Talc

Genomic DNA from Xoo was isolated using Phenol/Chloroform extraction, digested with BamHI and separated on a 0,8% agarose gel at 90V for 24h. The chemiluminescent detection of genomic TALE sequences on Southern blot was done using a digoxigenin-labelled (Roche Applied Science, Mannheim, Germany) probe derived from 500bp of the 3′ part of talC from Xoo BAI339 (link) that hybridizes to TALE genes.

To assess the degree of Si isotope fractionation that is associated with the bulk-rock serpentinisation of mafic and ultramafic rocks, a suite of variably serpentinised abyssal peridotites have been analysed for their whole-rock Si isotope compositions. The samples were taken from cores drilled on the Ocean Drilling Program Leg 209 cruise⁴⁰ , whose main aim was to drill mantle peridotites and associated mafic rocks from fracture zones along the Mid-Atlantic Ridge⁴¹ The samples were taken from two cores, Holes 1274 A and 1268 A. Briefly, Hole 1274 A is composed of harburgites, dunites, and gabbroic rocks, all of which had undergone 60–100% serpentinisation at low temperatures (100–200 °C). Major element analyses suggests that the serpentinisation was isochemical, i.e., the rocks from Hole 1274 A were hydrated, but no other metasomatism took place. Hole 1268 A also returned a mixture of harzburgites, dunites and gabbros; in contrast to Hole 1274 A, the rocks from 1268 A were 100% serpentinised before undergoing a late-stage, high temperature (>200 °C) metasomatic event (associated with a gabbroic intrusion) which resulted in the variable desilicification of some rocks to form talc (steatisation).
To specifically study the potential effect of serpentisation (both isochemical and metasomatic) on the Si isotope composition of ultramafic rocks, only variably altered, serpentinised harzburgites were chosen from both cores for analysis. Six samples were taken from Hole 1274 A and eleven samples were taken from Hole 1268 A. Samples were provided in powder form, and prepared for Si isotope analysis via solution MC-ICP-MS following the methods first described in^{42 (link)} and further detailed in^{43 (link)}.
Sample powders were dissolved using alkali fusion, whereby ~10 mg of sample powder was weighed into silver crucibles along with ~200 mg of NaOH flux (semiconductor grade, Merck). The crucibles were placed into a muffle furnace heated to 720 °C for 15 min to perform the fusion. Subsequently, the crucibles and fusion cake were placed into individual PFA vials filled with 20 ml of MQ-e water and left overnight to equilibrate. The fusion cake was then transferred via pipette to precleaned PP bottles, diluted with enough MQ-e to reach a final Si concentration of between 10 and 25ppm, and acidified with enough conc. HNO₃ to bring the pH of the solution to ~2. Final Si concentrations of the sample solutions were ascertained via the Heteropoly Blue method using a photospectrometer.
Samples were purified for Si isotopes using a single stage cation exchange procedure. Samples were loaded into BioRad Polyprep columns filled with 1.8 ml of AG50W X12 cation exchange resin (200–400 mesh, BioRad; the resin was cleaned in the column before samples were loaded—see^{43 (link)} for the cleaning method). Silicon is in either anionic or neutral forms in solution at low pH and so can be directly eluted using 5 ml of MQ-e water. Post-column, the samples were acidified to ~0.22 M HNO₃. Total procedural blanks of the whole chemical preparation procedure were measured at less than 100 ng of Si, which is approximately 0.35% of the total measured Si signal and considered negligible.
Silicon isotopes were measured on a Neptune Plus MC-ICM-MS instrument (ThermoFischer Scientific, Bremen, Germany), running at medium resolution (M/∆M ~7500) to avoid significant molecular interferences on the ²⁹Si and ³⁰Si beams. Samples were introduced into the instrument using a 100 μl min-1 PFA ESI (Elemental Scientific, Omaha, USA) microflow nebulizer running into the SIS spray chamber. Silicon isotopes were measured in the L3 (²⁸Si), C (²⁹Si), and H3 (³⁰Si) Faraday cups, and depending on instrumental conditions, a 2ppm Si beams typically gave a total analyte signal of ~7 V. Isotope ratios were measured in static mode with each measurement consisting of 25 cycles with a ~8 sec integration time.
Silicon isotope measurements were calculated using the standard-sample bracketing protocol relative to the NBS28 standard. Variations in Si isotopes are defined using the delta notation (δ³⁰Si _NBS28) as described before. Each measurement session consisted of 10 samples, two of which were always the external standards BHVO-2 and Diatomite. Long-term δ³⁰Si error on the NBS28 bracketing standard over the analytical sessions is 0.00 ± 0.10‰ (2 s.d.).
The δ¹⁸O values used in this study are those from different serpentinite samples but of the same ODP holes measured by ref. ^{43 (link)}.

The culture compatibility among the bacterial isolates was assessed by cross streaking method using NA medium. The bacterial strain was streaked at one end of the plate followed by streaking the other bacterial strains perpendicular to it and incubated at 30°C for 24–48 h. The inhibition in growth between the cultures was noted and the compatible microbes were selected for further confirmation. The selected compatible strains were streaked in a triangular pattern so that all the streaks overlap each other as a confirmation test. Any two perpendicular steaks that showed inhibition were incompatible and the absence of inhibition proved that the cultures were compatible (Thomloudi et al., 2019 (link)).
The elite compatible microbial isolates were mass multiplied as pure cultures in the NA media for further formulation of the microbial consortium. The mass culturing was carried out in 250 mL Erlenmeyer flask, which serves as seed culture for further multiplication. The culture broth was autoclaved in 15 lbs for 20 min and after cooling the microbial isolates were inoculated as loopful cultures in their respective broths prepared. The inoculated flasks were maintained as shake flask cultures by incubating them at room temperature at 120 rpm for 2 days for mass production. The 48 h old cultures were serially diluted and plated to count the population in the broth. To attain a uniform population in the consortium, 5 mL inoculum was centrifuged at 10,000 rpm for 15 min at 10°C and resuspended in 10 mL of deionized water to obtain 4 × 10⁹ CFU (Jain and Srivastava, 2012 (link)). The OD 660 nm value was also recorded for the resuspended water used for consortium development. Talc was used as carrier material for the consortium due to its local availability and high Magnesium and Calcium content. Talc is a fine, light-weight powder that is easily soluble in water and has been shown to retain viable bio-inoculant propagules (Tripathi et al., 2015 (link)). A 100 g of talc was taken in autoclavable plastic bags and autoclaved. A 10 mL of the resuspended solution of each selected isolate was mixed to obtain the consortium. A total volume of 30 mL of the bacterial isolates was added to the carrier material and left for drying in shade (1 day), after which it was evaluated as a soil inoculant.