Advantages of using thermophiles in biotechnological processes: expectations and reality

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Sano, K., Takinami, K. and Patte, J-C. Biotechnology Letters. 3, 461-464 24 Miwa, K., Terabe, M., Ishida, M., (1982) Eur. J. Appl. Microb. Biotechnol. 31 Ruklisha, M., Marauska, D., Shivinka, Matsui, H. and Momose, H. (1981) 15, 227-231 Japan patent 56-160997 J., Toma, M. and Galynina, N. (1981) 25 Debabov, V. G. (1982) Proc. 4th Int. 28 Lebeual, J. M. and Patte, J-C. (1982) Biotechnol. Lett. 3, 465-470 Proc. 4th Int. Symp. Genet. Ind. Symp. Gent. Microorg. p. 9 32 Fukumura, T. (1977)Agric. Biol. Microorg. p. 113 26 Richaud, F., Richaud, C., Haziza, C. Chem. 41, 1327-1330 and Patte, J-C. (1981) C. R. Acad. Sc. 29 Akashi, K., Shibai, H. and Hirose, Y. (1979) Agric. B i o L Chem. 43, 33 Ahmed, S. A., Esaki, N., Soda, K. and Paris sdrieIII 293, 507-512 Yotsumoto, K. (1981) Seikagaku (J. 2087-2092 27 Reverend, B. D., Boitel, M., Japan. Biochem. Soc.) 53, 841 Deschamps, A. M., Lebeanlt, J-M., 30 Hilliger, M. and H~nel, F. (1981)

Advantages of using thermophiles in biotechnological processes: expectations and reality Bemhard Sonnleitner and Armin Fiechter When thermophilic organisms were first considered for use in biotechnology, certain advantages were expected. The extraordinarily high reaction rates and insensitivity of processes to contaminations have not been experienced in practice, but over the last decade increasing interest has been shown in the possibility of deriving a wide variety of bioproducts from thermophiles. In addition to their high thermo. stability they also promise to have greater tolerance to organic solvents and a longer useful life. The possibility of recovering volatile products directly from a culture provides the opportunity to develop simplified, elegant bioprocesses. However, a series of engineering problems remain to be solved. Thermophilic organisms The term 'thermophilic' is rather illdefined and since it is central to this article, it must be specified further. During the last decade the expression 'thermophilic' has been increasingly restricted to organisms which can grow and/or form products at temperatures ~65°C. In 1953, Allen described at least six different species of the genus Bacillus as being 'thermophilic'. In Bergey's Manual of Determinative Bacteriology, 1975, however, Gibson and Gordon accepted B. stearothermophilus as the only true thermophilic Bernhard Sonnleitner and A r m i n Fiechter a r e a t the Department of Biotechnology, Swiss Federal Institute of Technology, ETH-Ziirich Hoenggerberg, C H 8093 Z i i r i c h , Switzerland.

species in this genus. B. coagulans, which has a maximal growth temperature of below or around 65°C, was classified as 'thermotolerant'. Growth or product formation at this temperature is currently used to distinguish thermophiles and non-thermophiles. If growth occurs at temperatures ) 7 5 ° C the respective organism is termed 'extremely thermophilic' or 'caldoactive'. However, this terminology is likely to be revised in the near future, since organisms capable of growth at temperatures exceeding 100°C have been found in the last few years (see Table 1). Many organisms that can be cultivated at temperatures only little over 37°C are also often named 'thermophilic', for instance several strains ofB. subtilis or B. licheniformis, because they are often used for the

