jaycor 1985 - immobilized algae systems

8/4/2019 Jaycor 1985 - Immobilized Algae Systems

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SERI/STR-231-2798UC Category: 61cDE85016890

Review and Evaluation ofImmobilized Algae Systems forthe Production of Fuels fromMicroalgaeA Final Subcontract Report

JAYCORAlexandria, Virginia

November 1985

Prepared under Subcontract No. XK-4-04124-01SERI Technical Monitor: Robins McintoshSolar Energy Research InstituteA Division of Midwest Research Institute1617 Cole BoulevardGolden, Colorado 80401-3393Prepared for theU.S. Department of EnergyContract No. DE-AC02-83CH10093


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NOTICEThis report was prepared as an account of work sponsored by the United States Government. Neither theUnited States nor the United States Department of Energy, nor any of their employees. nor any of theircontractors, subcontractors. or their employees, makes any warranty, expressed or implied, or assumes anylegal liability or responsibility for the accuracy, completeness or usefulness of any information, apparatus,product or process disclosed, or represents that its use would not infringe privately owned rights.

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Price: Microfiche A01Printed Copy AOSCodes are used for pricing all publications. The code is determined by the number of pages in the publication.Information pertaining to the pricing codes can be found in the current issue of the following publications,which are generally available in most libraries: Energy Research Abstracts, (ERA); Government ReportsAnnouncements and Index (GRA and I); Scientific and Technical Abstract Reports (STAR),'and publication,NTIS-PR-360 available from NTIS at the above address.


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FOREWORD

This report reviews and evaluates the use of immobilized algae systems. This work wassupported through SERI subcontract XK-4-04124-01 as part of the Aquatic SpeciesProgram. The SERI Aquatic Species Program is funded through the Biomass EnergyTechnology Division of the U.S. Department of Energy.

Robins P. McIntoshAqua tic Species Program

Approved forSolar Energy Research Institute

Eric H. Dunlop, Di-Peetor ISolar Fuels Research Di sion

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SUMMARY

In this paper the use 0 f immobiliza tion systems in research for industrial application isreviewed. Algal immobilization systems, while being examined in the laboratory, havenot, as yet, reached the commercial stage. Therefore, the examination ofimmobilization for energy production is treated as theoretical exercise.The possibility of immobilizing algal enzymes for lipid production was initiallyexamined. This would require the growth of sufficient algae to develop sufficiently largeimmobilization systems to produce the base quantities of lipid required for energyproduction. The economics of growing algae, extracting and immobilizing the multi-enzymes involved in lipid production was rejected as being prohibitively expensive for theproduction of a product as cheaply valued as fuel.The next option considered and rejected was the idea of immobilizing whole algal cells ina traditional immobilized system. In order for whole cells to be effective lipid producersin an immobil ized system, they must excrete the lipid through the cell wall (and throughthe immobilization matrix where they are encapsulated). At the present , no algae havebeen i sola ted or selected which produce lipids at high photosynthetic efficiencies andhigh volume and at the same time excrete lipid. That is not to say that this combinationof traits could no t be developed in the future with genetic manipula tion and a betterunderstanding of lipid production triggers and transport.Immobilization could also be used as part of the biomass production and/or harvestingsystem. While this analysis would indicate that the economics of such a system (at leastin this scheme) are prohibitive, it is important to point out that the hypothetical systemdid not take into account any improvement in cost or materials. There may well be arole for cell immobilization either for harvesting or production.One of the major advantages of going to an immobilized system is to increase culturedensities. Although one cannot ultimately reduce the land area required to capture acertain amount of solar energy (there is no way to increase the flux of energy per unitarea), thi s energy can, through a variety of mechanisms (solar collectors, etc.) be focusedon a small volume of growing algae. Reduction in the volume of growing algaeconsequently dec reases the required water - a key factor in growing algae in thesouthwest desert. Immobilization technology may be part of a system of reduced watervolume and high light intensity.

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CHAPTER I .

CHAPTER I I .

CHAPTER III .

CHAPTER IV.

CHAPTER V.

CHAPTER VI.

INDEX

Concept of Immobilized Cell systems l

Use of Immobilized Cell Reactors in Biotechnology 9

Industr ia l Applications 49

Economic Analysis 56

Conclusions 66

References 68


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CHAPTER IConcept of Immobilized Cell Systems

The purpose of this paper is to review and evaluate the use of immobi-lized algae systems. I t is our finding that commercial immobilized algaesystems are not in operation at this time but with research could certainlybecome so. The use of immobilized algae will depand on, as in a ll commercialsystems, the economic value of the product. In this paper we have reviewedthe technical feasibil i ty of immobilization as i t applies to algae. Finally,we investigated the economics of possible immobilized algal systems that wouldproduce l iq uid fue ls .

A major development in the pharmaceutical and chemical processing indust r ies in the past d ~ c a d e has been the introduction of large sc ale immobilizedenzyme and immobilized cell reactor systems. Industrial use of such immobi-lized cel l bioreactors suggests the possibi l i ty that this approach also couldhave applications for biotechnical proces se s involving algae. This paperexamines the potential for using immobilized cel l technology for large scaleproduct ion of fuels from microalgae.

Immobilization techniques involve the physical confinement or local i zation of cells (or enzymes) in a particular region of space with retention ofcatalyt ic activity (and viabil i ty in some cases) in order to permit repeatedor continuous use. The idea is to convert the reaction from a homogeneous( i .e . , freely suspended cells or dissolved enzymes) to a heterogeneouscondi t ion. This is done by developing a cat al yt ic al ly a ct ive solid phase ofmacroscopic dimensions to be placed in contact with a catalyst-free solutionof reactants.

Two important characteristics of an immobilized biocatalyst are: (a ) i tslevel of catalyt ic activi ty, and (b) i t s s tab i l i ty in use and during storage.Stabil i ty generally is indicated in terms of half- l i fe , th e time for a 50%decrease in activi ty. Conditions for immobilizing cells with good retentionof enzymatic function and viabil i ty have been devised, and in many cases, the

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stabi l i ty of th e immobilized cells is considerably greater than fo r freecells. Half-lives up to 12 months have been achieved in pharmaceutical andchemical bacterial processes.

Various methods fo r immobilizing cells are available and the appropriateprocedure to use fo r any particular cell and process must be determined empirical ly; what is fou.nd su i table in one case may be unsuitable fo r another.Immobilization methods include:

(I) Physical entrapment of cells in a (porous) matrix, such as agarose,alginate, carageenan, polyacrylamide, polyurethane, etc.(2) Enclosure or separation of the cells from the reaction mixture by encapsulation or thin films (dialysis membranes, etc .) .(3) Adsorpt ion onto the surface of an insoluble material. This may involve

ionic or non-ionic interaction of cells with some substratum, such assynthetic ion exchange resi ns, dext ran or porous glass beads, t i tani urnoxide particles , wood chips, etc.(4) Covalent bonding between the cell and a support surface, or direct covalen t c rossl inking of the cells without any carrier material.

A common practice in preparing immobilized cells is to f i rs t grow thecells in a fermenter under suitable condi t ions, harvest the cells by centr i -fugation and then immobilize them at a very high cell densi ty . By use ofpre-grown cells suspended (entrapped) in epoxy resin, cell densities of0.7-1.0 g wet cells/ml of matrix can be obtained. Alternatively, a few cellsmay be entrapped in a matrix and then allowed to grow after they are immobi-l ized. Another procedure is to inoculate a culture vessel containing l iquidg ro wth medium and suitable support materials or carr iers (e.g. , porous glassbeads, diatomaceous earth, etc.) and allow the cells to colonize the carriersduring growth. Recently, there has been interest in immobilizing living cellsin the hollow fibers of ultradialysis apparatus.

Advantages of Immobilized Cell Reactors

Immobilized cell reactors are used in two different types of s i tu-ations. One of these is the use of immobilized whole cells instead of animmobilized purified enzyme to catalyze a single-step enzymatic reaction. Inthis case, the whole cell is simply a convenient and less expensive source of

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(crude) enzyme. The second type of process is th e substitution of immobilizedcells for a conventional fermentation process (batch or contin uous flow) thatuses free cells . These processes often involve mUlti-step reactions.

Immobilized cells used as enzymes have th e same advantages over free(soluble) enzymes as do immobilized purified enzymes. These include:(1) Enhanced stabil i ty of the enzyme;(2) Ability to recover and re-use th e enzyme in batch operations;(3) Ability to use the enzyme for continuous flow operation;(4) Use of sma ll s imple reactor design;(5) Product can be obta ined in high yield, which simplifies purification.

Use of the whole cel l instead of purified enzyme is particularly indicated when th e enzyme involved is intracel lular , or when the extracted enzymeis unstable during and after immobilization. Often enzymes retain betteractivity when present in the whole cel l . Whole cells may be used as an enzymesou rce provided th e substrates and products are not high molecula r weightcompounds (cell wall and cell membrane are bar r iers to high molecular weightsubstances), and provided the cell does not have interfering enzyme reactions.

When compared to convent iona l fermentat ion processes using free cel ls ,the immobilized cell systems have certain advantages. Among these are:

(1) I t is possible to obtain high cell density without high viscosity. Thisaffords' better mass transfer and easier mixing.(2) Immobilized cells often show greater stabi l i ty than free cel ls.(3 ) With immobilized cells biomass can be retainedoperation. In conventional con tinuous fermenter swith the out flowing spent medium.

in continuous flowthe cells are lost

(4) Immobilized cells can be used in a continuous flow system undernon-growth conditions. In a conventional continuous fermenternon-growing cultures are washed out. Fermentation of some productsreq uire s condition s different from those needed fo r growth, sonon-growing cells are needed. Use of non-growth conditions simplifiesth e purification of products by eliminating growth substrates from thereaction mixture.

