Growth, Products, and Application
4.1 Maximal Specific Growth Rate
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).
4 Kinetics and Regulation
favorable conditions), even though they have not been observed to do so in their natural habitat (where there may be suboptimal or even less optimal conditions)
1,146,195)
4.2 Yield
4.2.1 Yx/S (cell mass per substrate)
To avoid further confusion, it would be of value and meaningful to define the yield coefficient Y before its values are discussed. As the 'effective' or true yield coefficient (Yeff), the ratio of produced biomass (x—XQ) to consumed substrate (s0—s) will be used since values one the absolute biomass production rate (rx) or substrate utilization rate (rs) to correctly calculate Y = rx/-rs are not available for thermo- philes. As an 'economic yield coefficient' (Yec0), the ratio of produced biomass to supplied substrate (s0) will be used. This differentiation is necessary because incomplete substrate utilization has been observed in a few cases (see Sect. 4.5).
In general, the yields determined for thermophiles do not greatly differ from those measured for mesophiles, provided that the substrates are utilized completely.
However, the values, if determined, are a small fraction lower than is expected from comparable mesophiles. This will be considered in Sect. 4.4 and is most likely due to the increased maintenance requirements of thermophiles.
In many cases, the addition of complex substrates as source of growth factors has been reported to be an absolute requirement for growth (see Sect. 3.2.2.1).
Only in a few cases complex substances do not function as a carbon or an energy source, as for example, in cultures of Thermoanaerobacter ethanolicus138), Thiobacillus TH1 64), or Clostridium thermohydrosulfuricum 35). However, the yeast extract con- centration in the medium has been shown to influence the final yield, but has not been linearity correlated 35). This allows one to calculate yields correctly. The optimal concentrations of yeast extract and tryptone, without any defined carbon source for growth of Thermomicrobium roseum is 0.5 % each, if final yield is to be optimized 26). At 70 °C 0.5 g 1_1 of brainheart infusion are sufficient to achieve the same yield of Bacillus caldotenax (YT-G) from 1 g 1_ 1 pyruvate at 70 °C, whereas 1 g l- 1 of BHI is required at 80 °C65).
A decrease in yield (final yield) is often used as an argument for inhibition of growth. Although this is good for qualitative decisions, it should always be evaluated in combination with the effect of the concentration of the inhibiting substance(s) on the specific growth rate (for qpantitative characterization). This is most easily achieved using chemostat culture, as shown, for example, for Thermus thermophilus 72), but is nonetheless practicable in batch, too, as has been shown for other Thermus strains 71).
Of special technological interest is the increase of yield by shifting concentrations of regulatory agents as with Thermoanaerobium brockii. Supplying 100 mM of acetone to the culture increases not only the yield but also the specific growth rate twofold 14). Growth yields are also influenced by the products formed during cultivation.
Growth of Clostridium thermoaceticum is inhibited by high concentrations of acetic acid, but product formation is not inhibited to the same extent. During exponential growth, the expected Y = 0.1 is closely approached, failing to values of as low as 0.03 in the stationary phase 190).
In this context many researchers should be encouraged to quantitatively determine yield rather than use '—', ' + ', or ' + ' for the characterization of utilization and effectiveness of substrates.
4.2.2 Yp/S (product formed per substrate)
Product yields have been described at least as rarely as the growth yields of thermophiles. This may be the consequence of the following characteristics:
a) Many 'ordinary' products are more or less volatile and are 'lost' at temperatures above 60 °C via exhaust gas unless special equipment is supplied. Therefore these products are not accounted for in carbon balances which usually give less than
100%
b) Among products of technical interest that are presently known to be produced by thermophiles are ethanol, acetic acid, and methane, products that are nearly exclusively formed by anaerobes where the contribution of complex substrates is often not clear.
c) (Thermostable) enzyme yields are not characterized on a mass basis (as is Y).
The more meaningful activities that will be considered here are not compatible with the usual Y-values.