© 1983,ElsewerSciencePubhshersB.V.,Amsterdam0166-9430/83/$01.00

production of thermostable technical enzymes. Many thermophilic organisms are known today, among them bacteria, algae and fungi. The group of true thermophiles, i.e. organisms that require high temperatures and are not active at ambient temperatures, comprises only a few species of eubacteria and most of the species now known as archaebacteria. Among them are strains of Bacillus, Thermus, Clostridium, Thermoanaerobacter, Thermoanaerobium, Thermobacteroides and of Methanobacteriales, Sulfolobales, Thermoplasmales, and Thermoproteales. They are either neutrophilic and acidophilic strains, but no thermophiles have been found so far among the alkalophiles. The absolute maximal temperature for the growth of eukaryotic cells seems to be around 6 2 ° 0 and for photosynthetic algae and bacteria around 75°O a. However, such statements must be made with caution; in the case of methanogens, it was believed that all were killed above 75 oC due to a disintegration of their inner membrane systems, but since 1981 this view has had to be revised. The isolation of Methanothermus fervidus with an optimal temperature of 97°C by Stetter et al. 3 and the findings of Baross et al?'6 that bacterial communities sampled from super-heated, deepsea waters (temperatures of up to 330°C) could form methane at 100°C (and grow under high pressure at up to 300 °C) overthrew the previous notions about the temperature range of methanogens. From today's knowledge, we must expect to find the most extreme thermophiles within the group of archaebacteria. At present, biotechnologists suffer from a substantial lack of knowledge about the metabolism and natural environments of these extreme thermophiles. Their technical exploita-

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Table 1. Current nomenclature of'thermophily' Technical term

Typical representative

Mesophilic Escherichia coli

Thermotolerant Bacillus coagulans

Thermophilic Bacillus stearothermophilus

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?????

Thermus thermophilus Methanothermus fervidus Isolates of Stetter (Ref. 4) Isolates of Baross et al. (Ref. 5 and 6)

tion, however, promises a range of (qualitatively) new products.

New bio-materials Thermophiles - especially extreme thermophiles - and thermotolerant organisms are expected to be and/or to produce qualitatively new biocatalysts. Since they can grow at high temperatures their cellular constituents such as enzymes, nucleic acids, and lipids or organized structures of these materials such as ribosomes or membranes must function at these same high temperatures. The questions why? and how? are interesting (and often not yet answered), but in this context we will focus on how new the thermophilic biocatalysts are and how they can be made and used. Our expectations (or speculations) need not be limited to biocatalysts that operate at or below the boiling point of aqueous solutions at atmospheric pressure (around 100°C) since, as indicated above, life can be found at even higher temperatures. Today, the most promising thermophilic biocatalysts are thermophilic enzymes, not only because of their enhanced thermostability, but also because they are more resistant to denaturing agents and more tolerant to higher solute (reactant) concentrations. Whether these features are causally linked to thermotolerance or not is still unclear.

Mass-produced biocatalysts The most famous and best investigated thermostable enzyme is probably thermolysin, the protease from Bacillus thermoproteolyticus which retains 86%

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of its activity after 30 h at 70°C. The cultivation of this bacterium takes place at 53-55°C and one would not, therefore, consider this strain a thermophile, but the product is thermostable. Other thermotolerant strains such as B. licheniformis or B. subtilis have also been described (and advertised) as producing enzymes that perform with maximal activity at 60 to 80°C, or even higher. Most often, however, the high activity is at the expense of the amount of active enzyme: at the temperature of maximal activity most enzymes are rapidly inactivated. Therefore, the 'useful temperature range' of the enzymes, i.e. where they are not completely inactivated within a few minutes, is always some 10 to 20°C lower than the activity maximum. Unfortunately, in practice these enzymes are used near their activity maximum if they are available in large quantities and at a low cost and thus the opportunity of recovering the enzyme, after the completion of the reaction, is lost. The advantage of enzymes from true thermophiles is that not only do they exhibit high activity at high temperatures, but they also exhibit high stability. Caldolysin, for example, a Thermus protease has a halflife of 30 h at 80°C, 30 times longer than thermolysin under the same conditions. The aqualysins, proteases from another Thermus strain, exhibit similar thermostability and are, in addition, extremely stable towards denaturing agents 7,s. The situation for amylases is similar. Conventional bacterial amylases are more thermostable than fungal or cereal enzymes with activity maxima