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Reactor Types

One advantage of cell immobilization is that i t allows the selection ofa number of different reactor configurations. These have different limitat ions and advantages for particular types of processes. The basic types ofreactors used for immobilized cell processes inc lude the following (see Figure1 ) :

(1) Packed bed reactors;(2) Stirred tank reactors: batch and continuous;(3) Fluidized bed reactors;(4) Miscellaneous reactors: hollow fiber reactors; tubular reactors;dialysis fermenters: cell-based electrodes.

The packed bed reactor has the advantages of low construction costs andsimple operation. In one simple form th e immobilized cells (cast as beads orchips) are placed in a length of glass tubing and confined by a mesh screen ateither end. The substrate solution is pumped through the bed, often in anupward flow to counteract compression by gravity. Such an arrangement allowshigh mass transfer rates under conditions that approach plug-flow. Forreactions that are not substrate-inhibited the packed bed reactor gi ves highreaction rates, being particularly advantageous fo r product-inhibitedreactions. Packed bed reactions are prone to clogging with particulates orgrowth-associated product s of cells (slime, capsules) and to compressioneffects (especially with non-rigid gels and small particles) . Additionalproblems include difficul t ies of providing adequate oxygen and nutrients tot he dense ly packed biomass, and eff icient CO 2 removal.

In a st irred tank reactor the immobilized cells (i n bead form, etc.) areagitated to obtain a well-mixed condition. I t is easier to obtain adequateaeration and pH control in such well-mixed vessels, bu t the shear fo rc es fromth e agitations tend to disintegrate th e immobilized cell preparations.Batch-type st irred tank reactors are seldom used for immobilized cells, andth e continuous flow stir red tank reactors (CFSTR) also have limited uses. Inth e CFSTR the level of substrate remains relatively low, so this type of

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reactor is advantageous for a substrate-inhibited reaction. Product levels ina CFSTR are relatively high, so i t is less suitable for product-inhibitedreactions than a packed bed reactor.

A fluidized bed reactor uses an upward flow of liquid and a ir to keepth e immobilized cells (beads, chips, e t c , ) from sett l ing out, but the shearforces are less than with a stir red tank. A flared shape is employed to causethe particles to lose velocity as the rise to th e top, thus preventingwash-out. This type of reactor is well suited fo r providing aeration and gasmixing fo r pH and temperature control, and for the use of high flow rates.Fluidized bed reactors are not particularly susceptible to plugging because ofcompression, use of viscous or particulate substrates or formation of capsularslimes.

CONSTRAINTS ON APPLICATION

Mechanical and Hydraulic Factors

In packed bed reactors there is a problem of compression of th e immobi-lized cells, even i f a relatively rigid matrix .i s used. In a CFSTR theshearing by the impellar blades tends to disintegrate the immobilized cellpreparation. Both these effects are minimized in a fluidized bed reactor bu tpreci se con trol of flow rates is needed to balance the density of the immobi-lized cells. Such control is complicated by poss ib le den sity changes overtime due to gas bubble formation, l ipid accumulation or other alterations.

Maintenance of Viability and/or Catalytic Activity

Some immobilizing techniques tend to cause loss of viabili ty . This isparticularly true of methods involving covalent bonding of cells to a supportor to other cel ls . Chemicals used for covalent attachment or for polymerizinga matrix often are strongly inhibitory. If viabili ty or half-l i fe areunsat isfactory, less harsh immobili zation methods may be possible. For somepurposes i t is suffic ient that a particular enzyme or enzyme system remainactive, even i f the cells are no longer viable.

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Gas ExchangeGas exchange is a gene ral problem in a ll aqueous systems due to the

limited solubili ty of oxygen in water. Immobiliza tion o f cells at highconcentrations further complicates the problem by imposing additionaldiffusion barriers along with increased 02 demand. Packed bed anaerobicreactors are particularly i l l -sui ted fo r dealing with the need for gasexchange (providing. 02 and removing CO 2 from respiring cel ls ) . When gasexchange is i nvolved, the fluidized bed reactor is more appropriate.

Nutrient Supply

In a packed bed reactor nutrients may be used completely as the reactionmixture passes through the f i rs t part of th e bed, leaving nothing for the rest .

General Constraints

Certain general problems are encountered with immobilized Li ving cellsthat are not necessarily found with non-viable immobilized cel ls . Theseinclude pH changes, foaming, and problems with microbial con tamina tion . Useof growth promoting nutrients in the reaction mixture requires use of s ter i letechniques to avoid growth of contaminants. Live cells produce pH shifts dueto fermation of acids ( lactic, acetic, et cv ) or ammonia. Aeration of l ivecultures in media containing proteins, peptone, or excreted biosurfactantsleads to foaming whether the cells are free or immobilized.

For various reascns i t is probably advantageous to l imit the size ofimmobilized cell reactors and use several smaller reactors instead of a singlelarger one. The use of several r eactor s arranged in parallel would allow th eshut-down or replacement of one without affecting the others.

Specific Cons traint s for Algae

In addition to the general consideration a lready descr ibed , there aretwo specific requi rements for immobilized algae: th e l ight supply and th e

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CO 2 supply. A fluidized bed reactor should perm it adequate amounts of CO 2to be supplied, but i t is not clear how i t would be possible to supplysuff icient l ight to a dense algal biomass to allow high photosyntheticefficiency. I t appears that those conducting laboratory experimen ts w ithimmobilized algae have been content to demonstrate that photosynthesis occurs ,but there is no indication that i t is occurring at a rate anywhere approachingmaximum efficiency.

Our analysis revealed that providing an immobilized algal system with asuff icient amount of l ight substantia l ly increased the cost of producingl ipids for fuel. Research to increase l ight availabili ty or reduceimmobilization capi tal costs could in longer terms improve immobilized systemeconomics. Alternatively, using immobilization techniques to harvest fUllygrown, l ipid rich cells shows promise as a cost reducing innovation.

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CHAPTER 2Use of Immobilized Cell Reactors in Biotechnology

A. General features of immobil ized cell systems.

1. Development of immobilized cel l systems.

a. Origin of th e concept

Many kinds of b io log ica l r eac to rs are available fo r m ic robia lprocesses, but a l l of these can be considered as using t he mic robi al cells inone of two conditions: free or fixed. In free cell processes, such as thosethat have been used fo r beer and wine f ermentat ions s ince ant iqui ty , the cellsare freely suspended in the reaction mixture. These have been, and s t i l lremain, the most commonly used processes, but during the 19th century a fixedcel l procedure for rapid vinegar production was introduced. This involvedpassing the reaction mixture over a film of microbial cells attached to acarr ie r or matrix. In such ftquick vinegar generators, ft beachwood shavingswere loosely packed in a container and the alcoholic substrate was t r ickleddown over the shavings unt i l a film or coating of Acetobacter developed. Oncethe active bacteria l film had developed, a packed bed reactor of this sor t wasc apab le of con tinuous production of vinegar for an extended period. This sametype of " f i xed film ft reactor , in the form of the s o-c alle d " t r LckLe f i l te r , ftlong has been used for small scale wastewater treatments. In th is process,set t led sewage is sprayed intermit tent ly over a bed of stones. AS th e wastewater percolates down through the bed, th e m ic robia l film that develops on th estones removes nutrients from th e waste stream for conversion to carbondioxide by respirat ion.

The fixed-film reactor I as examplif ied by the quick vinegar generator,is well adapted for continuous-flow operations because the ce l l s remain inplace, separated from the reaction mixture. The advantage of the fixed ce l lfilm is that th e act i ve biocatalyst is retained in the reactor , instead ofbeing sent out with the spent reaction mixture and, therefore, lo s t . I f the

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reaction is incomplete, the stream simply is recycled through the reactoragain. Such reactors are particularly well suited for soluble substrates thatare converted to primary metabolites.

Because of their small size, free cells of microorganisms, particularlybacteria, are diff icul t to separate from the reaction mixture. This meansthat th e cells will be lost when a continuous flow system is used. For thisreason, p roce ss es that use free cells tend to be operated in a batch moderather than continuously, and the reactions are allowed to go to complet ionbefore "harvesting" the product. Such batch processes can be used even forinsoluble substrates, and are well adapted for production of secondary metabo-l i tes and products that are no t directly growth-associated.

