NOTE: End date changed to 9/30/2018 per F. Hernandez/ARC (Ed., 3/23/17)

The objective of the proposed study is to determine the effect of different gravity levels on the nitrogen cycle leveraging experiments to be flown on DLR's Eu:CROPIS mission. This is of importance to NASA because The National Research Council’s Plant and Microbial Biology Decadal Survey (2011) states that there is a need for understanding the role of gravity on microbe-microbe interactions and microbe-plant interactions. The research proposed here will do just that. Nitrogen is an essential element for life. It is present in all living systems, occurring in several important molecules including proteins and nucleic acids. Without nitrogen life as we know it could not exist. Thus, the nitrogen cycle is important to supporting life whether it is on Earth, in space, or on other planets or moons. Because only Earth has a 1 x g environment understanding how the nitrogen cycle operates as a function of gravity is key to sustaining life off of Earth. To change the gravity levels the spacecraft will be maneuvered (by spinning) to produce three different gravity regimes during the courser of the mission. The three gravity regimes will be 0.01 x g - 0.1-x g (essentially microgravity); 0.16 x g (Moon gravity); and 0.38 x g (Mars gravity). Each gravity regime will last for six months. Eu:CROPIS will be used in reducing organic waste and in the development of efficient life support systems. Its core element is a microbiological trickling filter of lava rock – the habitat of a multitude of microorganisms that purify and decontaminate water. The development aims at a wet composting system that may be used in closed life support systems such as waste water recovery. A key component of the system is the nitrogen cycle. So, modeling the nitrogen cycle of the system is essential to understanding how the system functions. It will be the first time nitrogen-transformation reactions will be measured as a function of gravity. NASA has an excellent opportunity to participate in the DLR’s Eu:CROPIS mission that allows us to obtain data by leveraging their laboratory work and hardware at a fraction of what it would cost if funded/supported solely by NASA.

Eventually, space travel will require the ability for self-sufficiency. Once mission profiles extend beyond short trips to the lunar surface, the duration of each mission will mean it will no longer remain cost-effective -- or indeed feasible -- to dispose of all waste and resupply oxygen, water, and food to crew members from Earth. NASA has acknowledged this reality for more than two decades with programs exploring the development of both physicochemical and bioregenerative life support systems. The program on bioregenerative capabilities arose from observations that the only truly long-term, self-sustaining life support system that has a demonstrated stability and efficacy relies upon biological systems for its function; that system is the life support afforded by Earth. Since bioregenerative life support systems are not high on the NASA priority list at this time it was stated in the report: Because international collaborations will be essential to make rapid progress with these aims, NASA should support collaborations, where appropriate, with partners that are already pursuing these goals, such as European scientists….

Eu:CROPIS is a clear example that fits in with this statement. It allows NASA to obtain this data at little cost by using the laboratories, the hardware, and the spacecraft paid for by the DLR. The Eu:CROPIS (Euglena: Combined Regenerative Organic-food Production In Space) experiment will test the feasibility and technology in the areas of life support systems and gravitational biological research. The mission offers for the first time the opportunity of analyzing coupled biological life support systems under different levels of gravity (space, moon, Mars) utilizing state-of-the-art methods for image and molecular analysis. It combines the C.R.O.P. system plant growth water purification system developed at the DLR in Cologne, Germany with the well studied Euglena gracilis space flight system.

Euglena gracilis is a motile, photosynthetic, unicellular flagellate living in ponds and lakes. It uses gravity and light as hints to reach and stay in regions of the water column optimal for photosynthesis and growth. At low light irradiances, Euglena swims toward and at higher irradiances cells swim away from a light source (positive and negative phototaxis). In addition, Euglena typically orients away from the center of acceleration (negative gravitaxis). Euglena is considered a ‘professional gravi-sensing organism,’ a term that was coined by ESA (European Space Agency). In the past 15 years, Euglena has been established as a model organism for studying gravity perception of single cells. A model for gravitaxis was created by the combination of physiological, biochemical, and molecular biological methods. In this context substantial contributions came from microgravity experiments in space.

NOTE: End date changed to 9/30/2018 per F. Hernandez/ARC (Ed., 3/23/17)

The objective of the proposed study is to determine the effect of different gravity levels on the nitrogen cycle leveraging experiments to be flown on DLR's Eu:CROPIS mission. This is of importance to NASA because The National Research Council’s Plant and Microbial Biology Decadal Survey (2011) states that there is a need for understanding the role of gravity on microbe-microbe interactions and microbe-plant interactions. The research proposed here will do just that. Nitrogen is an essential element for life. It is present in all living systems, occurring in several important molecules including proteins and nucleic acids. Without nitrogen life as we know it could not exits. Thus, the nitrogen cycle is important to supporting life whether it is on Earth, in space, or on other planets or moons. Because only Earth has a 1 x g environment understanding how the nitrogen cycle operates as a function of gravity is key to sustaining life off of Earth. To change the gravity levels the spacecraft will be maneuvered (by spinning) to produce three different gravity regimes during the courser of the mission. The three gravity regimes will be 0.01 x g - 0.1-x g (essentially microgravity); 0.16 x g (Moon gravity); and 0.38 x g (Mars gravity). Each gravity regime will last for six months. Eu:CROPIS will be used in reducing organic waste and in the development of efficient life support systems. Its core element is a microbiological trickling filter of lava rock – the habitat of a multitude of microorganisms that purify and decontaminate water. The development aims at a wet composting system that may be used in closed life support systems such as waste water recovery. A key component of the system is the nitrogen cycle. So, modeling the nitrogen cycle of the system is essential to understanding how the system functions. It will be the first time nitrogen-transformation reactions will be measured as a function of gravity. NASA has an excellent opportunity to participate in the DLR’s Eu:CROPIS mission that allows us to obtain data by leveraging their laboratory work and hardware at a fraction of what it would cost if funded/supported solely by NASA.

