Chapter 10:Sterilization and Bioreactor OperationDavid ShonnardDepartment of Chemical EngineeringMichigan Technological UniversityDavid R. ShonnardMichigan Technological University1Sterilization Methods and Kinetics: 10.4Sterility: the absence of detectable levels of viable organisms in aculture medium or in a gasReasons for Sterilization1. Economic penalty is high for loss of sterility2. Many fermentations must be absolutely devoid of foreignorganisms3. Vaccines must have only killed viruses4. Recombinant DNA fermentations - exit streams must besterilizedDavid R. ShonnardMichigan Technological University21

Sterilization Agents1. Thermal - preferred for economical large-scale sterilizations ofliquids and equipment.2. Chemical - preferred for heat-sensitive equipment ethylene oxide (gas) for equipment 70% ethanol-water (pH 2) for equipment/surfaces 3% sodium hypochlorite for equipment3. Radiation - uv for surfaces, x-rays for liquids (costly/safety)4. Filtration membrane filters having uniform micropores depth filters of glass woolDavid R. ShonnardMichigan Technological University3Kinetics of Thermal Sterilization (Death)Practical considerations:1. Not all organisms have identical death kinetics. (increasing difficulty; vegetative cells spores virus)2. Individuals within a population of the same organism mayrespond differentlyFrom Probability Theory:p(t) the probability that an individual cell is still viable at time t.-kdtp(t) eDavid R. Shonnard(simplest form assuming 1st ordeMichigan Technological University42

Kinetics of Thermal Sterilization (cont.)E[N(t)] expected value (E) of the numbof individual organisat time t after sterilization starts.-k tNo e d No p(t) whereo is Nthe initial number of individualsN(t)No-k t edDavid R. ShonnardorN(t)ln -kd t survival"curNo5Michigan Technological UniversityTemperature Effects on theKinetics of Thermal SterilizationincreasingArrhenius Equation-kdkd α e-E od / RTα constant (time )-1lnN(tNoR gas constantT absolute temperatureE od activation energy for deatht(50 -150 kcal / g - mole) for spores(2 - 20 kcal / g - mole) for vitamins / growth factorsDavid R. ShonnardMichigan Technological University63

Population Effects on theKinetics of Thermal SterilizationMost Thermal Sterilizations are at 121 COrganismkd (min-1)Vegetative cells 1010Spores0.5 to 5.0Spores are the primary concern during thermal sterilizationDavid R. ShonnardMichigan Technological University7System Variables for Thermal SterilizationPrimary System Variables in Thermal Sterilization1. Initial concentration of organisms2. Temperature, T3. Time (t) of exposure at temperature T.[1-Po(t)Probability of an Unsuccessful FermeNo[1-Po(t)] 1-[1-p(t)]-kdt N o 1-[1-eDavid R. Shonnard](for a homogeneous populMichigan Technological University84

Sterilization Chart“Bioprocess Engineering:Basic Concepts”Shuler and Kargi,Prentice Hall, 2002David R. ShonnardMichigan Technological University9System Variables for Thermal SterilizationUse of Sterilization Charts:1. Specify 1-Po(t) which is acceptable (e.g. 10-3)2. Determine No in the system.3. Read kdt from the chart.4. Knowing kd for the spores (or cells), obtain the required time, t.David R. ShonnardMichigan Technological University105

Scale-up of Sterilization in batch sterilization, scale-up of small-scale sterilization datato a much larger scale will result in unsuccessful sterilization41-Liter Vessel154/Lno 10 spores10154 10 spores/Lno4N o (1 L)(no)o-kd t no[1-Po(t)] 1-[1-e] (10 L)(no)N-kd t 104 no-Po(t)][1 1-[1-e]-14 1-5x10 .003David R. Shonnard-L VesselMichigan Technological University 111Batch vs. Continuous SterilizationBatch1. Longer heat-up/cool down time2. Incomplete mixing“Bioprocess Engineering:Basic Concepts”Shuler and Kargi,Prentice Hall, 2002David R. ShonnardMichigan Technological University126

Batch vs. Continuous SterilizationContinuous1. Shorter time2. Higher temperature“Bioprocess Engineering:Basic Concepts”Shuler and Kargi,Prentice Hall, 2002David R. Shonnard13Michigan Technological UniversitySterilization of Gases aerobic fermentations require 0.1 to 1.0 (L air / (L liquid min)) 50,000 L fermenter requires 7x106 to 7x107 L air/day microorganism concentrations in air are about 1-10 / L airMethods for Air Sterilization at Inlet1. Adiabatic compression, 220 C for 30 seconds2. Continuous Filtration: depth filters (glass wool filters) surface filters (membrane cartridges)Exit gas mustbe filtered pathogenic recombinantDNA cells3. Economics 25% of production costs for air systemDavid R. ShonnardMichigan Technological University147

