Diverse thermophilic anaerobes from Indian hot springs show potential for bioenergy production from lignocellulosic biomass.

Thermophilic anaerobic bacteria degrade lignocellulosic biomass and produce biofuels under high-temperature, oxygen-limited conditions, yet their cultivable diversity and functional potential in Indian hot springs remain underexplored. Water and sediment samples were collected from ten geographically distinct Indian hot springs spanning a temperature range of 25-85 °C, and the sites were characterised using standard physicochemical measurements. Of the four substrates tested, cellulose, xylan, and neutral detergent fibre supported notable hydrogen-producing enrichments at 55-85 °C, while lignin yielded no positive enrichments at any temperature. Using anaerobic roll bottle isolation and genotypic screening by Restriction Fragment Length Polymorphism (RFLP), 83 strains were obtained. Sequencing and phylogenetic analysis of 16S rRNA genes identified representatives of 13 genera and 9 families, together with strains assignable only to the family level. Thermoanaerobacterium and Caloramator were the most frequently recovered genera, while Caldibacillus was obtained from several geographically distinct springs, alongside multiple putatively novel taxa. Numerous isolates exhibited cellulase, xylanase, and esterase activities on untreated rice and wheat straw, demonstrating the capacity to deconstruct lignocellulosic biomass without prior pretreatment. Enzymatic activities and fermentation profiles varied substantially among strains. Caloramator coolhaasii XMU5567-13 was the most effective ethanol producer, reaching 79.14 mM on wheat straw, whereas Thermoanaerobacter wiegelii XMU7067-6 exhibited the highest hydrogen production (14.05 mM) and strong lignocellulolytic activity on the same substrate. Several strains, including Caldibacillus thermoamylovorans, Thermoanaerobacter wiegelii, and Thermoanaerobacterium spp., coupled growth on untreated straw with concurrent enzyme production and fermentation, highlighting their potential for consolidated bioprocessing. This systematic culture-based survey highlights the ecological diversity of Indian geothermal anaerobes and their potential for bioenergy production from agricultural residues.