Biomass Production

Bruce Bugbee and Raymond Wheeler, Co-Chairs

Platform Presentations – Ivy Room, January 17
File# Title Authors
301 Biomass Production R. M. Wheeler and B. Bugbee

033 Candidate Crop Evaluation for Advanced Life Support R. M. Wheeler, N. C. Yorio, G. D. Goins,
G. W. Stutte, N. A. Cranston, L. M. Ruffe

034 Crop Optimization and Failure Analysis for Advanced Life B. Bugbee, T. Dougher, J. Frantz, S.
Support Klassen, D. Muhlestein, C. Mackoviak, G.
Ritchie, D. Pinnock

035 Techniques for Identification and Rate Measurement of W. Lertsiriyothin, B. K. Khoo, J. Lech,
Volatile Organic Compounds (VOCs) Emission from J. A. Hogan, R. M. Cowan, T. G. Hartman
Living Plants and Food Extrusion Processes in
Conjunction to NASA’s ALS Systems

036 Plant Growth Experiments in Zeoponic Substrates: D. W. Ming, J. E. Gruener, K. E.

Applications for Advanced Life Support Systems Henderson, S. L. Steinberg, D. J. Barta,
C. Galindo, Jr., D. L. Henninger

037 Microgravity Plant Nutrient Experiment: Year 1 Activities H. G. Levine and T. W. Dreschel

038 Plant Nutrient Delivery G. D. Goins

039 Performance of Sweetpotato for Bioregenerative Life D. J. Barta, K. E. Henderson, D. G.

Support Mortley, D. L. Henninger

040 Enhanced Protein Content and Quality in Sweetpotato M. Egnin, R. Pace, C. Prakash, J.
Engineered with a Synthetic Storage Protein Gene for Jaynes, K. Nakamura
Improved Human Nutrition and Health

041 Regulation of ADP-Glucose Pyrophosphorylase Subunit T. J. Gianfagna, X. Li, J. Xing, Y. Luo, J.

Expression – A Key Enzyme for Starch Biosynthesis in W. Janes
Plants

042 Lighting Technology Development for Bioregenerative J. C. Sager, R. M. Wheeler, G. Goins, S.

Components of the Advanced Life Support Project Young

043 Performance of Salad-Type Plants Grown Under Narrow- G. D. Goins

Spectrum Light-Emitting Diodes in a Controlled
Environment

044 Hybrid Solar and Artificial Lighting (HYSAL) for Space J. L. Cuello, Y. Yang, E. Ono, K. Jordan,
Life Support D. Larson, J. O’Leary, T. Nakamaru

045 Plant Growth Chamber Experiments for the Crop J. Cavazzoni, D. H. Fleisher, K.
Modeling Objectives of the NJ NSCORT Jumasaka, H. Janes, T. Gianfagna, L
Logendra

046 Modeling of Assimilation and Transpiration in Soybean F. Zee

Plants Using Artificial Neural Networks

047 Models for Scheduling and Control for Advanced Life D. Fleisher, K. C. Ting
Support Systems
048P Self Reporting Space Crops: Edible Biosensors M. Ayalew, V. Cardoza, B. Kivela, C. N.
Stewart, Jr.

049P Optimization of Root Zone Substrates for Reduced G. E. Bingham, D. Or, S. B. Jones, R. C.
Gravity Experiments Morrow

050P Use of Induced-Fluorescence Imaging and Green A. C. Schuerger, R. J. Ferl, K. A. Corey,

Fluorescent Proteins to Monitor the Health of Terrestrial T. Murdoch, W. Wells
Plants Under Simulated Martian Environments
 BIOMASS PRODUCTION
Raymond M. Wheeler, Ph.D.
Bruce Bugbee, Ph.D.

INTRODUCTION
The concept of using plants for life support systems has been discussed and studied for nearly 40 years.
The underlying principle involves the process of photosynthesis, where carbon dioxide (CO2) is removed from the
air and fixed into organic compounds. Simultaneously, oxygen (O2) is released as a byproduct, thus providing two
fundamental life support needs, i.e., a supply of O2 and CO2 removal from the air. By selecting plant species that
generate edible biomass (crops), a source of food is then added. On a stoichiometric basis, this process of “biomass
production” is the exactly the opposite of human respiration, where food and O2 are consumed creating CO2 as a
waste product (Fig. 1).
Plants can also be used to purify water, where for example wastewater (gray water and urine) is added to
the plant root-zone. Through the process of transpiration, plants take up and filter the water, which evaporates from
their leaves creating humidity (water vapor), which can then be condensed and recycled to humans (Fig. 1). The
organic components in the wastewater are then degraded to CO2 by microorganisms in the root-zone or by the plants
themselves. The plants can also recycle valuable nutrients from the wastewater stream (e.g., nitrogen from the urine).

Humans: C (H2O) + O2 CO2 + H2O (respiration)

Clean Water Waste Water

light
Plants: CO2 + H2O C (H2O) + O2 (photosynthesis)
Waste Water Clean Water (transpiration)

Figure 1. Simplified equations showing the opposite (balancing) effects of plant photosynthesis and human
respiration. The key input to drive photosynthesis is light energy, while the evaporative potential (vapor pressure
deficit) surrounding the plant leaves strongly controls transpiration rates.

Biomass production research sponsored by NASA in the 1980s and early 1990s focused largely on
controlled environment production tests of several candidate crops, especially crops rich in carbohydrate and protein,
e.g., “staple” crops. The studies typically involved the use of recirculating hydroponic culture systems, electric
lighting systems, and careful management of the ambient environment to optimize growth and yields. Similar
horticultural and physiological studies have continued through the 1990s under NASA’s Advanced Life Support
(ALS) Program. Testing in recent years has also included integration studies, where plants were linked with waste
treatment /resource recovery processes, studies of plant growth and management in atmospherically closed systems,
and definition studies for growing plants in a spaceflight environment, such as development of gravity-independent
watering systems.

SUMMARY OF PRESENTATIONS

Detailed overviews of biomass research from the past year can be found in abstracts in this proceedings.
This research might be classified into five general categories:

Horticulture

From NASA Kennedy Space Center, NASA Biomedical Office, Kennedy Space Center, FL (R. Wheeler) and Utah
State University, Department of Plants, Soils, and Biometeorology, Logan UT (B. Bugbee).
 a) Studies to refine cultural procedures for candidate staple crops (i.e., rice, sweetpotato, peanut, and bean).
b) Studies to define growing procedures for candidate salad crops (i.e., tomato, radish, lettuce, spinach,
chard, and carrot) for near-term missions, where fresh vegetables could provide dietary supplements for the crew. In
particular, tests focused on germination and plant establishment procedures, plant spacing, and cropping duration.

Environmental physiology

a) Studies to determine sub-optimal, optimal, and super-optimal concentrations of CO2, light intensity,
photoperiod, and temperature for candidate crops.
b) Studies of crop growth at the reduced atmospheric pressures that might be used for future missions.
c) Comparisons of different electric light sources (e.g., high-pressure sodium, microwave sulfur lamps, and
LEDs) to determine their effects on plant growth and the overall electrical efficiency of the lighting system.
d) Measurements of production rates and effects of volatile organic compounds (e.g., ethylene) on plants.
e) Studies of water and nutrient delivery concepts, including the effects of different ratios of NH4 : NO3 in
hydroponic systems, and the use of nutrient-loaded zeolite growing media that would only require addition of water
to sustain crop growth.

Crop selection / genetic improvement

a) Continued studies with sweetpotatoes that have been genetically altered for increased storage protein.
b) Studies to introduce green fluorescent protein to indicate environmental stresses in canola and
Arabidopsis.
c) Studies of ADP glucosepyrophosphorylase, a key carbohydrate metabolism enzyme in tomato.
d) Continued breeding and selection of short stature (dwarf) varieties of wheat, rice, and tomato for more
efficient use of volume in plant growing systems.

Crop modeling and systems analysis

a) Studies on the effects of environmental perturbations and failures on crop growth.
b) Use of neural net approaches to control and optimize growing environments.
c) Software for simulating and compensating for the effects of environmental perturbations to crop
production systems.
d) Continued development of crop growth models for predicting yields under optimal and sub-optimal
environments. For example, could plants tolerate a 14-d long “night” cycle in a lunar production system that used
solar light?

Definition studies for spaceflight tests

a) Tests of rooting systems to optimize delivery of water and oxygen to root surfaces and comparisons of
different watering techniques for microgravity. A current technique uses porous tubes to deliver water to plant roots,
which is then taken up by the plants through capillary action.
b) Extrapolation of the data from the physiological studies to determine the effects of reduced pressure,
elevated CO2 concentrations, and the build-up of ethylene in spaceflight for near-term space missions.

Participating universities and institutions in biomass research for the past year included:

Utah State University Kennedy Space Center

Rutgers University Johnson Space Center
Tuskegee University Stevens Institute
University of Arizona Dynamac Corp.
Texas A & M University Jet Propulsion Lab
 University of North Carolina, Greensboro

IMPLICATIONS FOR FUTURE RESEARCH

Near-Term

Develop crop handbook for BIO-Plex

Many handbooks are available to provide guidelines for growing crops in field settings. But plants in
controlled environments (growth chambers) never see sunlight or touch the soil. Thus, unique guidelines are needed
to grow crops under the controlled conditions of BIO-Plex (Bioregenerative Life Support Systems Complex) and
related ALS systems. An ALS crop handbook should be developed to provide recommendations on horticultural
approaches for crop establishment, planting densities, harvest procedures, and environmental set-points (i.e.,
photoperiod, temperature, CO2, and light intensity) for each of the candidate crops. Specific chapters of this
handbook could be written by investigators who have direct experience with individual crops and could include
descriptions of the underlying physiology related to the different environmental recommendations. In many cases
multiple authors will be needed to contribute to the recommendations for each crop.
Increased collaboration / interaction with food & waste processing groups
Plant biomass produced from controlled environments can have different biochemical characteristics than
biomass from field settings. In some cases, the nutritional value and food processing characteristics can be better in
controlled environment grown plants. On the other hand, anti-nutritional factors such as nitrate, and oxalic and
phytic acid can be higher in controlled environment grown plants. Clearly more research and collaboration are
needed, particularly for determining what changes in the environment are important for improving the quality of
harvested food. Changes in the environment might also be used to make inedible biomass easier to degrade. For
example, environments might be managed to reduce lignin content or the inorganic mineral content of inedible
biomass. Reduced lignin could facilitate biological processing of inedible plant materials, while reduced mineral
content could minimize slagging effects during combustion treatments. Combined approaches using biological
pretreatment (stirred-tank bioreactors) to remove minerals prior to combustion need continued study, particularly
with regard to their potential for recycling nutrients directly back to plant growing systems.

Cultivar selections for high yield, dwarf growth, and short life cycles

Smaller, quicker growing plants are needed for Advanced Life Support applications, where time, energy,
and volume will be limiting factors. Dwarf cultivars exist for many crop plants, but they must be identified through
careful comparison of multiple genetic lines in an optimal environment. Yields usually decrease with super dwarf
plants, but increased planting density can often overcome any yield reduction. Selection and study of dwarf cultivars
is especially important for spaceflight testing, where growing volumes are limited.

Salad crop testing and yield optimization

The first opportunities to grow plants for life support applications will likely involve small-scale systems to
grow vegetables or salad crops as dietary supplements. Tomato, lettuce, spinach, onion, carrot, radish, chard, and
cabbage have been identified for initial testing, yet we know relatively little about controlled environment physiology
and culture for some of these crops. For example, carrot production in hydroponics has not been rigorously tested,
and carrot, onion, chard, and cabbage are not typically grown in controlled environments. Each crop will likely
provide unique challenges, as will optimizing approaches for growing multiple species in common environments.

Refine capability to grow plants in microgravity and the spaceflight environment

 Biologists have successfully grown some plants through entire life cycles in space (e.g., Arabisopsis,
Brassica, and wheat). Thus microgravity does not appear to pose any fundamental impediments to plant growth.
But many details must be refined before we can grow a wide range of plants reliably in space. In addition to
microgravity, the effects of other factors typical of the spaceflight environment such as super-elevated CO2, elevated
ethylene, low to moderate irradiance, and poor aeration of root zones need continued study. In many cases, these
ancillary factors may present bigger challenges than micro- or hypogravity.
Collect existing data on psychological value of plants

Evidence continues to mount for plants having positive psychological effects on humans living in closed
environments (see Flagler and Poincelot, 1994). But much of this evidence is anecdotal, e.g. reports from
cosmonauts on the Mir space station, and the use of hydroponic systems for Antarctic habitats (see Sadler, 1995).
Some additional evidence from closed life support experiments is available, for example from Russian studies and
Biosphere II, and efforts are needed to assimilate and assess these observations in an unbiased, scientific manner.

Long-Term

Improved efficiency of lighting systems

Significant improvements in electrical efficiency are needed in lighting systems. Possible improvements
include changes in radiation quality (to maximize photosynthesis while satisfying photomorphological needs),
development of efficient, low-mass light delivery systems, use of solid-state (electronic) ballasts, and improvements
in reflector designs and efficiencies.

Genetic modification

Efficient food production systems on Earth were developed by manipulating both environmental and
genetic factors to improve crop yields. We cannot be efficient in space without manipulating both genetics and the
environment. Both classical plant breeding and molecular approaches will be important to matching genetic
characteristics with environmental constraints. For example, it appears that we can select plants for tolerance to high
ethylene levels that will probably occur in space. We should also be able to modify short-day crop plants (e.g.
potatoes, soybeans, rice) so that they can be grown in continuous light, and develop plants that grow well under
efficient, narrow-spectrum light sources.
Non-optimal environmental responses – failure analysis

It probably will not be cost effective to provide the exact environmental conditions needed to maximize
yields of each individual species. System efficiency will most likely be optimized by providing near optimal
conditions for all the crops that share a common environment. The effects of non-optimal conditions have not yet
been fully characterized. Furthermore, failures of environmental control systems will occur and we need to
understand the tolerance of plants to these failures. In some cases it will be more cost effective to replant rather than
to keep growing a crop that has been stressed by failure of the environmental control systems.

