U 5100 uv vis spectrophotometer

The E. coli DH5α strain was utilized for DNA cloning and plasmid preparation. A lysogeny broth (LB) medium, containing 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride per litre, was used for the cultivation of the E. coli strain, with appropriate antibiotics (100 μg/mL of ampicillin or 50 μg/mL of kanamycin).
Methylomonas sp. DH-1 (KCTC13004BP) was used as a parental strain of the engineered strains. It was cultured in a nitrate mineral salt (NMS) medium [14 (link)] supplemented with 30% (v/v) methane at 30 °C with shaking at 250 rpm. For genetic integration, the cells were transformed with plasmid-containing homology arms by electroporation [19 (link)] and grown in NMS plates with appropriate antibiotics (100 μg/mL of ampicillin and/or 10 μg/mL of kanamycin). To measure cell growth, Methylomonas sp. DH-1 cells were grown until the stationary phase was reached. They were then diluted to OD₆₀₀ = 0.03 with a fresh NMS medium and 30% (v/v) methane. For repeat methane feeding, gas substitution was performed using a gas-tight syringe. The headspace was refreshed daily. Cell growth (OD₆₀₀) was monitored for 5 days using a Hitachi U5100 UV–Vis spectrophotometer (Tokyo, Japan).

Lipid peroxidation was determined by measuring the total malondialdehyde (MDA) content, a marker of the lipid peroxidation process in algal cells, as previously described by Heath and Packer [45 (link)] and detailed by Piotrowska-Niczyporuk, et al. [39 (link)]. The level of hydrogen peroxide (H₂O₂) in D. armatus cells (10 mL) was measured using the method outlined by Alexieva, et al. [46 (link)]. For MDA and H₂O₂ determination, algal cells were disintegrated in 0.1% (w/v) trichloroacetic acid (TCA) and homogenized in a bead mill (50 Hz, 10 min, TissueLyser LT; Qiagen GmbH, Düsseldorf, Germany). Algal extracts were used for spectrophotometrical (Hitachi U-5100 UV-Vis spectrophotometer; Hitachi High-Tech Science Corporation, Tokyo, Japan) measurement of oxidative marker levels [34 (link),40 (link)].

The total ascorbate content in D. armatus cells was determined using the method described by Kampfenkel, et al. [47 (link)]. Cells were disrupted using 0.1 M phosphate buffer solution (pH = 2.0) in a bead mill (50 Hz, 10 min, TissueLyser LT; Qiagen GmbH, Düsseldorf, Germany). The extraction procedure of proline (Pro) started with homogenization of algal cells in 100 mM of phosphate buffer (pH 7.5) in a bead mill. The level of Pro in algal extracts was determined spectrophotometrically (Hitachi U-5100 UV-Vis spectrophotometer; Hitachi High-Tech Science Corporation, Tokyo, Japan) at a wavelength of 546 nm after reaction with an acidic 2.5% ninhydrin [48 (link)]. The proline concentration was calculated based on the cell number.

The monosaccharides content was measured spectrophotometrically (Hitachi U-5100 UV-Vis spectrophotometer; Hitachi High-Tech Science Corporation, Tokyo, Japan) using the methods of Nelson [75 (link)] and Somogyi [76 (link)] with modifications. For this purpose, 0.5 g of W. arrhiza powder was extracted in 5 mL of 62.5% (v/v) MeOH in a water bath (30 min at 60 °C) [77 (link)]. The standard sample was 30 mg of glucose dissolved in 62.5% (v/v) MeOH. The blind sample was distilled water. A 0.5 mL volume of all samples was mixed with 0.5 mL of copper reagent and placed in a boiling water bath for 20 min. Then, 0.5 mL of arsenomolybdate reagent was added, and after 5 min the extract was diluted in 3.5 mL of water and mixed. The absorbance measurement was performed at 540 nm.