Potential of Subterranean Microbes: High‐Throughput Screening for Industrially Relevant Enzymatic Activities in Dinaric Caves

Enzymes derived from extremophiles, or extremozymes, possess unique properties that enable them to function under extreme environmental conditions. Microbial communities in subterranean ecosystems have evolved specialized metabolic pathways to survive, leading to the discovery of bioactive molecules with diverse biotechnological and industrial applications as well as the development of sustainable methods for habitat restoration. This study aimed to identify cultivable microorganisms producing industrially relevant enzymes, such as laccases, proteases, and urethanases, from extremophiles in the Dinaric Karst subterranean ecosystems, which are known as biodiversity hotspot. A total of 40 samples were collected from six caves and an abandoned railway tunnel, now a key roost for a large Myotis myotis maternity colony. Cave samples were taken from the entrance, twilight, and dark zones, including soil, sediments, moonmilk, mineral deposits, bedrock deposits, insect remains, entomophagous fungi, wall biofilm, and guano from various bat species. Following microbial cultivation, 207 colonies were screened for enzymatic activity using substrate-specific assays. Functional analysis identified one microorganism exhibiting strong laccase activity, seven capable of degrading polyurethane, and numerous protease-producing colonies. Notably, this study constitutes the inaugural report on discovering polyurethane-degrading microorganisms in karst caves. Molecular identification revealed microbial genera, including Bacillus, Pseudomonas, Serratia, Paenibacillus, and Priestia. These findings underscore the biotechnological potential of subterranean extremophiles and highlight the importance of conserving these ecosystems. Further characterization of these enzymes may drive advancements in environmental remediation, waste recycling, and sustainable industrial processes.