Long-term reliability testing

Most crop life cycles are relatively short (2 to 3 months), but the hardware and control systems that support
crop production can fail. Microbial growth can degrade solution flow rates and electronic components, and pathogen
outbreaks can occur. Our best long-term reliability data come from the biomass production chamber at Kennedy
Space Center where multiple crops were grown back-to-back in a large, atmospherically closed chamber for over
400 days. But more testing is needed to assess the long-term reliabilities of these systems and make informed
decisions on their life support applications.

Study psychological value of plants

 After the existing data have been collected and analyzed (see short-term goals above), NASA should
carefully consider controlled studies to assess the effects of plants and plant growing systems on humans living in
confined habitats. This could include the effects of bright light and visual stimuli from plant systems, aromas
associated with plants, as well as the contribution of fresh food to the human diet.

Quantify long-term water purification capability of plants

Crop plants have been successfully grown using wastewater from humans, but the long-term ability of plants
to filter water without reducing the yield, quality, or reliability of the systems has not yet been established. Water is
the largest mass consideration in any regenerative life support system and more thorough understanding of the
capacity of plant systems to process and purify water is needed.

Mission Opportunities and Considerations for Plant Research

A primary objective for near-term research with plants for NASA will be the BIO-Plex project, which is a
large-scale, integrated test-bed being developed at Johnson Space Center for the ALS Program. In addition, several
spaceflight experiments with plant growing systems for Shuttle and the International Space Station (ISS) are in
development (Fig. 2). Although no official project has yet been initiated, development of a “Vegetable Production
Unit” (VPU) should be considered for ISS to gather data and experience with operating plant production systems in
space (Kliss and MacElroy, 1990). A possible timeline for developing and deploying a VPU is shown in Fig. 2. In
addition, suggested timelines for development of a VPU for Mars transit vehicles and an inflatable greenhouse for
planetary surface missions are recommended (Fig. 2). Research for meeting these objectives should begin soon to
provide sufficient time to gather critical data and gain operational experience.

Figure 2. Mission considerations for implementing plant systems in space. Other than BIO-Plex1 “Research and
Development” and “Integration” tests and several named flight experiments, table timelines and missions represent
unofficial goals that might be considered for biomass production research.
2000 2005 2010 2015 2020 2025
BIO-Plex 1
Research and Development

BIO-Plex
Integration Tests X X X X

Vegetable Production Unit

(VPU)
Research and Development

Vegetable Production Unit X X (?)

Subsystem Flight Tests 2
WONDER

Zone O2
Root

Vegetable Production Unit

Placement on ISS X (?)

Humans to Moon or Mars

Transit Vehicle VPU R & D

X (?)
Surface Deployable Greenhouse R & D
Related Plant Testing X X X Others?
Fundamental Biology
Program 2

RASTA
PASTA
PESTO
1
BIO-Plex = Bioregenerative Life Support Systems Complex be developed for NASA’s Advanced Life Support Program
2
WONDER, PESTO, PASTA, RASTA indicate existing plant research projects scheduled for spaceflight-testing.
3
Schedule suggestions and mission concepts also provided by Dr. Dan Barta of NASA JSC.

REFERENCES:

Advanced Technology for Human Support in Space. 1997. Chapter 2. Advanced Life Support systems. National
Research Council. National Academy Press.
Flager, J. and R.P. Poincelot (eds.). 1994. People-plant relationships: Setting research priorities. Food Products
Press, Binghamton, NY. 444 pages
Kliss, M. and R.D. MacElroy. 1990. Salad machine: A vegetable production unit for long duration space missions.
SAE Tech. Paper 901280. Williamsburg, VA, USA. July 1990.
Sadler, P.D. 1995. Plant hydroponics in Antarctica. Amer. Soc. of Agricul. Eng. Paper No. 957661. Chicago, IL
June 1995.
 CANDIDATE CROP EVALUATION FOR ADVANCED LIFE SUPPORT
R.M. Wheeler1, N.C. Yorio2, G.D. Goins2, G.W. Stutte2, N.A. Cranston2, and L.M. Ruffe2.
1
NASA Biological Sciences Branch and 2Dynamac Corp., Hangar L, Kennedy Space Center, FL.

INTRODUCTION
Numerous crops have been studied for advanced life support (ALS), with crop selections based on their
nutritional traits, productivity, and horticultural and processing requirements (Tibbitts and Alford, 1982).
Studies of ALS crops typically are carried out in controlled environments using electric lighting, some form
of hydroponic culture, and elevated CO2 concentrations (to accelerate growth). In previous studies with
potato, soybean, and radish, we have noted that CO2 concentrations near 400 ppm are suboptimal for total
biomass yields, while 1000-1500 ppm appears optimal. But depending on the crop, elevating CO2 beyond
1500 ppm can result in decreased growth and seed yields, and increased water use (Wheeler et al., 1993,
1999; Mackowiak and Wheeler, 1996; Grotenhuis and Bubgee, 1997). We have initiated similar series of
tests to study CO2 response trends in two cultivars of bean. Testing in our laboratory has also shown that
many crops perform well in hydroponic culture, including subterranean crops like potato (Wheeler et al.,
1990), but when the same nutrient solution is used for successive plantings, potatoes can become stunted and
prematurely induced to tuberize. We speculate this might be due to a build-up of tuber-promoting
compounds in the solution (Wheeler et al., 1995). Depending on the horticultural approach, this factor may
be useful for early tuber set, but it might be have to be controlled to sustain predictable yields.

CURRENT STATUS OF RESEARCH

Methods
Bean (Phaseolus vulgaris L cvs. Etna and Hystyle) plants were grown in chambers with fluorescent lamps at
350 µmol m-2 s-1 of irradiance and an 18 light / 06 dark photoperiod. Temperatures were cycled at 28°C light
/ 24°C dark and all plants were grown in nutrient film culture with nutrient solution maintained at 1200 µS
cm-1 and pH of 5.8. In separate tests, the atmosphere of the growth chamber was maintained at 400, 1200,
4000, and 8000 ppm CO2 to study CO2 effects on growth and water use.

Potatoes (Solanum tuberosum L. cv. Norland) were grown in chambers with fluorescent lamps at 400 µmol
m-2 s-1 of irradiance and a 12 light / 12 dark photoperiod. Temperatures were maintained at 20°C light /
16°C dark and CO2 levels were maintained at 1200 ppm throughout the test. Sequential generations (91 days
each) of plants were grown in recirculating nutrient film culture. Four nutrient solution management
strategies were compared: 1) continuous production in the same nutrient solution; 2) fresh nutrient solution
for each generation; 3) alternate plantings with wheat; 4) activated carbon filtering of nutrient solution for 7
days at the start of each planting. For each treatment, water volume and nutrients were replenished daily.

Results
The highest biomass for bean plants occurred at 1200 ppm CO2, but seed yields showed no consistent effects
between treatments (Table 1).

Table 1. Effect of CO2 concentration on growth of bean (Phaseolus vulgaris).

CO2 400 ppm 1200 ppm 4000 ppm 8000 ppm
Cultivar
(kg DM / m2) (kg DM / m2) (kg DM / m2) (kg DM / m2)
cv. Etna Seed 0.33 0.37 0.42 0.44
Total 1.08 1.36 1.30 1.25
cv. Hystyle Seed 0.25 0.22 0.25 0.28
Total 1.04 1.25 1.24 1.13
DM = dry mass.

Providing fresh nutrient solution for each planting of potato resulted in the greatest yields (Table 2).
Continuously using the same solution resulted in early tuber initiation but smaller shoot / canopy growth
and reduced light interception; consequently, yields were reduced. Rotating crops with wheat or filtering the
solution with activated charcoal for 7 days at the start could not reliably prevent the stunted shoot growth
effect (Table 2), and subsequent studies have shown that the carbon filtering can sometimes reduce yields.

Table 2. Effect of nutrient solution management on potato yield in nutrient film technique.
Treatment Fresh Continuous Rotated Carbon
Nutrient Use With Filtered
Solution Solution Wheat Solution
(kg DM / m2) (kg DM / m2) (kg DM / m2) (kg DM / m2)

First Planting Tuber DM 1.56 ± 0.19 1.64 ± 0.15 1.86 ± 0.06 1.60 ± 0.35
Total DM 2.68 ± 0.09 2.78 ± 0.01 2.97 ± 0.03 2.67 ± 0.52
Harvest Index (%) 58 % 58 % 63 % 60 %
Second Planting Tuber DM 1.637 ± 0.01 1.42 ± 0.22 1.18 ± 0.12
Total DM 2.29 ± 0.04 1.88 ± 0.37 (wheat crop) 1.61 ± 0.18
Harvest Index (%) 71 % 75 % 74 %
Third Planting Tuber DM 1.51 ± 0.04 0.91 ± 0.30 1.41 ± 0.16 1.12 ± 0.34
Total DM 2.15 ± 0.07 1.20 ± 0.41 1.87 ± 0.27 1.54 ± 0.48
Harvest Index (%) 79 % 76 % 75 % 73 %
± Values indicate 1 standard deviation; DM = dry mass; harvest index = (Tuber DM) / (Total DM); for the first
planting, all treatments were equivalent to the “fresh nutrient solution” treatment.

CONCLUSION
Like other crops tested for ALS systems, beans showed increased biomass production when CO2 was
elevated from ~400 to 1200 ppm, but unlike other species, there was no clear effect of CO2 on edible
biomass. Nonetheless, results suggest that maintaining CO2 near ~1000-1500 ppm (0.10 - 0.15 kPa) should
be acceptable for a wide range of crops (Wheeler et al., 1993, 1999; Mackowiak and Wheeler, 1996).
Hydroponic (NFT) studies with potato showed that good growth and tuber yields are achievable, but that
continual use of the same solution (with regular water and nutrient replenishment) can result in stunted shoot
growth and early tuber initiation. Changing the nutrient solution after each planting can avoid this, but use of
crop rotation or selective filtration (e.g. with carbon) to remove the “inductive” effects from the old solution
need further study to optimize management and conserve nutrient solution.

FUTURE PLANS
We plan to test bean growth at 16,000 ppm CO2 to determine if very high levels become toxic to the plants.
Even if such high concentrations are undesirable, they might occur in tightly closed habitats of space.
Increased emphasis in crop testing for ALS systems will be placed on “salad” species, that could provide
fresh, flavorful foods for near-term missions, such as Mars transit and early surface missions. These crops
include: lettuce, tomato, spinach, cabbage, radish, onion, chard, carrot, and possibly some fresh herbs.
Initial testing in the ALS BIO-Plex facility will likely include a range of these salad crops to simulate
possible plant contributions for early Mars missions.

REFERENCES
Grotenhuis, T.P. and B. Bugbee. 1997. Crop Sciencec 37:1215-1222.
Mackowiak, C.L. and R.M. Wheeler. 1996. J. Plant Physiol. 149:205-210.
Tibbitts, T.W. and D.K. Alford. 1982. NASA Conference Publication 2231.
Wheeler, R.M., C.L. Mackowiak, J.C. Sager, W.M. Knott, and C.R. Hinkle. 1990. Am. Potato J. 67:177-187.
Wheeler, R.M., C.L. Mackowiak, L.M. Siegriest, and J.C. Sager. 1993. J. Plant Physiol. 142:173-178
Wheeler, R.M., G.W. Stutte, C.L. Mackowiak, N.C. Yorio, and L.M. Ruffe. 1995. HortScience 30:790.
Wheeler, R.M., C.L. Mackowiak, N.C. Yorio, and J.C. Sager. 1999. Annals Bot. 83:243-251.

INDEX TERMS
Advanced Life Support, ALS, CELSS, controlled ecological life support systems, bioregenerative life
support, plant, photosynthesis, transpiration, food, salad crop, BIO-Plex
 CROP OPTIMIZATION AND FAILURE ANALYSIS FOR ADVANCED LIFE
SUPPORT

Bruce Bugbee, Tracy Dougher, Jonathan Frantz, Steve Klassen, Dawn Muhlestein, Cheryl Mackoviak, Glen Ritchie,
and Derek Pinnock
Crop Physiology Laboratory, Utah State University, Logan, UT 84322-4820.

INTRODUCTION
This talk will summarize our studies in 5 areas:
1. Failure analysis: The effect of prolonged darkness on growth and yield of salad crops
2. Respiration efficiency: plant adaptation to changing temperatures
3. Ethylene sensitivity: establishing a threshold level for tolerance
4. Recycling NH4: tolerance of plants for ammonium
5. Digital imaging of radiation capture.

CURRENT STATUS OF RESEARCH

Results
1. Failure analysis: crop plants can tolerate at least 2 weeks of darkness. This means that they can be grown on the
dark side of the moon. Low light levels (PPF = 10 umol m-2 s-1) are extremely useful if the plants cannot be chilled
during the dark period.

2. Respiration efficiency: Plants adapt to a wide range of night temperatures in long term studies.

3. Ethylene sensitivity: Growth reductions occur at only 50 ppb ethylene in all crops studied so far. Reducing the
temperature may reduce the sensitivity to ethylene.

4. High NH4 in hydroponics. Plants grow as well as all nitrate controls with 85% of the N supplied as ammonium.
However, the pH must be controlled above 5.

5. Digital imaging of radiation capture: A digital camera and appropriate software are all that is needed to quantify
radiation capture by a plant community.

CONCLUSION
Crop plants are more sensitive to some environmental parameters and less sensitive to others than we once thought.
These studies are reshaping our paradigms about plant response to the environment.

