Anaerobic biodegradation of Miocene lignites from an opencast mine by autochthonous microorganisms stimulated under laboratory conditions

Abstract. The supplementation and provision of appropriate nutrients to microorganisms, which are often lacking in the natural environment are essential and critical for microbial growth. One such element is nitrogen, most of which is found in the Earth's atmosphere. In this study, we present evidence of nitrogen processing and anaerobic N₂-fixation by microorganisms naturally present in sedimentary organic matter. Miocene detritic lignite from the opencast mine was incubated under anaerobic conditions in the dark (headspace atmosphere 85 % N₂, 10 % CO₂, 5 % H₂) for three years. The natural microbial community of these coal materials was stimulated for growth through the addition of trace elements, vitamins, and carbon-bearing compounds such as yeast extract, nutrient broth, methanol, and sodium acetate. A visual indicator of microbial activity was observed as the color of the fermentation solutions changed over time: from colorless to light yellow (after 3 months), dark brown (after 6 months), and finally black (after more than 1 year). This progression suggests the dissolution of fulvic and humic acids. At the end of the cultivation period, the total nitrogen (TN) and total inorganic nitrogen (TIN) contents in the solutions were significantly reduced whereas in incubations with sodium acetate, total organic nitrogen (TON) content significantly increased compared to the initial levels. In most cases, total carbon (TC) content significantly increased due to biodegradation, except for the incubations where methanol was added. A GC-MS analysis of the total extracts from lignite revealed that the main macromolecule decomposed by microorganisms was lignin, along with its diagenetic derivatives. The biogas released during the process contained CO₂ and trace amounts of CH₄ (up to 50 ppm). Isotopic data indicated the occurrence of anaerobic CH₄ oxidation. Notably, 16S rRNA gene sequencing identified the presence of N₂-fixing microorganisms in all investigated samples, members of the order Rhizobiales (families Beijerinckiaceae, Rhizobiaceae). Our findings demonstrate that N₂-fixation may play a pivotal role in coal decomposition under anaerobic conditions.