Use of halophytic plants for recycling NaCl in human liquid waste in a bioregenerative life support system

The purpose of this work was to develop technology for recycling NaCl containing in human liquid waste as intrasystem matter in a bioregenerative life support system (BLSS). The circulation of Na⁺ and Cl⁻ excreted in urine is achieved by inclusion of halophytes, i.e. plants that naturally inhabit salt-rich soils and accumulate NaCl in their organs. A model of Na⁺ and Cl⁻ recycling in a BLSS was designed, based on the NaCl turnover in the human–urine–nutrient solution–halophytic plant–human cycle. The study consisted of (i) selecting a halophyte suitable for inclusion in a BLSS, and (ii) determining growth conditions supporting maximal Na⁺ and Cl⁻ accumulation in the shoots of the halophyte growing in a nutrient solution simulating mineralized urine. For the selected halophytic plant, Salicornia europaea, growth rate under optimal conditions, biomass production and quantities of Na⁺ and Cl⁻ absorbed were determined. Characteristics of a plant production conveyor consisting of S.europaea at various ages, and allowing continuity of Na⁺ and Cl⁻ turnover, were estimated. It was shown that closure of the NaCl cycle in a BLSS can be attained if the daily ration of fresh Salicornia biomass for a BLSS inhabitant is approximately 360 g.     

Testing anti-fungal activity of a soil-like substrate for growing plants in bioregenerative life support systems

The object of this research is to study a soil-like substrate (SLS) to grow plants in a Bioregenerative Life Support System (BLSS). Wheat and rice straw were used as raw materials to prepare SLS. Anti-fungal activity of SLS using test cultures of Bipolaris sorokiniana, a plant-pathogenic fungus which causes wheat root rot was studied. Experiments were conducted with SLS samples, using natural soil and sand as controls. Infecting the substrates, was performed at two levels: the first level was done with wheat seeds carrying B. sorokiniana and the second level with seeds and additional conidia of B. sorokiniana from an outside source. We measured wheat disease incidence and severity in two crop plantings. Lowest disease incidence values were obtained from the second planting, SLS: 26% and 41% at the first and the second infection levels, respectively. For soil the values were 60% and 82%, respectively, and for sand they were 67% and 74%, respectively. Wheat root rot in the second crop planting on SLS, at both infection levels was considerably less severe (9% and 13%, respectively) than on natural soil (20% and 33%) and sand (22% and 32%). SLS significantly suppressed the germination of B. sorokiniana conidia. Conidia germination was 5% in aqueous SLS suspension, and 18% in clean water. No significant differences were found regarding the impact on conidia germination between the SLS samples obtained from wheat and rice straw. The anti-fungal activity in SLS increased because of the presence of worms. SLS also contained bacteria stimulating and inhibiting B. sorokiniana growth.     

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 and hydroponic growth of potato cultivars for bioregenerative life support systems

As part of the ESA-funded MELiSSA program, Ghent University and the Université catholique de Louvain investigated the suitability, growth and development of four potato cultivars in hydroponic culture under controlled conditions with the aim to incorporate such cultivation system in an Environmental Control and Life Support System (ECLSS). Potato plants can fulfill three major functions in an ECLSS in space missions: (a) fixation of CO₂ and production of O₂, (b) production of tubers for human nutrition and (c) production of clean water after condensation of the water vapor released from the plants by transpiration. Four cultivars (Annabelle, Bintje, Desiree and Innovator) were selected and grown hydroponically in nutrient film technique (NFT) gullies in a growth chamber under controlled conditions. The plant growth parameters, tuber harvest parameters and results of tuber nutritional analysis of the four cultivars were compared. The four potato cultivars grew well and all produced tubers. The growth period lasted 127 days for all cultivars except for Desiree which needed 145 days. Annabelle (1.45 kg/m²) and Bintje (1.355 kg/m²) were the best performing of the four cultivars. They also produced two times more tubers than Desiree and Innovator. Innovator produced the biggest tubers (20.95 g/tuber) and Desiree the smallest (7.67 g/tuber). The size of Annabelle and Bintje potatoes were intermediate. Bintje plants produced the highest total biomass in term of DW. The highest non-edible biomass was produced by Desiree, which showed both the highest shoot and root DW. The manual length and width measurements were also used to predict the total tuber mass. The energy values of the tubers remained in the range of the 2010 USDA and Souci-Fachmann-Kraut food composition databases. The amount of Ca determined was slightly reduced compared to the USDA value, but close to the Souci-Fachmann-Kraut value. The concentration of Cu, Zn and P were high compared to both databases.     

