BIOREGENERATIVE LIFE-SUPPORT SYSTEMS
Placing a man in space requires a complete life-support system capable of supplying sufficient oxygen, food, and water and removing excess carbon dioxide, water vapor, and human body wastes. In addition, the oxygen, carbon dioxide, and pressure must be maintained at a suitable level. Any accumulated toxic products and noxious odors must be removed.
In the spacecraft the human is confined in a restricted environment in which it is necessary to establish a balanced microcosm or closed ecological system. This is an enormous biological and bioengineering problem. Weight, size, simplicity of operation, and reliability particularly are important factors.
For relatively short missions involving one or several astronauts, food, oxygen, and water can be stored and made available as required, and the various waste products can be stored. On longer missions, particularly those involving more than one astronaut, efficient chemical or biological regenerative systems will be required. Any regenerative system introduces a fixed cost in weight of processing equipment and energy requirements.
Chemical, or partially regenerative, methods for providing breathing oxygen by the regeneration of metabolic products such as water vapor and carbon dioxide include the thermal decomposition of water and CO2, photolysis and radiolysis of water, electrolysis of fused carbonates and aqueous solutions, and the chemical reduction of CO2 with H2, followed by electrolysis of the water formed. Chemical regenerative systems have been developed to remove excess carbon dioxide and water vapor from the atmosphere. Nonbiological regenerative systems are time limited by the amount of food, water, and oxygen that can be carried or recovered. These physical-chemical processes show great potential, but they also present many difficulties, including requirements for extremely high temperatures and considerable amounts of power, the formation of highly toxic materials, and high susceptibility to inactivation. None of the presently studied nonbiological processes can function as completely as a bioregenerative system. All these nonbiological systems have unrealistic supply requirements and produce unusable wastes. Consequently, for long planetary missions the bioregenerative systems, though also beset with problems, are potentially far superior to their physical and chemical counterparts.
[Table VIII] shows average daily metabolic data for a 70-kg astronaut. A man breathes about 10 cubic feet of air per minute, or 400 000 liters, daily. The expired air contains about 4 percent carbon dioxide. Man normally breathes air containing 0.03 percent CO2, but can withstand comfortably about 1.5 percent CO2. Anything in excess of 1.5 percent will produce labored breathing, headaches, and, if greatly exceeded, death. A man exhales about 1.1 pounds of water per day and this, in addition to water from perspiration and other sources, must be removed from the air.
O2 input, kg | 0.862 |
CO2 output, kg | 1.056 |
Drinking water, liters | 2.5 |
Food rehydrating water, liters | 1 |
Caloric value of food, kcal | 3000 |
Water output: | |
Urine, liters | 1.6 |
Respiration andperspiration, liters | 2.13 |
Feces, kg | 0.09 |
Total heal output, Btu | 11 100 |
Two types of biological regenerative systems have been proposed. The photosynthetic closed ecological system was proposed as early as 1951. This involves the use of single-celled algae or higher plants, including floating aquatic and terrestrial plants, and requires the interaction of light energy with CO2 and H2O to produce O2 and plant cells. Another system, proposed in 1961, involves electrolysis of water into oxygen and hydrogen, and the concurrent use of Hydrogenomonas bacteria which take up hydrogen, some oxygen, carbon dioxide, and urine yielding water and bacterial cells.
System | Requirements/1 man[4] | Requirements/3 men(270 man-daymission)[5] | ||
|---|---|---|---|---|
Weight,kg | Power,kW | Weight,kg | Power,kW | |
Partial chemoregenerative | [7] 332 | 1.75 | ||
LiOH | 125 | 1.40 | ||
NaOH | 155 | 7.68 | ||
CO2-H2 | 34 | .36 | ||
Full bioregenerative—algae: | ||||
Artificial illumination | 116 | [6] 10.40 | 591 | 25.00 |
Solar illumination | 103 | 1.70 | 356 | .60 |
Electrolysis-hydrogenomonas | 55 | .25 | 129 | 2.60 |
The values given in [table IX] indicate relative weights and powers required by various systems to provide the gaseous environment for manned space cabins. If one considers operating temperatures and hazards, other systems may offer advantages which offset the weight and power advantages of the hydrogen reduction of LiOH systems.
