NASA's Phoenix lander measured high perchlorate concentrations (0.5% to 1.0%) in the regolith of Mars. In contrast, perchlorate is widely distributed on Earth, but typically occurs at only very low abundances (<<0.01%). However, it is hazardous at low concentrations to humans, and exposure limits of 24 ppb have been established for drinking water. Perchlorate is also mutagenic for some bacteria, and can have adverse impacts on a variety of animals. Thus, it clearly poses challenges for both human habitation and exploration of Mars, as well as for terraforming based on the use microbes that might prove perchlorate-sensitive. Thus, it is essential to identify bacteria that can detoxify perchlorate, and that also can provide additional ecosystem services through biogeochemical functions that contribute to terraforming. TECHNICAL IMPLEMENTATION Bacteria capable of reducing perchlorate to non-toxic chloride have been documented previously, but most of these isolates have limited application for Mars, since they require reductants, such as hydrogen, acetate or other complex organics, that do not occur at significant levels in regolith. Furthermore, high salt concentrations, which are common at Mars' surface, inhibit most known perchlorate reducers. Recently, however, several common extremely halophilic archaea have been identified that reduce perchlorate to chloride; in addition, several closely related extremely halophilic archaea have also been shown to use carbon monoxide (CO) as a metabolic substrate. Since CO occurs at very high concentrations (> 800 ppm) in Mars' atmosphere, we propose to investigate the feasibility of using a novel process, i.e., halophilic CO-dependent perchlorate reduction, for perchlorate bioremediation. We expect to establish this process quickly, since there is no biological reason that the two should be mutually exclusive, and since the two functions now appear to be relatively common among the halophilic euryarchaeota. INNOVATION STATEMENT The feasibility of detoxifying perchlorate-contaminated groundwater has been demonstrated previously in bioreactors using acetate or hydrogen as a source of reductant. However, the suitability of CO as a reductant has not yet been pursued. The work proposed here will thus represent the first attempt to employ a novel microbial process to detoxify perchlorate using an atmospheric resource abundant on Mars. We also anticipate that the outcome of our work will have broad applications for terraforming and Mars habitation. TECHNICAL READINESS LEVEL (TRL) TRL (10/1/14): TRL 2 TRL (9/30/15): TRL 4 PARTNERSHIPS Brad Bebout will conduct analyses of CO uptake and perchlorate reduction in previously collected saline soils/sediments and microbial mats already in his possession from a number of field collections, as well as field sites to be visited as part of the work proposed here (Task 1). This will serve to identify target sites for enrichment/isolation efforts and to characterize possible relevant ecological/environmental controls. He will construct engineered communities of organisms capable of perchlorate reduction and organisms capable of generating reductant necessary for this process (cyanobacteria) (Task 4). Gary King will screen existing and new isolates for CO oxidation/perchlorate reduction using molecular and physiological assays (Task 2) and assess environmental tolerances of the isolates for CO-based perchlorate reduction (Task 3). He will obtain material for new isolations at a promising new field locality at Bonneville Salt Flat, near Salt Lake City, Utah. DELIVERABLES Task 1. Characterize CO uptake and perchlorate reduction in natural communities of extreme halophiles. Expected completion: 10/1/14 - 3/30/15. Details: We will assess both CO uptake and perchlorate reduction by natural communities of extreme halophiles using two model systems: salt crusts and salt-saturated sediments of the Bonneville Salt Flat (UT) and arid, saline soils from Wendover, UT south of Bonneville. We will amend crusts, sediments and soils with perchlorate under a variety of conditions and manipulate CO availability to determine the capacity of these systems to reduce perchlorate, and to couple reduction to CO oxidation. Results will help identify systems to target for enrichment and isolation, and will provide novel insights about controls of perchlorate reduction relevant for understanding multi-species communities that might ultimately be assembled for use on Mars. Task 2. Identify novel extremely halophilic CO-oxidizing perchlorate reducers. Expected completion: 10/1/14 - 9/30/15. Details: We will screen new and existing collections of extreme halophiles for CO oxidation, and identify perchlorate-reducing isolates. New collections will be derived from ongoing efforts to characterize halophiles in saline arid soils and salt flats; existing isolates will be selected from a wide range of sources, including solar salterns, and saline temperate, tropical and Antarctic lakes. Task 3. Assess environmental tolerances for CO-based perchlorate reduction. Expected Completion: 1/1/14 - 9/30/15. Details: Isolates obtained from Task 2 will be used to determine the range of environmental conditions under which extreme halophiles reduce perchlorate with CO as a substrate. Variables will include salt, perchlorate, nitrate, oxygen and CO concentrations, temperature, and pressure among others. Task 4. Using a Synthetic Ecology Approach to Build Communities Capable of Using the Sun's Energy to Reduce Perchlorate. Expected Completion: 4/1/14 - 9/30/15. Details: We will construct model microbial communities of extremely halophilic CO-oxidizers, and assess their capacity to persist using CO. Isolates from Task 2 will be grown individually on basalt cinders, sand or Mars regolith analog following protocols we have used successfully with Alkalilimnicola ehrlichii MLHE1. We will also use additional isolates to develop more functionally complex communities. For example, the EPS-producing moderately halophilic Halomonas maura (Argandoña et al., 2006) will be used to add a capacity for nitrogen fixation, and MLHE1 will be used to introduce complexity to nitrogen transformations (nitrate respiration versus denitrification) and an alternate pathway for CO2 fixation (CBB; Hoeft et al., 2007). Total densities of mixed populations will be equivalent to those of individual populations. We will then subject both individual and mixed populations to water potential and temperature stresses with varied oxidant regimes to assess impacts on CO uptake and population and community persistence. To measure persistence, we will use growth-based recovery assays combined with 16S rRNA abundance measurements for mixed populations. The use of cyanobacteria to provide acetate (via fermentation) as a reductant for PRB will be investigated. Cyanobacteria isolated from microbial mats are known to support the growth of various heterotrophic bacteria via fermentation of their exuded photosynthate. We will combine these cyanobacteria with PRB.