Dynamics of microorganism populations in recirculating nutrient solutions.

This overview covers the basic microbial ecology of recirculating hydroponic solutions. Examples from NASA and Soviet CELSS tests and the commercial hydroponic industry will be used. The sources of microorganisms in nutrient solutions include air, water, seeds, plant containers and plumbing, biological vectors, and personnel. Microbial fates include growth, death, and emigration. Important microbial habitats within nutrient delivery systems are root surfaces, hardware surfaces (biofilms), and solution suspension. Numbers of bacteria on root surfaces usually exceed those from the other habitats by several orders of magnitude. Gram negative bacteria dominate the microflora with fungal counts usually much lower. Trends typically show a decrease in counts with increasing time unless stressed plants increase root exudates. Important microbial activities include carbon mineralization and nitrogen transformations. Important detrimental interactions include competition with plants, and human and plant pathogenesis.     

Biodegradation pathway of an anionic surfactant (Igepon TC-42) during recycling waste water through plant hydroponics for advanced life support during long-duration space missions.

The degradation of an anionic surfactant (Igepon TC-42) was investigated as part of an integrated study of direct recycling of human hygiene water through hydroponic plant growth systems. Several chemical approaches were developed to characterize the degradation of Igepon and to measure the accumulation of intermediates such as fatty acids and methyl taurine. Igepon was rapidly degraded as indicated by the reduction of methylene blue active substances (MBAS) and component fatty acids. The Igepon degradation rate continued to increase over a period of several weeks following repeated daily exposure to 18 micrograms/l Igepon. The accumulation of free fatty acids and methyl taurine was also observed during decomposition of Igepon. The concentration of methyl taurine was below detection limit (0.2 nmol/ml) during the slow phase of Igepon degradation, and increased to 1-2 nmol/ml during the phase of rapid degradation. These findings support a degradation pathway involving initial hydrolysis of amide to release fatty acids and methyl taurine, and subsequent degradation of these intermediates.     

Proximate composition of CELSS crops grown in NASA's Biomass Production Chamber.

Edible biomass from four crops of wheat (Triticum aestivum L.), four crops of lettuce (Lactuca sativa L.), four crops of potato (Solanum tuberosum L.), and three crops of soybean (Glycine max (L.) Merr.) grown in NASA's CELSS Biomass Production Chamber were analyzed for proximate composition. All plants were grown using recirculating nutrient (hydroponic) film culture with pH and electrical conductivity automatically controlled. Temperature and humidity were controlled to near optimal levels for each species and atmospheric carbon dioxide partial pressures were maintained near 100 Pa during the light cycles. Soybean seed contained the highest percentage of protein and fat, potato tubers and wheat seed contained the highest levels of carbohydrate, and lettuce leaves contained the highest level of ash. Analyses showed values close to data published for field-grown plants with several exceptions: In comparison with field-grown plants, wheat seed had higher protein levels; soybean seed had higher ash and crude fiber levels; and potato tubers and lettuce leaves had higher protein and ash levels. The higher ash and protein levels may have been a result of the continuous supply of nutrients (e.g., potassium and nitrogen) to the plants by the recirculating hydroponic culture.     

Comparison of aerobically-treated and untreated crop residue as a source of recycled nutrients in a recirculating hydroponic system.

This study compared the growth of potato plants on nutrients recycled from inedible potato biomass. Plants were grown for 105 days in recirculating, thin-film hydroponic systems containing four separate nutrient solution treatments: (1) modified half-strength Hoagland's (control), 2) liquid effluent from a bioreactor containing inedible potato biomass, 3) filtered (0.2 micrometer) effluent, and 4) the water soluble fraction of inedible potato biomass (leachate). Approximately 50% of the total nutrient requirement in treatments 2-4 were provided (recycled) from the potato biomass. Leachate had an inhibitory effect on leaf conductance, photosynthetic rate, and growth (50% reduction in plant height and 60% reduction in tuber yield). Plants grown on bioreactor effluent (filtered or unfiltered) were similar to the control plants. These results indicated that rapidly degraded, water soluble organic material contained in the inedible biomass, i.e., material in leachate, brought about phytotoxicity in the hydroponic culture of potato. Recalcitrant, water soluble organic material accumulated in all nutrient recycling treatments (650% increase after 105 days), but no increase in rhizosphere microbial numbers was observed.     

Use of biologically reclaimed minerals for continuous hydroponic potato production in a CELSS.