Eventually, space travel will require the ability for self-sufficiency. Once mission profiles extend beyond short trips to the lunar surface, the duration of each mission will mean it will no longer remain cost-effective -- or indeed feasible -- to dispose of all waste and resupply oxygen, water, and food to crew members from Earth. NASA has acknowledged this reality for more than two decades with programs exploring the development of both physicochemical and bioregenerative life support systems. The program on bioregenerative capabilities arose from observations that the only truly long-term, self-sustaining life support system that has a demonstrated stability and efficacy relies upon biological systems for its function; that system is the life support afforded by Earth. Since bioregenerative life support systems are not high on the NASA priority list at this time it was stated in the report: Because international collaborations will be essential to make rapid progress with these aims, NASA should support collaborations, where appropriate, with partners that are already pursuing these goals, such as European scientists….

Eu:CROPIS is a clear example that fits in with this statement. It allows NASA to obtain this data at little cost by using the laboratories, the hardware, and the spacecraft paid for by the DLR. The Eu:CROPIS (Euglena: Combined Regenerative Organic-food Production In Space) experiment will test the feasibility and technology in the areas of life support systems and gravitational biological research. The mission offers for the first time the opportunity of analyzing coupled biological life support systems under different levels of gravity (space, moon, Mars) utilizing state-of-the-art methods for image and molecular analysis. It combines the C.R.O.P. system plant growth water purification system developed at the DLR in Cologne, Germany with the well studied Euglena gracilis space flight system.

Euglena gracilis is a motile, photosynthetic, unicellular flagellate living in ponds and lakes. It uses gravity and light as hints to reach and stay in regions of the water column optimal for photosynthesis and growth. At low light irradiances, Euglena swims toward and at higher irradiances cells swim away from a light source (positive and negative phototaxis). In addition, Euglena typically orients away from the center of acceleration (negative gravitaxis). Euglena is considered a ‘professional gravi-sensing organism,’ a term that was coined by ESA (European Space Agency). In the past 15 years, Euglena has been established as a model organism for studying gravity perception of single cells. A model for gravitaxis was created by the combination of physiological, biochemical, and molecular biological methods. In this context substantial contributions came from microgravity experiments in space.

The satellite was successfully placed in orbit at an altitude of 600 kilometres. First radio contact with the satellite to the German Space Operations Center (GSOC) in Oberpfaffenhofen took place about one hour and 15 minutes after the launch. Two weeks after launch the SOC commissioned the satellite in space and tested all functions. Seven weeks after launch, the first of two greenhouses was scheduled to go into operation. However, a software malfunction occurred and communication with the payload was lost. Since then diagnostic tests were made from the ground to the satellite and on the ground using the ground simulation facility. The ground control is working perfectly. During the week of October 23rd 2019 new software will be uploaded and the mission restarted soon thereafter.

NOTE: End date changed to 9/30/2018 per F. Hernandez/ARC (Ed., 3/23/17)

The objective of the proposed study is to determine the effect of different gravity levels on the nitrogen cycle leveraging experiments to be flown on DLR's Eu:CROPIS mission. This is of importance to NASA because The National Research Council’s Plant and Microbial Biology Decadal Survey (2011) states that there is a need for understanding the role of gravity on microbe-microbe interactions and microbe-plant interactions. The research proposed here will do just that. Nitrogen is an essential element for life. It is present in all living systems, occurring in several important molecules including proteins and nucleic acids. Without nitrogen life as we know it could not exits. Thus, the nitrogen cycle is important to supporting life whether it is on Earth, in space, or on other planets or moons. Because only Earth has a 1 x g environment understanding how the nitrogen cycle operates as a function of gravity is key to sustaining life off of Earth. To change the gravity levels the spacecraft will be maneuvered (by spinning) to produce three different gravity regimes during the courser of the mission. The three gravity regimes will be 0.01 x g - 0.1-x g (essentially microgravity); 0.16 x g (Moon gravity); and 0.38 x g (Mars gravity). Each gravity regime will last for six months. Eu:CROPIS will be used in reducing organic waste and in the development of efficient life support systems. Its core element is a microbiological trickling filter of lava rock – the habitat of a multitude of microorganisms that purify and decontaminate water. The development aims at a wet composting system that may be used in closed life support systems such as waste water recovery. A key component of the system is the nitrogen cycle. So, modeling the nitrogen cycle of the system is essential to understanding how the system functions. It will be the first time nitrogen-transformation reactions will be measured as a function of gravity. NASA has an excellent opportunity to participate in the DLR’s Eu:CROPIS mission that allows us to obtain data by leveraging their laboratory work and hardware at a fraction of what it would cost if funded/supported solely by NASA.