Despite th e early introduction of th e idea of fixed film bacterialreactors, these have played almost no part in the development of the modernfermentation industry. Indeed, the modern industrial use of immobilized wholecell reactors has only recently developed directly out of immobilized enzymereactor technology, and not from th e old vinegar generators, etc. During thedevelopment of th e modern fermentation industry, from the days of theacetone-butanol-ethanol process ("ABE" fermentation) to th e a ntibo tic era,there has been an almost exclusi ve reliance on use of free cells in a batchmode. Before 1970 there appears to have been l i t t l e or no use of fixed filmor other immobilized cell systems fo r continuous processes. Probably onereason for this is that batch processes are well suited fo r secondary metabo-l i tes like antibiotics .

b. Immobilized enzyme reactors

Large scale production of microbial enzymes in recent yearshas provided a wide array of purified enzymes for use as catalysts in chemicalprocessing. The high cat alyt ic act iv it y and exquisite specificity of enzymesmake them attractive, but there are some problems with th e use of enzymes asindustrial catalysts. Two major drawbacks to the use of enzymes are theircost and lack of stabil i ty compared to chemical catalysts. If a solubleenzyme is added to a reaction mixture, i t is vi rtually impossible to recover

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th e enzyme fo r re-use, so i t will be lost during the product recovery step.Such batch mode, single-use enzyme processes a re unneces sa rily wasteful,because enzymes are not consumed in the reaction and thus they could be usedrepeatedly. This expensive loss of catalyst could be avoided if the enzymewere present in an easily recoverable insolubilized form. Also, in l abora to ryexperiments i t had been found that immob ilizati on of enzymes sometimesincreased their stabil i ty. In the case of activity losses due to thermaldenaturation or self-aggregation, i t seemed reasonable that a ttachment o f theenzyme to some carrier might improve i t s stabil i ty. Anchoring of an enzyme toa surface, particularly by multiple covalent linkages, would be expected toprevent unfolding and inter-molecular association. These incentives led tothe development of a large number of ways to immobilize enzymes with retentionof cat al yt ic a ct iv it y, and now over 100 methods have been reported (23, 43).The availability of these effective means of enzyme immobilization opened th eway for their use in industrial processes.

Possibly th e f i rs t industrialization of immobilized enzyme technologywas t he int roduct ion in 1969 of the use of an immobilized aminoacylase for theresolution of optical isomers of amino acids (10). Chemical synthesis ofamino acids is cheaper and faster than product ion by alternati ve means (fermentation or isolation from protein hydrolysates), bu t i t results in a racemicmixture of the D and L isomers. Only the L-isomers have biological activity,so if chemical synthesis is used, i t becomes necessary to separate theisomers. Ordinarily, th e L-amino acid is obtained from the DL-mixture byf i rs t converting the amino acid to an acyl derivative, then selectivelyhydrolyzing the L-isomer using an aminoacylase and finally separating th e freeL-amino acid from the acyl deri vati ve of the O-isomer. The success with animmobilized enzyme in this case was soon followed by the application ofimmobilized enzyme technology to other industrial processes, such as production of high fructose syrup using immobilized g lucose isomerase , p roduct ion of6 -aminopen ic il lanic acid from penicillin G using immobilized penicillinamidase, and hydrolysis of lactose in cheese Whey using immobilizedbeta-galactosidase (23). Numerous experimental reports have been publishedand various patents issued fo r other immobilized enzyme processes, bu t i t isuncertain which of these may be in actual commercial use.

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c. Modern immobilized cell reactors.

A further development has been th e construction ofimmobilized enzyme reactors using whole cells as the source of the enzymesinstead of using purified cell-free enzymes (10, 13, 25, 26, 40). The incent ives for this were the same considerations t ha t o ri gi na ll y led to the use ofimmobilized enzymes instead of free enzymes, namely the lowering of costs andth e improving of enzyme s tabi l i ty . In those situations where competing enzymereactions are not present in the cel l , or can be inactivated if they are present, the whole cell can be immobilized, thereby eliminating th e costsassociated with the extraction and purification of the enzyme. Also, someenzymes prove to be more stable when they are r et ained ins ide the cell insteadof being extracted and purified.

One of the f i r s t uses of such immobilized cell reactors in industry wasthe process for continuous production of L-aspartic acid from ammonium fumarate by cells of Escherichia coli that was introduced by Chibata and hiscolleagues in 1973 (10). For this purpose whole cells of ! . coli wereentrapped in a polyacrylamide gel and were found to retain good aspartaseactivity . An immobilized cell reactor prepared using the polyacrylamideentrapped cells were eff icient and produced aspartic acid from 1 M fumarate ina 95% yield.

A similar industrial process using immobilized cells then was devisedfor the production of L-malic acid from fumaric acid using th e fumarasereaction of Brevibacter ium ammoniagenes (10). In this case, the cells wereentrapped in polyacrylamide and then treated with bile to increase activityand to suppress an undes irable s id e reaction leading to the formation ofsuccinic acid as a by-product. The half-l i fe of this reactor was 52 days andi t produced L-malic acid from fumaric acid in a 70% yield. A third processwas SUbsequently developed and put into production to prepare L-a lanine fromL-aspartic acid using th e cells of Pseudomonas dacunhae as a crude aspartatedecarboxylase. All three of these industrial processes use an immobilizedenzyme reactor in which cells are serving simply as crude , non-v iable enzymesources.

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2. Types of reactors.

A large number of different configuratioins have been described forreactors that use immobilized cells (39). These can be grouped into thefollowing categories: packed bed reactors; batch-type and continuous-flowstir red tank reactors fluidized bed reactors biocatalytic membranes; mem-brane barrier systems. There are certain advantages and certain inherentproblems with each of these, and a knowledge of these factors can assist indeciding which design is more suitable for a particular application. Of thevarious reactor types available only two of these appear to have been used inindustrial processes (12): these are the st irred tank reactor (batchprocesses) and the packed bed reactor (see Table 1).

a. Packed bed reactors

This is probably th e most commonly used immobilized cellreactor because of i t s simplicity and ease of operation. The previouslydescribed immobilized cel l process of Chibata and co-workers (10) for production of L-aspa rt ic acid from fumarate employed a packed-bed reactor.

In most cases th e packed bed reactor consists simply of a section oflarge tubing or pipe (often glass) with perforated retaining plates at eachend. The cells are fab rica te d in to a form suitable for packing into thetube. Typ ical ly the cells are p rese nt in or on a matrix in the form of smallbeads, chips, blocks, threads, Sheets, pellets or discs. A reaction mixturewith the appropriate pH, substrate concentrations, etc. is prepared, passedthrough the bed of biocatalyst, and th e product is recovered from th e effluentstream.

This type of reactor involves relat ively simple and low costconstruction consisting only of some piping and pumps. Charging the reactorwith the biocatalyst and operation of the reactor is easy, and once i t is setup there is l i t t le labor cost fo r ope ra ti ng the reactor. Such a system can beautomated readily, and because i t is usually operated in a continuous flowmode, a small reactor can be used to process a large amount of material . For

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Reactor Type

Table 1. Commercial Processes Using ImmobilizedEnzyme and Immobilized Cell Reactors (12, 28).

Process Immobilized Catalyst Company

Stirred tank(batch system)

High fructose syrup Glucose isomerasefrom corn syrup

Novo Industri A/S

Lactose-free milk beta-galactosidase snamprogetti

6-aminopenicillanic penicillin acylaseacid from penicillin G

squibb: Astra:Beecham

Packed bed(continuousflow system)

L-amino acids

L-aspartic acidfrom fumarate

L-malic acidfrom fumarate

High fructose syrup

6-aminopenicillamicacid

aminoacylase

! . .oli cells( fo r aspar tase )

Brevibacteriumammoniagenes cell(for fumarase)

glucose isomerase

pennicillin acrylase

14

Tanabe Seiyaku

Tanabe Seiyaku

Tanabe Seiyaku

Novo: ClintonCorn Processing

Snamprogetti


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effic ient product formation and ease of product recovery i t is desi rable touse high substrate concentrat ions in the reaction mixture. For this reasonthe packed bed reactor is best suited fo r reactions that do not involvesubstrate inhibit ion. The continuous removal of the product from the bedminimizes the effects of product inhibit ion, so this type of reactor is advantageous for product- inh ib ited reactions.

Packed bed reactors are subject to certain mechanical difficul t ies.Particulate or insoluble materials in the reactor mixture tend to clog th ereactor with a tte nden t lo ss of flow and increases in the pressure-drop acrossthe bed. There is always a tendency for the bed to become compacted anddeformed because of the pressures from pumping and gravity, even in theabsence of clogging by particulates. Compacting and deformat ion producesfurther dec reases in flow and increases in pressure. Ideally, th e reactionmixture passes uniformly through a ll portions of the bed to achieve so called"plug flow." Defective flow characterist ics may result from development ofchannels or dead spots in the bed. To minimize compaction, rigid packingmaterial is preferred, and th e reactors are operated with an up-flow of thesolution to off-set th e effects of gravity.

Packed bed reactors are not well suited fo r effective gas exchange.There is limited gas-liquid surface area available, so cell respiration easilyleads to depletion of dissolved oxygen (DO) and accumulation of carbondioxide. When l ive, respiring cells are used, i t is diff icult to maintain anadequate DO except in very small l aboratory sca le reactors and/or at undesirably low cell densities. sim ilarly, there will be a depletion of othernutrients as the reaction mixture passes a packed bed of live cel ls .

b. eaten-type stir red tank reactors.

In batch operations a large vessel equipped with a stir r ingmotor is charged with the reaction mixture, inclUding the immobilized cells orenzyme, and the reaction allowed to go to completion. The con tents a re harvested and th e immobilized catalyst is recovered for re-use. The cost savingsfrom re-use of th e catalyst is the major difference between this and

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convent ional processes using free enzyme. This type of reactor con tinues tobe used in industry (Table 1), possible more because of the availability oft he appara tus than because of i ts intr insic merits.

c. Continuous-flow stir red tank reactor (CSTR).

