Diverse Thermophilic Anaerobes from Indian Hot Springs Exhibit High Potential for Bioenergy Production from Lignocellulosic Biomass

Diverse Thermophilic Anaerobes from Indian Hot Springs Exhibit High Potential for Bioenergy Production from Lignocellulosic Biomass | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Diverse Thermophilic Anaerobes from Indian Hot Springs Exhibit High Potential for Bioenergy Production from Lignocellulosic Biomass Sai Suresh Hivarkar, Prashant K. Dhakephalkar, Sumit Singh Dagar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8018409/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 15 You are reading this latest preprint version Abstract Thermophilic anaerobic bacteria are crucial for degrading lignocellulosic biomass and producing biofuels under high-temperature, oxygen-limited conditions, presenting industrial relevance. However, their diversity and function from Indian hot springs remain underexplored. In this study, water and sediment samples were collected from ten geographically distinct Indian hot springs (25–85°C), and physicochemical parameters were measured to characterise environmental heterogeneity. Enrichments were performed using cellulose, xylan, neutral detergent fibre, and lignin, which demonstrated significant hydrogen production, primarily at thermophilic temperatures (55–85°C). Using anaerobic roll bottle isolation and redundancy reduction by RFLP, 83 distinct strains were obtained. Phylogenetic analysis of the 16S rRNA gene identified 19 species across 13 genera and 9 bacterial families, including Caldibacillus , Caloramator , Clostridium , Thermoanaerobacterium , and Sporanaerobium . Numerous strains exhibited notable cellulase, xylanase, and esterase activities on untreated rice and wheat straw. Distinct strain-level variations were noted in enzyme activities and metabolite profiles. Isolates produced high yields of ethanol, hydrogen, and volatile fatty acids, including acetic, butyric, and propionic acids. Notably, strains of Caldibacillus thermoamylovorans , Thermoanaerobacter wiegelii , and Thermoanaerobacterium spp. showed promise for consolidated bioprocessing applications. This represents the first comprehensive systematic study of lignocellulolytic thermophilic anaerobes from Indian geothermal ecosystems, highlighting their ecological diversity and significant potential for bioenergy production from agricultural residues. Bacteria Diversity Ecology Phylogeny Taxonomy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Thermophilic anaerobic bacteria represent a distinct and ecologically significant group of microorganisms capable of thriving in extreme temperatures (45–80°C) under oxygen-free conditions. Their significance lies primarily in their ability to break down complex organic materials, particularly lignocellulosic biomass such as rice straw, wheat straw, corn straw, and sugarcane bagasse, predominantly consisting of lignin, cellulose, and hemicellulose. Research indicates that cellulose and hemicellulose constitute approximately 50–75% of this biomass, while lignin represents the remaining fraction (Rasool and Irfan 2024 ; Kukreti et al. 2024 ; Woźniak et al. 2025 ). Sugars released from the hydrolysis of these polymers can be biologically converted into biohydrogen, bioethanol, and other industrially relevant bio-based products (Svetlitchnyi et al. 2022 ; Mokhtarani et al. 2025 ). Thermophilic anaerobic bacteria are essential in decomposing organic matter and play a critical role in carbon cycling within geothermal ecosystems. Hot springs, characterised by high temperatures, diverse mineral compositions, and variable pH levels, create unique habitats that host specialised microbial communities adapted to these extreme conditions (Yabe et al. 2017 ; Oliverio et al. 2018 ). While much research has focused on mesophilic anaerobic bacteria, particularly those associated with the digestive systems of ruminants (e.g., Fibrobacter succinogenes , Ruminococcus flavefaciens , and others), the potential of thermophilic anaerobes remains largely underexplored (Awan et al. 2023 ; Mokhtarani et al. 2025 ; Tjo et al. 2025 ; Hsin et al. 2025 ). Prominent thermophilic anaerobic lignocellulose degraders include genera such as Bacillus , Caldibacillus , Caldicoprobacter , Caldicellulosiruptor , Clostridium and Thermoanaerobacter , recognised for their robust enzymatic profiles suitable for high-temperature industrial applications (Mhuantong et al. 2015 ; Brunecky et al. 2018 ; de Souza et al. 2025 ). In India, there is a rich array of geothermal activity with over 300 hot springs located across seven provinces, namely, the Himalayan, Northeast, Son-Narmada-Tapti (SONATA) lineament belt, the Sahara Valley, the West Coast, the Godavari and Mahanadi basins (Yadav et al. 2024 ). Each of these environments possesses unique physicochemical properties that support diverse microbial life forms. However, the microbial diversity within these hot springs, particularly thermophilic anaerobic lignocellulolytic bacteria, is understudied, largely because past investigations have centered on aerobic thermophiles or general microbial surveys (Saxena et al. 2017 , 2025 ; Verma et al. 2022 ; Soy et al. 2023 ; Priyadharshini et al. 2023 ; Sharma and Kumar 2024 ). Existing