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Soil organic matter (SOM) biomarker methods were utilized in this study to investigate the responses of fungi and bacteria to freeze–thaw cycles (FTCs) and to examine freeze–thaw-induced changes in SOM composition and substrate availability. Unamended, grass-amended, and lignin-amended soil samples were subject to 10 laboratory FTCs. Three SOM fractions (free lipids, bound lipids, and lignin-derived phenols) with distinct composition, stability and source were examined with chemolysis and biomarker Gas Chromatography/Mass Spectrometry methods and the soil microbial community composition was monitored by phospholipid fatty acid (PLFA) analysis. Soil microbial respiration was also measured before and during freezing...