Labspec

In the collection of spectra, a cube of hyperspectral data with dimensions M × N × λ, with M and N corresponding to spatial information and λ to spectral information, was generated. The first step was to transfer the data from the equipment software (LabSpec v. 6.7.1.10, Horiba) to the MATLAB workspace (Mathworks, Natick, MA, USA, v. 8.3, 2014). Then, the data cube was unfolded into a two-dimensional array of dimensions MN × λ. In the spectra pre-processing step, the asymmetric least-squares algorithm was used (AsLS; λ = 10⁵ and p = 0.001) [33 (link)], cosmic peaks were removed using the algorithm developed by Sabin et al. (k = 11) [34 (link)], and the spectra were normalized by the unitary length vector. Chemical maps were generated using classical least squares (CLS) with the PLS-Toolbox (Eigenvector Research, Manson, WA, USA, v. 8.6.2).

Raman spectra of individual cells in the artificial community or MMB enrichments were acquired using a LabRAM HR Evolution Confocal Raman microscope (Horiba Jobin-Yvon) equipped with a 532 nm laser and 300 grooves/mm diffraction grating. Spectra of cells in the mock community and the MMB enrichments were acquired using a 100× dry objective (NA = 0.90) in the range of 200–3200 cm⁻¹, with 2–4 acquisitions of 10 s each, and a laser power of 4.5 mW. Spectra were processed using LabSpec version 6.5.1.24 (Horiba). The spectra were preprocessed with a Savitsky-Goly smoothing algorithm, baselined, and finally normalized to the maximum intensity within the 2800–3100 cm⁻¹ region. To analyze the degree of deuterium substitution in C-H bonds (%C-D, i.e., (C-D_area/(C-D_area + C-H_area)*100)), the bands assigned to C-D (2040–2300 cm⁻¹) and C-H (2800–3100 cm⁻¹) were calculated using the integration of the specified regions [20 (link)]. Greigite (Fe₃S₄) was identified by its characteristic peak at 350 cm⁻¹ [39 (link)]. Neither magnetite (Fe₃O₄; 303, 535, and 665 cm⁻¹ [39 (link)]), nor its laser-induced oxidative product, hematite (Fe₂O₃; 225, 245, 291, 411, and 671 cm⁻¹ [39 (link)]), were observed.

SERS spectra were measured by a Renishaw inVia (Renishaw plc, New Mills, UK) with a He-Ne laser at 632.8 nm. Each Raman spectrum was obtained using 5% laser power, one accumulation and the acquisition time was typically 10 s. To detect Hg²⁺ ion, 1 M mercuric nitrate solution of was diluted to 10⁻¹–10⁻¹⁰ M. Secondly, the probes were marked on the surface and SERS spectra of 3–5 points were recorded (The error bar in all figures is equal to the standard deviation of relative intensity drop of the 3–5 points.). Then, the probes were immersed in 60 µL of 0.1 nM to 100 μM of mercuric nitrate solution, respectively. After 5 min standing, the probes were purged with N₂ and SERS spectra of the same points above were recorded. The background correction was processed by LabSpec (HORIBA, Ltd., Kyoto, Japan). To correspond with the actual application situation, every probe was used once and corresponded to one certain concentration.