Research is being conducted by NASA on life-support-system technology applicable to missions planned for 20 years in the future. Life-support systems include the requirements for supplying breathing gases, control of contaminants in the cabin atmosphere, water reclamation, food supply, and personal hygiene. The disciplines involved in such systems include biology and microbiology, cryogenic fluid handling at zero g, heat transfer, and thermal integration with other systems, such as power. The physiological, psychological, and sociological problems of the crew are also being considered.
Photosynthetic System
Green plants contain chlorophyll which captures light energy thermodynamically required to convert carbon dioxide and water into carbohydrate which can subsequently be transformed into other foods such as protein and fat. During this process, carbon dioxide is consumed, and an approximately equal amount of oxygen gas is liberated. As a first approximation, photosynthesis is the reverse of the oxidative metabolism of animal life:
Oxidation
C6H12O6 + 6O2 ———————> 6CO2 + 6H2O + heat
Photosynthesis
6CO2 + 6H2O + light ———————> C6H12O6 + 6O2
The photosynthetic process in plants and respiration during photosynthesis have been studied intensively, and several metabolic pathways have been elucidated. Mechanisms are being studied to explain the inhibitory effect of strong visible light on this process. This program may lead to the use of chloroplasts or chlorophyll without cells in future photosynthetic bioregenerative systems for long-term space travel.
One of the prime considerations of a closed ecological system is that the environmental gases shall remain physiologically tolerable to all of the ecologic components. Ideally, a photosynthetic gas exchange organism should possess a high ratio of gas exchange to total mass (considering all equipment and material incidental to growth, harvesting, processing, and utilization); and a controllable assimilation rate to maintain steady-state gas composition. It should also be (1) amenable to confining quarters which may be imposed by inflexibility of rocket or space station design; (2) genetically and physiologically stable and highly resistant to anticipated stresses; (3) edible and capable of supplying most or all human nutritional requirements; (4) capable of utilizing raw or appropriately treated organic wastes; and (5) amenable to water recycling as demanded by other components of the ecosystem.
Higher Plants
Efforts to utilize multicellular plants as photosynthetic gas exchangers have been somewhat neglected, since it has been assumed by many that algae would be more efficient. The family Lemnaceae (duckweeds) are small primitive aquatic plants with a minimum of tissue differentiation. Practically all of the cells of the plant contain chlorophyll and are capable of photosynthetic activity. They reproduce principally by asexual budding of parent leaflike fronds. They can be grown readily on moist surfaces ([ref.177]) on almost any medium suitable for the growth of autotrophic plants. With duckweeds the problems of gaseous exchange and harvesting are simplified and the volume of medium can be greatly decreased as compared with algae.
Ney ([ref.177]) obtained a very high gas exchange rate with duckweeds. Using small cultures under controlled optimal conditions of temperature, light (600-1000 ft-c), and CO2, concentration, he estimated that 2.3 m2 of frondal surface of duckweed, at a gas exchange rate of 10.8 liters m2/hr would provide sufficient gas exchange for one man. This would produce about 25 grams of dry plant material per hour.
A few nutritional studies have been carried out with duckweeds. Nakamura ([ref.178]) considered Wolffia as a possible source of food for space travel and found that it contained carbohydrate 25-60 percent, protein 8-10 percent, fat 18-20 percent, minerals 6-8 percent (all dry weights), and vitamins B2, B6, and C, with C the most abundant.
One of the desirable features of a duckweed system is that the gas exchange is direct between the atmosphere and the plant and does not require dissolving the respiratory gases in a bulky fluid system which introduces special engineering difficulties in zero- or low-gravity conditions.
In the design of equipment for photosynthetic studies, careful consideration should be given to the material used in the construction of the unit. Most plastic materials are subject to photo-oxidative degradation, with CO as one of the products. When air is recirculated through plastic tubing and transparent rigid plastics in the presence of light, considerable quantities of CO are given off. With high-intensity illumination such as sunlight, a CO buildup of several hundred parts per million is not uncommon. Also, plant pigments such as the carotenoids and chlorophylls will react similarly when exposed to light of high intensity. If the plants die, then CO is released quite rapidly.
