Development of the microfluidic Microbial Activity Microassay for detection and characterization of extant microorganisms from extreme environments
Abstract
Despite a major focus of planetary exploration being the search for life outside Earth current instruments available for astrobiology space missions are focused on identification of habitable environments or on detecting biosignatures. No direct life detection instrumentation has been deployed in space since the Viking missions in the 1970s. The microfluidic Microbial Activity MicroAssay ($\mu$MAMA) is one such direct life detection instrument and is being developed as part of a larger life detection payload capable of detecting unambiguous signs of life. As redox dye analyses are simple, low-cost, highly sensitive methods to determine and characterize in-situ extant microbial activity, they are amongst the best candidates to be automated for unambiguous microbial metabolic detection. The $\mu$MAMA, with its own microfluidic inoculation system, can detect and metabolically characterize microbial life in the laboratory, and extant microbial life from very low biomass cryoenvironments, using redox dye chemistry to detect microbial activity. The redox dyes are reduced, producing a detectable color change, if extant microorganisms in an environmental sample can oxidize the provided substrate (either organic or inorganic). We identified the BIOLOG Dye Mix G plus the BIOLOG IF-0a buffer, and the AlamarBlue dye plus the BIOLOG IF-0a buffer as the best combinations of redox dye/inoculation buffer, in terms of 1) detection limits, 2) robustness, and 3) autonomy. Metabolic activity was detected with as low as 100 cell/ml (bacteria/yeast) using pure cultures of Rhodotorula sp. JG-1b and Planococcus halocryophilus and in a variety of astrobiology analogue samples from polar sites including permafrost, unique Arctic cold saline springs, glacial and sea ice using organic substrates as electron donors and, significantly, for the first time, detecting lithoautotrophic metabolic activity using an autotrophic nitrate-reducing Fe(II)-oxidizing culture. The redox dyes were shown to be very robust as they did not provide false positive redox reactions when exposed to a range of temperatures (-10 to 37$^\circ$C), pH (4 to 13), salinities (0-30% NaCl, perchlorates), and Mars analog soil. In 2019, a semi-automated µMAMA card prototype, was successfully field tested with the two dyes in the Canadian high Arctic and showed detection of metabolic activity from various astrobiology analogue samples. To further develop this technology into a high technology readiness level life detection instrument testing is ongoing in 4 major areas. 1) We're continuing to test the detection capabilities of $\mu$MAMA with organisms employing diverse autotrophic and heterotrophic metabolisms. 2) Due to the ability of terrestrial microorganisms to become dormant or form spores under extreme environmental conditions we're evaluating the $\mu$MAMAs ability to detect low levels of metabolic activity produced by cells in this state. 3) The $\mu$MAMA is being developed to contain 16 wells per card. This design allows us to account for metabolic activity from a wide range of metabolisms thus increasing our chances of detection. Like the labelled release experiment flown on the Viking missions a rigorous test campaign will be required to test and select substrates to be included in the wells. Organic and inorganic substrates will be selected based on their long-term stability, structure, and no unspecific reactivity to the dyes. 4) We hope to further improve upon the overall design of the device and field test future prototypes in various astrobiology analogue sites such as those found in the Canadian High Arctic.
- Publication:
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43rd COSPAR Scientific Assembly. Held 28 January - 4 February
- Pub Date:
- January 2021
- Bibcode:
- 2021cosp...43E.204O