literature reveals gaps in understanding the metabolic capabilities of the anaerobes, which may present significant opportunities for industrial biomass conversion (Chukwuma et al. 2021 ). Lately, studies have been increasingly focused on exploring hot springs for novel anaerobic bacterial isolates, given their promising bioenergy applications. Strains belonging to genera such as Caldicellulosiruptor , Clostridium , and Thermoanaerobacter have shown significant potential due to their efficiency in lignocellulose degradation (Liu et al. 2020a ; Rodionov et al. 2021 ; Orlygsson and Scully 2024 ; Le et al. 2024 ; Hsin et al. 2025 ). Building upon valuable prior contributions from Indian researchers who isolated key thermophilic anaerobes like cellulolytic Clostridium thermocellum and xylanolytic Thermoanaerobacter strains (Singh et al. 2017 , 2018 ), a significant opportunity remains to explore the untapped microbial diversity within India's numerous, yet understudied, hot springs. Many of these unique geothermal niches have not been systematically investigated for their potential to harbour novel bacteria capable of efficient lignocellulose breakdown (Poddar and Das 2018 ). This study aims to bridge this knowledge gap by systematically investigating previously unexplored Indian hot springs. Employing targeted anaerobic cultivation strategies, our objectives were to (1) isolate and identify novel thermophilic anaerobic bacteria possessing lignocellulolytic capabilities, (2) conduct comprehensive physiological characterisation, evaluating their substrate degradation profiles (cellulose, hemicellulose), fermentation end-products, key enzymatic activities, and optimal growth parameters, and (3) perform robust phylogenetic and genomic analyses to elucidate their taxonomic position, evolutionary relationships, and the genetic determinants underpinning their lignocellulolytic potential and adaptation to thermophilic conditions. By addressing these objectives, this work seeks to uncover novel microbial taxa from India's unique geothermal environments. We anticipate that the detailed characterisation of the isolates will reveal unique enzymatic machinery and metabolic pathways that are valuable for converting lignocellulosic biomass into sustainable bioenergy. Ultimately, this research is poised to contribute unique biocatalysts and robust microbial chassis, thereby advancing the development of agricultural biomass-based renewable energy solutions in India. Materials and Methods Sample collection and environmental factors’ characterisation Sediment and water samples were collected from various hot springs across different geothermal provinces of India, located in Ladakh (North-Western Himalaya), Maharashtra (West Coast), and Meghalaya (North-Western Himalaya), using Whirl-Pak® High-Temperature bags. The pH, temperature and conductivity of the water were measured using a portable multiparameter (HANNA™ Instruments, USA). An aliquot of the collected sample was filtered through a 0.2 µm syringe filter for the determination of ionic composition. All the samples were transported to the lab in an expanded polystyrene insulated box and immediately processed for setting up the enrichments. The filtered water sample aliquots were subjected to anion determination using an Ion Chromatograph (Metrohm Compact IC plus, Switzerland) equipped with a conductivity detector. Metrosep A Supp 5 (250/4.0) column was used with sodium carbonate and bicarbonate (1.5 mmol/L: 2 mmol/L) mobile phase at a 0.5 ml/min flow rate. Organic disturbance in the sample was suppressed by adding 10% (v/v) acetonitrile in the eluent. To reduce the background conductivity of the eluent and enhance the conductivity of the analytes, 50 mM H 2 SO 4 and 0.45% oxalic acid solution were passed through the Metrohm Suppressor Module (MSM). The temperature of the column was kept at ambient temperature. Multielement Ion Chromatography Anion Solution (89886, Supelco, Merck, USA) was used as an anion standard, which contained 10.0 mg/kg ± 0.2% of F − , Cl − , Br − , NO 3− , PO 4 3− , SO 4 2− anions. For analysis, a 20 µl sample volume was used. The data acquisition and analysis were performed using MagIC Net IC 2.0 software. Enrichment, isolation and cryopreservation The collected samples were flushed with N 2 gas, mixed with sterile anaerobic diluent and transferred to a 125-ml sterile N 2 -flushed serum bottle. The anaerobic diluent consisted of (litre − 1 ) 150 ml each of solution 1 [0.3% K 2 HPO 4 ], solution 2 [0.3% KH 2 PO 4 , 0.6% (NH 4 ) 2 SO 4 , 0.6% NaCl, 0.06% MgSO 4 .7H 2 O and 0.06% CaCl 2 ], 1 ml resazurin (0.1%), 2 ml hemin (0.05%), 1 g L-cysteine-HCl and pH of the medium was buffered by addition of Na 2 CO 3 to 7.0 ± 0.2 in the presence of N 2 :CO 2 (80:20) as the headspace gas (McSweeney et al. 2005 ). After adequate flushing, the bottles were sealed to maintain the positive gas pressure. For enrichments, basal salt medium with four different substrates, i.e., Neutral Detergent Fibre (NDF) (Wu et al. 2025 ), cellulose mixture [microcrystalline cellulose (avicel) and carboxymethyl cellulose (CMC)], xylan, and lignin (471003, Sigma-Aldrich, USA) was used at a final concentration of 1% (w/v). The basal salt medium consisted of (litre − 1 ) 150 ml solution 1, 150 ml