ASTROCULTURE (TM) root metabolism and cytochemical analysis.

Physiology of the root system is dependent upon oxygen availability and tissue respiration. During hypoxia nutrient and water acquisition may be inhibited, thus affecting the overall biochemical and physiological status of the plant. For the Astroculture (TM) plant growth hardware, the availability of oxygen in the root zone was measured by examining the changes in alcohol dehydrogenase (ADH) activity within the root tissue. ADH activity is a sensitive biochemical indicator of hypoxic conditions in plants and was measured in both spaceflight and control roots. In addition to the biochemical enzyme assays, localization of ADH in the root tissue was examined cytochemically. The results of these analyses showed that ADH activity increased significantly as a result of spaceflight exposure. Enzyme activity increased 248% to 304% in dwarf wheat when compared with the ground controls and Brassica showed increases between 334% and 579% when compared with day zero controls. Cytochemical staining revealed no differences in ADH tissue localization in any of the dwarf wheat treatments. These results show the importance of considering root system oxygenation in designing and building nutrient delivery hardware for spaceflight plant cultivation and confirm previous reports of an ADH response associated with spaceflight exposure.

Wheat response to differences in water and nutritional status between zeoponic and hydroponic growth systems.

Hydroponic culture has traditionally been used for controlled environment life support systems (CELSS) because the optimal environment for roots supports high growth rates. Recent developments in zeoponic substrate and microporous tube irrigation (ZPT) also offer high control of the root environment. This study compared the effect of differences in water and nutrient status of ZPT or hydroponic culture on growth and yield of wheat (Triticum aestivum L. cv. USU-Apogee). In a side-by-side test in a controlled environment, wheat was grown in ZPT and recirculating hydroponics to maturity. Water use by plants grown in both culture systems peaked at 15 to 20 L m-2 d-1 up to Day 40, after which it declined more rapidly for plants grown in ZPT culture due to earlier senescence of leaves. No consistent differences in water status were noted between plants grown in the two culture systems. Although yield was similar, harvest index was 28% lower for plants grown in ZPT than in hydroponic culture. Sterile green tillers made up 12 and 0% of the biomass of plants grown in ZPT and hydroponic culture, respectively. Differences in biomass partitioning were attributed primarily to NH4-N nutrition of plants grown in ZPT compared with NO3-N in hydroponic nutrient solution. It is probable that NH4-N-induced Ca deficiency produced excess tillering and lower harvest index for plants grown in ZPT culture. These results suggest that further refinements in zeoponic substrate would make ZPT culture a viable alternative for achieving high productivity in a CELSS.

Calcium localization and tipburn development in lettuce leaves during early enlargement.

Tissue concentrations of Ca, Mg, and K were determined across immature leaves of lettuce (Lactuca sativa L. 'Buttercrunch') at different stages of enlargement using electron microprobe x-ray analysis. The analysis was with a wavelength dispersive spectrometer to permit detection of low concentrations of Ca. Patterns of mineral accumulation in immature leaves that were exposed were compared to patterns of accumulation in leaves that were enclosed within a developing head. The leaves developing without enclosure were free to transpire and developed normally whereas leaves developing with enclosure were restricted in transpiration and developed an injury that was characteristic of Ca deficiency. In the exposed leaves, Ca concentrations increased from an average of 1.0 to 2.1 mg g-1 dry weight (DW) as the leaves enlarged from 5 to 30 mm in length. In the enclosed leaves, Ca concentrations decreased from 1.0 to 0.7 mg g-1 DW as the leaves enlarged from 5 to 30 mm in length. At the tips of these enclosed leaves a larger decrease was found, from 0.9 to 0.3 mg g-1 DW during enlargement. Necrotic injury first became apparent in this tip area when the concentration was approximate to 0.4 mg g-1 DW. Magnesium concentrations across the exposed leaves were similar to concentrations across the enclosed leaves, and did not change with enlargement. Magnesium concentrations averaged 3.5. mg g-1 DW in both enclosed and exposed leaves during enlargement from 5 to 30 mm. In both exposed and enclosed leaves, K concentrations increased during enlargement from 40 to approximate to 60 mg g-1 DW. Potassium concentrations were highest toward the leaf apex and upper margin where injury symptoms occurred, and this may have enhanced injury development. This research documents the critical low levels of Ca (0.2 to 0.4 mg g-1 DW) that can occur in enclosed leaves of plants and which apparently leads to the marginal apex necrosis of developing leaves seen frequently on lettuce and other crops.

