Biochemical Engineering BIE 6810
Utah State University
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This 11 page Class Notes was uploaded by Jerrell Nitzsche on Wednesday October 28, 2015. The Class Notes belongs to BIE 6810 at Utah State University taught by Ronald Sims in Fall. Since its upload, it has received 18 views. For similar materials see /class/230406/bie-6810-utah-state-university in Biology at Utah State University.
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Date Created: 10/28/15
Chapter 6 7 HOW CELLS GROW 61 p 155 Introduction 7 1 Substrates Cells Extracellular Products More Cells 61 ZS X 2P n 3 Net Speci c Growth Rate Mm 1 62a X it um g 7 kd 62b 5 Cell growth in terms of cell number concentration MR l m MR net specific replication rate 63 N dt M R indicates that kd 0 cell death unimportant or ignored 62 p 156 Batch Growth 621 Quantifying Cell Concentration 6211 Determine Cell Number Density 1 Petroff7Hausser slide or hemocytometer 2 Petri dishes agar 7 viable cells 7 plate countquot method 3 Particle counter Fig 61 6212 Determine Cell Mass Concentration 1 Direct methods 7 11 Dry weight 12 Packed cell volume centrifuge fermentation broth 13 Optical density turbidity using spectrometer for light transmission 2 Indirect methods 7 fermentation process molds S or P 21 lntracellular components RNA DNA protein Fig 62 211 DNA 8L protein concs remain fairly constant 212 Cell Protein 7 total amino acids Biuret Lowry Kjeldahl 22 lntracellular ATP concentration mg ATPmg cells 7 constant luciferin 02 777777777 779 light 64 luciferase ATP conc of a typical bacterial cell 1 mg ATP g dry7weight cell 23 Nutrients used for cell mass production N P S O 2 carbon source Note This relates to the stoichiometry material covered previously 24 Products of cell metabolism 7 241 7 anaerobic conditions 7 ethanol lactic acid 242 7 aerobic conditions 7 C02 243 7 changes in pH eg nitrification denitrification 244 7 viscosity change 7 due to extracellular polysaccharide 62 160 Growth Patterns and Kinetics in Batch Culture 1 Batch growth curve7Eigure 63 phases include lag log deceleration stationary death 2 Lag 7 adaptation of cells to a new environment Note 1 Many commercial fermentation plants rely on batch culture 2 To obtain high productivit the lag phase must be as short as possible 21 Low concs of some nutrients and growth factors can extend the lag phase 22 Example 7 Enterobacter aerogenes 7 lag phase increases as Mg 2 is decreased Mg2 is a activator of the enzyme phosphatase 23 Example 7 heterotrophic cells require C02 fixation and excessive sparging removes metabolically generated COz too rapidy 24 Age of inoculum culture how long maintained in a batch condition minimize 7 cells are adapted to growth mediumconditions before inoculation 7 cells should be exponential phase 7 inoculum size should be large 5 to 10 by volume 25 Multiple lag phases 7 diauxic growth see Example 41 3 Log or exponential phase 7 balanced growth 7 all cells grow at same rate average composition remains constant 7 31 Net Specific Growth Rate remains the same during this phase 32 Exponential growth rate is First Order with respect to X g um X XX0 at t0 65 dt lntegration yields 11 X mt l or XX0 umtt 66 0 Where X and X0 are cell cons at time t and t0 33 Doubling time time required to double microbial mass or number td ln 2 0693 67 Hnet Hnet t d ln 2 68 H R 4 Deceleration phase 7 1 Depletion of one or more essential nutrients or 2 Accumulation of toxic byproducts of growth 3 Changes occur over a very short period of time log to deceleration phase 4 Cell physiology under conditions of nutrient limitations are studies in continuous culture 5 Stationary phase 7 net growth rate 0 1 Growth rate death rate 2 Metabolites 7 primary growth7related 7 secondary nongrowth7related 3 Phenomena occurring during Stationary phase7 1 Total cell mass constant but number of viable cells decrease 2 Cell lysis mass decrease 2 growth on lysis products 3 Secondary metabolites 7 produced as results of deregulation 4 Endogenous metabolismcatabolism7cell reserves for building blocks 8L energy 1 g 7kdX or XXsoe kdt 69 it XSD cell mass conc at beginning of Stationary phase Because S 0 ug 0 in Stationary phase Z If inhibitory product is produce and accumulates ethanol7yeast manage by 1 Dilution oftoxified medium 2 Add unmetabolizable chemical compound to complex the toxin 3 Simultaneous removal of toxin 6 