up to 110°C. A Bacillus licheniformis amylase is only active at this temperature for 7 to 11 min 9. We have isolated a series of extremely thermophilic Bacilli with maximal growth temperatures of 80°C or above, that produce amylases with half-lives of 1 to 2 h at 95°C (unpublished). Fig. 1 schematically. compares these products with the equivalent enzymes from fungi, cereal plants and non-thermophilic bacteria, providing a justification for being termed 'new bio-materials'. Most of the enzymes mentioned so far have been reported to exhibit good thermostability only in the presence of Ca 2+ions. This is mostly not significant in practice where crude enzyme preparations are used and the solvent is tap water rather than deionized water. It should be noted that a higher degree of thermophilicity of an organism does not necessarily mean that pure enzymes derived from such organisms will always be very thermostable. For example, while the glucose isomerases produced by thermophilic and thermotolerant Bacilli are quite thermostable, the most thermostable glucose isomerase comes from a mesophilic Actinoplanes which retains activity at 90°C for 20 min. The fact remains, however, that one has a better chance of finding more thermostable or more chemoresistant enzymes in (extreme) thermophiles than in (thermotolerant) mesophiles. Processes using thermophiles still lack the maturity of the classical processes using bacteria or yeasts; this is obviously a consequence of the meagre knowledge of metabolic control and special requirements of the thermophiles, of engineering problems at higher cultivation temperatures, and of the comparative lack of strain development. In a series of cases strains ofthermophiles used in biotechnological pro-

Glossary archaebacteria - are distinguished from eubacteria ('true' bacteria) by certain criteria, notably: (I) no peptldoglycan cell walls, (2) ether-hnked liplds, (3) characteristic tRNAs and rRNAs and (4) all known types live ~n 'unusual' habitats, eurythermal - a broad temperature range. specific growth rate (/~) - is correlated with doubling time (td) as follows' /a = In 2/t d