Microbial colonization of a closed growth chamber during hydroponic cultivation of lettuce.

The goal of this study was to characterize sessile and planktonic microbiota that developed during two successive hydroponic cultures of lettuce in a closed chamber system. Coupons of polyvinyl chloride (PVC) placed in the nutrient solution lines were removed periodically, as were samples of the nutrient solutions and condensate from the air-handling system. The bacteria and fungi present on the coupons and in fluid samples were enumerated by direct plate counts. Disinfecting the hydroponic system with 0.1% hypochlorite and 0.1 N nitric acid reduced the bacterial densities in biofilm samples from 1 x 10(7) CFU/10 cm2 to 1 x 10(1) CFU/10 cm2 and eliminated culturable fungi; Staphylococcus sp., Pseudomonas sp., and Micrococcus sp. survived this procedure. Bacterial and fungal concentrations in all samples returned to predisinfection levels after 2 days of plant growth. Pseudomonas and Acremonium predominated both before and after disinfection. Fungal concentrations never exceeded 7 x 10(2) CFU/10 cm2. The coupon microbiota differed from that of the rhizoplane at harvest. Overall, the greatest numbers of species were found on the rhizoplane samples collected during the second crop. The microbial community changed little during individual crops or between successive crops. Diversity indices remained relatively constant for all samples.

Atmospheric leakage and method for measurement of gas exchange rates of a crop stand at reduced pressure.

The variable pressure growth chamber (VPGC) was used in a 34-day functional test to grow a wheat crop using reduced pressure (70 kPa) episodes totalling 131 hours. Primary goals of the test were to verify facility and subsystem performance at 70 kPa and to determine responses of a wheat stand to reduced pressure and modified partial pressures of carbon dioxide and oxygen. Operation and maintenance of the chamber at 70 kpa involved continuous evacuation of the chamber atmosphere, leading to CO2 influx and efflux. A model for calculating CO2-exchange rates (net photosynthesis and dark respiration) was developed and tested and involved measurements of chamber leakage to determine appropriate corrections. Measurement of chamber leakage was based on the rate of pressure change over a small pressure increment (70.3 to 72.3 kPa) with the pump disabled. Leakage values were used to correct decreases and increases in chamber CO2 concentration resulting from net photosynthesis (Ps) and dark respiration (DR), respectively. Composite leakage corrections (influx and efflux) at day 7 of the test were 9% and 19% of the changes measured for Ps and DR, respectively. On day 33, composite corrections were only 3% for Ps and 4% for DR. During the test, the chamber became progressively tighter; the leak rate at 70.3 kPa decreasing from 2.36 chamber volumes/day pretest, to 1.71 volumes/day at the beginning of the test, and 1.16 volumes/day at the end of the test. Verification of the short-term leakage tests (rate of pressure rise) were made by testing CO2 leakage with the vacuum pump enabled and disabled. Results demonstrate the suitability of the VPGC or conducting gas exhange measurements of a crop stand at reduced pressure.

Photosynthesis and respiration of a wheat stand at reduced atmospheric pressure and reduced oxygen.

