Bioreactors: Design and OperationCharles L. Cooney

Biotechnology can be considered to bethe integration of several disciplines, in-cluding microbiology, biochemistry, mo-lecular biology, and chemical engineer-ing, for the purpose of utilizing biologicalsystems for manufacturing or environ-mental management. Bioreactors serve acentral role in biotechnological process-es by providing a link between startingmaterials and final products, as shown inFig. 1. It is in this area that multidis-

changed from the calf stomach to tem-perature-controlled vats. In addition, ashortage of calf rennin has led to the useof less expensive microbially derived en-zymes in some applications. Anotherearly bioreactor was used in bread mak-ing. Bread dough provides a matrix thatentraps yeast; this matrix (an early formof immobilized whole cells), placed on aflat, hot pan in an oven, traps carbondioxide generated from the metabolic

Summary. The bioreactor provides a central link between the starting feedstockand the product. The reaction yield and selectivity are determined by the biocatalyst,but productivity is often determined by the process technology; as a consequence,biochemical reaction engineering becomes the interface for the biologist and engi-neer. Developments in bioreactor design, including whole cell immobilization, immobi-lized enzymes, continuous reaction, and process control, will increasingly reflect theneed for cross-disciplinary interaction in the biochemical process industry.

ciplinary efforts are focused and value isadded to a less expensive materialthrough synthesis of a product or render-ing of a service. In this article I examinethe strategy for selection and design ofbioreactors and identify the limits andconstraints in their use. A biotechnologi-cal process is an integrated set of unitoperations involving not only productsynthesis and bioconversions but alsoproduct recovery. The bioreactor mustbe designed to meet the specific needsand constraints of a particular process,and its design will affect both cost andquality of the final product or service.Although the term bioreactor is rela-

tively new, the concept is quite old.Some examples of early bioreactors in-clude the calf stomach, used in ancienttimes for storage of milk. It was discov-ered that an active ingredient in the calfstomach transformed the milk intocheese. This active ingredient is rennin,a protease that catalyzes a specific andlimited hydrolysis of milk proteins andpromotes their coagulation into cheese.Calf rennin is still used for cheese pro-duction. However, the bioreactor has

action of yeast and rises to form a loaf.With advances in technology we learnedto place the dough in a pan and, whilenot changing the biocatalytic process,change the form and quality of the finalproduct. The use of closed containers topromote anaerobic yeast metabolism inalcohol production is another early ex-ample of the evolution of a bioreactor tomeet the needs of a particular biocatalyt-ic process. Pasteur demonstrated the im-portance of oxygen for efficient produc-tion of bakers' yeast; this discovery hada considerable impact on the bakers'yeast industry and is an early example ofprocess control. A major development inbioreactor design occurred in the 1940's,when investigators learned how to growmolds such as Penicillium chrysogenumin submerged culture for the productionof antibiotics such as penicillin. Beforethis, fermentations were carried out onsolid substrates, which were difficult tocontrol and keep aseptic (1). Thus, themodern bioreactor in the form of a fer-mentor used in large-scale manufac-turing of pharmaceutical products is arelatively new innovation in the fermen-tation industry.The fermentation industry has expand-

ed considerably in scope. Today it en-

compasses much more than fermentationand is more properly called the biochem-ical process industry, by analogy withthe chemical process industry. A discus-sion of the modern biochemical processindustry by Perlman (2) gives an over-view of a multibillion-dollar industry thatinvolves 145 companies throughout theworld and produces more than 250 differ-ent products spanning the pharmaceuti-cal, chemical, and agricultural indus-tries. Furthermore, biological processesare used in such diverse applications aswaste treatment, the production of deter-gent enzymes, and the enzyme-catalyzeddegradation of the anticoagulant heparinin blood (3). Considering the central roleof bioreactors in a diverse industry, it isnot surprising that they are emerging in awide variety of shapes, sizes, and forms.The goals in bioreactor design, however,are common; the purpose of a bioreactoris to minimize the cost of producing aproduct or a service. The evolution ofbioreactors, whether for cheese produc-tion or synthesis of antibiotics, is drivenby a need to increase the rate of productformation and the quality of the productor service. Bioreactor design has fo-cused on improved biocatalysts; betterprocess control, more recently withcomputers; better aseptic design and op-eration; and innovative ways to over-come rate-limiting steps, especially forheat and mass transfer.