Gas exchange rates of potato stands for bioregenerative life support

Plants can provide a means for removing carbon dioxide (CO₂) while generating oxygen (O₂) and clean water for life support systems in space. To study this, 20 m² stands of potato (Solanum tuberosum L.) plants were grown in a large (113 m³ vol.), atmospherically closed chamber. Photosynthetic uptake of CO₂ by the stands was detected about 10 DAP (days after planting), after which photosynthetic rates rose rapidly as stand ground cover and total light interception increased. Photosynthetic rates peaked ca. 50 DAP near 45 μmol CO₂ m⁻² s⁻¹ under 865 μmol m⁻² s⁻¹ PPF (average photosynthetic photon flux), and near 35 μmol CO₂ m⁻² s⁻¹ under 655 μmol m⁻² s⁻¹ PPF. Short term changes in PPF caused a linear response in stand photosynthetic rates up to 1100 μmol m⁻² s⁻¹ PPF, with a light compensation point of 185 μmol m⁻² s⁻¹ PPF. Comparisons of stand photosynthetic rates at different CO₂ concentrations showed a classic C₃ response, with saturation occurring near 1200 μmol mol⁻¹ CO₂ and compensation near 100 μmol mol⁻¹ CO₂. In one study, the photoperiod was changed from 12 h light/12 h dark to continuous light at 58 DAP. This caused a decrease in net photosynthetic rates within 48 h and eventual damage (scorching) of upper canopy leaves, suggesting the abrupt change stressed the plants and/or caused feedback effects on photosynthesis. Dark period (night) respiration rates increased during early growth as standing biomass increased and peaked near 9 μmol CO₂ m⁻² s⁻¹ ca. 50 DAP, after which rates declined gradually with age. Stand transpiration showed a rapid rise with canopy ground cover and peaked ca. 50 DAP near 8.9 L m⁻² d⁻¹ under 860 μmol m⁻² s⁻¹ PPF and near 6.3 L m⁻² d⁻¹ under 650 μmol m⁻² s⁻¹ PPF. Based on the best photosynthetic rates from these studies, approximately 25 m² of potato plants under continuous cultivation would be required to support the CO₂ removal and O₂ requirements for one person.     

Tolerance of chufa (Cyperus esculentus L.) plants,representing the higher plant compartment in bioregenerative life support systems,to super-optimal air temperatures

Plants intended to be included in the photosynthesizing compartment of the bioregenerative life support system (BLSS) need to be studied in terms of both their production parameters under optimal conditions and their tolerance to stress factors that might be caused by emergency situations. The purpose of this study was to investigate tolerance of chufa (Cyperus esculentus L.) plants to the super-optimal air temperature of 45 ± 1 °C as dependent upon PAR (photosynthetically active radiation) intensity and the duration of the exposure to the stress factor. Chufa plants were grown hydroponically, on expanded clay, under artificial light. The nutrient solution was Knop’s mineral medium. Until the plants were 30 days old, they had been grown at 690 μmol m⁻² s⁻¹ PAR and air temperature 25 °C. Thirty-day-old plants were exposed to the temperature 45 °C for 6 h, 20 h, and 44 h at PAR intensities 690 μmol m⁻² s⁻¹ and 1150 μmol m⁻² s⁻¹. The exposure to the damaging air temperature for 44 h at 690 μmol m⁻² s⁻¹ PAR caused irreversible damage to PSA, resulting in leaf mortality. In chufa plants exposed to heat shock treatment at 690 μmol m⁻² s⁻¹ PAR for 6 h and 20 h, respiration exceeded photosynthesis, and CO₂ release in the light was recorded. Functional activity of photosynthetic apparatus, estimated from parameters of pulse-modulated chlorophyll fluorescence in Photosystem 2 (PS 2), decreased 40% to 50%. After the exposure to the stress factor was finished, functional activity of PSA recovered its initial values, and apparent photosynthesis (P_apparent) rate after a 20-h exposure to the stress factor was 2.6 times lower than before the elevation of the temperature. During the first hours of plant exposure to the temperature 45 °C at 1150 μmol m⁻² s⁻¹ PAR, respiration rate was higher than photosynthesis rate, but after 3–4 h of the exposure, photosynthetic processes exceeded oxidative ones and CO₂ absorption in the light was recorded. At the end of the 6-h exposure, P_apparent rate was close to that recorded prior to the exposure, and no significant changes were observed in the functional activity of PSA. At the end of the 20-h exposure, P_apparent rate was close to its initial value, but certain parameters of the functional activity of PSA decreased 25% vs. their initial values. During the repair period, the parameters of external gas exchange recovered their initial values, and parameters of pulse-modulated chlorophyll fluorescence were 20–30% higher than their initial values. Thus, exposure of chufa plants to the damaging temperature of the air for 20 h did not cause any irreversible damage to the photosynthetic apparatus of plants at either 690 μmol m⁻² s⁻¹ or 1150 μmol m⁻² s⁻¹ PAR, and higher PAR intensity during the heat shock treatment enhanced heat tolerance of the plants.     