Plant-derived nutrients were successfully recycled in a Controlled Ecological Life Support System (CELSS) using biological methods. The majority of the essential nutrients were recovered by microbiologically treating the plant biomass in an aerobic bioreactor. Liquid effluent containing the nutrients was then returned to the biomass production component via a recirculating hydroponic system. Potato (Solanum tuberosum L.) cv. Norland plants were grown on those nutrients in either a batch production mode (same age plants on a nutrient solution) or a staggered production mode (4 different ages of plants on a nutrient solution). The study continued over a period of 418 days, within NASA Breadboard Project's Biomass Production Chamber at the Kennedy Space Center. During this period, four consecutive batch cycles (104-day harvests) and 13 consecutive staggered cycles (26-day harvests) were completed using reclaimed minerals and compared to plants grown with standard nutrient solutions. All nutrient solutions were continually recirculated during the entire 418 day study. In general, tuber yields with reclaimed minerals were within 10% of control solutions. Contaminants, such as sodium and recalcitrant organics tended to increase over time in solutions containing reclaimed minerals, however tuber composition was comparable to tubers grown in the control solutions.     

Ethylene production by plants in a closed environment.

Ethylene production by 20-m2 stands of wheat, soybean, lettuce and potato was monitored throughout growth and development in NASA's Controlled Ecological Life Support System (CELSS) Biomass Production Chamber. Chamber ethylene concentrations rose during periods of rapid growth for all four species, reaching 120 parts per billion (ppb) for wheat, 60 ppb for soybean, and 40 to 50 ppb for lettuce and potato. Following this, ethylene concentrations declined during seed fill and maturation (wheat and soybean), or remained relatively constant (potato). Lettuce plants were harvested during rapid growth and peak ethylene production. The highest ethylene production rates (unadjusted for chamber leakage) ranged from 0.04 to 0.06 ml m-2 day-1 during rapid growth of lettuce and wheat stands, or approximately 0.8 to 1.1 nl g-1 fresh weight h-1. Results suggest that ethylene production by plants is a normal event coupled to periods of rapid metabolic activity, and that ethylene removal or control measures should be considered for growing crops in a tightly closed CELSS.     

Controlled environments alter nutrient content of soybeans.

Information about compositional changes in plants grown in controlled environments is essential for developing a safe, nutritious diet for a Controlled Ecomological Life-Support System (CELSS). Information now is available for some CELSS candidate crops, but detailed information has been lacking for soybeans. To determine the effect of environment on macronutrient and mineral composition of soybeans, plants were grown both in the field and in a controlled environment where the hydroponic nutrient solution, photosynthetic flux (PPF), and CO2 level were manipulated to achieve rapid growth rates. Plants were harvested at seed maturity, separated into discrete parts, and oven dried prior to chemical analysis. Plant material was analyzed for proximate composition (moisture, protein, lipid, ash, and carbohydrate), total nitrogen (N), nonprotein N (NPN), nitrate, minerals, amino acid composition, and total dietary fiber. The effect of environment on composition varied by cultivar and plant part. Chamber-grown plants generally exhibited the following characteristics compared with field-grown plants: 1) increased total N and protein N for all plant parts, 2) increased nitrate in leaves and stems but not in seeds, 3) increased lipids in seeds, and 4) decreased Ca:P ratio for stems, pods, and leaves. These trends are consistent with data for other CELSS crops. Total N, protein N, and amino acid contents for 350 ppm CO2 and 1000 ppm CO2 were similar for seeds, but protein N and amino acid contents for leaves were higher at 350 ppm CO2 than at 1000 ppm CO2. Total dietary fiber content of soybean leaves was higher with 350 ppm CO2 than with 1000 ppm CO2. Such data will help in selecting of crop species, cultivars, and growing conditions to ensure safe, nutritious diets for CELSS.     

Excess nutrients in hydroponic solutions alter nutrient content of rice, wheat, and potato.

Environment has significant effects on the nutrient content of field-grown crop plants. Little is known, however, about compositional changes caused by controlled environments in which plants receive only artificial radiation and soilless, hydroponic culture. This knowledge is essential for developing a safe, nutritious diet in a Controlled Ecological Life-Support System (CELSS). Three crops that are candidates for inclusion in a CELSS (rice, wheat, and white potato) were grown both in the field and in controlled environments where the hydroponic nutrient solution, photosynthetic photon flux (PPF), and CO2 level were manipulated to achieve rapid growth rates. Plants were harvested at maturity, separated into discrete parts, and dried prior to analysis. Plant materials were analyzed for proximate composition (protein, fat, ash, and carbohydrate), total nitrogen (N), nitrate, minerals, and amino-acid composition. The effect of environment on nutrient content varied by crop and plant part. Total N and nonprotein N (NPN) contents of plant biomass generally increased under controlled-environment conditions compared to field conditions, especially for leafy plant parts and roots. Nitrate levels were increased in hydroponically-grown vegetative tissues, but nitrate was excluded from grains and tubers. Mineral content changes in plant tissue included increased phosphorus and decreased levels of certain micronutrient elements under controlled-environment conditions. These findings suggest that cultivar selection, genetic manipulation, and environmental control could be important to obtain highly nutritious biomass in a CELSS.     

