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.

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.

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.

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—

1. A pure oxygen atmosphere at a pressure which will provide an alveolar oxygen partial pressure equal to that provided by air at sea level

2. 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 33000 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 26000 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—

1. Individual susceptibility

2. The extent to which the nitrogen (or other inert gas) concentrations of tissues and body fluids have been reduced

3. The magnitude and rate of the inert-gas, partial pressure change on decompression

The probability of getting bends is reduced by—

1. Selection of bends-resistant individuals

2. Thorough denitrogenation before flight

3. Limitation of decompressive pressure changes by appropriate choice of cabin atmosphere pressure and composition

4. 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.

General Studies of Biological Rhythmicity

The effects of weightlessness on the organism as a whole may be manifested by important changes in certain integrated behavioral patterns having an inherently rhythmic character. Modifications in basic behavioral patterns and performance may occur as disruptions of rhythmic physiological phenomena, which are themselves the end product of interrelated functional activity in a number of physiological systems, such as the neuroendocrine, cardiovascular, and central nervous systems.

Measurements of interdependent components of biological rhythmicity are beginning to be analyzed by methods well established in physics—including correlation and spectral analyses, and phase modulation and variance in rhythmic processes. A wide variety of physiological functions can be treated as periodic variables in the analysis, including rhythmicities in cardiac output and blood pressure, respiration, brain waves, and the slower tides of appetite, and sleep-wakefulness. The importance of such investigations argues for their inclusion in forthcoming flight programs. Their experimental simplicity is an additional advantage. Biorhythms have been discussed in more detail in the section on "Environmental Biology."

Effects of Weightlessness on the Cardiovascular System

Earlobe oximetry, indirect measurements of blood flow and of blood pressure by finger plethysmography or impedance plethysmography, and ballistocardiographic techniques have potential application to manned space flight.

Adaptation to prolonged exposure to weightlessness or to lunar gravity may cause difficulties when the astronaut is exposed again to reentry forces and terrestrial gravity. It is possible that these adaptive changes may thus produce unacceptable effects on performance or cause risk to life. It is important to obtain experimental evidence on this subject.

It is common knowledge that following a stay in bed, dizziness, faintness, and weakness characterize arising, and that a feeling of general weakness may persist for several days. The phenomenon has been investigated in a number of laboratories. One approach has been to put healthy young subjects to bed, and even in extensive casts for periods of 2 or 3 weeks or more. Two major findings have emerged from these studies. First, a substantial adjustment in the blood circulatory system occurs, which is termed the "hypodynamic state." Second, there is a large decrease in the skeletal and muscle mass of the body.

There are two kinds of evidence for the hypodynamic state: measurement of parameters of circulatory function, and measurement of the response of the individuals to a quantitatively imposed mild gravitational load. After 3 weeks in bed, otherwise healthy persons exhibit an increase of more than 20 percent in heart rate; a reduction of 10 to 20 percent in total blood volume, primarily as a result of reduction of plasma volume; and a decrease in heart size of about 8 percent. Coupled with these cardiovascular changes is a reduction of 10 percent in the basal metabolic rate. It appears as though the circulation and metabolism are reset to a lower functional level commensurate with the reduced demands placed on the whole organism.

After 3 weeks of bed rest, all of the subjects tested showed pronounced orthostatic hypotension. After tilting, the average heart rate increased by 37 beats per minute, the systolic blood pressure fell some 12-mm Hg, and some of the subjects fainted. The measurements were continued for 16 days after the bed-rest period, and it was round that recovery was not quite complete when the experiment was terminated.

There is little question that in prolonged exposures to the weightless state, there is a fair probability of extensive circulatory adjustments, the seriousness of which cannot yet be foretold. While it is likely that the astronauts will adapt successfully to long periods of weightlessness at some new circulatory functional level, the remote possibility exists that the circulatory changes may be progressive to the point of ultimate failure.

Central Nervous System Functions in Weightlessness

The wide range of individual tolerances to the disturbing effects of vestibular stimulation has emphasized the importance of this factor in astronaut selection. At the same time, vestibular functions must be considered jointly with visual task performance, since both have special significance for such maneuvers as vehicle docking. Vestibular function in the weightless state remains almost completely unknown. Limited evidence from animal and manned space flights suggests that head turning, resulting from vestibular stimulation, may seriously interfere with visuomotor performance, but that susceptibility to these disturbances is significantly different between individuals and that partial adaptation occurs relatively quickly.

NASA is currently collecting extensive baseline electroencephalogram data under controlled conditions in a form suitable for mathematical analysis. Data are being taken from about 200 subjects in major national and overseas centers. It is intended that this study will assist in astronaut selection and monitoring in space.

