Some Origins of Biotechnology
Prof. R.K. Finn, School of Chemical Engineering, Olin Hall, Cornell University, Ithaca, N.Y.
14853-5201, USA
On the occasion of Professor A. Fiechter’s 65th birthday it is appropriate to reflect on developments in biotechnology over the past fifty years. It was in 1940–41 that Florey, Chain and their collaborators demonstrated the full therapeutic value of Fleming’s penicillin [1]. Thereby was ushered in the «age of antibiotics» and with it modern biotechnology–a fermentation industry capable of maintaining strict aseptic conditions on a large scale in stirred, aerated tanks. This short discourse sets down in chronological order some events and achievements of modern biotechnology from its beginnings, with the production of antibiotics in the early forties, to the advent of the «new biotechnology» with genetically engineered cells in the mid-seventies.
Earlier roots
From our vantage point fifty years later is it difficult to appreciate the radical implications of the submerged culture of molds. The basis for this methodology was laid by Kluyver and his student Perquin in their 1933 paper from Delft on the use of shake–flasks for physiological studies [2]. Nevertheless there was grave doubt that sterile air and sterile seals on rotating shafts could be maintained over week–long periods on an industrial scale–not just in the controlled environment of the laboratory. Consequently surface culture continued in use during the first three years of penicillin production in the United States; it was simply an extension of the method used for citric acid production.
Prior to this time, the only experience in deep tanks had been with fermentations to make alcoholic beverages, baker’s yeast, or lactic acid. None of these required more than pasteurization of the mash and steaming of the equipment. The sole exception was in the manufacturing of acetone and butanol by solventogenicClostridium species, where phage infections had nearly proved disastrous. That industry was able to recover however by establishing strict asepsis; and since the fermentations did not require either aeration or agitation this was relatively easy to accomplish.
Equally important, as a deterrent to developing a modern fermentation industry, was the widespread view that once their chemical structure was known, most natural products would be more economically synthesized by organic chemists than by microorganisms. Precedent had been established not only with the popular sulfa drugs but also with the various vitamins: thiamine, pantothenate, ascorbic acid, even the relatively complex molecule of riboflavin.
The forties
The manufacture of penicillinwas followed rapidly by that of streptomycin. Although the Waksman patent was not issued until 1948 [3], production of streptomycin was already in full swing from the Merck plant, built in Elkton, Virginia. Figures for 1947 to 1949 are shown in Table 1.
Table 1: Production of Penicillin and Streptomycin in kg from 1947 to 1949 in the United States (Source: US Dept of Commerce).
Penicillin
Streptomycin / 24 856
9 676 / 57 513
37 709 / 80 078
83 699
Other antibiotics also became prominent during this period: chloromycetin, aureomycin from Lederle Laboratories, bacitracin, neomycin, polymyxin. Tetracycline itself was not approved for use until 1953.
An important development for establishing the validity of microbial technology was the discovery of vitamin B-12 in 1948. It was not only of therapeutic value in treating pernicious anemia, but turned out to be the long-sought animal protein factor (APF) and attained widespread use in animal feeds. By 1952 the total sales (of 61 pounds, all produced by fermentation) amounted to over $5.5 million [4].
Traditional fermentations waned during these years, especially as the petrochemical industry developed. Thus the raw material for production of acetone/butanol was shifted from molasses to grain, and feed-grade riboflavin from the fermentation residues became almost more valuable than the solvents themselves.
During mostof the forties, research and development on the fermentations was conducted by biologists. Chemical engineers were restricted to working on methods for recovery and purification, despite growing recognition that submerged cultivation of cells involved numerous aspects of heat and mass transfer. In 1944, there appeared a seminal paper on the use of sulfite oxidation to characterize the aeration capacity of stirred tanks [5] and during this period bench-scale fermentations were developed in industry and at the Northern Regional Research Laboratories (NRRL), US Department of Agriculture, in Peoria, Illinois.