INDEX TERMS
wheat, soybeans, lettuce, rice, tomatoes, respiration, photosynthesis, nitrogen, ammonium, nitrate, radiation capture,
biomass production, bioregenerative life support.
TECHNIQUES FOR IDENTIFICATION AND RATE MEASUREMENT OF VOLATILE
ORGANIC COMPOUNDS (VOCs) EMISSION FROM LIVING PLANTS AND FOOD
EXTRUSION PROCESSES IN CONJUNCTION TO NASA’s ALS SYSTEMS

Weerasak Lertsiriyothin1, Bin K. Khoo1, Joseph Lech1, John A. Hogan2, Robert M. Cowan2, and
Thomas G. Hartman1
Center for Advanced Food Technology, Department of Food Science, Rutgers, The State
University of New Jersey1,
Department of Environmental Sciences, Rutgers, The State University of New Jersey2

INTRODUCTION
Long duration human exploration of the solar system calls for an extensive regenerative life
support system to supply food and recycle air, water and wastes. Neither the resupply of large
amounts of food, water and atmospheric gases nor the use of physico-chemical regeneration of
the water and air from waste as practiced currently for a short-term space mission is feasible for a
long- term one. A conceptual bioregenerative life support system, which involved the
regeneration of food, water and air from waste using biological processes, together with waste
processing is being tested for its ability to sustain human life under NASA’s Advanced Life
Support (ALS) systems. Basically, the bioregenerative life support system utilizes plants to
remove CO2 and generate O2, and edible foods through the photosynthesis process. A self-
sustaining ALS requires the integration of a plant growth module, a food-processing module, a
waste processing module and a crew module. In ALS, air quality is crucial for mission crew life
and plant life. VOCs from plants and food extrusion processes affect air quality. Therefore, the
major objective of this research is to identify VOCs and determine the rate of VOCs emission
from NASA’s selected living plants (tomato, wheat, and lettuce) and during food extrusion of
wheat and rice flours.

CURRENT STATUS OF RESEARCH

Techniques of VOCs sample collection and chemical analysis for both living plant and food
processing are discussed here. For living plants’ VOCs, a plant-section cuvette, based on close
system gas exchange measurement is used for collecting gaseous sample from tomato plant. A
purge and trap thermal desorption-gas chromatography-mass spectrometry or flame ionization
(P&T-TD-GC-MS or FID) is used as VOCs analytical techniques throughout this experiment. We
developed an analytical procedure for quantifying ethylene as low as 0.5 ppb (v/v) by using TD-
GC-FID detector and analytical procedures for identification and quantification of other VOCs by
using TD-GC-MS. Our studies showed that tomato plants release ethylene at the rate of 0.0002-
0.0014 µg/g dry biomass.min depending on the plant age (40-120 days after planting). In addition
to ethylene, tomato plant generates more than 30 VOCs with the total concentration of about 17.2
µg/g dry biomass. A discussion of a new design for plant VOCs collection (whole-plant vessel
system) based on an open system is also presented. The whole-plant vessel system is capable of
well maintaining environmental conditions (CO2, %RH and Temperature) within one-day
experimental period for small plant size (wheat and lettuce). For food extrusion VOCs, chemical
profiles of gas samples during extrusion were determined for wheat and rice for two levels of
moisture contents of 16% and 18%. The lower moisture content, the reactions appear to favor the
production of N-containing, S-containing compounds. The total rate of VOCs emission from
18% MC rice extrusion is nearly two times of the one from18% MC high gluten wheat extrusion.
The results of this research will provide NASA a means of estimating the VOCs effect on air
quality and take into consideration when designing air purification system and modeling
automated plant growth chamber. The developed VOCs collecting technology can be transferred
to use in various researches on plant biogenic compounds.
Future Plans
The standard protocols for collecting and analysis of plants’ VOCs are continued to test for their
reliability and robustness. The whole-plant vessel system for collecting small plants’ VOCs will
be used to study the rate of VOCs generation from wheat and lettuce plants. A List of VOCs
produced from living plants (tomato, wheat and lettuce) and food extrusion process is being
made. This database can be used for extending the list of chemicals in the SMACs (Spacecraft
maximum allowable concentrations).
 PLANT GROWTH EXPERIMENTS IN ZEOPONIC SUBSTRATES:
APPLICATIONS FOR ADVANCED LIFE SUPPORT SYSTEMS

D. W. Ming1, J. E. Gruener2, K. E. Henderson1, S. L. Steinberg3, D. J. Barta1, C. Galindo, Jr.2, and D. L. Henninger3

1
NASA Johnson Space Center, Houston, Texas 77058; 2Hernandez Engineering, Inc., Houston, Texas; 3Liberated
Services, Houston, Texas

INTRODUCTION
A zeoponic plant-growth system is defined as the cultivation of plants in artificial soils, which have zeolites as a
major component (Allen and Ming, 1995). Zeolites are crystalline, hydrated aluminosilicate minerals that have the
ability to exchange constituent cations without major change of the mineral structure. Recently, zeoponic systems
developed at the National Aeronautics and Space Administration (NASA) slowly release some (Allen et al., 1995)
or all of the essential plant-growth nutrients (Ming et al., 1995). These systems have NH4- and K-exchanged
clinoptilolite (a natural zeolite) and either natural or synthetic apatite (a calcium phosphate mineral). For the natural
apatite system, Ca and P were made available to the plant by the dissolution of apatite. Potassium and NH4-N were
made available by ion-exchange reactions involving Ca2+ from apatite dissolution and K+ and NH4+ on zeolitic
exchange sites. In addition to NH4-N, K, Ca, and P, the synthetic apatite system also supplied Mg, S, and other
micronutrients during dissolution (Figure 1).

The overall objective of this research task is to develop zeoponic substrates wherein all plant growth nutrients are
supplied by the plant growth medium for several growth seasons with only the addition of water. The substrate is
being developed for plant growth in Advanced Life Support (ALS) testbeds (i.e., BioPLEX) and microgravity plant
growth experiments. Zeoponic substrates have been used for plant growth experiments on two Space Shuttle flight
experiments (STS-60; STS-63; Morrow et al., 1995). These substrates may be ideally suited for plant growth
experiments on the International Space Station and applications in ALS testbeds. However, there are several issues
that need to be resolved before zeoponics will be the choice substrate for plant growth experiments in space. The
objective of this paper is to provide an overview on recent research directed toward the refinement of zeoponic plant
growth substrates.

CURRENT STATUS OF RESEARCH

The first report of wheat seed yields from zeoponic substrates were considerably lower than from control substrates.
Gruener et al. (2000) reported seed yields for wheat grown in a zeolite-synthetic apatite substrate diluted with a
potting soil (peat-vermiculite-perlite) to be less than 30 % of the controls watered with ½-strength Hoagland’s
nutrient solution. They attributed the low seed yield to: (1) above normal contents of N and P in the plant tissue, (2)
the NH4-N source (i.e, NH4-induced Ca deficiency), and (3) a wheat variety (var. Superdwarf) that performs poorly
in non-ideal growing conditions.

Although the synthetic apatite and nutrient-enriched clinoptilolite substrates supplied adequate levels of the essential
nutrients, N and P levels in plant tissues were slightly higher than required by wheat (see Gruener et al., 2000). In a
subsequent test (Henderson et al., 2000), plant tissue N and P concentrations were lowered into the expected nutrient
ranges for wheat by the addition of dolomite (CaMg(CO3)2), ferrihydrite, and nitrifying bacteria to the zeoponic
substrate containing synthetic apatite. Seed yields were about 30% greater in the dolomite-amended zeoponic
substrate inoculated with nitrifying bacteria compared with a peat-vermiculite-perlite control substrate watered with
½-strength Hoagland’s nutrient solution. The dolomite addition probably reduced the solubility of the synthetic
apatite due to a common ion effect (i.e., Ca2+), and the nitrifying bacteria aided in the conversion of NH4+ to NO3-,
which appeared to enhance seed production. The addition of dolomite reduced the P content of wheat from 1.4 wt.
% in treatments without dolomite additions to 1.1 wt. % in substrates containing dolomite. The addition of the
nitrifying bacteria further reduced the P plant tissue content to about 0.8 wt %. Grain yields increased from 4 to 8
grams/pot after the addition of nitrifying bacteria.

Steinberg et al. (2000) conducted a side-by-side comparison of wheat (var., USU-Apogee) growth and yield in a
hydroponic system and a zeoponic system in a controlled environment chamber. The hydroponic system was a
nutrient-film technique (i.e., seeds were place between fiberglass wicks inserted into a recirculating nutrient
solution) with a modified ½-strength Hoagland’s nutrient solution; the zeoponic substrate consisted of K- and NH4-
exchanged clinoptilolite, synthetic apatite, dolomite, and nitrifying bacteria (described by Henderson et al., 2000).
The substrate was diluted with 70 vol. % porous ceramic (heat-expanded clay) soil and watered with deionized
water in a microporous tube irrigation system that maintained the soil matric potential at -0.5 kPa (Steinberg and
Henninger, 1997). Temperature, light, and humidity were kept constant throughout the experiment at 23°C, 1700
µmol/m2/s, and 70%, respectively, with a 24-hr photoperiod. The seed yield and harvest index at 64 days for the
zeoponic substrate was 1.3 ± 0.2 kg/m2 and 37% vs. 1.8 ± 0.3 kg/m2 and 51% for the hydroponic system. Thus, the
wheat seed production in the zeoponic substrate was equivalent to about 200 bushel/acre.

The formulation for zeoponic substrates was further refined by independently adding three calcium minerals (calcite,
dolomite, and wollastonite). Calcium minerals were added to determine their effect on calcium and phosphorous
uptake by wheat (var., USU-Apogee). Seed yield (13-22 g/pot) and harvest index (41-54 %) of plants grown in the
zeoponic substrates were not significantly different from plants grown in control substrates watered with ½-strength
Hoagland’s solution (16-24 g/pot seed yield and 51-57 % harvest index). Wheat grown in zeoponic substrates
amended with more soluble Ca minerals (e.g., calcite) had higher plant calcium.

FUTURE PLANS
Experiments are currently underway to evaluate the productivity of salad crops in zeoponic substrates. Several root
crops (e.g., carrot and radish) may be more productive when grown in solid substrates, such as zeoponics, as
compared to those grown in hydroponic culture systems. Recent experiments are directed towards supporting plant
growth in ALS testbeds (i.e., BioPLEX) and planetary transfer vehicles (e.g., microgravity salad machine on Mars
transfer vehicle).

REFERENCES
Allen, E.R. and Ming, D.W. (1995) Recent progress in the use of natural zeolites in agronomy and horticulture. In D. W. Ming and F. A.
Mumpton, Ed., Natural zeolites '93: Occurrence, properties, use. 477-490. International Committee on Natural Zeolites, Brockport, New
York.
Allen, E.R., Ming, D.W., Hossner, L.R., Henninger, D.L., and Galindo Jr., C. (1995) Growth and nutrient uptake of wheat in clinoptilolite and
phosphate rock substrates. Agron. J., 87, 1052-1059.
Gruener, J.E., Ming, D.W., Henderson, K.E., and Carrier, C. (2000) Nutrient uptake of wheat grown in diluted clinoptilolite-natural/synthetic
apatite substrates. In C. Colella and F. A. Mumpton, Ed., Natural Zeolite '97: 5th International Conference on the Occurrence, Properties, and
Utilization of Natural Zeolites, De Frede Editore, Napli, Italy.
Henderson, K.E., Ming, D.W., Carrier, C., Gruener, J.E., Galindo, Jr., C., and Golden, D.C. (2000) Effects of adding nitrifying bacteria, dolomite,
and ferrihydrite to zeoponic plant growth substrates. In C. Colella and F. A. Mumpton, Ed., Natural Zeolite '97: 5th International Conference
on the Occurrence, Properties, and Utilization of Natural Zeolites, De Frede Editore, Napli, Italy.
Ming, D.W., Barta, D.J., Golden, D.C., Galindo Jr., C., and Henninger, D.L. (1995) Zeoponic plant-growth substrates for space applications. In
D. W. Ming and F. A. Mumpton, Ed., Natural zeolites '93: Occurrence, properties, use. 505-513. International Committee on Natural
Zeolites, Brockport, New York.
Morrow, R. C., Duffie, N. A., Tibbitts, T. W., Bula, R. J., Barta, D. J., Ming, D. W., Wheeler, R. M., and Porterfield, D. M. (1995) Plant response
in the ASTROCULTURE Flight Experiment Unit. SAE Technical Paper Series, #951624. San Diego, California.
Steinberg, S.L. and Henninger, D.L. (1997) Response of the water status of soybean to changes in soil water potentials controlled by the water
pressure in microporous tubes. Plant Cell Environ., 20, 1506-1516.
Steinberg, S.L., Ming, D.W., Henderson, K.E., Carrier, C., Gruener, J.E., Barta, D.J., and Henninger, D.L. (2000) Wheat response to differences
in water and nutritional status between zeoponic and hydroponic growth systems. Agron. J., 92, 353-360.

Nutrient Uptake
By Plants

PO4 Ca Cl
Figure 1. Dynamic equilibria for NASA’s zeoponic Water
Cu Fe
Zn Syn. Apatite B
plant growth system. Plant growth nutrients are H2O Fe
B
K
PO4 Mn Mg MoO4 SO4
slowly released from synthetic, nutrient- Zn "Soil"
NO3
Cl
substituted apatite by dissolution reactions and Ca Solution Mn
Mg NH4
from the zeolite (clinoptilolite) by ion exchange Soil Air Cu SO4 Microbes
reactions. The reactions in soil solution (i.e., CO2 N2 O2
MoO4 NH4 NO3

nutrient release) are driven towards the root-soil

interface by the uptake of nutrients by the plant. Zeolite
Exchange Sites
K NH4
 Microgravity Plant Nutrient Experiment: Year 1 Activities

Howard G. Levine and Thomas W. Dreschel

Dynamac Corporation, Life Sciences Support Facility, Kennedy Space Center, FL 32899

There is a need for microgravity-based plant culture nutrient delivery systems (NDS's) for both
bioregenerative Advanced Life Support and plant research functions. The provision of adequate levels of
water (without causing water logging) and oxygen to the root zone are the most crucial components
deterring major advancements in this area. The dominance of the surface tension of water under
microgravity conditions has often been found to lead to either severe water-logging or excessive drying in
the root zone. Consequently, differences in plant growth responses between spaceflight experiments and
their ground controls are expected based merely upon differences in moisture distribution patterns between
the two conditions. This project will address the question of "comparability of environmental conditions"
between the spaceflight and ground control experiments for both a porous tube plant NDS and a substrate-
based NDS by employing 3-6 different wetness level treatments for both of these approaches. It is
anticipated that different pre-set wetness levels than those used on Earth will be required to support optimal
plant growth in space. Dry seeds will be loaded three days prior to Orbiter lift-off, and the system will be
initiated by the crew on-orbit. A minimum of 72 wheat (Triticum aestivum) seeds (for each of the two
NDS's) will be imbibed and germinated on-orbit. Time-lapsed video recording of the plants will monitor
growth over time. At recovery, the plants will be measured, and extensive tissue analyses relating to gene
expression and stress-associated metabolites will be undertaken.

