1. Industrial Microbiology-contents
Microbial Technology-Introduction
Microbial Metabolism and Its Control
Process Development
Traditional Microbial Products
Antibiotics
Vitamins
Amino acids
Enzymes
Alcohol beverages
5. Biotecnology
Insulin
Vaccines
Genetic engineering in agriculture
Genetic engineering in animal

2. 병원미생물학 Microbiota A collection or community of microbes. Microbiome
The full collection of genes of all the microbes in a community. The human microbiome (all of our microbes’ genes) can be considered a counterpart to the human genome (all of our genes). The genes in our microbiome outnumber the genes in our genome by about 100 to 1.
Some use “microbiome” to mean all the microbes in a community.

3. Human-microbe interaction
Microbes
Human
Beneficial interaction
Harmful interaction
virulence resistance (defense)
normal microflora
pathogen
1013
1014

4. Industrial microbiology
application of microbiological systems to the manufacturing and service industries
use microbial cells or microbial products (eg. enzymes)
microbial technology: major part of biotechnology

5. What Microbial Technology Can Provide For Commercial products
foods (mushrooms, fermented foods, SCP,...)
enzymes (protease, amylase,....)
microbial metabolites
- amino acids, vitamins
- ethanol, acetic acid, citric acid
- other industrial chemicals (butanol, isopropanol,
acetone, polysaccharides, lipids…)
- therapeutic agents (antibiotics, alkaloids, steroids, vaccines,….)
- fuels (methan, ethonol, hydrogen, ethane, propane)

6. fermented foods
Worldwide: alcohol, wine, vinegar, olives, yogurt, bread, cheese
Asia
East and Southeast Asia: amazake, belacan, burong mangga, com ruou, , doenjang, douchi, , lambanog, kimchi, kombucha, , narezushi, , miso, nata de coco, natto, prahok, ruou nep, sake, soy sauce, stinky tofu, tape, tempeh, zha cai,….
Central Asia: kumis (mare milk), kefir, (camel milk)
India: achar, appam, dosa, dhokla, dahi (yogurt), gundruk, idli, mixed pickle
Africa: fermented millet porridge, garri, hibiscus seed, hot pepper sauce, injera, , laxoox, oilseed, ogi, ogiri
Americas: chicha, elderberry wine, kombucha, pickling (pickled vegetables), sauerkraut, lupin seed, oilseed, chocolate, vanilla, tabasco, tibicos
Middle East: torshi, boza
Europe: rakfisk, sauerkraut, ogórek kiszony, surströmming, mead, elderberry wine, salami, prosciutto, cultured milk products such as quark, kefir, filmjölk, crème fraîche, smetana, skyr.
Oceania: poi, (rotten corn)

8. What Microbial Technology Can Provide For
Environment: Cleanup of environmental pollutants by organisms (bioremediation)
microbial-based waste water treatment system
conversion of domestic waste into fuels (methane, methanol, ethanol)
substitution of toxic chemicals: microbial pesticides (농약)
cleanup of oil spills

9. Primary treatment removes insoluble particulate matter from sewage by settling, screening, coagulation, or other procedures.
A primary sedimentation tank at a rural treatment plant

10. Secondary treatment oxidizes dissolved organic material
In secondary treatment, bacteria use the dissolved organic compounds as source of carbon and energy for growth.
Most of the organic material is converted to either CO2 or to microbial cells.
There are three main systems of secondary treatment.
trickling filters (살수여상법)
activated sludge (활성 오니) process
Anaerobic digestion: organics - CO2 and CH4

12. Aeration tank of
an activated sludge installation
Wastewater flow through an
activated sludge installation

14. Anaerobic digestion is a series of biological processes in which microorganisms break down biodegradable material in the absence of oxygen.
One of the end products is biogas, which is combusted to generate electricity and heat, or can be processed into renewable natural gas and transportation fuels.
A range of anaerobic digestion technologies are converting livestock manure, municipal wastewater solids, food waste, high strength industrial wastewater and residuals, fats, oils and grease (FOG), and various other organic waste streams into biogas.
An aerobic digester

16. An anaerobic digester

18. Tertiary treatment
The advanced treatment process, following secondary treatment of waste water, that produces high quality water.
Tertiary treatment includes removal of nutrients such as phosphorus and nitrogen and practically all suspended and organic matter from waste water.

