Abstract
The ability to travel long distances over extended periods of time in space depends on the capacity to sustain astronauts, providing them with food, potable water and a breathable atmosphere throughout the mission duration. Supplying the required resources for such an endeavor is currently impossible due to the long transporting times, size constraints and high costs for cargo transport. Therefore, technological development focuses on in situ recovery of waste streams and production of fresh food, water and oxygen. One promising type of technology are bioregenerative life support systems (BLSS), which use a combination of biological and physiochemical processes to recover food, water and oxygen from waste streams. A space vessel that utilizes a BLSS will require only limited amounts of provisions, thereby providing the opportunity to increase crewed space mission durations.
The Micro-Ecological Life Support System Alternative (MELiSSA) project of the European Space Agency (ESA) aims at the recovery of 98 - 100% of nutrients from waste generated by the crew. The MELiSSA closed loop consists out of 5 compartments populated by different types of (micro)organisms, each with a distinct function to achieve full recovery from waste streams. In compartment III (CIII), a nitrifying consortium of NH3 oxidizing Nitrosomonas europaea and NO2- oxidizing Nitrobacter winogradskyi converts NH4+ to NO3- in a two-step process. NO3- serves as a nitrogen source for cultivation of higher plants and cyanobacteria for food and O2 production in the subsequent compartment. Urea, the most abundant nitrogen-containing compound present in urine, cannot be converted to NO3- by this consortium. Urea requires urease-positive bacteria to convert urea to NH4+. Therefore, the heterotrophic urease-positive strain Comamonas testosteroni was selected as a possible addition to the current CIII nitrifying consortium.
For spaceflight applications, the feasibility of ureolysis and nitrification in space has to be assessed before advancing with development of a technology demonstrator. In the previous NITRIMEL (2014) and BISTRO (2015) spaceflight experiments, the ability to reactivate N. europaea and N. winogradskyi after spaceflight exposure was confirmed. The next step in the process is a proof-of-concept study of active urine nitrification in space (URINIS project), where the effects of spaceflight will be assessed on active cultures of C. testosteroni, N. europaea and N. winogradskyi and on a synthetic culture of all three constituents. This PhD dissertation, as part of the URINIS project, mainly focuses on the whole transcriptomic responses of these bacteria exposed to simulated space conditions by means of RNA sequencing (RNA-Seq).
In Chapter 2, we discuss the optimization of an RNA extraction process to enable the extraction of high-quality RNA in adequate yields for downstream RNA-Seq. Due to their autotrophic nature, nitrifying bacteria exhibit slow growth rates and limited biomass generation. These constraints, combined with a limited amount of culturing volume due to restrictions in a spaceflight experiment, requires an optimal amount of RNA to be extracted from limited material. In the RNA extraction optimization process, the performance of standard commercial silica-column based RNA extraction kits amended by ultrasonication or enzymatic lysis was assessed. Optimization was performed on N. europaea cultures and was followed up by a validation on N. winogradskyi. First, commercial RNA extraction kits were tested by determining the RNA yields of the various kits without any additional pretreatment, but no clear differences were found. By adding an ultrasonication step, the NucleoSpin XS RNA (NS XS) kit generated significantly more RNA than the other kits, but RNA was highly degraded (low quality). Modifications to the ultrasonication procedure did not improve the resulting RNA extracts. On the other hand, enzymatic lysis through lysozyme digestion generated minimally degraded (high-quality), high-yield RNA samples. Subsequent RNA-Seq analysis of RNA samples from the low-biomass producing nitrifiers using lysozyme digestion and RNA extraction with the NS XS kit was successful and in accordance with quality thresholds. The RNA extraction protocol also proved to extract high amounts of qualitative RNA from the fast-growing heterotrophic C. testosteroni cultures. This optimized RNA extraction procedure could be used for terrestrial experiments to assess the effects of the simulated spaceflight environment as well as for the URINIS spaceflight experiment in future work.
