The Chemical En.&eeriw Journal, 50 (1992) B9-B16

The role of chemical engineering in biotechnology

Cohn Webb Department of Chemical Engineering, University of Manchester Institute of Science and Technology, PO Box 88, Manchester M60 l&D CUK)

Bernard Atkinson Brewing Research Foundation, LgteU Hall, Coopers Hill Road, Nu@dd, Suwey RHI 4HY (Ua

(Received January 1, 1992)

Abstract

Depending on which deCnition is chosen, biotechnology can be considered to be one of the oldest industrial technologies or one of the newest. For the chemical engineer, however, the major distinction is in terms of the scale of operation. The majority of products of the new biotechnology are extremely high ,value, low volume biochemicals. Thus recovery (separation) processes for such products may be relatively costly and energy intensive, because of the small quantities involved, but at the same time must be highly efficient in order to minimize loss of valuable product. This contrasts with the more traditional biotechnological processes of the food and beverage industries, antibiotic and medium value pharmaceuticals production, and wastewater treatment. Here, the problems for the chemical engineer are more like those in the chemical or process industries.

In this paper a typical biological process is compared with its counterpart chemical process and the role of the chemical engineer in designing and developing such a process is examined through a series of examples. Just as the chemical industry for decades was dominated by the chemist, so the bioprocess industry is still dominated by the industrial microbiologist. There are consequently many areas in which improvements can be made to established industrial biological processes by the application of simple engineering concepts.

B9

1. Introduction

Although it is often thought of as a new technology, biotechnology can be considered to be one of the oldest of industrial technologies. To the biochemical engineer, the intentional use of micro-organisms to produce beer, wine and cheese is biotechnology. So too is the biological treatment of sewage and wastewaters. Even the word itself is not as new as many would think. It was first used by a Hungarian, named Ereky, in a book published in 1919, to describe all lines of work by which products are produced with the aid of living organisms [ 11. This was addressed particularly to agriculture but it was around the same time that Chain-r Weizmann (at the University of Manchester) had developed an industrial process for the mass production of acetone by fermentation, a process which fitted into the definition given by Ereky.

Biotechnology continued to be synonymous with industrial fermentation technology and was defined

as such in an editorial article for the new journal Biotechnology and B?Ioengine&w by Elmer Gaden, Jr., in 1962. The same basic definition was used when the European Federation of Biotech- nology was established in 1978 but, just 1 year later, the word was redefined for a genetic engi- neering journal, to describe “scientific and financial developments in the field of genetics”. This latter definition was considered, by the US trademark commission, to be so different from the previous definition that it could be used as a trademark by the journal [2].

As more and more of the results, and products, of genetic engineering have made their way into the consciousness of industrialists, so the distinction between the two definitions has faded. Thus it is no longer clear, when the word biotechnology is used, whether the science of genetic manipulation or the industrial exploitation of living systems is being referred to. However, for the chemical en- gineer, the key feature of industrial biotechnological

0300-9467/92/$5.00 0 1992 - Elsevier Sequoia. All rights reserved

BIO C. Webb, B. Atkinson / Role of chemical engineering in biotech,mlogy

processes is the use of living organisms to produce commercially viable products, regardless of whether those organisms are genetically manipulated or nat- urally occurring. None the less, there is a major distinction between many of the products of the “new biotechnology” and those of “traditional bio- technology” in that the former are often extremely high value products, which are required in minute quantities (usually for medical or diagnostic pur- poses) while the latter are generally of low to medium value and are produced in quantities which require large-scale processing equipment.

These latter, relatively large-scale biotechnolog- ical processes provide a fairly traditional role for the chemical engineer. They involve similar problems of fluid mechanics, heat and mass transfer, and reaction and separation processes to those in the “core” chemical industries. The key difference, of course, is that they involve living systems and so the chemical engineer working with them must also have an understanding of life processes. This is the specialization of the biochemical engineer.

On the contrary, the problems associated with the extremely small-scale processes of the “new” biotechnology are not generally in the traditional areas of chemical engineering and many are unique to individual processes. Often, engineering efficiency can be sacrificed because of the scale of the equip- ment being used, and the role of chemical engi- neering is not so clear. It is likely that most such processes will remain the problem of the biotech- nologist rather than the biochemical engineer.