At Colorado State University the responses of plants to high-intensity radiation (ultraviolet to infrared) are being studied. Plants from high mountaintops that are exposed to greater ultraviolet light are being studied for specialized adaptations. The effect of temperature on photosynthesis is being explored. Various plants are also being studied under germ-free conditions.
Screening of higher plants for possible use in bioregenerative systems at Connecticut Agriculture Experiment Station resulted in the selection of corn, sugarcane, and sunflower. Under optimal conditions it has been shown that 100 to 130 ft2 of leaf surface are required to support an astronaut.
Plants considered as possible food sources include soybeans, peanuts, rice, and tomatoes, which can be combined with algae to give a well-balanced and reasonably varied diet. Hydroponic systems use large quantities of water, but progress is being made in reducing this.
The possibility of using animals in the closed ecological system is open to question, particularly in the absence of gravity, and much work remains to be done on using plant materials as animal food and on the disposal of wastes. Animals which have been considered are crustaceans, fish, chickens, rabbits, and goats.
Algae
Algae have the fastest growth rate and are among the most efficient plants for oxygen and food production. It has been amply demonstrated by Myers ([ref.179]) and other workers that Chlorella can be used in a closed ecological system to maintain animals such as mice and a monkey. The use of algae for supplying O2 and food, and for removing CO2 and odors has been considered by many authors for use in spacecraft, space platforms, and for establishing bases on the Moon or Mars.
Estimates of total efficiency are based on extrapolated laboratory data and vary widely, since many different types of data have been used as a basis for these estimates.
The respired air containing about 4-5 percent CO2 is bubbled into the Chlorella culture, at either atmospheric or increased pressure. Air containing a high percentage of oxygen and saturated with moisture is released from the algal system.
The use of algae for several purposes might require from one to three separate algal systems. For food production, Chlorella produces 50 percent protein and 50 percent lipids in high-nitrogen media. In low-nitrogen media, it produces 85 percent lipids. Proper choice of Chlorella strains and media will produce not only the necessary calories but also the necessary specific nutrients required. Certain strains are more effective in O2 production, and others in the use of urine and other wastes.
Some of the early estimates, using Chlorella grown at 25° C, for supplying these requirements for a single man in space include the following: 168 kg of algal suspension ([ref.179]), 200 kg of algal suspension and 50 kg of equipment including pumps (refs. [ref.180] and [ref.181]), and 100 kg of algal suspension and 50 cubic feet for equipment and gas exchange ([ref.182]). Using the blue-green alga Synechocystis, 600 kg of algal suspension would be required, according to Gafford and Craft. These estimates are based on preliminary studies, are quite high, and are not of real practical value.
Other studies have indicated an extremely efficient algal system which offers a real potential for a practical and effective gas exchanger ([ref.183]). A thermophilic strain of Chlorella with an optimum growth temperature of 39° C and an optimum temperature for photosynthesis of about 40° C can increase its cell mass 10 000-fold per day. When operating at one-half maximum efficiency, this alga produces 100 times its cell volume of oxygen per hour. Burk et al. ([ref.183]) state: "Future engineering development should lead to a space requirement, per adult person, of no more than 3 to 5 cubic feet of algal culture, equipment, and instrumentation for adequate purification of air." The requirements of this system would require additional energy in the form of light and of small amounts of nitrogenous and mineral material for the algae. The light source used by Burk et al. ([ref.183]) is a tungsten filament quartz lamp the size of a pencil, which has a long life, produces a luminous flux 5-10 times greater than sunlight on Earth, and operates at a 10-12 percent light efficiency.
Research is being carried out on algal regenerative systems by about 40 or 50 laboratories in the United States. NASA is supporting several basic studies on photosynthesis, the physiology of algae, and engineering pilot-plant development. Much of the research on algae is being supported by the Air Force.
Most algal studies have been carried out in small units and the data obtained have been used as a basis for extrapolating logistic values for the use of these organisms in manned space vehicles. Myers ([ref.179]) has shown that the quantity of algae necessary to support a man (with an assumed O2 requirement of 625 liters per day) would yield about 600-700 grams dry weight of new cells per day. If algal growth in mass cultures could be maintained in a steady-state concentration of 2.5 gram dry weight per liter with such a growth rate as to yield 10 grams weight per liter per day, the volume of algal culture would be 60-70 liters and the total mass of the system would approximate 200-250 pounds.