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preservability, a series of enzymes from thermophiles is going to replace the mesophilic enzymes currently used for ).. analytical purposes. Some examples Iare: alcohol dehydrogenase, glucokinase and glucose-6-P dehydrogenase, l--0 asparaginase, or modification methyle 50" ases. With respect to long-term stability bacterial (conventional)and usability in immobilized systems Z the enzymes from thermophiles seem Z to be superior to those from mesophiles. The immobilized fl-galactoILl sidase from Caldariella acidophila can r~ be stored for more than 8 months without any loss of activity and, under T (*C) Fig. 1. Characterization of heat stability of amylases from different organisms. Amylases operating conditions (70°C) its half-life from thermophiles are significantly more thermostable. Our findings are indicated by the is 30 days with an efficiency of 73%. point in the middle of the thick (= estimated) line. This would justify the term Entrapment of whole ceils resulted in a '(qualitatively) new bio-materials'. Figure adapted from Ref. 9 where the ordinate is termed: further increase of enzyme activity 'activity under suppliers analytical conditions'. (compared with free cells) which was not changed even upon acetone treatAlcohol dehydrogenases from ther- ment~6'~7; such systems can, therefore, cesses have been observed to lose the ability to produce the desired pro- mophiles have a broad substrate spec- be easily cleaned with organic solvents. trum including linear and branched duct(s). This brief sketch of the 'new alcohols and linear and cyclic ketones. qualities' of products from thermoThe enzyme from Thermoanaerobium philes shows the opportunities for these Biocatalysts made in small brockii shows highest activity with products to replace currently used bioquantities The above-mentioned enzymes are secondary alcohols and lowest activity catalysts that are derived from mesoproduced in large quantities (up to 500 with primary alcohols. The enzyme of philes. However, the development of tons worldwide per year; see Ref. 10). If Clostridium thermohydrosulfuricum is economic processes remains to be done they were replaced by enzymes from very similar. The enzymes tolerate high and it must be decided whether the (extreme) thermophiles significantly solvent concentrations and are of prac- 'new product with improved quality' longer useful lives could be achieved. tical use with up to 4 g 1-I alcohol or would really upgrade the particular Whether such a substitution would ketone at 60°C. The NADP-linked system or not. render the enzyme-catalysed processes activity was found to be oxygen-insensialso economical is not clear at present tive and not inhibited by NAD. With Higher growth rates are expected because experience of industrial-scale 2% of 2-pentanone a stereospecific re- from thermophiles According to Arrhenius' law, an inor at least pilot-scale operations is duction could be performed at 25°C lacking. Besides higher thermostability with a 90% yield of D-2-pentanol. The crease in temperature speeds up the expected advantages of thermo- reversibility of the enzymes provides chemical and enzymatic reactions and, philic enzymes are: increased chemo- an elegant means of regenerating therefore, microbial growth and resistance, a different substrate N A D P H without employing an extra product formation. The latter has been spectrum than comparable mesophilic enzyme: 2-butanone can be reduced shown true for a series of microenzymes and/or a different stereospeci- using only low concentrations of organisms as long as the optimal temficity, a longer useful life, less con- NADP in the presence of isopropanol perature is not exceeded. The expectatamination problems if the enzyme re- which is concomitantly oxidized to ace- tion is, however, that this holds true not action takes place at high temperature. tone ~3'~4.Using a continuous culture of only for a particular organism, but for These trends will be shown with a few Thermoanaerobium at 72°C we were the sum of all microbes in general; in able to reduce ethyl acetoacetate to the other words Fig. 2b would describe examples: The extracellular cellulase from Clos- ethyl ester of the S-(+)-3-hydroxybu- reality rather than Fig. 2a. Such a tridium thermocellum is stable at 70°C tyric acid with more than 90% optical generalization is difficult to make for 45 min. It does not show concomi- purity (to be published). The enzyme because a comparison of different types tant proteolytic activity (when not ex- from Thermoanaerobacter ethanolicus is of microbes would hardly be meaningtensively purified) and is not inhibited even more stable (over at least 2 days at ful. We will try to answer the question by Ca 2+, Mg 2+, or Mn 2+ at concentra- 70°C) and has its activity maximum at on a fair basis by comparing mesophilic and thermophilic Bacilli and methanotions greater than l0 mM, as is the 95°C 15. Because of their longer useful life gens: Specific growth rates ofthermoTrichoderma cellulase. The clostridial enzyme is not inhibited by the end- hydrogenases from thermophiles have philic Bacilli can easily reach and product, glucose, nor by moderate con- been proposed for the regeneration of exceed 3 h -~, and the highest reported centrations ofcellobiose. Further, it has N A D H or N A D P H in immobilized growth rates for mesophilic Bacilli are a significantly higher specific activity oxidoreductase systems. For the same around 1.5 h-L Mesophilic methanoreason, namely excellent stability and gens hardly exceed the value of0.15 hthan the fungal enzyme ",~2. 400-

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organisms from different thermoclasses with an activation energy (/3)less than that for a single organism (a). In this context, another phenomenon should be mentioned. The very high growth rates are often gained at the expense of yield. It is notable that aquatic thermophiles, e.g. Thermus strains, seem to grow either very rapidly (in continuous culture), but with an extremely low yield, or they grow very slowly, but utilize the substrates completely and yield biomass concentrations similar to those of mesophiles. An explanation for this meagre growth is still awaited. One speculation is that these organisms may have adopted a mechanism for survival which is different to sporulation: a very low affinity for substrate forces both the substrate uptake and growth to obey first-order kinetics with respect to substrate concentration. It is reasonable to suppose that the most rapidly growing bacteria which cannot form spores must adapt their growth rate to the environment to avoid 'suicide'18.