1.1. The future development of biotechnological processes

That the biotechnology industry will continue to expand is certain, although there is a long way to go before its predicted dominance over the con- ventional chemical industry is realized. One of the major reasons for such confidence in this continued expansion is that biotechnological processes are based on renewable resources. These will therefore gain in importance as conventional non-renewable feedstocks dwindle. Ultimately, all products will have to be manufactured from such renewable carbo- hydrate materials and particularly those which cur- rently constitute agricultural and food industry wastes (e.g. surplus food mountains). Such wastes provide ideal substrates for biological processes and the economics of converting them by biotechnol- ogical means is already considerably better than for chemical processing. In addition, global political considerations currently favour “natural” produc- tion of products wherever possible. This, by im- plication, means that biotechnological production

is likely to be preferred wherever it provides a viable alternative.

A further reason for the continued expansion of biotechnology is the ever-increasing range of val- uable products which can be produced through traditional biological methods and through genetic manipulation. The full range of possible products can, currently, only be guessed at. It is now, for example, almost routine to grow human tissue cells in mass cultivation in relatively simple fermentation equipment. The possible products of such cultures are only now being explored. Table 1 shows the likely range of future biotechnological processes [ 31. These are in addition to the traditional products of the biological process industries, namely alcoholic beverages, fermented foods, bulk enzymes, anti- biotics and wastewater treatment.

Many of the high value products referred to in Table 1 are speciality chemicals required in minute quantities, often of the order of a few kilograms per year. The economics of production is not likely therefore to be a limiting factor in their development. Natural product replacements, such as single cell protein, on the contrary, must compete with con- ventional agriculture if they are to succeed. Likewise, synthetic product replacements, such as organic acids and solvents, which were economically viable before petroleum was readily available, must prob- ably await the demise of this cheap alternative feedstock. This is also true of the large-scale direct petrochemical replacements such as fuel alcohols which, to be economical, will have t,o be produced at much larger scale than is currently practiced for any biological process except wastewater treatment.

The last category in Table 1 provides the greatest challenge for the biochemical engineer, and yet the current state of the biotechnology industry is com- parable with that of the chemical industry in the early decades of the century. It is generally operated by technologists rather than engineers and relies heavily on “black-art” techniques. Whilst this may be sufficient for the small-scale high value products where there is no alternative to the biotechnological route, it is inadequate for the marginally economic larger-scale processes, where cost-effective “engi- neering” can mean the difference between success and failure. Unfortunately, many biochemical en- gineers have been swept along by the biotechnology boom into becoming biotechnologists, concentrating on product-oriented technological research rather than on the broader aspects of process engineering. Whilst there is, of course, an important place in the development of the industry for the technologist, it is clear that the role of chemical engineering in

C. Webb, B. Atkinson / Role of chemical engineering in biotechnology

TABLE 1. The future range of biotechnological processes [S]

Bll

Small volume, high value products

Medium volume natural product replacements

Medium volume synthetic product replacements

Large volume petrochemical replacements

Medicinals Enzymes

Protein feedstuffs Oils Fats Polysaccharides

Organic acids Solvents Glycols Higher alcohols

Methane, hydrogen ethanol etc. and derived products

Established. Expanding range

Full-scale plants. Economics marginal but likely to improve

Many established. Economics marginal but likely to improve

New process concepts required. Economics political

biotechnology should be that of the biochemical engineer and not the biotechnologist.

2. Improving bioprocesses through better engineering

Although biotechnological processes are basically similar to chemical processes, in that they consist of three essential stages, namely raw materials prep- aration, reaction, and product recovery, there are some very important differences. The most important of these latter is the almost infinite range of products which might be produced from a given raw material, since this is merely a food source (substrate) for the growth of micro-organisms. The target product is usually a mere waste product of the microbial growth process.

It follows that there is no pre-defined reaction based on a particular set of reactants. What governs which product is produced is the micro-organism which is chosen to carry out the reaction. Even this does not guarantee specificity since the same micro- organism grown on the same substrate might pro- duce, for example, ethanol or lactic acid, or a particular enzyme, or an antibiotic. Only careful control of the physical conditions or the choice and timing of certain additions will ensure that the desired product is the one produced. Furthermore, the key component of the “reaction” mixture, the micro-organism, is both a catalyst for the reaction and a product of the reaction and cannot simply be provided in a large quantity at the beginning of the reaction.