Eventually, space travel will require the ability for self-sufficiency. Once mission profiles extend beyond short trips to the lunar surface, the duration of each mission will mean it will no longer remain cost-effective - or indeed feasible - to dispose of all waste and resupply oxygen, water, and food to crew members from Earth. NASA has acknowledged this reality for more than two decades with programs exploring the development of both physicochemical and bioregenerative life support systems. The program on bioregenerative capabilities arose from observations that the only truly long-term, self-sustaining life support system that has a demonstrated stability and efficacy relies upon biological systems for its function; that system is the life support afforded by Earth. Since bioregenerative life support systems are not high on the NASA priority list at this time it was stated in the report: Because international collaborations will be essential to make rapid progress with these aims, NASA should support collaborations, where appropriate, with partners that are already pursuing these goals, such as European scientists….

Eu:CROPIS is a clear example that fits in with this statement. It allows NASA to obtain this data at little cost by using the laboratories, the hardware, and the spacecraft paid for by the DLR. The Eu:CROPIS (Euglena: Combined Regenerative Organic-food Production In Space) experiment will test the feasibility and technology in the areas of life support systems and gravitational biological research. The mission offers for the first time the opportunity of analyzing coupled biological life support systems under different levels of gravity (space, moon, Mars) utilizing state-of-the-art methods for image and molecular analysis. It combines the C.R.O.P. system plant growth water purification system developed at the DLR in Cologne, Germany with the well studied Euglena gracilis space flight system.

Euglena gracilis is a motile, photosynthetic, unicellular flagellate living in ponds and lakes. It uses gravity and light as hints to reach and stay in regions of the water column optimal for photosynthesis and growth. At low light irradiances, Euglena swims toward and at higher irradiances cells swim away from a light source (positive and negative phototaxis). In addition, Euglena typically orients away from the center of acceleration (negative gravitaxis). Euglena is considered a ‘professional gravi-sensing organism,’ a term that was coined by ESA (European Space Agency). In the past 15 years, Euglena has been established as a model organism for studying gravity perception of single cells. A model for gravitaxis was created by the combination of physiological, biochemical, and molecular biological methods. In this context substantial contributions came from microgravity experiments in space.

Eu:CROPIS (Euglena: Combined Regenerative Organic-food Production In Space) is a closed ecosystem housed in a satellite that functions as water purifying and plant growth system. At its core is a lava rock trickling filter inhabited with microorganisms that breakdown urea in wastewater and produce nitrate for tomato growth. The Euglena produce oxygen and consume excess ammonium. An integral part of this system is the nitrogen cycle. Nitrogen is an essential element for life. Thus, the nitrogen cycle is important to supporting life whether it is on or off Earth. The objective of the study is to determine if the gravity levels of the Moon and Mars affect the nitrogen cycle of the system. The concentration of oxygen, urea, ammonium, nitrate, and nitrite in the system is monitored as a function of time.

Computer simulation of the filter column and the Eu:CROPIS-system

The simulation was performed with the GameMaker Software 8.1. (YoYo Games, Dundee, Scotland) . Cell types as well as substances were programmed as instances of objects with defined properties. The filter column as well as the subsequent compartments were programmed as 2 D-objects (representing a section of a “real column”). Substances were “flushed” into the system at the top of the column, where they migrated through the column, through the algae/plant compartment, and back to the column. The system enables performance of a “batch-run,” where an initial amount of substances is flushed into the system and subsequently degraded, “pulsed runs,” where preset amounts of substances are flushed into the system in regular time intervals and a combination of the two methods. In addition, substances can be added manually during an actual run. All instance numbers were constantly recorded, displayed on the screen, and stored. Only the cycles of carbon and nitrogen were simulated; other elements (phosphorous, potassium, trace elements) were not.

Experimental setup for determination of the filter column (full system without plants)

The Eu:CROPIS-test module was constructed mainly with same hardware components as the flight module. In order to allow detailed analysis of the filter-bacteria/algae-interaction no tomatoes were integrated in the system. It turned out in preliminary experiments that a head space of air is important for development of the filter column and Euglena gracilis. For this reason, an air reservoir with the size of the algae greenhouse was attached. A container with dormant states of Euglena gracilis cells was attached to the alga circuit. Activation of the pump of the algae section flushed the cells into the algae tank. The filter column was filled with small lava rocks which were primed among others with nitrifying bacteria (the rocks were part of a CROP-column before and provided from the DLR). The filter material was dry so that the experiments start with desiccated states of the bacteria. Temperature of the system was maintained at 20°C. Algae were constantly illuminated with a photon flux of 83 µmol photons/m2 provided from a mixed array of blue (peak wavelength 470 nm) and red (peak wavelength 660 nm)-high power LEDs. In order to allow initial growth of Euglena gracilis the algae section was started 10 days before the filter circuit was engaged.