Studies on many effects of weightlessness on nervous functions require monitoring of the autonomic nervous system, including such autonomic effects as gastrointestinal activity, secretion, lacrimation, salivation, sweating, and the central control of respiration. Urinary estimations of catecholamines and 5-hydroxyindoleacetic acid would provide important data on autonomic system activity if collected in flight and compared with preflight and postflight controls.

Major areas have been outlined in which prolonged weightlessness may be expected to interfere with performance, judgment, and, ultimately, chances of survival. These include cardiovascular, metabolic, central nervous, psychophysiological, and biorhythmic effects. They have been dealt with separately and in sequence, but have not been intended to be viewed as hierarchic. The relative scarcity of data necessarily precludes such an evaluation.

Soviet experience with zero gravity and weightlessness has increased their emphasis on this space-flight factor and was an important topic at the May 1964 COSPAR meeting. Discussion of the postflight medical status of Bykovsky (5-day flight) and Tereshkova (3-day flight) revealed a concern for the significance of prolonged weightlessness and the presence of postflight physical debility and fatigue following Vostok flights 3 through 6. These changes persisted for several days. Among the physiological conditions singled out for mention were—

1. Body fluids— Cosmonauts have shown a postflight weight loss of 1.9 to 2.4 kg apparently resulting from a redistribution of body fluid in response to elimination of the hydrostatic pressure gradients caused by Earth gravity. There is the suggestion that this redistribution is complete within the first 24 hours of flight. Titov is reported to have been dehydrated alter his flight with early hemoconcentration. These findings directly support predictions made from ground-based research.

2. Cardiovascular— Postflight orthostatic tachycardia is reported for Titov as long as 23 hours after landing; at 48 hours there was significant residual intolerance to the upright posture. Cosmonauts have demonstrated a 20- to 35-percent increase in oxygen consumption during the standard postflight exercise test.

In both of these areas there was a return to normal within the postflight period of study. The Soviets have continued their biological experiments in space with the Vostok/Voshkod series. Fixing of histologic specimens in flight by Bykovsky demonstrated a critical role for man and made possible an expanded experimental program. Biopackages have become more complex with each succeeding flight.

With the exception of postflight orthostatic intolerance after the third and fourth Mercury flights, changes as a result of exposure to a zero-gravity environment have not been noted by U.S. investigations in space. Ground-based research proceeds here at an advanced pace and is supported in large measure by both the USAF and NASA. A study of the relationships among renal and systemic hemodynamics, neurohumoral cardiovascular regulation, and renal excretory function in differently positioned subjects is underway, as are studies of acceleration tolerance.

Water Requirements

Water requirements are extremely critical and the amount supplied should not under any circumstances be kept to a minimum. Rather, a large margin of safety should be allowed.

Present data on water requirements show a very strong dependence upon suit inlet temperatures. In the absence of an accurately controlled suit temperature, water requirements can easily double. If this should occur, the mission would probably have to be aborted, since it is doubtful if electrolyte balance would be maintained at such high rates of water loss. Normal or even extreme conditions of the terrestrial environment usually include diurnal variation in temperature which may modify water needs. These conditions will not be obtained in the spacecraft.

In addition to ground-based experiments, measurements of water intake should be made under actual flight conditions. Data from short-term flights should be used for extrapolation to longer missions.

Waste

The problem of waste production is intimately related to nutrition and can be solved or simplified by dietary changes. Any diet should be adjusted for the minimum production of feces, before and during even short flights. Water will be sequestered by accumulation in the feces, and the net loss, under normal conditions, would be approximately 40 to 60 grams per man per day. Flatus can be a serious problem, since considerable concentrations of toxic gases may accumulate. The purification system for the recirculated atmosphere must be able to remove these, although the diet should be planned to minimize the problem. The collection of urine and its storage is of importance, particularly on short-term flights, and individual packaging and labeling of urine specimens will be necessary for the analyses.

Metabolism

An accurately measured intake of nutrients, calories, and water is necessary for determining metabolic demands imposed in any space flight. There is insufficient knowledge to predict total metabolic requirements under the numerous stresses which can be anticipated. Simulator studies are of great importance even for short-duration flights.

The two most important variables to be considered in establishing the minimal diet are protein and energy requirements. NASA is supporting research at the University of California (Berkeley) to determine these requirements and to estimate individual variation in healthy young men. The possibility of minimizing need through biological adaptation is being explored.

It is difficult to estimate the minimum protein requirement of an adult man. The generally accepted criterion of minimum adequate protein nutrition in the adult is the maintenance of nitrogen balance at minimum intake. The minimum protein requirements depend on endogenous nitrogen loss. Analysis of the little data available indicates a best estimate of 2 mg of nitrogen per kilocalorie of basal energy expenditure. However, this figure is higher than that noted in experiments in some human subjects.