Higher productivities are expected from thermophiles 28

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Fig. 2. Presentation of Arrhenius diagrams from various thermo-classes of bacteria: natural logarithm of specific growth rate versus inverse temperature are correlated linearilyover a wide range of temperature. (a) Under the assumption that the Arrhenius correlation holds only for single organisms. A typical activation energy (= slope of the linear part of the curve, a) for a bacterium (in this example Bacillus caldotenax) would be 70 to 80 kJ mol-1. It is further assumed that the growth rates of organisms in a higher thermo-class are generally not higher than those of organisms in a lower thermo-class, i.e. thermophiles (T), mesophiles (M), and psychrophiles (P) cannot be distinguished on the basis of their maximal specific growth rates. (b) Basically as under (a): the Arrhenius relation holds for each single organism, but it can also be applied for a set of different organisms from different thermo-classes: thermophiles (T) are assumed to grow more rapidly than mesophiles (M) which again grow faster than psychrophiles (P). Historically, the first expectations were that the corresponding activation energy ~) would be of the same magnitude as for a single, distinct organism, i.e. /3~a. This opinion can no longer be sustained. The figure represents a realistic situation: 0
philes have mostly been determined in continuous culture experiments; it must be remembered that the most rapidly growing variants (or mutants) are enriched in such cultures Is. Such selections have also been reported for mesophiles. Finally, from present knowledge, the expectations with respect to reaction rates shown in Fig. 2b can be considered realistic, i.e. the Arrhenius equation may formally be applied for a set of comparable

Productivity is unfortunately mostly given in terms of yield coefficients only and not in terms of specific product formation rates, a parameter which allows easy and direct comparisons between organisms. From the data presented in the literature, we estimated maximal specific production rates by assuming that the reported yields would also hold true at the maximal specific growth rate. I f the examples listed in Table 2 are compared with mesophilic ethanol producers such as Zymomonas mobilis or yeasts, one is disappointed to find that, contrary to expectations, thermophiles in general do not have higher product formation rates than mesophiles. In the case of mass products such as ethanol it is the product formation rate that counts; in the case of products derived from expensive substrates the yields have to be considered as well. The advantages of thermophilic ethanol production have been shown with a facultative anaerobe (Bacillus stearothermophilus). Evaporation of volatile products at high temperature is an elegant means for both directly recovering the product and removing potentially inhibiting products from the culture. For example, simply by gassing the culture with an inert gas

78

Trends in Biotechnology, Vol. 1, No. 3, 1983

Table 2. Comparison of specific ethanol production rates (%) and product-yield (YP/s) Organisms Clostridium thermocellum C. thermohydrosulfuricum Thermoanaerobium brockff Thermobacteroides acetoaethylicus Thermoanaerobacter ethanolicus Zymomonas mobilis Saccharomyces cerevisiae

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aUnits ofqp: g ethanol (g biomass)-lh -~ and of YP/S:mol ethanol formed (mol substrate)- k bExtrapolated as qp =/ama~ x Yp/slYxJs, where/ama~ is the maximal specific growth rate, YP/sis the product yield: product formed per substrate consumed, and Yyasis the biomass yield: biomass formed per substrate consumed. For more details and literature references see Ref. 18. and applying a low grade vacuum a 76-fold enrichment of ethanol in the 'distillate' could be achieved and the concentration of ethanol in the culture was clearly below inhibiting levels 19,2°. Similar principles should apply to the obligately anaerobic thermophiles which are more likely to be used to produce volatile products on a large scale. All the thermophilic ethanol producers form acetic acid as a by-product, sometimes in greater amounts than ethanol. The only really promising acetogenic thermophile is Clostridium thermoaceticum. However it grows best at neutral pH where the concentration of the volatile acid is less than 1% of the non-volatile acetate ion. To take full advantage of the volatility of the product at the cultivation temperature of about 60°C the cultivation should be operated at acidic pH. Schwartz and Keller 21'2~have selected variants of that strain which can grow at pH 4.5, i.e. below the pKa of acetic acid. However, the acidic-pH tolerance is gained at the expense of specific growth (only some 0.019 instead of 0.14 h-l) and product formation rates; also, less acetic acid is produced and glucose is no longer fully depleted. It should be possible to improve these features by continuously removing acetic acid from the culture since the free acid is more inhibitory than the ion. The productivities achieved at pH 7 (about 2 g 1-1h-~) and at pH 4.5 (0.086 g 1-Zh-1) are clearly below the limit of 5 g 1-1h-I which is regarded as economic.

