94 Fermentation and Biochemical Engineering Handbook 5.0 FERMENTER COOLING When designing a fermenter, one primary consideration is the removal of heat. There is a practical limit to the square feet of cooling surface that can be achieved from a tank jacket and the amount of coils that can be placed inside the tank. The three sources of heat to be removed are from the cooling of media after batch sterilization, from the exothermic fermentation process, and the mechanical agitation. The preceding topic about the design of a continuous sterilizer empha- sized reduced turnaround time, easier media sterilization, higher yields and one speed agitator motors. The reduced turnaround time is realized because the heat removal after broth sterilization is two to four times faster in a continuous sterilizer than from a fermenter after batch sterilization. The cooling section of a continuous sterilizer is a true countercurrent design. Cooling a fermenter after batch sterilization is more similar to a cocurrent heat exchanger. Assuming that all modern large scale industrial fermentation plants sterilize media through a continuous sterilizer, the heat transfer design of the fermenter is only concerned with the removal of heat caused by the mechani- cal agitator (if there is one) and the heat of fermentation. These data can be obtained while running a full scale fermenter. The steps are as follows: 1. Heat Loss by Convection and Radiation a. Perry's Handbook:[14] U = 1.8 Btu/hr/"F/ft2 (No insulation; if tank is insulated determine proper constant .) b. Calculate tank surface area =A c. Temp. of Broth = Tl d. Ambient Air Temp. = T, Q, = uA (Tl - T2) = Btukr Fermentation Design 95 Convection and radiation depend upon whether the tanks are insulated or not, and the ambient air temperature, especially during the winter. Measurements of convection and radiation heat losses are, on average, 5% or less of total heat of fermentation (winter and uninsulated tanks). 2. Heat Loss by Evaporation a. Iffermenters have level indicators, the average evapo- ration per hour is easily determined. b. Calculate pounds of waterhour evaporated from psychometric charts based on the inlet volume and humidity of air used, and at the broth temperature. The exhaust air will be saturated. Determine heat of vaporization from steam tables at the temperature of the broth = HEV = Btu/lb. Q2 = HEV x (lb water evaphr) = Btuh Evaporation depends upon the relative humidity of the compressed air, temperature of the fermentation broth and the aeration rate. It is not uncommon that the loss of heat by evaporation is 15 to 25% ofthe heat of fermentation. Modern plants first cool the com- pressed air then reheat it to 70-80% relative humidity based on summertime air intake conditions. Conse- quently, in winter the air temperature and absolute humidity of raw air are very low and the sterile air supply will be much lower in relative humidity than summer conditions. Therefore, in the winter more water is evaporated from the fermenters than in the summer. (Water can be added to the fermenter or feeds can be made more dilute to keep the running volume equal to summer conditions and productivity in summer and winter equal.) 3. Heat Removed by Refrigerant a. This is determined by cooling the broth as rapidly as possible 5°F below the normal running temperature 96 Fermentation and Biochemical Engineering Handbook and then shutting off all cooling. The time interval is then very carefully measured for the broth to heat up to running temperature (AT and time). b. Assume specific heat of broth = 1 .O BtuAb-OF c. Volume of broth by level indicator (or best estimate) = gal Q3 = Sp.Ht. x broth vol. x 8.345 x AT + time (hr) 4. Heat Added by Mechanical Agitation a. Determine or assume motor and gear box efficiency b. Measure kW of motor (about 0.92) Q4 = kW x 3415 x efficiency = Btuihr 5. Heat of Fermentation = AHf e, + Q2 + Q3 - Q4 = AHf The heat of fermentation is not constant during the course of the fermentation. Peaks occur simultaneously with high metabolic activity. Commercial fermentation is not constant during the course of the fermenta- tion. Commercial fermentations with a carbohydrate substrate may have peak loads of 120 Btu/hr/gal. The average AHf for typical commercial fermentations is about 60 Btu/hr/gal. The average loss of heat due to evaporation from aeration is in the range of 10 to 25 BtuMgal. Fermenta- tions with a hydrocarbon substrate usually have a much higher Mf than carbohydrate fermentations. Naturally, most companies determine the AHr for each product, especially after each major medium revision. (Typically, data are collected every eight hours throughout a run to observe the growth phase and production phase. Three batches can be averaged for a reliable AHf.) In this manner, the production department can give reliable data to the engineering department for plant expansions. Fermentation Design 9 7 The following is how the heat transfer surface area could be designed for a small fermenter. The minimum heat transfer surface area has been calculated (based on the data below) and presented in Table 2. Assume: S/S fermenter capacity Agitator Heat of fermentation (peak) Heat of agitation Heat transfer, U coils Heat transfer, U jacket Safety factor Chilled water supply Chilled water return Broth temperature (28°C) Table 2. The Heat Transfer 30,000 gal 15 hp/ 1,000 gal 100 Btu/hr/gal 38 Btu/hr/gal 120 Btu/hr/sq A 80 Btu/hr sq A No Btu lost in evaporation 50°F 60°F 82°F Surface Area (A2) Required for Tank with: Coils Only Jacket Only Mechanical agitation 200 Air agitation only 150 5 6 After the heat transfer surface area requirements are known, various shaped (height to diameter) tanks should be considered. Table 3 illustrates parameters of 30,000 gallon vessels of various WD ratios. 