Primary Author: Howard G. Levine

Affiliation/Address: Gravitational Biology Laboratory
Life Sciences Support Facility
Mail Code DYN-3
Kennedy Space Center, FL 32899

Telephone Number: 321-476-4321

Fax Number: 321-853-4220

email Address: Howard.Levine-1@ksc.nasa.gov

 PLANT NUTRIENT DELIVERY

Gregory D. Goins
Dynamac Corporation, Mail Code DYN-3, Kennedy Space Center, Florida 32899

INTRODUCTION
Unique growing procedures are needed to effectively cultivate plants in space. Fluids behave differently in
microgravity, and therefore, many plant nutrient delivery systems used on earth will not function effectively in
space. In a microgravity climate, a plant nutrient delivery system (NDS) must provide adequate amounts and
uniform distribution of water, nutrient, and oxygen levels in the root zone, while at the same time, prevent release of
free nutrient solution to the atmosphere. Solid media such as, vermiculite mixtures and various gelling agents that
contain predetermined amounts of water and nutrients have been successfully used in passive plant nutrient delivery
systems for brief stays in orbit. However, for extended plant cultivation in space, root-zone media will require more
than just an initial loading of water and nutrients due to losses from plant evapo-transpiration and nutrient uptake.
The ultimate goal is to design a nutrient delivery system that is capable of sustaining plants for long periods under
hypogravity, yet require minimal system maintenance and limited demands on crew time. Testing in our laboratory
has evaluated several types of nutrient delivery system (NDS) hardware and procedures designed for growing plants
in gravitational research as well as for planetary bioregenerative life support for humans during long-term space
missions. Concepts currently being tested at Kennedy Space Center (KSC) include microporous tubes with and
without various types of solid media, and gravity-dependent systems such as the hydroponic nutrient film technique
(NFT).

CURRENT STATUS OF RESEARCH

Methods
At KSC, the performance of each nutrient delivery concept has been based largely upon the yield response of
Advance Life Support staple crops such as wheat (Triticum aestivum L.) and sweetpotato (Ipomoea batatas L.). In a
series of ground-based investigations with wheat, our primary focus was to characterize the microporous tube
system with and without zeoponic substrate in terms of overall plant growth, nutrient uptake patterns, and water use.
Nutrient solution was constantly circulated under a slightly negative hydrostatic pressure (or suction) through the
central cavities of hydrophilic, microporous, ceramic filter tubes (0.45 µm nominal pore diameter, OD 2.3 cm, ID
2.1 cm, 2 mm flow channel diameter). Nutrient solution moved through the porous wall of the tube into the rooting
environment by capillary attraction. In sub-irrigated microporous tube systems we used stainless-steel tubes
(nominal pore size 30 µm, OD 0.953 mm, ID 0.635 mm), covered with zeoponic substrate from Johnson Space
Center. The sub-irrigated solid growing matrix provides root anchorage and a buffered source of nutrients. Zeoponic
substrates are being developed to supply all essential macro- and micronutrients (e.g. slow-release fertilization) to
plants over several growth cycles.

In gravity-dependent hydroponic systems at KSC, a general NFT nutrient management approach is applied to all
ALS crops. The KSC approach calls for daily replenishment of minerals to maintain a nutrient solution formula
essentially equivalent to modified 0.5-X strength Hoagland's solution (i.e., electrical conductivity ˜1200 µS/cm). In
addition nitrate is the sole source of nitrogen, and nitric acid and potassium hydroxide are used to constantly
maintain pH at approximately 5.8. Past tests at KSC with a generalized NFT protocol has generally produced
excellent yields with most ALS crops. However, the standard KSC nutrient management approach tends to promote
disproportionate foliage growth relative to storage organs (i.e, potato and sweetpotato). A major difference between
the KSC and Tuskegee University nutrient solution formulations is with nitrogen nutrition. The generalized KSC
modified 0.5-X strength Hoagland's contains approximately twice the amount of nitrogen found Tuskegee
University's nutrient solution. In addition, the KSC protocol uses nitrate at the sole source of N, whereas the
Tuskegee protocol uses a mixed source of N in the form of a 6:1 ratio of KNO3:NH42PO4.

Results
In terms of aboveground biomass production, seed yield, and harvest index, the drip-irrigated peat vermiculite
system was the most successful among the compared nutrient delivery systems (Table 1). The systems using
zeoponic substrate produced wheat with excessive seedless tiller formation as compared to wheat produced with the
microporous tubes only or drip-irrigated peat-vermiculite. Plants in the sub- and drip-irrigated zeoponics systems
displayed as many as 8 tillers, which failed to produce seed. The plants grown in zeoponic substrate were exposed
to a mixed-nitrogen source (NH4 and NO3), that may have promoted greater tiller formation in those plants.
Conversely, wheat grown in microporous tube only and drip-irrigated peat vermiculite systems were supplied with
NO3-N alone, and these plants produced a maximum of 2-3 tillers per plant. However, it is important to note that
the particular wheat growth cycle reported here was accomplished with the first generation of zeoponic substrate.

In recent tests with sweetpotato, we observed significantly less foliage growth and more storage root production
when nutrient solution N concentration was reduced (Table 2). Previous research by others has shown that although
nitrogen stimulates both sweetpotato foliage yields and storage root production, high available nitrogen (e.g. KSC
treatments) results in vegetative growth that occurs at the expense of storage root production. Overall, the method of
pH control did not appear to have a significant role in total biomass production or partitioning within either the KSC
or Tuskegee University protocols. Total biomass production was greatest in the KSC treatments, which can be
attributed to the daily replenishment of nutrients, especially the constant supply of N. However, in these same
treatments storage root production was extremely low. The Tuskegee treatments had significantly less foliage
growth and more storage root production.

Table 1. Wheat harvest data summary ± SE (per tray)1

Porous Tubes Porous Tubes Drip-Irrig. Drip-Irrig.
+Zeoponics Zeoponics P. Vermiculite
Aboveground DM (g)2 96.9 ± 3.5 90.9 ± 0.6 126.8 ± 5.0 148.9 ± 0.6
Straw DM (g) 33.7 ± 2.0 48.1 ± 0.1 78.8 ± 2.0 61.0 ± 0.3
Spike DM (g) 63.2 ± 1.7 44.7 ± 0.5 59.2 ± 3.2 98.9 ± 0.6
Chaff DM (g)3 23.0 ± 1.2 35.3 ± 1.1 46.2 ± 1.3 31.6 ± 0.3
Seed DM (g) 40.3 ± 0.9 8.5 ± 1.5 13.1 ± 1.9 67.2 ± 0.7
Spike No. 104 ± 6 218 ± 6 247 ± 6 155 ± 1
Harvest Index (%)4 41.5 9.4 10.3 45.1
1Means from 45 plants per tray which was equivalent to approximately 900 plants per m2.
2Aboveground dry matter (DM) = Spike DM + Straw DM; 3Chaff DM = Spike DM - Seed DM; 4Harvest index does not include roots

Table 2. Biomass yield comparison from sweetpotato plants grown under different nutrient and pH control management regimes.
Treatment Total Biomass Edible Biomass

(kg/m2) (g/m2 d) (g/mol PAR) (kg/m2) (g/m2 d) (g/mol PAR)

KSC HNO3/KOH 3.07 25.59 0.68 0.13 1.11 0.03
KSC HCl/NaOH 3.22 26.87 0.72 0.14 1.16 0.03
TU HCl/NaOH 2.38 19.84 0.53 0.90 7.46 0.20
TU HNO3/KOH 2.48 20.67 0.55 0.72 5.97 0.16

CONCLUSION
In ground-based studies, each NDS sufficiently supported wheat biomass production to varying degrees and showed
distinct patterns for plant nutrient uptake, water use, and partitioning between vegetative and edible tissues. Tests to
date confirm that reduced nitrogen availability in the nutrient solution would appear as a plausible solution to limit
the potential for large shoot growth by sweetpotato in NFT culture.

FUTURE PLANS
Nutrient delivery system tests at KSC are continuing using refinements of each system and improved generations of
solid substrates in preparation for future microgravity missions on shuttle (WONDER) and space station (PESTO).
In ground-based experiments, a study is planned to outline the baseline N-supply requirements for potato production
in our NFT systems. Perhaps potato (analogous to sweetpotato) would exhibit significantly less foliage growth and
more tuber production with a nutrient management regime that limits N-supply in NFT systems.

INDEX TERMS
Microporous tube, microgravity, hydroponic, zeolite, NFT, nutrient thin film, sweetpotato, Hoagland's solution,
nutrient solution culture, growth media, soil, potting media, N-nutrition, zeoponics, peat-vermiculite
 Performance of Sweetpotato for Bioregenerative Life Support
Daniel J. Barta1, Keith E. Henderson1, Desmond G. Mortley2, and Donald L. Henninger1.
1
NASA Johnson Space Center, Houston, TX, 77058; 2Tuskegee University Center for Food and Environmental
Systems for Human Exploration of Space, Tuskegee, AL, 36088

Introduction
Sweetpotato (Hill et al. 1992) was grown to harvest maturity within NASA Johnson Space Center's
Variable Pressure Growth Chamber (Barta and Henninger 1996) to characterize crop performance for potential use
in advanced life support systems as a contributor to food production, air revitalization and resource recovery.
Characterization of crop performance on a large-scale in closed atmosphere chambers is essential to advance the
readiness level of crop plants as candidate biotechnologies for advanced life support (Wheeler and Strayer 1997).
Data obtained from this research will be useful for systems modeling and analysis in support of development of
biomass production systems for the Bioregenerative Planetary Life Support Systems Test Complex (Barta et al.
1999) as a stepping stone to the development of technologies for long duration human exploration of space.

Methods
Stem cuttings of breeding clone "TU-82-155" were grown hydroponically at a density of 17 plants m-2
using a modified pressure-plate growing system (Patent No. 4860-490, Tuskegee University) within the Variable
Pressure Growth Chamber (Figure 1). Lighting was provided by HPS lamps at a photoperiod of 12h light:12h dark.
The photosynthetic photon flux was maintained at 500, 750 and 1000 µmol m-2 s-1 during days 1-15, 16-28 & 29-
119, respectively. Canopy temperatures were maintained at 28°C light:22°C dark. During the light period relative
humidity and carbon dioxide were maintained at 70% and 1200 µl l-1, respectively. Nutrient solution was manually
adjusted 2 to 4 times per week by addition of 10X concentrated modified half-strength Hoagland nutrient salts and
NaOH to return the electrical conductivity and pH to 1.2 mS cm-1 and 6.0, respectively.

Results & Discussion

At 17 weeks (119 days) from transplanting, a total of 56.5 kg fresh mass of storage roots (84.1% moisture)
were harvested from the 11.2 m2 chamber resulting in a yield 5.0 kg m-2 (Table 1, Figure 2). Harvest index, based
on fresh mass, was 38.6%. Proximate nutritional composition of the storage roots was generally similar to values
reported for field-grown sweetpotato except moisture and ash content were higher (USDA, see also Wheeler 1999).
Once canopy closure occurred, canopy net photosynthesis averaged 0.5 kg CO2 d-1 over the entire 11.2 m2 crop
which would support approximately one-half the air revitalization requirement for one person. The transpiration
rate, following canopy closure, ranged from 300 to 450 ml m-2 hr-1 during the light period and 100 to 150 ml m-2 hr-1
during the dark period. Disruption of the crop canopy during chamber entries to take measurements and during
episodes of nutrient stress affected gas exchange and transpiration.

Conclusions
Sweetpotato was successfully grown to harvest maturity in a large-scale atmospherically-closed controlled-
environment chamber. Yield of edible biomass and capacity for contributing to air revitalization and water recovery
were documented. Yield was slightly less than that found in smaller-scale studies, but this is not unusual (Wheeler
1999). Continued work is suggested to improve control of storage root initiation, bulking and vine growth.

Literature Cited
Barta, D.J. and D.L. Henninger. 1996. Johnson Space Center’s Regenerative Life Support Systems Test Bed. Adv.
Space Res. 18(1/2):211-221.
Barta, D.J., J.M. Castillo, and R.E. Fortson. 1999. The Biomass Production System for the Bioregenerative
Planetary Life Support Systems Test Complex: Preliminary Designs and Considerations.
Hill, W.A., C.K. Bonsi and P.A. Loretan. 1992. Sweetpotato Technology for the 21st Century. Tuskegee University,
Tuskegee, Alabama, 607 pages.
U.S. Department of Agriculture, Agricultural Research Service. 1999. USDA Nutrient Database for Standard
Reference, Release 13. Nutrient Data Laboratory Home Page, http://www.nal.usda.gov/fnic/foodcomp
Wheeler, R.M. and R.F. Strayer. 1997. Use of Bioregenerative Technologies for Advanced Life Support: Some
Considerations for BIO-Plex and Related Testbeds. NASA Technical. Memorandum. 113229, Kennedy
Space Center, Florida
Wheeler, R.M. 1999. Bioregenerative Life Support and Nutritional Implications for Planetary Exploration. In:
“Nutrition in Spaceflight and Weightlessness Models”, H.W. Lane and D.A. Schoeller, eds. CRC Press,
Boca Raton, Florida.

Index Terms Advanced Life Support, Biomass Production, Sweetpotato, Photosynthesis, Yield, Transpiration

Table 1. Biomass partioning in sweetpotato at harvest (17 weeks from transplanting).

Crop Component Fresh Mass (kg m-2) Dry Mass (kg m-2)
Storage Roots 5.05 0.80
Leaves, Stems, Vines 5.37 1.23
Fiberous Roots 2.14 0.11
String Roots 0.12 0.01
Stem & Stem Waste 0.27 0.04
Total Crop Biomass 12.95 2.18
Harvest Index 0.39 0.37
No. of Storage Roots* 281 281
*Includes all storage roots greater than 1 cm. in diameter

Table 2. Proximate analysis of sweetpotato storage roots at harvest.