Tolerance of thermophiles to their products

('concentration limitation'). Though their specific product formation rates do not differ by orders of magnitude from those ofmesophiles, the presently known thermophilic (wild type) strains must be considered inferior to the classical mesophiles. Acetobacter strains produce up to 15% of acetic acid whereas pronounced product inhibition limits the concentrations so far achieved with Clostridium thermoaceticum to 2% at neutral pH and only 0.5°7o at pH 4.5. Thermophilic ethanol producers are usually inhibited by as little as 1% ethanol and even tolerant mutant strains are inhibited by 2 to 8% 1~'23. By contrast Zymomonas produces up to 12 or 16% ethanol and yeasts up to 20%. Even if one bears in mind that practically no strain- and process-development has been done for the thermophiles, the expected higher productivities have not been achieved in practice.

This makes it all the more necessary to take full advantage of the high temperature for easy removal and cheap recovery of the volatile products from the cultures. This feature can render a whole process more economical. With only one significant exception, namely gaseous substrates and products, the reactants in microbial processes become more soluble at higher temperatures. This would allow one to use higher substrate concentrations and consequently to upgrade the volumetric productivity. With the exception of the typically aquatic Thermus strains and, of course, the obligate autotrophs which are inhibited by relatively low concentrations of organic material is, no thermophiles have so far been reported to be inhibited by high substrate concentrations. Although media concentrations higher than 20% have yet to be tested, the chances seem good that these organisms could tolerate even higher substrate concentrations. As shown in Fig. 3 the solubility of gases drastically decreases with increasing temperature. This can represent a serious problem, especially for aerobic thermophilic cultures. Firstly, the specific oxygen uptake rates of thermophilic obligate aerobes tend to be higher than those of comparable mesophiles. Secondly, the critical oxygen partial pressure may be higher for thermophiles than for mesophiles (shown only for one Thermus strain) but this must be checked for more organisms in the future. These facts necessitate higher oxygen transfer

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Trends in Biotechnology, VoL 1, No. 3, 1983

capacities of the reaction systems. Consequently, strict aerobic thermophiles can only be cultured to acceptable densities in high-performance bioreactors. Fig. 4 shows this trend, assuming that a certain percentage of dissolved oxygen concentration (1 and 50% of saturation, given in terms of equilibrium oxygen partial pressure) must be maintained. The curves would be even steeper if a constant oxygen concentration in the culture had to be maintained throughout the temperature range. The dependence of kLa on temperature has been neglected. But one also has to keep in mind that gaseous products must be removed from cultures, especially if they are inhibitory. Hydrogen gas, for example, inhibits growth of Thermoanaerobium brockff totally at a partial pressure of less than 1 bar; Thermoanaerobacter ethanolicus is even more pronouncedly inhibited. Carbon dioxide also inhibits a series of mesophiles at greatly differing partial pressures24; data on thermophiles are rare. Inhibitory gas concentrations in anaerobic cultures can be avoided by gassing with inert gases or by controlling their formation rate. The latter approach may be much cheaper, is more elegant and has been shown to work. The addition of acetone as an electron acceptor to a culture of Thermoanaerobium brockff reduced hydrogen formation and could speed up the specific growth rate significantly; a similar effect was obtained if hydrogen was scavanged by a thermophilic methanogen cocultured with Thermoanaerobiurn 25 or with a Clostridiurn. However, the wider applications of this kind of trick are limited. Engineering and process control A high portion of all the studies with thermophiles have, unfortunately, been conducted using tubes or flasks only. Experiences with controlled labscale or even large-scale bioreactors are rare. However, they show that the stress of the high temperatures on 'biotechnology hardware' must not be under-estimated. Leaching of trace elements or plasticizers from steel or peripheral equipment has been reported to cause serious inhibition of growth ofthermophiles 26. To take full advantage of controllable bioreactors a series of sensors have to be used. The performance of these instruments deteriorates more rapidly at high