Design and Operation of BioreactorsTypes of Bioreactors1. Reactors with Mechanical Agitation see Fig. 10.1Aa) disperse gas bubbles throughout tankb) increase residence time of bubblesc) shear large bubbles to smaller bubblesd) disk type or turbine type (dI 0.3 dT)e) provide high kLa valuesf) baffles (4) augment mixing ( 0.1 dT)2. Bubble Columnsee Fig. 10.3see Fig. 10.1Ba) disperse gas bubbles throughout tankb) perforated plates enhance gas dispersion and mixingDavid R. ShonnardMichigan Technological University15Figure 10.1A“Bioprocess Engineering:Basic Concepts”Shuler and Kargi,Prentice Hall, 2002David R. ShonnardMichigan Technological University168

Figure 10.3(1st Edition)RushtonImpeller(Disk-type)Axial flowhydrofoilImpeller lower energy demand comparable gas transfer superior axial mixing lower shear stress“Bioprocess Engineering:Basic Concepts”Shuler and Kargi,Prentice Hall, 2002David R. ShonnardMichigan Technological University17Design and Operation of Bioreactors (cont.)Types of Bioreactors3. Loop Reactors see Fig. 10.1 C, D, Ea) bubble rising in draft tube causes mixingb) mixing enhanced by an impeller or a jet pumpMaterials of Construction:Glass Vessels:Stainless Steel Vessels:Volume 500 LitersAll Volumes316 ss for vessel314 ss for covers & jacketsDavid R. ShonnardMichigan Technological University189

Figure 10.1B“Bioprocess Engineering:Basic Concepts”Shuler and Kargi,Prentice Hall, 2002David R. ShonnardMichigan Technological University19Reactor Geometry and LayoutFigure 10.2:a) height to diameter ratio of 2 to 3b) sterile air inlet and spargerc) baffle plates & impellersd) cooling coilse) foam breakerf) working volume (liquid capacity) 0.75 vessel volumeDavid R. ShonnardMichigan Technological University2010

Figure 10.2Height to DiameterRatio of 2 - 3VL 0.75 VR“Bioprocess Engineering:Basic Concepts”Shuler and Kargi,Prentice Hall, 2002David R. ShonnardMichigan Technological University21Reactor Types in IndustryNonstirred/Nonaerated Vessels:a) most fermentations in terms of total volumeb) food fermentations (beer, wine, diary products)Stirred / and (or) Aerated Vessels:a) most fermentations in terms of numbers of unitsDavid R. ShonnardMichigan Technological University2211

Aeration and Heat TransferAeration and heat transfer requirements often limit thedesign of commercial reactorsA multiplication signAeration Design Equation: (OUR OTR)Oxygen Uptake Rate: OUR XqO2X cell concentration (g cells / L) - ranges from 1 to 5q O 2 specific O 2 uptake rate (Yield) [mmol O 2 / (g cells hr)](2 to 90; bacteria, yeast, molds)David R. ShonnardMichigan Technological University23Table 10.1“Bioprocess Engineering:Basic Concepts”Shuler and Kargi,Prentice Hall, 2002David R. ShonnardMichigan Technological University2412

Oxygen Transfer RateOxygen Transfer Rate: OTR k L a (C * -C)k La volumetric mass transfer coefficient (hr-1 )C * O2 concentration in water at the bubble / water interface PO 2HO2partial pressure of O2 in air (Pa)Henry's Law Constant for O2 (Pa / (mole O 2 / L))C L O2 concentration in the bulk water (mole O2 / L) temperature, pressure, & salt concentration affect C* vessel geometry, operation, and fluid properties affect kLaDavid R. ShonnardMichigan Technological University25kLa for Stirred TanksOxygen Transfer Rate: OTR k L a (C * -C) Pg k La k VR 0.4(vS )0.5 (N) 0.5see equation 10.2ak empirical constant (fluid and reactor - specific)Pg power requirement for an aerated bioreactorVR bioreactor volumevS superficial gas exit speed (Fa / A)Fa volumetric flow rate of airUnitsdependuponcorrelationdataA bioreactor cross - sectional areaN impeller rotation speedDavid R. ShonnardMichigan Technological University2613