Using an 8-liter system, Ward et al. ([ref.176]) have produced algal concentrations of 5-7 grams of dry algae per liter with a high-temperature algal strain. The maximum growth rate observed with the culture was 0.375 gram dry weight per liter per hour, or 9 grams dry weight per liter per day. This was accomplished by using 1-centimeter layers of culture and a light intensity of 8000 foot-candles. The culture system consisted of a rectangular plastic chamber having an area of 0.5 square meter and illuminated on each side to an intensity of 4000 foot-candles (cool-white). To produce 25 liters of oxygen per hour, an area of 8.3 square meters (85 square feet) would be required.
The major problem in large-scale production of algae is that of illumination. Conversion of electricity to light has an efficiency of only 10 to 20 percent. In addition, the maximum efficiency of light utilization by Chlorella algae lies in the range of 18-22 percent. This results in a maximum efficiency of only 4 percent for photosynthetic systems. Another problem involved in conversion of electricity to light is the production of heat which has to be removed even with thermophilic algae. With a human demand of 600 liters of oxygen per day, the minimum electrical requirement becomes 4 kW. No large-scale culture has yet been managed at anything close to this minimum figure.
Another problem is the poor penetration of light into concentrated cultures of algae. This necessitates construction of large tanks of only about ¼-inch thickness. This results frequently in fouling of the surfaces of the tank by algae and makes the removal of the excess algae difficult. Production of 1 liter of oxygen results in the production of 1 gram dry weight of algae. Although a small amount of CO is produced by some algae, it can probably be removed by catalytic oxidation. Other problems include mutation and genetic drift of the algae and the necessity for maintaining bacteria-free cultures. There are also difficulties in maintaining a sterile culture if urine is to be used as a nitrogen source. While there is a potential for using algae as food, more research is required before it can be determined what quantity and methods of processing can be used. Research and development on algae is much greater than on both the higher plants and the electrolysis-Hydrogenomonas systems together.
The difference between the photosynthetic and electrolysis-chemosynthetic systems is the way electrical energy is made available to the organisms. In the photosynthetic system, electrical energy is converted to light which the algae or plants transform into chemical energy. In the chemosynthetic process, electrical energy is transformed into the chemical energy of hydrogen gas which is used by the bacteria. Both organisms use the chemical energy available to them to synthesize cell material with similar degrees of efficiency. The problem is to make the conversion of electricity to available chemical energy as efficient as possible.
In photosynthetic systems much energy is lost in the conversion of electricity to light, a process only 10-20 percent efficient at best. When this is combined with the loss from the inefficient use of light by plants, an overall efficiency of about 4 percent is obtained. In the electrolysis-Hydrogenomonas system, the two steps are very efficient. Electrolysis cells can operate at up to 85 percent efficiency and the overall efficiency can be up to seven times that of a photosynthetic system.
ELECTROLYSIS-HYDROGENOMONAS SYSTEM
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 H2 is required for the conversion of 1 mole of CO2 (the hourly production of a man). The removal of this amount of CO2 would thus require the cleavage of 4 moles of water. In addition, to supply oxygen for human respiration (at a rate of 1 mole of O2 per 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 in [table VIII] show that the CO2 of 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 Culture
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].
| Culture parameter | Optimum value |
|---|---|
| Cell density, g (dry weight)/liter | 10 |
| Temperature, °C | 35 |
| Pressure, atm | 1 |
| pH (phosphate buffer) | 6.8 (6.4-8.0) |
| H2, percent | 75 |
| O2, percent | 15 |
| CO2, percent | 10 |
| Urea CO(NH2)2, g/liter | 1 |
| 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 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.
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.
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.
Bacterial Composition and Nutrition
Hydrogenomonas bacteria 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 of Hydrogenomonas eutropha is shown in [table 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.
Hydrogenomonas cells contain about 75 percent water. Of the dry weight, about 74 percent is protein, calculated as 6.25 times the nitrogen content. [Table XI] shows 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 System
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 in [table 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-Hydrogenomonas system 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.
