Effects of a spaceflight environment on heritable changes in wheat gene expression.

Once it was established that the spaceflight environment was not a drastic impediment to plant growth, a remaining space biology question was whether long-term spaceflight exposure could cause changes in subsequent generations, even if they were returned to a normal Earth environment. In this study, we used a genomic approach to address this question. We tested whether changes in gene expression patterns occur in wheat plants that are several generations removed from growth in space, compared to wheat plants with no spaceflight exposure in their lineage. Wheat flown on Mir for 167 days in 1991 formed viable seeds back on Earth. These seeds were grown on the ground for three additional generations. Gene expression of fourth-generation Mir flight leaves was compared to that of the control leaves by using custom-made wheat microarrays. The data were evaluated using analysis of variance, and transcript abundance of each gene was contrasted among samples with t-tests. After corrections were made for multiple tests, none of the wheat genes represented on the microarrays showed a statistically significant difference in expression between wheat that has spaceflight exposure in their lineage and plants with no spaceflight exposure. This suggests that exposure to the spaceflight environment in low Earth orbit space stations does not cause significant, heritable changes in gene expression patterns in plants.

Crop yield and light/energy efficiency in a closed ecological system: Laboratory Biosphere experiments with wheat and sweet potato.

Two crop growth experiments in the soil-based closed ecological facility, Laboratory Biosphere, were conducted from 2003 to 2004 with candidate space life support crops. Apogee wheat (Utah State University variety) was grown, planted at two densities, 400 and 800 seeds m-2. The lighting regime for the wheat crop was 16 h of light-8 h dark at a total light intensity of around 840 micromoles m-2 s-1 and 48.4 mol m-2 d-1 over 84 days. Average biomass was 1395 g m-2, 16.0 g m-2 d-1 and average seed production was 689 g m-2 and 7.9 g m-2 d-1. The less densely planted side was more productive than the denser planting, with 1634 g m-2 and 18.8 g m-2 d-1 of biomass vs. 1156 g m-2 and 13.3 g m-2 d-1; and a seed harvest of 812.3 g m-2 and 9.3 g m-2 d-1 vs. 566.5 g m-2 and 6.5 g m-2 d-1. Harvest index was 0.49 for the wheat crop. The experiment with sweet potato used TU-82-155 a compact variety developed at Tuskegee University. Light during the sweet potato experiment, on a 18 h on/6 h dark cycle, totaled 5568 total moles of light per square meter in 126 days for the sweet potatoes, or an average of 44.2 mol m-2 d-1. Temperature regime was 28 +/- 3 degrees C day/22 +/- 4 degrees C night. Sweet potato tuber yield was 39.7 kg wet weight, or an average of 7.4 kg m-2, and 7.7 kg dry weight of tubers since dry weight was about 18.6% wet weight. Average per day production was 58.7 g m-2 d-1 wet weight and 11.3 g m-2 d-1. For the wheat, average light efficiency was 0.34 g biomass per mole, and 0.17 g seed per mole. The best area of wheat had an efficiency of light utilization of 0.51 g biomass per mole and 0.22 g seed per mole. For the sweet potato crop, light efficiency per tuber wet weight was 1.33 g mol-1 and 0.34 g dry weight of tuber per mole of light. The best area of tuber production had 1.77 g mol-1 wet weight and 0.34 g mol-1 of light dry weight. The Laboratory Biosphere experiment's light efficiency was somewhat higher than the USU field results but somewhat below greenhouse trials at comparable light levels, and the best portion of the crop at 0.22 g mol-1 was in-between those values. Sweet potato production was overall close to 50% higher than trials using hydroponic methods with TU-82-155 at NASA JSC. Compared to projected yields for the Mars on Earth life support system, these wheat yields were about 15% higher, and the sweet potato yields averaged over 80% higher.

Soil and crop management experiments in the Laboratory Biosphere: an analogue system for the Mars on Earth(R) facility.