Objectives in Bioreactor DesignThe primary objective in the design of

a bioreactor or any component of a bio-technological process is to minimize thecost of producing a high-quality productor service. Because the bioreactor pro-vides a link between the starting materi-als and the product, it plays a critical rolein the economics of biotechnologicalprocesses. To place this in perspective,it is useful to consider the manufacturingcosts for different products. These aresummarized in Wable 1 for a commodityproduct, ethanol, and a specialty prod-uct, penicillin (4, 5). It is apparent thatthe primary cost is for the raw materialand that other major costs include capitalinvestment and utilities, mostly energy.In the case of older plants, where thecapital is depreciated, maintenancechanges will increase and become moresignificant. The magnitude of the capitalinvestment is also a strong function ofinterest rates and method of financing.The cost of energy, since 1978, has risenat an annual rate of 16 percent, while rawmaterials and labor costs have risen at 6and 10 percent respectively (6). As a

SCIENCE, VOL. 219

Charles L. Cooney is a professor in the Depart-ment of Chemical Engineering, Massachusetts Insti-tute of Technology, Cambridge 02139.

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result, the energy cost has become asmuch as 35 percent of the manufacturingcost for some products (6). For thisreason there is increased interest in mini-mizing energy demands in bioreactor op-eration and subsequent product recov-ery.

Processes that require bioreactors andtypical products are summarized in Ta-ble 2. It is useful to separate these pro-cesses itito those that are conversioncost-intensive and those that are recov-ery cost-intensiVe; although in both cas-es it is important to minimize the cost ofraw materials for conversion, in conver-sion cost-intensive processes the volu-metric productivity, Qp (grams of prod-uct per liter per hour), is of paramountimportance and in recovery cost-inten-sive processes the product concentra-tion, P (grams per liter), is the dominantcriterion for cost minimization.

Biological Reaction Kihetics

Several major differences betweenbiological and chemical reaction kineticsare important in bioreactor design. Inmost biological systems there is a com-plex series of reactions that must beoptimized and coordinated. Since thecell is able to facilitate this coordinationin an elegant manner, growing cells areoften used as a means of synthesizingcomplex metabolites, proteins, polysac-charides, and other products. Fermenta-tion processes are autoc thecatailyst coiciiiiration as well as theenvironment changes with tiiiiie-;thisleads to-'s-one -unique process controlproblems. Furthermore, the reactionsare conducted under moderate condi-tions of pH (near neutrality) and tem-perature (for instance, 200 to 65C).While low temperature is often cited asan advantage of biocatalysis, it frequent-ly causes a problem because heat remov-al is difficult at low temperature (7).Essentially all processing is carried outin an aqueous phase and product streamsare relatively dilute (typically 0.2 to 20percent), making recovery operationsexpensive.

Despite the apparent complexity ofbiocatalytic processes, there is usually asingle rate-limiting step controlling thereaction as well as a few secondary limi-tations, and these rate limitations pro-vide a basis for process design. The ratelimitation may be biologically imposed,as is often the case in complex reactionsequences for antibiotic, organic acid, oramino acid synthesis, where a singleenzyme step may limit metabolic flux.Productivity may be improved either by11 FEBRUARY 1983

Table 1. Comparison of manufacturing costsas percent of total for penicillin (4) and etha-nol (5) made by fermentation. Ethanol costsare based on corn as the starting material.

Peni- Eth-Category cillin anol

Raw materials cost 35 62Operating cost 26

Utilities 15Labor 4Maintenance I IPlant and overhead 8

Capital costs 22* 12**Includes depreciation, taxes, and insurance.

genetically alleviating the rate-limitingstep in the cell or increasing the cellconcentration. This is exemplified in Fig.2, which shows a plot of Qp versus cell orenzyme concentration, X, in a bioreac-tor. Productivity increases with increas-ing catalyst concentration and specificactivity or productivity, Sa, which re-flects the catalytic capacity of the cell orenzyme. Increasing the catalyst concen-tration is generally a biochemical engi-neering problem, while improving specif-ic activity is generally a biological one.Productivity limitations may be external-ly imposed, either by an engineeringconstraint such as mass and heat transferor by the need to limit a substrate con-centration. With growing cells, limitationof a nutrient such as carbon, nitrogen, orphosphate may be required to overcomecatabolite repression problems, in whichone of those nutrients, when above acritical concentration, prevents synthe-

sis of enzymes required for product for-mation (8). With an enzyme, substratelimitation may be required to avoid sub-strate inhibition.The goal in bioreactor design is to