Summary

The computer model seems to reflect what is occurring in the ground module of the system. We will continue to gather more data from the ground module to refine the model in anticipation of the flight experiment.

NOTE: End date changed to 9/30/2018 per F. Hernandez/ARC (Ed., 3/23/17)

The objective of the proposed study is to determine the effect of different gravity levels on the nitrogen cycle leveraging experiments to be flown on DLR's Eu:CROPIS mission. This is of importance to NASA because The National Research Council’s Plant and Microbial Biology Decadal Survey (2011) states that there is a need for understanding the role of gravity on microbe-microbe interactions and microbe-plant interactions. The research proposed here will do just that. Nitrogen is an essential element for life. It is present in all living systems, occurring in several important molecules including proteins and nucleic acids. Without nitrogen life as we know it could not exits. Thus, the nitrogen cycle is important to supporting life whether it is on Earth, in space, or on other planets or moons. Because only Earth has a 1 x g environment understanding how the nitrogen cycle operates as a function of gravity is key to sustaining life off of Earth. To change the gravity levels the spacecraft will be maneuvered (by spinning) to produce three different gravity regimes during the courser of the mission. The three gravity regimes will be 0.01 x g - 0.1-x g (essentially microgravity); 0.16 x g (Moon gravity); and 0.38 x g (Mars gravity). Each gravity regime will last for six months. Eu:CROPIS will be used in reducing organic waste and in the development of efficient life support systems. Its core element is a microbiological trickling filter of lava rock – the habitat of a multitude of microorganisms that purify and decontaminate water. The development aims at a wet composting system that may be used in closed life support systems such as waste water recovery. A key component of the system is the nitrogen cycle. So, modeling the nitrogen cycle of the system is essential to understanding how the system functions. It will be the first time nitrogen-transformation reactions will be measured as a function of gravity. NASA has an excellent opportunity to participate in the DLR’s Eu:CROPIS mission that allows us to obtain data by leveraging their laboratory work and hardware at a fraction of what it would cost if funded/supported solely by NASA.

Eventually, space travel will require the ability for self-sufficiency. Once mission profiles extend beyond short trips to the lunar surface, the duration of each mission will mean it will no longer remain cost-effective - or indeed feasible - to dispose of all waste and resupply oxygen, water, and food to crew members from Earth. NASA has acknowledged this reality for more than two decades with programs exploring the development of both physicochemical and bioregenerative life support systems. The program on bioregenerative capabilities arose from observations that the only truly long-term, self-sustaining life support system that has a demonstrated stability and efficacy relies upon biological systems for its function; that system is the life support afforded by Earth. Since bioregenerative life support systems are not high on the NASA priority list at this time it was stated in the report: Because international collaborations will be essential to make rapid progress with these aims, NASA should support collaborations, where appropriate, with partners that are already pursuing these goals, such as European scientists….

Eu:CROPIS is a clear example that fits in with this statement. It allows NASA to obtain this data at little cost by using the laboratories, the hardware, and the spacecraft paid for by the DLR. The Eu:CROPIS (Euglena: Combined Regenerative Organic-food Production In Space) experiment will test the feasibility and technology in the areas of life support systems and gravitational biological research. The mission offers for the first time the opportunity of analyzing coupled biological life support systems under different levels of gravity (space, moon, Mars) utilizing state-of-the-art methods for image and molecular analysis. It combines the C.R.O.P. system plant growth water purification system developed at the DLR in Cologne, Germany with the well studied Euglena gracilis space flight system. Euglena gracilis is a motile, photosynthetic, unicellular flagellate living in ponds and lakes. It uses gravity and light as hints to reach and stay in regions of the water column optimal for photosynthesis and growth. At low light irradiances, Euglena swims toward and at higher irradiances cells swim away from a light source (positive and negative phototaxis). In addition, Euglena typically orients away from the center of acceleration (negative gravitaxis). Euglena is considered a ‘professional gravi-sensing organism,’ a term that was coined by ESA (European Space Agency). In the past 15 years, Euglena has been established as a model organism for studying gravity perception of single cells. A model for gravitaxis was created by the combination of physiological, biochemical, and molecular biological methods. In this context substantial contributions came from microgravity experiments in space.

• The computer model of nitrogen transformation reactions has been and is continuing to be refined as we obtain more data from the ground control.

• Gas leaks in the laboratory system hhave been minimized.

• The integratated Eu:CROPIS flight system has been tested and is working properly.

