By: Puan Nurul Ain Harmiza 1 CHAPTER 6 : CELL GROWTH KINETICS: BATCH & CONTINUOUS CULTURES [Page 133] Shuler, M. L. and Kargi. (2002). Bioprocess Engineering: Basic Concept. 2 nd Ed. Upper Saddle River, NJ: Prentice Hall PTR PTT203: BIOCHEMICAL ENGINEERING SEMESTER 1 (2014/2015)
CHAPTER 6 : CELL GROWTH KINETICS: BATCH & CONTINUOUS CULTURES COURSE OUTCOME 2: Ability to categorize the metabolic pathways in microorganisms and analyze the growth kinetics in both batch and continuous reactors
Content • Introduction • Batch Culture • Quantifying Growth Kinetics • Continuous Culture
INTRODUCTION
Cell growth • Microbial growth is an autocatalytic reaction. The rate of growth is directly related to cell concentration. Substrate Cells Extracellu lar Products More Cells S X P nX S: substrate concentration (g/L); X: cell mass concentration (g/L); P: product concentration (g/L); n: increased number of biomass.
Cell growth • Characterized by the net specific growth rate: (1/time) t: the time 1 dX net X dt
Cell growth • Net specific growth rate (1/time): net g k d : Gross specific growth rate (1/time) g The rate of loss of cell mass due to cell death k : d or endogenous metabolism • Endogenous metabolism: during the stationary phase, the cell catabolizes cellular reserves for new building blocks and for energy-producing monomers.
Cell growth • Microbial growth can also be described in terms of cell number concentration, N. • So, the net specific replication rate (1/time): 1 dN R N dt k ' d R R N : Cell number concentration (cell number /L) : ' Gross specific replication rate (1/time) R k : The rate of cell death (1/time) d
BATCH CULTURE • QUANTIFYING CELL CONCENTRATION • GROWTH PATTERNS AND KINETICS • HOW ENVIRONMENTAL CONDITIONS AFFECT GROWTH KINETICS • HEAT GENERATION BY MICROBIAL GROWTH
Batch growth • Refers to culturing cells in a vessels with an initial charge of medium that is not altered by further nutrient addition or removal.
Quantifying Cell Concentration • Either cell mass or cell number can be quantified. • Purpose : • For determination of the kinetics and stoichiometry of microbial growth. • Two categories: • Direct – not feasible due to the presence of suspended solids or other interfering compounds in the medium. • Indirect
Quantifying Cell Concentration DETERMINING CELL NUMBER DENSITY Hemocytometer 1. • Direct microscopic count • Counts all cells present (viable and non-viable) • Immediate result Agar plates 2. • Counts only living cells • Delayed result • Assumption: each viable cell will yield 1 colony • Results expressed in CFUs(colony-forming units) Particle counters 3. • Counts all cells present (viable and non-viable) • Suitable for discrete cells in a particulate-free medium • Can distinguish between cells of different sizes
Hemocytometer
Viable Cell Count
Coulter Particle Counter
Quantifying Cell Concentration DETERMINING CELL MASS CONCENTRATION: DIRECT 1. Dry cell weight (DCW) • A sample of fermentation broth is centrifuged, washed, and dried at 80 ° C for 24hrs • Off-line measurement; wet cell weights (WCW) can performed in- process 2. Packed cell volume • Like wet cell weight, but measures cell pellet volume 3. Optical density (OD) • Turbidity – based on the absorption of light by suspended cells in culture media
Quantifying Cell Concentration DETERMINING CELL MASS CONCENTRATION: INDIRECT • When direct method is inapplicable. (mold solid state fermentation) • Indirect methods are therefore employed, based on the measurement of substrate consumption and/or product formation. • Cell mass can be determined by measurement of protein, DNA or ATP. e.g. 1mg ATP/g dry weight bacterial cell. • If 100 mg ATP/L is measured, then the cell mass: • 100 mg (ATP/L)/1 mg ATP/g dry cells=100 (g dry weight cells/L)
Growth Patterns and Kinetics
Growth Patterns and Kinetics Growth Phases Lag 1. Exponential 2. Deceleration 3. Stationary 4. Death/Decline 5.