A 34-day functional test was conducted in Johnson Space Center's Variable Pressure Growth Chamber (VPGC) to determine responses of a wheat stand to reduced pressure (70 kPa) and modified partial pressures of carbon dioxide and oxygen. Reduced pressure episodes were generally six to seven hours in duration, were conducted at reduced ppO2 (14.7 kPa), and were interrupted with longer durations of ambient pressure (101 kPa). Daily measurements of stand net photosynthesis (Pn) and dark respiration (DR) were made at both pressures using a ppCO2 of 121 Pa. Corrections derived from leakage tests were applied to reduced pressure measurements. Rates of Pn at reduced pressure averaged over the complete test were 14.6% higher than at ambient pressure, but rates of DR were unaffected. Further reductions in ppO2 were achieved with a molecular sieve and were used to determine if Pn was enhanced by lowered O2 or by lowered pressure. Decreased ppO2 resulted in enhanced rates of Pn, regardless of pressure, but the actual response was dependent on the ratio of ppO2/ppCO2. Over the range of ppO2/ppCO2 of 80 to 200, the rate of Pn declined linearly. Rate of DR was unaffected over the same range and by dissolved O2 levels down to 3.1 ppm, suggesting that normal rhizosphere and canopy respiration occur at atmospheric ppO2 levels as low as 11 kPa. Partial separation of effects attributable to oxygen and those related to reduced pressure (e.g. enhanced diffusion of CO2) was achieved from analysis of a CO2 drawdown experiment. Results will be used for design and implementation of studies involving complete crop growth tests at reduced pressure.

Mission simulation as an approach to develop requirements for automation in Advanced Life Support Systems.

This paper examines mission simulation as an approach to develop requirements for automation and robotics for Advanced Life Support Systems (ALSS). The focus is on requirements and applications for command and control, control and monitoring, situation assessment and response, diagnosis and recovery, adaptive planning and scheduling, and other automation applications in addition to mechanized equipment and robotics applications to reduce the excessive human labor requirements to operate and maintain an ALSS. Based on principles of systems engineering, an approach is proposed to assess requirements for automation and robotics using mission simulation tools. First, the story of a simulated mission is defined in terms of processes with attendant types of resources needed, including options for use of automation and robotic systems. Next, systems dynamics models are used in simulation to reveal the implications for selected resource allocation schemes in terms of resources required to complete operational tasks. The simulations not only help establish ALSS design criteria, but also may offer guidance to ALSS research efforts by identifying gaps in knowledge about procedures and/or biophysical processes. Simulations of a planned one-year mission with 4 crewmembers in a Human Rated Test Facility are presented as an approach to evaluation of mission feasibility and definition of automation and robotics requirements.

Johnson Space Center's Regenerative Life Support Systems Test Bed.

The Regenerative Life Support Systems (RLSS) Test Bed at NASA's Johnson Space Center is an atmospherically closed, controlled environment facility for human testing of regenerative life support systems using higher plants in conjunction with physicochemical life support systems. The facility supports NASA's Advanced Life Support (ALS) Program. The facility is comprised of two large scale plant growth chambers, each with approximately 11 m2 growing area. The root zone in each chamber is configurable for hydroponic or solid media plant culture systems. One of the two chambers, the Variable Pressure Growth Chamber (VPGC), is capable of operating at lower atmospheric pressures to evaluate a range of environments that may be used in a planetary surface habitat; the other chamber, the Ambient Pressure Growth Chamber (APGC) operates at ambient atmospheric pressure. The air lock of the VPGC is currently being outfitted for short duration (1 to 15 day) human habitation at ambient pressures. Testing with and without human subjects will focus on 1) integration of biological and physicochemical air and water revitalization systems; 2) effect of atmospheric pressure on system performance; 3) planetary resource utilization for ALS systems, in which solid substrates (simulated planetary soils or manufactured soils) are used in selected crop growth studies; 4) environmental microbiology and toxicology; 5) monitoring and control strategies; and 6) plant growth systems design. Included are descriptions of the overall design of the test facility, including discussions of the atmospheric conditioning, thermal control, lighting, and nutrient delivery systems.

Microbiological characterization of a regenerative life support system.

A Variable Pressure Plant Growth Chamber (VPGC), at the Johnson Space Center's (JSC) ground-based Regenerative Life Support Systems (RLSS) test bed, was used to produce crops of soil-grown lettuce. The crops and chamber were analyzed for microbiological diversity during lettuce growth and after harvest. Bacterial counts for the rhizosphere, spent nutrient medium, heat exchanger condensate, and atmosphere were approximately 10(11) Colony Forming Units (CFU) g-1 10(5) CFU ml-1, 10(5) CFU ml-1, and 600 CFU m-3, respectively. Pseudomonas was the predominant bacterial genus. Numbers of fungi were about 10(5) CFU g-1 in the rhizosphere, 4-200 CFU ml-1 in the spent nutrient medium, 110 CFU ml-1 in the heat exchanger condensate, and 3 CFU m-3 in the atmosphere. Fusarium and Trichoderma were the predominant fungal genera.

