The quality of soil organic matter plays a central role in soil structure, carbon sequestration and pollutant immobilization. The effect of 16–23 years of fertilization on the quality of soil organic matter was studied in field experiments at ten experimental sites in Central Europe. Soil samples were collected in 2016 after barley harvest. Six crops were rotated: pea–canola–winter wheat–spring barley–beet/potato–spring barley. Six treatments were studied: unfertilized control, mineral fertilization (NPK), farmyard manure, farmyard manure + NPK, straw incorporation, and straw incorporation + NPK. Although carbon input did not significantly correlate with any soil organic carbon fractions, the C/N ratio of applied organic fertilizers significantly correlated with the content of humic acid carbon (C-HA), the C-HA/C-FA ratio and humification index in soil. The combination of farmyard manure + NPK resulted in a higher humic acid carbon content in soil, humification rate, and humification index compared to the application of NPK, straw return, and the combination of straw return + NPK. Although straw return led to a lower E4/E6 (A400/A600, Q4/6) ratio compared to farmyard manure application, the C-HA/C-FA ratio was unchanged among these treatments. The application of farmyard manure with and without the addition of NPK led to higher values of carbon sequestration efficiency in soil compared to the straw return with and without the addition of NPK.
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