1. Cell growth and metabolism Yong-Cheol Park (ycpark@kookmin.ac.kr) Department of Advanced Fermentation Fusion Science & Technology

2. 2/16  Growth ( 증식, 생장, 성장 ) - Population increment of cells by using various nutrients (C,H,O,N)  Metabolism ( 대사 ) - Cellular chemical reactions for destruction or synthesis of organic compounds ‘Metabolism by Growth’ = ‘Growth for Metabolism’  Issuing point in this part (1) How to count cell number or measure cell concentration (2) Understanding of cell growth curve (3) Environmental factors related with microbial growth (4) Energy production process by carbon metabolism (5) Significant efforts for survival  ethanol and lactic acid production Overview

3. 3/16 Quantitative assay of cell population 1. Cell number density 1) Hemocytometer (Petroff-Hausser Slide) - Microscopic counting

4. 4/16 Quantitative assay of cell population 2) Plate count (colony count) - Viable cell counting (bacteria/yeast) - not for mold - colony-forming unit (CFU, number/volume)

5. 5/16 Quantitative assay of cell population 2. Cell mass concentration 1) Direct method A. Cellular dry weight (or dry cell mass) ~ The most commonly used direct method ~ In solids-free medium ~ Cell harvest by centrifugation or filtration  washing the cells  drying the washed wet cell at 80oC for 24 hr  measurement

6. 6/16 Quantitative assay of cell population B. Optical density (OD) ~ The absorption of light by suspended cells at a visible wavelength (600 ~ 700 nm) ~ Fast, inexpensive, simple 2) Indirect method - For mold fermentation - DNA, RNA, protein concentration  cell concentration - Viscosity of culture broth for extracellular polysaccharide formation (PHB, PHA...) Optical density (600nm) Dry cell mass (g/L) Y(DCM,g/L) = slope*OD slope

7. 7/16 2. Microbial growth (1) Population growth  Growth curve ( 생장곡선 ) : A mathematical graphic display showing the changes of cell number or concentration in terms of time - How to grow : Bacteria (binary fission), Yeast (budding), Fungi (hyphae growth & mitosis) - All organisms have their own specific growth curves. - Cell growth can be predicted by mathematical modeling. Time Cell mass A B C

8. 8/16 2. Microbial growth (1) Population growth  Cell growth phase (1) Lag phase (유도기) - No population increase (new formation=death) - Stage for adaption to new environment (temperature, acidity), and for biosynthesis of metabolic enzymes and cell components (2) Exponential (log) phase (지수생장기) - Stage for cell growth and division at the maximum possible rate (formation ≫ death) - A bacterial population doubles at regular intervals called the generation (or doubling) time. - The individual cells contain similar contents of intracellular components. - Because of the uniform composition, cell growth can be analyzed by mathematical modeling at the exponential phase. (3) Stationary phase (정지기) - No population increase (new formation=death) - Reason: nutrient depletion, environmental change by-product (growth inhibitor) formation - Stage for production of antibacterial agents (4) Death phase (사멸기) - Decrease in cell population (new formation≪death) - Spore formation at harsh conditions

9. 9/16 2. Microbial growth (2) Water & temperature  Importance of water and temperature - Water : Major component of cell, Most enzymes can react in the presence of water. - Temperature : Significant effects on enzyme activity * Lower temp. : slow enzyme reaction * Higher temp. : rapid reduction of reaction rate, permanent denaturation  Classification by growth temperature (1) Psychrophiles (저온성 미생물) - Well grown at 0℃ ~20℃ - polar region, deep sea - growth in refrigerator (5℃)  Streptococci (milk spoilage), Staphylococci (toxin production) (2) Mesophiles (중온성 미생물) - Well grown at 20℃ ~45℃ - Most microbes able to be cultured in laboratory - Most of human pathogenic M/O (human temp.,37℃) (3) Thermophiles (고온성 미생물) - Can grow at over 45℃, even 100℃ - Most archaea (고세균) - hot spring, volcano, etc

