necessary if the test strain requires. It should be noted that although low NaCl
practically do not affect pH, high concentrations can lower the pH value by
suppressing the dissociation of bicarbonate/carbonate. The effect of NaCl on the
dissociation constant of the carbonate buffer is gradual; therefore, an extra check of
the pH may be required. A carbonate-free buffered medium with the optimal pH and
supplemented with an optimal concentration of NaCl can be used to determine the
strain’s requirement for carbonates. To be sure that the strain does not need carbonates, up to five successive transfers on this medium are usually enough. Growth in the
fifth transfer means that given culture does not require carbonates.
The amount of substrate when determining the pH range should be minimal. This
is especially true for anaerobic alkaliphiles, fermenting sugars to organic acids. The
molar concentration of sugar should not exceed (less is better) the molar concentration of the buffer; otherwise, the initial pH will last only for a short time of the
growth curve, depending on the buffer capacity of the medium. This applies to any
fermentable substrate, when ionic products are excreted to the medium. Readers can
find in Sorokin’s letter [138] other precautions that should be taken into
account when determining the pH range for growth of alkaliphiles. Features of pH
measurement at elevated temperatures, which are required when working with
alkalithermophiles, can be found in the publication of Mesbah and Wiegel
[139]. Pouring anaerobic media with various pHs is conveniently carried out in a
specially designed conical flask (Fig. 3). The anaerobic medium with the initial pH,
freed from oxygen (by vacuum or boiling), is pumped under a slight nitrogen
pressure into a flask with attached accessories. The flask should be previously
purged by nitrogen. Depending on the direction of the pH change, the plastic syringe
is filled with concentrated HCl or NaOH and attached to a thin flexible tubing to
accurately supply the titrant to the bulk of the medium. After stabilization of reading,
the medium with the set pH is poured over the tubes in a nitrogen flow.
Determining the sodium concentration range for growth of alkaliphiles can
possibly be achieved by two methods. The first one is the same as that used for
halophiles, i.e., the calculated weights of dry NaCl are put into the culture tubes and
brought to the mark by a 0.1–0.2 M buffered (by carbonate for alkaliphiles) medium
with optimal pH. Accordingly, sodium, introduced with carbonates, should be taken
into further account. In the case of natronophiles with a high demand for carbonates,
it is preferable to use the second method. It implies the mixing of solutions I and II in
various proportions. Solution I is a saturated solution of carbonate buffer with the
optimal pH for a given strain plus all other constituents. Solution II is an aqueous
solution of all other constituents except for carbonates. The disadvantage of this
method is the strong dependence of the solubility of the saturated sesquicarbonate
solution on pH and, consequently, the inability to reach high concentrations of
dissolved sodium. The solubility of sodium carbonate is worse (sodium bicarbonate
is even much worse) than that of NaCl, and its addition to the saturated solution of
the former leads to salting out of bicarbonate or carbonate, depending on the
pH. After finding the optimum concentration for sodium, regardless of the method
72
V. V. Kevbrin
practically do not affect pH, high concentrations can lower the pH value by
suppressing the dissociation of bicarbonate/carbonate. The effect of NaCl on the
dissociation constant of the carbonate buffer is gradual; therefore, an extra check of
the pH may be required. A carbonate-free buffered medium with the optimal pH and
supplemented with an optimal concentration of NaCl can be used to determine the
strain’s requirement for carbonates. To be sure that the strain does not need carbonates, up to five successive transfers on this medium are usually enough. Growth in the
fifth transfer means that given culture does not require carbonates.
The amount of substrate when determining the pH range should be minimal. This
is especially true for anaerobic alkaliphiles, fermenting sugars to organic acids. The
molar concentration of sugar should not exceed (less is better) the molar concentration of the buffer; otherwise, the initial pH will last only for a short time of the
growth curve, depending on the buffer capacity of the medium. This applies to any
fermentable substrate, when ionic products are excreted to the medium. Readers can
find in Sorokin’s letter [138] other precautions that should be taken into
account when determining the pH range for growth of alkaliphiles. Features of pH
measurement at elevated temperatures, which are required when working with
alkalithermophiles, can be found in the publication of Mesbah and Wiegel
[139]. Pouring anaerobic media with various pHs is conveniently carried out in a
specially designed conical flask (Fig. 3). The anaerobic medium with the initial pH,
freed from oxygen (by vacuum or boiling), is pumped under a slight nitrogen
pressure into a flask with attached accessories. The flask should be previously
purged by nitrogen. Depending on the direction of the pH change, the plastic syringe
is filled with concentrated HCl or NaOH and attached to a thin flexible tubing to
accurately supply the titrant to the bulk of the medium. After stabilization of reading,
the medium with the set pH is poured over the tubes in a nitrogen flow.
Determining the sodium concentration range for growth of alkaliphiles can
possibly be achieved by two methods. The first one is the same as that used for
halophiles, i.e., the calculated weights of dry NaCl are put into the culture tubes and
brought to the mark by a 0.1–0.2 M buffered (by carbonate for alkaliphiles) medium
with optimal pH. Accordingly, sodium, introduced with carbonates, should be taken
into further account. In the case of natronophiles with a high demand for carbonates,
it is preferable to use the second method. It implies the mixing of solutions I and II in
various proportions. Solution I is a saturated solution of carbonate buffer with the
optimal pH for a given strain plus all other constituents. Solution II is an aqueous
solution of all other constituents except for carbonates. The disadvantage of this
method is the strong dependence of the solubility of the saturated sesquicarbonate
solution on pH and, consequently, the inability to reach high concentrations of
dissolved sodium. The solubility of sodium carbonate is worse (sodium bicarbonate
is even much worse) than that of NaCl, and its addition to the saturated solution of
the former leads to salting out of bicarbonate or carbonate, depending on the
pH. After finding the optimum concentration for sodium, regardless of the method
72
V. V. Kevbrin