The fifties
In the year 1950, a number of significant papers appeared which set the tone for process engineering during the ensuing decade. These included the work on aeration and agitation by Gaden at Columbia University [6] along with similar studies from the team at Merck & Co., under the guidance of Professor Wilhelm of Princeton [7]. Students led by Professor Marvin Johnson at Wisconsin and by Professors Halvorson and Piret at Minnesota began to publish their studies on controlled fermentations. In that same year the chemostat was born (Monod and Novick & Szilard) [8].
In fact, the theme of continuous fermentation was important throughout the decade, led by groups in Scandinavia, Czechoslovakia, and at Porton in England [9]. In 1951, the First International Symposium on Chemical Microbiology was held in Rome and at the time an international research center was inaugurated there, under the direction of Professor E.B. Chain. A «First Symposium on Continuous Cultivation of Cells» was held in Prague 1958. The VIIth International Congress for Microbiology held in Stockholm in August of that same year included symposia not only on continuous culture but also on «Recombination mechanisms in bacteria» and «Role of protein in the nucleic acid synthesis and role of nucleic acid in protein synthesis». The American Society for Microbiology began publication of Applied Microbiology in 1953 (now Applied and Environmental Microbiology).
This was also the time when the Institute of Biochemistry in Uppsala, Sweden, led by Tiselius, was germinating many of the techniques still being used for separation of proteins and other macromolecules. These included preparative electrophoresis, variations on column chromatography, and partitioning between immiscible aqueous phases. Co-workers included leaders such asPorath, Albertsson, and Flodin.
Meanwhile, an exciting new development came from the pharmaceutical industry when the Upjohn Company announced simplification in the synthesis of adrenal cortex hormones. In 1952, a hydroxy group was introduced at the 11-alpha position of progesterone in yields of 85% [10]. In 1953 independent work by the Squibb and Ciba groups showed that microorganisms can introduce double bonds into the steroid nucleus at specific sites. The word bioconversion began to take on added significance.
The sixties
The First International Fermentation Symposium held in Rome during May 9–14, 1960 was the culmination of two decades of rapid development. It was a memorable occasion, attended by 130 scientists and engineers from around the world plus 50 from Italy itself. Many of the topics discussed then are still under investigation – aeration, shear, continuous culture. Oxygen electrodes were described as well as pH measurements. A special panel discussion was devoted to genetics.
Many other institutions were founded also in this decade when modern biotechnology came into full flower. In the United States, the Division of Microbial Chemistry became a separate Division of the American Chemical Society in 1963. The first textbook on bioprocess engineering, by Aiba, Humphrey and Millis, appeared in 1965. The Journal of Biochemical and Microbiological Technology and Engineering (now Biotechnology and Bioengineering) had been established a little earlier, in 1959.
This was, above all, the decade of microbial enzymes. The commercial availability of glucoamylase in 1958 made possible the completely enzymatic conversion of corn starch to glucose during the sixties. Microbial proteases were first used in detergents during this period. Although there were early scattered reports of covalently immobilized enzymes, Manecke in Germany and Katchalski in Israel, along with Crook and others in England developed techniques whereby catalytic activity was largely retained. Dunnill and Lilly established a center for enzyme study at University College in London. Some expected an even greater impact of enzyme engineering than did indeed occur (just as, a decade earlier, the assessment of continuous fermentation was sometimes too sanguine). In any case, during this period considerable support for enzyme research was provided by the National Science Foundation in the United States. By the end of the decade, immobilized enzymes were used for resolution of optical isomers of acyl amino acids and by 1970 immobilized glucose isomerase was used to produce high fructose corn syrup. Finally, an important conference on «Enzyme Engineering» was held at Henniker, New Hampshire, USA in 1971. The study of immobilized cells, either encapsulated in gels or retained in hollow fibers or other membrane devices, also began at about this time.
The discovery of Kinoshita et al. of bacteria that excreted large amount of glutamic acid [11] led to the rapid exploitation of commercial methods for producing natural amino acids. Monosodium glutamate, widely used a flavor enhancer, was manufactured from molasses by fermentation more economically than in the prior art, namely extraction and hydrolysis of precursors derived from agricultural residues.