minimize cost. Maximizing volumetricproductivity allows one to minimize cap-ital investment. For this reason it isusually desired to operate a reactor at itsmaximum physical limits, which are of-ten constrained by mass or heat transfer(9). The superposition of this engineeringconstraint on top of biological capabilityis shown in Fig. 2 by dotted lines, whichrepresent the operating limits for a biore-actor. The vertical line is the maximumcatalyst concentration, which is about200 grams per liter for cells or cellularorganelles that have a moisture contentof 80 percent and are packed ti*tly withlittle interstitial void volume; for en-zymes it is the maximum protein loadingtimes the specific activity. The horizon-tal line represents a heat or mass transferlimitation that is stoichiometrically relat-ed to product formation. When the spe-cific activity or productivity of a micro-organism or biocatalyst is improved, lesscatalyst is required to achieve the maxi-mum productivity, Qp max~for the reac-tor. This will be reflected in a reducedcatalyst cost. Improvements in engineer-ing design raise the value of Qp max,Thus, even though engineering con-straints limit productivity, improve-ments in the biological system can have asubstantial impact on the cost of biocon-version.

Fig. 1. Schematicoverview of a bio-technological processshowing the centralrole of a bioreactor inlinking starting mate-rials and the finalproduct. This over-view is typical of aer-obic fermentationprocesses and unitoperations such as aircompression and me-dium sterilizationmay not be requiredin all processes.

Table 2. Examples of products in different categories in the biochemical process industry.Category Example

Cell mass* Bakers' yeast, single-cell proteinCell componentst Intracellular proteinsBiosynthetic productst Antibiotics, vitamins, amino and organic acidsCatabolic products* Ethanol, methane, lactic acidBioconversion* High-fructose corn syrup, 6-aminopenicillanic acidWaste treatment Activated sludge, anaerobic digestionService Heparin degradation, optical isomer resolution*Typically conversion or feedstock cost-intensive processes. tTypically recovery cost-intensive process-es.

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Types of Bioreactors

In view of the importance of engineer-ing constraints and the need to integratebiological and engineering improve-ments, it is useful to consider the formsof bioreactors that are commonly usedtoday. There are three major types ofbioreactors and two forms of biocata-lysts. Bioreactors may be batch, semi-continuous (fed-batch), or continuous re-actors; furthermore, continuous bioreac-tors may be continuous stirred tank reac-tors (CSTR) or plug flow reactors. Thebiocatalysts may be individual enzymes,used as soluble or immobilized catalysts,or whole cells, and the cells may be in agrowing or nongrowing state. Combiningthese types of reactors and catalystsprovides the choice of operating systemssummarized in Fig. 3. The literature onnovel and alternative bioreactor configu-rations is quite extensive (10); however,to tap this body of knowledge and designa bioreactor to meet specific need-sre-quires a few simple decisions. The first isthe -choice-between enzymes and wholecells- as the biocatalyst. Today, the use ofone or a few enzymes in cell-free form islimited to simple and generally degrada-tive-that is, hydrolytic or oxidative-reactions which do not require energycoupling. Although in vitro biosynthesis,which requires energy coupling, hasbeen demonstrated in the laboratorywith cell-free enzymes (11), it is general-ly more practical to use whole cellswhenever there are requirements for co-factor regeneration or energy coupling.When using enzymes, one can choosebetween soluble and immobilized formsthat can be employed in a variety ofbatch and continuous reactors. The de-sign of immobilized enzyme systems hasbeen reviewed (12) and a variety of im-mobilization methods based on physicalentrapment, covalent cross-linking, co-valent attachment to a matrix, and ad-sorption have been described (13). Im-mobilized enzyme processes used com-mercially are summarized in Table 3(14).When products or services require

more complex reaction sequences, it isbest to use whole cells. These may beeither growing cells, as used in the syn-thesis of most fermentation products to-day, or nongrowing cells, which can beused in concentrated suspensions or asimmobilized cells for the catalysis ofrelatively simple reactions. Normally,with nongrowing cells the catalysts areimmobilized by incorporation into a ma-trix or retention by a membrane. Suchsystems are being increasingly used forindustrial applications, as shown by

730

Catalyst concentration, XFig. 2. Volumetric productivity (grams ofproduct per liter per hour) in a bioreactor as afunction of catalyst concentration (grams perliter) and specific activity (grams of productdivided by grams of catalyst per hour). Physi-cal constraints on productivity are shown bydashed lines and are discussed in the text.

