Chapter Ten: Selection, Scale-up Operation, and Control of Bioreactors

Factors for Consideration in Reactor Design Heat Removal: Foam Control: Providing oxygen: Sterilization: Why and How?

Factors for Consideration in Reactor Design Heat Removal: Foam Control: Providing Oxygen: Sterilization: Cellular metabolism produces heat, removed by internal coils or reactor jackets. Cellular metabolism produces compounds that promote foaming. Controlled by mechanical foam breakers and chemical additives. Cellular respiration requires oxygen. Sparged (pumped) air or impeller makes smaller bubbles and increases residence time. Respiration is a series of metabolic processes by which living cells produce energy through the oxidation of organic matters. Elimination of undesirable viable organisms using for example steam or filtration.

What Factors Limit Size of Reactors? Ability to provide oxygen and efficiency of heat removal.

Reactor Types A. Stirred-tank B. Bubble column C. Airlift D. Propeller Loop E. Jet Loop

Reactor Type Comparison Stirred-tank Good oxygen mass transfer High energy requirement for mixing High shear environment Seal to maintain and keep sterile Bubble Column Low shear environment No seal needed Restricted to low viscosity Less mixing than stirred-tank

Reactor Type Comparison Loop reactors (Airlift, Propeller Loop, Jet Loop): Better mixing than bubble column with same low shear and energy requirements and lack of seal Work with higher viscosity liquids than bubble columns Still less mixing than stirred-tank

Oxygen Mass Transfer Profile Bulk gas phase oxygen concentration Partitioning into the liquid phase (C* at saturation) Transfer across stagnant liquid layer to cell O2 Concentration concentration at cell Transfer across stagnant gas layer distance Bulk liquid concentration (CL) Transfer across stagnant liquid layer

Oxygen Mass Transfer For a steady state operation: (1) Oxygen mass transfer equation: For a steady state operation: OTR is not the rate at which you provide air to the bioreactor. You will actually provide much more oxygen to the bioreactor than is transferred to the cells. (2) X= the cell density (or biomass concentration) qO2= the specific oxygen uptake rate kL= the oxygen mass transfer coefficient a=the interfacial area per unit reactor volume kLa= the volumetric mass transfer coefficient C*= the concentration of O2 in the liquid at the gas-liquid interface (often assumed to the saturation value) CL=the concentration of O2 in the “bulk” liquid

Estimating O2 volumetric mass transfer coefficient (kLa) Correlations can be used to predict the volumetric mass transfer coefficient kLa Many correlations can be found in the literature. Shuler and Kargi (the textbook we use) give the general form: (3) k= an empirical constant that depends on the reactor type and bubble diameter, among other things Pg= the power required for an aerated reactor VR= the reactor volume vs= the superficial gas exit speed (volumetric exit flow rate divided by vessel cross-sectional area times the volume of gas per volume of reactor) N= the impeller rotational speed

Experimental Determination of O2 Mass Transfer Medium components, temperature, and pressure can affect kLa and oxygen solubility Simple experiments can be done to measure kLa Unsteady state, steady state, dynamic and sulfite are methods to measure kLa

Unsteady State Method Fill the reactor with medium only – no cells. Remove any dissolved oxygen from the medium by sparging with N2. Sparge oxygen (or air) into the bioreactor, and measure DO concentration in the medium. (4) Separate and integrate (5) Plot ln(C*-CL) versus t, slope is –kLa and y-intercept is lnC*

Steady-State Method Requires an oxygen gas analyzer for the effluent air. Perform an O2 mass balance to obtain OUR. Difficulty in both methods (steady and unsteady state) is that C* is a function of pressure (height of liquid and high pressure aeration gas). (6)

Example In an experiment, the dissolved oxygen in bioreactor was measured in the absence of cells as a function of time and the obtained data are tabulated below: t (min) CL (mg/L) ln(C*-CL) 1.974081 5 1 1.84055 20 2 1.667707 30 3 1.458615 40 4 1.193922 50 4.7 0.955511 60 0.832909 a) If the saturation concentration of oxygen (C*) is 7.3 mg/L, calculate kLa using the unsteady state method. b) At t= 90 min some cells were added and the dissolved oxygen concentration approached steady state immediately. Calculate the biomass concentration (X) if the specific oxygen uptake rate (qO2) is 0.04 mg O2/(g cells.min).

Example a) kLa= -Slope= -(-0.0196)=0.0196 min-1. b) From eq. (5): Since the dissolved oxygen concentration approached steady state immediately after the addition of cells, therefore:

Sulfite Method In presence of , the sulfur in is oxidized to sulfate ( ) in a zero-order reaction. The reaction is very rapid and CL approaches zero. Thus, Therefore: (7) (8) 16

Dynamic Method Utilizes a fermentor with actively growing cells. Requires only a DO meter. The air to the fermentor is shut off, and the DO decreases due to consumption by the microorganisms. The air is then turned on, and the DO increases.

Dynamic Method (9) When the air is off, kLa = 0, so the slope of DO vs. time is equal to –qO2X. The slope of the ascending curve with time can be determined from tangents to the curve. The slope of the plot dCL/dt + qO2X vs (C*-CL) will be kLa.

Example 10.1 Use the data in Fig. 10.5 (see below) to estimate kLa from the dynamic method. Also, if the cell dry weight has been measured as 2 g/L, evaluate the specific respiration (qO2, also known as the specific rate of oxygen consumption) rate of the culture. Slope~0.8 Slope=dCL/dt~0.1 CL~1.65 mg/L

Example 10.1: Solution When the aerator is off, we can use the declining part of the curve in Fig. 10.5 to estimate OUR. For the straight-line part of the curve (0.5 to 3 min after the aerator is off) we estimate OUR as: To estimate kLa, we use the ascending curve formed during reaeration: The best method to estimate kLa is to plot versus (the slope= kLa) but you can just make use of single points at individual times. In this problem kLa=0.16 min-1. the specific respiration rate is estimated as:

Some Terminologies Sanitize- to clean with the purpose of removing possible biological and nonbiological threats to human health. Disinfect- to greatly reduce the number of living organisms. Sterilize- to eliminate all viable organisms present (often our goal in bioprocesses).

Sterilization Sterilization = the absence of detectable, viable organisms. Sterilization is probabilistic: some portion of the population is more resistant to sterilizing agents than other portions. Would you use the same procedure (i.e. T, time) to sterilize a large scale reactor as a small scale reactor?

Nature of the Problem Chemostat- a faster growing contaminating organism can outgrow the desired organism and cause washout of the desired organism. Batch- the product can be biologically contaminated (could be lethal) or the purity profile could be significantly affected.

Methods of Sterilization Filtration: Used to sterilize heat sensitive liquids and gases. Most common for gases - P important. Heating: Most common for liquids and equipments. Typically steam at 121oC is used. Time and T are both important. Risk: degrading medium components. Radiation: To sterilize surfaces. Chemical: To sterilize surfaces. The risk is contamination of equipment with toxic chemical residues.

Specific Agents Thermal sterilization is a common method. Media that cannot be heat-sterilized (heat labile vitamins, proteins, sugars) must be filter-sterilized using filters with narrow pore-size distributions. Ethylene oxide is common for the insides of equipments that cannot be heat-sterilized or steam-sterilized. 70% v/v ethanol in water with HCl to pH 2 is a good sterilizing fluid. Weak (3%) sodium hypochlorite (HCLO) solution is commonly used to sterilize filtration equipment.

End of Chapter Ten