CABIN ATMOSPHERES[9]
In the first U.S. manned space flight program, Project Mercury, and in the face of very severe weight limitations, a cabin atmosphere of pure oxygen at one-third atmospheric pressure was adopted. This choice probably represented the greatest simplification which could be achieved readily and, at the same time, provide protection against some of the risks of rapid decompression. Although breathing pure oxygen at higher pressures was known to be attended by some undesirable physiological effects, the short duration of the flights to be undertaken, and the low pressure employed, suggested that no harmful results would result in this case. That these expectations were generally borne out is now history. Preparations for space flights of longer duration—many weeks or months—present similar problems and require special attention to phenomena which may be either undetectable or of trivial significance on a time scale of a few days.
Physiological Criteria in the Choice of Cabin Atmosphere
If maintenance of normal respiratory function were the only consideration, a cabin atmosphere of about sea-level composition and pressure might be an ideal and straightforward choice for manned spacecraft. In fact, this atmosphere has been used in the manned space flights conducted by the U.S.S.R. No other atmosphere has been shown to be more satisfactory from the physiological point of view, and the tedious respiratory studies which should accompany the use of other atmospheres can be avoided. Nevertheless, the formidable problems of spacecraft design and the necessary precautions for safeguarding the crew from accident require that other atmospheric compositions and pressures be considered. For example, if a cabin at 1-atm pressure were decompressed to space suit pressure (0.3 atm), the occupants would develop decompression sickness; i.e., "bends."
Several engineering considerations argue for low cabin pressures and pure oxygen composition. Among these are structural design, weight of atmospheric gas storage and control equipment, and the difficulty of contriving pressure suits which allow operation at pressures near one atmosphere. Such departures from the normal human gaseous environment, however, require the demonstration of an acceptable level of safety and physiological performance.
The limits of the composition and pressure of acceptable cabin atmospheres are then set by—
- A pure oxygen atmosphere at a pressure which will provide an alveolar oxygen partial pressure equal to that provided by air at sea level
- A mixed gas (oxygen and inert gas) atmosphere having a pressure and composition that will allow decompression to the highest acceptable suit pressure without the risk of bends
A numerical value for the lower limit (1) is approximately 0.2 atm of pure oxygen. The upper limit (2) is determined by the operating pressure and composition of the space-suit atmosphere and may be of the order of 0.5 atm for a cabin atmosphere of 50 percent oxygen. It is necessary to determine the astronaut's ability to survive and perform his duties in any atmosphere selected.
Atelectasis and Pulmonary Edema
Localized or diffuse collapse of alveoli in the lungs may, if the condition persists, lead to arterial hypoxia which may be extremely undesirable under the stresses of space flight. The alveoli are probably unstable when pure oxygen is breathed; they tend to collapse if there is blockage of the airways, especially at low pressures. This collapse occurs because each of the gases present in the alveoli (oxygen, water vapor, and carbon dioxide) is subject to prompt and complete absorption from the alveoli by the blood.
The alveoli are normally stabilized against collapse by the presence of inert and relatively insoluble gas (nitrogen) and an internal coating of lipoprotein substances with low surface tension.
Theoretical and experimental results strongly suggest the desirability of using oxygen-inert gas atmospheres for long missions to avoid atelectasis and other gas absorption phenomena, such as retraction of the eardrum. However, further experimental evidence is required both to confirm this point and to establish its upper limit of suitability of pure oxygen atmospheres.
At Ohio State University in 1962, scientists studied the effect on young rats exposed for 27 days to 100 percent oxygen (with no nitrogen), at a reduced barometric pressure equivalent to 33 000 feet altitude. The rats showed no difference in growth rate, oxygen consumption, food and water intake, or behavior from control rats in air at 1 atm.
Oxygen Toxicity
It has long been known that breathing pure oxygen at normal atmospheric pressure often produces pulmonary irritation and other toxic effects both in man and animals. This knowledge has occasioned concern over the use of pure oxygen atmospheres in spacecraft.
The effect of 100 percent oxygen at a simulated altitude of 26 000 feet for 6 weeks was studied using white rats at Oklahoma City University under a NASA grant. Radioactive carbon techniques revealed a 15-percent reduction of metabolism in the 100-percent oxygen-exposed rats, compared with rats in air at 1 atmosphere. There was a 20-percent decrease in lipid metabolism in the liver compared with controls, but no decrease in heart metabolism. There was no gross change in body weight.
