ELECTROLYSIS-HYDROGENOMONASSYSTEM

ELECTROLYSIS-HYDROGENOMONASSYSTEMElectrolysis is carried out in a closed unit containing an electrolyte (KOH solution) with an anode and a cathode. These cells produce a maximum yield (60-80 percent or more) in gas production per unit of power consumption. According to Dole and Tamplin ([ref.184]), a unit capable of producing enough oxygen to sustain one man would be highly reliable, weigh approximately 18 kg, and require a power input of 0.25 kW.One approach to zero-gravity operation is to rotate the electrolysis cell as described by Clifford and McCallum ([ref.185]) and Clifford and Faust ([ref.186]). The smallest known electrolysis cell under development uses this artificial gravity to separate oxygen from the anode and electrolyte, while the dry hydrogen gas permeates through the foil cathode,fabricated from palladium-silver alloy. This electrolysis cell, which would provide breathing oxygen for three men, has a volume of 1.4 liters, weighs 4.5 kg, and requires 0.67 kW, excluding auxiliary equipment, and has an efficiency of 84 percent.The chemosynthetic conversion is carried out by the hydrogen bacteria. By the oxidation of molecular hydrogen, supplied from the electrolysis of water, energy is made available for biosynthesis. The generation of this "biological energy" is mediated by the stable enzyme hydrogenase which is present in the bacteria. On the average, the oxidation of 4 moles of H2is required for the conversion of 1 mole of CO2(the hourly production of a man). The removal of this amount of CO2would thus require the cleavage of 4 moles of water. In addition, to supply oxygen for human respiration (at a rate of 1 mole of O2per hour) the cleavage of two additional moles of water is required. Therefore, the chemosynthetic regeneration and human respiration together would require, on the average, the splitting of 6 moles of water per hour.The material balance for electrolysis, biosynthesis, and human metabolism, with gram molecular weights in parentheses, are shown in equations (1) to (3), respectively:6H2O ———————> 3O2+6H2(108) ———————> (96) + (12) (1)The bacterial synthesis requires 6 moles of H2, 2 moles of O2, and 1 mole of CO2(from the astronaut), as shown in equation 2:6H2+ 2O2+ CO2———————> CH2O + 5H2O(12) + (64) + (44) ———————> (30) + (90) (2)The respiration of the astronaut requires 1 "food" mole (CH2O) representing about 120 kcal, and 1 mole of O2, as shown in equation 3:CH2O + O2———————> CO2+ H2O(30) + (32) ———————> (44) + (18) (3)The metabolic data intable VIIIshow that the CO2of the astronaut and the bacteria must balance at about 1.056 kg per day.The water relations are not completely balanced, but are fairly close. About 2.6 liters per day of water are split by electrolysis. The astronaut has an intake of 3.5 liters of water per day, 2.5 liters for drinking and 1 liter for preparing dehydrated food. The output is about 1.6 liters of urine and 2.1 liters of water of respiration and perspiration per day, or a total output of 3.7 liters, with the 0.2-liter excess due mainly to water of metabolism. The bacteria-produced water, amounting to 2.2 liters per day, and the excess from the astronaut would supply 2.4 liters toward balancing the 2.6 liters of water electrolyzed.Bacterial CultureHydrogen bacteria are characterized by their ability to metabolize and multiply in a strictly inorganic medium, when supplied with H2, CO2and O2in 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 ofHydrogenomonas eutrophaby 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 forHydrogenomonas eutrophaare shown intable X.Table X.—Optimum Growth Requirements ofHydrogenomonas eutrophaCulture parameterOptimum valueCell density, g (dry weight)/liter10Temperature, °C35Pressure, atm1pH (phosphate buffer)6.8 (6.4-8.0)H2, percent75O2, percent15CO2, percent10Urea CO(NH2)2, g/liter1MgSO4·7H2O, g/liter0.1Fe(NH4)2(SO4)2, g/liter0.008The effects of temperatures ranging from 20° to 42.5° C on the growth rates ofHydrogenomonas eutrophawere 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.Hydrogenomonasrequires, 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 CO2partial 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. At0.2 mMoxygen 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 CO2conversion 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 CO2is adequate. Upon a further increase in cell concentration, the conversion rate still increases but not linearly. The highest amount of CO2taken 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 CO2per hour. With a suspension at a density of approximately 10 grams, the conversion of 1.1 liters of CO2per liter per hour is obtained. At a CO2output 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-growingH. facilis, the volume of suspension required to support one man is about 500 liters. UsingH. 