To ensure that the correct micro-organism, and therefore product, is produced, the substrate must be seeded with a small quantity of the chosen micro- organism and subsequently protected from com- petitors. Prior to this, all other microbial life forms

must be removed from the substrate since they offer direct competition and some would most probably proliferate more successfully. This need for isolation from competitors is generally met by initial steri- lization of the substrate followed by aseptic operation throughout the reaction.

Most biological reactions are considerably slower than chemical reactions and this generally leads to batch rather than continuous operation, unlike in the chemical industry where continuous processes prevail. Many biological reactions become product inhibited at relatively low product concentrations and this again leads to a preference for batch operation. Unlike most chemical reactions, the rates of biological reactions cannot be increased by raising the temperature or pressure and most must be carried out under rather mild conditions, close to ambient temperature. Similarly, many of the prod- ucts of interest are heat labile and must be separated from the reaction mixture under relatively mild conditions to avoid their destruction.

Despite the differences from conventional chem- ical processes there are many areas in which the @io)chemical engineer can contribute to the design and operating performance of biological processes. The following represent a few examples from work in the laboratories of the Department of Chemical Engineering at University of Manchester Institute of Science and Technology.

2.1. Reducing bioprocess variability through better inoculum preparation

Biological processes are inherently variable, and so variability in industrial bioprocess performance is generally accepted as being inevitable. Never- theless, improvements can often be made through simple process engineering. For example, because of the batchwise operation of bioprocesses, there is a continual need for the production of good

B12 C. Webb, B. Atkinson / Role of chemical en&eering in biotechnology

quality inocula to seed the fermentation vessels. This need is usually met through quality control or microbiology laboratories. However, it is not just consistent quality which is required but also con- sistent quantity, since the inoculum must usually pass through several stages of scale-up, prior to the production vessel.

The autocatalytic nature of microbial growth re- sults in an exponential increase in the population with respect to time when there is no restriction to growth, i.e.

dx - = px dt

or x=x0 exp(&

where x is the cell concentration at tune t, x0 is the initial cell concentration and p is the specific growth rate of cells. Thus, since cells are usually transferred between stages before the population has reached a maximum value, any variability in the initial stages can lead to considerable differences in the effective amount of inoculum (i.e. x0) going into the final production vessel.

The first of all the inoculum preparation stages usually involves the transfer of cells from an agar culture. Table 2, column A, shows the level of variability which results from making such transfers using the conventional microbiological wire-loop technique. Although this can be substantially im- proved by the simple introduction of a written protocol (column C), it is still inherently variable. By devising a more controllable technique, such variations in inoculum size - which occur through- out the bioprocess industries - can be removed

effectively (column E). The liquid transfer technique offers the further advantage that it lends itself to automation [ 4 1.

2.2. Improving process control through rheological monitoring

The variability and unpredictability of most bio- processes, together with the batchwise nature of their operation, make estimation of progress through the course of the fermentation difficult. For this reason it is desirable to have on-line feedback control mechanisms to enable identification of key points in the tune course, e.g. points at which additions should be made or the point at which maximum product concentration is achieved. Generally, this cannot be done by direct monitoring of the chemical composition of the broth since on-line sensors for most bioproducts are not yet available. However, during the course of many fermentations, physical parameters such as broth rheology change consid- erably and these can be used to give good indirect estimates of the progress of the fermentation.

An example of such monitoring-control is the dextran fermentation. Figure 1 shows the time course of a typical dextran fermentation together with rheological data for the same fermentation, obtained from a full-scale industrial plant [ 51. From the point of view of production, the “end” of the batch is reached when the dextran concentration has attained its maximum level. Beyond this time the quality of the dextran deteriorates if the fer- mentation is allowed to proceed. The “end” point is normally identified via the flattening of the pH curve, since there is no means of determining,

TABLE 2. Variability in the number of cells transferred from agar slants using various techniques

A ( X 1 OS cells)

B ( X 10” cells)

C ( X IO* cells)

D (X lo6 cells ml-l)

E (X lo7 cells ml-‘)

0.16 0.14 0.13 1.3 5.05 0.24 0.17 0.16 1.9 5.1 0.26 0.18 0.19 2.2 5.11 0.4 0.20 0.20 2.5 5.15 0.9 0.24 0.20 2.8 5.2 0.95 0.29 0.28 2.9 5.2 0.99 0.30 0.32 3.3 5.25 1.87 0.32 0.40 3.4 5.3

0.34 4.0 0.35 4.0

D 0.72 0.25 0.24 2.83 5.17 o- 0.54 0.07 0.085 0.84 0.078 2, 1069% 150% 208% 208% 5%

A, conventional loop transfer carried out by different persons; B, conventional loop transfer carried out repeatedly by the same person; C, loop transfer carried out by different persons (as A) using a written protocol; D, liquid transfer from slants inoculated by wire loop; E, liquid transfer from slants inoculated by liquid transfer. b, average value; o, standard deviation; II, range of variation.

