Growth, Products, and Application
6.5 Stability of Organisms
Inherent stability or the proper means to stabilize microorganisms are absolute requirements for their technical application. From the data published on instability of characteristic properties of thermophiles, one might assume that besides other kinetic parameters, the probability of mutation or adaptation is also increased.
No conclusive answers are possible since the molecular bases of the observed alterations are not clear at present. However, metabolic regulation should — by definition — be reversible. Exact reversibility of physiological and/or kinetic changes has not been reported. On the other hand, during the same period of time, a higher number of generations of the more rapidly growing thermophiles has been observed than of mesophiles; a higher number of mutants would consequently be found even at the same frequency of mutation. But all these speculation's do not provide satisfactory explanations as to why the variations of characteristic properties occur either in continuous culture after a relatively low number of generations has been grown or upon repeated subcultivation on solid or liquid media. This fact supports the theory of enrichment of 'specialists' which are either present in the parent culture or arise from mutation due to a selection pressure, the character of which is not known to the experimentator.
Thermus aquaticus could be grown as a pure yellow culture only in batch systems.
Upon starting a chemostat, white cell types emerged, increased in number and could overgrow the yellow cells within a few volume changes (independent of dilution rate).
The resulting pure or mixed cultures were either stable or unstable in chemostat as shown by the x-D-diagrams, with no hysteresis (stable population) or with an 'open hysteresis' (change of population) 7 3 , 1 4 8 ). Although the populations were kinetically quite different, on the basis of their biochemical characteristic properties and the typical cross hatched structure of the outer membrane, it was concluded that all the observed populations were Thermus aquaticus, t o o7 3 , 8 4 ). On the other hand, T.
thermophilus has been found to be stable in chemostat cultures over a period as long as 3 months 7 2 ). T. thermophilus has been described as having quite different growth rates (on very similar media): μmax was found to be about 0.15 h-1 1 5 2 ), 0.8 h-1 1 9 4 ), 2.2 h-1 2 1 9 ) and up to 2.7 h-1 72). The lysine auxotrophy found for T. thermophilus 1 3 1 ), support of growth through vitamins and amino acids 2 8 , 2 1 9 , 3 4 9 ), and substrate inhi
bition only at relatively high concentrations 1 4 7 ) could not be verified in chemostat studies 72).
The loss of exoenzyme productivity of Bacillus subtilis strain NRRL-B3411 observed in the early phase of a chemostat on different media was interpreted as due to the takeover by mutants 2 8 5 ). The originally pure (cloned) culture of a thermophilic Bacillus producing a-amylase was found to segregate into two types of cells in an early phase of chemostat culture. Both cell types could grow on nutrient broth, but only one, the transparent type, could grow on starch alone. The second type was able to grow in the vicinity of the transparent type on starch agar plates because it could utilize the reducing sugars produced by the readily diffusing a-amylase of the transparent type from starch 7 3 ). The ability of B. stearothermophilus strain
1503-4 to produce high amounts of a-amylase has been irreversibly lost, only less than 1 % of the originally reported quantity2 8 4 ) could be detected even upon reisolation of the original rough strain 35C).
Clostridium thermocellum, originally requiring more than 0.1% of yeast extract in the medium for growth, could be 'adapted' by a few serial transfers to grow equally well in the presence of only 0.02% yeast extract. A spontaneous mutation of the glucose transport system was assumed to be the reason 11). However, the glucose transport system of C. thermocellum is under metabolic control 3 5 1 ).
For 'thermoadaptation' in the case of Bacillus caldotenax, as postulated earlier 1 0 4 ), no indication could be found using continuous culture technique 6 7 ).
The problem of instability of thermophilic strains is, of course, a biological one which remains to be clarified, but it obviously is not restricted to thermophiles.
It therefore should be regarded instead as a technical problem, and its solution depends on labor efforts and time; mesophilic production strains have not been developed within only a few years; they now have histories of up to several decades of continuous improvement, stabilization and preservation.,
6.6 Cultivation Systems
Many microbiologists have tried to carry out their experiments on solidified media or in standing or shaking flasks because these methods are not complicated and are thus easy to use. However, the disadvantages of these systems must be carefully con
sidered. Low heat and mass transfer and incomplete mixing may cause a significant amount of artifacts if the reaction rates of the organisms are high, e.g., even
dilute cultures of obligate aerobes may be oxygen limited and/or affected by carbon dioxide. Evaporation must be compensated for either by the addition of sterile water or by calculations. Since p02, pH and redox potential cannot be controlled, unknown shifts of these parameters can easily produce incomplete or misinter- preted results, such as 'growth did not cease due to substrate depletion'. On the other hand, the metabolic properties of thermophiles, such as the utilization of special substrates or the excretion of enzymes, are sometimes not expressed in submerged culture 131, 278-279) where influences from the relatively inert materials of vessels and supports are not expected. Deficiencies in measurement and control are easily eliminated by using controlled bioreactors. This first step of process development should be made as early as possible, if necessary, in combination with uncontrolled culture systems to find out the significant differences (parameter identification).
Two inherent disadvantages of discontinuous systems are the continuous changes of the chemical conditions and physiological state, and the limited range of observable generations may become significant. Many contaminating organisms may be cultivated and remain undetected since they are not sufficiently diluted out or selected during the short period of growth. Or if adaptation to different concentrations is necessary, as in the case of substrate inhibition, unpredictable, long' lag times can occur or the actual growth and turnover rates of substrates and products will never meet the maximal capacity of the organism. The use of continuous culture systems can prevent these problems. Steady states are independent of time and the specific growth rate of the organism can be chosen and preset by the experimentator. These techniques provide a powerful means for measuring the kinetics of fast reactions such as the growth and product formation of thermophiles, for investigating metabolic control, testing the stability of an organism, selecting and detecting improved strains
Fig. 7. Dependence of specific growth rate on the culture system. Data from,72,73,148) for Thermus aquaticus and T. thermophilus. Growth media were: 3 g l- l peptone and 5 g l- 1 meat extract.
Growth conditions: pH 7, 75 °C, in controlled bioreactors: p 02 80% of air saturation at 75 °C.
Only the white type cells of T. aquaticus were grown on a defined glucose medium, see 84)
(mutants or new organisms), and last but not least, for developing and optimizing of appropriate media.
Continuous cultivation is advantageous not only for basic research and process development, but also for production purposes if one takes into account that many potentially useful thermophiles are inhibited by either substrates or products. The respective concentrations can be almost arbitrarily chosen, preset and controlled (within the given limits) in a continuous culture. How much this explains why
Thermus strains can be cultivated in chemostat at more than 5 times greater specific growth rates than in batch systems is not clear (Fig. 7). In any case, these findings undoubtedly indicate that Thermus strains show great potential for use in continuous cultivation systems only.
7 Future Prospects
During the last years, in particular, the great technical potential of thermophilic microorganisms has been shown by studies on the formation of thermostable enzymes, immobilization of thermophilic whole cells and enzymes, the production of chemicals, solvents and fuels, and the utilization of abundant renewable substrates by thermo- philes. Undoubtedly, the spectrum of products formed and substrates utilized will be increased in the future as new organisms are isolated and/or constructed. From the point of microbial diversity, rapid development and prosperity of thermophilic biotechnology can be expected.
Although many of the expected benefits of thermophilic processes can at present be regarded as realistic, some of them have been overestimated and some disadvantages have probably been underestimated. It would be of practical use to discuss these features from three different points of view: (7.1) technical realization and equipment, (7.2) regulation and metabolic control, and (7.3) genetics.