98 Fermentation and Biochemical Engineering Handbook Table 3. Maximum Heat Transfer Surface Area Available (ft2) on 80% of the Straight Side H/D F (ft) D (ft) Jacket Coils* 2 27.3 13.7 938 1,245 2.5 31.7 12.7 1,010 1,340 3 35.8 11.9 1,070 1,400 3.5 39.7 11.3 1,130 1,455 4 43.4 10.8 1,180 1,150 *Coil area is based on 3.5 inch o.d., 3.5 inch spacing between helical coils and 12 inches between the tank wall and the center line of the coil. It can be seen by comparing Tables 1 and 2 that if mechanical agitation is used and ajacket is desired, then additional internal coils are required. The internal coils can be vertical, like baffles, or helical. Agitation experts state that helical coils can be used with radial turbines if the spaces between the coil loops are 1 to 1.5 pipe diameters. Once helical coils are accepted, Why use ajacket at all? Reasons in favor of coils (in addition to the better heat transfer coefficient) are: 1. Should stress corrosion cracking occur (due to chlorides in the cooling water), the replacement of coils is cheaper than the tank wall and jacket. 2. The cost of a fermenter with helical coils is cheaper than a jacketed tank with internal coils. 3. Structurally, internal coils present no problems with continuous sterilization. However, if batch sterilization is insisted upon, vertical coils are one solution to avoiding the stress between the coil supports and tank wall created when cooling water enters the coils while the broth and tank wall are at 120°C. Notice that the method of media sterilization, batch or continuous, is related to the fer- menter design and the capital cost. Fermentation Design 99 6.0 THE DESIGN OF LARGE FERMENTERS (BASED ON AERATION) 6.1 Agitator Effectiveness Laboratory scale work frequently reports aeration rates as the volume of air at standard conditions per volume of liquid per minute, or standard cubic feet of air per hour per gallon. Production engineers realized that the scale-up of aeration for a large range of vessel sizes was by superficial linear velocity (SLV), or feet per second. Large scale fermenters, for energy savings in production equipment, use air-agitated fermenters. The cost savings are not apparent when comparing the cost of operating a fermenter agitator to the cost of the increased air pressure required. However, when the total capital and operating costs of fermentation plants (utilities included) for the two methods of fermentation are compared, the non-mechanically agitated fer- menter design is cheaper. The questions are, How much mixing horsepower is available from aeration, versus how much turbine horsepower is effective for aeration and mixing? D. N. Miller[”] of DuPont, describing his results of scale-up of an agitated fermenter states, “both &a and gas hold-up increase with an increasing gas rate and agitator speed. Gas sparging is the ‘stronger’ effect and tends to be increasingly dominant as gas rate increases. At superficial gas velocities, 0.49 Wsec and higher, very little additional mass transfer improvement can be gained with increased mechanical energy input.” Otto Nagel and associates[12] found in gas-liquid reactors that the mass transfer area of the gas in the liquid is proportional to the 0.4 power in the energy dissipation. Thus for a 50 hp agitator, 12 hp directly affects the mass transfer area of oxygen. The upper impellers mainly circulate the fluid and contribute very little to bubble dispersion and oxygen transfer. Most of the agitator’s power is spent mixing the fluid. (To understand mixing theories see Brodkey, Danckwerts, Oldshue or other texts.[I3]) The primary function of mixing for aerobic fermentations is to increase the surface area of air bubbles (the interfacial surface area) to minimize the bubble diameter. The fermenter is not the same as a chemical reactor where first and second order reactions occur between soluble reactants. The dissolution rate of oxygen into fermentation broth is controlled by diffusion. The consumption of soluble oxygen by the organism is an irreversible reaction and unless sufficient oxygen diffuses across the air-liquid surface area, the fermentation will cease aerobic metabolism. Methods of forcing more air into solution are: more interfacial surface area, more aidoxygen, higher air pressure, reduced cell volume, or controlling metabolism by reduced carbohydrate feed rates. 100 Fermentation and Biocltemical Engineering Handbook Not all of these options are practical because of shear, foaming and control devices. 6.2 Fermenter Height The height-to-diameter (WD) ratio of a fermenter is very important for oxygen transfer efficiency. Tall, narrow tanks have three major advantages compared to short, squat fermenters. Bubble residence time is longer in taller vessels than shorter ones. The air pressure is greater at the sparger resulting in higher dissolved oxygen in taller vessels. The third advantage is shown in Table 4, namely that for a vessel of constant volume, as the WD ratio increases, the volume of air required is reduced even though the superficial linear velocity remains constant. At the same time, bubble residence time and sparger air pressure increase. For larger volume fermenters, even greater vertical heights are used. The conclusion is that fermenter height is the most important geometrical factor in fermenter design. Conversely, shorter vessels need more air and/or more mechanical agitation to effect the same mass transfer rate of oxygen. The majority of industrial fermenters are in the H/D range of 2-3. The largest sizes are about 10' liters. It is thought that the cost of compressing air sufficient for air agitation alone is prohibitive. However, as seen in Table 5, if the fermenters are tall, the power consumption is less than for short squat tanks. Carehl selection of