Shaking incubator

A completely randomized design (CRD) was adopted to elucidate the effects of overliming conditions (detoxification time and temperature) on sugar recovery and fermentation performance. Three detoxification times (1, 5, and 16 h) and two temperatures (30 and 50 °C) formed six treatments, and each treatment had three replicates, which led to a total of eighteen runs. The detoxification was carried out in 500 mL media bottles (Wheaton Industries, Millville, NJ). Ca(OH)₂ was added in the liquid hydrolysate until the pH reached 10. The bottles were placed in a shaking incubator (Thermo Scientific, Odessa, TX) at 150 rpm (2.5 Hz) according to the targeted detoxification time and temperature.
After detoxification, the pH of the detoxified hydrolysate was adjusted back to 6 using 20% (w/w) H₂SO₄. The detoxified hydrolysate was centrifuged at 7025g for 10 min to separate the liquid hydrolysate from the residues. Approximately 2 mL of the hydrolysate was filtered through a 0.22-µm polyethersulfone membrane filter for sugar analysis. The remaining detoxified liquid hydrolysate was stored in the −20 °C freezer for the following fermentation test.

Overnight bacterial cultures (E. coli O157:H7 or BL21, ~10⁹ CFU mL⁻¹) grown in TSB were diluted to 1:10, 1:100, 1:1000, and 1:10,000 in PBS. For each dilution and the original overnight culture, 10 replicates of 200 μL bacterial solution were added to a sterile 96-well plate. A 10 μL aliquot of the phage microgel suspension was then added to each of the first three replicates as the sample group (named “With microgels”). A 10 μL drop of sterile PBS was added to the remaining three replicates as the control group (named “No microgels”). Afterwards, the 96-well plate was placed in a shaking incubator (Thermo Scientific, 37 °C, 180 rpm) for 9 h, and the bacterial titer count of each sample at the end point was calculated.

A commercially available S. cerevisiae library of ∼4,500 nonessential genes was used. All the deletion strains in this library contain a specific gene deleted, with KanMX resistance cassette. Primary culture was obtained by growing these strains overnight in 200 μl of YPD media containing G418, in 96-well format. Then, 5 μl of this culture was transformed to another 96-deep-well plate containing 400 μl of SC media, with and without 1 mM cysteine. The strains were then grown at 30°C in a shaking incubator (Thermo Fisher Scientific), with 200 rpm, for 12 h, and then, cell density was measured using multimode reader.

The chemicals that are used in the present study were purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India. The pathogens Bacillus subtilis (MTCC 10403), Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 424), Micrococcus luteus (MTCC 1809), Staphylococcus aureus (MTCC 1144), Salmonella enterica typhimurium (MTCC 98), Klebsiella pneumonia (MTCC 10309), Bacillus cereus (MTCC 1272), Klebsiella aerogenes (MTCC 2822), and Pseudomonas fluorescens (MTCC 667) were received from Microbial Type Culture Collection (MTCC) and Gene Bank, Chandigarh, India. In the current study, the instruments used were microplate reader (Multiskan FC, Thermo Fisher Scientific, Mumbai, India), centrifuge (Remi CPR-30 plus, Mumbai, India), Vortex Shaker (SPINIX, Kolkata, India), PVDF syringe filter (0.22 μm; Thermo Fisher Scientific, Mumbai, India), CO₂ incubator (Thermo Fisher Scientific, Mumbai, India), and shaking incubator (Thermo Fisher Scientific, Mumbai, India).

Extractions were carried out using a shaking incubator (Thermo Scientific, Illkirch-Graffenstaden, France) for 24 h. An agitation speed of 180 rpm was applied at 30 °C. First, the effect of the solvent composition was studied using ethanol from 0 to 100% (v/v) in water, with a solid–liquid ratio of 1/50 (g_DM/mL solvent). Second, the effect of the solid–liquid ratio was studied in the range of 1/10 to 1/200, using the best solvent composition found in the first step. The range for the solid–liquid ratio was selected based on preliminary experiments.
Samples were taken to be analyzed by UHPLC and to determine the chlorogenic acid content (mg/g_DM).