Growth Patterns and Kinetics Turbidity (optical density) 9.0 8.0 Stationary Death Optical density Log CFU/ml Exponential 7.0 Log CFU/ml Optical Density 10 6.0 5.0 4.0 g a L Lag Time
Lag Phase • Occurs immediately after inoculation and is a period of adaptation for the cells to their new environment. • New enzymes are synthesized, synthesis of other enzymes is repressed. • Intracellular machinery adapts to the new conditions. • May be a slight increase in cell mass and volume, but no increase in cell number. • The lag phase can be shortened by high inoculum volume, good inoculum condition (high % of living cells: 5-10% by volume), age of inoculum, nutrient-rich medium.
Influence of [Mg 2+ ] on Lag Phase Duration in E. aerogenes Culture E. aerogenes requires Mg 2+ to activate the enzyme phosphatase, which is required for energy generation by the organism The concentration of Mg 2+ in the medium is indirectly proportional to the duration of the lag phase
Log/Exponential Growth Phase • In this phase, the cells have adjusted to their new environment • At this point the cells multiply rapidly (exponentially) • Balanced growth – all components of a cell grow at the same rate • Growth rate is independent of nutrient concentration, as nutrients are in excess • The first order exponential growth rate expression is: dX X where X X at t 0 net 0 dt X t ln t or X X e net net 0 X 0
Log/Exponential Growth Phase • An important parameter in the exponential phase is the doubling time (time required to double the microbial mass) • A graph of ln X versus t produces a straight line on a semi-logarithmic plot: ln 2 0 . 693 d max max • The doubling time based on cell number is expressed as: ' ln 2 d R
Exponential phase Nutrients and conditions are not limiting growth = 2 n or X = 2 n X 0 2 0 2 0 2 0 2 0 2 0 2 0 Where X 0 = initial number of cells 2 1 2 1 2 1 2 1 2 1 2 1 X = final number of cells 2 2 2 2 2 2 2 2 2 2 2 2 n = number of generations 2 3 2 3 2 3 2 3 2 3 2 3 2 4 2 4 2 4 2 4 2 4 2 4 2 n 2 n 2 n 2 n 2 n 2 n
Log/Exponential Growth Phase t
Deceleration Phase • Very short phase, during which growth decelerates due to either: • Depletion of one or more essential nutrients, or, • The accumulation of toxic by-products of growth (e.g. Ethanol in yeast fermentations) • Period of unbalanced growth: td=td’ • Cells undergo internal restructuring to increase their chances of survival • Followed quickly by the Stationary Phase
Stationary Phase • Starts at the end of the Deceleration Phase, when the net growth rate is zero (no cell division, or growth rate is equal to death rate) • Cells are still metabolically active, and can produce secondary metabolites • Primary metabolites are growth-related products, while secondary metabolites are non-growth-related • Many antibiotics and some hormones are produced as secondary metabolites • Secondary metabolites are produced as a result of metabolite deregulation growth = death (dX/dt = 0)
Stationary Phase • During this phase, one or more of the following phenomena may occur: • Total cell mass concentration may stay constant, but the number of viable cells may decrease • Cell lysis may occur, and viable cell mass may drop. A second growth phase may occur as cells grow on lysis products from the dead cells (cryptic growth) • Cells may not be growing, but may have active metabolism to produce secondary metabolites
Stationary Phase • During the stationary phase, the cell catabolizes cellular reserves for new building blocks and for energy-producing monomers • This is called endogenous metabolism • The cell must expend maintenance energy in order to stay alive • The equation that describes the conversion of cellular mass into energy, or the loss of cell mass due to lysis during the stationary phase is: dX k t k t or X X e d d SO dt
Death Phase Cell lysis (spillage) may occur 1. Rate of cell decline is first-order 2. where: – k d = 1 st order death rate constant, X s = conc. of cell at end of stationary phase Growth can be re-established by transferring to fresh 3. media
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