Growth, Products, and Application
2.2 Taxonomy
According to the latest edition of Bergey's Manual of Determinative Bacteriology 68), only two obligate thermophilic species can be distinguished among the neutrophilic Bacilli: B. stearothermophilus and B. coagulans. However, some strains of this latter species have been found to fail to grow at 65 °C. There is one species under the acidophilic Bacilli: B. acidocaldarius. The caldoactive Bacilli isolated and described by Heinen 65'66>, as well as B. thermodenitrificam, and B. schlegelii are not considered there but seem to be accepted in the literature as separate species. However, Heinen and Heinen 65) have differentiated between the three isolates and B. stearo- thermophilus on the basis of a) temperature range, b) fatty acid pattern, and c) ultra- structure. Each of these groups can be differentiated further according to a) temperature optima, b) sporulation behavior, and c) ultrastructure. B. caldotenax (YT-G) could be grown only at temperatures up to 72 °C to 75 °C on all tested carbon sources except on pyruvate, where growth at 84 °C was reported. These findings, however, have not been reproduced thus far. B. caJd&tyticus and B.
caldotenax (YT-G) have been reported to grow prototrophically, whereas B. caldovelox
required methionine (Sharp and Atkinson 6 9 )). In contrast, an absolute requirement of B. caldotenax for methionine and biotin has been demonstrated 67) and a maximal temperature of 72 °C has been found on all carbon sources tested, including pyruvate (unpublished results).
As suggested by these examples, a classical taxonomy of thermophilic Bacilli appears to be problematic. In Bergey's Manual68) B. stearothermophilus is described as markedly heterogeneous. There are uncertainties about its demarcation. The emphasis to grow at 65 °C results in an exclusion of organisms with a maximal growth temperature between 55 and 60 °C from this species although they cannot be distinguished by any other property. Either the distinguishing criteria have been improperly chosen or the stability of the expression of the 'typical' properties has been so low that the resulting taxonomy is meaningless. Changes in properties that previously had been regarded as typical for a particular organism seem to be at least partly responsible for this rather chaotic situation. However, such findings are rarely published (Zuber, personal communication and7 0 ~73)) due to the lack of reliable means for distinguishing between contamination and 'variations' or mutations.
Among genera that contain only a few known strains, such problems are, of course, much less confusing than for the genus Bacillus. Presently taxonomic problems of thermophilic bacteria cannot be considered as solved 74).
Taxonomies based on the guanosine and cytosine (GC) content of the DNA or on the denaturation temperature of ribosomes as compiled by Aragno 21) and Wolf and Sharp 69) may work for a species (or a family); however, there are so many interspecies
differences that a general definition of thermophily based on either of these taxonomies would not be meaningful. Thiobacillus strains even show an inverse correlation: the thermophilic strain TH1 has a lower GC coniont (48%) than the mesophilic T. thiooxidans (51%), strain A2 (69.5%), and other non-iron oxidizing
Thiobacilli (58—66%)63>. This has also been found for the genus Clostridium 75). Attempts to develop a practical approach for the classification or characterization of thermophilic Bacilli have usually consisted of grouping similar strains together.
Allen 20) classified more than 100 strains according to morphological (cells, sporangia) and biochemical characteristics, which resulted in four groups. Klaushofer and Hollaus
76) recorded 68, mostly biochemical characteristics of nearly 90 thermophilic aerobic spore formers, and they calculated simple matching coefficients (according to Sokal and Michener77). On this basis, they constructed a dendrogram and an ordered similarity matrix from which four groups could be distinguished with less than 70 % similarity between the groups, some of which could be substructured even further.
A similar study exists for isolates of the genus Thermus71). Wolf and Barker 78>
and Walker and Wolf79) determined three major groups based on the studies of biochemical, physiological, and serological properties. Successful attempts to set up a taxonomy on the basis of RNA polymerase structure have also been undertaken within the group of archaebacteria 80-81>.
Taxonomy is, of course, not the domain of biotechnologists, but it is definitely a necessary tool for the identification and recognition of organisms of interest. It is important because of the possible instability of characteristic properties of strains, which has been observed in several cases with Bacillus stearothermophilus, B.
caldotenax, Thermus aquaticus and several thermophilic isolates (to be discussed in detail in Sect. 6.5).
3 Requirements for Growth at High Temperatures 3.1 Requirements Fulfilled by the Cells
To take part in reactions of growth and product formation at specific temperatures, all of the constituents of cells must be both stable and active. This has been shown in many cases for enzymes, ribosomes, nucleic acids, and membrane systems.
