Significant Achievements in Space Bioscience 1958-1964 Part 11

Higher Plants

Efforts to utilize multicellular plants as photosynthetic gas exchangers have been somewhat neglected, since it has been a.s.sumed 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 princ.i.p.ally by as.e.xual 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 m of frondal surface of duckweed, at a gas exchange rate of 10.8 liters m/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 s.p.a.ce 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 quant.i.ties 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 ft 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 quant.i.ties 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 s.p.a.cecraft, s.p.a.ce platforms, and for establis.h.i.+ng 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 s.p.a.ce 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 ma.s.s 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 s.p.a.ce 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 s.p.a.ce vehicles. Myers ([ref.179]) has shown that the quant.i.ty of algae necessary to support a man (with an a.s.sumed O2 requirement of 625 liters per day) would yield about 600-700 grams dry weight of new cells per day. If algal growth in ma.s.s 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 ma.s.s 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 -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 quant.i.ty 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 quant.i.tation 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.

Table X.-_Optimum Growth Requirements of_ Hydrogenomonas eutropha

--------------------------------------------------------- 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 --------------------------------------------------------- MgSO47H2O, 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.