Composition Based on Dry Mass

Moisture Ash Protein Carbohydrate Fat
Grade % % % % %
No. 1 (5.1-8.9 cm diameter) 83.8 4.3 3.7 91.4 <0.6
Jumbo (>8.9 cm diameter) 85.2 4.0 4.7 90.6 <0.7
Canner (2.5-5.1 cm diameter) 84.4 4.4 5.1 89.2 1.3
Culls (1-2.5 cm diameter) 84.9 5.3 5.3 88.8 <0.7
Field-grown (USDA) 72.0 3.3 5.9 89.6 1.1

Figure 1. View through open door of the VPGC shortly Figure 2. Harvested sweetpotatoes from
after planting. There are eight growing areas, four on each of one-half of the chamber (5.6 m2)
side of the chamber, in two levels. Perforated aluminum
sheet was to contain the sweetpotato vines during growth.
 ENHANCED PROTEIN CONTENT AND QUALITY IN SWEETPOTATO
ENGINEERED WITH A SYNTHETIC STORAGE PROTEIN GENE FOR IMPROVED
HUMAN NUTRITION AND HEALTH

Marceline Egnin1, Ralphenia Pace1, Channapatna Prakash1, Jesse Jaynes2, and Kenzo Nakamura3. 1Center for Plant
Biotechnology Research, Tuskegee University, Tuskegee, AL. 2Demegen BioTechnologies, Inc., Pittsburgh, PA.
3
Nagoya University, Japan. (megnin@tusk.edu).

It is essential that human diets are endowed with those amino acids that the body cannot synthesize (essential amino
acids, EAA) and nitrogen in the form of non-essential amino acids (NEAA). Both EAA and NEAA are required for
the biosynthesis of protein and other nitrogen-compounds vital for homeostasis and human growth. Thus the quality
of a food crop protein is of considerable importance to crop improvement. We sought to improve the nutritive
quality of sweetpotato proteins, a NASA candidate crop, by enhancing its ‘essential amino acids’ (EAA) content. A
novel storage protein (asp-1) rich in many EAA and with improved protein stability was rationally developed. The
synthetic asp-1 gene under the control of CaMV 35S promoter was introduced into sweetpotato (Ipomoea batatas L)
using the Agrobacterium gene transfer system. Transgenic sweetpotato plants grew normally in a field trial, and in
an unanticipated twist, showed 300 - 500% increase in the total protein content in their storage roots (‘tuber’).
Levels of many EAA such as methionine, threonine, isoleucine, and lysine also increased proportionally, while
tryptophan increased by several orders of magnitude. Although transgenic plants expressed the asp-1 protein
detectable by immunoblot analysis, the increased protein content in the roots was primarily due to enhanced levels
of several native proteins, especially sporamin and ß-amylase. The increase in protein as well as EAA levels in
transgenics remained steady over a period of three field trials. Protein accumulation in the transgenic plants follows
a temporal fashion in a time course study. The yields of two transgenic lines were comparable to that of control
plants while the other three lines exhibited yield penalty. Animals (golden Syrian hamsters) fed with transgenic
sweetpotato had 56% more live body weight over the control-fed animals and exhibited lowered total cholesterol,
triglycerides, and LDL-cholesterol levels in their plasma and liver. The animal feeding also showed the superiority
of asp-1 sweetpotatoes in terms of the true protein digestibility, nets protein utilization and biological value of the
protein. The corrected ‘protein efficiency ratio’ of transgenic sweetpotato (3.71) was comparable to that of soy
protein (3.72) and higher than control sweetpotato (2.57) or casein (2.49). The animal histopathological studies with
brain, liver, kidney, intestine, and bone showed that transgenic sweetpotato lines did not have any detectable toxic
effects. Our research now is testing various hypotheses for the causes of dramatic increase in the total protein
content in sweetpotato transformed with the asp-1 gene. Research supported by NASA and USDA.
 REGULATION OF ADP-GLUCOSE PYROPHOSPHORYLASE SUBUNIT
EXPRESSION –-A KEY ENZYME FOR STARCH BIOSYNTHESIS IN PLANTS

T.J. Gianfagna, X. Li, J. Xing, Y. Luo and H.W. Janes

Plant Science Department, Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901-8520

INTRODUCTION
Starch is the predominant carbon sink of storage organs and leaves of many plants. Substantial evidence from the
analysis of starch-deficient mutants, transgenic plants, and enzyme kinetics suggest that ADP-glucose
pyrophosphorylase (AGPase) catalyzes a key step for starch biosynthesis in both photosynthetic and non-
photosynthetic tissues. Fruit quality in tomato is largely a function of the soluble solids content of the fruit, and there
is a direct relationship between starch levels early in fruit development and the soluble solids content at maturity.
Moreover, the ability to synthesize starch shortly after fruit set, may be critical for successful organ development.
Increased starch biosynthesis in tomato fruit may also lead to increased assimilate partitioning into fruit, and a
decrease in non-edible biomass, reducing waste processing needs in the BLSS. Thus, AGPase appears to be an
attractive biotechnological target for increasing starch synthesis in plants, both on earth and in space. The enzyme is
a heterotetramer containing two S and two B subunits. We have identified three distinct genes for the S subunit and
one gene for the B subunit (Chen et al., Plant Science 136:59-676, 1998). The purpose of this research is to
determine the biochemical basis for regulation of AGPase activity in tomato, and to design novel ways of increasing
starch content in plants by biotechnological manipulation of the gene(s) for this enzyme.

CURRENT STATUS OF RESEARCH

Methods
Total RNA was extracted from young tomato fruit, mRNA separated, and a cDNA expression library created by
standard techniques. Identification of the multiple forms of the S subunit transcripts was accomplished by PCR
amplification using the following degenerate primers: T(GG)AGAGG (A)AAC(A)C(T)GC(T) AATGT and
AAGTA(GA)AACC(ACG)GTA(AA)CAA(TG)ATACC, followed by 3’and 5’ RACE (rapid amplification of
cDNA ends) to create full length cDNAs. PCR products were cloned and sequenced.

Agp S1 was isolated from a lambda phage genomic library from tomato fruit using standard techniques. The 8.2 kb
insert was cut with restriction enzymes, sub-cloned and sequenced. Truncated promoter regions were cloned into
Agrobacterium tumefaciens LBA 4404 using pBI101, which contains the GUS gene. The results from three
truncated promoters (0.8, 1.3 and 3.0 kb), all beginning upstream from the structural gene, are reported upon here.
Leaf discs of tobacco were transformed with A. tumefaciens containing the three truncated promoter regions of agp
S1 and plants were regenerated using antibiotic resistance for selection of transgenic plants. For tomato plants,
cotyledons and hypocotyls were maintained on media supplemented with tobacco cell suspension cultures before
and after transformation with A. tumefaciens.

For agp S1 promoter analysis, leaves, stem sections, roots and flowers were treated with GUS staining solution
containing 5-bromo-4-chloro-3-indolyl β-D-glucuronide for 6 h at 37C. Tissues were washed with water. Leaves
were bleached with ethanol before staining. Sucrose induction experiments were carried out with normal tomato
fruit and excised tobacco leaves from transgenic plants transformed with the three truncated promoters (0.8, 1.3 and
3.0 kb) of agp S1.

Results
1. Expression of subunits and effect of sucrose
Complete sequences for three S subunit cDNAs (agp S1, agp S2, and agp S3; genebank accession numbers U81033,
U81034, and U85497) and one for the B subunit (agp B) were identified. Northern analysis revealed some major
differences in the expression profiles of the isoforms between different tissues, and between tomato and potato. In
the fruit, the expression of agp B and agp S1 was very strong, agp S2 was expressed more weakly, while agp S3 was
very low or absent. In the leaves, agp B and agp S3 were the highly expressed isoforms, whereas expression of agp
S1 was moderate and agp S2 very low. No obvious differences in transcript levels were observed for any isoform
between mature and young leaves. In roots, neither agp B or agp S3 were detected, although there was moderate
expression of agp S1 and S2.

1
When excised leaves were incubated in sucrose there was a significant induction of transcription for agp B, agp S1,
and agp S2 within 8 h after incubation. Transcript levels remained elevated for at least 16 h. In contrast, agp S3 was
not stimulated by sucrose. In fact, transcript levels declined by 16 h. When sucrose was injected into 10-d-old fruit,
there was an increase in the level of agp S1 transcription within 3 h. The response to sucrose reached a maximum by
6 h and was maintained for 24 h after injection. When water alone was injected into fruit, activity declined relative
to fruit that had not been injected.

2. Cloning of agp S1 and analysis of the promoter region

A lambda phage clone that hybridized with the AGPase S1 probe contained a tomato DNA insert with 8,200 base
pairs (bp). Based upon comparison with the cDNA isolated from tomato fruit, the transcription initiation site is at bp
3178, the termination site at bp 7808 and there are 14 introns in the gene. The translation start codon (ATG) is at bp
3826 and the translation termination signal (TGA) at bp 7484. There is a TATAA box, a common eukaryotic
regulatory sequence, at bp 3123, 55 bps upstream from the transcription initiation site.

Using transgenic tobacco with the agpS1 promoter and its truncated versions ligated to the GUS reporter gene, we
will describe the histological location of agpS1 expression in all the organs of the plant, throughout their
development. In the leaves e.g., agp S1 is located primarily in the guard cells and is conspicuously absent in the leaf
mesophyll. All of the constructs (0.8, 1.3, and 3.0 kb) caused GUS expression. There were no differences between
constructs; therefore, only the 0.8 kb region is required for guard cell expression. We determined the location of
possible cis-acting elements that confer sucrose sensitivity. Only guard cells from plants that contained the 3.0 kb
region responded to sucrose treatment. There were no changes in expression when sucrose was fed to leaves
containing the 0.8 or 1.3 kb promoter fragments, implying that the sucrose sensitive regulatory region is located in a
1.7 kb region upstream from the 1.3 kb fragment. There are four elements in the 1.7 kb segment of the 3.0 kb
construct that are very similar to sucrose regulatory regions in other genes. It is likely, therefore, that one of the
functions of this region of the agp S1 promoter is to confer responsiveness to sucrose.

Agp S1 is highly expressed in fruit tissue. In cv. Laura, fruit development from anthesis to red-ripe fruit takes
approximately 60 d. Significant levels of agp S1 transcript appear about 10 d after anthesis. Transcript levels
increase to a maximum about 15 d after anthesis and then decline significantly. Transcription of agp S1 is no longer
detectable in fruit more than 35 d after anthesis. Both the B and S2 genes are also activated during the same period
of fruit development as agp S1. There is, however, some additional transcriptional activity of the latter two genes in
red fruit. This may be localized to the seed.

CONCLUSIONS
There are significant differences in S gene expression between the different plant organs throughout their
development. Agp S1 and S2, but not S3, are activated by sucrose. Transcriptional activity is consistent with the
pattern of starch accumulation in tomato fruit, and suggests that AGPase is a key enzyme on the pathway in which
sucrose is converted to starch.

FUTURE DIRECTIONS
Our transgenic tomato plants have begun to set fruit. We will extend our characterization of agp S1 expression in
fruit using anti-sense constructs. In addition, and perhaps of greater significance, we can now use the agp S1
promoter to drive other genes that may increase carbon partitioning to the fruit, and perhaps produce plants with
higher yield and nutritional quality for cultivation in space and on earth. The gene for isopentenyladenine transferase
(ipt) is a good candidate. When ligated to the agp S1 promoter, the ipt gene should increase cytokinin production
and cell division in the fruit only during the early stages of fruit development.

INDEX TERMS
Transgenic plants, tomato, starch, gene promoter

2
 LIGHTING TECHNOLOGY DEVELOPMENT FOR BIOREGENERATIVE
COMPONENTS OF THE ADVANCED LIFE SUPPORT PROJECT

J. C. Sager1, R.M. Wheeler1, G. Goins2, and S. Young2

1
Biological Science Branch and 2 Dynamac Corporation, Kennedy Space Center, FL 32899

INTRODUCTION
Bioregenerative Life Support Systems (BLSS) are enabling technologies for applications in microgravity, e.g.,transit or flight
experiments, and in hypogravity, e.g.,Lunar/Mars surface habitat. The lighting of plants/crops to provide the life support
elements of water, air, and food is critical to these bioregenerative life support technologies. The high-energy demand to
provide for biomass production is the primary obstacle to developing feasible bioregenerative systems. The primary
objective is to significantly improve the efficiency of converting electrical energy into edible plant biomass. The methods to
reduce high energy use are defined by a range of factors, including low electrical conversion efficiency, poor transport and
distribution of the light to the plant/crops, and inefficient absorptance and conversion of the light energy by the plant system
(photosynthesis). In the near term these technologies will be applicable to flight experiments on STS and ISS and surface
applications in BIO-Plex and related ground testbeds. Current electric lamps are 15-35% efficient in conversion of electrical
energy to light, but promising technologies with sources (light emitting diodes (LEDs), microwave lamps), solar collectors,
and light pipe, fiber optic, and holographic distribution systems offer the potential to improve on these efficiencies. It is
essential to develop new technologies and horticultural methodologies to significantly reduce the energy demands for these
systems without excessive penalties in mass, volume, other resources, or production of the life support commodities. Current
technologies will be discussed, a developmental schedule presented, and anticipated deliverables listed.

CURRENT STATUS OF RESAERCH

Methods
Currently available technologies are being evaluated, and promising technologies developed and integrated into the crop
production systems at KSC and, ultimately, into human testbeds at JSC (BIO-PLEX Project) and plant flight hardware for the
Shuttle and International Space Station. Technology development efforts focus on obtaining sources to increase electrical
conversion efficiency of lighting systems and methods of collecting, transporting and distributing photosynthetically active
radiation (PAR) to the crop canopy. Studies also address techniques to improve efficiency in the use of radiant energy by the
plant to produce biomass through photosynthesis by cultivar development and cultural improvements. Technology
development is primarily through SBIR, STTR and NRA programs. Evaluation of systems with performance tests of
selected sources and horticultural tests of these technologies will determine their effects on crop development and
productivity. Testing of the new technologies will include quantifying photosynthetically active radiation from lamps,
quantifying radiant conversion efficiencies, and evaluating photosynthetic efficiencies and plant growth under the different
lamps to define the system efficiency and optimal configuration.