compressors with high efficiencies will keep power costs at a minimum. Table 4. Effect of Air Requirements on Geometric Fermenter Design Bubble Sparger WDF D scfm Residence Time Pressure 2 27.3 13.7 3,522 1 12.3 3 35.8 11.9 2,683 1.3 16.0 4 43.4 10.8 2,219 1.6 19.4 Constant: 30,000 gal tank; 24,000 gal run vol; 0.4 ft/sec SLV. Fermentation Design IO1 Table 5. Air Compressor Horsepower per Fermenter 30 psig 50 psig compressor compressor m sch (hP) (hP) 2 3,522 429 609 3 2,693 327 464 4 2,2 19 270 3 84 Constant: 30,000 gal tank; 24,000 gal run vol; 0.4 Wsec SLV. Note: Basis of hp is (8 hp)/(0.7) 6.3 Mixing Horsepower by Aeration The theoretical agitation effect of aeration alone can be easily calcu- lated. There are two separate forces, the first caused by the free rise of bubbles. The bubbles rise from the sparger at a pressure equal to the hydrostatic pressure ofthe liquid and as they rise to the surface, the gas bubble pressure remains in constant equilibrium with the hydrostatic pressure above it until it escapes from the liquid surface. The temperature of the air in the bubble is equal to the fermentation temperature and remains constant due to heat transfer from the fermentation broth. These conditions describe an isothermal expansion ofgas; gas pressure and gas volume change at constant temperature. Using the formula from Perry and Chilt~n,['~I the theoretical horsepower for the isothermal expansion of air can be calculated. p2 1,000 sch 4 hp = 4.36P2 In - where: P, is the hydrostatic pressure (absolute) P, is the (absolute) pressure above the liquid Figure 8 shows the curves at different superficial linear velocities and the relationship ofhorsepower to height ofliquid in a fermenter. These curves are the mixing energy (power per unit volume) released by rising bubbles to the liquid. 102 Fermentation and Biochemical Engineering Handbook w 'C Fermentation Design 103 Thus in a fermenter: 1. The horsepower per 1000 gallons (P/v) can be increased by adding more air. 2. The effect of aeration scale-up by superficial linear velocity (SLV) is not proportional to (PlV). However, by using these curves scale-up at constant (PR) can be used to determine the required SLV. 3. Experience indicates that the PN relationship is not affected by non-Newtonian fluids below 6000 cps apparent viscosity. 4. If the air temperature at the bottom of the fermenter is less than the liquid temperature, there is a gain in PN. This is due to the fact that at a lower temperature, the air density is greater, and heat is transferred from the broth to the bubbles (isothermal expansion) resulting in more work (Pm or kinetic energy imparted to the broth by turbulence. 5. If the fermenter vent valve is restricted to increase the pressure above the broth, it has the effect of reducing PN, but oxygen transfer increases due to the greater partial pressure of oxygen. There have been reports of air dispersion with improved oxygen transfer using static mixers attached to the air ring. Two papers on static mixers were given by Smith and Koch at the Mixing (Engineering Founda- tion) Conference in Rindge, NH (1 977). Additional papers can be found in the waste treatment field. There is additional energy to be gained from aeration. In order for the air to enter a tank below the liquid surface, the pressure in the sparging device must exceed the static head pressure. Thus the mass of air has a determinable velocity through the orifices of the sparger. The force exerted against the liquid is F=@/2g. That is, for a fixed mass flow rate of air, the force varies as the velocity squared. The velocity of air through a nozzle is a function of the (absolute) pressure ratio on each side ofthe orifice, and it can be increased to sonic velocity. The time of flow through an orifice is so short there is no heat transferred from the broth to the air and the air temperature drops. The expansion of air at sonic velocity is isentropic (adiabatic). The horsepower obtained by the isentropic expansion of air (at any pressure ratio) is (see Perry and Chilton.)[14]: 104 Fermentation and Biochemical Engineering Handbook k-1 hp = "(L)q&[(z) Pz -T -11 33,000 k - 1 where: V, = initial volume before orifice (e3) P, = initial pressure (absolute) P2 = (absolute) pressure after expansion k = CJC,, Assuming that k = 1 SO, then: Figure 9 illustrates the adiabatic hp/1000 sch in fermenters. Impor- tant features of using high velocity aeration are the following: 1. Increasing the liquid volume in the fermenter, such as feeding, reduces the horsepower. Conversely, removal of portions of broth will increase the horsepower. 2. The curves show the horsepower range at the air orifice from zero to sonic velocity which can be obtained by knowing the ungassed liquid height (differential pressure cell), the air pressure upstream of the orifice, and the sch of air used. 3. Increasing the air pressure above the liquid reduces the horsepower (see Fig. 10). The total theoretical horsepower of mixing by aeration alone is the sum ofthe isothermal and isentropic horsepower. At normal operating conditions, it is possible to double the agitation (PN) by increasing the velocity of the air without increasing the sch. It is easy to calculate the size of the orifices to give any desired velocity (up to sonic velocity), and the mixing horsepower. Conversely, one can scaleup aeration by horsepower per unit volume and determine the air required, i.e., it is possible to scaleup mechanical horse- power used in a pilot scale fermenter to a production vessel which is not mechanically agitated. Fermentation Design I05 90 80 10 I- W W Y, n W v) 40 v) d u Z 3 30 20 10 0 5 10 15 20 25 30 35 40 ADIABATIC HP/1000 S.C.F.M. Figure 9. Isentropic horsepowedl000; scfm. I06 Fermentation and Biochemical Engineering Handbook 1100 1000 900 5- 700 