Metabolt: An In-Situ Instrument to Characterize the Metabolic Activity of Microbial Soil Ecosystems Using Electrochemical and Gaseous Signatures

Metabolt is a portable soil incubator to characterize the metabolic activity of microbial ecosystems in soils. It measures the electrical conductivity, the redox potential, and the concentration of certain metabolism-related gases in the headspace just above a given sample of regolith. In its current design, the overall weight of Metabolt, including the soils (250 g), is 1.9 kg with a maximum power consumption of 1.5 W. Metabolt has been designed to monitor the activity of the soil microbiome for Earth and space applications. In particular, it can be used to monitor the health of soils, the atmospheric-regolith fixation, and release of gaseous species such as N2, H2O, CO2, O2, N2O, NH3, etc., that affect the Earth climate and atmospheric chemistry. It may be used to detect and monitor life signatures in soils, treated or untreated, as well as in controlled environments like greenhouse facilities in space, laboratory research environments like anaerobic chambers, or simulating facilities with different atmospheres and pressures. To illustrate its operation, we tested the instrument with sub-arctic soil samples at Earth environmental conditions under three different conditions: (i) no treatment (unperturbed); (ii) sterilized soil: after heating at 125 °C for 35.4 h (thermal stress); (iii) stressed soil: after adding 25% CaCl2 brine (osmotic stress); with and without addition of 0.5% glucose solution (for control). All the samples showed some distinguishable metabolic response, however there was a time delay on its appearance which depends on the treatment applied to the samples: 80 h for thermal stress without glucose, 59 h with glucose; 36 h for osmotic stress with glucose and no significant reactivation in the pure water case. This instrument shows that, over time, there is a clear observable footprint of the electrochemical signatures in the redox profile which is complementary to the gaseous footprint of the metabolic activity through respiration.


Electrical Design and System Software
The core of the electronic box is the Whitebox Labs Tentacle Shield for the Arduino and an Arduino Mega 2560 Rev 3. The Tentacle Shield houses the three Atlas Scientific EZO™ circuits: an EC, and the two ORP circuits that are galvanically isolated from each other.
These circuits were programmed to communicate with the ATmega328 processor sequentially via a universal I 2 C bus. However, from the two experiment containers, we have two EC signals and six ORP signals. To avoid stacking multiple tentacle shields to accommodate more Atlas Scientific EZO™ circuits that would eventually increase the weight, power and cost, Metabolt uses the sequential signal routing approach with the 2:1 and 4:1 bidirectional multiplexer/demultiplexers. The routing part of the circuit was built on a prototyping breadboard and mounted in the casing of the experiment containers, such that only the signal from one of the sensors/probes is transmitted at a time. Redox potential signals from each of the experiment containers were routed to a different ORP circuit to avoid any possible ground looping while the EC circuit was shared. The data lines of the two DS18B20 temperature sensors, one from each experiment, were connected to a single digital pin of the Arduino and programmed to communicate via a universal I 2 C bus. The oxygen and the carbon dioxide sensors communicate directly with the Arduino by the TX/RX TTL serial communication via the three external TX/RX pins and the two software serial ports.
In addition, the electronic box houses a 4 x 1.5 V AA battery pack, serially connected as a 6 V equivalent power supply, that runs through a slider switch for the user power mode selection and 2 then to the Vin pin of the Arduino, where it is regulated to a 5 V. A micro-SD shield was integrated, providing the user with access to the unprocessed raw data of the experiments and the three status LEDs to monitor the progress. The experiment containers and the electronic box share the electrical connections through a male-female DB-25 cable that plugs into the female DB-25 ports installed on the experiment containers casing and the electronic box.
The ATmega328 processor of the Arduino Mega 2560 Rev 3 controls the entire commanding, processing and the data management protocols. The source code of the system software was developed in the Arduino IDE and can be updated real-time by connecting the Arduino USB port to a PC. The system software was designed systematically to be executed in four stages. 1) Sensor setup, 2) Calibration, 3) Data acquisition and transmission, and 4) Hibernation. Sensor setup prepares the sensors by sending the proper commands regarding the sensor status, output format, etc. Calibration stage assigns the calibration values to the sensors and circuits, if any. It also test-runs the sensors for a few seconds to make sure that everything is in order and ready to conduct the experiments. Data acquisition and transmission is handled by sending the data request commands to the sensors and circuits and after a short processing time delay, the individual variables receive the data in a specified format. Once all the eight measurements were carried out, the data were formatted as the comma-separated values and transmitted to the PC and/or stored in the micro-SD card. After a few allowed iterations at a specified frequency of the data acquisition and transmission, the instrument was hibernated for a specified time to save power and automatically woke up to continue the process. The timing, delays and the counters were managed by the in-built Arduino library function, millis().

Data Management Modes
Metabolt supports the two modes by which the data from the experiment are utilized. Depending on the user preferences, nature of the operation, and the mobility and power constraints, the experiment data can either be transmitted to a PC through the USB data connection and/or can be stored locally in the in-built memory (.csv/.txt/etc.).
The local storage mode is simple and straight-forward but allows accessing the data after the completion of the experiments or during the hibernation time to avoid any data loss. On the other hand, the PC mode allows visualizing the data real-time with any serial data acquisition tool and saving in any suitable format (.csv/.txt/etc.). A copy of the unprocessed data is also maintained in the micro-SD storage in this mode.
The data storage requirements for the Metabolt are small: less than 500 kB for a 10-day experiment.
3 Figure S1. Results of (a) Electrical Conductivity (EC), (b) Redox potential (Eh), (c) Oxygen concentration, (d) Carbon dioxide concentration measurements with no soil in experiment containers for 70 min during the afternoon between 15:47 and 16:57. Electrical conductivity shows no variation due to absence in contact between electrodes, redox potential just showing arbitrary values with exposure to air and the oxygen and carbon dioxide concentrations reaching equilibrium with the ambient environment and a gradual lowering in temperature. The color bar represents the air temperature (°C). 5 Figure S4. Comparison of the water activity (aw) and the concentration of CaCl2. Also highlighted (circles) is the predicted water activity for our experimental condition with the 25% concentration of CaCl2 and its correlation with the achieved relative humidity. The experiment shows a delayed response after 4 days of incubation when the metabolic process in the mine sample is activated enough to be measurable. After activation, the diurnal variation as a function of daily temperature changes could be observed.