19. What Microbial Technology Can Provide For
Soil improvement
microbial fertilizer (biofertilizer)
a substance which contains living microorganisms which, when applied to seeds, plant surfaces, or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant
Etc
Leaching(제련) of valuable minerals from mining waters (광산수) and low grade ores (광석): 제련(광석의 금속 함량을 높임)의 한 방법

20. Bioleaching is the extraction of metals from their ores through the use of living organisms. Several methods are used to recover copper, zinc, lead, arsenic, antimony, nickel, molybdenum, gold, silver, and cobalt.

21. Arrangement of a leaching pile and reactions involved
in the microbial leaching of
copper sulfide minerals
to yield Cu0 (copper metal)
By bacteria
By bacteria and chemical process
By chemical process
Reaction #3 Is the most important in most mining operations
Leptospirillum ferrooxidans
Acidithiobacillus ferrooxidans
Oxidation pond

22. Products of industrial microbiology

23. interdisciplinary (복합학문적) nature
microbiology
biochemistry
chemical engineering

24. Metabolism and metabolite
Metabolism of a cell
all the reactions in the metabolism are catalyzed by enzymes (<--- genes in DNA)
physicochemical environments
affect metabolism via various metabolic regulatory mechanisms
--->
cells do not generally
oversynthesize a specific metabolite.

25. and Secondary metabolite
Primary metabolite
and Secondary metabolite
Alcohol fermentation in yeast Penicillin production
by Penicillium chrysogenum

26. Primary metabolites:
formed during the primary growth phase
produced by organism as part of it’s normal growth
required for cells to survive and replicate
e.g. amino acids, proteins, nucleic acids, lipids,…
production of primary metabolites matches the growth in population
Secondary metabolites:
formed near the end of the primary growth phase, frequently at or near the stationary phase
no direct involvement in fundamental metabolite processes.
primary metabolism is generally similar in all cells.
secondary metabolism shows distinct differences from one organism to another.

28. Characteristics of Secondary Metabolites (SM)
1) each SM is only formed by a relatively few organisms.
2) SMs are seemingly not essential for
growth and reproduction.
3) the formation of SMs is extremely
dependent on growth conditions.
4) SMs are often produced as a group of closely related structures.
(a strain of Streptomyces produce
32 related anthracycline antibiotics)
5) it is often possible to get
dramatic overproduction of SMs.
Various secondary metabolite antibiotics containing aromatic rings

29. How to increase the production yield of
a specific metabolite?

30. Steps where eucaryotic gene expression can be controlled
the main control site
for most genes
Steps where eucaryotic gene expression
can be controlled

31. Switching genes on and off with a repressor protein
Trp repressor
always present in the cell (constitutive gene expression)
an allosteric protein: (repressor + trp) complex can bind to DNA.

32. Feedback inhibition
a form of metabolic control in which the end product of a metabolic pathway reduces the activity of an enzyme early in the pathway

34. How to increase the production yield of a specific metabolite?
to obtain transcriptional control-insensitive mutant
mutations in operator regions or a regulatory gene (no repressor or deficient repressor) could greatly enhance transcription and thus the production of a metabolite (amino acid, vitamin,...)
a slight alteration of a promotor sequence could increase the rate of transcription and thus the production of an enzyme

35. 2. to obtain a feedback-resistant mutant
3. to induce excretion of the amino acid into the culture medium generally, cells do not excret essential metabolites such as amino acids

36. *Corynebacterium glutamicum
biotin (Vitamin B7, cofactor in fatty acid biosynthesis) deficiency leads to cell membrane damage (as a result of poor phospholipid production), and under these conditions, intracellular glutamic acid is excreted into media.
culture in biotin-rich condition ---> good growth
culture in biotin-deficient condition ---> excretion of glutamic acid

37. Process development strategies
Aims of process development: maximizing three factors
the yield of product per gram of substrate
the concentration of the product
the rate of product formation
Obtaining microorganisms
Determination of culture conditions
3. Modification of the genetic structure
4. Cell cultivation system