10. 10/16 2. Microbial growth (3) Oxygen & acidity  Importance of oxygen and acidity on microbial growth - Oxygen : be supplied or removed for microbe’s survival - Acidity (pH) : influences enzyme activity and reaction rate  Classification of M/O by oxygen requirement (1) Aerobic M/O ( 호기성 ) - Has to be supplied for metabolism - Many microbes, animals, plants (2) Anaerobic M/O ( 혐기성 ) - Growth without oxygen, death with oxygen - some lactic acid bacteria,, methanogenic bacteria (3) Facultative M/O ( 통성 혐기성 ) - Can grow irrespective of oxygen - Rapidly adapts the aerobic or anaerobic condtions - Most microbes  Classification by growth pH (1) pH6~8 is suitable for the growth of most M/O (2) pH5.0 for budding yeast (alcohol yeast) (3) Acidophilic M/O : can grow at acidic conditions - lactic acid bacteria - Helicobacter pylori : causing stomach ulcer

11. 11/16 3. Metabolism Catabolism vs. Anabolism Metabolic networks in cell  Metabolism : all chemical reactions in the cell - Basic chemical reactions for the growth of cells and maintenance of life (e.g.) nutrient consumption, transfer, decomposition waste secretion, energy production, cell component synthesis - Category : Catabolism & Anabolism

12. 12/16 3. Metabolism (1) Enzyme  Enzyme : Organic molecules that act as catalysts for biological reactions - Proteins catalyzing the reactions of cellular metabolisms - Substrate specificity : An enzyme acts on a substrate specifically. - Enzymes can lose their activities by protein denaturation.

13. 13/16 3. Metabolism (2) Energy storage  Main purpose of metabolism : maintenance of life by the endless supply of energy  Adenosine triphosphate (ATP) : main energy storage molecule - Similar to rechargeable battery - Used for various reactions: nutrient delivery, waste removal, protein synthesis - Produced mostly by respiration or photosynthesis - Composition : Adenine + Ribose + Phosphates - Energy storage by hydrolysis and linkage of phosphorus groups  Nicotinamide adenine dinucleotide (NAD) - A storing molecule of electron erupted in sugar metabolisms - Moves to electron transport system and is used for ATP synthesis.

14. 14/16 3. Metabolism (3) Glycolysis  Glycolysis ( 해당과정 ) - Metabolic pathway where glucose (C6) is disassembled into pyruvate (C3). - Glucose is a main substrate containing chemical energy. - Takes place in the cytoplasm and does not require oxygen for reaction. - Glucose is broken down by oxidation-reduction reaction. - During the glycolysis, electrons erupting from glucose are stored into nicotinamide adenine dinucleotide (NAD). - Overall reaction equation of glycolysis Glucose (C 6 H 12 O 6 ) + 2NAD + + 2ADP +2Pi  2 pyruvate (C 3 H 4 O 3 ) + 2ATP + 2NADH +2H + + 2 물 (H 2 O)

15. 15/16 3. Metabolism (4) Fermentation  Fermentation ( 발효 ) - Glycolysis without oxygen supply - Mechanisms for ethanol, lactate and acetate production (1) Sufficient oxygen  NADH from glycolysis moves to full oxidation process and is used for ATP production. NAD produced is recycled in the glycolysis (2) Oxygen depletion  Full oxidation process stops  By- products (ethanol, lactate, acetate) are produced by using pyruvate  NADH is consumed and regenerated NAD is recycled in the glycolysis. The fate of pyruvate when oxygen is used up Recycle of NADH and NAD

16. 16/16 3. Metabolism (5) Krebs cycle & electron transport system  Krebs cycle (Tricarboxylic acid cycle, TCA) - Full oxidation of pyruvate - 1 pyruvic acid  3 CO 2 + 4NADH + 1ATP - Does not require oxygen - Main pathway for amino acid production Electron transport system The Krebs cycle  Electron transport system ( 전자전달계 ) - Where electron energy stored on NADH in the glycolysis and TCA is transferred into ATP. - Electrons combine with oxygen - Producing water from hydrogen and oxygen - Finally, large amount of ATP is produced. (1 glucose molecule  36~38 ATP) - In the cytoplasm (prokaryotes) or the mitochondria (eukaryotes)