Urine nitrification with a synthetic microbial community.

During long-term extra-terrestrial missions, food is limited and waste is generated. By recycling valuable nutrients from this waste via regenerative life support systems, food can be produced in space. Astronauts' urine can, for instance, be nitrified by micro-organisms into a liquid nitrate fertilizer for plant growth in space. Due to stringent conditions in space, microbial communities need to be be defined (gnotobiotic); therefore, synthetic rather than mixed microbial communities are preferred. For urine nitrification, synthetic communities face challenges, such as from salinity, ureolysis, and organics. In this study, a synthetic microbial community containing an AOB (Nitrosomonas europaea), NOB (Nitrobacter winogradskyi), and three ureolytic heterotrophs (Pseudomonas fluorescens, Acidovorax delafieldii, and Delftia acidovorans) was compiled and evaluated for these challenges. In reactor 1, salt adaptation of the ammonium-fed AOB and NOB co-culture was possible up to 45mScm-1, which resembled undiluted nitrified urine, while maintaining a 44±10mgNH4+-NL-1d-1 removal rate. In reactor 2, the nitrifiers and ureolytic heterotrophs were fed with urine and achieved a 15±6mg NO3--NL-1d-1 production rate for 1% and 10% synthetic and fresh real urine, respectively. Batch activity tests with this community using fresh real urine even reached 29±3mgNL-1d-1. Organics removal in the reactor (69±15%) should be optimized to generate a nitrate fertilizer for future space applications.

Reactivation of Microbial Strains and Synthetic Communities After a Spaceflight to the International Space Station: Corroborating the Feasibility of Essential Conversions in the MELiSSA Loop.

To sustain human deep space exploration or extra-terrestrial settlements where no resupply from the Earth or other planets is possible, technologies for in situ food production, water, air, and waste recovery need to be developed. The Micro-Ecological Life Support System Alternative (MELiSSA) is such a Regenerative Life Support System (RLSS) and it builds on several bacterial bioprocesses. However, alterations in gravity, temperature, and radiation associated with the space environment can affect survival and functionality of the microorganisms. In this study, representative strains of different carbon and nitrogen metabolisms with application in the MELiSSA were selected for launch and Low Earth Orbit (LEO) exposure. An edible photoautotrophic strain (Arthrospira sp. PCC 8005), a photoheterotrophic strain (Rhodospirillum rubrum S1H), a ureolytic heterotrophic strain (Cupriavidus pinatubonensis 1245), and combinations of C. pinatubonensis 1245 and autotrophic ammonia and nitrite oxidizing strains (Nitrosomonas europaea ATCC19718, Nitrosomonas ureae Nm10, and Nitrobacter winogradskyi Nb255) were sent to the International Space Station (ISS) for 7 days. There, the samples were exposed to 2.8 mGy, a dose 140 times higher than on the Earth, and a temperature of 22°C ± 1°C. On return to the Earth, the cultures were reactivated and their growth and activity were compared with terrestrial controls stored under refrigerated (5°C ± 2°C) or room temperature (22°C ± 1°C and 21°C ± 0°C) conditions. Overall, no difference was observed between terrestrial and ISS samples. Most cultures presented lower cell viability after the test, regardless of the type of exposure, indicating a harsher effect of the storage and sample preparation than the spaceflight itself. Postmission analysis revealed the successful survival and proliferation of all cultures except for Arthrospira, which suffered from the premission depressurization test. These observations validate the possibility of launching, storing, and reactivating bacteria with essential functionalities for microbial bioprocesses in RLSS.

Membrane stripping enables effective electrochemical ammonia recovery from urine while retaining microorganisms and micropollutants.