Thermophilic enzymes are not much different from mesophilic ones. It is difficult to find out the structural and compositional differences that make an enzyme thermostable since the differences of enzymes from different species, that catalyze the same reaction, have the same order of magnitude as the changes of a protein that render it thermostable. Comparison of proteins from similar organisms is therefore necessary. A relatively high content of hydrophobic and ionic interactions contribute to enhanced thermal stability. On the other hand, the enzymes must be sufficiently flexible at high temperatures to do their duty, but consequently, they are not suffi- ciently flexible at low temperatures; therefore, their activities are most often negligible at room temperature. Nucleotides in nucleic acids from thermophiles are more frequently modified than those of mesophiles. The purpose is again to achieve higher thermal stability. Membranes must be in a liquid-cristalline state to fulfill their func- tions. Organisms adapt the fluidity of their membranes to the actual growth temper- ature by changing the pattern of fatty acids ('homeoviscous adaptation') or by syn- thesizing lipids that have been regarded as nontypical for bacteria. Since biochemical and/or molecular biological aspects are not discussed in this review, the following section gives a list of excellent reviews concerning this aspect.
3.2.1 Nutrients
and tryptone for growth. A maximal yield is observed when each is provided at 0.5 %, but a maximal specific growth rate occurs with 0.3% of each, and inhibition of growth at concentrations of each at more than 1 % 26).
Thermobacteroid.es acetoaethylicus and Thermoanaerobacter ethanolicus both require yeast extract22,138); in the latter case it cannot be replaced by tryptone, casein hydrolysate, beef extract or ashed yeast extract (strains JW200 and JW201). But yeast extract is not reported to function as carbon and energy source or at least its functions are very limited 138). Thermoanaerobacter ethanolicus grows in media with up to 10gl"1 glucose. Mass balances have not yet been evaluated in highly concentrated media u>. Yeast extract, and no substitute, is also necessary for growth of Thermoanaerobiwn brockii36). Growth factor requirements of thermophilic Clostridia vary. Clostridium thermocellum strains (ATCC 27405, LQRI, and NCIB 10682) can grow well on defined media that are supplemented with biotin, pyridoxamine, vitamin B12, and p-amino benzoic acid. Either growth factor of vitamin B12 and p-amino benzoic acid can be replaced by methionine but not both together. Using defined medium, slightly more lactate is produced than in a medium containing yeast extractI55). For some strains of C. thermohydrosulfuricum, yeast extract or tryptone have been reported to be necessary for growth, but do not serve as a carbon source. The complex substrates do not reduce growth rates up to 2 g I"1. Lactate, a product, is inhibitory 35). Methanobacterium thermoautotrophicum is an obligate autotroph, and only cysteine is known to stimulate growth. The addition of a cysteine-sulphide reducing agent decreases the minimal doubling time at optimal growth conditions from 5 to 3 h 37). Whether this is a consequence of cysteine addition or of lowering the redox potential is not clear. Yeast extract is not absolutely necessary for growth of Thermoproteus tenax. Addition of 0.1 to 0.2 g l- 1 effected a 5 fold increase of the growth rate but 0.5 g 1_1 were found to be inhibitory. A vitamin mixture cannot replace yeast extract for acceleration of growth 156).
Bacillus acidocaldarius strains have been reported to grow prototrophically 157), yet yeast extract is normally included in the media 1 5 7 _ 1 6 0). Growth is inhibited by citrate and acetate. However, these nutritional studies were done on agar solidified media using replica plating techniques, and not in liquid culture 157). Some other strains isolated from Japanese hot springs require 2 mg l- 1 of biotin for growth in submerged culture 161).
The facultative chemolithoautotrophic Bacillus schlegelii does not require growth factors. Growth is totally inhibited in the presence of 1 % glycine and is strongly inhibited when cultures are shaken, regardless of whether autotrophic or heterotrophic conditions are given 162).
Sulfolobus and Caldariella strains grow heterotrophically on yeast extract as energy source. This is not necessary for autotrophic growth 1 0 9 - 1 6 3 - i 6 5 ) practically all of the thermoacidophiles that are able to leach metals from ores require yeast extract for heterotrophic growth 166). Thermoplasma acidophilum absolutely requires yeast extract which inhibits growth at concentrations higher than 0.5% (optimal is 0.2%)
167-169) yeast extract cannot be replaced by casamino acids, peptone, or glucose.
