Fastprep 24 high speed benchtop homogenizer

Metagenomic DNA was extracted from soil using three methods that rely on mechanical and/or chemical/enzymatic lysis^{64 (link)–66 (link)}. The first technique used the Hoffman and Winston lysis solution to lyse the microbial cells^{64 (link)}, the second used lysozyme to lyse the cell walls based on the method developed by Sambrook and Russell⁶⁵ , and the third used a thermo/mechanical technique based on the method developed by Valenzuela-Encinas et al.^{66 (link)}. The soil was homogenized using a FastPrep24 high-speed benchtop homogenizer (MP Biomedicals, Solon, OH, USA) at 4 m s⁻¹. The three techniques were used to extract DNA twice from 0.5 mg soil samples from the three plots of each of the six treatments (n = 18), and then pooled to create a metagenomic sample. As such, a total of 3 g soil was extracted per plot, i.e. three extraction techniques applied twice to a 0.5 g soil subsample.
The V3-4 hypervariable region of the 16S rRNA gene (about 490 bp amplicon size) was amplified using 8-bp fused barcode primers 341-F (5′-CCTACGGGIGGCWGCAG-3′) and 805-R (5′-GACTACHVGGGTATCTAATCC-3′)^{67 (link)} with a two-step PCR protocol “16S metagenomic sequencing library preparation” published by Illumina Inc (15044223 Rev. B). Triplicate PCR amplification reactions per metagenomic DNA were done in a MultiGene OptiMax thermal cycler (Labnet International Inc.) under conditions previously reported^{19 (link)}. The PCR amplification conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 25 cycles of denaturation at 95 °C for 45 s, annealing at 53 °C for 45 s, extension at 72 °C for 1 min and a final extension at 72 °C for 10 min. A no-template control (negative PCR reaction) was included each time a PCR was done. The triplicate PCR reactions (12.5 µL each) were pooled and cleaned using FastGene™ columns (Nippon Genetics, Co., Ltd). Pooled and cleaned PCR products were quantified using the Invitrogen’s PicroGreen^® assay with a NanoDrop™ 3300 fluorospectrometer (Thermo Fisher Scientific Inc., Suwanee, CA) and standardized at equal molar amounts for later sequencing. As such, a total of 288 metagenomic DNA samples, i.e. one DNA sample from each soil sample, were PCR-amplified and sequenced. Both library normalization and sequencing process was done by Macrogen Inc. (Seoul, Korea). Amplicon libraries were paired-end sequenced using the MiSeq v3 platform (2 × 300 cycle kit) at Macrogen Inc. (Seoul, Korea).

Total proteins were extracted from Pt3 cultures enriched in one specific and dominant morphotype. Microalgae cells from the different cultures were recovered by centrifugation at 4,500 × g for 5 min.
Cell pellets were washed twice with 10% SW in order to decrease salt concentration. For each culture, a pellet of approximately 1.10⁸ cells was harvested and immediately re-suspended in 500 μL of D2R2 protein extraction buffer {7 M urea, 2 M thiourea, 2 mM tributyl phosphate (TBP), 0.5% 3-(4-heptyl) phenyl-3-hydroxypropyldimethylammoniopropanesulfonate (C7BzO), and 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (Chaps)}. The cell suspension was stored at –80°C until further use. For protein extraction, the cell suspension was transferred into a 2 mL tube of lysing matrix E (MP Biomedicals, Fisher Scientific, Illkirch, France), and cells were lysed by 4 runs of 30 s at 6.5 m.s^–1 with the FastPrep^®- 24 high-speed benchtop homogenizer (MP Biomedicals^®). Cell lysates were centrifuged at 10,000 × g for 10 min in order to remove cellular debris. The supernatants containing proteins were collected. The remaining pellet was re-suspended in 500 μL of D2R2 protein buffer and extracted once more. Finally, the supernatants were pooled in a unique fraction called IP for intracellular proteins.

Fungal biomass from MTPS and 1.5 L cultivations were filtered through a Whatman No. I filter paper in a vacuum setup (GE Whatman, Maidstone, UK), while in case of the 25 L cultivations, a 75-μm aperture test sieve was used (Endecotts, London, UK) for biomass separation. After filtration, the fungal biomass was washed thoroughly with distilled water. In case of microalga C. cohnii, the biomass was separated from the medium by centrifugation at 3000 rpm and it was washed once with distilled water. In the next step, the fungal and algal biomass was frozen at − 20 °C and then was lyophilized overnight in an Alpha 1-2 LDPlus freeze-dryer (Martin Christ, Osterode am Harz, Germany) at − 55 °C and 0.01 mbar pressure. The freeze-dried biomass was also used to calculate cell dry weight (CDW, g/L). Approximately 10 mg of freeze-dried biomass was transferred into 2-mL screw-cap tubes containing 500 μL distilled water and 250 ± 30 mg acid-washed glass beads (800 μm, OPS Diagnostics, Lebanon, USA). Biomass was then homogenized for 1–2 min in a FastPrep-24 high-speed benchtop homogenizer (MP Biomedicals, USA) at 6.5 m s⁻¹. This homogenized fungal suspension was used for HTS-FTIR analysis. Lipid extraction protocol was performed according to previously described protocol (Kosa et al. 2017 (link)).