some examples in Table 3. While thebiochemical process industry is charac-terized primarily by the use of growingcells in aqueous systems, some recentwork has been done with growing immo-bilized cells. The apparent contradictionbetween growth and immobilization isresolved when the latter is considered asa form of cell recycle. Immobilization byentrapment in a gel or adsorption on asupport provides a form of internal cellrecycle. Alternatively, cell recycle canbe achieved by devices external to thebioreactor, such as centrifugal separa-tors, membrane devices, or settlingtanks, which collect concentrated cellsfrom a fermentation effluent and returnthem to the fermentor. It is more com-mon today, however, to use growingcells in batch and semicontinuous or fed-batch fermentors, but this will not neces-sarily be the case in the future.

Approaches in Bioreactor Design

A bioreactor is a biocatalyst in a con-tainer. Its engineering design is based onminimizing process constraints such asheat and mass transfer and allowing opti-mal control of biocatalytic activity whileminimizing total process cost. Since amajor cost in most biological processesis that of the raw materials, an importantobjective in bioreactor design is to maxi-mize the conversion yield of startingmaterials to final product, Yp,, (grams ofproduct divided by grams of substrate),and this reflects the catalyst selectivity.

Selectivity is important not only formaximizing conversion yield but also forminimizing by-product formation, whichincreases problems of product recovery.While the maximum conversion yield isconstrained by thermodynamics (15), theactual yield is constrained by bioconver-sion mechanisms and the requirementfor biocatalyst synthesis. The objectivefunctiofi to be maximized is Rp/xg theratio of product per unit of biocatalyst,

fc~~~~~~~RpIX=- Yp/s 0 S,(t) dt (1)

where Sa(t) is the specific activity as afunction of time for the cell or enzymecatalyst (grams of substrate reacted di-vided by grams of catalyst per unit time)and t, is the effective catalyst life (oftenexpressed as a half-life). Values of RPIXcan vary greatly for different processes.For enzyme-catalyzed reactions, such ashigh-fructose corn syrup (HFCS) pro-duction with immobilized glucose isom-erase, this ratio can be 2 2500 g pergram of enzyme preparation. For com-modity products, such as single-cell pro-tein and ethanol, Rp/x varies from 1 to 10g of product per gram of cells, respec-tively. For specialty products, such aspenicillin, values of about I g per gram ofcells are typically observed.

In the case of enzyme-catalyzed reac-tions, improvements in Rp,X are made byusing enzymes with high Sa, increasingthe enzyme stability (t,), and increasingthe yield of active enzyme per unit massof organism used for enzyme production.In the biosynthesis offermentation prod-ucts, RP,X is improved by controlling thecell's use of raw materials (primarily thecarbon source) for the synthesis of cellmass, the desired product, by-products,and cell maintenance in order to maxi-mize Yp,, (9). This can be done genetical-ly, environmentally, or by reusing thebiocatalyst as many times as possible asin cell recycle or immobilization sys-tems. Solutions to the problem of maxi-mizing RP,X involve a combination ofbiological and chemical engineering ap-proaches to increasing specific activity,selectivity, and effective catalyst life.As shown in Fig. 2, bioreactors are

designed and operated to achieve a maxi-mum volumetric productivity. The im-portance of productivity is reflected inthe need to minimize capital investment;even if a biological process has attractiveoverall economics, it is difficult toachieve a high return on investment ifthe capital investment per unit weight ofproduct is exceptionally high. Volumet-ric productivity is improved by increas-ing the specific activity of the enzyme orspecific productivity of the growing cell

SCIENCE, VOL. 219

and increasing the concentration of en-zyme or cells (or both) in the reactor.The use of immobilized enzymes makesit possible to achieve high catalyst con-centrations in the reactor and to reusethe catalyst many times. With solubleenzymes the catalyst capacity of thereactor is often limited by the solubilityof the protein catalyst and it is difficult torecover the catalyst after use. With im-mobilized enzymes it is possible to ex-ceed this solubility limit since the cata-lyst is coupled to a solid support matrix.Then catalyst loading becomes limitedby the specific activity of the immobi-lized enzyme preparation and the loadingcapacity of the immobilizing matrix. Im-mobilized enzyme systems such aspacked columns may have protein con-centrations of 50 g/liter (16). The specificactivity of enzymes can vary (17) from 1to 10,000 units per milligram of protein(where I unit is I micromole of substratereacted per minute under optimal condi-tions). With highly active enzyme prepa-rations, one could expect to use biocatal-ysis to achieve product formation ratesgreater than 100 moles per liter per hour.This is difficult to achieve, however,because of engineering limitations andphysical constraints.The volumetric productivity in fer-

