Preface to the Second Edition ThesecondeditionoftheFermentationandBiochemicalEngineer ing Handbook, likethe previous edition,is intendedto assist the develop ment, design and production engineerwho is engaged inthe fermentation industry. Particularemphasisisgivetothoseunitoperationsmostfrequently encounteredinthecommercialproductionofchemicalsandpharmaceuticals via fermentation, separation, and purification. Sometheory isincludedto provide the necessaryinsightintothe unit operation but isnot emphasized. Rather, the emphasis is placed on the practical aspects of development, design and operation-how one goesaboutcollectingdesign data,what arethescale-upparameters,how to select the right piece ofequipment, where operating problems arise, and how to troubleshoot. Thetext iswrittenfrom apracticaland operatingviewpoint,and all of the contributing authors have been chosen because of their industrial background and orientation. Several ofthe chapters which were in the first edition have been either deleted or replaced by other chapters which are more germane to current fermentation practice. Those chapters which were retained have been updated or have been rewritten to reflect current practice. Several new chapters were intro duced to reflect current emphasis on cell cultures, nutritional require ments, statistical methods for fermentation optimization, cross-flow filtration, environmental concerns, and plant design vi PrefacetotheSecondEdition vii TheeditorswishtoexpresstheirgratitudetoMrs. ConnieGaskill ofHeinkel FilteringSystems,Inc., forthewordprocessingassistanceshe gave to this edition. Scotch Plains, NewJersey Henry C. Vogel Bridgeport, New Jersey CelesteL. Todaro September, 1996 Preface to the First Edition This bookisintendedtoassistthedevelopment, designand produc tion engineer who is engaged in the fermentation industry. Particular emphasisisgiventothose unitoperationsmostfrequently encounteredinthe commercialproductionofchemicals and pharmaceuticalsviafermentation, separation, and purification. Sometheory isincludedtoprovidethenecessaryinsightintotheunit operation but is not emphasized. Rather, the emphasis is placed on the practicalaspectsofdevelopment,designandoperation-howonegoesabout collecting design data, what are the scale-up parameters, how to select the right piece of equipment, where operating problems arise and how to troubleshoot. Thetextiswritten from apracticalandoperatingviewpoint, andall ofthe contributing authors have been chosen because oftheir industrial backgroundandorientation. Sincethehandbook concerns fermentationand oftentheengineers involvedinfermentation are notversed inmicrobiology, itwasthoughtadvisabletointroducethissubjectatthebeginningofthebook. Similarly,sincemuch offermentation deals withtheproductionofantibiot ics,itwasdeemedadvisabletoincludesomechaptersspecificallyorientedto the productionofsterile products. The engineering using this handbook may wish that other unit operationsordifferentpiecesofequipmenthadbeenincludedotherthanthose viii Prefacetothe First Edition ix selected. Theselectionwas basedontheindividualcontributorsandmyown experience, over many years ofwork in the field, with unit operations and pieces ofequipment that have been the backbone and workhorses ofthe industry. The editor wished to express his thanks to Mr. Stanley Grossel of Hoffmann-LaRocheand Mr. JohnCarneyofDavy McKee Corporationfor reviewingandeditingthedraftcopies. HealsothanksMiss MaryWatsonof DavyMcKee Corporationfor typing assistance,and Mr. Michael Garzeof Davy McKee Corporationfor hishelpinproducingmanyofthegraphsand illustrations. Dr. SolBarer,theauthorofthemicrobiologychapteracknowl edges the valuable input to the Celanese Biotechnology Department, and especiallythanks Miss MariaGuerraforherpatienceintyping and retyping the manuscript. Berkeley Heights, New Jersey Henry C. Vogel June 1983 Contributors Michael J. Akers Edwin O. Geiger Eli Lilly and Company PfizerInc. Indianapolis, IN Groton, CT Giovanni Bellini Stephen