Regenerative life support systems--why do we need them?

Human exploration of the solar system will include missions lasting years at a time. Such missions mandate extensive regeneration of life support consumables with efficient utilization of local planetary resources. As mission durations extend beyond one or two years, regenerable human life support systems which supply food and recycle air, water, and wastes become feasible; resupply of large volumes and masses of food, water, and atmospheric gases become unrealistic. Additionally, reduced dependency on resupply or self sufficiency can be an added benefit to human crews in hostile environments far from the security of Earth. Comparisons of resupply and regeneration will be discussed along with possible scenarios for developing and implementing human life support systems on the Moon and Mars.

Evaluation of light emitting diode characteristics for a space-based plant irradiation source.

Light emitting diodes (LEDs) are a promising irradiation source for plant growth in space. Improved semiconductor technology has yielded LED devices fabricated with gallium aluminum arsenide (GaAlAs) chips which have a high efficiency for converting electrical energy to photosynthetically active radiation. Specific GaAlAs LEDs are available that emit radiation with a peak wavelength near the spectral peak of maximum quantum action for photosynthesis. The electrical conversion efficiency of installed systems (micromole s-1 of photosynthetic photons per watt) of high output LEDs can be within 10% of that for high pressure sodium lamps. Output of individual LEDs were found to vary by as much as 55% from the average of the lot. LED ratings, in mcd (luminous intensity per solid angle), were found to be proportional to total photon output only for devices with the same dispersion angle and spectral peak. Increasing current through the LED increased output but also increased temperature with a consequent decrease in electrical conversion efficiency. A photosynthetic photon flux as high as 900 micromoles m-2 s-1 has been produced on surfaces using arrays with LEDs mounted 7.6 mm apart, operating as a current of 50 mA device-1 and at an installed density of approximately 17,200 lamps m-2 of irradiated area. Advantages of LEDs over other electric light sources for use in space systems include long life, minimal mass and volume and being a solid state device.

Calcium localization in lettuce leaves with and without tipburn: comparison of controlled-environment and field-grown plants.

An electron microprobe was used to determine tissue concentrations of Ca across 20-mm-long leaves of 'Green Lakes' crisphead lettuce (Lactuca sativa L.) with and without tipburn injury. Concentrations within the fifth and 14th leaves, counted from the cotyledons, from plants grown under controlled-environment conditions were compared to concentrations within similar leaves obtained from plants grown under field conditions. Only the 14th leaf from plants grown under controlled-environment conditions developed tipburn. Injured areas on these leaves had Ca concentrations as low as 0.2 to 0.3 mg g-1 dry weight. Uninjured areas of tipburned leaves contained from 0.4 to 0.5 mg g-1 dry weight. Concentrations across the uninjured 14th leaf from field-grown plants averaged 1.0 mg g-1 dry weight. Amounts across the uninjured fifth leaves from both environments averaged 1.6 mg g-1 dry weight. In contrast, Mg concentrations were higher in injured leaves than in uninjured leaves and thus were negatively correlated with Ca concentrations. Magnesium concentrations averaged 4.7 mg g-1 dry weight in injured leaves compared with 3.4 mg g-1 dry weight in uninjured leaves from both environments. Magnesium concentrations were uniform across the leaf. Potassium concentrations were highest at the leaf apex and decreased toward the base and also decreased from the midrib to the margin. Potassium averaged 51 mg g-1 dry weight in injured and uninjured leaves from both environments. No significant differences in K concentration were present between injured and uninjured leaves. This study documented that deficient concentrations of Ca were present in areas of leaf tissue developing tipburn symptoms and that concentrations were significantly higher in similar areas of other leaves that had no symptoms. This study also documented that Ca concentrations were significantly lower in enclosed leaves that exhibited tipburn symptoms than in exposed leaves that did not exhibit tipburn. Also, the amounts of Ca in plants that developed tipburn in controlled environments were lower than in plants of the same cultivar that did not develop tipburn in field plantings. The reduced levels of Ca in plants grown in controlled environments were associated with faster development rates compared with field-grown plants.