mentation or immobilized whole cell sys-tems is limited by cell density (recall thatthe moisture content of cells is typically80 percent) and by the concentration of arate-limiting enzyme in a metabolic path-way. Bioreactors containing immobi-lized Escherichia coli and the enzymeaspartase can synthesize 3 moles of as-partic acid per liter per hour in 98 per-cent yield from 1.OM ammonium fuma-rate (18). Immobilized cell reactors con-taining Saccharomyces cerevisiae or Zy-momonas mobilis can produce ethanol atrates greater than 1.5 moles per liter perhour (19). By comparison, with fermen-tations for antibiotics such as penicillin

Fig. 3. Decision treeshowing the optionsin selecting biocata-lysts and bioreactorconfigurations.

Biocatalysis

Cells Enzymes

Nongrowing Growing Soluble Immobilized

Suspension Immobilized Batch Continuous

Matrix Membrane Adsorption Entrapped Cross- Chemretention linked attach

Gel Adsorption Chemicalattachment

BBatch ContinuousFermentation

Batch Semi- Continuousbatch

the volumetric productivity is less than Imillimole per liter per hour (20), and thechemical industry often achieves I to 10moles of product per liter per hour.Once a product is formed, it must be

isolated and purified. The cost of recov-ery is, in general, proportional to theamount of water that must be processed.Thus it is important to maximize theconcentration of product leaving thebioreactor in order to minimize both thecapital investment and the operating costof the recovery system. Antibiotics, en-zymes, and, more recently, proteins pro-duced by genetically engineered cells areexamples of recovery cost-intensiveproducts because they are often formedin dilute solution or coproduced withsimilar materials from which they mustbe purified. Recovery of highly purifiedintracellular protein from bacteria fortherapeutic use is an example (21).

In a continuous process, the relationbetween product concentration, P, andoperating conditions is

p SaVXF

where V is the volume of the reactor andF is the flow rate through the reactor.Product concentration can be maximizedby decreasing the flow rate or increasingthe activity and concentration of thecatalyst. In the traditional fermentation

Immobilized

Recycle Gel Adsorptionsystem

Plug CSTRflow

industry-exemplified by antibiotic,amino acid, and organic acid produc-tion-batch and fed-batch processes arecommonly used, in part because theyallow high product concentrations to ac-cumulate by letting the flow rate ap-proach zero. Continuous processes oftenhave higher volumetric productivitiesthan batch processes, but the productconcentration is lower. This limitationcan be overcome by using high concen-trations of active catalyst; examples arethe use of immobilized enzymes in theconversion of penicillin to 6-aminopeni-cillanic acid, the conversion of glucoseto HFCS, and the use of cell recycle inthe conversion of sugar to ethanol.There are other criteria to be consid-

ered in bioreactor design. Product quali-ty must be maximized, and this is oftenachieved by increasing process repro-ducibility and minimizing by-productformation. Energy costs for air compres-sion, mixing, temperature control, andsteam sterilization have become increas-ingly important. The major energy costassociated with a bioreactor is that foraeration and mixing. As a consequence,considerable effort over the past 30 yearshas gone into the design of mass transfersystems for bioreactors that can achievehigh mass transfer rates at good energyefficiency. In most biochemical process-es the labor cost is relatively low, less

Table 3. Commercial immobilized enzyme reactors (14).Immo- Reactor Operating Start-

Enzyme Product bilizing type mode Company ingmethod tpmoedate

Aminoacylase L-Amino acids Adsorbed Packed bed Continuous Tanabe Seiyaku 1969Aspartase* Aspartate Entrapped Packed bed Continuous Tanabe Seiyaku 1973Fumarase* Fumarate Entrapped Packed bed Continuous Tanabe Seiyaku 1974Glucose isomerase HFCSt Adsorbed Packed bed Continuous Clinton Corn 1972

Covalent Stirred tank Batch Novo 1974Packed bed Continuous Novo 1975

Lactase Lactase-free milk Entrapped Stirred tank Batch Snamprogetti 1977Penicillin acylase 6-Amino penicillanic Adsorbed Stirred tank Batch Squibb 1966

acid Covalent Stirred tank Batch Astra 1973Covalent Stirred tank Batch Beecham 1974Entrapped Packed bed Continuous Snamprogetti 1975