M. Glasgow 3V Cogeim S.P.A. Union Carbide Chemical and Dalmine, Italy Plastics Co., Inc. South Charleston, WV Ramesh R. Bhave U.S. Filter Corporation Elliott Goldberg Warrendale, PA Consultant FortLee, NJ FrederickJ. Dechow BiocrystPharmaceuticals Inc. Yujiro Harada Birmingham, AL K. F. Engineering Co., Ltd. Tokyo, Japan Barry Fox Niro Inc. Willem Kampen Columbia, MD Louisiana State University Agriculture Center Howard L. Freese Baton Rouge, LA A1lvac Monroe, NC x Contributors xi MarkKeyashian Shinsaku Takayama CENTEON Tokai University Kankakee, IL Numazu, Shizuoka Pref., Japan John P. King Celeste L. Todaro Foxboro Company Heinkel Filtering Systems, Inc. Rahway, NJ Bridgeport,NJ MaungK. Min David B. Todd Gemini ManagementConsultants Todd Engineering New York, NY Princeton, NJ James Y.Oldshue Henry C. Vogel Mixing Equipment Co., Inc. Consultant Rochester, NY Scotch Plains, NJ Laura Pellegrini MarkR. Walden Politecnicodi Milano EliLilly Company Milano, Italy Indianapolis, IN Russell T. Roane Bechtel Engineering San Francisco, CA Kuniaki Sakato Kyowa Hakko Kogyo Co., Ltd. Tokyo, Japan Seiji Sato KyowaMedex Co., Ltd. Sunto-gun, Shizuoka Pref., Japan Allan C. Soderberg Fort Collins, CO Curtis S. Strother Eli Lilly Company Indianapolis, IN 1 Fermentation Pilot Plant Yujiro Harada, Kuniaki Sakata, Seiji Sato andShinsaku Takayama PROLOGUE(by YujiroHarada) The rapiddevelopmentofbiotechnologyhas impacteddiverse sectors oftheeconomy overthelastseveral years. The industriesmostaffected are the agricultural, fine chemical, food processing, marine, and pharmaceuti cal. Inorderforcurrentbiotechnologyresearchtocontinuerevolutionizing industries, newprocesses must bedeveloped to transformcurrentresearch intoviablemarketproducts. Specifically,attentionmust bedirected toward the industrialprocessesofcultivationofcells, tissues,and microorganisms. Althoughseveral suchprocessesalreadyexist(e.g., r-DNAandcellfusion), more are needed and it isnot even obvious which ofthe existingprocesses is best. To develop the most cost efficient process, scale-up data must be collected by repeating experiments at thebench and pilot scale level. These datamustbeextensive. Unfortunately,thecollectionisfarmoredifficultthan it would be in the chemical and petrochemical industries. The nature of working withlivingmaterialmakescontaminationcommonplaceand repro ducibility ofdata difficult to achieve. Such problems quickly distort the relevant scale-up factors. In this chapter,three research scientistsfrom Kyowa Kogyo Co. Ltd. have addressed the problems of experimentation and pilot scale-up for 1 2 Fermentation andBiochemicalEngineeringHandbook microorganisms, mammalian cells, plantcells, and tissue. It is our sincere hope that the reader will find this chapter helpful in determining the best conditionsforcultivationandthecollectionofscale-updata. Hopefully,this knowledgewill,inturn, facilitatethetransformationofworthwhileresearch programs into commercially viable processes. 1.0 MICROBIAL FERMENTATION(by KuniakiSakato) Chemical engineers are still faced with problems regarding scale-up and microbial contamination in the fermentation by aerobic submerged cultures. Despitemanyadvancesinbiochemicalengineeringtoaddressthese problems,theproblemsneverthelesspersist. Recently, manyadvanceshave beenmadeintheareaofrecombinantDNA, whichthemselveshave spunoff newand lucrativefields intheproductionofplantand animal pharmaceuti cals. Acarefulstudy ofthis technology isthereforenecessary, not onlyfor the implementationofefficient fermentation processes, but also for compli ance with official regulatorybodies. There are several major topics to consider in scaling up laboratory processestothe industrial level. Ingeneral, scale-up isaccomplishedfor a discrete system through laboratory and pilot scale operations. The steps involvedcanbebrokendownintoseventopicsthatrequiresomeelaboration: 1. Strain improvements 2. Optimizationofmediumcompositionandculturalcondi tions such as pH and temperature 3. Oxygen supply