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

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.

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

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

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)

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)

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

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

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

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

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

An anaerobic digester

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.

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

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.

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

Products of industrial microbiology

interdisciplinary (복합학문적) nature microbiology biochemistry chemical engineering

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.

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

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.

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

How to increase the production yield of a specific metabolite?

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

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.

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

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

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

*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

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

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

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

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

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

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

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

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)

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

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

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 3.7 % casein 2.7 % lactose 4.9 % whey protein 0.7 % whey ash 0.7 % water 87.2 %

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

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

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

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

Aerobic respiration

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

Continuous culture

Semi-continuous culture

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

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

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

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

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

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

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 10,000-500,000 liters In all stages, O2 dynamics are carefully measured at each step to determine how volume increases affect O2 demand in the fermentation

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