LL 0 t 500 400 300 0 10 20 30 40 50 60 FEET OF WATER OVER THE SPARGER Figure 10. Isentropic horsepower reduction by back pressure. Fermentation Design 107 6.4 Air Sparger Design Air sparger design of large fermenters is one of the least discussed topics of the fermentation industry. Most companies design their own air spargers. Some companies have designed and tried a wide variety of ideas. Agitator manufacturers insist that the air ring emit the air bubbles at the optimum radius ofthe first turbine, However, in air-agitated fermenters, the engineer must be creative to consider both the best mixing and oxygen transfer effects to be obtained from an air sparger. Not much is gained in mixing or mass transfer by having more than one orifice per 10 ft2 of cross- sectional area. 6.5 Comparison of Shear of Air Bubbles by Agitators and Jets Visualize a filled fermenter with a variable speed agitator at rest with a Rushton turbine. When aeration is started, large bubbles will rise and impinge on the underside of the turbine disc and escape around the perimeter. When the agitator is rotated at very slow speed, the large bubbles will accumulate behind the turbine blades and small bubbles will trail off into the liquid. If aeration and the agitator speed are increased, the volume of air behind each blade becomes larger, and smaller bubbles trail off into the liquid from the mass of air behind each blade. Due to the design of a Rushton turbine, the liquid and the small air bubbles move horizontally (or radially) into the liquid. As the speed of the turbine increases, the fluid velocity caused by the pumping action of the turbine produces a profile of shear stresses. Theoretically, the turbine speed could be increased to obtain any fluid velocity up to sonic velocity, but the power cost would be high since the energy must move the mass of liquid. Similarly, a limit is reached by increasing the aeration under a rotating turbine. The limit results when the air rotating behind each turbine blade fills all the space in the arc back to the front face of the next turbine blade. All the blades then are spinning in an envelope of air, or the impeller is flooded. For details see Klaas van? Riet.[lsI Now visualize the action of a submerged jet of air in liquid. At very low air flow velocities, the bubbles are large. They rise as independent bubbles at the orifice. When the velocity of air through the orifice increases, the air projects as a cone into the liquid and small bubbles shear off. The maximum velocity is reached when the ratio ofabsolute hydrostatic pressure outside the orifice divided by the absolute air pressure in the orifice is 0.528. This determines sonic velocity. Four regions of the air cone or jet are conceptually drawn in Fig. 1 1. Region I is called the potential core of air with a uniform velocity. The outermost annular cone, Region 11, is an intermittency zone in 108 Fermentation and Biochemical Engineering Handbook which flow is both turbulent and non-turbulent. Region I11 lies between the potential core and the liquid and is characterized by a high velocity (shear) gradient and high intensity ofturbulence. Region IV is the mixing zone where the fluid and air merge beyond the potential core. It is a totally turbulent pattern (see Brodkey).[16] A cloud of very fine bubbles is produced. Oxygen mass transfer is enhanced because of the increased surface area of the very small bubbles. In low viscosity fluids, much more coalescence of bubbles will occur than in high viscosity fluids. Figure 11. Region of mixing and shear for a submerged jet. Comparing now the bubble dispersion produced by agitators and jets, it should be clear that agitators shear large air bubbles by moving fluid, and jets produce fine bubbles by forcing high velocity air past relatively stationary fluid. This explains the high energy cost and low efficiency of mechanical agitation. Another benefit of air agitation is that it is a variable horsepower device depending upon the quantity of air and the velocity of the jet. Fermentation Design I09 6.6 The Effect of Shear on Microorganisms The effect of shear on microorganisms must be determined experimen- tally. Most bacteria and yeast can withstand very high shear rates. On the other hand, filamentous organisms are less predictable; some are stable and others are ruptured very easily. In the latter case, the air velocity must be reduced below the “critical” shear rate the organism can tolerate. The shear rate of Rushton turbines can be reduced by lowering the speed andor moving the vertical blades nearer to the shaft. The average bubble diameter will become larger and more air (SCFM) may have to be added for adequate dissolved oxygen. Unfortunately, laboratory and/or pilot plant experiments cannot easily test air agitation because it requires long bubble residence time (tall vessels) with minimal wall drag. The short vessels of the laboratory and pilot plant are precisely where mechanical agitation is absolutely required to achieve good fermentation performance compared to tall air-agitated fermenters. If anyone with large (tall) mechanically agitated fermenters wants to experi- ment with air agitation, it will be necessary to remove the turbines from the agitator shaft. Ifthe vessel has cooling coils, the mixing section is somewhat like a “drafttube”giving good top to bottom mixing. The air sparger will have to be replaced. Normally, air velocities of 0.75 Mach provide sufficient levels of increased agitation, mass transfer and shear. There is a reasonable body of literature, although scattered, about air agitation, frequently under gas- liquid reactor design and occasionally in bioengineering journals. D. G. Mercer read a paper at the Second World Congress of Chemical Engineering. Dr. M. Charles (Lehigh