**Background:** Lignocellulose is the most abundant biomass on Earth, with approximately 180 billion tons of cellulose produced annually. Termites can digest lignocellulose with high efficiency through a combination of their own cellulases and gut symbiotic microorganisms. Lower termites like Reticulitermes grassei harbor a diverse microbiota including bacteria, archaea, protozoa, yeasts, and fungi. This study aimed to isolate and characterize microorganisms associated with R. grassei in southern Spain and quantify their cellulolytic activity, while also preliminarily evaluating microorganism dispersion by termites.
**Methods:** Three ecosystems colonized by R. grassei were sampled around Cordoba, Spain (UTM coordinates provided) between spring and autumn 2018. Samples included degraded wood from Eucalyptus, Pinus, and Populus spp., and termites collected via baiting stations with sterilized corrugated cardboard. Microorganism isolation was performed from surface-disinfected wood fragments plated onto nutrient agar (NA) and agar sabouraud (AS), and from PBS suspensions of 10 termite specimens plated onto the same media. Two laboratory assays evaluated microorganism dispersion: Assay I placed 150 termite workers with sterilized wood (Pinus pinea or Populus alba) or filter paper for one week, then incubated degraded substrates without termites; Assay II re-exposed colonized substrates to new termite groups, observing fungal structure degradation at 24, 48, and 120 hours. Molecular characterization used ITS region (ITS1-5.8S rDNA-ITS2), beta-tubulin (TUB), and partial 28S rDNA (LSU) for fungi, and 16S rDNA for bacteria. Cellulolytic activity was evaluated on carboxymethylcellulose (CMC) agar, with enzymatic index (EI) calculated as the ratio of hydrolysis zone diameter to colony diameter. EI > 1.0 indicated potential cellulase producers.
**Key Results:** A total of 23 microorganism isolates were obtained: 6 (26.1%) from field-degraded wood, 3 (13.0%) from termite exoskeleton resuspension, and 14 (60.9%) from laboratory-exposed substrates. Fungi were more prevalent (72.7%) than bacteria (27.3%). Molecular identification revealed 10 fungal species: Aspergillus flavus (2 isolates), A. niger (2), Chaetomium globosum (2), Doratomyces stemonitis (1), Mucor circinelloides (2), M. fragilis (1), Penicillium citrinum (1), Pichia guilliermondii (1 yeast), Trichoderma asperellum (4), and T. longibrachiatum (1). Four bacterial species were identified: Burkholderia kirstenboschensis (1), Klebsiella aerogenes (1), Lactococcus lactis subsp. cremoris (1), and Serratia marcescens (3). Optimum growth temperatures for fungi ranged from 22.2°C (C. globosum MZC-9) to 32.1°C (T. longibrachiatum MZC-4). Maximum growth rates (MGR) ranged from 1.8 mm/day (P. citrinum MZC-21) to 33.6 mm/day (T. longibrachiatum MZC-4). All 16 filamentous fungal isolates grew on CMC agar, confirming cellulase production. The positive control P. chrysogenum showed the highest EI (1.50 ± 0.011), followed by P. citrinum MZC-21 (EI = 1.34 ± 0.020). A. flavus MZC-20 (EI = 1.11), T. longibrachiatum MZC-4 (EI = 1.09), A. flavus MZC-17 (EI = 1.08), A. niger MZC-14 (EI = 1.06), and D. stemonitis MZC-16 (EI = 1.05) showed EI values above 1.0. Eight isolates (A. niger MZC-18, C. globosum MZC-2 and MZC-9, M. circinelloides MZC-5 and MZC-23, M. fragilis MZC-1, and T. asperellum MZC-8, MZC-10, MZC-11, and MZC-24) showed EI = 1.0. No hydrolysis halos were observed for bacterial or yeast isolates. In Assay I, macroscopic fungal structures appeared only on cellulose sources exposed to termites, with fungi (64.3%) isolated more frequently than bacteria (35.7%). In Assay II, progressive degradation of fungal mycelia was observed at 24, 48, and 120 hours after re-exposure to termites.
**Clinical Implications:** This study demonstrates that R. grassei-associated microorganisms, particularly fungi, are promising sources of cellulolytic enzymes with potential biotechnological applications in biofuel production and the food industry. The symbiotic relationship between termites and cellulolytic fungi—where termites disperse fungi and fungi provide nutritional supplementation and potentially hygienic benefits—represents a natural model for lignocellulose degradation. The identification of D. stemonitis from termites is a first report. The findings support the hypothesis that termite habitats are valuable targets for discovering novel cellulolytic microorganisms. However, further quantitative enzyme assays and studies on the specific interactions between R. grassei and individual fungal species are needed to fully elucidate the mechanisms and potential applications.