Chapter 3 describes the effects of simulated microgravity (SMG) conditions on the three pure strains and on the tripartite community. Two microgravity-analogue devices were used; the rotary cell culture system (RCCS) and the random positioning machine (RPM). The RCCS simulated microgravity by rotating continuously in a 2D-plane parallel to the gravity vector, creating an environment where the bacteria are in continuous free-fall. The RPM, on the other hand, rotates in a 3D-plane at random speeds and in random directions, confusing the biological specimens’ perception of gravity. By filling the vessels to capacity, leaving no headspace, solid body rotation is achieved in both microgravity-analogues. Because of significant air bubble production by C. testosteroni due to its CO2 production during ureolysis, the use of the conventional high aspect ratio vessels (HARVs) for the RCCS and RPM had to be reconsidered. These air bubbles have a detrimental effect on the fluid dynamics within a vessel and, consequently, on the SMG environment. In this PhD project, a PermaLife (PL)-70 cell culture bag mounted on in-house designed, 3D-printed holders was introduced to cultivate C. testosteroni and the tripartite culture in an SMG environment. The use of cell culture bags substantially reduced air bubble formation. These cell culture bags can also be used for future
experiments that study the effects of SMG on gas-producing bacteria. Gene expression changes after growth in the SMG environment indicated that fluid dynamics in SMG caused nutrient and O2 limitations due to mass transfer issues. Genes involved in urea hydrolysis and nitrification were minimally affected but denitrification-related gene expression was increased. The findings highlight potential obstacles for nitrogen recovery in space because denitrification results in a loss of recoverable nitrogen as NO3-.
Chapter 4 explores the effects of ionizing radiation (IR) on the synthetic microbial community and its constituents in monoculture. In particular, we assessed the radiotolerance of C. testosteroni, N. europaea and N. winogradskyi after exposure to acute γ-irradiation. After exposure to increasingly higher doses, the activity, survival and growth kinetics of the bacteria were assessed. The strains could all be categorized as radiosensitive strains. In a second segment of the study, bacteria were exposed to a high energy Cf-252 neutron source with an average dose rate of 5.09 × 10-1 mGy h-1. In an ‘accelerated life test’ setup, the samples were subjected to a total absorbed dose (DT) equivalent to a 4-month stay onboard of the International Space Station, over the course of three days. A comprehensive whole transcriptome analysis indicated that the IR environment elicited nuanced and varying responses across the bacterial strains. Importantly, no significant impact on ureolysis and nitrification genes was observed, indicating that nitrogen recovery may not be impacted by the increased IR in space.
Whole transcriptomic analysis of C. testosteroni, N. europaea and N. winogradskyi exposed to simulated space conditions allowed insights into the impact on gene expression and a validation that the bacteria were active. This research marked a milestone in paving the way for the proof-of-concept URINIS spaceflight study focused on nitrification processes in space and highlighted potential challenges of nitrification for spaceflight applications.
The Micro-Ecological Life Support System Alternative (MELiSSA) project of the European Space Agency (ESA) aims at the recovery of 98 - 100% of nutrients from waste generated by the crew. The MELiSSA closed loop consists out of 5 compartments populated by different types of (micro)organisms, each with a distinct function to achieve full recovery from waste streams. In compartment III (CIII), a nitrifying consortium of NH3 oxidizing Nitrosomonas europaea and NO2- oxidizing Nitrobacter winogradskyi converts NH4+ to NO3- in a two-step process. NO3- serves as a nitrogen source for cultivation of higher plants and cyanobacteria for food and O2 production in the subsequent compartment. Urea, the most abundant nitrogen-containing compound present in urine, cannot be converted to NO3- by this consortium. Urea requires urease-positive bacteria to convert urea to NH4+. Therefore, the heterotrophic urease-positive strain Comamonas testosteroni was selected as a possible addition to the current CIII nitrifying consortium.
For spaceflight applications, the feasibility of ureolysis and nitrification in space has to be assessed before advancing with development of a technology demonstrator. In the previous NITRIMEL (2014) and BISTRO (2015) spaceflight experiments, the ability to reactivate N. europaea and N. winogradskyi after spaceflight exposure was confirmed. The next step in the process is a proof-of-concept study of active urine nitrification in space (URINIS project), where the effects of spaceflight will be assessed on active cultures of C. testosteroni, N. europaea and N. winogradskyi and on a synthetic culture of all three constituents. This PhD dissertation, as part of the URINIS project, mainly focuses on the whole transcriptomic responses of these bacteria exposed to simulated space conditions by means of RNA sequencing (RNA-Seq).