Ethanol is formed in significant amounts by anaerobic sporeformers as well as by non-sporeformers and also — to a smaller extent — by the facultative (? 68>) aerobic Bacillus stearothermophilus 196). Thermophilic cultures for ethanol formation have been recently reviewed by Zeikus et al.14). Most important is Clostridium thermo- hydrosulfuricum since this bacterium utilizes glucose with the highest yield thus far reported for thermophiles to give ~ 1.9 mol ethanol per mol glucose (strain 39E)13). Variations of Yp/S between 1 and 1.5 (mol ethanol mol- 1 glucose) have been observed with C. thermohydrosulfuricum 40). Therefore, this organism seems to be of potentially practical use, especially because it easily sustains high substrate concentrations as 20 % sucrose 197). Comparable is the yield obtained with Thermo- anaerobacter ethanolicus, which has been reported to produce 1.8 (mol ethanol mol- 1
glucose) on 0.5% medium 138,198>. It is also able to grow on media containing as much as 20 % starch 40). However, wild type strains presently have the disadvantage of a pronounced ethanol sensitivity; it can be overcome by the production and
selection of tolerant mutants. This is a prerequisite for the use of maximum YP/S
in technical processes and has been started, for example, with T. ethanolicus strain JW200: Strain JW200 wt forms a maximum of ethanol in only 1 % carbohydrate medium, whereas in the case of strain JW200Fe, the production of ethanol increases up to 14% carbohydrate concentration (no further decrease shown)40).
Another possibility for increasing the achievable yield is the use of cocultures as demonstrated with Clostridium thermocellum and C. thermohydrosulfuricum. The former degrades P-l,4-xylans and glucanes, whereas the latter utilizes Cs- and C6- sugars, as well as dimers, more rapidly, and does not allow a significant accumulation of reducing sugars. The very stable coculture produces ethanol at a rate that is three fold higher and at a fifteen times higher ethanol/acetate ratio than that produced by a C. thermocellum monoculture. The coculture reaches essentially the same yield (Y = 0.58 g ethanol g_ 1 cellulose) as does the monoculture of C.
thermohydrosulfuricum in combination with C. thermocellum cellulase in a 0.7%
cellulose medium at 100% substrate consumption14). The ethanol yield of C.
thermocellum (mutant strain S-4) on solka-floc SW-100 is dependent on the nature of the buffer used, 0.22 (gg- 1) in NaHC03 buffer and 0.14 (gg- 1) in phosphate buffer. With increasing temperature (50 to 60 °C), the yield increases from 0.08 to 0.20 (g g- 1), and the maximal specific growth rate from 0.033 to 0.107 h- 1 50).
Another product of interest produced by thermophiles in large amounts is acetic acid. However, product recovery from neutral culture broths is the problem, although strains grow and produce very well at neutral pH. Clostridium thermoaceti- cum wild type strains produce about 2 moles of acetate per mol glucose — the theoretical yield is 3: 'homofermentation'185) — and reach final concentrations ranging from 4 to 15 g l- 1. The highest determined values of 20 g l- 1 (at pH 7) may also be inhibitory both for product formation and for growth. However, an efficiency of glucose conversion to acetate of 90% (Yp/S = 0.9, Yx/S = 0.1) can be reached with mutant strains (S3 and 1735) even at pH 6 and final product concentrations of 15 g l- 1 190). C. thermoaceticum (strain 1745), if 'adapted', can grow at pH 4.5 with a doubling time of 36 h and reach a final acetic acid concentration of 4.5 g l- 1. It is expected that further increase of specific growth rate at this low pH can be achieved using chemostat culture. At a pH of 4.5, the criterion of easy product recovery is fulfilled 189,199).
Thermophilic production of methane gas is of great economic importance since the thermophilic methanogens presently known grow much more rapidly than the mesophilic ones. This implies shorter mean residence times and/or smaller volumes needed for biogas formation. Currently only 3 species of thermophilic methanogens are identified: Methanobacterium thermoautotrophicum (for comparison of strains, see 200)), Methanococcus thermolithotrophicus 177) and Methanothermus fervidus 55). If the rate of methane formation is not limited by both H2 and C02, methane yield is independent of the growth rate (Yp/X = 0.63 mol g- 1 or Yx/P = 1.6 g mol- 1) in Methanobacterium thermoautotrophicum (strain Marburg), but Yp/X decreases to 0.33 mol g-1 (or Yx/P increases to 3 g mol- 1) if either H2 or C02 limit growth.