During the years 2002 and 2003, three closed system experiments were carried out in the "Laboratory Biosphere" facility located in Santa Fe, New Mexico. The program involved experimentation of "Hoyt" Soy Beans, (experiment #1) USU Apogee Wheat (experiment #2) and TU-82-155 sweet potato (experiment #3) using a 5.37 m2 soil planting bed which was 30 cm deep. The soil texture, 40% clay, 31% sand and 28% silt (a clay loam), was collected from an organic farm in New Mexico to avoid chemical residues. Soil management practices involved minimal tillage, mulching, returning crop residues to the soil after each experiment and increasing soil biota by introducing worms, soil bacteria and mycorrhizae fungi. High soil pH of the original soil appeared to be a factor affecting the first two experiments. Hence, between experiments #2 and #3, the top 15 cm of the soil was amended using a mix of peat moss, green sand, humates and pumice to improve soil texture, lower soil pH and increase nutrient availability. This resulted in lowering the initial pH of 8.0-6.7 at the start of experiment #3. At the end of the experiment, the pH was 7.6. Soil nitrogen and phosphorus has been adequate, but some chlorosis was evident in the first two experiments. Aphid infestation was the only crop pest problem during the three experiments and was handled using an introduction of Hyppodamia convergens. Experimentation showed there were environmental differences even in this 1200 cubic foot ecological system facility, such as temperature and humidity gradients because of ventilation and airflow patterns which resulted in consequent variations in plant growth and yield. Additional humidifiers were added to counteract low humidity and helped optimize conditions for the sweet potato experiment. The experience and information gained from these experiments are being applied to the future design of the Mars On Earth(R) facility (Silverstone et al., Development and research program for a soil-based bioregenerative agriculture system to feed a four person crew at a Mars base, Advances in Space Research 31(1) (2003) 69-75; Allen and Alling, The design approach for Mars On Earth(R), a biospheric closed system testing facility for long-term space habitation, American Institute of Aeronautics and Astronautics Inc., IAC-02-IAA.8.2.02, 2002).

Atmospheric dynamics in the "Laboratory Biosphere" with wheat and sweet potato crops.

Laboratory Biosphere is a 40-m3 closed life system equipped with 12,000 W of high pressure sodium lamps over planting beds with 5.37 m2 of soil. Atmospheric composition changes due to photosynthetic fixation of carbon dioxide and corresponding production of oxygen or the reverse, respiration, are observed in short timeframes, e.g., hourly. To focus on inherent characteristics of the crop as distinct from its area or the volume of the chamber, we report fixation and respiration rates in mmol h-1 m-2 of planted area. An 85-day crop of USU Apogee wheat under a 16-h lighted/8-h dark regime peaked in fixation rate at about 100 mmol h-1 m-2 approximately 24 days after planting. Light intensity was about 840 micromoles m-2 s-1. Dark respiration peaked at about 31 mmol h-1 m-2 at the same time. Thereafter, both fixation and respiration declined toward zero as harvest time approached. A residual soil respiration rate of about 1.9 mmol h-1 m-2 was observed in the dark closed chamber for 100 days after the harvest. A 126-day crop of Tuskegee TU-82-155 sweet potato behaved quite differently. Under a 680 micromoles m-2 s-1, 18-h lighted/6-h dark regime, fixation during lighted hours rose to a plateau ranging from about 27 to 48 mmol h-1 m-2 after 42 days and dark respiration settled into a range of 12-23 mmol h-1 m-2. These rates continued unabated until the harvest at 126 days, suggesting that tuber biomass production might have continued at about the same rate for some time beyond the harvest time that was exercised in this experiment. In both experiments CO2 levels were allowed to range widely from a few hundred to about 3000 ppm, which permitted observation of fixation rates both at varying CO2 concentrations and at each number of days after planting. This enables plotting the fixation rate as a function of both variables. Understanding the atmospheric dynamics of individual crops will be essential for design and atmospheric management of more complex CELSS which integrate the simultaneous growth of several crops as in a sustainable remote life support system.

Technical review of the Laboratory Biosphere closed ecological system facility.

Laboratory Biosphere is a 40 m3 closed life system that commenced operation in May 2002. Light is from 12,000 W of high pressure sodium lamps over planting beds with 5.37 m2 of soil. Water is 100% recycled by collecting condensate from the temperature and humidity control system and mixing with leachate collected from under the planting beds. Atmospheric leakage was estimated during the first closure experiment to be 0.5-1% per day in general plus about 1% for each usage of the airlock door. The first trial run of 94 days was with a soybean crop grown from seeds (May 17, 2002) to harvest (August 14, 2002) plus 5 days of post-harvest closure. The focus of this initial trial was system testing to confirm functionality and identify any necessary modifications or improvements. This paper describes the organizational and physical features of the Laboratory Biosphere.