Chlorella vulgaris culture as a regulator of CO2 in a bioregenerative life support system

It is the primary task for a bioregenerative life support system (BLSS) to maintain the stable concentrations of CO₂ and O₂. However, these concentrations could fluctuate based on various factors, such as the imbalance between respiration/assimilation quotients of the heterotrophic and autotrophic components. They can even be out of balance through catastrophic failure of higher plants in the emergency conditions. In this study, the feasibility of using unicellular Chlorella vulgaris of typically rapid growth as both “compensatory system” and “regulator” to control the balance of CO₂ and O₂ was analyzed in a closed ecosystem. For this purpose, a small closed ecosystem called integrative experimental system (IES) was established in our laboratory where we have been conducting multi-biological life support system experiments (MLSSE). The IES consists of a closed integrative cultivating system (CICS) and a plate photo-bioreactor. Four volunteers participated in the study for gas exchange by periodical breathing through a tube connected with the CICS. The plate photo-bioreactor was used to cultivate C. vulgaris. Results showed that the culture of C. vulgaris could be used in a situation of catastrophic failure of higher plant under the emergencies. And the productivity could recover itself to the original state in 3 to 5 days to protect the system till the higher plant was renewed. Besides, C. vulgaris could grow well and the productivity could be affected by the light intensity which could help to keep the balance of CO₂ and O₂ in the IES efficiently. Thus, C. vulgaris could be included in the design of a BLSS as a “compensatory system” in the emergency contingency and a “regulator” during the normal maintenance.     

Selection and hydroponic growth of bread wheat cultivars for bioregenerative life support systems

As part of the ESA-funded MELiSSA program, the suitability, the growth and the development of four bread wheat cultivars were investigated in hydroponic culture with the aim to incorporate such a cultivation system in an Environmental Control and Life Support System (ECLSS). Wheat plants can fulfill three major functions in space: (a) fixation of CO₂ and production of O₂, (b) production of grains for human nutrition and (c) production of cleaned water after condensation of the water vapor released from the plants by transpiration. Four spring wheat cultivars (Aletsch, Fiorina, Greina and CH Rubli) were grown hydroponically and compared with respect to growth and grain maturation properties. The height of the plants, the culture duration from germination to harvest, the quantity of water used, the number of fertile and non-fertile tillers as well as the quantity and quality of the grains harvested were considered. Mature grains could be harvested after around 160 days depending on the varieties. It became evident that the nutrient supply is crucial in this context and strongly affects leaf senescence and grain maturation. After a first experiment, the culture conditions were improved for the second experiment (stepwise decrease of EC after flowering, pH adjusted twice a week, less plants per m²) leading to a more favorable harvest (higher grain yield and harvest index). Considerably less green tillers without mature grains were present at harvest time in experiment 2 than in experiment 1. The harvest index for dry matter (including roots) ranged from 0.13 to 0.35 in experiment 1 and from 0.23 to 0.41 in experiment 2 with modified culture conditions. The thousand-grain weight for the four varieties ranged from 30.4 to 36.7 g in experiment 1 and from 33.2 to 39.1 g in experiment 2, while market samples were in the range of 39.4–46.9 g. Calcium levels in grains of the hydroponically grown wheat were similar to those from field-grown wheat, while potassium, magnesium, phosphorus, iron, zinc, copper, manganese and nickel levels tended to be higher in the grains of experimental plants. It remains a challenge for future experiments to further adapt the nutrient supply in order to improve senescence of vegetative plant parts, harvest index and the composition of bread wheat grains.     