The CSTR for use with immobilized enzymes or cells is similarto a conventional chemostat or cont inuous fermenter except that the biocatalyst is retained in the vessel instead of being lost with the exit stream.Fresh reaction mixture in continuously fed into a vessel that is equipped witha st irr ing device (turbine and impeller) to keep the contents well mixed. Theinput of fresh medium displaces an equivalent volume of reactor contents, andth e flow rate is adjusted to obtain a maximal concentration of product in theexit stream. Such vessels can be aerated, so respiration is not a majorfactor. These reactors are relatively easy to construct or adapt from convent ional reactors and are not complicated to operate. A major advantage is thatthe immobilized catalyst can be replaced easily without shutting down th eoperation.

In a well-mixed CSTR th e concentrations should be uniform throughout th ereactor and they will be the same as those in the exit stream. Because ofth is , the substrate concentration is maintained at a low level and hence aCSTR is more suitable than a packed-bed reactor fo r reactions that aresubstrate-inhibited. probably on of the major reasons why this type ofreactor has been used only to a lim ited ex tent is that the high shearingforces produced by the st irr ing require the immobilized biocatalyst to beexceptionally robust. This type of reactor is no t particularly well suitedfor immobilized l ive (growing) cells.

d. Fluidized-bed reactors.

These represent a sort of blending of the packed bed and CSTRto obtain the benefits of both types. The c at aly st p ar ti cl es are kept inconstant motion by an upward flow of liquid and/or air . Typical ly the reactorvessel is narrow at th e bottom and flared ou t at the top. In this way the

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upward velocity of the fluid is greater in the lower part of the reactor sothat th e catalyst part ic les are carried upward. Near the top the f lu idvelocity fal ls off due to the flared design, so the part icles tend to fal lback and do no t wash out of th e reactor . By adjust ing the flow rate thepart icles can be kept agitated ("f luidized") , but are no t los t .

Such a f luidized bed reactor does not suffer from the clogging bypart icula tes, no r are the problems with compacting and deformation seen withthe packed bed. This type of reactor is well suited for gas exchange andprovision of oxygen. Also, the mixing is less violent than with a CSTR, sothe biocatalyst part icles are no t s ub je cte d to as much mechanical damage.

One important requirement is that there should be a signif icant densitydifference between the biocatalyst part ic les and the suspending l iquid. Thisis part icularly a problem when hydrogels l ike agar, alginate and carrageenanare used to immobilize the cells , because these materials have densi t iessimilar to that of water. In actual operations the control of a f luidized bedreactor is s omewhat more compl icat ed than for a packed bed or CSTR, becauseflow rates must be adjusted i f (when) the density of the c at al ys t p ar ti cl eschanges. Although, to our knowledge, f luidized bed bioreactors have not beenused as yet in commercial operations, they have features that are part icularlyfa vo rable fo r their use with immobilized growing ce l ls .

e. Other types of reactors.

In some types of reactors the cells are no t actuallyimmobilized, but instead some sort of membrane barrier is used to keep asuspens ion o f free cells localized in a part icular space. Examples are theseare the hollow f iber reactors and th e dialysis membrane fermenter. St i l lanother type of reactor is the flow-through biocatalyst membrane reactor inwhich the reaction mixture is passed directly through a thin film of theimmobilized material .

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(1) Hollow fiber reactors.

Recently, hollow-fiber untradialysis systems have beenadapted to the immobilization of cells or enzymes (20). In these devices thef i l t e r membrane presents a mechanical barrier to the escape of the cells , butallows exchange of substrates and products between a mobile phase and anon-mobile phase. The cells can be entrapped inside the fibers and thereaction mixture passed through the space surrounding th e fibers, orvice-versa. Both methods have been used successfully. Reactors prepared fromsuch dev ices should be useful for immobilized living cells because no deleterious chemicals are needed for the immobilization. One problem is that i t isdifficul t to control the uni formity of cell loading or density in hollow fibersystems. Also, i t would seem that maintaining adequate DO levels wouldpresent problems.

(2) Dialysis fermenters.

Gerhardt (3) has experimented with adaptations of ordinary stir red jar fermenters to accomodate high cel l density and nutrientreplenishment. The fermenter consists of two compartments separated by adialysis membrane. The culture is grown in one compartment and fresh mediumcan be passed through th e other. This system is analogous to th e hollow fiberreactor in that th e cells are mechanically separated from a mobile phase by asemi-permeable membrane which enables exchange of substrates and productswhile retaining the cells in th e fermenter. The hollow fiber system wouldseem to offer g re at er e ff ic ie ncy of exchange because of i t s greater surfacearea, but a dialysis fermenter would allow better aeration and mixing. Thedialysis fermenter has not been widely adopted, possibly because of thefragil i ty of dialysis membranes or other technical difficul t ies.

(3) Enzyme and cell-based electrodes.

The great specificity of enzymes long has been employedfor analytical determinations, but now there is interest in constructingenzyme or c el l- ba sed e le ct rode s that would simplify such measurements.Miniaturization is important fo r such electrodes and actually they are onlyformally related to the la rg e sc ale reactors designed fo r chemical production.

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In one type of configuration a suspension of free cells (o r enzymesolution) is enclosed in th e reaction chamber of the electrode by a semipermeable membrane. When the elect rode is placed in th e sample solution, substrates diffuse through the membrane and are converted to products. Detectionof product fo rmation o r other changes in the reaction chamber as a consequenceof the reaction is made by anothe r dev ice (e .g. , pH electrode, ion selectiveelectrode, dissolved electrode, etc .) .

(4) Flow-through membrane reactors.

These have not been greatly used, bu t may present certainadvantages over other configurations. In this type of reactor the biocatalystis fabricated into a microporous membrane through Which th e reaction mixtureis passed using pressure (o r vacuum). To eliminate clogging by particulatesthe reaction mixture must be passed through pre-fi l ters before entering thebiocatalyst membrane. The advan tages of such a design over th e usual packedbed reactor are considered to be a potential reduction in reactor size, lackof mass transfer limitations, absence of substrate hold up and no channelling. The flow-through membrane may avoid some of the hydraulic problemsassociated with packed bed reactors.

In one report (14) a novel microporous sheet of polyvinyl chloridesil icawas devised as a support for enzyme immobilization. Purified enzymes, such asglucose isomerase, were covalently bonded in the pore of this plasticsupport. The resul ting b ioca ta ly st membrane was mounted in standard membranefi l trat ion holders to serve as a flow-through reactor. When the reactionmixture is pumped through th e membrane, i t passes through th e pores where th esubstrate comes in direct contact with th e bound enzyme through bulk flow notdiffusion through a matrix. Such a flow-through reactor was reported to havesuperior operating characteristics, including half- l i fe , compared to a packedbed reactor using a commercially available immobilized enzyme in bead form.I t seems likely that such a microporous sheet may no t be as useful forimmobilizing cells as for immobilizing enzymes.

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3. Advantages of immobilized cell reactors.

The reason that immobilized cell reactors are attractive fo rindust r ia l purposes is that they use a high concentration of acti ve biocatalyst to acnt eve a rapid and complete convers ion of substrate to product in acontinuous process. The key features for a favorable process are a longhalf-l ife fo r the immobilized cells and high conversion rates without lossesf rom competing side reactions. The overall eff iciency of these immobilizedcel l reactors results in significant cost savings and process improvementsover alternative methods.

Vieth and Venkatsubramanian (41) l i s t a number of potential advantagesof immobilized cel l systems over conventional free-cell fermentation, whichinclUde:

(1) Higher product yields.(2) Ability to use continuous processes instead of batch-type.(3) Operation at high flow ( dil ut ion) r at es without cel l wash-out.( 4 ) Ability to rechargenutrient feed thatnon-growing cells .

(reactivate) the system bywi11 induce growth and switching to areproduction of

(5) Eliminat ion or shortening of lag and growth phases for productsthat are not growth-associated.

(6) High reaction rates due to high cel l density.These authors (41) also present reasons (or condi ti on s) fo r using immobilizedwhole cel ls instead of immobilized purified (cell-free) enzymes:

(1) Eliminates cost of enzyme extraction and purification.(2) Provision of higher operational stabil i ty and resistance toenvironmental perturbations.(3) Retention of high enzyme activity after immobilization.(4) Provides for cofactor regeneration.(5) Allows use of multi-step processes.(6) Retention of structural integr i ty.

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a. Cost reduction.

(1) Continuous operation with high product yield.

To obtain maximum eff iciency, immobilized ce l l reactorsgenerally are operated in a continuous flow mode with a relat ively highsubstrate concentrat ion. This gives high product yields . When immobilizednon-growing cel ls are used, the reactor can be operated under condit ionsoptimal for the desired reaction, and these may be quite di f fe ren t from thoseneeded for growth. This contributes to the high eff iciency of these processes.

This can be contrasted with batch operations in which the reactionmixture is "harvested" afte r complet i on of the p ro cess, and the reactor thenis cleaned and re-charged. The clean-up and "down time" represent substant ia lcosts for a non-producti ve step. When growing up a batch culture of cel l s ,there is a lag period before growth a nd /o r p roduction begins. Such lagperiods also const i tute an unncessary expense that can be eliminated in acontinuous process.

(2 ) Simplified product purif icat ion.

With non-growing immobilized ce l l s a simple reaction mixturecan be used. The absence of extraneous materials means there is less toremove in the purif icat ion steps. The high concentrat ion of product aids inpurif icat ion. I t is usually desirable to recover the product by some simpleprocedure, such as prec ip i ta t ion by changing pH, and recovery of product ismore eff ic ient when i t is present at high concentrations. In general , thecost of product recovery is proportional to the amount of water that must beprocessed (12) , so i t is essent ial to achieve high product concentrations.