Results
The current range of lighting system efficiencies includes sources with electrical conversion efficiencies of 10 to 35
percent with expected improvement to about 50 percent with the microwave lamp. The light distribution
efficiencies to deliver the PAR the plant canopy range from 30 to 80 percent. The system efficiency, the product of
the two efficiencies, ranges from 3 to 30 percent with improvement to 40 percent anticipated. Marginal increases in
system efficiency by improvements in either conversion or distribution efficiencies are significant. Promising
technologies such as light emitting diodes (LEDs) and microwave lamps have shown these increased efficiencies.
Improved conversion of the microwave lamp is the goal of a contract with Fusion Lighting Inc. (Rockville, MD)
(SBIR-III). Development of a solar collector system with light pipes/fiber optic transport and distribution systems
are under development at the University of Arizona (NRA) and Physical Sciences Inc. (San Ramon, CA) (STTR-
III), and holographic distribution systems are being developed by Physical Optics Corporation (Torrance, CA)
(SBIR-II). In addition, the modification of the industry standard high pressure sodium (HPS) lamp with the addition
of a water jacket has been under development for 25 years and has recently been dramatically improved. The
improved conversion and distribution efficiencies, spectral quality, and reduce thermal output of sources are
increasing the efficiencies of the lighting systems and experiments on plant responses (salad and staple crops) are
validating the biological systems responses. Crop improvements are continuing with the most notable with wheat at
the Utah State University (NRA). Results will include data on new lamps and luminaires, evaluations of lighting
and transportation systems, and horticultural lighting methodologies for increased productivity.
Conclusions
High conversion efficiency and minimum equivalent system mass (ESM) are more critical in the development of
flight experiment hardware, and bioregenerative systems for transit and Mars scenarios than on Earth. Requirements
for each type system will be unique, but all systems will benefit from improved components. Poor transport and
distribution efficiency to the plant/crop canopy will be improved by using LEDs and/or fiber optics, and holographic
distribution systems. Improvement in crop productivity and development through more efficient cultivar breeding
and selection (e.g. developmental physiology and photosynthesis), and culture (e.g., spacing, nutrients, and
environment) are improving the bioregenerative systems. However, new approaches must be developed and
existing technologies improved to provide the enabling technologies for a BLSS.

FUTURE PLANS
The Advanced Life Support (ALS) Project must meet the challenges of utilizing new light sources or improving the
conversion efficiency, spectral quality, and thermal exchange/removal of existing light sources and approaches for
plant lighting. System design must improve distribution uniformity and interception by the crop, while reducing
light leakage between crop modules and providing for photoperiod isolation. The ESM must be reduced and
systems developed based on modularity of components. A looming challenge is the development of improved
cultivars and crops whose developmental physiology allows us to improve the biomass conversion and thus the
overall system efficiency.

INDEX TERMS
Conversion Efficiency
Distribution Uniformity
Light Interception
Light Sources
Lighting Systems
PAR
Quantum Efficiency
Spectral Absorptance
Spectral Quality
 PERFORMANCE OF SALAD-TYPE PLANTS GROWN UNDER NARROW-
SPECTRUM LIGHT-EMITTING DIODES IN A CONTROLLED ENVIRONMENT

Gregory D. Goins
Dynamac Corporation, Mail Code DYN-3, Kennedy Space Center, Florida 32899

INTRODUCTION
New lighting technologies must be developed and existing technologies improved to significantly increase the
efficiency of converting electrical energy into edible plant biomass. Light-emitting diodes (LEDs) represent an
innovative artificial lighting source with several appealing features specific for supporting plants, whether on space-
based transit vehicles or planetary life support systems. Because of their rugged design, small mass and volume, and
narrow spectral output, red and blue LEDs are particularly suited for outfitting plant growth hardware in spaceflight
systems. Appropriate combinations of red and blue LEDs have great potential for use as a light source to drive
photosynthesis due to the ability to tailor irradiance output near the peak absorption regions of chlorophyll.
Previous research has shown that red LEDs supplemented with blue light can adequately support various crop
plants. Certain mission scenarios depict salad-type plants (i.e., lettuce, spinach, radish, carrot, etc.) representing
crops which will be grown on-board space transportation vehicles on a comparatively small scale. With this
strategy, salad-type plants would provide a supplemental portion of food, and perhaps provide psychological benefit
to the crew. Data generated from these studies with various salad-type crops grown under LED sources provides
important information for the modeling and development of future applications in growth chambers onboard transit
vehicles or on planetary surfaces.

CURRENT STATUS OF RESEARCH

Methods
Experiments compared the performance and productivity of spinach (Spinacia oleracea L. cv. Noric IV), radish
(Raphanus sativus L. cv. Cherry Belle), and lettuce (Lactuca sativa L. cv. Waldmann's Green) grown under
conventional broad-spectrum lighting sources (high-pressure sodium and cool-white fluorescent lamps) versus LED
arrays at certain red wavelengths (660, 670, 680, 690, nm central wavelength) supplemented with blue LEDs (470
nm central wavelength). Plants were grown in recirculating hydroponics systems located under each light bank.
Radish and lettuce received an 18hr/6hr light/dark photoperiod, while spinach was grown under a 16hr/8hr
light/dark photoperiod with approximately equal photosynthetic photon flux 250 µmol•m-2•s-1. Growth chamber
air temperature and relative humidity for all treatments were maintained at 22°C and 70%, respectively. Carbon
dioxide concentration of the atmosphere was monitored and controlled at 1200 µmol•mol-1. Beginning at 14 days
after planting (DAP), six plants were harvested at pre-determined locations within 0.3 m2 plant trays located under
each light source. Harvesting continued on a weekly interval, with final harvest at 28 DAP.

Results
Overall, spinach grown under 690 nm LEDs and HPS lamps displayed the highest biomass yield per unit growth
area or mole of PAR (Table 1). Net leaf photosynthesis data suggested that the plants grown under or 690 nm LEDs
or HPS lamps had no clear dry matter accumulation advantage in terms of CO2 assimilation rate (Table 1). In the
case of HPS and 690 nm LED lighting sources, the low amount of blue light in combination with more far-red
radiation may have promoted more rapid stem elongation (Table 2). The relatively greater amounts of far-red
radiation (Table 2) emitted from HPS and 690 nm LED treatments appeared to promot differences observed in
growth through more light interception during early growth rather than increasing photosynthesis per se. Other
experiments at Kennedy Space Center have reported greater growth and stem elongation for plants grown in the
presence of HPS lamps when compared to plants grown under more blue-rich lighting sources. It is important to
note that HPS lamps were point-sources of radiation as opposed to CWF and LED light sources, which were
intrinsically more diffuse over the plant canopy as a result of lamp design and associated luminarie. Better light
distribution can be achieved (with electrical efficiency penalties) by locating the lamp greater distances away from
the canopy. However, small-scale plant growth chamber studies, such as the present study, often allocate limited
area for mounting lamps. Likewise, space-based plant growth chambers are usually designed with the light source
mounted as close as possible to the plant canopy. Hence, there is a need for further development of focusing
materials, which will efficiently and evenly distribute point-source light over plant canopies.
Table 1. Net rate of leaf photosynthesis and biomass yield for spinach plants at 28 DAP.
-2 -1
Lampa µmol CO2•m-2•s-1 kg FM•m g FM•mol PARc

CWF 6.8 6.0 24

HPSb 6.7 8.8 35
660 6.5 7.8 31
670 8.3 8.2 33
680 10.1 7.8 31
690 9.1 9.0 36

aAll lighting sources were measured over a 0.3m2 growing area.

bHPS lamp emission attenuated with Plexiglas™ barrier and screening.
cCalculated over 28 days with 16-h light/8- dark photoperiod with 250 µmol•m-2•s-1.

Table 2. Spectral data from lamp sources1,2.

LED λ (nm)
Measurement CWF HPS3 660 670 680 690
(µmol•m-2•s-1)
Photon flux (300-1100 nm) 272 401 251 253 264 296
PPF (400-700 nm) 250 250 250 250 253 249
YPF 218 229 225 221 212 196
Blue (400-500 nm) 54 16 22 22 22 21
Red (600-700 nm) 66 106 227 227 230 228
Far-Red (700-800 nm) 4 15 1 2 7 30
YPF:PAR 0.87 0.92 0.90 0.89 0.84 0.79
Red:Far-Red 10 4 226 78 20 5
PSS 0.84 0.85 0.88 0.87 0.84 0.76
1All lighting sources were 25 cm away from root-shoot barrier.
2All lighting sources were measured over a 0.3m2 growing area.
3HPS lamp emission attenuated with Plexiglas™ barrier and screening.

Conclusion
Tests to date confirm that LED lighting technologies are plausible alternatives to conventional plant lighting
sources. The combination of far-red radiation and higher light intensity boosted by near PAR wavelengths appeared
to promote increased growth in the 690 nm LED treatment. The yield increase observed under the 690 nm LED
array, therefore, was largely a consequence of more efficient light interception during early growth as opposed to
increased quantum yield or increased photosynthesis per se. These findings suggest that improvement of crop
productivity may be achieved by optimizing canopy radiation capture through more efficient light delivery and
interception through plant spacing and developmental physiology.

FUTURE PLANS
We plan to continue evaluations of salad crops responses and photosynthetic conversion efficiencies under the
various lamp sources. Following completion of work with spinach, lettuce, and radish, we plant to also evaluate
other species of salad crops such as carrot and onion. Future research needs to consider development of a reliable
low maintenance nutrient delivery system which allows rapid and straightforward planting and harvesting.

INDEX TERMS
Light-emitting diodes, LEDs, red light, blue light, ALS, CELSS, plant, photosynthesis, salad crop, plant lighting,
photomorphogeneis, express rack, mid-deck locker, electric lamps
 HYBRID SOLAR AND ARTIFICIAL LIGHTING (HYSAL) FOR
SPACE LIFE SUPPORT

J.L. Cuello1, Y. Yang1, E. Ono, K. Jordan1, D. Larson1, J. O’Leary2, H. Watanabe3 and

T. Nakamura4
Department of Agricultural and Biosystems Engineering, The University of Arizona,1
Department of Plant Sciences, The University of Arizona,2 Mitsubishi Chemical Corporation,3
Physical Sciences Inc. 4

INTRODUCTION
Since plants are central to a biologically-based or bioregenerative life support system (BLSS), and since controlled-
environment plant production demands significant electrical-energy inputs, reducing the electrical-power load
required for plant production assumes immediate primacy in the development of a dependable and ultimately
feasible BLSS. A Solar Irradiance Collection, Transmission and Distribution System (SICTDS) for harnessing
available solar irradiance was combined with selected electric lamps to design a reliable and more energy-efficient
hybrid BLSS plant lighting system.

CURRENT STATUS OF RESEARCH

Methods
The mirror-based optical waveguide SICTDS units that were used (Figure 1) were positioned on the roof top of the
Subterranean Plant Growth Facility (Figure 1) at The University of Arizona in Tucson, AZ (32° 16’ 49’’ N). Figure
2 shows how the fiberoptic cables from either SICTDS penetrated through the ceiling of the SPGF, and entered
through the top of the controlled-environment growth chamber inside the SPGF. Figure 3 shows how the ends of the
cables’ individual optical fibers were distributed in a rectangular array through a frame over the plant growing area
inside the BPC. The frame was at a distance of 11.4 cm above the plant growing area, which measured 76.2 cm x
48.3 cm. The BPC had a total volume of 1 m3, had its interior walls covered with aluminum foil, had hydroponic
trays connected to a nutrient-solution reservoir, and was provided with an air ventilation system. Figures 3 and 4
show the configuration for the hybrid lighting design with parallel light-emitting cables or fibers (Figure 4)
transmitting the irradiance from xenon-metal halide lamps, while Figures 5 and 6 show the configuration with light-
emitting diodes (LEDs). The former configuration is named HYSLEF, while the latter is named HYSLED.

Results
The resultant spectral distributions for the HYSLEF and HYSLED are shown in Figures 7 and 8, respectively. Each
SICTDS unit had an efficiency for transmitting photosynthetic photon flux (PPF) of 40.5%. The overall PPF
transmission efficiency for the plant growing system, however, with the SICTDS as the sole light source was 28.4%.
The HYSLEF configuration yielded a low overall PPF transmission efficiency of only 0.2%, while HYSLED
yielded 21.4%.

FUTURE PLANS
The thermal loading and effective volume of the HYSLED configuration are currently being analyzed and compared
with those for a reference chamber equipped with high-pressure sodium (HPS) lamp. Growth studies using lettuce
are also being conducted. The effect on the overall efficiency of replacing the current glass optical cables of the
SICTDS with new liquid-based optical cables will also be investigated.
 FIGURE 1 FIGURE 2

FIGURE 3 FIGURE 4

FIGURE 5 FIGURE 6
250.00
Collectors + LED's inside HYSAL Chamber
400
RELATIVE INTENSITY

350 200.00

300 Average

250 150.00
Intensity

200
150 100.00

100
50 50.00

0
330 430 530 630 730 830 930 0.00
300 400 500 600 700 800 900
Wave Length
WAVELENGTH (nm)

FIGURE 7 FIGURE 8
 PLANT GROWTH CHAMBER EXPERIMENTS FOR THE CROP MODELING
OBJECTIVES OF THE NJ-NSCORT
James Cavazzoni, David H. Fleisher, Konomi Kumasaka, Harry Janes, Tom Gianfagna, Logan Logendra
Department of Plant Science, Rutgers, the State University of New Jersey
New Brunswick, New Jersey

INTRODUCTION
Crop production in Advanced Life Support Systems will most likely take place in controlled environment,
hydroponic growth chambers under elevated CO2 concentrations. Multiple crops (e.g. wheat, soybean, white potato,
etc.) will be grown to fulfill life support system demands including food production, air revitalization and water
recovery. Crop models are useful for production scheduling of multiple crops, optimization of growth and
development processes, design of environment control systems, and decision support. To further the crop modeling
efforts at the NJ-NSCORT (New Jersey-NASA Specialized Center of Research and Training), hydroponic
experiments for soybean, white potato, and tomato are being conducted in plant growth chambers at the New Jersey
Agriculture Experiment Station Horticulture Greenhouse Facility at Rutgers University, New Brunswick, New
Jersey. The objective is to provide specific data for model development, modification, calibration and validation, by
locating modeling and associated experimentation within the same research facility.