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 30liters 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 intable 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 ofHydrogenomonasBacteriaGrowth 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 occursunder 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 ofHydrogenomonasbacteria, 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 ofHydrogenomonas eutrophaconsumed hydrogen produced at the cathode of an electrolysis cell built into a specially constructed Warburg flask. Attempts to immobilizeHydrogenomonascells 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 integratedsystem 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 eutrophahas 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.Bacterial Composition and NutritionHydrogenomonasbacteria can be used for at least part of the astronauts' diet. The washed bacteria have a mild taste and are being studied for their total energy content, protein and lipid digestibility, and vitamin content. Carbon and nitrogen balances, and respiratory quotient are to be determined in animals fed the bacteria as their sole food source. No toxic constituents have been discovered. Sonicated and cooked bacteria, when fed to white rats as 12 percent of the solids of a nutritionally balanced diet, were eaten readily and produced no ill effects. Net utilization of the protein appears to be somewhat lower than casein and about the same as legume proteins.The composition ofHydrogenomonas eutrophais shown intable XI. The composition of the bacteria varies with the age and growth phase of the cells and with the medium and gas available. It is possible to modify the growth conditions to grow the type of bacteria desired for nutritive purposes.Hydrogenomonascells contain about 75 percent water. Of the dry weight, about 74 percent is protein, calculated as 6.25 times the nitrogen content.Table XIshows the amino acid composition to be comparable with other bacterial proteins, except for higher tryptophan and methionine values.Table XI—Analysis ofHydrogenomonas eutrophaCells Grown in Continuous Culture[From[ref.194]]ConstituentPercent by weightMoisture74.55Fat.44Ash1.73Nitrogen3.02(wet)11.87(dry)Protein (N × 6.25)18.90(wet)74.26(dry)Amino acids (dry weight)8Alanine4.47Arginine3.41Aspartic acid4.32Cystine.08Glutamic acid7.67Glycine2.76Histidine.95Isoleucine2.17Leucine4.04Lysine2.65Methionine1.14Phenylalanine2.20Proline2.06Serine1.80Threonine2.15Tryptophan.78Tyrosine1.79Valine3.03The lipid content of rapidly growing cells is normally quite low (0.45 to 2.3 percent crude ether extractable lipids). The most important lipid is poly-beta-hydroxybutyric acid, which is stored under the growing conditions of insufficient nitrogen or oxygen supply (refs.[ref.187]and[ref.191]). Under these conditions, this unusual polymer constitutes up to 80 percent of the dry weight. While the monomer itself, beta-hydroxybutyric acid, is rapidly and efficiently used in cell metabolism, the nutritive value of the polymer is yet to be determined. The fatty acids found include lauric, myristic, palmitic, palmitoleic, heptadecaenoic, C17 saturated(?), stearic, linoleic, and linolenic(?) ([ref.195]).Application to Spacecraft SystemA bioregenerative life-support system will be required in long manned space flight, especially with several astronauts such as would be required for a manned mission to Mars in the 1980 time period. While almost 15 years is a long leadtime, the biological research and engineering problems are formidable, and a system would have to be developed at least 5 years before the mission.The power and weight requirements for both chemical and biological regenerative life-support systems were presented intable VIII. These should be considered tentative best estimates based on present data.The use of bioregenerative systems in spacecraft systems has been studied by Bongers and Kok ([ref.175]) who put the electrolysis-Hydrogenomonassystem in proper perspective with the following statement:The bioregenerative systems are more or less in a transitory phase between research and development. The power data can be considered fairly accurate, at least within ±20 percent. The postulated weight data, however, represent approximations, particularly with respect to auxiliary equipment and construction materials. Also omitted are the weight penalties most probably involved in the processing of the solid output of the exchangers, elegantly defined as potential food. Further research is required in this area to evaluate the regenerative systems, especially the bacteria, with respect to this potential. Furthermore, as yet there is no experimental proof that the growth rates of the heavy bacterial suspensions can be realized in a large design, determined on a relatively small scale with fairly precise control of physiological conditions and gas exchange. This aspect may affect considerably the weight involved in a chemosynthetic balanced system. Nevertheless, at present, this approach still seems most promising.