Earth applications of closed ecological systems: relevance to the development of sustainability in our global biosphere.

The parallels between the challenges facing bioregenerative life support in artificial closed ecological systems and those in our global biosphere are striking. At the scale of the current global technosphere and expanding human population, it is increasingly obvious that the biosphere can no longer safely buffer and absorb technogenic and anthropogenic pollutants. The loss of biodiversity, reliance on non-renewable natural resources, and conversion of once wild ecosystems for human use with attendant desertification/soil erosion, has led to a shift of consciousness and the widespread call for sustainability of human activities. For researchers working on bioregenerative life support in closed systems, the small volumes and faster cycling times than in the Earth's biosphere make it starkly clear that systems must be designed to ensure renewal of water and atmosphere, nutrient recycling, production of healthy food, and safe environmental methods of maintaining technical systems. The development of technical systems that can be fully integrated and supportive of living systems is a harbinger of new perspectives as well as technologies in the global environment. In addition, closed system bioregenerative life support offers opportunities for public education and consciousness changing of how to live with our global biosphere.

Initial experimental results from the Laboratory Biosphere closed ecological system facility.

An initial experiment in the Laboratory Biosphere facility, Santa Fe, New Mexico, was conducted May-August 2002 using a soil-based system with light levels (at 12 h per day) of 58-mol m-2 d-1. The crop tested was soybean, cultivar Hoyt, which produced an aboveground biomass of 2510 grams. Dynamics of a number of trace gases showed that methane, nitrous oxide, carbon monoxide, and hydrogen gas had initial increases that were substantially reduced in concentration by the end of the experiment. Methane was reduced from 209 ppm to 11 ppm, and nitrous oxide from 5 ppm to 1.4 ppm in the last 40 days of the closure experiment. Ethylene was at elevated levels compared to ambient during the flowering/fruiting phase of the crop. Soil respiration from the 5.37 m2 (1.46 m3) soil component was estimated at 23.4 ppm h-1 or 1.28 g CO2 h-1 or 5.7 g CO2 m-2 d-1. Phytorespiration peaked near the time of fruiting at about 160 ppm h-1. At the height of plant growth, photosynthesis CO2 draw down was as high as 3950 ppm d-1, and averaged 265 ppm h-1 (whole day averages) during lighted hours with a range of 156-390 ppm h-1. During this period, the chamber required injections of CO2 to continue plant growth. Oxygen levels rose along with the injections of carbon dioxide. Upon several occasions, CO2 was allowed to be drawn down to severely limiting levels, bottoming at around 150 ppm. A strong positive correlation (about 0.05 ppm h-1 ppm-1 with r2 about 0.9 for the range 1000-5000 ppm) was observed between atmospheric CO2 concentration and the rate of fixation up to concentrations of around 8800 ppm CO2.

Development and research program for a soil-based bioregenerative agriculture system to feed a four person crew at a Mars base.

For humans to survive during long-term missions on the Martian surface, bioregenerative life support systems including food production will decrease requirements for launch of Earth supplies, and increase mission safety. It is proposed that the development of "modular biospheres"--closed system units that can be air-locked together and which contain soil-based bioregenerative agriculture, horticulture, with a wetland wastewater treatment system is an approach for Mars habitation scenarios. Based on previous work done in long-term life support at Biosphere 2 and other closed ecological systems, this consortium proposes a research and development program called Mars On Earth(TM) which will simulate a life support system designed for a four person crew. The structure will consist of 6 x 110 square meter modular agricultural units designed to produce a nutritionally adequate diet for 4 people, recycling all air, water and waste, while utilizing a soil created by the organic enrichment and modification of Mars simulant soils. Further research needs are discussed, such as determining optimal light levels for growth of the necessary range of crops, energy trade-offs for agriculture (e.g. light intensity vs. required area), capabilities of Martian soils and their need for enrichment and elimination of oxides, strategies for use of human waste products, and maintaining atmospheric balance between people, plants and soils.

Light, plants, and power for life support on Mars.

