Glycine betaine assay kit

The archaea cultured to the stationary phase (A₆₀₀ value no longer changed with time, about 0.5) in YCM1 media was collected and transferred to YCM2 medium after being washed 3 times with 250 g/L NaCl solution. Cells were cultivated in different salinities in the media YCM2 for 24 h with or without adding extra 1% trehalose and 1% glycine betaine. Cells were lysed by ddH₂O. Osmoprotectants were identified and quantified in cell-free extracts. Concentration of intracellular K⁺, trehalose and glycine betaine measured using K⁺ turbid assay kit, colorimetric trehalose assay kit and glycine betaine assay kit (Solarbio, Beijing, China). The principle of K⁺ detection was that K⁺ reacted with Na₂B₄O_7·xH₂O to produce K₂B₄O₇·5H₂O which was insoluble in water, and the turbidity was proportional to the K⁺ concentration within a certain range. The detection of trehalose utilized the anthrone spectrophotometric methods. The detection principle of glycine betaine was that glycine betaine reacted with Reinecke salt under strong acid conditions to form a precipitate, which can be dissolved in acetone to form a red solution with a characteristic absorption peak at 525 nm.

The content of lignin was measured using a Lignin Content Test Kit (MZS-2-G, Suzhou Comin Biotechnology Co., Ltd., China) according to the manual. The total sugar was measured using the sulfuric acid-phenol method, as described previously. The glycine betaine content was measured using a glycine betaine assay kit (BC3130, Solarbio, Beijing, China) according to the manufacturer’s instructions (Tao et al., 2019 (link)).