This can be explained by the tighter coupling of growth and methanogenesis under substrate limiting conditions; however, the reasons for this are not yet understood 54). Furthermore, the concentration of NH4 + (nitrogen source) triggers methane yield.
In limited cultures, the highest productivity is achieved with YP/X = 0.47 to 0.53 mol g- 1 (or: Yx/P = 1.9 to 2.2 g mol- 1) versus 0.44 to 0.46 mol g- 1 (or: 2.2 to 2.3 g mol-1) with excess NH4 + . Growth rate, but not explicitly methane formation, of M. thermoautotrophicum strain AH is unaffected by 0.2M NH4 + . This and higher concentrations are reported to inhibit methane production in anaerobic digestors 201). Biomethanation of acetic acid 51) and acid waste water containing acetate and furfural188) has been successfully carried out at 60 °C using a granular 'consortium' of methanogens and non-methanogens. At a hydraulic retention time of 43 h (dilution rate = 0.023 h- 1) , 6-8 1 of biogas l- 1 d- 1 containing 55% CH4
(4.2 1 methane l- 1 d- 1) can be produced from 20 g l- 1 acetic acid (almost equimolar to utilized acetate) at constant pH 6.8. Only part of the granules were found to actively gas at a rate of approximately 1 volume gas per pellet volume per minute 51). In the previously mentioned waste water, 0.1 to 0.3 1 gas l- 1 h- 1 could be produced at a dilution rate of 0.015 h- 1. The authors regard such a process as feasible on a technical scale 188).
Methane production at the high temperatures >65 °C is possible 55) up to 97 °C using Methanothermus fervidus or using still unidentified methanogens at even higher temperatures5); however, no quantitative data on yields are available at present.
In the future, thermophilic leaching will possibly provide a rapid method of extracting metal from (minor quality) ores 163). At present, some thermophilic Thiobacillus isolates are known that solubilize 4 g l- 1 Fe from 1 0 g l- 1 pyrite within 10—15 days. However, strain THl oxidizes iron at a rate which is only half the rate of the mesophilic Thiobacillus ferrooxidans202), but this thermophile is highly resistant to high concentrations of Cd (> 1000 ppm) 63). No differences between a thermophilic acidophile at 50 °C and T. ferrooxidans at 30 °C have been observed with respect to rate and yield during copper leaching from chalcopyrite waste 60). Sulfobacillus thermosulfidooxidans leaches 2 to 2.1 g l- 1 iron from 2% copper-zinc- pyrite ore supplemented with 0.1 % glucose or glutamine within 120 h 204). The above mentioned strains do not effectively leach at temperatures > 50 °C. Strains of the genus Sulfolobus, which are able to grow at significantly higher temperatures (up to 87 °C), have been shown to leach 51 % of the copper from chalcopyrite containing 29% Cu within 60 days, that is, 43% more than what can be extracted only chemically, i.e., without organisms, under these conditions (60 °C, pH 2—3). Mixed cultures of Sulfolobus acidocaldarius and Ferrolobus leached 38 % of the Cu from chalcopyrite containing 0.31 % Cu in 161 days at an average rate of 21 mg 1-1 d-1
but only small amounts of Zn and Ni (1 %) and minimal amounts of Pb (2.6 ppm) were extracted biologically 163).
The product yield of enzymes from thermophiles is difficult to compare with yields from mesophilic organisms because of the changed qualities of the enzymes. Enhanced thermal and chemical resistance may be desired much more than equal or even higher yields on a mass basis. For example, Clostridium thermocellum (LQRI) cellulase has a higher endoglucanase/exoglucanase activity ratio than Trichoderma reesei (QM 9414) cellulase, exhibits total stability at 60 °C, and is not oxygen labile 42). Cellulase is produced growth-associated by Clostridium thermocellum (strain LQ8) with a yield of 125 endoglucanase units and 300 exoglucanase units per g cellulose MN300 degraded in cultivations at 60 °C with 0.7 % initial cellulose concentration over 4 days41).