Electrolysis is carried out in a closed unit containing an electrolyte (KOH solution) with an anode and a cathode. These cells produce a maximum yield (60-80 percent or more) in gas production per unit of power consumption. According to Dole and Tamplin ([ref.184]), a unit capable of producing enough oxygen to sustain one man would be highly reliable, weigh approximately 18 kg, and require a power input of 0.25 kW.

One approach to zero-gravity operation is to rotate the electrolysis cell as described by Clifford and McCallum ([ref.185]) and Clifford and Faust ([ref.186]). The smallest known electrolysis cell under development uses this artificial gravity to separate oxygen from the anode and electrolyte, while the dry hydrogen gas permeates through the foil cathode,fabricated from palladium-silver alloy. This electrolysis cell, which would provide breathing oxygen for three men, has a volume of 1.4 liters, weighs 4.5 kg, and requires 0.67 kW, excluding auxiliary equipment, and has an efficiency of 84 percent.

The chemosynthetic conversion is carried out by the hydrogen bacteria. By the oxidation of molecular hydrogen, supplied from the electrolysis of water, energy is made available for biosynthesis. The generation of this "biological energy" is mediated by the stable enzyme hydrogenase which is present in the bacteria. On the average, the oxidation of 4 moles of H2is required for the conversion of 1 mole of CO2(the hourly production of a man). The removal of this amount of CO2would thus require the cleavage of 4 moles of water. In addition, to supply oxygen for human respiration (at a rate of 1 mole of O2per hour) the cleavage of two additional moles of water is required. Therefore, the chemosynthetic regeneration and human respiration together would require, on the average, the splitting of 6 moles of water per hour.

The material balance for electrolysis, biosynthesis, and human metabolism, with gram molecular weights in parentheses, are shown in equations (1) to (3), respectively:

6H2O ———————> 3O2+6H2(108) ———————> (96) + (12) (1)

6H2O ———————> 3O2+6H2

(108) ———————> (96) + (12) (1)

The bacterial synthesis requires 6 moles of H2, 2 moles of O2, and 1 mole of CO2(from the astronaut), as shown in equation 2:

6H2+ 2O2+ CO2———————> CH2O + 5H2O(12) + (64) + (44) ———————> (30) + (90) (2)

6H2+ 2O2+ CO2———————> CH2O + 5H2O

(12) + (64) + (44) ———————> (30) + (90) (2)

The respiration of the astronaut requires 1 "food" mole (CH2O) representing about 120 kcal, and 1 mole of O2, as shown in equation 3:

CH2O + O2———————> CO2+ H2O(30) + (32) ———————> (44) + (18) (3)

CH2O + O2———————> CO2+ H2O

(30) + (32) ———————> (44) + (18) (3)

The metabolic data intable VIIIshow that the CO2of the astronaut and the bacteria must balance at about 1.056 kg per day.

The water relations are not completely balanced, but are fairly close. About 2.6 liters per day of water are split by electrolysis. The astronaut has an intake of 3.5 liters of water per day, 2.5 liters for drinking and 1 liter for preparing dehydrated food. The output is about 1.6 liters of urine and 2.1 liters of water of respiration and perspiration per day, or a total output of 3.7 liters, with the 0.2-liter excess due mainly to water of metabolism. The bacteria-produced water, amounting to 2.2 liters per day, and the excess from the astronaut would supply 2.4 liters toward balancing the 2.6 liters of water electrolyzed.

Bacterial CultureHydrogen bacteria are characterized by their ability to metabolize and multiply in a strictly inorganic medium, when supplied with H2, CO2and O2in 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 ofHydrogenomonas eutrophaby 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 forHydrogenomonas eutrophaare shown intable X.Table X.—Optimum Growth Requirements ofHydrogenomonas eutrophaCulture parameterOptimum valueCell density, g (dry weight)/liter10Temperature, °C35Pressure, atm1pH (phosphate buffer)6.8 (6.4-8.0)H2, percent75O2, percent15CO2, percent10Urea CO(NH2)2, g/liter1MgSO4·7H2O, g/liter0.1Fe(NH4)2(SO4)2, g/liter0.008The effects of temperatures ranging from 20° to 42.5° C on the growth rates ofHydrogenomonas eutrophawere 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.Hydrogenomonasrequires, 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 CO2partial 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. At0.2 mMoxygen 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 CO2conversion 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 CO2is adequate. Upon a further increase in cell concentration, the conversion rate still increases but not linearly. The highest amount of CO2taken 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 CO2per hour. With a suspension at a density of approximately 10 grams, the conversion of 1.1 liters of CO2per liter per hour is obtained. At a CO2output 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-growingH. facilis, the volume of suspension required to support one man is about 500 liters. UsingH. 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 30liters 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 intable 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.

Hydrogen bacteria are characterized by their ability to metabolize and multiply in a strictly inorganic medium, when supplied with H2, CO2and O2in 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 ofHydrogenomonas eutrophaby 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 forHydrogenomonas eutrophaare shown intable X.