(3 ) Catalyst recovery.

P roduct ion o f the microbial cells const i tutes a major expense,so i t is important to use them with maximum eff iciency. Immobilized ce l lreactor systems achieve a hig h d eg ree of efficiency as the ce l ls are retained

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in the reactor for use as catalysts fo r long periods of time in a continuousoperation. In constrast , the production phase is of relatively limiteddura t ion in a batch fe rmente r with free cells , and then the cells are simplydiscarded. In the usual continuous fermenter with free cells , the cells arecont inuously discarded from the fermenter although sometimes they are used ina second stage reactor. In neither of these situations is there a particularly eff icient use of the cells.

(4) Simplified reactor design.

In many cases relatively small and simple reactors can be usedfor immobilized cell processes, and this reduces capital costs. In the simplest si tuat ion a packed bed reactor fo r immobilized cells may consist of asection of glass pipe and some pumps, This can be contrasted with the usualindustr ia l batch fe rmenter constructed of steel to withstand steam pressureand equipped with a large motor to mix the viscous culture.

Some immobilized cell reactors are operated in a non-sterile fashion,using conditions that minimize growth of contaminants (high temperature,unfavorable pH, absence of nutrients). The abili ty to dispense with s ter i l i -zation results in g reat s impl if ic at ion of the apparatus and major costsaVings. This would no t always be possible, particularly where the processinvolves l ive, growing cells that must be supplied with nutrients.

b. Process enhancement.

(1) Stabilization of enzyme systems.

Although immobilization may not be a general method forincreasing enzyme s tabi l i ty , there are, in fact , many instances whereimmobilized enzymes have proven to be more stable in use or storage. Withpurified cell-free enzymes i t is found often that there is loss of activityduring an immobil ization procedure, bu t once immobilized, the enzyme exhibitsincreased stabil i ty, i . e . , retains activity for a much longer time than th efree enzyme. This has been interpreted as indicating that immobilization hasimposed restr ictions of changes in tert iary structure of the enzyme and that

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such res tra ints confer greater resistance to denaturation by heat, agita t ion,etc .

In some cases i t is advantageous to retain the enzymes inside the ce l linstead of purifying them. This would apply especial ly to membrane-boundenzymes that are no t ea si ly sol ub il iz ed and purif ied, and to highly unstableo r oxygen- sens it iv e enzymes (hydrogenase, nitrogenase). For these, th e ce l l sare immobilized, with or without t reatments designed to increase access ofsubst rates (rupture or permeabilization of the ce l l membrane). Increaseds tabil ization against thermal denaturation by immobilization is suggested byexperiments in which free or immobilized ce l ls were incubated a t elevatedtemperatures (see Table 2). Regardless of the mechanisms involved, there aremany examples of increased operational s tab i l i ty of immobilized cel l s ( i . e . ,under conditions of use), some of which are summarized in Table 3.

(2) Optimal reac t ion cond i tions .

In immobilized cel l systems, part icular ly continuous flowreactors, the conditions used are those optimal for product formation, becausegrowth does not need to occur. Operating conditions may i nvolve t emperaturesor pH values that are no t optimal for growth. In convent iona l bat ch fermenta t ions for secondary metaboli tes th e ini t i a l growth phase (trophophase) isfollowed by th e p roductio n phase (idiophase), but both of these occur in thesame vessel . Conditions favoring growth often are deleterious to secondarymetabolite formation. For example, phosphate concentrations benef icial forgrowth of ten ' are found to give poor antibiot ic production (29). In thesecases empi r ically developed fermentation media undoubtedly represent a com-promise between conditions needed for growth and those needed for productformation.

(3) High cel l density without high Viscosity.

t ionaltheres t i r

When extremely high cel l densi t ies are employed in a convenst i rred fermenter, part icularly in the case of mycelial organisms,

is a problem with high Viscosity. Viscous cultures are dif f icul t to(high power requirements) and they are dif f icul t to aerate (poor

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Table 2. Increased Heat s t a b i l i t y of Enzymesin Immobilized Cells (adopted from reference 13)

Percent of I n i t i a l ActivityOrganism and Enzyme Treatment Free Cells Immobilized c e l l sEscherichia fre u n d ii 80 min at 50 C 4 20(phosphatase)Escherichia c o l i 30 min a t 60C 10 25(peni ci l l i n-i C yl ase)Escherichia c o l i 30 min a t Ssc 8 40(a spa r t a se r -Brevibacterium 180 min at 60C 2 25ammoniagenes(CoA sy n th e sis)

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Table 3. Increased Operational Stabili ty of Enzyme systemsIn Immobil ized Microbial Cells Compared To Free CellsUnder Use Conditions

Enzyme or Free Cells Immobilized ImmobilizationOrganism Enzyme System (non-immob.) Cells Method ReferenceEscherichia aspartase 11 120 polyacrylamide 10coli

Escherichia pennicil l in 1 40 epoxy resin 22coli acylaseBrevibacterium fumarase 5 53 polyacrylamide 10ammoniagenes

Erewinia conversion of 4 359 alginate 8rhapontici sucrose toisomaltuloseAchromobacter NAD kinase 6 20 polyacrylamide 28aceris

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gas-liquid mixing and oxygen transfer). If the same cell mass is immobilized( i .e . , in bead or pellet form), the viscosity i f low and the problem iseliminated.

4. Theoretical and technical constraints.

a. General.

Klibanov (23) describes three areas of difference between freeand immobilized catalysts that may be considered as general constraints.These are the par ti tion ing e f fec ts resulting from the conversion of a homogen-eous to a heterogeneous (an isot ropic) condi tions; the l imitations imposed bydiffusion rates; and the effects of steric hindrance.

(1) Partition effects.

Par ti ti on e ff ec ts arise from the creation of a two phasesystem. The immobilized cells or enzymes are present in one phase which hasdifferent physiochemical properties form the bulk aqueous phase. All the com-ponents of the reaction process , includ ing substrates, products, cofactors,hydrogen ions, inhibitors, etc. , are parti t ioned between these two' phases.

This can lead to creation of microenvironments in the immobilized phasethat are quite different from that of the bulk aqueous phase, and these couldexplain apparant Ralterations of k inetic p rope rt ie s. Thus, if the localenvironment of an immobilized enzyme is enriched in protons because of th eelectrostatic attraction of a negatively charged support, the apparent pHoptimum would be different from that of the free enzyme. Similarly, a hydrophobic support could cause a localized concentration of hydrophobic moleculesaround the immobilized catalyst . The same situation could obtain forinteraction of charged substrates (or products) with an oppositely chargedsupport, and this could be important for reactions showing substrate (o rproduct) inhibition. To some extent these effects could be minimized byproper selection of the support material and the appropr ia te reactor design.For reactions exhibiting Michaelis-Menton kinetics i t is generally considered

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that a plug-flow continuous reactor (packed or fluidized bed) is preferable toa continuous flow st irred tank reactor (CFSTR) because less catalyst is neededfor the same deg ree of conversion (39, 20). Only in th e case of substrateinhibited reactions does th e CFSTR ha ve more favorable kinetics.

(2) Diffusion and mass transfer effects.

These are of two types: external and internal. A cel limmobilized on th e surface of a support shows only the "external" masstransfer effects. These can be considered as involving the existence of astagnant layer surround ing the part ic le , and the fact that substrates andproducts have to be transported across this layer by diffusion, which isdriven by the concentration differences between the surface and bulk phase.For cells immobilized within a porous matrix, there is an additional difusionstep within the pore ("internal" effect) before substrates from the bulk phasecan reach the cel l . Concentration gradients develop within the pores as th esubstrate concentration decreases with distance from the surface.

Diffusional l imitations reduce the catalytic eff iciency and need to beminimized. This is approached by: decreasing the size of th e immobilizedpart ic le: optimizing i ts geometry: increasing substrate concentrations: andincreasing flow rates (20).

(3) Steric hindrance.

Immobilized cells and enzymes usually are not suitable forreactions involving insoluble materials (cellulose, keratin) or very largemolecules (high molecular weight polysaccharides, proteins, nucleic acids).Cells or enzymes attached to the surface of a support may show poor acti vi tywith large molecules that cannot approach the active sites because of sterichindrance by the support. For cells entrapped in a matrix, macromoleculesdiffuse poorly into th e matrix as would be expected.

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(4) Physical or mechanical properties of th e immobilized cells .

The immobilized biocatalyst must have suitable physical ormechanical f eatures that will allow i ts use in a reactor. Some of the charac-ter i s t ics that must be considered include: size, shape, rigidity, porosity,density, resistance to abras ion; and resistance to chemical attack.

The size of the immobilized material is important fo r good flow charac-teris t ics and maximum catalytic activity . For attachment of cells to a sur-face, smaller size part ic les provide greater surface area per unit volume; fo rcells imbedded in a matrix, a smaller particle size will improve diffusion andmass transfer rates. The optimal size will differ not only for the materialand method used fo r immobilization, bu t also on the type of reactor used. Fora packed bed reactor smaller part ic les will give a larger pressure drop acrossthe bed and will rest r ic t flow rates. Smaller particles are more susceptibleto clogging, bu t if the part ic les are too large, there may be more tendencyfor channelling to occur.

