2.5. Measurement of NH4+ and O2 Fluxes by NMT

Net fluxes of NH₄⁺ and O₂ were measured noninvasively using the noninvasive microtest technique (NMT-100 series System, Younger USA LLC, Amherst, MA, USA; Xuyue Sci. & Tech. Co., Ltd., Beijing, China). Prior to NH₄⁺ flux determination, a prepulled and silanized microsensor (Φ1.5 ± 0.5 μm, XY-CGQ-02, Younger, USA) was first filled with a backfilling solution (100 mM NH₄Cl) to a length of approximately 1 cm from the tip. The micropipette was front filled with 50–60 μm columns of selective liquid ion-exchange cocktails (NH₄⁺ LIX, XY-SJ-NH4, Younger, USA). An Ag/AgCl wire microsensor holder (YG003-Y11, Younger, USA) was inserted in the back of the microsensor, to make electrical contact with the electrolyte solution. YG003-Y11 was used as the reference microsensor. For calibration of the NH₄⁺ microsensor, we used measuring solution (0.1 mM NH₄Cl, 0.1 mM CaCl₂, 0.3 mM MES, 0.2 mM Na₂SO₄, pH 6) with different concentrations of NH₄⁺ (0.05 mM, 0.1 mM and 0.5 mM) to choose the qualified one with a Nernstian slope at 58 ± 5 mv/decade. The microsensor was then placed in the blank measuring solution for testing by X-10 until the NH₄⁺ flux was near the baseline, at which time the microsensor can be used [30].

To detect dissolved oxygen, the Pt/Ir polarographic oxygen microsensor (tip diameter 20 ± 5 μm, XY-CGQ-501, Younger USA) was used under -750-mV polarization voltage. A reference microsensor was also used to complete the circuit. Prior to O₂ flux measurement, the microsensor should be calibrated with measuring solution (0.1 mM KCl, 0.1 mM CaCl₂, 0.1 mM MgCl₂, 0.5 mM NaCl, 0.3 mM MES, 0.2 mM Na₂SO₄, pH 6) containing different concentrations of O₂ (N-saturated and control cultural media). Only when the Std Curve is between −2000~−9000 pA/mM can the microsensor be placed in the blank measuring solution for polarization for 1 h, until the net O₂ flux is near baseline, at which time the microsensor can be used [31].

During formal measurement, the fluxes of NH₄⁺ and O₂ were determined by measuring six similar samples separately. The potential difference was obtained by moving the microelectrode repeatedly from one point to another, in a direction perpendicular to the surface of the individual cell (Polar X-10), and fluxes were calculated automatically by Fick’s law of diffusion: J = −D(dc/dx). The steady-state flux measurements were continuously recorded for 6–10 min, and each measurement was repeated three times at different positions of the cell.