University)[17] will be publishing his experimental work soon. Also, see Shapiro, A. H.;[l8I Townsend, A. A.;[”] Forstall and Shapiro. L20] 6.7 Other Examples of Jet Air/Liquid Mixing The Buss loop reactor is a system to increase the dissolving of gas into a liquid which contains a dissolved chemical and a catalyst. Normally the reaction is first order and the reaction rate is dependent upon gas diffusion rate. An example is the hydrogenation of glucose to sorbitol. The rate of reaction and yield is increased as follows. The liquid is pumped from the bottom ofthe reactor, externally up to and through an eductor and discharged subsurface into the agitated vessel contents. The reason for its success is that a high velocity eductor mixes and shears a gas into very fine bubbles of very 110 Fermentation and Biochemical Engineering Handbook large surface area better than sparging beneath an agitator. The Buss company, of Bern, Switzerland, designs these systems. A variation of this design is a production fermenter which has several eductors welded to discharge horizontally and tangentially just above the bottom dish. Broth is continuously pumped from the bottom of the fermenter to all eductors simultaneously. Sterile air is provided to the suction side of the eductor (exterior to the tank). The fermenter has an agitator of very minimal horsepower to keep the solids suspended during sterilization. The pressure of the air supply is low (about 8 psig). A low horsepower blower is used to overcome the pressure drop over the air filter and line losses. Not too surprising, however, the total horsepower of the blower, the circulation pump, and tank agitator have the same power consumption per unit liquid volume as fermenters with conventional agitators, turbines, i.e., about 15 hp/ 1000 gallons of liquid is minimal for the average commercial aerobic fermentation. Therefore, pumping non-compressible water through eductors at high velocity to shear air into small bubbles is no more efficient than agitator mixing. The reverse case of compressing a gas to expand isentropi- cally at high velocities into a liquid does have significant advantages for large volume fermenters. (See Bailey and Ollis.[21]) 6.8 Mechanical Versus Non-mechanical Agitation In summary, the need for mechanical agitation beyond oxygen mass transfer is not clearly understood in fermentation broths which have a viscosity before inoculation of aNewtonian fluid, but change to pseudoplastic (non-Newtonian) after growth starts. Air-agitated fermenters exist in industry today for a wide range of products. It is a viable alternative to mechanically agitated systems. The advantages are the following: 1. Improved sterility because of no top- or bottom-entering agitator shaft. 2. Construction of very large fermenters is possible because the design is not limited by motor size, shaft length and its weight. 3. Refrigeration requirements are reduced 20 to 35% be- cause of no mechanical agitation-see Table 2. 4. Since no agitator, gear box or crane rail is needed, less structural steel is used and cheaper fermenter design results. Fermentation Design 11 1 5. No maintenance of motors, gear boxes, bearings or seals. 6. The air-agitated fermenter is a variable mixing power unit, like a variable speed drive with no motor and drive noise. 7. Air compressors can be steam driven to reduce power cost and continue to operate during power outages in large plants that have minimal power generation for controls. 7.0 TROUBLE SHOOTING IN A FERMENTATION PLANT The art oftroubleshooting is acquired by an alert individual after much experience actually operating a fermentation department. There are electri- cal, instrumental, mechanical and other physical problems arising from time to time which must be solved. However, the usual source of trouble in a fermentation department is a foreign, or contaminating, microorganism or bacteriophage in the fermentation media. This results in one or more of the following problems: 1. Inoculum cannot be used for the fermenters. 2. Complete or varying degrees of inhibition of product formation in the fermenters. 3. Possibly the product is produced, but contaminating products cannot be separated from the final product. 4. The fermentation broth cannot be filtered or otherwise processed due to physical broth characteristics. A fermentation department attempts to operate its culture lab, seed and main fermenters at 100% aseptic conditions. Good performance is when contamination occurs in 1 % or less of the batches. Naturally, any occurrence of such problems raises the cost ofproduction, andthe worst situation is when production is brought to a stop due to contamination. First, it is necessary to have standard operating procedures which are rigidly followed. Second, it is important to have well-trained operators who report back all problems, errors and irregularities, and third, the supervisors and engineer must listen to the operators, and react or explain the information, as the case may be, to close the communication loop and motivate the operators. I I2 Fermentation and Biochemical Engineering Handbook The basic sources of contamination in a fermentation department usually fall into the following categories: 1. Contamination of the plant stock culture due to poor techniques. 2. Contamination by the raw materials used in the fermenta- tion. 3. Contamination attributed to inadequate sterilization of the equipment, air or media involved in a fermentation plant. 4. Contamination attributed to inadequate procedures, or procedures not being followed, or insufficient operator training. 5. Contamination caused by lack of a definite routine main- tenance schedule on all the fermentation equipment. 