After minimum nitrogen requirements and minimum amino acid requirements have been established, studies will be directed toward investigating caloric restriction and adaptation to restriction of calories. It has been suggested that caloric restriction in animals and man results in apparent decreased energy need for the same activity. This apparent paradox has never been explained. It has been shown that there is adaptation to repeated episodes of caloric restriction both in animals and man, so that subsequent periods of caloric restriction result in decreased rate of weight loss, nitrogen loss, and longer survival.

Additional experiments are urgently required to determine the metabolic demands for minerals—in particular, the metabolic balance of calcium, potassium, sodium, and phosphorus. Under conditions of high water consumption, large mineral losses are to be expected. Failure to replace these can cause an imbalance which could impair the efficiency of the individual to the extent of endangering the flight.

Analysis of samples taken in flight, both of urine and feces, should be made. Respiratory quotients can be determined in flight, blood samples should be taken before and immediately after flight for analyzing selected components (in simulator studies these could be taken periodically), and nutritional intakes (which would be facilitated by formula diets) must be measured and analyzed.

Short-Range Technology

There are many practical difficulties in providing for food storage and accessibility in spacecraft. The packaging of food materials, both dehydrated and liquid, has proceeded satisfactorily under the supervision of the Food and Container Institute. If packaging materials are to be made to withstand very high relative humidities and large variations in temperature, additional investigations are required, since such containers are not yet available. In packaging, serious consideration must be given to the ease with which the food may be reached and eaten.

If dehydrated formula foods are to be fed on short-term missions, additional work is required on the rehydration of such formulas. Present methods of water measurement under weightless conditions are not satisfactory, and better methods will have to be contrived.

Long-Term Nutritional Problems

There is a dearth of metabolic information, even for short-duration flights, without which changes in metabolic patterns to longer flights cannot be extrapolated. However, using scattered information, certain changes which may be encountered can be hypothesized. Decalcification of bone and changes in water-holding capacity of the body may be anticipated. It is also possible that changes in proportion of fat to lean body mass could be experienced and should be considered in nutritional planning. Nutritional requirements depend on size, particularly lean body mass, sex, physiological state, and individual metabolic rates. Therefore, individuals for space flight should be screened with these factors in mind if it is desirable to minimize food intake in long flights. The factors which influence the total nutritional requirements of the individual also influence his mental and physical responses to stress.

Synthetic Foods

The development of food materials other than those derived directly from animal or vegetable origin is of interest. Advantages of such diets may be low residue, ease of storage, rehydration, and manipulation. Experiments with chemically defined synthetic diet for humans have been carried out by Medical Sciences Research Foundation, San Mateo, Calif. The complete liquid diet is composed of required amino acids, fat, carbohydrate, vitamins, and minerals. A cubic foot of the diet (50 percent solids in H₂O) supplies 2500 calories per day for 1 month, and has been given a variety of artificial flavors.

This synthetic diet has been fed to human volunteers for 6 months in a pilot study at the California Medical Facility, Vacaville, Calif., and the results are being reviewed. Schwarz Bioresearch, Inc., is studying the storage, stability, and packaging of chemically defined synthetic diets for human and animal flights.

Food Production in Space

Long-term feeding in space depends upon a payload of stored food unless food is produced during flight. If sufficient propulsive energy is available, the duration of missions using stored food may be quite long. However, in emergencies in which a mission lasts longer than planned, survival may depend on the ability to produce food extraterrestrially. Eventually it will be desirable or necessary to produce food beyond the confines of Earth.

The nutritional requirements of the crew will be influenced by such factors as activity, physical and psychological stress, individual size of the members, and individual metabolic rates. The food intake will have to be adjusted to meet these requirements. It is necessary to know the nutritional requirements of each astronaut and the way in which these are altered by the conditions of space flight in order to estimate needs on long missions. Without this information, the food supplies for the longer flights may be too much, too little, or improperly balanced. Where dependence would not be on stored food alone, but on food produced en route, more exact information on requirements is needed to determine the capacity of food production units.

In the discussion of bioregenerative systems, it was suggested that food materials could be produced by photosynthetic organisms (e.g., algae, duckweed, and other higher plants) or by nonphotosynthetic organisms (e.g., Hydrogenomonas). In contrast to the use of living organisms, reprocessing waste materials by chemical treatment or the actual synthesis of high-energy compounds has been suggested. No chemical system has yet been demonstrated as workable for the economical production of food in space, and the systems considered produce materials which may be converted to food, but are not food as such.

Algal cultures have had the most extensive investigation as food in space, but the technical problems of using this material as a food source have not yet been solved. It is apparent from the investigations to date that algae will require treatment before they can be used as food. In limited trials, difficulties have been experienced with amino acid deficiencies, digestibility, high residues, and gastric distress. Processing methods which would be applicable in space travel and the possibility of secondary conversion by other animals or plants should be systematically investigated.