Ammonia recovery from urine avoids the need for nitrogen removal through nitrification/denitrification and re-synthesis of ammonia (NH3) via the Haber-Bosch process. Previously, we coupled an alkalifying electrochemical cell to a stripping column, and achieved competitive nitrogen removal and energy efficiencies using only electricity as input, compared to other technologies such as conventional column stripping with air. Direct liquid-liquid extraction with a hydrophobic gas membrane could be an alternative to increase nitrogen recovery from urine into the absorbent while minimizing energy requirements, as well as ensuring microbial and micropollutant retention. Here we compared a column with a membrane stripping reactor, each coupled to an electrochemical cell, fed with source-separated urine and operated at 20 A m-2. Both systems achieved similar nitrogen removal rates, 0.34 ±â€¯0.21 and 0.35 ±â€¯0.08 mol N L-1 d-1, and removal efficiencies, 45.1 ±â€¯18.4 and 49.0 ±â€¯9.3%, for the column and membrane reactor, respectively. The membrane reactor improved nitrogen recovery to 0.27 ±â€¯0.09 mol N L-1 d-1 (38.7 ±â€¯13.5%) while lowering the operational (electrochemical and pumping) energy to 6.5 kWhe kg N-1 recovered, compared to the column reactor, which reached 0.15 ±â€¯0.06 mol N L-1 d-1 (17.2 ±â€¯8.1%) at 13.8 kWhe kg N-1. Increased cell concentrations of an autofluorescent E. coli MG1655 + prpsM spiked in the urine influent were observed in the absorbent of the column stripping reactor after 24 h, but not for the membrane stripping reactor. None of six selected micropollutants spiked in the urine were found in the absorbent of both technologies. Overall, the membrane stripping reactor is preferred as it improved nitrogen recovery with less energy input and generated an E. coli- and micropollutant-free product for potential safe reuse. Nitrogen removal rate and efficiency can be further optimized by increasing the NH3 vapor pressure gradient and/or membrane surface area.

Anaerobic ureolysis of source-separated urine for NH3 recovery enables direct removal of divalent ions at the toilet.

Source-separated urine is of interest for nutrient recovery. Most nitrogen recovery technologies rely on ammonia (NH3) as input, which requires ureolysis. As urease positive bacteria are widespread, source-separated urine is unstable, not only leading to NH3 release but also loss, odor nuisance, and downstream scaling. Hence, ureolysis ideally occurs in a closed controlled environment close to the toilet. We characterized microbial-induced ureolysis, subsequent divalent cation precipitation, and fermentation in anaerobic sequencing batch reactors (SBRs) at 15 °C and 28 °C. Temperatures were a proxy for urine hydrolysis in a wet well at street level or in the toilet, respectively. The need for inoculation and the metabolic stability was assessed by inoculation with autofermented urine or a mixture of anaerobic digestion and fermentation sludge. The highest specific ureolysis rates in the SBRs were achieved at 28 °C: 2107 ±â€¯395 and 1948 ±â€¯1121 mg N g VSS-1 d-1, for the mixed and autofermented inoculum, respectively. For Ca2+ and Mg2+ precipitation, and organics fermentation, autofermented urine at 28 °C performed best with 47.9 ±â€¯16.4 mg Ca2+ g VSS-1 d-1, 8.2 ±â€¯4.6 mg Mg2+ g VSS-1 d-1, and 623 ±â€¯129 mg VFA-COD g VSS-1 d-1, respectively. This indicates the hydrolysis reactor should be close to the toilet. The selected inoculum did not impact ureolysis, whereas both Ca2+ and Mg2+ precipitation and fermentation were better in the SBRs with autofermented urine. Ureolysis was identified as the only process significantly impacting the microbial community, indicating external inoculation would not be required. A urine hydrolysis reactor in the toilet without external inoculation could thus serve as a controlled environment to release NH3 and remove divalent cations to prevent scaling in downstream transport and processing. For practical implementation in a household toilet, the reactor should be designed for user-friendly precipitate discharge and odor control.

Electrochemical Ammonia Recovery from Source-Separated Urine for Microbial Protein Production.

