Ox 25

A Unisense oxygen measuring system 1-CH (Unisense, OXY METER) equipped with a micromanipulator (Unisense, MM33), motorized micromanipulator stage (Unisense, MMS), motor controller (Unisense, MC-232), and a 25-μm Clark type/amperometric oxygen sensor (Unisense, OX-25) were used to quantify oxygen above and within colony biofilms. The readings were automated and analyzed by using SensorTrace Suite software v3.1.151 (Unisense, STSUITE). Colony biofilms were point inoculated with 1,000 spores in 0.002 ml on glucose minimal medium with 1.5% agar and cultured in normal oxygen or hypoxia (0.2% O₂) with 5% CO₂ at 37° for 72 h. The calibrated oxygen sensor was positioned 1.2 or 0.5 mm above the agar surface and measurements in technical duplicates were acquired every 0.1 mm over a period of 5 s with a 5-s wait period at each new depth. Colony biofilms were analyzed in a minimum of biological triplicates.

A 25-µm-tip oxygen microsensor (Unisense OX-25) was used to measure oxygen concentrations within biofilms during the first 2 days of development, grown as described above. For oxygen profiling on 3-day-old colonies (Figure 4), biofilms were grown as for the thin sectioning analyses. To calibrate the oxygen microsensor, a two-point calibration was used. The oxygen microsensor was calibrated first to atmospheric oxygen using a calibration chamber (Unisense CAL300) containing water continuously bubbled with air. The microsensor was then calibrated to a ‘zero’ point using an anoxic solution of water thoroughly bubbled with N₂; to ensure complete removal of all oxygen, N₂ was bubbled into the calibration chamber for a minimum of 30 min before calibrating the microsensor to the zero calibration point. Oxygen measurements were then taken throughout the depth of the biofilm using a measurement time of 3 s and a wait time between measurements of 5 s. For 6-hr-old colonies, a step size of 1 µm was used to profile through the entire colony; for 12 hr and 24 hr colonies, 2 µm; for 48 hr colonies, 5 µm. A micromanipulator (Unisense MM33) was used to move the microsensor within the biofilm and profiles were recorded using a multimeter (Unisense) and the SensorTrace Profiling software (Unisense).

The profiles of the O₂ concentration as a function of depth in the microbial mat were measured in situ by using a Clark-type O₂ microsensor (OX25; Unisense, Aarhus, Denmark) with a tip diameter of <25 µm, low stirring sensitivity (<1–2%) and fast response time (t₉₀ < 0.5 s). The O₂ microsensor was mounted on a motorized micromanipulator (Unisense, Aarhus, Denmark) and connected to a PC-interfaced pA-meter (Unisense, Aarhus, Denmark), both of which were controlled by dedicated data acquisition, profiling, and positioning software (SensorTrace Pro, Unisense, Aarhus, Denmark). The micromanipulator was mounted on a metal stand placed next to the hot spring, allowing for vertical insertion of the microsensor tip into the microbial mat under natural flow, temperature and light conditions. The microsensor tip was carefully positioned at the mat surface (defined as 0 µm) by manual operation of the micromanipulator. Subsequently, O₂ microprofiles were recorded automatically every 15 min for 24 h starting at 18:00 on 3 November 2016. In each profile, O₂ measurements were made in 100 µm increments from the water-phase and into the mat. One measurement was taken per depth and, for each measurement a 10 s wait period was applied, to ensure steady O₂ signal, and the O₂ signal was then recorded averaged over a 1 s period.