Death phase 7 rate of death usually follows first7order kinetics g 7de or N Nse dk W 610 it where NS is the concentration of cells at the end of the Stationary phase and k d is the first7order death rate constant 7 Yield Coefficient Growth Yield in a fermentation is YXs i M 611 Apparent Growth Yield at end of a fermentation batch growth period AS ASassimilation ASassimilated into ASgrowth ASmaintenance 612 into biomass extracell prod energy energy Other yield coefficients YxO2 AX YpS Q 613 614 A 02 AS Table 61 p 167 lists values of YXg and YXO2 for substrates 8L organisms Maintenance Coefficient specific rate of substrate uptake for cell maintenance M7 dSdtm 615 X 1 During Stationary phase 7 endogenous metabolism is used for quotMquot 2 Maintenance 7 repair damage components transfer nutrients inout of cells motility adjust osmolarity of cell interior 8 Microbial products are classified into three major categories Fig 66 p 168 1 Growth7associated products are produced simultaneously with microbial growth 01p l YPx Hg 616 X it eg production of a constitutive enzyme 2 Nongrowth7associated product formation occurs during Stationary Phase Specific rate of Product formation is constantquot qp B constant 617 eg Secondary metabolites 7 antibiotics eg penicillin 3 Mixed7growth7associated product formation occurs during slow growth 8L Stationary phases Specific rate of product formation qp ocug 3 618 eg lactic acid fermentation xanthan gum 8L some secondary metabolites 9 Example 61 7 Growth rate amp Yield 7 Solution 1 Max Specific New Growth Rate net lfl 7 ll 01 hi1 l2 l1 2 Apparent Growth Yield Y M 41 7 04 g cellg subtrate AS 063 7 100 3 Maximum cell concentration if 150 g glucose used with same inoculum size Xmax X0 YSO g cellsL 623 p 169 7 Now Environmental Conditions affect Growth Kinetics 1 Temperature above optimal level 7 specific replication rate Fig 67 m M R715d N 619 1 Both 12 and k d vary with temperature Arrhenius Eq H R Ae7EaRT 7 k d Aie7EdRT 63920 Ea activation energy for thermal growth 10720 kcalmol Ed activation energy for thermal death 60780 kcalmol 2 Temp 7 affects Yield coefficient Yxs eg single 7cell protein production 1 Affects Maintenance coefficient m Em 15720 kcalmol 3 Temp 7 affects rate 7limiting step in fermentation process 1 Rate of bioreaction gt rate of diffusion immobilized cell system 2 Ea for molecular diff 6 kcalmol Ea for bioreactions gt 10 kcalmol 2 pH 7 bacteria 378 animal cells 65775 non7optimum gt quotmquot increase NOg39 utilization causes increase in pH Organic acid production decreases pH C02 production affects pH 3 Dissolved Oxygen 7 consumption rate gt supply rate at high cell concentration 1 Growth rate can be proportion to Oz conc MichaeliseMenten relation 2 Fig 69 172 7 growth rate as fOz while mass is determined by S 3 Critical 02 as DOW 571 0 bacteria yeast 1050 mold gtlt 4 Rate of Oz transfer NO2 mg Ozlehr kLaC CL 62l kL oxygen transfer coefficient cmh a gaseliquid interfacial area cmZcms kLa volumetric oxygen transfer coefficient h l CSat CLDo conc 5 Oxygen Uptake Rate OUR mg Ozh qo2X ugx 622 YXO2 6 OTR OUR g X kLaCrCL 623 YXO2 Or 7 YXo2 kLaC CL 624 dt p 173 8 Redox Potential Eh E O M log 1302 23 log H 625 4F F 9 lonic Strength 7 l affects transport of nutrients in and out of cells and 2 solubility of Oz 3 l l 2 CiZiZ 626 eg NaCl inhibitory at gt 40 gL osmotic pressure max conc glucose 100 gL ethanol 50 gL 624 Heat Generation by Microbial Growth you read 63 p 175 Quantifying Growth Kinetics 631 Introduction Structured model 7 divides cells mass into components Unstructured model 7 assumes fixed cell mass composition balanced growth Valid in single7stage steady7state continuous culture 8L log phase of batch culture Fails during any transient condition Segregation 7 divide culture into individual units cells7 differ from each other 632 Unstructured Nonsegregated Models to Predict Specific Growth Rate 6321 Substrate7limited growth p176 uy 11ES Fig 611 MONOD EQN 630 Ks S 7 Michaelis 7 Menten kinetics for enzymes applied to whole cells for growth 7 If endogenous metabolism is not important then mt MY p 177 For rapidly growing dense cultures W umS 631 K50 SO S Where So is the initial substrate concentration and K30 is dimensionless Skip equations 633 through 638 6322 Models with growth inhibitors High S P and with inhibitory substances growth rate depends on inhibitor concentration 1 Substrate inhibition 7 skip 2 Product