Conventional plant and meat protein production have low nitrogen usage efficiencies and high energy needs. Microbial protein (MP) is an alternative that offers higher nitrogen conversion efficiencies with low energy needs if nitrogen is recovered from a concentrated waste source such as source-separated urine. An electrochemical cell (EC) was optimized for ammonia recovery as NH3/H2 gas mixtures usable for MP production. Undiluted hydrolyzed urine was fed to the caustic-generating cathode compartment for ammonia stripping with redirection to the anode compartment for additional ammonium extraction. Using synthetic urine at 48 A m-2 the nitrogen removal efficiency reached 91.6 ± 2.1%. Tests with real urine at 20 A m-2, achieved 87.1 ± 6.0% and 68.4 ± 14.6% requiring 5.8 and 13.9 kWh kg N-1 recovered, via absorption in acid or MP medium, respectively. Energy savings through accompanying electrolytic H2 and O2 production were accounted for. Subsequently, MP was grown in fed-batch on MP medium with conventional NH4+ or urine-derived NH3 yielding 3.74 ± 1.79 and 4.44 ± 1.59 g CDW L-1, respectively. Dissolution of gaseous NH3 in MP medium maintained neutral pH in the MP reactor preventing caustic addition and thus salt accumulation. Urine-nitrogen could thus be valorized as MP via electrochemical ammonia recovery.

The microbiome as engineering tool: Manufacturing and trading between microorganisms.

The integration of microbial technologies within the framework of the water-energy nexus has been taking place for over a century, but these mixed microbial communities are considered hard to deal with 'black boxes'. Process steering is mainly based on avoiding process failure by monitoring conventional parameters, e.g., pH and temperature, which often leads to operation far below the intrinsic potential. Mixed microbial communities do not reflect a randomised individual mix, but an interacting microbiological entity. Advance monitoring to obtain effective engineering of the microbiome is achievable, and even crucial to obtain the desired performance and products. This can be achieved via a top-down or bottom-up approach. The top-down strategy is reflected in the microbial resource management concept that considers the microbial community as a well-structured network. This network can be monitored by means of molecular techniques that will allow the development of accurate and quick decision tools. In contrast, the bottom-up approach makes use of synthetic cultures that can be composed starting from defined axenic cultures, based on the requirements of the process under consideration. The success of both approaches depends on real-time monitoring and control. Of particular importance is the necessity to identify and characterise the key players in the process. These key players not only relate with the establishment of functional conversions, but also with the interaction between partner bacteria. This emphasises the importance of molecular (screening) techniques to obtain structural and functional insights, minimise energy input, and maximise product output by means of integrated microbiome processes.

Microbial community redundancy in anaerobic digestion drives process recovery after salinity exposure.

Anaerobic digestion of high-salinity wastewaters often results in process inhibition due to the susceptibility of the methanogenic archaea. The ability of the microbial community to deal with increased salinity levels is of high importance to ensure process perseverance or recovery after failure. The exact strategy of the microbial community to ensure process endurance is, however, often unknown. In this study, we investigated how the microbial community is able to recover process performance following a disturbance through the application of high-salinity molasses wastewater. After a stable start-up, methane production quickly decreased from 625 ± 17 to 232 ± 35 mL CH4 L-1 d-1 with a simultaneous accumulation in volatile fatty acids up to 20.5 ± 1.4 g COD L-1, indicating severe process disturbance. A shift in feedstock from molasses wastewater to waste activated sludge resulted in complete process recovery. However, the bacterial and archaeal communities did not return to their original composition as before the disturbance, despite similar process conditions. Microbial community diversity was recovered to similar levels as before disturbance, which indicates that the metabolic potential of the community was maintained. A mild increase in ammonia concentration after process recovery did not influence methane production, indicating a well-balanced microbial community. Hence, given the change in community composition following recovery after salinity disturbance, it can be assumed that microbial community redundancy was the major strategy to ensure the continuation of methane production, without loss of functionality or metabolic flexibility.