required by cells to achieve the proper metabolic activities 4. Selection ofan operative modefor cultureprocess 5. Measurementofrheological properties ofcultural broth 6. Modelling and formulation ofprocess control strategies 7. Manufacturingsensors, bioreactors,andotherperipheral equipment Items 1and2shouldbedeterminedinthelaboratoryusingshakeflasks or smalljarfermenters. Items 3-7are usuallydetermined inthepilot plant. The importanceofthe pilot plantis,however, not limitedto steps 3-7. The pilot plant also provides the cultured broths needed for downstream FermentationPilotPlant 3 processing and can generate information to determine the optimal cost structureinmanufacturingandenergyconsumptionaswellasthetestingof various raw materials inthe medium. 1.1 Fermentation Pilot Plant Microorganisms such as bacteria, yeast, fungi, or actinomycete have manufacturedamino acids, nucleic acids, enzymes, organic acids, alcohols and physiologically active substances on an industrial scale. The "New Biotechnology"ismakingitincreasingly possible to use recombinantDNA techniquestoproduce manykindsofphysiologically active substancessuch as interferons, insulin,and salmongrowth hormonewhichnowonlyexist in small amounts inplants and animals. This sectionwilldiscuss thegeneral problemsthatarise inpilot plant, fermentation and scale-up. The sectionwill focus onthree maintopics: (i) bioreactors and culture techniques, (ii) the application of computer and sensingtechnologies to fermentation, and (iii) the scale-up itself. 1.2 Bioreactors and Culture Techniques for Microbial Processes Currentbioreactorsaregroupedintoeitherculturevesselsandreactors usingbiocatalysts(e.g.,immobilizedenzymes/microorganisms)orplantand animal tissues. The latter is sometimes used to meanthe bioreactor. Table 1shows a number ofaerobic fermentation systems which are schematically classified into (i) internal mechanical agitation reactors, (ii) external circulation reactors, and (iii) bubble column and air-lift loop reactors. This classification is based on both agitation and aeration as it relatestooxygensupply. Inthistable, reactor1isoftenusedattheindustrial leveland reactors (a)2, (b)2, (c)2,and (c)3, can befittedwith draughttubes to improve both mixing and oxygen supply efficiencies. Culture techniques can be classified into batch, fed-batch, and con tinuousoperation(Table2). Inbatchprocesses,allthenutrientsrequiredfor cell growth and product formation are present in the medium prior to cultivation. Oxygenissuppliedbyaeration. Thecessationofgrowthreflects the exhaustion of the limiting substrate in the medium. For fed-batch processes, the usual fed-batch and the repeated fed-batch operations are listed inTable 2. Afed-batch operationisthatoperationinwhichoneormorenutrients are addedcontinuouslyor intermittentlytothe initialmediumafterthestart ofcultivation or fromthehalfway point through the batch process. Details 4 Fermentation andBiochemicalEngineeringHandbook offed-batchoperationare summarizedinTable3. Inthetable thefed-batch operationisdividedintotwobasicmodels,onewithoutfeedback control and the other with feedback control. Fed-batchprocesseshave been utilized to avoid substrateinhibition, glucoseeffect, and cataboliterepression, as well as for auxotrophic mutants. Table 1. ClassificationofAerobic Fermentation Systems (a) Internal mechanicalagitation reactors 1. Turbine-stirring installation 2. Stirredvesselwithdrafttube 3. Stirredvesselwith suctiontube (b) External circulationreactors 1. Waterjet aerator 2. Forcedwaterjet aerator 3. Recyclingaeratorwithfritteddisc (c) Bubblecolumnandair-loopreactors 1. Bubblecolumnwithfritteddisc 2. Bubblecolumnwithadrafttubeforgyrationflow 3. Airlift reactor 4. Pressurecyclereactor 5. Sieveplatecascadesystem Table2. ClassificationofFermentationProcesses 1. Batchprocess 2. Fed-batchprocess(semi-batchprocess) 3. Repeatedfed-batchprocess(cyclicfed-batchprocess) 4. Repeatedfed-batchprocess(semi-eontinuousprocessorcyclic batchprocess) 5. Continuousprocess