Hydrogen bacteria are characterized by their ability to metabolize and
multiply in a strictly inorganic medium, when supplied with H₂, CO₂ and
O₂ in required amounts. They can be grown in batch culture or in
continuous culture using different methods of supplying entire medium or
components on a demand feed system.
A medium was developed for batch culture of _Hydrogenomonas eutropha_ by
Repaske ([ref.187]) with quantitation of a number of components
including trace minerals. Experiments by Bongers ([ref.188]) showed that
a simplified medium, using laboratory-grade chemicals, could be used. A
definite requirement was found for magnesium and ferrous iron (Fe⁺⁺).
The optimal growth requirements observed for _Hydrogenomonas eutropha_
are shown in table X.
Table X.—_Optimum Growth Requirements of_
Hydrogenomonas eutropha
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Culture parameter Optimum value
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Cell density, g (dry weight)/liter 10
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Temperature, °C 35
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Pressure, atm 1
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pH (phosphate buffer) 6.8 (6.4-8.0)
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H₂, percent 75
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O₂, percent 15
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CO₂, percent 10
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Urea CO(NH₂)₂, g/liter 1
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MgSO₄·7H₂O, g/liter 0.1
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Fe(NH₄)₂(SO₄)₂, g/liter 0.008
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The effects of temperatures ranging from 20° to 42.5° C on the growth
rates of _Hydrogenomonas eutropha_ were studied by Bongers ([ref.189]),
and the optimal temperature was found to be about 35° C. Experiments at
25° and 35° C indicated that the efficiency of energy conversion was
essentially identical at both temperatures. _Hydrogenomonas_ requires,
as part of its substrate, a mixture of three gases: hydrogen, oxygen,
and carbon dioxide. Experiments were performed by Bongers ([ref.189]) to
determine the toleration limits of the three gases. Growth rates were
found to be identical when hydrogen varied from 5 to 80 percent. Nearly
identical growth was obtained when CO₂ partial pressures were 5 to 60
percent, being slightly lower at higher partial pressures. The organism
was highly sensitive to oxygen concentration. Dissolved oxygen
concentrations above 0.13 mM were found to inhibit cell division; energy
utilization was also affected by oxygen concentration. At 0.2 mM oxygen
concentration, the efficiency of energy conversion was approximately
half the value observed with 0.05 mM.
Another parameter of importance is the total volume of suspension which
would be required to balance the metabolic needs of one man. The volume
of suspension is determined by the conversion capacity of a unit volume.
This capacity is a function of the cell concentration; hence, the more
cells that can be packed in a unit volume of suspension (and adequately
provided with H₂, O₂, and CO₂), the less the volume of suspension
required.
Results of experiments by Bongers (refs. [ref.190] and [ref.191]) on
conversion capacity-density relationships show that the rate of CO₂
conversion obtained with suspensions up to approximately 10 grams (dry
weight) per liter is linear with relation to density. This indicates
that the supply of H₂, O₂, and CO₂ is adequate. Upon a further increase
in cell concentration, the conversion rate still increases but not
linearly. The highest amount of CO₂ taken up per liter of suspension was
approximately 2 liters per hour. At these very high cell concentrations,
the relationship between rate of conversion and density is no longer
linear. This is demonstrated when the conversion rate is calculated per
unit cell weight instead of per unit suspension volume. The rate per
gram dry weight per liter decreases from 146 to 68 ml of CO₂ per hour.
With a suspension at a density of approximately 10 grams, the conversion
of 1.1 liters of CO₂ per liter per hour is obtained. At a CO₂ output of
22 liters per man per hour, 20 liters of suspension would be sufficient
to balance the gas exchange needs of one man.
At higher cell concentrations, less volume of suspension would suffice
if gas equilibration could be maintained at the higher consumption rates
to avoid anaerobic conditions which could lead to a shift in metabolism.
In the final analysis, the technical problem of gas transfer from the
gas to the liquid phase determines the optimal cell concentration and,
therefore, the required suspension volume.
From data presently available, it can be concluded that, using the
slow-growing _H. facilis_, the volume of suspension required to support
one man is about 500 liters. Using _H. eutropha_, Schlegel ([ref.192])
calculated a suspension volume of 66 liters with 1 gram dry weight of
bacteria per liter.
In recent NASA-supported research, the amount of culture medium has been
estimated using improved cultivation methods and conditions. For batch
culture, the data show that from 10 to 66 liters would be required per
man, with a best practical estimate of 20 liters at 9 to 10 grams dry
weight of bacteria per liter ([ref.191]). For continuous culture using
the turbidostat, the present data indicate a demand for some 30 liters
of suspension, and a volume of 20 liters (at approximately 10 grams dry
weight of bacteria per liter) as a realistic goal.
In the foregoing section, the material balance for gases and water was
discussed. It was shown that a close match could be obtained with these
components of the closed environment.
Less abundant, though no less important, are the nonwater components of
urine and feces. The urine is important for the content of fixed
nitrogen and other products of man’s metabolism and serves as a very
effective substrate for cultivation of hydrogen bacteria. Maximum
closure of the system necessitates utilization of the urea in urine as a
nitrogen source.
The average man produces 1.2 to 1.6 liters of urine per 24-hour period.
This contains about 0.00005 gram per liter of iron, 0.113 gram per liter
of magnesium, and 24.5 grams per liter of urea ([ref.193]). As shown in
table X, each liter of bacterial medium requires 0.008 gram per liter of
Fe(NH₄)₂(SO₄)₂, about 0.1 gram of MgSO₄·7H₂O, and 1.0 gram per liter of
urea. In comparing the daily urine output with the estimated required
ingredients of a bacterial medium, a relatively close balance is
observed, with the exception of iron.
For the fixation of 24 moles of CO₂ (288 grams of C) produced per man
per day, the production of about 640 grams dry bacterial mass is
required. At an average N-content of 12 percent, the nitrogen
requirement would be some 100 grams. A comparison of daily output
(urine) and daily requirement by the bacterial suspension reveals that
only 10 to 33 percent of this amount could be recovered from average
urine. To obtain a material balance, either the man must be fed a
protein-rich diet or the bacterial suspension must be grown under
conditions which lead to the production of a cell mass relatively low in
protein content. Experiments have indicated that nitrogen starvation of
the bacterial culture might be a promising solution. Culture "staging"
(cultivation under nitrogen-rich conditions, followed by cultivation in
the absence of substrate nitrogen and subsequent harvesting for food
processing) will probably be the most promising means of nitrogen
economy in the closed environment. As discussed in a following section,
a biomass of relatively high lipid content can be obtained under
conditions of nitrogen starvation.