Of the several hundred compounds tested as substitutes, none support growth, with the exception of a few peptones, ferredoxins and aged glutathione, each of which give only meager responses. Fractionation of yeast extract results in only a minor
natural habitat from which the organism was isolated is 8 ppm. When grown heterotrophically, this organism requires other reduced sulphur sources23*. Of special interest for industrial application are the findings that Clostridium thermo- hydrosulfuricum is 'relatively tolerant' to high heavy metal ion concentrations, acid and base shocks, as well as to vacuum and 02 U ).
3.2.2.3 pH
Brock 18>187> has collected data from hot springs all over the world and found a bimodal distribution with respect to the pH values of the springs: Two peaks, one at pH ~3 and a'broader one from pH ~7 to ~8.5, with a minimum in between at pH ~5.5. It may be surprising that all thermophilic bacteria known today, and not only those found in an aquatic habitat, fall into the following two groups:
— Thermoacidophiles, which include strains of Bacillus acidocaldarius, Caldarie/la, Desulfurococcus, Sulfolobus, Thermoplasma, Thermoproteus, Thiobacillus, and the
— thermophilic neutrophiles, which include strains of Bacillus, Clostridium, Methano- bacterium, Methanococcus, Methanothermus, Thermus, Thermomicrobium, Ther- moanaerobium, Thermobacteroides, Thermoanaerobacter, and Thermothrix.
The first group has pH optima of between 1.5 and 4, the latter between 5.8 and 8.5.
True alkalophilic thermophiles have not been isolated so far.
Data concerning pH minima, optima, and maxima in the literature should be critically evaluated. Often the conditions of pH determination are not given, i.e., whether the pH of a medium was set to distinct values prior to sterilization or was measured during the cultivation, or whether the pH was measured using an electrode or indicator paper. Papers may also give incorrect results at higher temperatures.
Readings from pH-electrodes are only useful if additional information is given: the temperature of measurement and temperature of calibration of the electrode are the minimum requirements. Several suppliers of calibration buffers give the respective pH values at a given temperature between 0 and 100 °C. Various buffers exhibit different temperature coefficients. Not obeying these simple rules or disregarding the dependence of pH on temperature may easily lead to errors of as much as 1 pH unit.
3.2.2.4 Oxygen; Partial Pressure and Dissolved Gas Concentration
One of the advantages of thermophilic anaerobic processes over mesophilic ones is that they are easier to handle and to control due to reduced oxygen solubility 13). On the other hand, this is a real problem for thermophilic aerobic cultures. Fig. 2 shows the saturation concentration of 02 in pure water at 1 atm of air. In the natural habitats, oxygen is transported into the medium only by diffusion. Provided that an aerobic population utilizes this low amount of oxygen, the percentage of total medium allowing growth of strict aerobes is relatively small, as has been shown for a natural ecosystem 144), and provides sufficient oxygen for only very dilute cultures of strict aerobes 18).
At present, very little quantitative data are available on oxygen sensitivity and/or requirements with respect to oxygen partial pressure or dissolved oxygen concen-
Pig. 2. Temperature dependence of gas solubility in pure water for oxygen, hydrogen, carbon dioxide, and methane at partial pressures of 1 atm, for oxygen also at a partial pressure of 0.21 atm, equivalent to 1 atm of air. Plotted from 381)
3.2.2.5 Hydrogen; Partial Pressure and-Redox Potential
Unfortunately, very few quantitative measurements of redox-potential for cultures of thermophiles are available51,188-191). Indirect information may be drawn from experiments using resazurin, which is generally implemented in media for anaerobic organisms to indicate redox potential below or above +0.042 V at neutral pH 192). However, resazurin color is not only dependent on the redox potential of the solution; it is also used as pH indicator and changes from violet at pH 5.8 to orange at pH 3.8. Only a rough estimate of the reduced state of a culture should be drawn from resazurin color. An indication of more than +0.042 V may often be only
an explanation for why a culture no longer grows, since the growth and product formation of many strict anaerobes is dependent on redox potentials of less than
—0.33 V 192). The critical redox potential that must not be exceeded is not a constant for a given organism; it varies with inoculum size and richment of the medium 193).
At present most available information must be drawn from data on hydrogen partial pressure. However, this can be troublesome if the organisms are inhibited by hydrogen. Total inhibition of growth of Thermoanaerobacter ethanolicus occurs if 75 % or more of the gaseous atmosphere consists of hydrogen, the inhibition being detectable from 10% upwards 138). Thermoanaerobium brockii is totally inhibited in the presence of 1 atm hydrogen 14), whereas Thermobacteroides acetoaethylicus is not affected by even 2 atm 22) and Clostridium thermocellum is not affected by 1 atm of hydrogen gas 14).