Culture parameter

Optimum value

Cell density, g (dry weight)/liter

Temperature, °C

Pressure, atm

pH (phosphate buffer)

6.8 (6.4-8.0)

H2, percent

O2, percent

CO2, percent

Urea CO(NH2)2, g/liter

MgSO4·7H2O, g/liter

0.1

Fe(NH4)2(SO4)2, g/liter

0.008

The effects of temperatures ranging from 20° to 42.5° C on the growth rates ofHydrogenomonas eutrophawere 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.Hydrogenomonasrequires, 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 CO2partial 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. At0.2 mMoxygen 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 CO2conversion 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 CO2is adequate. Upon a further increase in cell concentration, the conversion rate still increases but not linearly. The highest amount of CO2taken 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 CO2per hour. With a suspension at a density of approximately 10 grams, the conversion of 1.1 liters of CO2per liter per hour is obtained. At a CO2output 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-growingH. facilis, the volume of suspension required to support one man is about 500 liters. UsingH. 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 30liters 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 intable 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 ofHydrogenomonasBacteriaGrowth 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 occursunder 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 ofHydrogenomonasbacteria, 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 ofHydrogenomonas eutrophaconsumed hydrogen produced at the cathode of an electrolysis cell built into a specially constructed Warburg flask. Attempts to immobilizeHydrogenomonascells 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 integratedsystem 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 eutrophahas 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.

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 occursunder 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 ofHydrogenomonasbacteria, 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 ofHydrogenomonas eutrophaconsumed hydrogen produced at the cathode of an electrolysis cell built into a specially constructed Warburg flask. Attempts to immobilizeHydrogenomonascells 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 integratedsystem 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 eutrophahas 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.

Bacterial Composition and NutritionHydrogenomonasbacteria can be used for at least part of the astronauts' diet. The washed bacteria have a mild taste and are being studied for their total energy content, protein and lipid digestibility, and vitamin content. Carbon and nitrogen balances, and respiratory quotient are to be determined in animals fed the bacteria as their sole food source. No toxic constituents have been discovered. Sonicated and cooked bacteria, when fed to white rats as 12 percent of the solids of a nutritionally balanced diet, were eaten readily and produced no ill effects. Net utilization of the protein appears to be somewhat lower than casein and about the same as legume proteins.The composition ofHydrogenomonas eutrophais shown intable XI. The composition of the bacteria varies with the age and growth phase of the cells and with the medium and gas available. It is possible to modify the growth conditions to grow the type of bacteria desired for nutritive purposes.Hydrogenomonascells contain about 75 percent water. Of the dry weight, about 74 percent is protein, calculated as 6.25 times the nitrogen content.Table XIshows the amino acid composition to be comparable with other bacterial proteins, except for higher tryptophan and methionine values.Table XI—Analysis ofHydrogenomonas eutrophaCells Grown in Continuous Culture[From[ref.194]]ConstituentPercent by weightMoisture74.55Fat.44Ash1.73Nitrogen3.02(wet)11.87(dry)Protein (N × 6.25)18.90(wet)74.26(dry)Amino acids (dry weight)8Alanine4.47Arginine3.41Aspartic acid4.32Cystine.08Glutamic acid7.67Glycine2.76Histidine.95Isoleucine2.17Leucine4.04Lysine2.65Methionine1.14Phenylalanine2.20Proline2.06Serine1.80Threonine2.15Tryptophan.78Tyrosine1.79Valine3.03The lipid content of rapidly growing cells is normally quite low (0.45 to 2.3 percent crude ether extractable lipids). The most important lipid is poly-beta-hydroxybutyric acid, which is stored under the growing conditions of insufficient nitrogen or oxygen supply (refs.[ref.187]and[ref.191]). Under these conditions, this unusual polymer constitutes up to 80 percent of the dry weight. While the monomer itself, beta-hydroxybutyric acid, is rapidly and efficiently used in cell metabolism, the nutritive value of the polymer is yet to be determined. The fatty acids found include lauric, myristic, palmitic, palmitoleic, heptadecaenoic, C17 saturated(?), stearic, linoleic, and linolenic(?) ([ref.195]).

Hydrogenomonasbacteria can be used for at least part of the astronauts' diet. The washed bacteria have a mild taste and are being studied for their total energy content, protein and lipid digestibility, and vitamin content. Carbon and nitrogen balances, and respiratory quotient are to be determined in animals fed the bacteria as their sole food source. No toxic constituents have been discovered. Sonicated and cooked bacteria, when fed to white rats as 12 percent of the solids of a nutritionally balanced diet, were eaten readily and produced no ill effects. Net utilization of the protein appears to be somewhat lower than casein and about the same as legume proteins.

