Growth of hydrogen bacteria in a batch culture, after an initial period
of adjustment, becomes steady and rapid during the exponential growth
phase. This steady state of growth is temporary and ceases when nutrient
substrate or gas concentrations drop to limiting values. For long
periods a continual supply of nutrients must be provided. Growth then
occurs under steady-state conditions for prolonged periods, and such
factors as pH, concentration of nutrient, oxygen, and metabolic products
(which change during batch culture) are all maintained constant in
continuous culture.
Two methods can be used for control of continuous cultures: the
turbidostat and the chemostat. In the turbidostat, regulation of medium
input and cell concentration is controlled by optically sensing the
turbidity of the culture.
The dilution rate varies with the population density of the culture and
maintains the density within a narrow range. Organisms grow at the
maximum rate characteristic of the organism and the conditions. The
growth rate can be changed by modifying the nutrient medium, gas
concentration, or incubation temperature. A disadvantage of the
turbidostat is that all nutrient concentrations in the culture chamber
are necessarily higher than the minimum, resulting in inefficient
utilization of nutrients.
The turbidostat system for continuous culture of _Hydrogenomonas_
bacteria, developed by Battelle Memorial Institute ([ref.194]), includes
electrolysis of water in a separate unit. Hydrogen and oxygen are fed
separately up to the point of injection into the culture vessel, and the
mixed volume is kept very small to minimize am possibility of explosion.
However, the two gases may be injected simultaneously if there is a
demand for both.
In the chemostat, growth of the organisms is limited by maintaining one
essential nutrient concentration below optimum. A constant feed of
medium, with one nutrient in limiting concentration and with constant
removal of culture at the same rate, is used to achieve the steady
state. The dilution rate is set at an arbitrary value, and the microbial
population is allowed to find its own level. By appropriate setting of
the dilution rate, the growth rate may be held at any desired value from
slightly below the maximum possible to nearly zero. This constitutes a
self-regulating system and allows selection of a desired growth rate.
A combined electrolysis-chemostat method, developed by Magna Corp.,
maintained the hydrogen-producing electrode of an electrolysis cell in
the bacterial culture. Resting cells of _Hydrogenomonas eutropha_
consumed hydrogen produced at the cathode of an electrolysis cell built
into a specially constructed Warburg flask. Attempts to immobilize
_Hydrogenomonas_ cells on a porous conductor were partially successful.
This system could lower the volume requirements compared with those for
the isolated subsystems. Disadvantages of this integrated system include
electrolysis of the bacterial medium, possibly resulting in toxic
breakdown products, and the possible effects of electric power and the
KOH electrolyte on the bacteria. The main disadvantage of an integrated
system would be the disparity between optimal conditions for efficient
electrolysis and efficient bacterial conversion, particularly
temperature and pH, with the combination possibly resulting in
considerably higher power and weight demands.
Both continuous-culture approaches are being studied with NASA support.
The turbidostat offers the greatest potential efficiency in weight and
volume, but uses nutrient materials less efficiently and is more
complex. The chemostat is less efficient in weight and volume, but has
greater simplicity and reliability.
_Hydrogenomonas eutropha_ has been grown in 15-liter batch cultures and
in 2.1-liter continuous cultures. A 20-liter continuous culture,
sufficient to balance the requirements of a man, is under development.
The potential problem areas in large-scale continuous production of the
bacteria include assuring genetic stability, preventing or controlling
bacteriophage and foreign bacterial contamination, and preventing
heterotrophic growth caused by exposure to organic material from the
urine. Genetics of hydrogen bacteria and phage infection have been
studied by DeCicco. Research on these problems indicates that they are
not of major importance, but cause significant effects and must be
eliminated or controlled.