lnhibition 7 a Competitive My 2 lm S 642 Ks1 PKp S b Noncompetitive MY 2 um i 643 l KgS l PKp Example Ethanol fermentation from glucose ETOH is inhibitor at concentrations above 5 3 Inhibition by toxic compounds 7 same as enzyme kinetic expressions a Competitive Mg lm S 646 Ksl lK1 S b Noncompetitive Mg gm 647 1 KgS 1 lKI c Uncompetitive Mg d include death term in rate expression Hg ims kyd KS S J33 K S l lK1 HIKr j 648 649 6323 The Logistic Equation To describe growth curve in Fig 63 combine growth equation 62 with Monod equation 630 and assume no endogenous metabolism l lnetx dt l lnetllms KS 8 To get J S X Clt KS S Relationship between microbial yield Y and substrate consumption is Y Q dX y d8 d8 lntegrate over X and S to get X 7 X0 sz SO 7 S See text for Equations 652 and 653 Logistic equations are a set of equations that characterize GROWTH in terms of 62a 630 650 651 CARRYING CAPACITY CC Specific growth is related to amount of unused CC pg k17XXoo x00cc Therefore M dx 9 RX 1 XXoo dt X dt Xo e 17 x9 16 Xoo Equ 656 is represented by the growth curve in Fig 612 182 X Study 7 Example 62 Logistic Equationz pages 1817182 654 655 656 6324 p 183 Growth models for filamentous organisms or submerged microbial pellet SKlP 633 p183 Models for Transient Behavior 7 shift in environmental or cultural conditions 6332 Chemically Structured Models Fig 614 1 All reaction should be expressed in terms of intrinsic concentrations amount of a compound per unit cell mass or cell volume Extrinsic concentration amount of a compound per unit reactor volume 7 cannot be used in kinetic expressions 2 The dilution of intrinsic concentration by growth must be considered 321 VRX r 660 dt 634 p 189 Cybernetic Models 7 Process is goal seeking eg maximization of growth rate 1 Initially motivated by desire to predict response of a microbial culture to growth on a set of substitutable carbon sources 2 Recently 7 identify regulatory structure of a complex biochemical reaction network eg cellular metabolism 3 Newest use 7 metabolic engineering 7 relating information on DNA sequences in an organism to physiologic function See Chapter 8 64 p 189 How Cells Grow in Continuous Culture 641 Introduction 7 1 Constant environmental conditions for growth product formation 2 Determine response of cells to the environment 642 Specific Devices for Continuous Culture 1 Chemostat Constant chemical environment 7 constant nutrient cell product 2 Turbidostat 7 cell concentration maintained constant Study environmental stress select cell variants or mutants with desirable properties 3 Plug Plow Reactor PPR 643 ldeal Chemostat CPSTR with pH and DO control units Fig 618 Material balance on cell concentration around the chemostat FXO 7 FX tth VR 7 kdX VR VR 664 dt Solve for g 97 g HEX VR 7 th VB 664a Dl VR VR VR VR F D dilution rate l Where 6 retention time in chemostat V From 664 FXO 7 FX ng VR 7 kdX VR g VR dt 0 7 FX HEX VR 7 0 0 steady state Therefore FX ng VR Divide by X F pg VR Solve for pg pg F l D 666 VR 6 Therefore setting the Dilution D rate sets the Growth Rate 1 g NOW substitute Monod equation pg D Emil S 667 Ks S Find max Kg 7 from plot of lug versus lS 1 K S Ks 5 K 11 Mg pm 8 MM S MM 8 HM S um Y In X b Use 667 to solve for relate S as a function of D D KS T max S D Ks DS umax S DKS l lmaxSTDSSlJ39maxTD Therefore S D K 668 max 7 Material balance on limiting substrate around the chemostat FSO HEX VR 1 quVRl ClS VR 669 YMWS Yps dt FSO 7 FS 7 ng VR 1 r 0 extracell prod 0 steady state Y XS F 80 i HEX VR 1 Y XS F Q 7 S D SO 7 S HEX 670 VR YMws Since Hg D at steady state if kd 0 then DSOrS DX Y gt03 Therefore X YMXS SO S 671 Using Equation 668 the steadyrstate cell concentration can be expressed as X YMXS SO D Kg 672 max 7 Now lets consider endogenous metabolism Equ 666 D pg becomes D pg 7 kd um 673a Or pg D kd 673b Substitute 673b into steady7state substrate balance and extracell 130 then Eqn 669 becomes DSO 7 S 7 D l5dX 0 673c Y gt03 Where YMWS denotes the maximum yield coef cient no maintenance or endogenous respiration Eqn 673c can be rearranged to DQQQ 7 1 2 ki 0 674 X Y XS Dp 7D ikd 70 67520 Y XS WW8 Y MS Divide by D and solve for lYAPXS 1 1ltd 675b YAPXs YMm YMws D l ms 676 YAPXs W D Y b m X Where ms kg 677 YMws While YMWS is a constant YAPXS varies with growth conditions if kd gt 0 Find YMX S and mg plot 1 YA versus lD slope ms intercept 1 Y XS
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