Fungal metagenome from soil treated with carboxylic acid: Citric acid
Soil Induced Respiration (SIR) - were measured using the MicroRespTM plate (Campbell et al. 2003). The data obtained had yield values for: (1) microbial biomass; (2) CO2 evolution without any additional substrate determined the basal respiration and with substrates addition determined substrated utilization and (3) microbial functional diversity. In order to do so fifteen different carbon sources (Table 1) of carbohydrates, carboxylic acids, amino acids and aromatic carboxylic acids was added to whole soil samples in deep well plates. CO2 evolution was measured by dye plates- a colorimetric reaction which absorbent alkali with the ability to measure carbon dioxide evolution, to determine the carbon utilization ability for each substrate. The plates with the soil and substrates added were left for one hour in order to allow acidic substrates to eliminate CaCO3-derived CO2 evolution (Oren and Steinberger, 2008). Soil sample from the same batch of soil used for SIR were used further for microbial community composition analysis.soil DNA was extracted from 0.5 g of soil sample from each sample using the GeneAll ExgeneTM mini Kit (Seoul, Korea), according to the manufacturer's instructions.  A pair of primers were used to express the ITS region. DNA was amplified using PCR using SimpliAmpTM thermal cycler (Thermo Fisher Scientific, Walham, MA, USA), by mixing 12.5 L HS Taq Mix Red (PCR Biosystems, London, UK), 9.5 L ultrapure water, 1.0 L extracted DNA (concetration of 100 ng/ul), 1 L CS1-ITS2, and 1 L CS2-ITS4. The thermal cycling program was set to: 95 C for 2 min, followed by 40 cycles at 95 C for 15 s, 50C for 30 s, 72C for 30 s, and after the cycles, 72C for 3 min.  For bacteria taxonomic analysis - were added primers: 1 L CS1_515F and 1 L CS2_806R. The amplified DNA was stored at 20 C until sequencing.  forward primer- CS1-ITS2F: ACACTGACGACATGGTTCTACAGCTGCGTTCTTCATCGATGC reverse primer- CS2-TS4R: TACGGTAGCAGAGACTTGGTCTTCCTCCGCTTATTGATATGC All final PCR products were run on an agarose gel to verify amplification specificity and quality, in parallel with a negative control. The final PCR products were conducted by Hylabs Inc., using the Fluidigm Access Array primers for Illumina to generate libraries compatible for sequencing on the Miseq. Samples were measured for concentration by Qubit and size by Tapestation and then sequenced on the Illumina Miseq using a Miseq V2 sequencing kit (500 cycles) to generate 2x250 paired end reads.