C. Webb, B. Atkinson / Role of chemical engineering in biotechnology B13

Dextrin 9

Time (hour)

Fig. 1. Results for a typical dextran fermentation [ 5) showing (b) the change in rheological properties K and 12 with time, as well as the (a) change in dextran concentration and pH.

rapidly, the dextran concentration. The former is observed usually at around 30 h, a considerable time after the dextran concentration has reached its maximum level. The rheological data, on the contrary, show a peak at about the same time as the maximum level of dextran is reached and also show quite clearly the deterioration of the dextran beyond this point.

It would be a relatively simple matter to insert an on-line viscometer into the dextran production vessel in order to monitor the progress of the fermentation. The viscometer need not even be calibrated since it is the change in apparent viscosity which is important.

2.3. Achieving process in&ns@icat&m through cell immobilization

As suggested earlier, bioprocesses are charac- terized by dilute aqueous solutions, by batch op- eration and by dispersions of microscopic cells. When a microbial population exists in the form of single cells dispersed throughout a fermentation medium, its physical behaviour is governed by the properties of the bulk liquid. In other words the individual cells behave as elements of the fluid within which they are suspended. Hence, when the liquid is removed from the vessel, so too are the cells. This represents a severe limitation to the operation

of such systems since it is often desirable that the cells be retained for further (continuous or repeated batch) use. In order that cells can be retained, they must be separated from the fermentation broth. This can most readily be achieved if the cells can be arranged so as to exhibit physical (hydrodynamic) characteristics which differ from those of the broth, in which case they can be thought of as being immobilized.

Cell immobilization then can be defined as the confinement or localization of cells to a certain defined region of space in such a way as to exhibit hydrodynamic characteristics which differ from those of the surrounding environment. This is most usually achieved by significantly increasing the ef- fective size or density of the cells by aggregation or by attachment of the cells to some support surface. Thus flocculated celIs in the form of large aggregates can be considered to be immobilized if the floes can be separated from the bulk liquid by, for ex- ample, coarse filters or rapid sedimentation. Equally, cells which are entrapped within a porous matrix of sufficient size or density and cells which are attached to a solid surface are examples of cell immobilization.

The methods by which cells can be immobilised are many and varied and have been reviewed ex- tensively, most recently in books by Webb et al. [ 61, Rosevear et al. [ 71, Tampion and Tampion [8] and Moo-Young [ 91. The immobilization can be a natural process or can be induced by chemical or physical means. Examples of natural immobilization include the formation of microbial films in waste- water treatment systems and in the production of vinegar by the “quick” process. In both these cases, recognition of the concept of immobilization was preceded by industrial exploitation of the phenom- enon. Only recently, through the development of techniques to effect cell immobilization artificially, has awareness of the advantages that immobilized cells can offer in the operation of fermentation systems become widespread.

2.3.1. The consequences of immobilization By immobilizing cells it becomes possible, at least

in principle, for biological particles of any size, shape and density to be produced for a wide variety of cell types. As a consequence, one of the major features of most immobilization processes is the very high cell concentrations that can be achieved and this, combined with the ability to handle im- mobilized cells distinguishably from the fluid phase, offers a number of potentially advantageous pos- sibilities for fermentation processes. These are sum marized in Fig. 2.

B14 C. Webb, B. Atkinson / Role of chemical enginee-rir~g in biotechnology

high cell density

accelerated internal cell mixed

reaction diffusmn cell/liquid

retention separation culture rates gradients

1

repeated reduced

broth

vlscoslty

+

* mazhYa::fer 1

Fig. 2. The consequences of immobilizing cells [lo].

u 40 80 120

Time (hours)

+ Repeated batches --W

.; 0.2

I/

8 o.o- 0 40 80 120

Time (hours)

Fig. 3. Results from repeated batch fermentations to oxidize Fe SO4 solutions using immobilized Thiobacillus ferrooxiduns cells (111.

