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Research on renewable biotechnology for renewable biofuel applications has reached new heights. This is highlighted by extensive biomining for novel enzymes to reduce the production costs from animal and insect gut microbiomes. This study explored the diversity and composition of hemicellulolytic fungi in the gut microbiota from dung beetles of the family Scarabaeidae (Pachylomerus femoralis, Anachalcos convexus and Euoniticellus intermedius). Two hundred and twenty-two filamentous fungi were isolated, purified and identified using rDNA sequencing of the ITS and D1/D2 regions. The fungal isolates were assigned to 12 genera and 25 species. Fungi associated with the genus Aspergillus was in abundance, with Hypocrea lixii predominantly isolated. Isolates that produced more than 3 U/mL of xylanase activity were evaluated further. The highest xylanase activity was of 23.6 and 23.5 U/mL for L1XYL9 (E. intermedius larvae) and Hypocrea lixii AB2A3 (A. convexus), respectively. Phylogeny of the fungal strains with xylanolytic activity was analysed using ITS rDNA sequences and revealed close genetic relatedness between isolates from the different dung beetle species. Fungal genera commonly found in the gut of both adult beetles and larvae included Aspergillus, Hypocrea, Talaromyces and Penicillium. The results obtained in this study suggest that the gut of Scarabaeidae dung beetles in South Africa is a rich source of xylanolytic fungi. [ABSTRACT FROM AUTHOR]