate steady state culture

Product is harvested from the outflow stream

Stable chemostat cultures can operate continuously forweeks or months.

7/27/2019 TekFer1(2013)

27/64

Component Parts of Fermentation Process

Formulation of media

Sterilization of medium, fermenter, equipmentProduction of active, pure culture

Optimization of cell growth for product formation

Extraction of products and purification

7/27/2019 TekFer1(2013)

28/64

7/27/2019 TekFer1(2013)

29/64

7/27/2019 TekFer1(2013)

30/64

7/27/2019 TekFer1(2013)

31/64

Fermentation Media Media must satisfy all nutritional requirements of the organism andfulfil the objectives of the process

Generally must provide

a carbon source (for energy and C units for biosynthesis)

Sources of nitrogen, phosphorous ans sulfur

Minor and trace elements Some require added vitamins e.g. biotinand riboflavin

Media generally contain buffers or pH controlled by adding acids /alkalis

Potential problems

Compounds that are rapidly metabolized may repress productformation

Certain compounds affect morphology

7/27/2019 TekFer1(2013)

32/64

C b

7/27/2019 TekFer1(2013)

33/64

Carbon sources

Molasses

Byproduct of cane sugar production

a dark viscous syrup containing 50% CHO

(sucrose) with 2% nitrogen, vitamins and

minerals Malt extract

Use aqueous extracts of malted barleyto

produce C sources for cultivation of fungi andyeasts

Contain 90% CHO, 5% nitrogen and proteins,

peptides and amino acids

7/27/2019 TekFer1(2013)

34/64

Carbon sources (2)

Whey

Aqueous byproduct of dairy industry

Contains lactose and milk proteins

Difficult to store (refrigerate) so freeze dried

Many MOs wont metabolize lactose but whey is used in

production of penecilluin, ethanol, SCP, xanthan gum etc

Alkanes and alcohols

C10 C20 alkanes, metane and methanol used for vinegar and

biomass production

Use is dependent on prevaling petroleum price

7/27/2019 TekFer1(2013)

35/64

Nitrogen Sources

Mos generally can use inorganic ororganic N

Inorganic sources: ammonia, ammonium salts

Organic sources: amino acid, proteins and

urea

Corn steep liquor

Yeast extract

Peptones Soya bean meal

7/27/2019 TekFer1(2013)

36/64

Fermentor design and construction

Laboratory fermentations: shake flasks

Industrial fermentors are custom designed

Design, quality, mode of operation depends on:

Production organism

Optimal operating conditions for product formation

Product value

Scale of production

Reliability Economics: must minimise running costs and capital

investment

7/27/2019 TekFer1(2013)

37/64

Lab Scale Fermentor

7/27/2019 TekFer1(2013)

38/64

Industrial Scale Fermentor

7/27/2019 TekFer1(2013)

39/64

Industrial Scale Fermentor

7/27/2019 TekFer1(2013)

40/64

7/27/2019 TekFer1(2013)

41/64

Fermentor Controls

Temperature control Critical for optimum growth

Aeration

Related to flow rate and stirrer speed

Dependent on temperature Mixing

Dependent on fermentor dimensions, paddle designand flanging

Controls aeration rate

May cause cell damage (shear) pH control

Important for culture stability/ survival

Foaming control

7/27/2019 TekFer1(2013)

42/64

Mixing

Efficient mixing is critical for: Homogeneous distribution of nutrients

Even temperature distribution

Rapid pH adjustment

Retention of air bubbles

Different designs of stirrer blades give different circulation

patterns

Mixing is assisted by the presence of flanges on the fermentor

walls

Stirrer tip speed dictates the degree of shear stress

Some cells types are very susceptible to shear stress

Excessive stirring can promote foaming

7/27/2019 TekFer1(2013)

43/64

pH control

Most cultures have narrow pH growth ranges The buffering in culture media is generally low

Most cultures cause the pH of the medium to rise

during fermentation

pH is controlled by using a pH probe, linked viacomputer to NaOH and HCl input pumps.

pH

Growth

2-3 pH units

pH

6.5

8.5

Uncontrolled

Controlled

7/27/2019 TekFer1(2013)

44/64

Foaming

Foaming is caused when

Microbial cultures excrete high levels of

proteins and/or emulsifiers

High aeration rates are used Excess foaming causes loss of culture volume and may

result in culture contamination

Foaming is controlled by use of a foam-breaker and/or

the addition of anti-foaming agents (silicon-basedreagents)

7/27/2019 TekFer1(2013)

45/64

7/27/2019 TekFer1(2013)

46/64

Medium Raw Materials

7/27/2019 TekFer1(2013)

47/64

Stock culture

Shake flask

Seed fermenter

Medium Sterilization

Medium Formulation

Production fermenter

Culture fluid

SeparationPurificationProduct

7/27/2019 TekFer1(2013)

48/64

7/27/2019 TekFer1(2013)

49/64

7/27/2019 TekFer1(2013)

50/64

7/27/2019 TekFer1(2013)

51/64

7/27/2019 TekFer1(2013)

52/64

7/27/2019 TekFer1(2013)

53/64

7/27/2019 TekFer1(2013)

54/64

7/27/2019 TekFer1(2013)

55/64

7/27/2019 TekFer1(2013)

56/64

7/27/2019 TekFer1(2013)

57/64

7/27/2019 TekFer1(2013)

58/64

7/27/2019 TekFer1(2013)

59/64

7/27/2019 TekFer1(2013)

60/64

7/27/2019 TekFer1(2013)

61/64

7/27/2019 TekFer1(2013)

62/64

7/27/2019 TekFer1(2013)

63/64

7/27/2019 TekFer1(2013)

64/64

Thank you