The objective of the proposed study is to determine the effect of different gravity levels on the nitrogen cycle leveraging experiments to be flown on DLR's Eu:CROPIS mission. This is of importance to NASA because The National Research Council’s Plant and Microbial Biology Decadal Survey (2011) states that there is a need for understanding the role of gravity on microbe-microbe interactions and microbe-plant interactions. The research proposed here will do just that. Nitrogen is an essential element for life. It is present in all living systems, occurring in several important molecules including proteins and nucleic acids. Without nitrogen life as we know it could not exits. Thus, the nitrogen cycle is important to supporting life whether it is on Earth, in space, or on other planets or moons. Because only Earth has a 1 x g environment understanding how the nitrogen cycle operates as a function of gravity is key to sustaining life off of Earth. To change the gravity levels the spacecraft will be maneuvered (by spinning) to produce three different gravity regimes during the courser of the mission. The three gravity regimes will be 0.01 x g - 0.1-x g (essentially microgravity); 0.16 x g (Moon gravity); and 0.38 x g (Mars gravity). Each gravity regime will last for six months. Eu:CROPIS will be used in reducing organic waste and in the development of efficient life support systems. Its core element is a microbiological trickling filter of lava rock – the habitat of a multitude of microorganisms that purify and decontaminate water. The development aims at a wet composting system that may be used in closed life support systems such as waste water recovery. A key component of the system is the nitrogen cycle. So, modeling the nitrogen cycle of the system is essential to understanding how the system functions. It will be the first time nitrogen-transformation reactions will be measured as a function of gravity. NASA has an excellent opportunity to participate in the DLR’s Eu:CROPIS mission that allows us to obtain data by leveraging their laboratory work and hardware at a fraction of what it would cost if funded/supported solely by NASA.

Eventually, space travel will require the ability for self-sufficiency. Once mission profiles extend beyond short trips to the lunar surface, the duration of each mission will mean it will no longer remain cost-effective - or indeed feasible - to dispose of all waste and resupply oxygen, water, and food to crew members from Earth. NASA has acknowledged this reality for more than two decades with programs exploring the development of both physicochemical and bioregenerative life support systems. The program on bioregenerative capabilities arose from observations that the only truly long-term, self-sustaining life support system that has a demonstrated stability and efficacy relies upon biological systems for its function; that system is the life support afforded by Earth. Since bioregenerative life support systems are not high on the NASA priority list at this time it was stated in the report: Because international collaborations will be essential to make rapid progress with these aims, NASA should support collaborations, where appropriate, with partners that are already pursuing these goals, such as European scientists….

Eu:CROPIS is a clear example that fits in with this statement. It allows NASA to obtain this data at little cost by using the laboratories, the hardware, and the spacecraft paid for by the DLR. The Eu:CROPIS (Euglena: Combined Regenerative Organic-food Production In Space) experiment will test the feasibility and technology in the areas of life support systems and gravitational biological research. The mission offers for the first time the opportunity of analyzing coupled biological life support systems under different levels of gravity (space, moon, Mars) utilizing state-of-the-art methods for image and molecular analysis. It combines the C.R.O.P. system plant growth water purification system developed at the DLR in Cologne, Germany with the well studied Euglena gracilis space flight system. Euglena gracilis is a motile, photosynthetic, unicellular flagellate living in ponds and lakes. It uses gravity and light as hints to reach and stay in regions of the water column optimal for photosynthesis and growth. At low light irradiances, Euglena swims toward and at higher irradiances cells swim away from a light source (positive and negative phototaxis). In addition, Euglena typically orients away from the center of acceleration (negative gravitaxis). Euglena is considered a ‘professional gravi-sensing organism,’ a term that was coined by ESA (European Space Agency). In the past 15 years, Euglena has been established as a model organism for studying gravity perception of single cells. A model for gravitaxis was created by the combination of physiological, biochemical, and molecular biological methods. In this context substantial contributions came from microgravity experiments in space.

• Demonstrated Euglena growth on NO3- as well as on NH4+. Significance: Complicates the interpretation of the N-transformation reactions and their rates in the primary payload.

• Demonstrated Euglena growth on NH4+ produced by cyanobacteria in co-culture in 2 types of media. Significance: Euglena able to grow on NH4+ produced by other organisms (ground control data for potential contaminants in system – the current prototype CROP system is full of cyanobacteria).

• Colorimetric assays for the various nitrogen species produced variable results leading to the decision to use ion-chromatography for the ground controls and flight experiment. Significance: Changes the hardware, its configuration, as well as mass, power and volume in the payload.

• Decision finalized to use gas sensors to measure atmospheric gases in the primary payload instead of a gas chromatograph. Significance: Gas sensors are simpler and less prone to error and failure. It also impacts the hardware configuration as well as resulting in a reduction in the mass and power requirements.

• Using the ion chromatograph we are monitoring the concentration of the ammonium, nitrate, and nitrite in the system as well as net rate of the reactions from a batch of 20% synthetic urine run through the CROP system.

• Computer simulations of the microbial and nitrogen species changes in the Eu:CROPIS system were refined to better incorporate the data obtained from the CROP portion of the system.

• We constructed an additional test module that isolates each component of the system (i.e., greenhouse for Euglena, tomato growing section, trickling filter, etc.), such that they can function independently. This module is fitted with gas inlets/outlets and sampling ports so that we can control the atmosphere in the system. This allows us to test each system at various O2 levels in a controlled manner.