Steroid dehydrogenase* Heat-treated Squibb 1964*Immobilized cells. tHigh-fructose corn syrup.

nicalhment

II FEBRUARY 1983 731

than 5 percent of the manufacturing cost.This is achieved by designing equipmentthat is simple to operate, has minimalmaintenance requirements, and has on-line process control. It is a characteristicof many biological processes that theequipment, particularly the bioreactor, isversatile and can be used for many dif-ferent products. This is generally nottrue in the chemical industry, but isimportant in biochemical process devel-opment since often a single plant may beused for several different products.

Limitations in BioreactorOperation and DesignWhile bioreactors can be loaded with

sufficient catalyst to achieve very highrates of product formation, there arephysical constraints in the design andoperation of the reactors that preventone from realizing these rates. Catalystconcentration and specific activity canbe improved by using high cell or en-zyme concentrations and catalysts withhigh specific activity. Ultimately, how-ever, the rate is limited by the problem ofmass or heat transfer or both. The prob-lem of mass transfer is particularly im-portant in processes that require oxygenbut also occurs when there is a water-insoluble nutrient requirement. Equation3 illustrates the supply and demand re-quirements for oxygen by a growing pop-ulation of microorganisms:

-

= kLa (C* - CL) (3)Y02

where ,u is the specific growth rate, X thecell concentration, and Yo2 the oxygenyield for growth and product formation.The rate of oxygen transfer is limited bythe liquid film around the air bubble (9).As a consequence, oxygen transfer rates

are improved by increasing the bubblesurface area, a; maximizing the liquidfilm mass transfer coefficient, kL; andmaximizing the driving force, C* - CL,where C* and CL are the equilibriumconcentration of oxygen in the bubbleand the concentration of dissolved oxy-gen, respectively. Many fermentor con-figurations have been proposed to im-prove the rate and efficiency of masstransfer (22). The demand term is con-trolled by the metabolic rate of themicroorganisms as characterized by thespecific growth rate, ,u; the density ofactively growing cells, characterized byX; and the efficiency with which oxygenis coupled to energy generation and bio-synthesis, as given by the value of YO2.In most aerobic bioreactors oxygen

732

transfer is the rate-limiting step durpart or all of the process, as seensolving Eq. 3 for X:

X = Y0 kLa (C* - CL)

In addition to improvements in equment design, it is possible to use proccontrol strategies to cope with the 0:gen transfer limitation. By reducingduring periods of high oxygen demandis possible to operate at a higher 4density and at the maximum producti'vas determined by mass transfer rates,to continue to accumulate and prodiproduct in the reactor for longer perilof time. This can be done by reducingtemperature of the process or by contiling metabolism by restricting the flomsome essential nutrient such as carbinitrogen, or phosphate. The impositof this restriction is important and allcCL to be maintained above a critivalue to avoid oxygen starvation ofculture, which often leads to decrea,product formation and conversion yieAn important aspect of bioreactor desis integration of the design with opeling strategy, and this has been a madriving force for investigations intouse of computer control in fermentatprocesses. In the application of proccontrol, the fermentation industry 1many years behind the chemical procindustry (23). This is because it is ofdifficult or impossible to measureprimary objective parameter-prodformation-in a bioreactor and becaithe use of this information to controlphysiology of the culture and achi4process optimization is poorly undstood. In addition, biological procesare generally operated in a batch or fbatch mode, whereas in the chemiindustry continuous processes are ncommon. These factors have slowedimplementation of computer controlbiological processes; however, procimprovements through modificationmicroorganisms and better controllikely to provide major improvement'bioreactor operation.