The ability to treat cells as a discrete phase facilitates cell-liquid separation and enables re- peated batch fermentations to be carried out. Hence, at the end of one batch, a large resident inoculum is made available for subsequent use. This can be particularly useful in cases where production is normally preceded by a lengthy growth phase but also provides a simple means of effecting inoculation in most other types of fermentation. Figure 3 shows results for the repeated batch oxidation of ferrous sulphate @art of a process currently being developed as a means of removing hydrogen sulphide from sour gases). The time of 40 h required for complete oxidation in the first batch is exactly the same as

it would be for freely suspended cells. However, it can be seen quite clearly that, by immobilizing the cells and enabling their re-use, the length of time required to achieve full oxidation is reduced con- siderably after the first batch. Maximum productivity is not reached until the sixth batch, by which time it is three times higher than for the conventional single-batch system.

Figure 4 shows results for the same fermentation operated continuously. Again, productivities con- siderably higher than for a conventional batch can be achieved. The grey band in the figure shows the approximate value of maximum specific growth rate for the organism, representing the dilution rate beyond which it would not be possible to operate a free cell system.

The combination of accelerated volumetric re- action rates, brought about by increased cell con- centrations, and the ability to operate fermenters continuously, at throughputs well in excess of normal cell washout can lead to much increased produc- tivities compared with those of conventional batch fermentation. Under such conditions, it is also often unnecessary to sterilize the feed stream and this can be economically very advantageous. However, the presence of cells in such high densities can present transport problems for substrates and prod- ucts, with reaction rates becoming limited by the rate at which certain compounds can diffuse to or from the cells. Diffusional limitations can create heterogeneity within particles, leading to the pos- sibility of undesirable byproduct formation, but can also prove advantageous under certain circumstan- ces, leading to favourable local reaction conditions.

A further consequence of confining the cells as a discrete phase dispersed throughout the fermen- tation medium is the potential for maintaining lower

2.0

7

_c 1.5 3 E bJl 5 10 x

.z

.;

2 05 a

2 a

0 0 0 1 02 0.3 04

Dilution rate (h-l)

Fig. 4. Results from continuous fermentations to oxidize Fe SO, solutions using immobilized T. fen-ooxidans cells [ 111.

C. Webb, B. Atkinson / Role of chemical engineering in biotechnology B15

bulk liquid viscosities than would prevail in normal free cell culture. This can be an important factor, particularly in fermentations involving filamentous organisms, in sustaining reasonable gas-liquid mass transfer rates. It can also make cell-liquid separation much easier since, when cells are to be recovered for disposal or processing, this can be achieved simply by draining the fermenter. Figure 5 shows that atration of a Penicillium chrysogenum broth is considerably easier when immobilized cells are used. The potential also exists for cell production and product recovery to be carried out in the same vessel, thus integrating the first downstream pro- cessing step with the fermentation.

In addition to the above there are a number of other features of immobilization which may lead to further possibilities. For example an immobilized cell system can withstand large fluctuations in liquid loading with no appreciable change in cell con- centration. In the limit, this can mean operating with extended periods of downtime (i.e. no flow). The immobilized cells, since they can be readily separated from the fluid phase, may thus provide a convenient form of storage for large quantities of cells which can be brought into action almost immediately as required. This aspect is demonstrated by the results shown in Fig. 6, in which a continuously operating beer fermenter was stored for a period of 1 month at 0 “C and then restarted with no apparent loss of activity. It is also possible that more than one species can be immobilized (inde- pendently if necessary) and used together in the same reactor so that a controlled mixed culture can be sustained. This offers the further possibility of the spatial location of different species in different parts of a reactor, performing different duties when the chemical composition of the medium also varies spatially owing to the combined effects of reaction and, say, plug flow or the judicious use of a recycle in multiple-tank continuous systems.

Raw medium Imm. celIs Free cells

Fig. 5. Results of filtration tests on fermentation broths for free and immobilized cell penicillin fermentations, after fermentation for 72 h [12].

1

Time (days)

Fig. 6. Results from a continuous brewing trial using immobilized yeast cells, inwhich operationwasinteruptd and the immobilized cells stored for a period of 1 month.