6. Contamination by bacteriophage. The following is a list of procedures, steps, or operations that need to be followed closely and checked routinely to prevent contamination in the six categories stated above. I. Contamination in the Culture Laboratory a. Check the stock culture used for the presence of foreign microorganisms and bacteriophages. b. Check the flask, etc., used to inoculate the seed fermenter for contamination. c. Check the sterilization procedures used on flasks, media, etc. d. Check the techniques of the operator who inoculated the flask, etc. e. Check the sterilizer, including the temperature and pres- sure gauges, and operation. Be sure all air is removed from the sterilizing chamber. Use controls. f. Check sterility of the sterile area where the culture was transferred by using exposure plates. Fermentation Design I13 g. Carry out all practical tests for contamination by the best possible techniques using agar plates, several different media and microscopic examination. Use both static tubes and shake flasks. Incubate samples at different temperatures. h. Keep the laboratories clean, especially work benches or hoods where transfers are made. Check the germicidal solution used to wash the benches for growth. i. Presterilize the cotton used for plugs. j. When using grains or other materials which do not dissolve, use a finely ground type to be sure there is no lumping, and that all the material is wetted and suspended. Dry lumps can present sterilizing problems. k. If raw materials are heavily contaminated with spores, prepare the media and incubate at 30°C for several hours. When the spores have germinated, sterilize the media. 2. Contamination in Raw Materials a. Purchase all dry materials in a finely ground form. b. For those materials which do not dissolve, suspending first in cold water usually prevents lumping. Use adequate agitation in the mix tank. c. Pump the blend over a vibrating or rotating screen to separate all lumps larger than the size that can be sterilized by the time-temperature limitation of the sterilization cycle. Discard the oversized lumps. (Calculate the maximum lump size by the Gurney, Laurie charts. See Perry and Chilton.[l4I) d. Batch sterilizing in fermenters is frequently a problem because the slow agitation speed is not adequate to keep all the suspended material equally dispersed. Some may settle out in poor circulation areas of the vessel; some materials may enter the air ring while filling the vessels. Solutions to these problems can be: prehydrolyze the starch or protein with enzymes; use a longer sterilizing time or higher temperatures; use a continuous sterilizer (abandon batch sterilization). I I4 Fermentation and Biochemical Engineering Wan dbook 3. Contamination from Equipment Contaminated inoculum tanks: a. Check inoculum used, and the procedures of inoculating the tank with the operator. b. Thoroughly inspect the tank inside for cleanliness, includ- ing the head. c. Check tank and accessories for leaks in gaskets, coils, jacket, hatches, air lines from the sterile side of the air filters, etc., by pressurizing the equipment at 20 psig air pressure and going over all joints with a soap solution on a brush. Leakage ofair produces foam and large bubbles. d. Calibrate the temperature and pressure gauges on the tank for accuracy. e. Inspect the bottom valve and gasket, sample line valve, and the inoculation fitting. J: Inspect the inside tank fittings such as instrument bulbs, gauges, spargers, brackets, ladders, nuts, bolts, hangers, etc., for dead spots, holes, pits, corrosion, or for sources of debris (old dried up mycelia, media, etc.) which has not been washed or boiled out of the tank. g. Inspect the sterile air system, the packing and condition of the filters, gaskets, lines, etc. If when steaming out an air filter dark brown media runs out, there has been a media blowback. The filter should be completely cleaned out, restored and sterilized. h. Talk to the operators about the actual process used for cleaning, sterilization, cooling, inoculation, tank opera- tion and sampling procedures. i. Check the operators’ skills and techniques in taking aseptic samples from the tank for sterility tests. Unless the tank is grossly contaminated on a slide, you may have had a bad sample, due to poor technique on their part. j. Check the anti-foam and the complete system for addition of the anti-foam, or other substances, such as for pH control, etc. Fermentation Design I1 5 Contaminated fermenters: a. Check thoroughly the inoculum tank used to inoculate the fermenter. b. Inspect the inoculum line or hoses used, and verify that the operator has used the correct technique. c. Check tank cleanliness and method of cleaning; person- ally inspect the inside of the tank for sources of debris in cracks, crevices, flanges, bolts, hangers, coils, ladders, top of the tank, etc. The tank should be spotless. d. Check all the accessories for leaks, while the tank is under 20 Ib. pressure, with a soap solution. e. If coils or jackets are used, test hydrostatically for pres- sure drop. J Check all sensors for temperature, pH, DO, differential pressure cells, etc. Sometimes a crack in a thermometer bulb will continually contaminate a tank due to internal pressure changes. g. Check out the air system, filters, lines, packing, packing density, and lower than normal pressure drop. h. Check the anti-foam system and determine if anti-foam is sterile. i. Check the operators’ techniques for running tanks, addi- tions, nutrient recharges, partial broth harvests, etc. j. Inspect bottom valve, gaskets, sample line and valve, vent line valves, vacuum breakers, etc. k. If the fermenter is fed continuously (or intermittently), with a sterile nutrient, check the nutrient feed tank as if it were a fermenter. 