79

temperatures, producing drift or noise on the signals. This is not a problem which is peculiar to thermophilic culture conditions but in these cases it does become obvious more rapidly. We hope that these problems will motivate both researchers and industry to improve the existing sensors and to develop new ones to control biotechnological processes. The 'cooling problem' has, of course, to be considered in this context. If the linear correlation between specific rate of heat production and specific growth rate or specific oxygen uptake rate holds true for thermophiles - which remains to be checked in detail - as it does for mesophiles 27, a similar or higher heat evolution must be expected during thermophilic cultivations. However, the higher the temperature of cultivation, the larger is the temperature gradient between culture and coolant and, hence, cooling is easier and cheaper. In reactors with a high surface/volume ratio extra thermal insulation may be required to prevent convectional temperature losses if dilute suspensions of anaerobic organisms (with a much lower specific heat production than aerobes) are cultured. This last problem, however, will be minimized with increasing scale. Evaporation must also be considered since it causes both the removal of

water (solvent) and volatile products and the removal of heat (of evaporation) from the system. As discussed above, this may be desirable for lowering the concentrations of inhibitory products, it also can be a cheap means of cooling. On the other hand, the volume of liquid in the reactor will not remain constant and the concentrations of reactants may increase significantly and this may be very undesirable. Evaporation must therefore be controlled. This could be done either by sparging with humidified gas or by adding sterile water or by using efficient reflux coolers; these are additional, complex and costly procedures. It has often been postulated that thermophilic bioprocesses would become contaminated rarely, if at all. The idea that ubiquitous and hence potentially infectious organisms are unable to grow under thermophilic conditions is wrong. Viable forms of thermophiles have been found everywhere on earth ~, thermophilic sewage and waste treatment processes function without human help and thermophiles occur in geographically very distant aquatic habitats that are fed by superheated geothermal waters at rates that would wash out the ceils. The experiences of researchers working with thermophiles show that their cultures are infected approximately as

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often as mesophilic cultures. Most in- 16 deRosa, M., Gambacorta, A., Lama, L., Nicolaus, B. and Buonocore, V. fections found in our lab were caused (1981) Biotechnol. Letters 3, 183-186 by spore-forming species. This experience is also shared by Zeikus who 17 Drioli, E., Iorio, G., Santoro, R., deRosa, M., Gambacorta, A. and reported the overgrowth of Thermus by Nicolaus, B. (1982)J. Mol. Catal. 14, Bacillus in improperly sterilized media 247-251 and even growth ofclostridial species in 18 Sonnleimer, B. Adv. Biochem. Eng. (in purely autotrophic cultures of Methpress) anobacterium2L T. D. Brock wrote in 19 Atkinson, A., Ellwood, D. C., Evans, 1969 that 'the quality of aseptic C. G. T. and Yeo, R. G. (1975)Biotech. techniques of people working with Bioeng. 17, 1375-1377 thermophiles deteriorates with time, 20 Hartley, B. S. and Pyle, D. L. (1983) in Proc. Biotech. '83, pp. 895-905 sometimes with disappointing results". High cultivation temperature per se 21 Schwartz, A. D. and Keller, F. A. (1982) Europ. Patent Applic., Publ. 0 must not be regarded as a patent 043 071 solution for improper or non-aseptic 22 Schwartz, R. D., Keller, F. A. (1982) working techniques.

Appl. Environm. Microbiol. 43, 13851392 Wiegel, J. (1980) Experientia 36, 1434-1446 Jones, R. P. and Greenfield, P. F. (1982) Enz. Microb. Technol. 4, 210-223 Zeikus, J. G., Ben-Bassat, A., Ng, T. K. and Lamed, R. J. (1981) Basic Life Sc~. 18, 441-461 Sonnleitner, B., Cometta, S. and Fiechter, A. (1982)Biotech. Bioeng. 24, 2597-2599 Cooney, C. L., Wang, D. I. C. and Mateles, R. I. (1968)Biotech. Bioeng. 11, 269-281 Zeikus, J. G. (1979) Enz. Microb. Technol. 1, 243-252