CURRENT STATUS OF RESEARCH

White-Potato
For white potato (cv. Norland), two 56 day and one 105 day experiment were completed for nominal environmental
conditions (irradiance = 407 µmol m-2 s-1, light cycle–dark cycle temperature regime of 20-16 ºC, [CO2] = 1020 µL
L-1, photoperiod = 12 h, and 70% relative humidity). The time series data provided by these experiments was used to
modify an existing white potato model, originally developed for field conditions. The data was first used to calibrate
crop growth and development parameters, and then to determine the modifications to the existing source code that
were needed in order to increase model accuracy (Fleisher, D.H., J. Cavazzoni, G.A. Giacomelli, and K.C. Ting.
2000. Adaptation of SUBSTOR for Hydroponic Controlled Environment Production of White Potato. ASAE Paper
No. 004089. ASAE, St. Joseph, MI, USA).

Soybean
Coupling non-destructive sensing of plant variables to crop models is potentially useful for diagnosing problems
with the plant growth system, or for feedback to the model for evaluation of plant scheduling and potential yield.
Work has been completed linking non-destructive measurements of soybean top projected canopy area and canopy
height to model predictions. Model simulations of soybean top projected canopy area and canopy height were
evaluated against data for hydroponic soybean grown under two separate light cycle-dark cycle temperature regimes
(23-19 °C and 26-22 °C). The results suggest that top projected canopy area and canopy height are potentially useful
variables for closed-loop plant production in controlled environments during the first few weeks of growth, before
canopy closure (Cavazzoni, J. and P. Ling. 1999. Coupling Machine Vision and Crop Models for Closed-Loop Plant
Production in Advanced Life Support Systems. Life Support & Biosphere Science, 6:279-285.)

Two crop life-cycle experiments have been completed for soybean (cv. Hoyt) under near-ambient (425 µL L-1) and
elevated (1100 µL L-1) [CO2], with two irradiance treatments (450 and 800 µmol m-2 s-1) per experiment. In order to
complement the destructive plant growth information obtained from these reach-in chamber experiments, four
controlled environment plant growth chambers have been constructed to monitor canopy net photosynthesis, dark
cycle respiration and transpiration of various advanced life support food crops. The plant chambers [0.55 m2; 0.43
m3] are housed within a walk-in growth chamber [9.3 m2; 28 m3], which provides the basic environment control for
plant growth. Gas-exchange experiments are being conducted to determine the response of soybean (cv. Hoyt)
canopies to the short- and long-term effects of temperature, CO2 concentration, and irradiance. The information
provided by both the destructive harvests, and the non-destructive gas-exchange rates will be used to evaluate the
leaf-photosynthesis components and source-sink relationships used in soybean canopy models.

Tomato
Tomato experiments are being conducted in reach-in growth chambers with the objectives of evaluating cultivars,
environment conditions and cultural tasks so as to provide recommendations for tomato production in the
Bioregenerative Planetary Life Support Systems Test Complex at the NASA Johnson Space Center, and to obtain
plant growth and development information to be applied toward mathematical models. The first experiment will
evaluate the productivity of a processing tomato (determinate growth habit, cv. BOS 3155) that may serve for both
processing and salads, and of an indeterminate “cluster” variety (Husky Red), using different light sources to alter
the red:far red ratio (a cool white fluorescent (CWF)/incandescent (INC) lamp combination versus CWF lamps
alone). It is expected that plants would develop normally under CWF lamps, but with shorter internodes than under
the INC/CWF combination, as observed for soybeans (J. Cavazzoni, unpublished data). Shorter plants with
comparable yields would be beneficial for advanced life support applications.

FUTURE PLANS
The growth chamber experiments for white potato and soybean have been completed. The tomato growth chamber
experiments will be continued, and may be extended to canopy gas-exchange experiments once those for soybean
are completed.

INDEX TERMS
Tomato, White potato, Soybean, Machine vision, Crop modeling, Controlled environment, Canopy Photosynthesis,
Hydroponic
 MODELING OF ASSIMILATION AND TRANSPIRATION IN SOYBEAN PLANTS
USING ARTIFICIAL NEURAL NETWORKS*
Frank Zee
Jet Propulsion Laboratory, California Institute of Technology, 4800 Oak Grove Dr., Pasadena, CA 91109-8099

INTRODUCTION
As part of the objectives for NASA’s Advanced Life Support program, future bioregenerative life support systems
providing a significant degree of self-sufficiency to crews for productive research and exploration of space will rely
on plants to perform several functions. Through the process of photosynthesis or assimilation, plants remove carbon
dioxide from the atmosphere and produce oxygen while incorporating carbon into biomass (food). Fresh water is
released via the process of transpiration. Understanding and optimally controlling the dynamic functions associated
with assimilation, transpiration, biomass accumulation and allocation, as well as the demands for resources
(resources recovered from wastes) is essential to achieving safe and efficient support of crews during long-term
space exploration or habitation.

In pursuit of these objectives, we are developing an artificial neural network control system to manage, control, and
optimize plant-based life support functions. This will allow efficient growth of crop plants to provide the maximum
amount of life essentials of air, water, and food to a human crew using the minimal amount of resources. Before
developing the control system, neural network models characterizing plant growth functions which will be able to
better interpolate between various environmental conditions and parameters and be able to simulate short-term (less
than a day) and long-term (plant life cycle) responses and performance of various plants are currently being
implemented. These models will provide a system identification and an understanding of plant behavior to develop
the neural network control system.

RESEARCH
Assimilation and transpiration are complex, nonlinear, dynamic, and multivariable plant processes where not all
relationships between various environmental conditions and input sensor parameters are well defined. Artificial
neural networks with their ability to learn and approximate arbitrary nonlinear input-output relationships from a
collection of examples are very suitable for characterizing these plant-based life support processes. A 2-layer
feedforward neural network architecture has been developed to model transpiration and assimilation for soybean
crops under various environmental conditions. The inputs to the neural network model are the crop type, stage of
growth, and the environmental conditions: carbon dioxide level, light intensity, air temperature. The model predicts
within a limited range the amount of water (transpiration rate) and net photosynthesis (assimilation rate) produced
under these conditions.

The neural network was trained by a modified error-backpropagation learning algorithm from experimental crop
data. In collaboration with Rutgers University (NJ-NSCORT), controlled environment soybean crop experiments
were conducted in their specially designed and constructed plant growth chamber, capable of monitoring canopy net
photosynthetic rates, night respiration, and water vapor flux. Short term tests, lasting 48 hours, consisting of a
factorial combination of two atmospheric carbon dioxide concentration levels (400 and 1000 ppm), three irradiances
(450 and 153 µmol/m2/s and no light), and three air temperatures (22, 26, and 30 °C) were conducted to measure the
crop responses at different environmental conditions. Although the neural network soybean model, trained on this
data, can interpolate between these environmental conditions and predict the assimilation and transpiration rates, it is
still limited to within these ranges. However, as more crop experiments are conducted, the neural network model can
easily be expanded and refined by further training. This soybean model demonstrates how neural networks can
model the complex plant processes of assimilation and transpiration.

INDEX TERMS
artificial neural networks, bioregenerative life support, modeling, control, soybean, assimilation, transpiration, food,
biomass, plants

*Sponsored by NASA, Office of Life and Microgravity Sciences and Applications

 MODELS FOR SCHEDULING AND CONTROL FOR ADVANCED LIFE SUPPORT
SYSTEMS

D. Fleisher1, K.C. Ting2

1
Bioresource Engineering, Rutgers University, New Jersey 08901-8500
2
Department of Food, Biological and Agricultural Engineering, Ohio State University, Ohio 43210-1057

INTRODUCTION
Methods to predict effects of and compensate for setpoint disturbances in biomass production chambers for ALS
systems need to be developed. Dynamic models that can predict both magnitude and direction of changes in crop
growth and development due to off-nominal conditions will be useful for these studies. For this project, an
explanatory field crop model for white potato was modified for controlled environment hydroponic production
under elevated CO2. This modified model was then combined with previously modified models for wheat and
soybean and used to simulate growth and development data under nominal and off-nominal environmental setpoints.
A multivariable polynomial regression (MPR) tool was used to fit 2nd order equations to the simulated time series
data. These MPR models are then incorporated into a software program that can predict effects of a given
perturbation on crop production and scheduling, and compensate for that perturbation using model-based control
logic.

CURRENT STATUS OF RESEARCH

Methods
There are four main steps for this project. The first step involved production of time series data on white potato
growth and development. A series of four growth chamber experiments were completed for this task with white
potato cv. Norland. The experiments used ebb and flood hydroponics under elevated CO2 (1020 ppmv) at a 12 hour
photoperiod, 407 µmol m-2 s-1 PPF (photosynthetic photon flux), 20 / 16 °C day / night temperature, and 70%
relative humidity. Data collected from the experiments included time-series dry weight measurements of leaf,
stem, root/stolon, and tuber organs at 28, 42, 56, and 105 days after transplanting, measurements of canopy light
attenuation, and developmental observations.

The next step involved modification of the existing white potato field model for hydroponic growth conditions in
controlled environments under elevated CO2. These modifications were implemented to the white potato field
model based on the in-house data obtained above and literature. Genetic coefficients and plant parameters were
calibrated first. Next, model source code was changed to include calculation of canopy absorbed light for prediction
of photosynthesis and a canopy CO2 response curve parameterized as a function of irradiance and photoperiod.
Source code changes were also incorporated which affected simulated potato developmental rates including
senescence and delay in the prediction of tuber initiation. A carbon mass balance was included as a check on the
model predictions. Modified model results were then verified using experimental data and gas exchange data from
Kennedy Space Center.

The modified white potato model was used to simulate total biomass and edible organ growth over time for nominal
and off-nominal environmental conditions. Similar simulations were conducted with models for wheat and soybean.
MPR (multivariable polynomial regression) models were fit to the time series simulations for each crop, where
relative growth rate of either total or edible biomass was the dependent variable and average cumulative daily air
temperature, light intensity, and carbon dioxide used as independent variables. To ensure appropriate fits, statistical
tests for significance (t-statistic) and regression fit (mean square error) were conducted.

Microsoft Visual Basic programming language was used to begin development of a software program in which
environmental perturbations in temperature, light intensity, and carbon dioxide can be simulated on wheat, soybean,
and white potato growth and development. The software tool uses the MPR models to predict effects of user-input
setpoint deviations. Methods for incorporating a model-based control logic using the MPR models are also being
investigated including feedback, feedforward, and optimization techniques.
Results
Time-series data from the growth chamber experiments was used to justify model modifications made to the white
potato field model. Model modifications discussed above were evaluated with this in-house set of data (figure 1)
and with data produced at Kennedy Space Center’s Biomass Production Chamber. Based on this evaluation, the
model has been verified for 400 to 900 µmol m-2 s-1 PPF, 16° to 18°C average air temperature, and 12 hour
photoperiod with elevated CO2 concentration.

Six 2nd order MPR equations were fit to the time series data simulated by the modified potato model and wheat and
soybean models. Thus, each complex crop model was reduced to six simple equations that predict development and
vegetative and reproductive growth as a function of cumulative air temperature, light intensity, and carbon dioxide
inputs. An example of a generic MPR model is given below:

−1
Ri = a1 + a2 ⋅ Wi −1 + a3 ⋅ cPPFi ⋅ cCO2i − a4 ⋅ cTi ⋅ Wi −1 + ...
2 2

where:
Ri - relative growth rate at time i
A1,Ax… - regression coefficient
Wi-1 - growth at time i-1
cPPFi, cCO2i, cTi - cumulative light intensity, carbon dioxide, or temperature at time i

A Visual Basic program has been developed to simulate multiple crop production under nominal and off-nominal
environmental inputs for wheat, soybean, and white potato crops. The software uses the MPR models for each crop
and allows the user to input environmental perturbations during at any time during the growth cycle. Graphing and
simple spreadsheet functions allow the researcher to investigate effect of disturbances on daily growth and final
yield values. An initial model-based controller was also developed and incorporated in the software using the MPR
models. Figure 2 shows the environmental input window and some simulated results.

9
8
LAI [m2 leaf m-2 ground]

7 c
6
b
5
4
3
2
1
a
0
0 20 40 60 80 100 120 140
DAT

Figure 1: Modified-SUBSTOR predictions for in-house data shown

for leaf area index (LAI) verse days after transplanting (DAT).
Figure 2: Window from software program. Growth curves for total
Curves: a – parameter calibration; b - absorbed light calculation + a;
biomass production of wheat are shown. Top curve is growth under
c - carbon balancing, partitioning, CO2 response, and senescence + b.
nominal conditions. Bottom curve shows growth with a 30% 20 day
Observed values (• ) are averaged results of three replicated
deviation in light intensity.
experiments except for DAT 105.

CONCLUSION
A method of compensating for and predicting effects of environmental disturbances on multiple ALS crop
production is being developed. The method is based on detailed, explanatory field crop models modified for
hydroponic controlled environments under elevated CO2 and sets of MPR models used to increase their portability.
A software tool incorporates the MPR models with a model-based control logic to facilitate analysis.

FUTURE PLANS
A finalized model-based controller using the MPR models will be incorporated into the Visual Basic program. The
feasibility of using such control to compensate for environmental perturbations in ALS biomass production
chambers will be investigated.

INDEX TERMS
Advanced life support, crop modeling, control, decision support, white potato, Visual Basic, regression.
 SELF REPORTING SPACE CROPS: EDIBLE BIOSENSORS

Mentewab Ayalew, Vinitha Cardoza, Brenda Kivela, C. Neal Stewart, Jr.

Department of Biology, UNC Greensboro, NC 27402-6174

INTRODUCTION
This newly funded project has, as it’s main objective, to develop an integrated system to
monitor plant health in real time and space employing a bright transgenic reporter system
based upon green fluorescent protein (GFP) for prolonged space flight applications.

Four lines of canola (Brassica napus) each containing one of four GFP constructs will be
produced and characterized for spatial and temporal GFP expression in response to
potential abiotic stressing agents.

The research will use the real-time in vivo power of GFP to produce fluorescent canaries
in a coalmine. The only differences are that the canaries will be edible green-fluorescing
plants, and the coalmine is a spacecraft. Just as coalminers of old used a living, toxic-
sensitive animal, a canary, to tell him when toxic gases were present in the coalmine,
self-reporting green fluorescent plants will alert the astronaut of tomorrow that space
crops are losing health; that there is something in the environment that is negatively
affecting the plant, and ultimately the astronaut’s health. One other difference: the
coalminer did not eat his canary.