In the multi-modular test system running without tomatoes the addition of urea needs to be balanced with biomass production. To that end we have modified the system and improved the O2 production rate compared to the data obtained, improving the connection filter between the algae tank and the lava-tricking filter. In this improved system when it is flushed with 500 µl of urea it results in a significant reduction of oxygen but a much shorter time for oxygen recovery (build-up) in the system. Currently, the algae tank seems to be providing sufficient oxygen for the whole system. The O2 level is in the range of 4-5 mg/l oxygen in the filter circuit (before and about 2 h after urea supplement).

• We have improved the lighting program so that 100 different intensities can be tested. This will be important, because the light intensity needs to be adjusted to match the increasing cell density (algal growth is the prerequisite of oxygen production).

• Construction of the flight unit was completed this year and testing is ongoing in preparation for a March 2017 launch.

• Refined the computer simulation model by obtaining and incorporating more data from the laboratory studies of the CROP system.

• Minimize the gas leak rate from the laboratory system.

• Test integrated automated Eu:CROPIS system.

The objective of the proposed study is to determine the effect of different gravity levels on the nitrogen cycle leveraging experiments to be flown on DLR's Eu:CROPIS mission. This is of importance to NASA because The National Research Council’s Plant and Microbial Biology Decadal Survey (2011) states that there is a need for understanding the role of gravity on microbe-microbe interactions and microbe-plant interactions. The research proposed here will do just that. Nitrogen is an essential element for life. It is present in all living systems, occurring in several important molecules including proteins and nucleic acids. Without nitrogen life as we know it could not exits. Thus, the nitrogen cycle is important to supporting life whether it is on Earth, in space, or on other planets or moons. Because only Earth has a 1 x g environment understanding how the nitrogen cycle operates as a function of gravity is key to sustaining life off of Earth. To change the gravity levels the spacecraft will be maneuvered (by spinning) to produce three different gravity regimes during the courser of the mission. The three gravity regimes will be 0.01 x g - 0.1-x g (essentially microgravity); 0.16 x g (Moon gravity) and 0.38 x g (Mars gravity). Each gravity regime will last for six months. Eu:CROPIS will be used in reducing organic waste and in the development of efficient life support systems. Its core element is a microbiological trickling filter of lava rock – the habitat of a multitude of microorganisms that purify and decontaminate water. The development aims at a wet composting system that may be used in closed life support systems such as waste water recovery. A key component of the system is the nitrogen cycle. So, modeling the nitrogen cycle of the system is essential to understanding how the system functions. It will be the first time nitrogen-transformation reactions will be measured as a function of gravity. NASA has an excellent opportunity to participate in the DLR’s Eu:CROPIS mission that allows us to obtain data by leveraging their laboratory work and hardware at a fraction of what it would cost if NASA had to pay for it.

Eventually, space travel will require the ability for self-sufficiency. Once mission profiles extend beyond short trips to the lunar surface, the duration of each mission will mean it will no longer remain cost-effective - or indeed feasible - to dispose of all waste and resupply oxygen, water, and food to crew members from Earth. NASA has acknowledged this reality for more than two decades with programs exploring the development of both physicochemical and bioregenerative life support systems. The program on bioregenerative capabilities arose from observations that the only truly long-term, self-sustaining life support system that has a demonstrated stability and efficacy relies upon biological systems for its function; that system is the life support afforded by Earth. Since bioregenerative life support systems are not high on the NASA priority list at this time it was stated in the report: Because international collaborations will be essential to make rapid progress with these aims, NASA should support collaborations, where appropriate, with partners that are already pursuing these goals, such as European scientists….

Eu:CROPIS is a clear example that fits in with this statement. It allows NASA to obtain this data at little cost by using the laboratories, the hardware, and the spacecraft paid for by the DLR. The Eu:CROPIS (Euglena: Combined Regenerative Organic-food Production In Space) experiment will test the feasibility and technology in the areas of life support systems and gravitational biological research. The mission offers for the first time the opportunity of analyzing coupled biological life support systems under different levels of gravity (space, moon, Mars) utilizing state-of-the-art methods for image and molecular analysis. It combines the C.R.O.P. system plant growth water purification system developed at the DLR in Cologne, Germany with the well studied Euglena gracilis space flight system. Euglena gracilis is a motile, photosynthetic, unicellular flagellate living in ponds and lakes. It uses gravity and light as hints to reach and stay in regions of the water column optimal for photosynthesis and growth. At low light irradiances, Euglena swims toward and at higher irradiances cells swim away from a light source (positive and negative phototaxis). In addition, Euglena typically orients away from the center of acceleration (negative gravitaxis). Euglena is considered a ‘professional gravi-sensing organism,’ a term that was coined by ESA (European Space Agency). In the past 15 years, Euglena has been established as a model organism for studying gravity perception of single cells. A model for gravitaxis was created by the combination of physiological, biochemical, and molecular biological methods. In this context substantial contributions came from microgravity experiments in space.

• Demonstrated Euglena growth on NO3- as well as on NH4+. Significance: Complicates the interpretation of the N-transformation reactions and their rates in the primary payload.

• Demonstrated Euglena growth on NH4+ produced by cyanobacteria in co-culture in 2 types of media. Significance: Euglena able to grow on NH4+ produced by other organisms (ground control data for potential contaminants in system – the current prototype CROP system is full of cyanobacteria).