Biological reactions are generally cried out at relatively low temperatui200 to 65C. As a consequence,removal is often a problem, particulEfor large bioreactors. Heat productincreases linearly with volume; hower, on scale-up, the surface-to-voluratio increases by the two-thirds pomSince heat is removed in proportiorthe available surface area, there iimaximum reactor size that can be cocwithout mechanical refrigeration,thus heat removal often limits the sc

ing up of large bioreactors. Improving theby conversion efficiency for raw materials

to products generally reduces the heatload (24); thus the problem of heat trans-

(4) fer limitation can be attacked by biologi-cal as well as mechanical means.

lip-essxy- Trends and Future Directions in

Bioreactor Development1, itcell It will continue to be important tolity develop catalysts with high specific ac-but tivity and selectivity. This mnay beace achieved at the molecular level by usingads genetic engineering techniques to alterthe the primary structure of enzyme cata-rol- lysts. An alternative is the use of "artifi-' of cial enzymes" as recently described byon, Breslow (25). This problem will also beion attacked with better methods of enzyme)ws immobilization, which will lead to higherical retention of activity and longer lifetimes.the For fermentation or whole cell systemssed with complex regulated and coordinated-ld. pathways, increases in specific activity,ign can be achieved by applying recombi-rat- nant DNA techniques to increase theLjor production of rate-limiting enzymes andthe alter metabolic regulatory schemes inion order to maximize the flow of raw mate-ess rials to desired products and away fromags undesired by-products. Such efforts,ess coupled with improved process controlten of fermentations and improved stabilitythe of immobilized cell systems, will resultuct in substantially lower catalyst costs.use However, bioreactor performance isthe usually ultimately limited by heat andeve mass transfer capabilities. As a conse-ler- quence, there is a need to improve meth-,ses ods and equipment for heat and massed- transfer in biological reactors. As theical ability to increase catalyst loading in theore reactor improves, the demands for pro-the cess technology to remove process heatin and transfer nutrients through diffusion

ess barriers will increase.of The processing of large volumes of

are water after product removal is expen-s in sive. To minimize this cost, especially

with commodity products such as etha-:ar- nol and single-cell protein, water recycleres, is being employed. As a means of mini-ieat mizing energy use, some processes areLrly being optimized by minimizing waterion use, and this trend is likely to increase.,ev- In ain effort to further minimize capitalime investment in biotechnological process-,er. es, there will be increased emphasis onto continuous processes. A major deterrent

s a to the use of continuous bioreactors isAled the problem of low product concentra-and tion. This problem can be minimized byale- using catalysts with high specific aFtivi-

SCIENCE, VOL. 219

ty. Keeping in mind the limits in carryingout a fermentation, one can envisionimmobilized whole cell systems provid-ing an opportunity to achieve high pro-ductivities and hence high product con-centrations. The unit operations in abiological process have a considerableeffect on each other, and it is axiomaticthat whatever you do upstream will havean impact on downstream processing.For this reason, it is important to careful-ly integrate bioreactors and all subse-quent product recovery operations. Bio-technology holds great promise for theefficient production of pharmaceutical,chemical, and agricultural products. Thecontinued success of biotechnology de-pends significantly on the developmentof bioreactors, which represent the focalpoint for interaction between the lifescientist and the process engineer.

References and Notes1. E. J. Lyons, AIChE Symp. Ser. 100, 31 (1970).2. D. Perlman, Am. Soc. Microbiol. News 43, 83

(1978).3. R. Langer, R. J. Linhardt, S. Hoffberg, A. K.

Larsen, C. L. Cooney, D. Tapper, M. Klein,Science 217, 261 (1982).

4. R. W. Swartz, Annu. Rep. Ferment. Processes3, 75 (1979).

5. Mitre Corporation, Comparative Economic As-sessment ofEthanolfrom Biomass (NTIS reportHCP/ET-2854, National Technical InformationService, Springfield, Va., 1978).

6. T. Murphy and S. Walsh, paper presented at theAmerican Chemical Society meeting, KansasCity, Mo., September 1982.

7. C. L. Cooney et al., Dev. Ind. Microbiol. 18,255 (1977).

8. J. F. Martin and A. L. Demain, Microbiol. Rev.44, 230 (1980).

9. D. I. C. Wang, C. L. Cooney, A. L. Demain, P.Dunnill, A. E. Humphrey, M. D. Lilly, Fermen-tation and Enzyme Technology (Wiley, NewYork, 1979).

10. B. Atkinson, Biochemical Reactors (Pion, Lon-don, 1974); S. Aiba, A. E. Humphrey, N. F.Millis, Biochemical Engineering (AcademicPress, New York, ed. 2, 1973).

11. J. G. Stramondo et al., AIChE Symp. Rev. 74(No. 172), 1 (1978).

12. R. A. Messing, Immobilized Enzymes for Indus-

trial Reactors (Academic Press, New York,1975).

13. I. Chibata, Immobilized Enzymes (Wiley, NewYork, 1978); 0. R. Zaborsky, Immobilized En-zymes (CRC Press, Boca Raton, Fla., 1973); K.Morbach, Methods Enzymol. 44 (1976).