38. Obtaining microorganisms
How to get a suitable strain for producing the desired product in high yield?
isolation from nature
purchase from a number of culture collections:
ATCC, KCTC, …
improvement of existing strain

39. Microbial Collections
Agricultural Research Service USDA
ALGAE a world catalogue of algal collections
ATCC American Type Culture Collection
BCCM Belgian Co-ordinated Collections of Microorganisms
BGSC Bacillus Genetic Stock Center
BCC BIOTEC Culture Collection, Thailand
CABRI CABRI (Common Access to Biological Resources and Information) by EC
CBS Centraalbureau voor Schimmelcultures
CCAP The Culture Collection of Algae and Protozoa
CCUG University of Goteborg, Sweden (>38000 strains)
CGSC E.coli Genetic Stock Center
Developmental, Cell and Molecular Biology Group Duke University
DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
Fungal Genetics at U. Texas Houston Medical School
MA (Real Jardin Botanico, Madrid) cryptogamic collections
HAMBI University of Helsinki, Finland
IEGM Institute of Ecology and Genetics of Microorganisms
IMI CABI Bioscience Genetic Resource Collection
INVAM The International Culture Collection of Arbuscular and VA Mycorrhizal Fungi
JCM Japan Collection of Microorganisms
JFCC a catalogue of strains preserved in Japan Federation for Culture Collection
MGD Microbial Germplasm Database
Matsushima Mycological Memoirs No.8
Microbial Information Network of China
MICH University of Michigan Fungus Collection
MSDN Microbial Strain Data Network mirrored in Japan
NCCB
NCIMB
Quinone Database
RDPII (Ribosomal Database Project II)
The Chlamydomonas Genetics Center
TISTR
UAMH
UKNCC
UNSW University of New South Wales
VKM All Russian Collection of Microorganisms
World Phytophthora Collection, University of California Riverside (US)
HBMMD HARBOR BRANCH MARINE MICROBE DATABASE

40. Desirable properties of a microbial culture
a) be a pure culture and be free of phages
b) be genetically stable
c) grow vigorously and rapidly
d) produce the required product within a short period of time
e) can protect itself against contamination
f) produce the product in a form readily seperable from all others
g) be amenable to change by certain mutagenic agents

41. 2. Determination of culture conditions: optimum values of nutritional requirements, temperature, pH and oxygen supply
Nutritional Requirements
energy source
carbon source: ~50% of biomass;
the largest ingredient in culture medium
other essential nutrients
the absence of inhibitors

42. microbes are amazingly flexible in their ability to alter their metabolism and composition in response to environmental change.
understanding of the relationship between chemical and physical environment and the regulation of metabolism is required for optimizing the culture conditions

43. Criteria for raw material selection
1. cost
2. availability
3. uniformity
4. solubility
5. rheology (유동성)
6. foaming property
7. material handling (smell, gas,...)
8. storage
9. government regulation

44. Organic materials C source for biomass and energy
phototrophs (Chlorella, Spirulina,...): CO2
heterotrophs: organic materials: sugars, starch, alcohols, organic acids, proteins, peptides, amino acids, ...
Organic materials
1. crude form
rice, barley, soy bean, sweet potato, corn,...
2. by-products
1) molasses (당밀)
2) corn steep liquor (옥수수 침지액)
3) sulfite waste liquor (아황산 펄프 폐액)
4) whey (유장)

45. 1) molasses (당밀)
by-product of sugar industry
sugar beet or sugar cane (사탕수수)
--> sugar + molasses (~50 % sugar)

46. 2) corn steep liquor
corn ----> starch + corn steep liquor
lactic acid, amino acids, glucose, vitamins, salts

47. 3) sulfite waste liquor
wood + calcium bisulfite -- heat, pressure --> cellulose pulp + SWL(2 % sugar)
4) whey (유장)
a by-product of the manufacture of cheese
(proteins and fat from milk )
milk fat %
casein %
lactose %
whey protein % whey
ash %
water %

48. 3. modification of the genetic structure to increase product formation
mutation:
any heritable change in the base pair sequence of an organism's DNA
traditional mutagenesis by several chemical or physical agents
site-directed mutagenesis