• Colorimetric assays for the various nitrogen species produced variable results leading to the decision to use ion-chromatography for the ground controls and flight experiment. Significance: Changes the hardware, its configuration, as well as mass, power, and volume in the payload.

• Decision finalized to use gas sensors to measure atmospheric gases in the primary payload instead of a gas chromatograph. Significance: Gas sensors are simpler and less prone to error and failure. It also results impacts the hardware configuration as well as resulting in a reduction in the mass and power requirements.

• Using the ion chromatograph we are monitoring the concentration of the ammonium, nitrate, and nitrite in the system as well as net rate of the reactions from a batch of 7% and 20% synthetic urine run through the CROP system. The data show an initial increase in ammonium (breakdown of urea) and nitrite (the first step of nitrification), followed by decreasing nitrite levels and a rise in the concentration of nitrate (from the second step of nitrification). At day 60 the nitrate decreases (due to denitrification). If on day 62 the system is drained and a new batch of synthetic urine is added the nitrate production occurs immediately. If the system is replenished on day 42 and ~ every 10 days thereafter nitrate increases rapidly with little nitrite detected. One explanation is that the system is primed with all of the organisms at a higher population density in the trickling filter to readily begin metabolism, so as soon as ammonium is produced it is transformed to nitrite that is immediately oxidized to nitrate. The rate of nitrification is faster if the pH is not controlled, but the total yield of NO3- is less, whereas if the system is buffered the rate of nitrification is slower, but the total amount of nitrate produced is greater.

These data indicate that nitrification is the major N transformation reaction in this system, and the efficiency of converting urea through NH4+ to nitrate is pH dependent.

The objective of the proposed study is to determine the effect of different gravity levels on the nitrogen cycle leveraging experiments to be flown on DLR's Eu:CROPIS mission. This is of importance to NASA because The National Research Council’s Plant and Microbial Biology Decadal Survey (2011) states that there is a need for understanding the role of gravity on microbe-microbe interactions and microbe-plant interactions. The research proposed here will do just that. Nitrogen is an essential element for life. It is present in all living systems, occurring in several important molecules including proteins and nucleic acids. Without nitrogen life as we know it could not exits. Thus, the nitrogen cycle is important to supporting life whether it is on Earth, in space, or on other planets or moons. Because only Earth has a 1 x g environment understanding how the nitrogen cycle operates as a function of gravity is key to sustaining life off of Earth. To change the gravity levels the spacecraft will be maneuvered (by spinning) to produce three different gravity regimes during the courser of the mission. The three gravity regimes will be 0.01 x g - 0.1-x g (essentially microgravity); 0.16 x g (Moon gravity) and 0.38 x g (Mars gravity). Each gravity regime will last for six months. Eu:CROPIS will be used in reducing organic waste and in the development of efficient life support systems. Its core element is a microbiological trickling filter of lava rock – the habitat of a multitude of microorganisms that purify and decontaminate water. The development aims at a wet composting system that may be used in closed life support systems such as waste water recovery. A key component of the system is the nitrogen cycle. So, modeling the nitrogen cycle of the system is essential to understanding how the system functions. It will be the first time nitrogen-transformation reactions will be measured as a function of gravity. NASA has an excellent opportunity to participate in the DLR’s Eu:CROPIS mission that allows us to obtain data by leveraging their laboratory work and hardware at a fraction of what it would cost if NASA had to pay for it.

Eventually, space travel will require the ability for self-sufficiency. Once mission profiles extend beyond short trips to the lunar surface, the duration of each mission will mean it will no longer remain cost-effective - or indeed feasible - to dispose of all waste and resupply oxygen, water, and food to crew members from Earth. NASA has acknowledged this reality for more than two decades with programs exploring the development of both physicochemical and bioregenerative life support systems. The program on bioregenerative capabilities arose from observations that the only truly long-term, self-sustaining life support system that has a demonstrated stability and efficacy relies upon biological systems for its function; that system is the life support afforded by Earth. Since bioregenerative life support systems are not high on the NASA priority list at this time it was stated in the report: Because international collaborations will be essential to make rapid progress with these aims, NASA should support collaborations, where appropriate, with partners that are already pursuing these goals, such as European scientists….

Eu:CROPIS is a clear example that fits in with this statement. It allows NASA to obtain this data at little cost by using the laboratories, the hardware, and the spacecraft paid for by the DLR. The Eu:CROPIS (Euglena: Combined Regenerative Organic-food Production In Space) experiment will test the feasibility and technology in the areas of life support systems and gravitational biological research. The mission offers for the first time the opportunity of analyzing coupled biological life support systems under different levels of gravity (space, moon, Mars) utilizing state-of-the-art methods for image and molecular analysis. It combines the C.R.O.P. system plant growth water purification system developed at the DLR in Cologne, Germany with the well studied Euglena gracilis space flight system. Euglena gracilis is a motile, photosynthetic, unicellular flagellate living in ponds and lakes. It uses gravity and light as hints to reach and stay in regions of the water column optimal for photosynthesis and growth. At low light irradiances, Euglena swims toward and at higher irradiances cells swim away from a light source (positive and negative phototaxis). In addition, Euglena typically orients away from the center of acceleration (negative gravitaxis). Euglena is considered a ‘professional gravi-sensing organism’, a term that was coined by ESA. In the past 15 years, Euglena has been established as a model organism for studying gravity perception of single cells. A model for gravitaxis was created by the combination of physiological, biochemical and molecular biological methods. In this context substantial contributions came from microgravity experiments in space.

