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Research in Lab

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Title: Research in Lab

1
? ? ? ? ? ? ? ? ? Antibiotics Fermentation
Production

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?????
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???????
?????

Introduction
Research in Lab
Strain improvement
Production processing
Fermentation control
Isolation purification

2
1. ? ? Introduction

Antibiotics
Any organic substances produced by various
organisms that have the power of arresting the
growth of other microorganisms or of destroying
them.

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???????????????,?????????????????????????????????

3
1. ? ? Introduction

History
Penicillin was discovered by Fleming in 1929.
World War II, fermented in large-scale
The first major antibiotic to be commercialized

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1929?,??????????
????,???????
?????????????

4
1. ? ? Introduction
? ? Classification

???????????? (Modes of Action of Antibiotics)
??????? (Cell Wall Synthesis) ???
??????? (Cell Membrane Damage) ??????
??????? (Protein Synthesis) ???
?????? (Nucleic Acid Synthesis) ????

5
??
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6
1. ? ? Introduction
? ? Classification

????????????
ß-???? (ß-lactam) ???? (penicillins)
????? (cephalosporins)
????? (aminoglycosides) ????????
????? (macrolides) ?????????
? ? ? (tetracyclines) ???????
? ? ? (polymyxin) ????

7

natural penicillin R
CH2-CO-
8
ß-??? ?
9
????
Antibiotics are so complex they can only be
synthesized in a living system
10
2.?????? Research in Lab

??????
?????
????????
????
???????

Screening of a strain
Antimicrobial trial
Isolation, purification and identification
Toxicological trial
Medicinal clinical trial

11
agar plate ?????
Agar diffusion assay
12
? ? ? ? Analytical instruments

??????
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?????
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??????
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Modern analytical instruments
Nuclear magnetic resonance (NMR)
Capillary electrophoresis (CE)
Gas chromatography (GC)
High performance liquid chromatography (HPLC)
Mass spectroscopy (MS)
Automation Sensitivity Accuracy
? ? ? ? ? ?

13
3. ???????? Strain Improvement

Conventional strain selection
Physical mutagen
eg. Ultraviolet Radiation
Chemical mutagen
eg. Nitrosoguanidine (NTG)
Improved strain can be selected empirically

?????
????X-??r-???
??????????????????????

14
3. ???????? Strain Improvement
Recombinant DNA Technology (DNA????)
???? Genetic Engineering
??

15
Strain Improvement Scale-up
Master cell bank
Working cell bank
Secondary shake flask evaluation different
media ?????? ?????
Lab fermentor evaluation Production Condition ??
??? ??????
Primary shake flask Evaluation ????
Agar plate ????
Strain ????
6000
Possible recycle
300
30
3
1
Inoculum Development ??????
Pilot plant Evaluation ????
Production Evaluation ??
16
(No Transcript)
17
Scale-up(????)
? ?
18
? ? ? ? Shake-flask fermentation

10-50 ml in 125-500 ml flask
Batch only
Limited control temperature
Slow metabolizing carbohydrate
Ambient pressure
Buffers needed to control pH
In-process sampling difficult
Volume decrease by evaporation
Solid growth on side walls
Antifoam not needed
Agitation limited shaker speed
No control of dissolved oxygen

???10 (250ml)
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?????pH
?????
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?????
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?????
???????

19
? ? ? ? ? Stirred tank fermentation

10-100,000???
???????
????pH,??
???????
2 ???
?????
?????
??,????
??????
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???????,??

10-100,000 liter vessels
Batch and feed possible
Continuous control pH, temperature
Ammonium salts or ammonia needed
Pressure at two atmospheres possible
No buffers necessary
In-process sampling easy
Volume increase by sugar feed
Very uniform growth
Antifoam often required
Wide variety of impellers and baffles
Dissolved oxygen controlled by
aeration, pressure

20
? ? ? ? Shake-flask
? ? ? ? ? Stirred tank

???10 (250ml)
????
?????
????????
??
?????pH
?????
????
?????
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?????
???????

10-100,000???
???????
????pH,??
???????
2 ???
?????
?????
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??????
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???????,??

21
4. ????????? Antibiotics Production Processing

?? ? ????
? ??
? ??????
? ?????
? ????

Strain ? Preparation of seed
? Fermentation
? Treatment of broth
? Isolation purification
? Package of product

22
A scheme of the material flow in an antibiotic
production
23
???
??
???
???
A scheme of the material flow in an antibiotic
production
24
?????? Materials for Fermentation

Carbon source
Beet cane molasses
Glucose, lactose, starch
Ethanol, methanol
Glycerol
Maltose syrup, corn syrup
Cottonseed oil, peanut oil, soy oil
Lard oil
Methyl oleate
Tallow

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25
?????? Materials for Fermentation

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?????

Nitrogen source
Cotton seed meal
Corn meal steep liquor
Soybean meal flour
Peanut meal
Rapeseed meal
Beef blood, Casein hydrolysate
Whey
Fish meal
Whole yeast
Yeast extract
Meat bone meal

26
? ? ? Media for Cell Growth Final production
Fermentation ?????
Tank seed ?????
Components Range ? ? Glucose/
sucrose 3.0-5.0 ???/ ?? Starch ??
3.0-5.0 Corn steep liquor
3.0-5.0 Calcium carbonate 0.5-1.0
??? Phosphate ??? 0.1-0.5 Ammonium
sulphate 0.1-1.0 ??? Urea ??
0.1-0.5 Oil ?
0.1-0.5
Components Range ?
? Glucose
0.5-1.0 Starch
0.5-5.0 Corn steep liquor 5.0-8.0
????? Soy flour
1.0-5.0 Phosphate
0.1-1.0 Ammonium sulphate 0.5-1.0
and fed periodically Oil
1.0
and fed continuously Corn syrup fed
continuously ???? ????
27
Stirred tank Fermentation ????
28
5. ? ? ? ? Fermentation Control
Sugar/Oil feed

Sample Analysis
pH
DO
Sugar
Ammonia
Phosphate
Sulphate
Products
Precursors
Contamination

Pressure probe
Level probe
Antifoam
pH probe
Acid/Base
Temp. probe
Cooling
DO probe
Air/agitation
29
Flow Rate Control ????
Supervisory Computer
Weight Signal
Pump Speed
Amplifier
Scale
Controller
30
? ? ? ? Fermentation control
31
? ? ? ? ? (1) Penicillin Fermentation