Increased BLSS closure using mineralized human waste in plant cultivation on a neutral substrate

The purpose of this work was to study the full-scale potential use of human mineralized waste (feces and urine) as a source of mineral elements for plant cultivation in a biological life support system (BLSS). Plants that are potential candidates for a photosynthesizing link were grown on a neutral solution containing human mineralized waste. Spring wheat Triticum aestivum L., peas Pisum sativum L. Ambrosia cultivar and leaf lettuce Lactuca sativa L., Vitaminny variety, were used. The plants were grown hydroponically on expanded clay aggregates in a vegetation chamber in constant environmental conditions. During plant growth, a determined amount of human mineralized waste was added daily to the nutrient solution. The nutrient solution remained unchanged throughout the vegetation period. Estimated plant requirements for macro-elements were based on a total biological productivity of 0.04 kg day⁻¹ m⁻². As the plant requirements for potassium exceeded the potassium content of human waste, a water extract of wheat straw containing the required amount of potassium was added to the nutrient solution. The Knop’s solution was used in the control experiments.     

Integration of crop production with CELSS waste management.

Lettuce plants were grown utilizing water, inorganic elements, and CO2 inputs recovered from waste streams. The impact of these waste-derived inputs on the growth of lettuce was quantified and compared with results obtained when reagent grade inputs were used. Phytotoxicity was evident in both the untreated wastewater stream and the recovered CO2 stream. The toxicity of surfactants in wastewater was removed using several treatment systems. Harmful effects of gaseous products resulting from incineration of inedible biomass on crop growth were observed. No phytotoxicity was observed when inorganic elements recovered from incinerated biomass ash were used to prepare the hydroponic solution, but the balance of nutrients had to be modified to achieve near optimal growth. The results were used to evaluate closure potential of water and inorganic elemental loops for integrated plant growth and human requirements.     

Microbiological characterization of a regenerative life support system.

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

Effect of salt stress on growth and physiology in amaranth and lettuce: Implications for bioregenerative life support system

Growing plants can be used to clean waste water in bioregenerative life support system (BLSS). However, NaCl contained in the human urine always restricts plant growth and further reduces the degree of mass cycle closure of the system (i.e. salt stress). This work determined the effect of NaCl stress on physiological characteristics of plants for the life support system. Amaranth (Amaranthus tricolor L. var. Huahong) and leaf lettuce (Lactuca sativa L. var. Luoma) were cultivated at nutrient solutions with different NaCl contents (0, 1000, 5000 and 10,000 ppm, respectively) for 10 to 18 days after planted in the Controlled Ecological Life Support System Experimental Facility in China. Results showed that the two plants have different responses to the salt stress. The amaranth showed higher salt-tolerance with NaCl stress. If NaCl content in the solution is below 5000 ppm, the salt stress effect is insignificant on above-ground biomass output, leaf photosynthesis rate, Fv/Fm, photosynthesis pigment contents, activities of antioxidant enzymes, and inducing lipid peroxidation. On the other hand, the lettuce is sensitive to NaCl which significantly decreases those indices of growth and physiology. Notably, the lettuce remains high productivity of edible biomass in low NaCl stress, although its salt-tolerant limitation is lower than amaranth. Therefore, we recommended that amaranth could be cultivated under a higher NaCl stress condition (<5000 ppm) for NaCl recycle while lettuce should be under a lower NaCl stress (<1000 ppm) for water cleaning in future BLSS.     

Selection of candidate salad vegetables for controlled ecological life support system

Higher plants, as one of the essential biological components of CELSS, can supply food, oxygen and water for human crews during future long-duration space missions and Lunar/Mars habitats. In order to select suitable leaf vegetable varieties for our CELSS Experimental Facility (CEF), five varieties of lettuce (“Nenlvnaiyou”, “Dasusheng”, “Naichoutai”, “Dongfangkaixuan” and “Siji”), two of spinach (“Daye” and “Quanneng”), one of rape (“Jingyou No. 1”) and one of common sowthistle were grown and compared on the basis of edible biomass, and nutrient content. In addition, two series of experiments were conducted to study single leaf photosynthetic rates and transpiration rates at 30 days after planting, one which used various concentrations of CO₂ (500, 1000, 1500 and 2000 μmol mol⁻¹) and another which used various light intensities (100, 300, 500 and 700 μmol m⁻² s⁻¹). Results showed that lettuce cvs. “Nenlvnaiyou”, “Siji” and “Dasusheng” produced higher yields of edible biomass; common sowthisle would be a good source of β-carotene for the diet. Based on the collective findings, we selected three varieties of lettuce (“Nenlvnaiyou”, “Dasusheng” and “Siji”) and one of common sowthistle as the candidate crops for further research in our CEF. In addition, elevated CO₂ concentration increased the rates of photosynthesis and transpiration, and elevated light intensity increased the rate of photosynthesis for these varieties. These results can be useful for determining optimal conditions for controlling CO₂ and water fluxes between the crops and the overall CELSS.     