Regardless of how well other growing conditions are optimized, crop yields will be limited by the available light up to saturation irradiances. Considering the various factors of clouds on Earth, dust storms on Mars, thickness of atmosphere, and relative orbits, there is roughly 2/3 as much light averaged annually on Mars as on Earth. On Mars, however, crops must be grown under controlled conditions (greenhouse or growth rooms). Because there presently exists no material that can safely be pressurized, insulated, and resist hazards of puncture and deterioration to create life support systems on Mars while allowing for sufficient natural light penetration as well, artificial light will have to be supplied. If high irradiance is provided for long daily photoperiods, the growing area can be reduced by a factor of 3-4 relative to the most efficient irradiance for cereal crops such as wheat and rice, and perhaps for some other crops. Only a small penalty in required energy will be incurred by such optimization. To obtain maximum yields, crops must be chosen that can utilize high irradiances. Factors that increase ability to convert high light into increased productivity include canopy architecture, high-yield index (harvest index), and long-day or day-neutral flowering and tuberization responses. Prototype life support systems such as Bios-3 in Siberia or the Mars on Earth Project need to be undertaken to test and further refine systems and parameters.

Evaluation of sterilization of dental handpieces by heating in synthetic compressor lubricant.

The Centers for Disease Control and Prevention and the American Dental Association guidelines recommend sterilization of dental handpieces after each use. Steam autoclaving is the most commonly used sterilization method. However, pressurized steam causes corrosion and partial combustion of the handpiece lubricant, leaving a sticky carbon residue on the turbine which must then be replaced after several usages. Replacement of autoclave-damaged dental handpieces represents a major expense for dentists that may be avoided through the use of less destructive sterilization techniques.

Food production and nutrition for the crew during the first 2-year closure of Biosphere 2.

Biosphere 2's finite natural resources: atmosphere, plants, water, and soil, and its unique increased rate of nutrient cycling, mandated a design for the agriculture that emphasized sustainability and high productivity. The results of the initial 2-year test of the agriculture system showed that it could provide a diet that was both nutritionally adequate and pleasing to the palate of the eight-member crew from September 1991 to September 1993. The agriculture design was developed from 1985 to 1991 at the Space Biospheres research greenhouses with consulting from the Institute of Ecotechnics (London) from its experiments in New Mexico, Australia, and France and the Environmental Research Laboratory (University of Arizona). During the 2-year mission this research was continued with the close collaboration of outside scientific consultants, particularly in the area of soil management and integrated pest management. The 2000-m2 cropping area provided approximately 81% of the overall nutritional needs of the crew. Initial results showed light to be the main limiting factor and the additional electric light was added after the first 2-year mission to increase the productivity for future experiments. The diet was primarily vegetarian supplemented with daily amounts of milk, and weekly meals of meat and eggs from the system's domestic goats, pigs, and chickens. Nontoxic methods of pest and disease control were used. The main pest problems were broad mite and root knot nematode. Inedible plant material, domestic animal wastes, and human waste water were successfully processed for nutrient return to the soil. Eighty-six varieties of crops were grown in Biosphere 2. Major staple crops included rice, sweet potato, beets, banana, and papaya. The African pygmy goats were the most productive of the domestic animals producing on average 1.14 kg of milk per day. The diet averaged 2200 calories, 73 g of protein, and 32 g of fat per person per day over the 2 years. The crew had a 10-20% weight loss, mostly occurring in the first 6 months of closure, after which weights stabilized with some increase in the second year. Agriculture field management took 25% of crew time, animal care required an additional 9% and food preparation accounted for 12% of crew labor.

Phenotypic expression of Pseudomonas syringae avr genes in E. coli is linked to the activities of the hrp-encoded secretion system.