The shape of the particles does not seem to be an overriding factor.Spherical beads are commonly used, but many other types have been usedsuccessfully ( i .e . , discs, chips, flakes, blocks, thin films, etc .) . Onlyfibrous forms seem to be poorly adapted to use in a reactor.

For packed bed reactors rigidity of the particles is very important inorder to minimize deformation and compacting of the bed. For use in fluidizedbed or st irred reactors th e particles of immobilized catalyst must be tough,rathar than rigid, in order to withstand the abr as ion caused by mixing. Also,in a fluidized bed reactor the density of th e material is important for main-taining fluidization. For a l l types of reactors th e porosity of th e particleis of great s igni fi cance when th e catalyst is imbedded within the matrix. Ina ll cases, the part ic les must withstand the chemical effects of the reactionmixture that would cause them to dissolve, soften or aggrega te .

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(5) Biological factors.

There are several factors relating to the biocatalyst thatgovern i ts ut i l i ty in an immobilized system. These include cell viabil i ty:enzyme activity and stabil i ty: and cell permeability.

Some immobil izat ion procedures may result in the death of cel ls ,particularly when chemicals like glu tara ldehyde are used in th e process. Forsimple one-step enzymatic reactions i t may not be important that the cellsremain alive. In f ac t, a ct iv it y of a particular enzyme may be retained longerin dead than live cells. possibly protein "turnover" involving degradation byproteases is less in the non-viable cells . In any case, very long"half-lives" are reported from some reactors using non-viable cells. If cellviabili ty is required, there are several alternatives: use an alternativeimmobilization method that does not kill th e cells: use a more resistant formof the cell (bacterial endospores, fungal or streptomycete conidia) forimmobil izat ion, then supply nutrients to induce germination; i f only some ofthe cells are killed by immobilization, supply nutrients to allow growth ofthe survivors to occur.

Obviously, th e relevant enzyme systems must be active after immobilization and shoUld remain active fo r a long period of time. I t is important tomaximize the level of enzyme activity in the cells to be immobilized. Thiswould include genetic modification of the culture and use of optimal growthconditions and harvest times . The cell envelope (cell wall and cell membrane)represent potential barriers to free passage of reactants and products. Ifthis appears to be a limiting factor, i t is sometimes possible to alter thesebarriers by treatment of th e immobilized cells with surfactants or solvents.

(6) Microbial contamination.

Deleterious effects of microbial contamination have not been amajor problem for reactors operating with non-viable cells under conditionsunfavorable for growth of contaminants. However, fo r complex biosynthesis

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with l iving immobilized cells , nutrients have to be supplied, and this wouldallow contaminants to grow also. Industrial scale preparation of immobilizedcells under aseptic conditions and operation of steri le reactors involvesconsiderable diff iculty , wh ic h u nd oU bted ly explains why there are no commer-cia l processes of this type yet.

Some immobilization procedures would be diff icult to accomplish underster i le conditions, and every "handling" step is a potential source of contamination. One approach that would m in imize "handling" is the use of steri le" ca rr ie rs " t ha t are colonized by a growing culture. s ter i le carriers, such asporous glass or ceramic beads, wire mesh balls , etc . , are added to a fermenterculture and are colonized by the cells. These biomass-filled carriers thenwould be pumped aseptically into a steri le reactor. Hollow fiber dialysissystems represent another "minimum handling" approach to avoid contaminationof liVing immobilized cultures.

b. Specific constrains for algal systems.

(I) Light.

The only real advantage for use of algae is the photosyntheticcapture of l ight energy. For economical fuel production, the algae mustphotosynthesize with an efficiency above rates observed in nature. Mostfeasibil i ty analyses presuppose very high photosynthetic efficiency, asdetermined by short-term m ea su re me nts on free cells in well mixed reactors.The provision of l ight will be a major constraint fo r the operation ofimmobilized algae systems for fuel production.

The underlying point of immobilized cell techniques is the use of veryhigh cel l mass to achieve a rapid reaction. I t is unclear how well l ight willbe able to penetrate layers of algal cells in immobilized systems. If thecel l layer is thin (a s in surface attachment) to p ro mo te good l ight penetrat ion, there is a relatively low ratio of biomass to carr ier . If th e cells areimbedded in a matrix to obtain a higher concentration of biomass, l ight penet ra t ion would be reduced and cells in the interior would not show goodefficiency.

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Furthermore, any materials used f or i mm obilization w i l l have to betransparent to permit good l i g h t penetration. polyurethane foams and agargels have been used with algae, bu t i t i s no t known whether these are the moste f f i c i e n t . Another p o t e n t i a l source of l i g h t loss i s refl ect i on from s u r -faces. Presumably t h i s would be minimal fo r s urfa ce s c oa ted w ith a lg a l c e l l s ,but i t might be s i g n i f i c a n t where c e l l s are re stra in e d inside microcapsules orhollow f i b e r s , or fo r porous beads where algae have colonized only th ei n t e r i o r .

(2) Gas exchange.

A second major component of th e photosynthetic system i scarbon dioxide, and pr ovi si on of adequate carbon dioxide will be a over r i di ngc o n stra in t for a ll a lg a l systems. Maximizing th e mass t r a n s f e r of gases(C02, 02) in to an aqueous phase is a bioengineering concern in many b io -technical processes and, as indicated pr evi ousl y, the flu id iz e d bed ands t i r r e d tank reactors are preferable fo r t h i s purpose. Gas exchange i s poorin packed bed re a c to rs, and probably would be a limitin g factor in hollowf i b e r reactors as well.

The concent r at i ng of a lg a l c e l l s into a densely packed condition ast y p i c a l l y used in i m mo bi li z at io n t e ch n iq u es would be expected to r e s u l t in apoor supply of CO 2 to c e l l s in the i n t e r i o r of th e mass. Use of c e l l s int hi n sur f ace layers would improve CO 2 penetration, bu t a t th e cost of usinglow biomass loading.

(3) Nutrients.

providing n u t r i e n t s such as ammonia, phosphate, etc . , toimmobilized algae would no t seem to present any p a r t i c u l a r problems beyondthose fo r fre e a lg a l c e l l s . The problems of diffusion and n u t r i e n t depletionin immobilized c e l l reactors have been alluded to previously, but should notbe of great sig n ific a n c e , and should no t be th e limitin g f a c t o r s .

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(4) Harvesting of biomass and l ipid extraction.

Using an algal system for fuel production demands productionand harvesting of a very large amount of biomass, so any immobil ized systemmust be adapted to easy recovery of the cells. In some systems i t may bedifficul t to recover the cells (for example, hollow f iber r eacto rs ), and inothers, l ipid extraction may be more difficul t than fo r free cells (e .g .,polymer-entrapped cel ls ) .

(5) waste products or by-products.

Production of dry algal biomass for fuel production wouldyield thousands of tons of extracted cells as the by-product. Some schemeshave envisioned recycling some of this fo r algal nutrients, or using th e cellsin catt le feeds, composts, etc. some immobilization processes might interferewith such prospects and create a waste disposal problem. For example, immobi-l ization methods using non-biodegradable plastics , ceramics or glass mightprevent compost ing or use of the cells in animal feeds. Use of non-biodegradable plastics or inorganic supports, such as titanium or aluminum oxides,might cause problems with recycling the cells for nutrients.

B. Cell immobilization methods.

Cell and enzyme immobilization has been an area of intense interest andactivity for two decades, and as a result many different methods andstrategies have been devised. These have been reviewed extensively (4 , 13,22, 25, 26) and th es e authors have presented var ious schemes for cat egor iz ingthese methods. We can use a simplified classif icat ion to recognize thefollowing types of cell immobilization:

(1) adsorption and adhesion methods(2) physical entrappment methods(3) chemical coupling methods, with or without a carr ier material

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1. Adsorption and adhesion methods.

Some authors view the undirected agglomeration, f1ocu1ation orpellet izing of cells that occurs during thei r growth as a form o f immobil iz a-t ion. Such cel l aggregates can resul t from adhesive slimes, from ionicadsorption effects between cel ls or from th e physical intertwining of f i l a -ments. Such macroscopic cel l masses can be used in b io reac to r p rocesses ,provided they can be formed in a reproduci b1e manner, and are suff ic ient lystable and have other suitable (mechanical) properties. In general this isnot a sat isfactory approach for the biotechno1ogist because the basis foraggregation often is unknown and the process of aggregation is ei ther uncon-t rol lable or dif f icul t to control (result ing in variabi l i ty of s ize , cel ldensity and act iv i ty ) . Attempts have been made to devise control led floccula-t ion procedures, and patents have been awarded for processes using polyelec-t rolytes to induce flocculation of the yeast , Sacchoromyces cerevisiae (U.S.Patent 3,821,086) and th e bac te rium, Ar th robac te r (U.S. Patent 4,060,456) foruse as sources of invertase and glucose isomerase , respectively.

A more widely used procedure is to induce adsorption or adherence ofcel l s onto the surface of a solid carr ier , or within the pores of a porouscarr ier . The cel l attachment may be achieved by several different mechanisms:adhesion due to a cel lular slime layer; ionic at t ract ion between cel l surfaceand the carr ier surface; weak interact ions (hydrogen bonding, chelat ioneffects , van der Waal forces) between cel l and carr ier .

The advant aqe of this type of cel l immobilization is that v ia bi li ty isgenerally retained. The diff icul ty is that the attachment between cel ls andcarr ier is relat ively weak, and cel ls tend to be released into the react ionmixture ("leakage").