1. Inspect closely and critically the inoculum and sterile feed transfer lines and procedures. Make sure when they are sterilized that no steam condensate accumulates in the line, but is bled off and all parts of the lines are up to sterilizing temperature. 11 6 Fermentation and Biochemical Engineering Handbook m. Be certain the relative humidity of the non-sterile fer- menter air before the “sterile” filter is and has been less than 85% at all times. n. Check that pH and DO probes have been removed and cleaned after every run, that the probe holder has been brushed and cleaned with a hypochlorite or a formalde- hyde solution. 0. When repeated runs in a vessel become contaminated while others remain sterile, it is customary to remove valves for cleaning or replacement. Replace gaskets. Remove and clean all instrument sensors; “high boiling” of the vessel with Na&O, or Na,PO, and possibly a germicide may be required. 4. Operating Procedures Training of personnel a. A training schedule and program should be standard for all new operators in fermentation departments. How and why every operation is important, and similarly, all procedures should be explained to each person. b. All operators should perform each task exactly alike and in the same sequence. No variation in procedures by individuals or shifts can be permitted. c. There should be a basic operating manual for each process. That is, if aplant makes antibiotics A, By and Cy there should be three manuals detailing all the steps necessary from seed fermenter media preparation to fermenter harvesting, with what to do if certain variables get out of control, and when a supervisor has to be notified for a decision. No deviations from standard procedures should be allowed except those in writing from the supervisor. Fermentation Design 11 7 d. Like regularly scheduled safety meetings, regular techni- cal meetings with operators are necessary to instruct and update technical and procedural changes. e. Operators must be encouraged to feed back their observa- tions, ideas and errors without fear (such as wrong material or quantity being used, a foam over, a blowback into the air filters occurring, pipe leaks, etc.). 5. Lack ofMaintenance as a Source of Contamination a. Braided packing on agitator shafts of sterile vessels is always a problem. Germicidal solutions are helpful. b. Mechanical seals on agitator shafts of sterile vessels usually have a sterilizing liquid circulating which acts as a lubricant as well. c. Calibration oftemperature and pressure gauges for accu- rate sterilizing temperatures are critical. d. Check all flanged sterile piping for leaks. Repeated sterilization cycles of lines will lengthen bolts and loosen flanges which can result in material getting between the gasket and flange. This might result in a leak and a source of contamination. The bolts on flanges should be tight- ened when the lines are hot, after an hour or so at 120°C. e. Check list for packed-bed type air filters: Filter should be properly packed to the correct density ( 12- 16 lb/ft3). Preweigh the exact amount of material for the volume of the filter. The filter bed should be inspected periodically, on a routine basis and new fiber added to the top if the rest of the fiber is all right, and no channeling is noted. Any time a filter is subjected to a blowback of media from the tank due to loss of pressure, or a vacuum, it should be cleaned out and repacked. Occasionally, check from the sterile side of the filter for leaks at the gaskets, valves, piping, threaded connection, etc. Also, check the spargers in the tank for debris or pockets. Make sure filters are dried out before being placed in actual service. I I8 Fermentation and Biochemical Engineering Handbook J: Repairs on the internals of the sterile vessels, such as bottom bearings, the air ring, coils, baffles, etc., must be done competently so they do not come apart during the vibration of sterilizing or operating. Work tools, gloves, etc., must not be left inside the vessel. g. Pressure reliefvalves andvacuum breakers must be tested for accuracy and routinely cleaned every run. h. If hoses are used to make sterile transfers, frequent inspection and replacements must be made. i. Each sterile vessel should have an inspection record that shows all maintenance and the date completed. 6. Bacteriophage Contamination How a bacteriophage enters the fermentation process is usually a matter of speculation, although it is known in some instances that it can accompany the culture itself. Bacteriophages affecting bacterial and fungal fermenta- tion can be found in soils, water, air, and even in the raw materials. They are widely distributed in nature. Their presence usually results in lysis of the cells which results in reduction of cell mass, no further product formation, no oxygen uptake, no heat production, etc. Confirmation of the presence of a bacteriophage can be made with phage plaque plates or by using ultra-filters and isolating the specific virus itself. The filtrate can be tested in the laboratory with the culture. The only immediate recourse to a bacteriophage attack is to substitute at once an immune strain if one is available. If one is not available, it is better to produce another product while the microbiologists in the culture labora- tory isolate a new strain ofculture which is resistant to the phage. This usually requires several weeks, which must include yield testing as well as new master lots of reserve culture. From a plant operation standpoint, the appearance of phage is treated as a bacterial contamination. Equipment checks and cleanup are no more or less rigid. Fermentation Design 119 8.0 GENERAL COMMENTS a. Usually if a tank is contaminated in the first 24 hours, it is due to contaminated inoculum, poor sterilization oftank accessories and contents, or unsterile air. If contamina- tion comes in after 24 