In the late fifties and early sixties, water soluble polysaccharides from bacteria were under investigation at the NRRL in Peoria, especially xanthan gum from common soil bacteria. This product was commercialized by the Kelco Company in San Diego, California during the period 1960 – 63 and has become a popular food additive.
The seventies
The search for unconventional sources of protein especially for animals, but also possibly for humans, is not a new venture. Nevertheless, in the late sixties and early seventies there was intensive effort to convert petroleum fractions to edible protein by algae, yeasts, molds or bacteria. At the time of the first international conference at the Massachusetts Institute of Technology (MIT) in 1967 the term single-cell protein or SCP was coined [12]. Most of the projects at that time were still in the experimental stage, but by the time of the second MIT conference in 1973 various large-scale plants around the world were being completed. The rise in oil prices during the mid-seventies combined with deep public concern over nutritional safety of the products quenched such early enthusiasm in most countries. Interest in using methanol as a feedstock for SCP production continued through the seventies but economic factors have mitigatedagainst large scale production of animal feeds in most Western countries.Spurred on though by such possibilities a number of sizeable research and pilot plans around the world were established during the seventies. Among the most notable were the GBF (GesellschaftfürBiotechnologischeForschungmbH) at Braunschweig-Stöckheim in Germany and of course Professor A. Fiechter’s Institute for Biotechnology at the ETH Zürich.
In 1973 it was first demonstrated that hybrid plasmids, constructed in vitroby restriction endonuclease cutting, annealing and ligation, could be functionally expressed in Escherichia coli[13]. The next year eucaryotic DNA was successfully cloned, and by the end of the decade synthetic human insulin was produced in Escherichia coli. As if to underscore revolutionary events, hybridoma cells were developed in 1975; these in turn led to the production of monoclonal antibodies. Surely the «new biotechnology» represents a qualitatively new era involving radical changes for medicine and agriculture. The most recent development has extended the genetic engineering approach to the economical manufacture of calf rennet – chymosin – from both Escherichia coli (Pfizer) and from yeast(Gist-Brocades).
Literature
[1] Chain, E., et al.: Lancet 2, 226 – 228 (1940); Abraham, E.P., et al.: Lancet 2, 177 – 189 (1941).
[2] Kluyver, A.J., Perquin, L.C.H.:Biochem. Z. 266, 68 – 81 (1933).
[3] Waksman, S.A., Schatz, A.: U.S. Patent 2, 449, 866 (Sept. 21, 1948).
[4] Hester, A.S. Ward, G.E.: Ind. Eng. Chem. 46, 237 – 243 (1954).
[5] Cooper, C.M. Fernstrom, G.A. Miller, S.A.: Ind. Eng. Chem. 36, 504 – 509 (1944).
[6] Hixson, A.W., Gaden, E. L. Jr.: Ind. Eng. Chem. 42, 1970 – 1801 (1950).
[7] Bartholomew, W. H., et al.: Ind. Eng. Chem. 42, 1801 – 1818; 1827 – 1830 (1950).
[8] Monod, J.: Ann. Inst. Pasteur 79, 390 – 410 (1950; Novick, A., Szilard, L.: Proc. Natl. Acad. Sci. USA 36, 708 – 719 (1950).
[9] Herbert, D., Elsworth, R., Telling, R.C.: J. Gen. Microbiol.14, 601 – 622 (1956).
[10] Peterson, D.H., Murray, H.C.: J. Am. Chem. Soc. 74, 1871 – 1872 (1952).
[11] Kinoshita, S. Nakayama, K., Shimono, M.: J. Gen. Appl. Microbiol.3, 193 – 205 (1957).
[12] Mateles, R.I., Tannenbaum, S.R. (eds): Single-Cell Protein; MIT Press, Cambridge, Mass., USA (1968).
[13] Cohen, S.N., et al.: Proc. Natl. Acad. Sci., USA 70, 3240 – 3244 (1973).
Reprinted from SWISS BIOTECH 7 (1989) No. 4, 15-17.
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