References 1 Brock, T. D. (1978) in Thermophilic microorganisms and life at high temperatures (Starr, M. P., ed.), SpringerVerlag 2 Langworthy, T. A. (Rapporteur) (1979) LifeSci. Res. Rep. 13, 489-502 3 Stetter, K. O., Thomm, M., Wimer, J., Wildgruber, G., Huber, H., Zillig, W., Janecovic, D., Koenig, H., Palm, P. and Wunderl, S. (1981) Zbl. Bakt. Hyg., I. Abt. Orig. C 2, 166-178 4 Stetter, K. O. (1982)Nature, 300, 258-260 5 Baross, J. A., Lilley, M. D. and Gordon, L. I. (1982)Nature 298 (5872), 366-368 6 Baross, J. A. and Deming, J. W. (1983) Nature 303 (5916), 423-426 7 Morgan, H. W., Daniel, R. M., Cowan, D. A. and Hickey, C. W. (1981) Eur. Patent Applic. 80302743.2; Publ. 0 024 182 8 Taguchi, H., Hamaoki, M., Matsuzawa, H. and Ohta, T. (1983) J. Biochem. 93, 7-13 9 Godfrey, T. (1983) in The application of enzymes in Industry (Godfrey, T. and Reichelt, J., eds), pp. 466-502, The Nature Press 10 Aunstrup, K., Andresen, O., Falch, E. A. and Nielsen, T. K. (1979) in Microbial Technology. Microbial Processes. I, (2nd edn) (Peppier, H. J. and Perlman, D., eds), pp. 281-309, Academic Press 11 Ng, T. K., Weimer, P. J. and Zeikus, J. G. (1977)Arch. Microbiol. 114, 1-7 12 Johnson, E. A., Reese, E. T. and Demain, A. L. (1982)J. Appl. Biochem. 4, 64-71 13 Lamed, R. J. and Zeikus, J. G. (1981) Biochem. ,,7. 195, 183-190 14 Zeikus, J. G. and Lamed, R. J. (1982) US Patent 4352885 15 Ljungdahl, L. G., Bryant, F., Carreira, L., Saiki, T. and Wiegel, J. ( 1981) Basic Life Sci. 18, 397-419

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Enzyme engineering: applications and promise William H. Rastetter Enzyme research and development have been shaped by the tools and concepts available for the production and utilization of enzymes. A new phase of enzymology has begun as recombinant DNA technology has made it possible to produce large quantities of pure, naturally occurring enzymes and to design modified enzymes with biotechnological uses. Recombinant DNA (rDNA) technology provides a means of producing natural or modified proteins with a facility unprecedented in conventional protein chemistry. The design and rDNA-directed alteration of enzymes, or enzyme engineering offer a wealth of information on the relationship of protein structure to enzyme function and activity. The structures of enzymes have evolved in response to the specific metabolic demands which these catalysts meet in vivo. Outside of the natural setting, however, an enzyme may not be well suited to an alternate use, e.g., as an industrial catalyst. Ideally and engineered enzyme will preserve or enhance the catalytic properties of the wild-type protein while bringing altered properties which are advantageous for industrial catalysts. For instance, improved thermal stability or an akered pH-activity profile would bring advantages to an enzymatic process stream which operates at nonphysiological temperature and pH. Thus, enzyme engineering entails the

tailoring of protein catalysts to meet the specific demands of the end user. More broadly, protein engineering may apply to all classes of protein products which emerge from the rDNA industry, including enzymes for therapeutic or diagnostic use, the interferons, growth hormones and a variety of other proteins of potential value in human and animal health care. Engineered proteins for pharmaceutical or veterinary use may display enhanced pharmacokinetics and altered activities, or increased stabilities as formulated products.

Study of enzyme structure and function To study enzyme structure and function systematically requires the means to make selected structural changes in the enzyme. Naturally occurring mutant enzymes provide a set of proteins for examination, but the number and variety of such proteins are limited. Moreover, the total chemical synthesis of enzymes and mutant enzymes from amino acids is impracticable since the chemical synthesis, of even small proteins is William H. Rastetteris Director, Chemical Sciences, Genentech, Inc. and Genencor, extremely laborious. Synthetic studies Inc., 460 Point San Bruno Boulevard, have focused, instead, on the chemical South San Francisco, CA 94080, USA. modification 1 of enzymes of natural © 1983,ElsewerSclencePubhshersB.V,Amsterdam 0166-9430/83t501.00