PROPOSED METHODS

We will produce canola (Brassica napus) plants that have four versions of the Aequoria
victoria GFP gene: two stable (mgfp5-er, egfp) and two unstable versions (mgfp4,
d2egfp) under the constitutive expression of the 35S promoter. The constitutive
expresssion of the GFP genes in plants under favorable conditions for plant growth will
cause green fluorescence: lights on. If conditions become unfavorable the lights will go
out, signifying trouble; that remediation is needed.

High expressing homozygous progeny of single lines of GFP and non-transgenic plants
will be planted in flats (2 inch) pots, and grown in growth chambers or greenhouse with
16 hour photoperiods and 22/16 C thermoperiods in a standard potting mix. The
following treatments will be implemented:
1. Complete fertilizer
2. –N
3. –NK
4. –micronutrients
5. Suboptimal water (2d)
6. Cold spell (8h, 10C)
7. Hot spell (8h, 30C)

Fluroscence data and western blot analyses will be performed on a 2-4 hour time course
intitially and then a daily time course over the following week. The power of GFP allows
us to physiologically monitor the molecular basis of plant health in real time. We will
also take other plant health and fitness data such as biomass and seed yield for correlation
analyses.

The purpose of these experiments at this pilot stage is not to exhaustively test all factors
that might affect plant health during space flight, but to select certain factors to
demonstrate this approach is feasible.

INDEX TERMS

Abiotic stress, Agriculture, Biotechnology, Brassica napus, Canola, Crops, Green

fluorescent protein (GFP), Remote sensing.
 OPTIMIZATION OF ROOT ZONE SUBSTRATES FOR REDUCED GRAVITY
EXPERIMENTS

Gail E. Bingham1, Dani Or1, Scott B. Jones1,. Robert C. Morrow2

1
Dept. of Plants, Soils and Biometeorolgy, Utah State University, Logan, UT 84341
2
ORBITEC, Inc. 1212 Fourier Drive, Madison, WI, 53717

INTRODUCTION
This flight experiment measures root zone gas exchange through important plant growth substrates at varying water
content levels in microgravity. This information is critical for proper water management and the prevention of root
zone hypoxia during plant growth experiments and ALS biomass production. Microgravity data suggest that
mechanisms such as enhanced hysterisis in water retention alters the gas diffusion process, changing the optimum
control setpoint. Measuring gas diffusion in microgravity will determine the maximum water content at which
plants can be grown without root zone oxygen stress, and will allow the development of models needed to determine
improved substrate properties and management protocols. These data will directly support substrate selection and
management for NASA’s initial ISS plant experiments PESTO and MPNE-02, guide BIO-Plex protocols and guide
NASA’s new generation flight growth chambers (PRU, etc.) development.

Plants require adequate water and oxygen supply (inversely related processes) and metabolic gas removal from their
root tissues. For terrestrial plants in coarse porous substrates, pores are readily drained after watering providing
adequate air-filled pore space for gas exchange with the root tissues. Under reduced gravity conditions, capillary
forces are dominant, creating water distribution profiles and non-uniformities not observed on earth. A few space
borne experiments have investigated water movement and control in µg, but none have investigated the region
where oxygen diffusion becomes the limiting factor for plant growth. The data are applicable to aerobic bioreactor
design.

Our experiment measures substrate gas exchange and develops critical space experiments protocols and theoretical
substrates models for reduced gravity responses. Validated models are required to allow substrate property
adjustment and hardware design. This experiment is suggested as a Mid-deck Locker style payload, and could be
hosted in the FHAME hardware being developed by ORBITEC, Inc. for an early 2001 flight in support of BPS
development.

CURRENT STATUS OF RESEARCH

This flight experiment was selected for definition studies in FY 00 and is in its first few months of effort. As
proposed, the experiment will utilize experiment specific measurement chamber hardware, controlled by the
ORBITEC, Inc. FHAME controller. The flight feasibility studies are being supported by prototype diffusion cell
hardware and laboratory studies.

We suggest that fundamental soil physical models developed on earth should apply in space to the extent that the
hydrostatic and hydrodynamic processes are preserved. Based on data collected in plant experiments on the Mir station,
we have demonstrated the ability to optimize the empirical parameters of a ground based water – substrate model, to
map physical characteristics of an optimal porous media. As illustrated in Figure 1 the optimization is a tradeoff
between the reduction in gas diffusion and concentration as θ increases with the reduction in hydraulic conductivity
and potential (more negative) as θ is reduced. This relationship is largely a function of transpiration/respiration rate
and the matric suction control system operation limits.
The maximum substrate depth and respiration rate assumed for modeling are two important factors constraining the
increase in optimal water content. The rapid reduction in gas concentration within a narrow range of water content
emphasizes the critical nature of controlling the root zone environment to avoid hypoxic conditions. The difference
in the water content distribution modeled in the Svet root module at 1 g and µg appear in Figure 2. Note that with
the large (1–2mm) particle sizes used to minimize hypoxia in space, vertical water content gradients approaching
20% per cm are observed at 1g. At µg, however, the gradient has a much different pattern. Using both flux and
water content information, we have modeled the 2 dimensional patterns of water content that developed under a
rapidly transpiring wheat crop in µg.
Our experiment is designed to measure the movement of oxygen through wet substrates in microgravity, and to
provide us with the data necessary to model these results. This is a fundamentally simple experiment, but one that
requires careful design and execution, backed up with careful scientific work. Figure 3 is a schematic illustrating key
components of our experiment hardware. The test article consists of a linear section of porous substrate, rectangular in
100 1

Matric
1 Hydraulic
Conductivity 2
Suction
0.1
10 1optimal
h *
Gas m
Content 0.01
Gas
1 Diffusion
0.001
Objective h o*
Function

0.1 0.0001
0 0.2 0.4 0.6 0.8 1
Water content ( 1 )

Figure 1: Root zone 1g optimum water content for a zeolite substrate as predicted by the substrate
optimization model (25). The model trades-off hydraulic conductivity and oxygen diffusion against
transpiration and root respiration rates.

Figure 2: The difference between the vertical root zone water content distributions modeled in µg and at 1g in
the 10 cm tall Svet root modules during the Greenhouse 2a (1995) experiment and its ground control. (Note:
0.60 g H2O / g Sub = 100 % RWC in the 1–2 mm zeolite substrate).
cross section, bounded on the upper and lower sides by porous plate water reservoirs. Negative pressure in the water
reservoirs control the water content of the porous substrate. Air reservoirs are found on each end of the substrate. The
substrate is constrained by screens at each end to prevent spilling into the air reservoir. Sensors monitor the oxygen
concentrations in each air reservoir. Using cabin level oxygen concentration as the source gas allows the experiment to
be conducted in the cabin, without pressurized or other hazardous gas supplies.

Figure 3: Experiment Concept: Measuring oxygen diffusion through wet substrates in µg

A typical measurement will begin with cabin concentration of O2 on one side of the diffusion cell, essentially O2
free air on the other. The N2 and O2 counter diffuse through the test cell, and tend to equilibrate the partial pressures
in the reservoirs at each end. The final, equalized concentration in both end reservoirs will be just less than half of
the concentration value of the high end at the start of the experiment. As the partial pressures differences decrease,
the diffusion slows down, and the measurement is terminated before complete equilibrium is achieved.
FUTURE PLANS
This program will be selected for full flight development in August, 2001, and will fly in 2003.
 USE OF INDUCED-FLUORESCENCE IMAGING AND GREEN FLUORESCENT
PROTEINS TO MONITOR THE HEALTH OF TERRESTRIAL PLANTS UNDER
SIMULATED MARTIAN ENVIRONMENTS

A0-99-HEDS-01-032

A. C. Schuerger1 (PI), R. J. Ferl2, K. A. Corey3, T. Murdoch4, and W. Wells4

1
Dynamac Corporation, Mail Code DYN-3, Kennedy space Center, FL 32899; 2University of Florida, Gainesville,
FL; 3University of Massachusetts, Amherst, MA; 4Bionetics Corporation, Kennedy space Center, FL

INTRODUCTION

Advanced Life Support (ALS) systems using higher plants have been proposed for Human missions to
Mars. The ALS systems could dramatically reduce launch costs from Earth by regenerating oxygen,
water, and food. However, no information is currently available on how terrestrial plants will grow
under simulated Martian environmental conditions. The proposed research will address the following
three questions. Is Martian regolith capable of supporting robust plant growth? Can low-pressure
environments be designed for optimizing plant growth? Can a miniaturized remote sensing system be
developed to monitor plant health within low-pressure environments in which plants are stressed by soil
factors likely to be found in Martian regolith? To answer these questions a series of experiments will be
conducted at Earth-normal (101 kPa) and low (10-40 kPa) pressures in which genetically engineered
plants of Arabidopsis thaliana will be grown in an analog Mars soil supplemented with different
stressing agents. Arabidopsis thaliana plants will be genetically modified with green fluorescent proteins
(GFP) linked to plant reporter genes in order to monitor specific physiological pathways in plants
affected by specific soil stressing agents.

CURRENT STATUS OF RESEARCH

A) Remote Sensing Imager: A remote sensing imaging (RSI) system will be developed to collect plant
stress data throughout these experiments. The RSI system will use a single charged-couple device (CCD)
camera fitted with five unique narrow-bandpass filters in a rotating filter-wheel. The CCD camera will
capture images at a resolution of 768 x 494 pixels. The RSI camera will be fitted with the following
filters: 440 (blue), 525 (green), 685 (red), 735 (far-red), or 750 (far-red) nm (± 10 nm). Images from the
five filters will provide data for a number of image-analysis approaches. First, images from the 440, 525,
and 685 nm filters will be processed to yield standard RGB color images of the plants. Second, images
from the 685 and 750 nm filters will be used to calculate standard spectral reflectance ratios (e.g., NDVI
values, R750/R685 ratios, and logR750/logR685 ratios) in order to estimate general plant stress in visible
wavelengths. [NDVI = normalized difference vegetation index.] Third, fluorescent images from the 685
and 735 nm filters will be processed to yield red:far-red ratio images for detecting general plant stress
under fluorescent illumination. Fourth, green fluorescent images will be collected with the 525 nm filter
under fluorescent illumination to measure green fluorescent protein (GFP) expression in transgenic
plants. The RSI system will be used to capture images from non-transgenic and transgenic lines of A.
thaliana grown under various stresses including low pressure and the presence of soil peroxides or heavy
metals.

B) Use of Green Fluorescent Proteins (GFP) as Marker Genes: The expression of GFP is controlled by
stress-induced reporter genes linked to specific GFP-constructs in transgenic lines of A. thaliana. By
carefully selecting the stress promoters, information will be obtained on the function of key physiological
pathways in plants that are affected by specific soil stressing agents. Furthermore, the use of these GFP-
tagged stress promoters in plants should allow the transgenic lines of A. thaliana to serve as bioassays for
specific classes of compounds in regolith during the Mars 2005 HEDS lander mission. The stress
reporter genes will be: Cat (catalase), Adh (general plant stress gene), and Hsp/Gst (heat-shock) which
are specific for the presence of soil peroxides, oxidants or anoxia, and heavy metals, respectively.

C) Effects of Low Pressure on Plant Growth: The surface ambient pressure on Mars is 6-8 mb
(approximately 0.7 kPa); a pressure that is not conducive to the growth of terrestrial plants. However, if
a low–pressure environment (e.g., 20-40 kPa) were identified in which normal plant growth can be
achieved; then the structural, volume, and gas-consumable requirements of a Mars lander plant growth
experiment might be reduced. Several plant functions are affected by growth under low pressures
including, photosynthesis, evapotranspiration, respiration and water use. But very little research has
been conducted on plant growth and development in pressures below 70 kPa (70% of the ambient
pressure on Earth). This research will study the limits of growth for the terrestrial plant, Arabidopsis
thaliana, under low atmospheric pressure (10-40 kPa) and altered gas compositions. Two main questions
will be addressed in this portion of the work: a) what is the minimum total ambient pressure and b) what
are the proper partial pressures of CO2 and O2 required to maintain normal plant growth.

RESULTS

The RSI system is currently in the initial design and testing phases of operation. The primary objective
for the first 6 months of this project has been to determine the sensitivity of each spectral band in both
the spectral reflectance and fluorescence mode. Results indicate that the CCD chip used in the RSI
system is sensitive enough in the spectral ranges used for these tests to accommodate all measurements
listed above. Plant biology experiments are currently ongoing, and results will be presented at the
Bioastronautics conference.

As of this writing, both the plant transgenic and low-pressure experiments were scheduled for the fall of
2000. Results of these experiments will be discussed at the Bioastronautics conference in January, 2001.

FUTURE PLANS

The research discussed above will be conducted in separate but parallel efforts. But the objective for
Year-2 (June, 2001-June, 2002) activities will be to develop a series of integrated experiments conducted
at low pressure within a 1 cubic meter Mars Simulation Chamber (MSC) located at the Kennedy Space
Center, FL. Experiments will be conducted in the MSC system in which GFP-transgenic plants will be
grown at pressures lower than terrestrial ambient conditions in Mars analog soils treated with peroxides
or heavy metals. The integrated experiments will be designed to confirm the effectiveness of the GFP
and fluorescence imaging system to measure plant stress induced by soil factors on plants grown at low
pressures. The primary soil factors selected for this phase of the research (soil peroxides and heavy
metals) are those anticipated (based on the literature) as posing significant risks to inducing plant stress
during a plant biology experiment on future Mars lander missions. Transgenic lines of A. thaliana will
be grown at the total atmospheric pressure (expected to be between 20-40 kPa) and partial pressures of
O2 and CO2 determined by the research described above. The expression of GFP genes (i.e., as they are
expressed while linked to reporter genes) are hypothesized to be capable of detecting soil stressing agents
at lower concentrations than measuring whole-plant physiological changes induced by the same stressing
agents and using standard methods of remote sensing (e.g., NDVI, R750/R685, logR750/logR685, or
red:far-red ratios).

INDEX TERMS
Remote sensing, leaf fluorescence, spectral reflectance, green fluorescent proteins, GFP, anoxia, hypoxia,
plant physiology, Arabidopsis thaliana, Mars, simulated soils, Mars regolith, low-pressure environments