Light-emitting diodes as a radiation source for plants.

Development of a more effective radiation source for use in plant-growing facilities would be of significant benefit for both research and commercial crop production applications. An array of light-emitting diodes (LEDs) that produce red radiation, supplemented with a photosynthetic photon flux (PPF) of 30 micromoles s-1 m-2 in the 400- to 500-nm spectral range from blue fluorescent lamps, was used effectively as a radiation source for growing plants. Growth of lettuce (Lactuca sativa L. Grand Rapids') plants maintained under the LED irradiation system at a total PPF of 325 micromoles s-1 m-2 for 21 days was equivalent to that reported in the literature for plants grown for the same time under cool-white fluorescent and incandescent radiation sources. Characteristics of the plants, such as leaf shape, color, and texture, were not different from those found with plants grown under cool-white fluorescent lamps. Estimations of the electrical energy conversion efficiency of a LED system for plant irradiation suggest that it may be as much as twice that published for fluorescent systems.

Use of electron microprobe x-ray analysis for determination of low calcium concentrations across leaves deficient in calcium.

An electron microprobe with wavelength-dispersive x-ray spectrometry (WDS) was found to be useful for the determination of Ca concentrations in leaf tissue deficient in Ca. WDS effectively detected Ca concentrations as low as 0.2 mg/g dry wt in the presence of high levels of K and Mg (120 and 50 mg/g dry wt, respectively). Leaf specimens were prepared for analysis by quick-freezing in liquid nitrogen and freeze-drying at -20 degrees C to maintain elemental integrity within the tissue. Because dry material was analyzed, sample preparation was simple and samples could be stored for long periods before analysis. A large beam diameter of 50 gm was used to minimize tissue damage under the beam and analyze mineral concentrations within several cells at one time. Beam penetration was between 50 and 55 microns, approximately one-third of the thickness of the leaf. For analysis of concentrations in interveinal areas, analyses directed into the abaxial epidermis were found most useful. However, because of limited beam penetration, analyses of veinal areas would require use of cross sections [correction of crosssections]. Solid mineral standards were used for instrument standardization. To prevent measurement errors resulting from differences between the matrix of the mineral standards and the analyzed tissue, concentrations in leaves were corrected using gelatin standards prepared and analyzed under the same conditions. WDS was found to be useful for documenting that very low Ca levels occur in specific areas of lettuce leaves exhibiting the Ca deficiency injury termed tipburn.

Effects of artificial enclosure of young lettuce leaves on tipburn incidence and leaf calcium concentration.

The young developing leaves of 20-day-old lettuce plants (Lactuca sativa L. 'Buttercrunch') were enclosed by aluminized polyethylene sheaths to decrease transpiration and reduce Ca transport. The plants were grown in recirculating solution culture system using a modified half-strength Hoagland's solution under cool-white fluorescent lamps with a photosynthetic photon flux of 350 micromoles s-1 m-2 in a 16:8-hr (light:dark) period. Air temperature and humidity were 20 degrees C and 65%, respectively. After 4 days of enclosure, 53% of the inner leavers (leaves one to 3 cm in length) were tipburned. After the same period, less than 1% of the inner leaves on control plants were tipburned. The concentration of Ca in enclosed inner leaves was 0.63 mg g-1 dry weight, compared to 1.48 mg g-1 dry weight in inner leaves that were not enclosed. The Ca concentration in transpiring outer leaves of all plants was 9.9 mg g-1 dry weight. The Mg concentration in enclosed inner leaves was 2.25 mg g-1 dry weight, compared to 2.34 mg g-1 dry weight in inner leaves that were not enclosed. This research documents that enclosures of leaves at the growing point, as would occur with normal head development, is sufficient to create a limiting concentration of Ca in the enclosed tissue and encourage tipburn development.