Bioreactors hold great promise for treating graywater in an advanced life support system for space applications. However, questions remain regarding the reproducibility and reliability of biological systems for long-term use. Although there have been numerous studies on ground-based biological systems, most studies focus on a single reactor or a simple (single carbon) waste stream. There have been very few studies on microbial communities in replicate reactors using a nonsterile, complex waste stream. In this report, we describe the characterization of five replicate denitrifying reactors receiving a complex feed, including urine and limb washes from donors at Johnson Space Center over a 100-day period. Denitrifying conditions were employed because of the ease in adding a terminal electron acceptor to the bioreactor. Bacterial populations were tracked by 16S rRNA and nosZ genes T-RFLP analysis to target the total and denitrifying microbial communities. The results demonstrated reproducible biological communities with nearly identical performance that slowly changed with time and exhibited low variability with respect to the bacterial community (T-RFLP peak area) in all reactors. These results suggest that, when designed for replication, bioreactors are not stochastic systems exhibiting chaotic behavior, but are biological systems that can be highly reproducible and reliable.

Bioregenerative life support systems may be necessary for long-term space missions due to the high cost of lifting supplies and equipment into orbit. In this study, we investigated two biological wastewater treatment reactors designed to recover potable water for a spacefaring crew being tested at Johnson Space Center. The experiment (Lunar-Mars Life Support Test Project-Phase III) consisted of four crew members confined in a test chamber for 91 days. In order to recycle all water during the experiment, an immobilized cell bioreactor (ICB) was employed for organic carbon removal and a trickling filter bioreactor (TFB) was utilized for ammonia removal, followed by physical-chemical treatment. In this study, the spatial distribution of various microorganisms within each bioreactor was analyzed by using biofilm samples taken from four locations in the ICB and three locations in the TFB. Three target genes were used for characterization of bacteria: the 16S rRNA gene for the total bacterial community, the ammonia monooxygenase (amoA) gene for ammoniaoxidizing bacteria, and the nitrous oxide reductase (nosZ) gene for denitrifying bacteria. A combination of terminal restriction fragment length polymorphism (T-RFLP), sequence, and phylogenetic analyses indicated that the microbial community composition in the ICB and the TFB consisted mainly of Proteobacteria, low-G؉C gram-positive bacteria, and a Cytophaga-Flexibacter-Bacteroides group. Fifty-seven novel 16S rRNA genes, 8 novel amoA genes, and 12 new nosZ genes were identified in this study. Temporal shifts in the species composition of total bacteria in both the ICB and the TFB and ammonia-oxidizing and denitrifying bacteria in the TFB were also detected when the biofilms were compared with the inocula after 91 days. This result suggests that specific microbial populations were either brought in by the crew or enriched in the reactors during the course of operation.A mission of the Advanced Life Support (ALS) Project is to develop bioregenerative life support systems to enhance the International Space Station and ultimately sustain a lunar base or a voyage to Mars. This necessitates creating a combination of physicochemical and biological systems that can regenerate resources (or materials) needed to sustain life for long time periods, possibly without the potential of resupply. Since bacteria perform recycling of various elements on Earth, it is reasonable to assume that a bioregenerative ALS system for a space vehicle or colony may also rely on bacterial activity. Understanding the microbial ecology in a biological treatment system is important for designing and operating it at optimal efficiency and to ensure reliability. In 1997, two fixed-film biological wastewater treatment reactors were tested by the National Aeronautics and Space Administration (NASA) at the Johnson Space Center (JSC) during a 91-day simulated longterm manned space mission. This experiment (Lunar-Mars Life Support Test Project-Phase III) consisted of four crew members confined in a test chamb...

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