The specific recognition of elicitors produced by plant pathogenic bacteria carrying avirulence (avr) genes is postulated to initiate cellular defense responses in plants expressing corresponding resistance genes. The biochemical functions of most avr genes, however, are not known. A heterologous system was developed to phenotypically express Pseudomonas syringae avr genes in Escherichia coli cells that required the P. syringae hrp cluster. E. coli MC4100 transformants carrying the plasmic-borne P. syringae pv. syringae Pss61 hrp cluster and p. syringae pv. glycinea avrB expressed from a triple lacUV5 promoter gained the ability to elicit the hypersensitive response in soybean cultivars expressing Rpg1 and in an Arabidopsis thaliana accession expressing RPM1. Inactivation of energy transducing or outer membrane components of the hrp-encoded secretion system blocked phenotypic expression expression of avrB in E. coli, but deletions abolishing harpinPSS production had little effect on the production of the AvrB phenotype by the E. coli transformants. Phenotypic expression of avrA, AvrPto, avrRpm1, avrRpt2, and avrPph3 in E. coli was also shown to require the hrp cluster. The results indicate that generation of the Avr phenotype in P. syringae strains is specifically dependent on the secretion activities of the hrp cluster.

Food production and nutrition in Biosphere 2: results from the first mission September 1991 to September 1993.

The initial test of the Biosphere 2 agricultural system was to provide a nutritionally adequate diet for eight crew members during a two year closure experiment, 1991-1993. The overall results of that trial are presented in this paper. The 2000 m2 cropping area provided about 80 percent of overall nutritional needs during the two years. Adaptation of the crew to the diet which averaged 2200 calories, 73 g. of protein and 32 g. of fat per person over the course of the two years. The diet was primarily vegetarian, with only small amounts of milk, meat and eggs from the system's domestic animals. The crew experienced 10-20 percent weight loss, most of which occurred in the first six months of the closure reflecting adaptation to the diet and lower caloric intake during that period. Since Biosphere 2 is a tightly sealed system, non-toxic methods of pest and disease control were employed and inedible plant material, domestic animal wastes and human waste-water were processed and nutrients returned to the soil. Crop pests and diseases, especially broad mites and rootknot nematode, reduced yields, and forced the use of alternative crops. Outstanding crops included rice, sweet potato, beets, banana, and papaya. The African pygmy goats were the most productive of the domestic animals. Overall, the agriculture and food processing required some 45% of the crew time.

The 73-kb pIAA plasmid increases competitive fitness of Pseudomonas syringae subspecies savastanoi in oleander.

Pseudomonas syringae subsp. savastanoi causes tumors on olive and oleander by producing the plant growth regulators indoleacetic acid (IAA) and cytokinins following infection of the plant. The contribution of IAA production to the ability of P. syringae subsp. savastanoi to grow and survive in oleander leaf tissue was studied. Bacterial strains differing only with respect to IAA production were characterized. Growth and survival of wild-type and two mutant strains of P. syringae subsp. savastanoi in oleander leaf tissue were monitored by weekly colony counts and IAA plate assays. Growth rate of the three strains in culture and in planta did not differ significantly. However, the wild-type strain reached a higher population density and maintained its maximum density at least 9 weeks longer than either mutant population. An insertion mutant containing the IAA plasmid (pIAA), but incapable of IAA production, did not maintain a higher population density than a strain cured of the IAA plasmid. The pIAA-cured strain maintained a higher population density when coinoculated with an IAA-producing strain than when inoculated alone. These results suggest that IAA production may contribute to the fitness of P. syringae subsp. savastanoi in oleander tissue and that the iaa operon alone may be responsible for the competitive advantage of cells harboring pIAA.

Rapid in situ assay for indoleacetic Acid production by bacteria immobilized on a nitrocellulose membrane.

We have developed a new assay that differentiates between indoleacetic acid (IAA)-producing and -nonproducing bacteria on a colony plate lift. Medium supplemented with 5 mM L-tryptophan is inoculated with isolates of interest, overlaid with a nitrocellulose membrane, and then incubated until bacterial colonies reach 1 to 2 mm in diameter. The membrane is removed to a filter paper saturated with Salkowski reagent and incubated until distinct red haloes form around the colonies. The colorimetric reaction to IAA is limited to a region immediately surrounding each colony, is specific to isolates producing IAA, occurs within 1 h after the membrane is placed in the reagent, and is sensitive to as little as 50 pmol of IAA in a 2-mm spot. We have used this assay for quantifying epiphytic and endophytic populations of IAA-producing isolates of Pseudomonas syringae subsp. savastanoi and for detecting IAA-producing colonies of other pseudomonads and Erwinia herbicola. The assay provides a rapid and convenient method to screen large numbers of bacteria.