is possible. Most vitamins are complex in structure and should be added as a separate
filter-sterilized solution (the only exceptions are nicotinic and p-aminobenzoic acids,
resistant to thermoalkaline hydrolysis) yet the parts of vitamin’s molecule can still
possess a stimulating effect [134].
4 Getting the Right pH and Na
+ Concentration Range
for Alkaliphiles
Finding the pH and Na
+ concentration range/optimum is an important part of
physiological characterization of newly isolated strains of alkaliphiles. At present,
it is mandatory for description of new taxon to include the data of pH and Na range/
optimum for growth. A routine task to determine a pH range for growth of a given
alkaliphilic strain sometimes may not be unequivocal. For example, for alkaliphiles
which are facultative aerobes, a search for the growth pH range should carry out on
fermentable rather than nonfermentable substrate as these alkaliphiles have different
pH ranges depending on the type of the used substrate: growth on nonfermentable
carbon sources (succinate) occurs in more narrow pH range than on fermentable
substrate (sucrose) [135]. For anaerobic alkaliphiles, pH optimum can shift
depending on the substrate used, if non-dissociated (methanol) or dissociated
(methylamine) as it has been shown earlier for Methanosalsum zhilinae [136]. A
generally accepted technique to get a pH range is to prepare the growth medium with
two or more buffer substances (one buffer is insufficient) which will embrace a full
possible growth pH range. For this purpose, Good’s buffers at a concentration of near
0.1 M or less are commonly used [137]. In addition to buffer substances, it is
necessary to leave in a medium a small amount of carbonates for anabolic purposes
since many alkaliphiles cannot grow in the complete absence of carbonates in the
medium. Some of the Good’s buffers are costly, but they have been specially
designed to be biological compatible, so the use of simple, inexpensive buffers
such as organic acids or amino acids should be used with caution, after making
sure that the test strain grows in the medium with 0.1 M or more of the test buffer.
Phosphate salts which are commonly used for cultivation of neutralophiles are
ineffective in the alkaline growth range. Although the isolation and cultivation of
alkaliphiles are carried out in carbonate buffer, its use to obtain the pH range is
possible but less convenient because of a significant shift in the pH of the medium
after autoclaving especially at the neutral region. In any case, when preparing a series
of tubes with different pHs, it is necessary to prepare one extra tube for each point
that will be used for the pH measurement after autoclaving. It is the pH value verified
after autoclaving that should be taken for further plotting. For maintenance and
further cultivation, after finding the optimal pH, the ratio of carbonate and bicarbonate is chosen in such a way as to provide this optimum pH. Addition of NaCl may be
Isolation and Cultivation of Alkaliphiles
71
filter-sterilized solution (the only exceptions are nicotinic and p-aminobenzoic acids,
resistant to thermoalkaline hydrolysis) yet the parts of vitamin’s molecule can still
possess a stimulating effect [134].
4 Getting the Right pH and Na
+ Concentration Range
for Alkaliphiles
Finding the pH and Na
+ concentration range/optimum is an important part of
physiological characterization of newly isolated strains of alkaliphiles. At present,
it is mandatory for description of new taxon to include the data of pH and Na range/
optimum for growth. A routine task to determine a pH range for growth of a given
alkaliphilic strain sometimes may not be unequivocal. For example, for alkaliphiles
which are facultative aerobes, a search for the growth pH range should carry out on
fermentable rather than nonfermentable substrate as these alkaliphiles have different
pH ranges depending on the type of the used substrate: growth on nonfermentable
carbon sources (succinate) occurs in more narrow pH range than on fermentable
substrate (sucrose) [135]. For anaerobic alkaliphiles, pH optimum can shift
depending on the substrate used, if non-dissociated (methanol) or dissociated
(methylamine) as it has been shown earlier for Methanosalsum zhilinae [136]. A
generally accepted technique to get a pH range is to prepare the growth medium with
two or more buffer substances (one buffer is insufficient) which will embrace a full
possible growth pH range. For this purpose, Good’s buffers at a concentration of near
0.1 M or less are commonly used [137]. In addition to buffer substances, it is
necessary to leave in a medium a small amount of carbonates for anabolic purposes
since many alkaliphiles cannot grow in the complete absence of carbonates in the
medium. Some of the Good’s buffers are costly, but they have been specially
designed to be biological compatible, so the use of simple, inexpensive buffers
such as organic acids or amino acids should be used with caution, after making
sure that the test strain grows in the medium with 0.1 M or more of the test buffer.
Phosphate salts which are commonly used for cultivation of neutralophiles are
ineffective in the alkaline growth range. Although the isolation and cultivation of
alkaliphiles are carried out in carbonate buffer, its use to obtain the pH range is
possible but less convenient because of a significant shift in the pH of the medium
after autoclaving especially at the neutral region. In any case, when preparing a series
of tubes with different pHs, it is necessary to prepare one extra tube for each point
that will be used for the pH measurement after autoclaving. It is the pH value verified
after autoclaving that should be taken for further plotting. For maintenance and
further cultivation, after finding the optimal pH, the ratio of carbonate and bicarbonate is chosen in such a way as to provide this optimum pH. Addition of NaCl may be
Isolation and Cultivation of Alkaliphiles
71