In Chapter 2, we discuss the optimization of an RNA extraction process to enable the extraction of high-quality RNA in adequate yields for downstream RNA-Seq. Due to their autotrophic nature, nitrifying bacteria exhibit slow growth rates and limited biomass generation. These constraints, combined with a limited amount of culturing volume due to restrictions in a spaceflight experiment, requires an optimal amount of RNA to be extracted from limited material. In the RNA extraction optimization process, the performance of standard commercial silica-column based RNA extraction kits amended by ultrasonication or enzymatic lysis was assessed. Optimization was performed on N. europaea cultures and was followed up by a validation on N. winogradskyi. First, commercial RNA extraction kits were tested by determining the RNA yields of the various kits without any additional pretreatment, but no clear differences were found. By adding an ultrasonication step, the NucleoSpin XS RNA (NS XS) kit generated significantly more RNA than the other kits, but RNA was highly degraded (low quality). Modifications to the ultrasonication procedure did not improve the resulting RNA extracts. On the other hand, enzymatic lysis through lysozyme digestion generated minimally degraded (high-quality), high-yield RNA samples. Subsequent RNA-Seq analysis of RNA samples from the low-biomass producing nitrifiers using lysozyme digestion and RNA extraction with the NS XS kit was successful and in accordance with quality thresholds. The RNA extraction protocol also proved to extract high amounts of qualitative RNA from the fast-growing heterotrophic C. testosteroni cultures. This optimized RNA extraction procedure could be used for terrestrial experiments to assess the effects of the simulated spaceflight environment as well as for the URINIS spaceflight experiment in future work.
Chapter 3 describes the effects of simulated microgravity (SMG) conditions on the three pure strains and on the tripartite community. Two microgravity-analogue devices were used; the rotary cell culture system (RCCS) and the random positioning machine (RPM). The RCCS simulated microgravity by rotating continuously in a 2D-plane parallel to the gravity vector, creating an environment where the bacteria are in continuous free-fall. The RPM, on the other hand, rotates in a 3D-plane at random speeds and in random directions, confusing the biological specimens’ perception of gravity. By filling the vessels to capacity, leaving no headspace, solid body rotation is achieved in both microgravity-analogues. Because of significant air bubble production by C. testosteroni due to its CO2 production during ureolysis, the use of the conventional high aspect ratio vessels (HARVs) for the RCCS and RPM had to be reconsidered. These air bubbles have a detrimental effect on the fluid dynamics within a vessel and, consequently, on the SMG environment. In this PhD project, a PermaLife (PL)-70 cell culture bag mounted on in-house designed, 3D-printed holders was introduced to cultivate C. testosteroni and the tripartite culture in an SMG environment. The use of cell culture bags substantially reduced air bubble formation. These cell culture bags can also be used for future
experiments that study the effects of SMG on gas-producing bacteria. Gene expression changes after growth in the SMG environment indicated that fluid dynamics in SMG caused nutrient and O2 limitations due to mass transfer issues. Genes involved in urea hydrolysis and nitrification were minimally affected but denitrification-related gene expression was increased. The findings highlight potential obstacles for nitrogen recovery in space because denitrification results in a loss of recoverable nitrogen as NO3-.
Chapter 4 explores the effects of ionizing radiation (IR) on the synthetic microbial community and its constituents in monoculture. In particular, we assessed the radiotolerance of C. testosteroni, N. europaea and N. winogradskyi after exposure to acute γ-irradiation. After exposure to increasingly higher doses, the activity, survival and growth kinetics of the bacteria were assessed. The strains could all be categorized as radiosensitive strains. In a second segment of the study, bacteria were exposed to a high energy Cf-252 neutron source with an average dose rate of 5.09 × 10-1 mGy h-1. In an ‘accelerated life test’ setup, the samples were subjected to a total absorbed dose (DT) equivalent to a 4-month stay onboard of the International Space Station, over the course of three days. A comprehensive whole transcriptome analysis indicated that the IR environment elicited nuanced and varying responses across the bacterial strains. Importantly, no significant impact on ureolysis and nitrification genes was observed, indicating that nitrogen recovery may not be impacted by the increased IR in space.
Whole transcriptomic analysis of C. testosteroni, N. europaea and N. winogradskyi exposed to simulated space conditions allowed insights into the impact on gene expression and a validation that the bacteria were active. This research marked a milestone in paving the way for the proof-of-concept URINIS spaceflight study focused on nitrification processes in space and highlighted potential challenges of nitrification for spaceflight applications.
Original language | English |
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Qualification | Doctor of Science |
Awarding Institution |
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Supervisors/Advisors |
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Date of Award | 19 Apr 2024 |
Publisher | |
Print ISBNs | 9789463577304 |
State | Published - 19 Apr 2024 |