The White Leghorn chick between 2 and 7 weeks old is markedly resistant to the toxic effects of 1 atm of O2. Continuous exposure (Ohio State University) for as long as 4 weeks did not cause deaths, obvious morbidity, or any signs of pulmonary damage on gross autopsy. Nevertheless, the hyperoxia had some adverse effects, primarily reducing the growth rate to between three-fourths to one-fourth of normal; reducing feed intake per unit body weight to three-fourths of normal; slowing respiratory rate by 30 percent; decreasing erythrocytes, hemoglobin, and hematocrit by 9 to 12 percent; and causing reversible histological changes in the lungs. Arterial O2 tensions were elevated over 300-mm Hg, but arterial pCO2 and blood pH were unaffected. No residual effects were noted upon return to air breathing. It is possible that the anatomical peculiarities of the avian lung play some role in the chicks' resistance to hyperoxia, but it is also possible that this resistance is a function of age, similar to the tolerance shown by the young rat but not the adult.
Carbon Dioxide Tolerance
Studies of CO2 tolerance in submarine crews indicate that no loss of performance is involved if the concentration in air at normal pressure does not exceed 1.5 percent with exposures of 30 to 40 days. However, biochemical adaptive changes were observed at this concentration.
Inert-Gas Components
If other investigations establish the need for an inert gas in manned spacecraft atmospheres, gases other than nitrogen may be considered. Compared with nitrogen, the physical properties or helium and neon offer advantages with respect to solubility in body fluids, storage weight, and thermal properties.
Studies at Ohio State University in 1964, under a NASA grant, showed that helium substituted for nitrogen in a closed container causes humans to feel "cold" at a normally comfortable temperature. Studies with animals have shown that in a helium atmosphere there is greater heat loss due to the increased conducting capacity and probably greater evaporative capacity. In 6 days at 21 percent oxygen and 79 percent helium at 1-atmosphere pressure, young rats grew at the same rate as controls, but drank more water, excreted more urine, and had a higher rate of food and oxygen consumption than controls in air at 1 atmosphere. Men are being tested on a bicycle ergometer in saturated and low relative humidity helium atmospheres to study heat balance.
Mice were exposed to 80 percent argon and 20 percent oxygen continuously at 1-atmosphere pressure for 35 days at Oklahoma City University. Carbon 14 studies of metabolism showed a slight slowing and a twofold to threefold increase in fat deposition.
Bends
Decompression, whether accidental (due to damage of the spacecraft) or intentional (as in the use of the pressure suit outside the capsule), carries the risk of bends if the inert gases dissolved in the tissues and body fluids come out of solution. The magnitude of this risk is determined to a very considerable extent by—
- Individual susceptibility
- The extent to which the nitrogen (or other inert gas) concentrations of tissues and body fluids have been reduced
- The magnitude and rate of the inert-gas, partial pressure change on decompression
The probability of getting bends is reduced by—
- Selection of bends-resistant individuals
- Thorough denitrogenation before flight
- Limitation of decompressive pressure changes by appropriate choice of cabin atmosphere pressure and composition
- Space-suit pressure setting
In some cases, further improvements might be obtained by using, in the cabin atmosphere, an inert-gas component which has a lower solubility in tissue and body fluids or less tendency than nitrogen to form bubbles.
Fire Hazard
Experience indicates that fires in pure oxygen atmospheres, even at low pressures (e.g., 1/3 atm), are extremely difficult to extinguish. While this phenomenon has nothing to do with respiratory physiology, the risk on flights of long duration may be so serious as to demand special measures. Unless effective countermeasures can be devised, this risk may argue very strongly against the use of such atmospheres in the future. Further experimental investigation is required.
Acceleration Effects on the Lungs and Pulmonary Circulation
Forces produced by high acceleration overdistend one part and compress another part of the lungs. Blood flow diminishes in some parts of the lungs and increases in others. Fluid leaks from the blood into the tissues and into the air sacs in parts of the lungs. These effects cause difficulty in breathing, low arterial oxygen saturation, and impaired consciousness during high sustained acceleration and, to a lesser extent, after its cessation. They must be considered when selecting the best gas to be breathed, since a high partial pressure of oxygen is favorable for consciousness, but a low inert-gas concentration during acceleration is unfavorable for rapid lung recovery afterward.