properties in the reference books or to assay its content by an appropriate analytical
technique (e.g., chromatographically or colorimetrically) before and after autoclaving. In particular, by measuring the content of total free amino acids that released into
the alkaline medium with albumin or caseinate after autoclaving, we found that half of
the amounts of these proteins added was destroyed and, hence, should be added
separately [131]. When isolating proteolytic alkaliphiles in our lab, sodium caseinate
turned out to be more convenient to use than egg albumin, since the aqueous solution
of the former could be safely autoclaved staying soluble. No free amino acids were
revealed. Sodium caseinate is a special processed casein to give the latter a good
solubility, but with the preservation of the polypeptide chain.
Agar is also destroyed during autoclaving, so the preparation of solid media for
alkaliphiles is possible only by the double concentration technique. It has been
proposed to use carrageenan as an agar substitute. This sulfated polysaccharide
withstands thermoalkaline hydrolysis and forms a strong gel even at a pH of 13.5
[132]. However, the use of labile substrates negates the benefits of carrageenan,
since it still requires separate preparation of the constituents.
In addition to the destruction of organic molecules, heating of aqueous alkaline
solutions causes a gradual erosion of glassware used for the cultivation of
alkaliphiles, and, over time, the test tubes become turbid (etched) and have to be
discarded. Unfortunately, manufacturers do not make glassware especially for
“alkalimicrobiologists,” yet alkali-resistant varieties of glass do exist, e.g., Schott
Glass 8436 has the highest an alkali resistance class A1. Although borosilicate
glassware (e.g., Duran or Pyrex brands) is more resistant to alkali than soda lime
glass, it is thermo- rather than alkali-resistant and has an alkali resistance class A2.
Divalent cations of alkaline-earth metals (Mg and Ca) precipitate out in alkaline
medium as insoluble carbonates; therefore, their content should be minimized, but
not completely eliminated as both relate to biogenic elements. The precipitation
threshold for each of them strongly depends on both the pH and the ionic strength of
the solution, and it is significantly lower for calcium than for magnesium. Unfortunately, because of too great a variety of combinations of sodium carbonate, bicarbonate, and chloride, it is impossible to calculate in advance the thresholds for
precipitation for each formulation, so, if needed, their selection is possible only
empirically. In our experience, the critical concentration for magnesium was 3 mM
and for calcium was 0.2 mM, and in practice, we use 0.5 mM for Mg
2+ and 0.1 mM
for Ca
2+ .
As noted by many microbiologists, alkaliphiles, regardless of the carbon source
used, often require some additives like yeast extract (usually up to 0.5 g/L) and/or the
mixture of vitamins different in content, and only some of them can grow in a
defined (minimal) medium. In this, alkaliphiles resemble halophiles where both
additives serve for anabolic purposes. Yeast extract is a source of B vitamins,
trehalose [133], and peptides of uncertain composition, and it can significantly
enhance the growth or be essential; therefore, at the step of isolation, it is better to
include it in the medium at a concentration of at least 0.1 g/L. An artificial mixture of
vitamins does not always replace yeast extract, and, in case of revealing of the
stimulating effect of yeast extract, it is desirable to check whether such a substitution
70
V. V. Kevbrin
technique (e.g., chromatographically or colorimetrically) before and after autoclaving. In particular, by measuring the content of total free amino acids that released into
the alkaline medium with albumin or caseinate after autoclaving, we found that half of
the amounts of these proteins added was destroyed and, hence, should be added
separately [131]. When isolating proteolytic alkaliphiles in our lab, sodium caseinate
turned out to be more convenient to use than egg albumin, since the aqueous solution
of the former could be safely autoclaved staying soluble. No free amino acids were
revealed. Sodium caseinate is a special processed casein to give the latter a good
solubility, but with the preservation of the polypeptide chain.
Agar is also destroyed during autoclaving, so the preparation of solid media for
alkaliphiles is possible only by the double concentration technique. It has been
proposed to use carrageenan as an agar substitute. This sulfated polysaccharide
withstands thermoalkaline hydrolysis and forms a strong gel even at a pH of 13.5
[132]. However, the use of labile substrates negates the benefits of carrageenan,
since it still requires separate preparation of the constituents.
In addition to the destruction of organic molecules, heating of aqueous alkaline
solutions causes a gradual erosion of glassware used for the cultivation of
alkaliphiles, and, over time, the test tubes become turbid (etched) and have to be
discarded. Unfortunately, manufacturers do not make glassware especially for
“alkalimicrobiologists,” yet alkali-resistant varieties of glass do exist, e.g., Schott
Glass 8436 has the highest an alkali resistance class A1. Although borosilicate
glassware (e.g., Duran or Pyrex brands) is more resistant to alkali than soda lime
glass, it is thermo- rather than alkali-resistant and has an alkali resistance class A2.
Divalent cations of alkaline-earth metals (Mg and Ca) precipitate out in alkaline
medium as insoluble carbonates; therefore, their content should be minimized, but
not completely eliminated as both relate to biogenic elements. The precipitation
threshold for each of them strongly depends on both the pH and the ionic strength of
the solution, and it is significantly lower for calcium than for magnesium. Unfortunately, because of too great a variety of combinations of sodium carbonate, bicarbonate, and chloride, it is impossible to calculate in advance the thresholds for
precipitation for each formulation, so, if needed, their selection is possible only
empirically. In our experience, the critical concentration for magnesium was 3 mM
and for calcium was 0.2 mM, and in practice, we use 0.5 mM for Mg
2+ and 0.1 mM
for Ca
2+ .
As noted by many microbiologists, alkaliphiles, regardless of the carbon source
used, often require some additives like yeast extract (usually up to 0.5 g/L) and/or the
mixture of vitamins different in content, and only some of them can grow in a
defined (minimal) medium. In this, alkaliphiles resemble halophiles where both
additives serve for anabolic purposes. Yeast extract is a source of B vitamins,
trehalose [133], and peptides of uncertain composition, and it can significantly
enhance the growth or be essential; therefore, at the step of isolation, it is better to
include it in the medium at a concentration of at least 0.1 g/L. An artificial mixture of
vitamins does not always replace yeast extract, and, in case of revealing of the
stimulating effect of yeast extract, it is desirable to check whether such a substitution
70
V. V. Kevbrin