Examples of some of the materials that have been used for immobilizationby a ds op rtion a re l is ted in Table 4. These include such diverse materials ashydrated zirconium and titanium oxides, porous glass beads ("control led poreglass") , crushed brick, ceramic materials of various composition, ion exchangeresins (e.g . , Dowex-1, DEAE cel lulose, etc . ) , anthracite coal and wood chips.

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Table 4. Partial Listing of Carrier Materials UsedFor Immobilizing Cells by Adsorption (o r Adherence) Methods (13, 25, 26).

Natural organicWood chipsAnthrac ite coa l

Modified orsynthetic organicDEAE-cellulolseeM-celluloseDEAE-sysbadexDowex-l resinPolyvinyl chloride

34

InorganicTitanium oxideZirconium oxideCeramics (various)Glass (various forms)Diatomaceous earthBrickClaySand


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In the case of hydrated zirconium oxide (and other transition metal oxides)the adsorption of cel ls is considered to result from replacement of hydroxylgroups of th e oxide by suitable ligands on the cell surface (hydroxyl or aminogroups of polysaccharides or proteins). Obviously, surface properties of th ecell playa major role in adsorption to the carrier surface; both bacteria andyeasts are negatively charged at neutral pH and would be expected to bind wellto sur faces car rying posi t i ve charges. In the case of s t rong ion exchangeresins the adsorption of cel ls presumably is due to ionic binding. For someof these carriers (e.g. , glass) th e mechanisms of adsorption are le ss c erta inand several may be acting simultaneously.

For porous carriers the pore size is important. With porous glassbeads th e optimal pore size differs with th e cel l size and type: for bacteriaand yeast maximal colonization and biomass accumulation occurred with a porediameter 1-5 times the largest cell dimension (31).

Loading capacity fo r adsorption methods varies with the material andculture. With the mold Penicillium chrysogenum an untreated frit ted glasscarrier material retained only 0.17 mg cel ls (dry wt )/g of carrier , but aporous spinel-zirconia material adsorbed 2.6 mg/g (26). Comparison of variousanion exchange resins using the yeast Saccharomyces cerevisiae showed the mosteffective one (XE-352 resin) to bind about 130 mg cells (dry wt)/g resin(22) With the yeast Saccaromyces carlsbergensis Dowex-l resin bound 24 mgcells (dry wt)/g, PVC chips 80 mg/g and wood chips, 248 mg/g (13).

2. Physical entrapment methods.

One of the most useful approaches to cell immobilization is th ephysical entrapment of the cel ls within a porous network or matrix (gel, fiberor solid) or inside microcapsules. The number of materials used and th e chemis try of th e processes involved in ext remely diverse (Table 5). A completediscussion of these methods is beyond th e scope of this review (consultreferences 13, 22, 43), and only selected examples will be mentioned toi l lustrate th e range of possibil i t ies available. Depending on the processrequirements, appropriate entrapment methods can ,be selected to maximizeparticular performance characterist ics, e.g. , chemical and mechanical

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Table 5. Examples of Entrapment Methods for Cell Immobilization (13, 22, 25, 26

Methods and MaterialsFiber Entrapment:Cellulose tr iacetate

Alpha-celluloseMicroencapsulation:Liquid membranes

Insoluble membranes Nylon

Basis of the procedure

Precipitation from solventsPrecipiation from solvents

Emulsification of cellsin mixture of hydrocarbon,surfactant and polyamine.Emulsification and interfacial polymerization.

Performance Characteristics

Chemical stabil i ty;viabil i ty loss.Chemical stabil i ty;viability loss.

Fragile; "leakage" of cell

Viability loss.

Mixed alginate-polylysine

Gel Entrapment:polymerization methods Polyacrylamide

Alginate beads formed byion replacement; interfacial ionic complexingwith polycation; liquefactionof internal alginate.

Free radical polymerizationand cross-linking of acrylamide with KfS20S'TMED and N,N -methylenebisacrylamide.

Viability retained.

Soft non-rigid gel; someviabill i ty loss; cell loading 20% w/v (wet weight).

Good mechanical propertiessome viability loss.Epoxy resins polycondensation of precursors; fo r poros it y,include alginate, thendissolve after polymerization.

Polyurethane foam

Network formed byionic replacement Alginate

Cross-linking of polyisocyanate prepolymer by aqueouscell suspension at low temperature; release of C02produces foam.Insolubilization of sodiumalginate by Ca++in phosphate.

Transparent; stable;elast ic; low cell loading;viabili ty loss.

Good mechanical propertieshigh cell loading; uns tabl

Chitosan Insolubi1ization by poly- Stable in phosphate.valent anions (polyphosphate.)Network formed by gelationof melted polymer -agar

Kapa-carrageenan

Gelation by cooling ofmelted polymer.

Gelation by cooling plusionic interactions.

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Soft non-r ig id gel.

Non-rigid; high loading;viability retained.


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stabi l i ty of the immobilizing network: control of size and shape; suitableporosity: high cell loading capacity: retention of c el l a ct iv it y or viabili ty .

Cells can be immobilized in fibers of cellulose derivatives (e.g . ,cellulose acetate) produced by precipitation from organic solvents. Thesefibers are r ela tiv ely s ta ble , bu t have poor flow properties and there issignificant kil l ing of cells by the solvents used (DMSO, formamide, acetone).

Microencapsulation of cells in liquid membranes or insoluble polymermembranes (ny lon, etc .) has been used to a limited extent. Liquid membranemicrocapsules are produced by emulsifying th e cell suspension in a mixture ofhydrocarbon plus surfactant and a polyamine. These are fragile structures andcells readily -leak- from th e microcapsules. Microcapsules with insolublenylon membranes have been produced by dispersion of aqueous droplets containing hexamethylenediamine in a solvent that contains sebacoyl chloride.Interfacial polymerization produces th e nylon membrane. The reagents andsolvents used produce cell growth, and in general microcapsules often do notpossess good mechanical properties for industrial applications. A newerprocedure (Encapcel method) has been developed that allows microencapsulationof living animal cel ls (17, 27). In this procedure th e cells a re d ispe rsed ina sodium alginate solution and cast in bead from by dropping into caC12solution. The beads are then transferred to a polycation solution (polylysi ne , etc .) , which produces an insoluble alginate-polylysine surface layer.Finally, the alginate from th e interior of the bead is solubilized by chelation of th e calcium ions, thus producing a microcapsule. Because of the mildconditions employed, cel l viabil i ty is maintained.

Numerous methods have been devised that are based on the polymerizationof a material in which the cells are dispersed. The approach most frequentlyused has been the polymeri zat ion of ac rylamide, but many othe r polymers havebeen used, such as polymethacrylamide, polys tyrene, polyvinyla lcohol, polyurethane, maleic polybutadiene (PBM), poly(ethyleneglycol), polyvinylpyrolidon,polymethylmethacrylate, etc. None of these appears to possess a ll of thedesirable properties.

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Polyacrylamide gels are easily formed and are chemically stable, but aresoft and non-rigid. There is some cell death and cell loading is limited toabout 20% wet weight of cells fo r bacteria (less fo r larger cells) . Severalfactors contribute to loss of viabili ty during immobilization of cells inpolyac rylamide. These include heat gene ration and the tox ie i ty of chemicalsused in the polyme ri zation and crosslinking reactions (ammonium persulfate ,TMED and N,N'-methylenebisacrylamide). Conditions (low temperature, briefexposures) can be arranged to minimize cel l death. Greater mechanicalstrength can be obtained with other monomers, such as methacry lamide, butoften these are more toxic.

Entrappment in polyurethane foams has proven to be an effective method.A thick cell paste is mixed with a suitable hydrophil ic urethane wprepolymer"(available commercially, for example, Hypol 4000 from W.R. Grace) and thepolymerization allowed to occur at low temperature. Evolution of CO 2results in a final product that is tough, compressible foam, in which a substantial number of cells have retained viabiil ty .

A method of considerable value is th e formation of an isoluble networkof a polyelectrolyte material by mul tiva lent ions. This has been used withvarious polyanions, such as alginate, copoly (styrene-maleic acid), copoly(acrylamide-acrylate), etc . , and with th e polycat ion, chi tosan . Alginate is anatural biopolymer of mannuronic and gUluronic acids, and can be used undermild conditions to immobilize liVing cells in a highly porous network withgood mechanical properties. Cells are dispersed in a solution of sodiumalginate, which is added dropwise to a solution of calcium chloride (o r othercounterion, such as aluminum, iron, etc.) to produce spherical beads ofin so luble c alc ium alginate. This is a simple and rapid method, and the onlysignificant problem is that phosphate or other calcium chelators causesolubilization an disruption of the matrix.

Use of melted agar to entrap cells is as old as the s cience of bacteriology, bu t i t has had limited application fo r immobilized cell reactorsbecause of the poor mechanical properties of th e gel. More interest has beenshown in the related material, kappa carrageenan. Cells are mixed into a

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melted carrageenan solution at 45C and gelation is produced by cooling ortreatment with NH: or K+ ions (or other materials). carrageenan gelsa re non -rig id , but with mechanical properties superior to agar, and there isgood cell survival.

3. Covalent bonding.a. Cel l- to-ce l l (carr ier -f ree) linkage.

Chemical bonding of cells to one another by means of glutaraldehyde has been used to produce insolubilized cells that retain enzymaticactivi ty. These insolubilized cells have maximal cell density, bu t generallyhave poor mechanical properties fo r reactor use and seldom retain Viability.The approach seems to hold l i t t le promise for use with living cell systems.

b. Chemical bonding of cells to a carrier .