Application of ISFETs in closed-loop systems for greenhouses.

In horticulture, growing in artificial substrates such as rockwool is more and more considered to be a sound alternative to growing in soil. This development enables the opportunity to create closed-loop systems which lower the waste of raw materials and reduce pollution of the environment. Applying closed-loop systems needs precise knowledge of the composition of the recirculating nutrient solution. This paper presents basic principles of a measuring system, which can monitor continuously the concentration of nutrients in water. The system is based on ion-selective field effect transistors (ISFETs). By appropriate calibration, a high accuracy is achieved for pH and potassium measurements in the nutrient solution. An accuracy of better than 10% (mMol/l) has been achieved.     

Biological degradation and composition of inedible sweetpotato biomass.

Many challenges are presented by biological degradation in a bioregenerative Controlled Ecological Life Support System (CELSS) as envisioned by the U.S. National Aeronautics and Space Administration (NASA). In the studies conducted with biodegradative microorganism indigenous to sweetpotato fields, it was determined that a particle size of 75 microns and incubation temperature of 30 degrees C were optimal for degradation. The composition of the inedible biomass and characterization of plant nutrient solution indicated the presence of potential energy sources to drive microbial transformations of plant waste. Selected indigenous soil isolates with ligno-cellulolytic or sulfate-reducing ability were utilized in biological studies and demonstrated diversity in ability to reduce sulfate in solution and to utilize alternative carbon sources: a lignin analog--4-hydroxy, 3-methoxy cinnamic acid, cellulose, arabinose, glucose, sucrose, mannitol, galactose, ascorbic acid.     

Impaired growth of plants cultivated in a closed system: possible reasons.

Plants in experiments on "man-higher plants" closed ecosystem (CES) have been demonstrated to have inhibited growth and reduced productivity due to three basic factors: prolonged usage of a permanent nutrient solution introduction into the nutrient medium of intra-system gray water, and closure of the system. Gray water was detrimental to plants the longer the nutrient solution was used. However, higher plant growth was mostly affected by the gaseous composition of the CES atmosphere, through accumulation of volatile substances.     

An overview of Japanese CELSS research activities.

Many research activities regarding Controlled Ecological Life Support System (CELSS) have been conducted and continued all over the world since the 1960's and the concept of CELSS is now changing from Science Fiction to Scientific Reality. Development of CELSS technology is inevitable for future long duration stays of human beings in space, for lunar base construction and for manned mars flight programs. CELSS functions can be divided into two categories, Environment Control and Material Recycling. Temperature, humidity, total atmospheric pressure and partial pressure of oxygen and carbon dioxide, necessary for all living things, are to be controlled by the environment control function. This function can be performed by technologies already developed and used as the Environment Control Life Support System (ECLSS) of Space Shuttle and Space Station. As for material recycling, matured technologies have not yet been established for fully satisfying the specific metabolic requirements of each living thing including human beings. Therefore, research activities for establishing CELSS technology should be focused on material recycling technologies using biological systems such as plants and animals and physico-chemical systems, for example, a gas recycling system, a water purifying and recycling system and a waste management system. Based on these considerations, Japanese research activities have been conducted and will be continued under the tentative guideline of CELSS research activities as shown in documents /1/, /2/. The status of the over all activities are discussed in this paper.     

Subcritical and supercritical water oxidation of CELSS model wastes.

Controlled-Ecological-Life-Support-System (CELSS) model wastes were wet-oxidized at temperatures from 250 to 500 degrees C, i.e., below and above the critical point of water (374 degrees C and 218 kg/cm2 or 21.4 MPa). A solution of ammonium hydroxide and acetic acid and a slurry of human urine, feces, and wipes were used as model wastes. Almost all of the organic matter in the model wastes was oxidized in the temperature range from 400 to 500 degrees C, i.e., above the critical conditions for water. In contrast, only a small portion of the organic matter was oxidized at subcritical conditions. Although the extent of nitrogen oxidation to nitrous oxide (N2O) and/or nitrogen gas (N2) increased with reaction temperature, most of the nitrogen was retained in solution as ammonia near 400 degrees C. This important finding suggests that most of the nitrogen in the waste feed can be retained in solution as ammonia during oxidation at low supercritical temperatures and be subsequently used as a nitrogen source for plants in a CELSS while at the same time organic matter is almost completely oxidized to carbon dioxide and water. It was also found in this study the Hastelloy C-276 alloy reactor corroded during waste oxidation. The rate of corrosion was lower above than below the critical temperature for water.