hours, one should check the air supply, nutrient recharges, anti-foam feeds, loss of pres- sure during the run, lumps in the media, media blowbacks, humid air (wet air filters), etc. b. The use of air samplers or bubblers on the sterile air system for detection of contamination is merely an exer- cise in futility. The result means absolutely nothing, as the sample tested is such a minute part ofthe total air supplied to the tank. c. Be sure sterile samples from the plant have the correct tank number, time and date. d. Use TempilstiksTM to check sterilizing temperatures. e. During a siege of contamination in the plant, it may also be advisable to make some blank tank runs. j Permanently installed feed systems can create problems. Some chemicals (such as urea, sugar, NaOH, CaCl,, etc.) tend to weep from valves and flanges. It is particularly important after new gaskets have been installed in flanges to tighten up all the bolts after sterilization and to check for leaks occasionally. Where possible, the feed should be kept hot (60°C). Reliable valves on feed lines and removable stainless steel strainers with bypass lines at all critical points will improve operations. g. Vacuum breakers, anti-foam meters, etc., need special attention during the turn around time of every tank. A germicidal rinse prior to placing the tank on the line is considered good practice. h. If a plant has a continuous sterilizer, keeping the cooling water pressure less than the media pumping pressure will result in media being forced into the cooling water section if leaks develop. The installation of a conductivity probe and a suitable alarm system on the exit cooling water line will detect an immediate leak. I20 Fermentation and Biochemical Engineering Handbook i. Checking for stress corrosion cracking of stainless steel should be carried out by the supervisor at suitable inter- vals. This occurs in coils and jackets where water is cooled by cooling towers. This can be prevented or minimized by regular water analysis checks for chlorides which are the usual causative agents. The use of chlorides in the media makeup can also cause this problem in the equipment itself. Cracking will occur from the side of metal which is in tensile stress. j. It is important to have good housekeeping throughout all fermentation areas. Good sanitation practices involve daily washdowns ofthe area and equipment, otherwise the work area will become infested with organisms which can outgrow the commercial cultures. k. Tanks which are used for batch sterilization of media and have internal cooling coils are subject to a rapid wearing ofthe coils on the hangers. This is because during heating, the coils expand relative to the tank wall. However, when water is added to the coils to cool the batch, the coils contract while the tank shell is still expanded; the resulting friction will wear away the coil and eventually cause a leak. 1. Thermowells should be welded on the inside ofthe tank so media cannot get into the threads. m. Sometimes media can get between the foam probe and its covering. After a contaminated run, check the foam probe and lining. Clean with a germicide before replacing. REFERENCES 1. Peppler, H. J. and Perlman, D. (eds.), Microbial Technology, Second Ed., 1:285-290, Academic Press, New York (1979) 2. Aiba, S., Humphrey, A. F., and Millis, N. F., Biochemical Engineering; pp. 223-237, New York, Academic Press (1965) 3. Bruno, C. F. andSzabo, L. A.,FermentationAirFiltrationUpgradingbyUse ofMembrane Cartridge Filters, American Chemical Society Meeting, New York (August 24, 1981) 4. Perkowski, C. A,, Fermentation Process Air Filtration via Cartridge Filters, American Chemical Society Meeting, New York (August 24, 1981) Fermentation Design I21 5. Wiseman, A. (ed.), Topics in Enzyme andFernentation Biotechnology, pp. 170-266, John Wiley, New York (1979) 6. Peppler, N. J., Microbial Technology, Reinhold Pub. Corp., New York (1 967) 7. Lin, S. H., A Theoretical Analysis of Thermal Sterilization in Continuous Sterilizer, J. Ferment. Technol., 53(2):92 (1 975) 8. Lin, S. H., Residence Time Distribution ofFlow in Continuous Sterilization Process, Process Biochem., 14(7) (July, 1979) 9. Ashley, M. H. J. and Moopan, J., Continuous Sterilization of Media, American Chemical Society Meeting, New York, (August 24, 1981) 10. Wang, D. I. C., Cooney, C. L., Demain, A. L., Dunnill, P., Humphrey, A. F., and Lilly, M., Fermentation and Enzyme Technology, pp. 138- 156, John Wiley and Sons, New York (1 979) 11. Miller, D. N., Scale-up ofAgitated Vessels Gas-LiquidMass Transfer, Am. Inst. Chem. Engrs. J., 20(3):445 (May, 1974) 12. Nagel, O., Energy Engineering Systems Seminars, 2:835-76 (1979) 13. General Texts: Danckwerts, P. V., Gas-Liquid Reactions, McGraw Hill (1 970) Ho, C. S., and Oldshue, J. Y., Biotechnology Processes Scale-Up and Mixing, American Institute of Chemical Engineers (1 987) 14. Perry, R. H. and Chilton, C. H. (eds.), ChemicaEngineers’Handbook, Fifth Ed.; pp. 6-16, McGraw Hill, New York (1973) 15. van’t Reit, K., Turbine Agitator Hydrodynamics and Dispersion Perfor- mance, Ph.D. Thesis, University of Delft, Netherlands (1 975) 16. Brodkey, R. S., Turbulence inMixing Operations, Theory andApplications to Mixing and Reaction, New York, Academic Press (1 975) 17. Charles, M., Oxygen Transfer and Mixing Characteristics ofthe DeepJet- Aeration Fermenter (personal correspondence). 18. Shapiro, A. H., Dynamics and Thermodynamics of Compressible Fluid Flow, McGraw Hill, New York (1 953) 19. Townsend, A. A,, The Structure of Turbulent Shear Flow, Cambridge University Press (1 956) 20. Forstall and Shapiro, Momentum andMassTransfer incoaxial Jets,J. Appl. Mech.; Vol. 29 (1967) 21. Bailey, J. E. and Ollis, D. F., BiochemicalEngineen’ng Fundamentals, Ch. 9, McGraw-Hill Book Co. (1 977)