This has been used ex te nsive ly for preparing insolubilizedenzymes, but has seen only l imited appl icat ion for cell immobilization becauseof the toxicity of th e coupling agents. Typical examples of this approach arebonding of cells to carbodiimide-activated organic carriers (e.g. , carboxymethyl cellulose) or to trialkoxysilane derivatized glass. The only approachthat appears to have had much success is the use of chemical bonding tostrengthen the matrix in the entrapment procedures. In those cases where abrief exposure to glutaraldehyde can introduce c ro ss -l in ks in to a network, i tis possible sometimes to icnrease the strength without complete loss of cel lviabil i ty .

4. Selection of appropriate methods fo r immobiliZing algae.

A cel l immobilization system suitable for algal biomass productionwill need to provide three characterist ics relating to growth and photosynthesis: cell viabil i ty must be retained; there must be good l ight penetration;there must be adequate gas exchange fo r CO 2 uptake or 02 evolution.

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a. Cell viability.

Procedures that allow good retention of viabili ty include thevar ious adsorpt ion methods, such as binding to ion exchange resins, titaniumor zirconium oxides or other carr iers , and a lso coloniz ation by growingcultures of performed porour carr iers , including polyurethane foam. Someentrapment methods (alginate, carrageenan, possible polyacrylamide or pOlyurethane) may also give satisfactory retention of c el l v iabi li ty during immobilization, and/or allow growth of entrapped cells to obtain high densities ofl ive cells.

b. Light.

Polyurethane has good transparency, as does polyacrylamide.Agar and carrageenan may be suitable also. Opaque carriers would tend tointerfere with l ight penetration, and this may rule out titanium and zirconiumoxides, as well as some ion exchangers.

c. Gas exchange.

Agar does not give particularly good gas exchange, andprobably this would be true also of carrageenan and polyacrylamide. Unlessth e material is rather porous, entrapment methods in general probably will notbe optimal for gas exchange. Surface adsorption and colonization of porouscarr iers would be expected to show better gas exchange properties.

C. Examples of immobilized cell systems.

1. Use of non-growing cells for single enzymatic reactions.

As stated previously, use of immobili zed cell reactor systems hasdeveloped out of immobilized enzyme technology, and quite naturally theimportant early commercialization applications have been fo r single-stepenzyme reactions. In these cases the cells are not actively growing, and i tis unimportant even to maintain viabili ty of the cells, provided enzyme

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activity and stabi l i ty are satisfactory. This allows a very wide latitude inselecting immobili zat ion methods that will meet reactor performance specif i -cation. The immobilized cells systems for glucose isomerase and aspartasereactions are examples of this category (10, 28).

2. Use of non-growing cells for complex metabolic pathways.a. Catabolic pathways.

Although the cells may not need to be actively growing, we cancons ider that they must remain al i ve in order to be useful for processesinvolving integrated metabolic pathways such as glycolysis. Various procedures have been used successfully to immobilize yeasts (Saccharomycescerevisiae, ~ carlsbegensis, Kluyveronmyces lactis) fo r p roduction o f beverage and industrial ethanol from sugars. Methods used include entrapment inagar, carrageenan, calcium alginate and polyacrylamide, as well as adsorptionon diatomaceous earth, PVC, ion exchange resin and wood chips (28).Similarly, immobilized Lactobacillus and streptococcus cells have been usedfo r conversion of sugars to lactic acid via glycolysis. In these situationslong term reactor operation seems to require that some cel l growth can occurto replenish ca ta lyt ic ac tiv ity lost as cells die.

b. Biosynthetic pathways.In many commercial fermentations the products (primary or

secondary metabolites) arise from complex biosynthetic pathways. Immobilizedcell systems have been used fo r such b iosynthe ti c r eact ions with varyingdegrees of success.

The bacterium, Corynebacterium glutamicum is used for direct fermentations to produce L-glutamic acid and certain other amino acids (e.g . ,L-lysine) from glucose. Cells of C. glutamicum immobilized in polyacrylamidewere able to produce glutamic acid, bu t the efficiency of the process was nothigh enough to be compe ti ti ve with th e conventional free-cell fermentation(28). Oxygen transfer l imitations probably were responsible.

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Immobilized cel l methods would seem to have more potential in th e production of secondary metaboli tes, such as ant ib io t ics . Some work has beenreported showing successful production of penicil l in G by immobilized geni cillium chrysogenum and bacitracin by Bacil lus species (20, 28). Althoughsuch processes appear to be feasible, they have a tt ra ct ed r el at iv el y l i t t l eindustr ia l in teres t , probably because of th e extremely high productivi ty ofconventioinal ant ib io t ic fermentations.

3. Use of growing cel ls .

While i t is possible that ce l l growth occurs in many of thereported applications of immobilized cel ls , in most cases there i s no specif icrequirement for the cel l s to be growing in order to accomplish th e p rocess.Growth of immobilized cel l s often has been observed when nutr ients aresupplied, and such growth can resul t in high biomass levels .

Growth of yeast cel l s entrapped in car rageenan was noted by Chibata e tale (11). A growing culture was immobilized in carrageenan beads at a lowcel l densi ty (about 3.5 x 106 cel ls /ml of gel ) , and these beads wereincubated 60 hours in growth medium. This resulted in a 1000 fold increase of9cel l s (to 5.4 x 10 cells/ml) and most of th e cel ls were found to havedeveloped near the surface of the bead.

Growth also occurs when cel l s adsorbed to a carr ier surface are suppliedwith nutr ients . Messing e t a t , (31) adsorbed Aspergillus niger conidia ontocontrol led-pore glass beads, which were the n in cub ated in growth medium toallow germination and growth of mycelium. Colonization of diatomaceous earthby Streptomyces cat t leya was studied by Baker e t ale (5). When s ter i le Celitewas incubated with a growing culture for 72 hours, the streptomyces myceliumcolonized the Celite part ic les to yield an immobilized cel l preparation withabout 0.2 g cel ls/g of Celi te . In repeated batch incubations using a "production medium", ant ibiot ic production by the immobil ized Strep tomyces wascomparable to that of free cel ls , and the immobilized cel ls could be kept inan active condition for over 190 days by intermit tent " feed ing" with nutr ients .

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Methods for process intersif icat ion of fermentations by use ofbiomass-filled "carrier" have been described by Atkinson et all (4). Onepromising procedure was the use of wire mesh "particles" as carriers. Sterilemesh carriers were incubated with growing cultures of bacteria, yeast or moldswhich coloniz ed the carr iers . After the carriers become f i l led with a mass ofcells , they were recovered and used as immobilized cells in bioreactors.

4. Immobilized algae systems.

Only a limited number of studies have been reported on th e use ofimmobilized algae (see Table 6). These include use of immobilized algal cellsfor photoproduction of hydrogen; fo r "normal" photosynthetic production ofoxygen (e.g. , to support oxygen-requiring bacterial processes); and fo r production of chemicals, such as ammonia or sulfated polysaccharides.

a. Photoproduction of hydrogen.

The heterocystous cyanobacterium Anabaena cylindrica canevo lve molecu la r hydrogen (H 2) from water when irradiated with l ight (37).Vegetative cells of this organism contain photosystem II (PSS II) and carryou t "normal" pho tosynthesi s to produce 02' but the th ick-wal led heterocystsdo not contain PS II and have an interior that is effectively anaerobic. Thisallows act i vi ty of an 02-labi Ie ni t rogenase , which not only can reduce N2for "nitrogen fixation", but also is cpable of an ATP-dependent generation ofH2 The involvement of n itrogena se explain s why photoproduction of H2production require illumination of an N2 starved culture incubated underargon-carbon dioxide with a trace of N2 or NH 4

Cells of Anabaena immobilized in agar have been used fo r continuousphotoproduction of hydrogen to operate a fuel cell to produce electr icalcurrent (19). Hydrogen production by immobilized cells was about 3 timesgreater than that of free algae. Cells of Anabaena sp , strain N-7363 wereimmobilized in agar at a level of 3.3 mg dry cel ls ( i .e . , 50 _G chlorophyll ~ pe r gram of gel (wet wt) and cut into I mm 3 blocks. Incubation of theseimmobilized cells under argon in a medium with to mM sodium carbonate (pH 8)

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Table 6. Summary o f Sele cted Immobil ized Algae Systems

Immobilizationorganism product Substrate MethodChlorella vulgaris plus H2 H20 Agar entrapmentClostridium butyricum

Anabaena sp. H2 H20 Agar entrapment

Anabaena cylindrica H2 H20 Glass beads

ProductionRate Referenc0.Z9-l.3 u 37wood/hr/mgchlorophyll0.0003 u mol/ 16hr/mg dry wt .0.014-0.135 u 16mol/hr/mgchlorophyll

Nostoc muscroum; HZchlorogloea f r i t schi i ;Mastigocladus laminosusScenedesmus obliquus 02

Chlorella pyridenoidosa 02

Anabaena sp. 02

Ascorbate Colonization ofpolyurethanefoamAlginateentrapmentAlginateentrapmentAlginateentrapment

3-26 u mol/hr/mgchlorophyll200 u mollhr/l0 9 cel ls2.1 u mol/hr/mg dry wt110-145 u mollhr/mg

jaycor 1985 - immobilized algae systems

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