Progress to date:

• Demonstrated Euglena growth on NO3- as well as on NH4+. Significance: Complicates the interpretation of the N-transformation reactions and their rates in the primary payload.

• Demonstrated Euglena growth on NH4+ produced by cyanobacteria in co-culture in 2 types of media. Significance: Euglena able to grow on NH4+ produced by other organisms (ground control data for potential contaminants in system – the current prototype CROP system is full of cyanobacteria).

• Colorimetric assays for the various nitrogen species produced variable results leading to the decision to use ion-chromatography for the ground controls and flight experiment. Significance: Changes the hardware, its configuration, as well as mass, power, and volume in the payload.

• Decision finalized to use gas sensors to measure atmospheric gases in the primary payload instead of a gas chromatograph. Significance: Gas sensors are simpler and less prone to error and failure. It also results impacts the hardware configuration as well as resulting in a reduction in the mass and power requirements.

• Using the ion chromatograph we are monitoring the concentration of the ammonium, nitrate, and nitrite in the system as well as net rate of the reactions from a batch of 20% synthetic urine run through the CROP system. Thus far these data show an initial increase in ammonium (presumably from ureases acting on the synthetic urine to produce NH4+) and nitrite (most likely from the first step of nitrification, i.e., the conversion of NH4+ to NO2-). As the concentration of nitrite decreases there is a rise in the concentration of nitrate (from the second step of nitrification, i.e., the conversion of NO2- to NO3). At day 60 the nitrate begins to decrease (presumably due to denitrification; the gas sensors were not yet installed when these data were collected). Reaction rates can be empirically derived from the slope of the line. At day 62 the system is drained and a new batch of synthetic urine is added. Here we see the production of nitrate occurring immediately. One potential explanation is that the system is primed with all of the organisms at a higher population density in the trickling filter to readily begin metabolism, so as soon as ammonium is produced it is transformed to nitrate which is immediately oxidized to nitrate.

• The first phase of a computer model to simulate the microbial and nitrogen species changes in the Eu:CROPIS system was developed. In a test of the computer simulated system is was shown that when there is no light (primary energy source) entering the system oxygen production ceases, CO2 is no longer reduced to organic compounds, accumulates, and the system becomes anaerobic. The anaerobic microbial population increases as they consume the available carbon, but because this is a closed system the microbes run out of nutrients and die and the system ceases to function due to lack of nutrients and energy. In the presence of a light dark cycle the simulated system functions optimally. Unlike when it is in the dark the organic carbon rises ahead of the rise in CO2. The organic carbon then falls as it is consumed by heterotrophs. Slightly delayed from the rise and fall in CO2 is the increase in O2 from photosynthesis which is accompanied by an increase in the Euglena (phototroph) population. These results show that the computer simulation tests were successful and it is reflecting what should happen in the laboratory Eu:CROPIS system.

The objective of the proposed study is to determine the effect of different gravity levels on the nitrogen cycle leveraging experiments to be flown on DLR's Eu:CROPIS mission. This is of importance to NASA because The National Research Council’s Plant and Microbial Biology Decadal Survey (2011) states that there is a need for understanding the role of gravity on microbe-microbe interactions and microbe-plant interactions. The research proposed here will do just that. Nitrogen is an essential element for life. It is present in all living systems, occurring in several important molecules including proteins and nucleic acids. Without nitrogen life as we know it could not exits. Thus, the nitrogen cycle is important to supporting life whether it is on Earth, in space, or on other planets or moons. Because only Earth has a 1 x g environment understanding how the nitrogen cycle operates as a function of gravity is key to sustaining life off of Earth. To change the gravity levels the spacecraft will be maneuvered (by spinning) to produce three different gravity regimes during the courser of the mission. The three gravity regimes will be 0.01 x g - 0.1-x g (essentially microgravity); 0.16 x g (Moon gravity); and 0.38 x g (Mars gravity). Each gravity regime will last for six months. Eu:CROPIS will be used in reducing organic waste and in the development of efficient life support systems. Its core element is a microbiological trickling filter of lava rock –the habitat of a multitude of microorganisms that purify and decontaminate water. The development aims at a wet composting system that may be used in closed life support systems such as waste water recovery. A key component of the system is the nitrogen cycle. So, modeling the nitrogen cycle of the system is essential to understanding how the system functions. It will be the first time nitrogen-transformation reactions will be measured as a function of gravity. NASA has an excellent opportunity to participate in the DLR’s Eu:CROPIS mission that allows us to obtain data by leveraging their laboratory work and hardware at a fraction of what it would cost if supported solely by NASA.