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.
Continuous Culture of Hydrogenomonas Bacteria
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.