Significant Achievements in Space Bioscience 1958-1964 Part 12

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 relations.h.i.+ps 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 relations.h.i.+p 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 s.h.i.+ft in metabolism.

In the final a.n.a.lysis, 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 MgSO47H2O, 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 ma.s.s 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 ma.s.s 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 bioma.s.s 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 Inst.i.tute ([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 const.i.tutes 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 a.s.suring 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 const.i.tuents 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.

Table XI-_a.n.a.lysis of_ Hydrogenomonas eutropha _Cells Grown in Continuous Culture_ [From [ref.194]]

----------------------------------------------------------------- Const.i.tuent 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 -----------------------------------------------------------------

8 Trace amounts of the following were also found: methionine sulfoxide, citrulline, alpha-amino-n-butyric acid, h.o.m.ocitrulline, glucosamine, galactosamine, methionine sulfoximine, ethionine, and ethanolamine.

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 const.i.tutes 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 s.p.a.cecraft System

A bioregenerative life-support system will be required in long manned s.p.a.ce 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 s.p.a.cecraft 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?

? Includes part of [ref.196].

In the first U.S. manned s.p.a.ce 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 s.p.a.ce 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 s.p.a.cecraft. In fact, this atmosphere has been used in the manned s.p.a.ce 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 s.p.a.cecraft 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 s.p.a.ce 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 s.p.a.ce-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