14. M. D. Lilly, DECHEMA-Monogr. 82, 1693(1978).

15. V. K. Eroshin and I. G. Minkevich, Biotechnol.Bioeng. 24, 2263 (1982).

16. K. Buchholz, DECHEMA-Monogr. 84, 1724(1979).

17. Th. E. Barman, Enzyme Handbook (Springer-Verlag, New York, 1969), vols. I and 2.

18. M. Fusee, W. E. Swann, G. J. Calton, Appl.Environ. Microbiol. 42, 672 (1981).

19. R. R. Bland et al., Biotechnol. Lett. 4, 323(1982).

20. C. L. Cooney, Process Biochem. 14 (No. 5), 13(1979).

21. D. V. Goedell et al., Nature (London) 287, 411(1980); T. Staehelin, D. S. Hobbs, H. Kung,C.-Y. Lai, S. Retska, J. Biol. Chem. 256, 9750(1981).

22. M. Moo-Young and H. Blanch, Adv. Biochem.Eng. 19, 1 (1981).

23. C. L. Cooney, Biotechnol. Bioeng. Symp. 9, 1(1979).24. _ , in Microbiological Growth on C,-Com-

pounds, (Society of Fermentation Technology,Japan, 1975), p. 183.

25. R. Breslow, Science 218, 532 (1982).

Production of Feedstock ChemicalsT. K. Ng, R. M. Busche, C. C. McDonald, R. W. F. Hardy

Photosynthetic products accumulatedover eons as natural gas, petroleum,coal, and related carbonaceous materialshave provided an immense resource forthe development of our highly industrial-

chemical industry from a fosbase to a renewable carbon tlikely to be rapid or easy asone generation. However, it isfor future generations that we

Summary. Renewable raw materials may be converted by biologicalfeedstocks for the chemical industry. Glucose from cornstarch is the currena substrate, although advances may enable the use of less expensive lignmaterals. The production of oxychemicals and their derivatives fromresources could amount to about 100 billion pounds annually, or about halfproduction of organic chemicals. Ethanol produced by fermentation iscompetitive with industral ethanol produced from fossil fuel. Biological routoxychemicals exist and are expected to be important in the future. Sevelrecovery methods may be used, but new energy-conserving methods willto make the engineering-biology combinations economical.

ized and energy-intensive civilization.However, we now recognize that thisreservoir of fossil carbon is finite and isalready limiting industrial growth. Wemust develop ways to rely more heavilyon products of current photosynthesisfor the basic feedstocks required by thechemical industry. Conversion of the11 FEBRUARY 1983

tion and support to initiating,ing out this conversion.There are three major appi

the transformation of biomassfeedstock chemicals: chemicaland biological. Hydrogenatiosis, and fermentation are respamples. In this article we r

current status and potential of fermenta-tive processing of biomass to feedstockchemicals (1-3). We discuss the nature,supply, and economics of biomass; themicrobial transformation of biomass todesired chemicals by fermentation pro-

/, cesses; and the recovery and purificationof these materials from the dilute aque-ous solutions in which they are pro-duced. We do not deal with fermentationroutes to chemicals for energy use, suchas ethanol. Ethanol may play a usefulrole as an octane enhancer in gasoline,

isil carbon but the amount potentially availablebase is not from biomass would not be a significantviewed by replacement for fossil fuel. Fermentationimportant processes for specialty chemicals suchgive atten- as antibiotics and vitamins (4) are well

established and are not reviewed in thisarticle.

means toIt choice asocellulosic Biomass Sourcesrenewableof the U.S. Biomass consists of collectible plant-now cost- derived materials that are abundant, in-es to other expensive, and potentially convertible toral product feedstock chemicals by fermentationbe needed processes. Biomass is found as starch in

corn, wheat, potatoes, cassava, the sagopalm, and other agricultural productsand as monomeric sugars or soluble

and carry- oligomers in corn syrup, molasses, rawsugar juice, sulfite waste liquors, and so

roaches to on. It also occurs as lignocellulose in thes to useful1, physical,n, pyroly-iective ex-eview the

T. K. Ng is a research biologist, R. M. Buscheplanning consultant, C. C. McDonald research man-ager, and R. W.-F. Hardy director of life sciences inthe Central Research and Development Department,E. I. du Pont de Nemours & Co., Wilmington,Delaware 19898.

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