Pg CorrelationPg P2 N D3 K u 0.56 i Qa0.45or Q Pg f a3 N Di Pu Q N A aeration number a 3 N Di K emperical constant (reactor geometry - specific)Pu power requirement for an ungassed bioreactorDi impeller diameterQa aeration rate (Fa / VR )David R. ShonnardUnitsdependuponcorrelationdata27Michigan Technological University“Biochemical Engineering”Blanch and Clark,Marcel Dekker, 1997Pu Correlation(Figure 5.20 of Blanch and Clark)David R. ShonnardMichigan Technological University2814

“Biochemical Engineering”Blanch and Clark,Marcel Dekker, 1997NA Correlation(Figure 5.22 of Blanch and Clark)XDavid R. ShonnardMichigan Technological University29Example ProblemA 10, 000 liter (of liquid) bioreactor contains 5 g / L of growing cellsq O2 20 mmole O 2 / (g cells hr)D T 2 m,CL 1 mg O2/LD i 1 m, (6 - blade turbine agitator) x 3 bladesFor 1 liquid volume per minute aeration rate (air), can theOTR OUR for N 100 rpm?David R. ShonnardMichigan Technological University3015

Example Problem SolutionPu : power requirement for ungassed reactorRe Reynold's Number ρ L 1,000 kg / m3ρ L N D 2iµLµL 10-3 Newton s / m2kg 100 -1 2 2 Newton 1,000 3s (1 m ) 1 m 60 (kg m / s2 ) Re 3 Newton s102m 1.67x106David R. ShonnardMichigan Technological University31Example Problem Solution (cont.)From Figure 5.20 of Blanch and ClarkPuPower number 4 ρ L N3 D5iPu 4 ( ρL N3 D5i )for 1 impeller22kg 100 -1 5 5 4 kg m / s 4 1,000 3s (1 m ) 1.852x10(Watts) sm 60 22 4 kg m / s4(Watts) 5.62x10 WattsPu (3 impellers) 3 1.852x10 s 74.5 HPDavid R. ShonnardMichigan Technological University3216

Example Problem Solution (cont.)Pg : NA (aeration no.) NA Qa3N Diliters 3 m3 10,000 10 min liter 0.10(100 min -1 )(1 m)3From Figure 5.22Pg 0.42 Pg (.42)(5.56x10 4 Watts)Pu 2.335x104 Watts 31.3 HPDavid R. Shonnard33Michigan Technological UniversityExample Problem Solution (cont.) Pg HP k La (mmole O2 / (l hr atm) 0.60 3 VR 10 liters Pg31.3 HPHP 3.13 3VR(10)(103 liters)10 liters0.4(v S )0.5(N(rpm))0.5 cm3 10 4 liters / min 10 3 liter cmvS 318.3πcm222min(2 m) (10)4mk La 0.60(3.13)0.4 (318.3)0.5 (200)0.5 169 (mmole O 2 / (l hr atm)David R. ShonnardMichigan Technological University3417

Example Problem Solution (cont.)g cells mmoles O2 20OUR X q O 5 2 liter g cells hr 100mmoles O2liter hrOTR k La(PO - P*)2P * for CL 1mg O 2 H O CL2liter 0.21 atm mg O 2 1 0.0263 atm mg O2 liter 8 liter David R. ShonnardMichigan Technological University35Example Problem Solution (cont.)OTR k La(PO - P*)2mmoles O 2(0.21 0.0263) atm 169liter hr atmmmoles O 2 31.05liter hrSince OUR OTR, we must modify the bioreactor operationin order to bring them into balance increase N use pure O2 rather than air.David R. ShonnardMichigan Technological University3618

Measurement of OTRN2O2 N2“Bioprocess Engineering:Basic Concepts”Shuler and Kargi,Prentice Hall, 2002O2CLt 0dC L kLa(C*-CL )dtt 0,CL 0ln(C* -CL ) -(kL a)David R. ShonnardMichigan Technological University37Heat Generation Rate: Aerobic GrowthQ GR 0.12 (OUR kca L hr David R. Shonnard mmol O2 L hr Michigan Technological University3819

Heat Generation Rate: AgitationQ agit Pg (power input aeratedVR (working volume of r 1 hp 100 ga David R. ShonnardMichigan Technological University39Heat BalanceHRR (Heat Removal Rate) U A TLMU overall heat transfer coefficientA surface area of heat transfer surface TLM log mean temperature difference betweenthe bioreactor fluid and cooling fluid(T - t1 ) (T - t 2 ) ln[(T - t1 ) /(T - t 2 )]T bioreactor fluid temperaturet1 cooling water inlet temperaturet 2 cooling water outlet temperatureDavid R. ShonnardMichigan Technological University4020

4 David R. Shonnard Michigan Technological University 7 Population Effects on the Kinetics of Thermal Sterilization Most Thermal Sterilizations are at 121 C Organism k