A biological method of including mineralized human liquid and solid wastes into the mass exchange of bio-technical life support systems

The main obstacle to using mineralized human solid and liquid wastes as a source of mineral elements for plants cultivated in bio-technical life support systems (BLSS) is that they contain NaCl. The purpose of this study is to determine whether mineralized human wastes can be used to prepare the nutrient solution for long-duration conveyor cultivation of uneven-aged wheat and Salicornia europaea L. plant community. Human solid and liquid wastes were mineralized by the method of “wet incineration” developed by Yu. Kudenko. They served as a basis for preparing the solutions that were used for conveyor-type cultivation of wheat community represented by 5 age groups, planted with a time interval of 14 days. Wheat was cultivated hydroponically on expanded clay particles. To reduce salt content of the nutrient solution, every two weeks, after wheat was harvested, 12 L of solution was removed from the wheat irrigation tank and used for Salicornia europaea cultivation in water culture in a conveyor mode. The Salicornia community was represented by 2 age groups, planted with a time interval of 14 days. As some portion of the nutrient solution used for wheat cultivation was regularly removed, sodium concentration in the wheat irrigation solution did not exceed 400 mg/L, and mineral elements contained in the removed portion were used for Salicornia cultivation. The experiment lasted 4 months. The total wheat biomass productivity averaged 30.1 g · m⁻² · day⁻¹, and the harvest index amounted to 36.8%. The average productivity of Salicornia edible biomass on a dry weight basis was 39.3 g · m⁻² · day⁻¹, and its aboveground mass contained at least 20% of NaCl. Thus, the proposed technology of cultivation of wheat and halophyte plant community enables using mineralized human wastes as a basis for preparing nutrient solutions and including NaCl in the mass exchange of the BLSS; moreover, humans are supplied with additional amounts of leafy vegetables.     

Efficacy of oxygen-supplying capacity of Azolla in a controlled life support system

Azolla shows high growth and propagation rates, strong photosynthetic O₂-releasing ability and high nutritional value. It is suitable as a salad vegetable and can be cultured on a multi-layered wet bed. Hence, it possesses potential as a fresh vegetable, and to release O₂ and absorb CO₂ in a Controlled Ecological Life Support System in space. In this study, we investigated the O₂-providing characteristics of Azolla in a closed chamber under manned, controlled conditions to lay a foundation for use of Azolla as a biological component in ground simulation experiments for space applications. A closed test chamber, representing a Controlled Ecological Life Support System including an Azolla wet-culture device, was built to measure the changes in atmospheric O₂ and CO₂ concentrations inside the chamber in the presence of coexisting Azolla, fish and men. The amount of O₂ consumed by fish was 0.0805–0.0831 L kg⁻¹ h⁻¹ and the level of CO₂ emission was 0.0705–0.0736 L kg⁻¹ h⁻¹; O₂ consumption by the two trial volunteers was 19.71 L h⁻¹ and the volume of respiration-released CO₂ was 18.90 L h⁻¹. Under 7000–8000 Lx artificial light and Azolla wet-culture conditions, human and fish respiration and Azolla photosynthesis were complementary, thus the atmospheric O₂ and CO₂ concentrations inside chamber were maintained in equilibrium. The increase in atmospheric CO₂ concentration in the closed chamber enhanced the net photosynthesis efficiency of the Azolla colony. This study showed that Azolla has strong photosynthetic O₂-releasing ability, which equilibrates the O₂ and CO₂ concentrations inside the chamber in favor of human survival and verifies the potential of Azolla for space applications.     