The composition ofHydrogenomonas eutrophais shown intable XI. The composition of the bacteria varies with the age and growth phase of the cells and with the medium and gas available. It is possible to modify the growth conditions to grow the type of bacteria desired for nutritive purposes.

Hydrogenomonascells contain about 75 percent water. Of the dry weight, about 74 percent is protein, calculated as 6.25 times the nitrogen content.Table XIshows the amino acid composition to be comparable with other bacterial proteins, except for higher tryptophan and methionine values.

Constituent

Percent by weight

Moisture

74.55

Fat

.44

Ash

1.73

Nitrogen

3.02

(wet)

11.87

(dry)

Protein (N × 6.25)

18.90

(wet)

74.26

(dry)

Amino acids (dry weight)8

Alanine

4.47

Arginine

3.41

Aspartic acid

4.32

Cystine

.08

Glutamic acid

7.67

Glycine

2.76

Histidine

.95

Isoleucine

2.17

Leucine

4.04

Lysine

2.65

Methionine

1.14

Phenylalanine

2.20

Proline

2.06

Serine

1.80

Threonine

2.15

Tryptophan

.78

Tyrosine

1.79

Valine

3.03

The lipid content of rapidly growing cells is normally quite low (0.45 to 2.3 percent crude ether extractable lipids). The most important lipid is poly-beta-hydroxybutyric acid, which is stored under the growing conditions of insufficient nitrogen or oxygen supply (refs.[ref.187]and[ref.191]). Under these conditions, this unusual polymer constitutes up to 80 percent of the dry weight. While the monomer itself, beta-hydroxybutyric acid, is rapidly and efficiently used in cell metabolism, the nutritive value of the polymer is yet to be determined. The fatty acids found include lauric, myristic, palmitic, palmitoleic, heptadecaenoic, C17 saturated(?), stearic, linoleic, and linolenic(?) ([ref.195]).

Application to Spacecraft SystemA bioregenerative life-support system will be required in long manned space flight, especially with several astronauts such as would be required for a manned mission to Mars in the 1980 time period. While almost 15 years is a long leadtime, the biological research and engineering problems are formidable, and a system would have to be developed at least 5 years before the mission.The power and weight requirements for both chemical and biological regenerative life-support systems were presented intable VIII. These should be considered tentative best estimates based on present data.The use of bioregenerative systems in spacecraft systems has been studied by Bongers and Kok ([ref.175]) who put the electrolysis-Hydrogenomonassystem in proper perspective with the following statement:The bioregenerative systems are more or less in a transitory phase between research and development. The power data can be considered fairly accurate, at least within ±20 percent. The postulated weight data, however, represent approximations, particularly with respect to auxiliary equipment and construction materials. Also omitted are the weight penalties most probably involved in the processing of the solid output of the exchangers, elegantly defined as potential food. Further research is required in this area to evaluate the regenerative systems, especially the bacteria, with respect to this potential. Furthermore, as yet there is no experimental proof that the growth rates of the heavy bacterial suspensions can be realized in a large design, determined on a relatively small scale with fairly precise control of physiological conditions and gas exchange. This aspect may affect considerably the weight involved in a chemosynthetic balanced system. Nevertheless, at present, this approach still seems most promising.

A bioregenerative life-support system will be required in long manned space flight, especially with several astronauts such as would be required for a manned mission to Mars in the 1980 time period. While almost 15 years is a long leadtime, the biological research and engineering problems are formidable, and a system would have to be developed at least 5 years before the mission.

The power and weight requirements for both chemical and biological regenerative life-support systems were presented intable VIII. These should be considered tentative best estimates based on present data.

The use of bioregenerative systems in spacecraft systems has been studied by Bongers and Kok ([ref.175]) who put the electrolysis-Hydrogenomonassystem in proper perspective with the following statement:

The bioregenerative systems are more or less in a transitory phase between research and development. The power data can be considered fairly accurate, at least within ±20 percent. The postulated weight data, however, represent approximations, particularly with respect to auxiliary equipment and construction materials. Also omitted are the weight penalties most probably involved in the processing of the solid output of the exchangers, elegantly defined as potential food. Further research is required in this area to evaluate the regenerative systems, especially the bacteria, with respect to this potential. Furthermore, as yet there is no experimental proof that the growth rates of the heavy bacterial suspensions can be realized in a large design, determined on a relatively small scale with fairly precise control of physiological conditions and gas exchange. This aspect may affect considerably the weight involved in a chemosynthetic balanced system. Nevertheless, at present, this approach still seems most promising.

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