3. Concluding remarks

The above examples illustrate some of the many ways in which the chemical, or process, engineer can contribute to the development of economic industrial biotechnology. There are many challenging opportunities for the chemical engineer in biotech- nology, the most far reaching of which are yet to be addressed. These are areas in which the engineer and biotechnologist must work hand in hand, firstly to identify the problems and then to develop so- lutions.

In general, by comparison with chemical pro- cesses, biological processes suffer from low volu- metric rates of reaction and low product concen- trations. Some improvement may already be possible by process intensification, through the use of cell immobilization, but this is limited. Many micro- organisms may not possess the ideal physical and physiological characteristics for immobilization, par- ticularly where viability and growth are essential for production. Some improvement might be possible if, for example, cells can be “engineered” to adhere to surfaces or to express products either intra- cellularly or extracellularly, or to release products after stimulation, according to the needs of the overall process.

By far the majority of commercially successful large-scale fermentation processes producing rel- atively low value products are those which can be operated non-aseptically. The costs (both capital and operating) of achieving sterilization and main- taming asepsis are considerable and any process for which these can be relaxed is clearly at an advantage. Such processes tend to be largely “nat- ural” fermentations in which the micro-organism of interest has evolved, in its growth environment, a competitive edge over possible infectants. It may

B16 C. Webb, B. Atkinson / Role of chemical engineering in biotechnology

be possible, through genetic manipulation, to confer similar advantages on currently less robust species.

It is also true that the more successful large-scale processes are those which have relatively simple downstream processing requirements. Although much attention is currently being given to the scale- up of highly specific separation processes, based on techniques often already available in the analytical laboratory, these are likely to remain expensive and therefore limited to high value products. The de- velopment of low cost separation processes or, alternatively, fermentations requiring little down- stream processing still remains a major challenge to chemical engineer and biotechnologist alike.

Finally, perhaps the most challenging role for the chemical engineer in biotechnology is associated with the waste that fermentation processes produce. Unlike chemical processes there are usually very few byproducts of an industrial fermentation pro- cess. Often the best that can be achieved is sale of spent biomass as a source of animal food. Low grade carbon compounds, such as COa, are produced at the expense of relatively high grade compounds such as sugars. In addition the processes are usually associated with the production of large quantities of low grade heat and substantial high biological oxygen demand wastewaters, little of which can currently be recovered, re-used or recycled. If bio- technology is to compete with chemical technology in anything other than high value speciality products, these are the problems which must be addressed.

References

1 M. .I. Kennedy, The evolution of the word biotechnology, Tr& Biotechnol., 9 (1991) 218-220.

2 R. Bud, Janus-faced biotechnology: an historical perspective, Trends Biotechnol., 7 (1989) 230-233.

3 B. Atkinson, The process engineering challenges of bio- technology, in B. Atkinson (ed.), Research and Innovation

for the 1990’s: The Chaica.1 Engineering Challenge, In-

4

5

6

7

8

9

10

11

12

stitution of Chemical Engineers, Rugby, 1986, pp. l-27.

C. Webb and S. P. Kamat, Improving fermentation consistency through better inoculum preparation, Biopractice, (1992), in press. R. C. S. Law, The rheology of dextran fermentation broths, MSc Dissertation, University of Manchester Institute of Science and Technology, 1990. C. Webb, G. M. Black and B. Atkinson, Process Engineering

Aspects oflmmobilised CellSystems, Institution of Chemical Engineers, Rugby, 1986. A. Rosevear, J. F. Kennedy and M. S. Cabral, Immobilised

Enzymes and Cells, Adam Hilger, Bristol, 1987. J. Tampion and M. D. Tampion, Immobilised Cells: Prin-

ciples and Applications, Cambridge University Press, Cam- bridge, Cambridgeshire, 1987.

M. Moo-Young, Bioreactor Immobilised Enzymes and

Cells: Fundamentals andApplications, Elsevier, NewYork, 1988.

C. Webb, The role of cell immobilisation in fermentation technology, Au&. J. Biotechnol., 3 (1) (1989) 50-62. H. Armentia and C. Webb, Ferrous sulphate oxidation using Thiobacillusfemooxidans cells immobilised in polyurethane foam support particles, Appl. Microbial. Biotechnol., 30 (1992) 609-613.

P. A. L. Rodrigues, Penicillin production by cells immobilised in foam BSPs, M.Sc. Thesis, University of Manchester In- stitute of Science and Technology, 1990.