Molecular analysis of the graviperception signal transduction in the flagellate Euglena gracilis: Involvement of a transient receptor potential-like channel and a calmodulin

Euglena gracilis, a unicellular, photosynthetic flagellate is a model system for environmentally controlled behavior responses. The organism shows pronounced negative gravitaxis. This movement is based on physiological mechanisms, which in the past had been only indirectly assessed. It was shown that mechano-sensitive calcium channels are involved in the gravitaxis response. Recent studies have demonstrated that members of the transient receptor potential (TRP) family function as mechano-sensitive channels in several different cell types. We have sequenced part of a TRP gene in Euglena and applied RNA interference (RNAi) to confirm that these channels are involved in graviperception. It was found that RNAi against the putative TRP channel abolished gravitaxis. The genes of three calmodulins were sequences in Euglena, one of which was previously known in its protein structure (cal 1). The other two were unknown (cal 2 and cal 3). Cal 2 has been analyzed in detail. The biosynthesis of the corresponding proteins of cal 1 and cal 2 was inhibited by means of RNA interference to see whether this blockage impairs gravitaxis. RNAi of cal 1 leads to a long-term loss of free swimming in the cells (while euglenoid movement persists). It induced pronounced cell form aberrations and the division of cells was hampered. After recovery from RNAi the cell showed precise negative gravitaxis again. Thus cal 1 does not seem to be involved in gravitaxis. In contrast, the blockage of cal 2 has no pronounced influence on motility and cell form but leads to a complete loss of gravitactic orientation for more than 30 days showing that this calmodulin is an element in the signal transduction chain. The data are discussed in the context of the current model of the gravitaxis signal transduction chain in Euglena gracilis.     

Cowpeas and pinto beans: Performance and yields of candidate space crops in the laboratory biosphere closed ecological system

An experiment utilizing cowpeas (Vigna unguiculata L.), pinto beans (Phaseolus vulgaris L.) and Apogee ultra-dwarf wheat (Triticum sativa L.) was conducted in the soil-based closed ecological facility, Laboratory Biosphere, from February to May 2005. The lighting regime was 13 h light/11 h dark at a light intensity of 960 μmol m⁻² s⁻¹, 45 mol m⁻² day⁻¹ supplied by high-pressure sodium lamps. The pinto beans and cowpeas were grown at two different planting densities. Pinto bean production was 341.5 g dry seed m⁻² (5.42 g m⁻² day⁻¹) and 579.5 dry seed m⁻² (9.20 g m⁻² day⁻¹) at planted densities of 32.5 plants m⁻² and 37.5 plants m⁻², respectively. Cowpea yielded 187.9 g dry seed m⁻² (2.21 g m⁻² day⁻¹) and 348.8 dry seed m⁻² (4.10 g m⁻² day⁻¹) at planted densities of 20.8 plants m⁻² and 27.7 plants m⁻², respectively. The crop was grown at elevated atmospheric carbon dioxide levels, with levels ranging from 300–3000 ppm daily during the majority of the crop cycle. During early stages (first 10 days) of the crop, CO₂ was allowed to rise to 7860 ppm while soil respiration dominated, and then was brought down by plant photosynthesis. CO₂ was injected 27 times during days 29–71 to replenish CO₂ used by the crop during photosynthesis. Temperature regime was 24–28 °C day/deg 20–24 °C night. Pinto bean matured and was harvested 20 days earlier than is typical for this variety, while the cowpea, which had trouble establishing, took 25 days more for harvest than typical for this variety. Productivity and atmospheric dynamic results of these studies contribute toward the design of an envisioned ground-based test bed prototype Mars base.     

Response of photosynthetic systems to salinity stress in the desert cyanobacterium Scytonema javanicum

The present study investigated the physiological and biochemical characteristics of Scytonema javanicum, a pioneer species isolated from desert biological crusts, under salinity stress. Pigment analysis showed that salinity decreased chlorophyll a and phycocyanin content, while low salinity increased carotenoid concentration and high salinity decreased carotenoid concentration. Salinity also inhibited CO₂ assimilation rate and photosynthetic oxygen evolution in this cyanobacterium. Chlorophyll a fluorescence transient parameters (φPo, φEo, ψO, RC/ABS, RC/CS, PI_ABS, and PI_CS) were decreased under salt stress, while dVo/dto(Mo), Vj and φDo were increased. The decrease of ETRmax and Yield and the change of chlorophyll a fluorescence transients showed that salt stress had an important influence on photosynthesis. These results indicated that the effects of salinity stress on photosynthesis in S. javanicum may depend on the inhibition of electron transport and the inactivation of the reaction centers, but this inhibition may occur in the electron transport pathway at the PSII donor and acceptor sites.