49. Induced mutations can be caused by
Chemicals
Nitrosoguanidine (NTG)
Hydroxylamine NH2OH
Base analogs (e.g. BrdU)
Simple chemicals (e.g. acids)
Alkylating agents (e.g. N-ethyl-N-nitrosourea (ENU))
Methylating agents (e.g. ethyl methanesulfonate (EMS))
Polycyclic hydrocarbons
DNA intercalating agents (e.g. ethidium bromide)
DNA crosslinkers (e.g. platinum)
Oxidative damage caused by oxygen(O) radicals
Radiation
Ultraviolet radiation (nonionizing radiation) excites electrons to a higher energy level. DNA molecules are good absorbers of ultraviolet light, especially that with wavelengths in the 260 to 280 nm range
UV light can induce adjacent thymine bases in a DNA strand to pair with each other, as a bulky dimer.
Ionizing radiation

50. 2) recombination
brings together genetic informations from two or more organisms in an individual organism
a) sexual hybridization of eucaryotes
b) bacterial recombination: transformation, conjugation, transduction
c) genetic engineering: restriction endonuclease, ligase, vector

51. 4. Cell cultivation system Fermentation:
Growth of cells within a fermenter or bioreactor (under aerobic and or anaerobic conditions) (in industrial microbiology)

52. Aerobic respiration

53. batch culture
continuous culture
flow rate is set at a particular value
and the microbial growth adjust to this flow rate
fed-batch culture
sequential addition of medium to an initial batch
without any withdrawal of reaction mixture
*dialysis culture

54. Continuous culture

55. Semi-continuous culture

56. Medium in reservoir
limiting nutrient
Flow rate (F), 50 ml/hr
Culture volume (V), 500 ml
Dilution rate (D=F/V), 0.1/hr
Culture vessel
Overflow

57. population density and growth rate can be controlled
Concentration of limiting nutrient
-----> cell density (growth yield)
Dilution rate (Flow rate) > growth rate

59. Dialysis culture inoculum dialysis tubing medium
extended exponential phase
concentrated biomass
easy recovery of macromolecules

60. Fermentor: laboratory scale: 1-10 liter pilot scale
plant (industrial) scale

61. Aerobic fermentor: Anaerobic fermentor: need cooling, mixing, aeration
Cooling jacket:
for medium sterilization and heat removal
steam or cooling water
external or internal
Aeration system
a critical part of a fermentor
tremendous O2 demand by a high-density
microbial population
sparger + impeller

62. Fermentation control and monitoring
to measure and control temperature, O2 concentration, pH,
cell mass, product concentration
use computers to process these data on-line and respond

63. Scale-up process 1. laboatory flask:
to derermine whether a process of commercial interest is possible
2. laboratory fermentor:
1-10 liters
to test variations in medium, temperature, pH, …
3. pilot plant stage
300-3,000 liters
4. commercial fermentor
10, ,000 liters
In all stages, O2 dynamics are carefully measured at each step to determine how volume increases affect O2 demand in the fermentation

64. Antibiotics Antimicrobial agent
chemical substances that are produced by microorganisms which kill or inhibit the growth of other microorganisms
(cf. an antibiotic is a chemotherapeutic agent with activity against microorganisms)
Penicillin의 놀라운 능력에 감명을 받아 다른 미생물의 성장을 억제하는 유용한 미생물을 흙으로부터 찾기 위해 일생을 바친 Selman A. Waksman과 그의 연구원들은 10,000번 이상의 흙을 배양하는 실
험을 수없이 반복하던 중, 1943년 병든 닭의 목에서 배양된 특이한 미생물이 결핵균의 성장을 억제하는 것을 관찰할 수 있었고, 이 물질을 streptomycin 이라고 명명하였다. Waksman은 흙 속의 곰팡이나 미생물로부터 생성되어 다른 미생물의 성장을 억제하거나 파괴시키는 화학물질을 총칭하여 “antibiotics”라는 용어를 처음으로 사용하였다
Antimicrobial agent
Chemical that kills or inhibits the growth of microorganisms