solution 2, 0.5 g yeast extract, 1 ml resazurin (0.1%), 1 ml 10x Pfennig trace element solution (McSweeney et al. 2005 ), 2 ml hemin (0.05%), 0.5 g L-cysteine-HCl and pH of the medium was buffered by addition of NaHCO 3 to 7.0 ± 0.2 in the presence of N 2 :CO 2 as the headspace gas. After sterilising the medium, a 10% v/v sample was inoculated into the medium and incubated at varying temperatures of 40 − 85°C with an interval of 15°C. Enrichments were monitored routinely for gas production and turbidity, and positive enrichments were sub-cultured every 7 − 10 days. In terms of individual gas production, the enrichments showing ≥ 10% hydrogen gas production were selected for regular sub-culturing. Positive enrichments were sub-cultured at least 3 − 4 times before carrying out isolations. The serum roll bottle method (Hungate 1969 ; Bryant 1972 ) was used to isolate pure cultures of thermophilic anaerobic bacteria from different dilutions. For this, enrichments were serially diluted up to 10 − 14 using the diluent medium. Briefly, 0.1 ml inoculum was added to 125-ml glass serum bottles containing 10 ml culture medium (pH 7.0 ± 0.2), which comprised of similar basal salt medium but supplemented with 0.5 g trypticase and agar (2%) or Gelrite (0.8%) as the gelling agent. The substrate used for isolation was avicel, CMC and xylan at a final concentration of 0.2% (w/v). The serum bottles were further incubated at their respective temperatures till bacterial colonies were observed. After incubation, the morphologically distinct colonies were picked under anaerobic conditions and inoculated into the fresh liquid culture medium containing simple sugars, viz. , glucose or xylose (1%, w/v), instead of a complex substrate to facilitate the better growth of isolates. The cultures were examined for purity, cell shape, size, motility, etc., using a phase-contrast microscope (Nikon Eclipse 80i, Japan) and Gram staining using a differential interference contrast (DIC) microscope (Olympus BX53, Japan) equipped with a digital camera (Olympus DP 73, Japan). Mixed cultures were purified by repeated serum roll bottle method till pure cultures were obtained. Pure cultures thus obtained were cryopreserved in 15% glycerol at − 80°C and − 196°C (liquid nitrogen) in duplicates. Molecular identification, phylogenetic analysis and ecological distribution The genomic DNA of the cultures were extracted using the Bacterial Genomic DNA Isolation Kit (RKN15-250D, Chromous Biotech, India) following the manufacturer’s instructions. The genomic DNA thus obtained were quality checked by gel electrophoresis, quantified by UV spectrophotometry (NanoDrop 2000 Spectrophotometer, Thermo Scientific, USA) and amplified using 27F and 1492R universal bacterial primers (Hivarkar et al. 2023 ). The amplified PCR products were subjected to restriction digestion using HaeIII restriction enzyme (R0108, New England Biolabs, USA) as per the manufacturer's protocol. The isolates showing different restriction patterns or having the same patterns but differing in sampling locations and growth conditions were outsourced for sequencing at 1st BASE, Singapore. The obtained 16S rRNA gene sequence of the isolates was deposited in the NCBI GenBank database and compared with sequences of other type strains using the BLASTn program (Altschul et al. 1990 ; Benson et al. 1999 ) to confirm their identities. For phylogenetic analyses, the 16S rRNA gene sequences of type strains of nearest matches were downloaded from the NCBI GenBank database. All the obtained sequences were aligned using the MAFFT (Katoh et al. 2019 ) with default settings. The aligned sequences were used to construct a maximum-likelihood-based phylogenetic tree in W-IQ-TREE (Minh et al. 2020 ) using ModelFinder (Kalyaanamoorthy et al. 2017 ) to estimate substitution models and tested by 1000 bootstrap replications. To determine the occurrence of our strains in other environmental habitats, the 16S rRNA gene sequences of our strains were queried with the GenBank dataset. All the closely related cultured and uncultured sequences were downloaded, and their phylogenetic positions were inferred using a maximum-likelihood-based phylogenetic tree, as described earlier. Next, we documented the ecological distribution of the nearest clades from the GenBank dataset. Lignocellulolytic activities, substrate utilisation profile and fermentation product analysis Based on the phylogenetic analysis, phylogenetically distinct thermophilic cultures were selected for the determination of lignocellulolytic activities, substrate utilisation profile and fermentation product analysis. Representative strains were selected based on their isolation location, substrate specificity, temperature and hydrogen production ability to study the strain-level variation. For all the characterisation experiments, strains were revived from the glycerol stock in basal salt medium with glucose to minimise the strain generation error. To determine the lignocellulolytic activities, the selected strains (OD 600 0.5; 1 ml) were inoculated in 9 ml of basal salt medium (pH 7.0 ± 0.1) containing 10 g/L rice straw or wheat straw as the sole carbon source and incubated at respective temperatures. After three days of incubation, the total produced gas was measured by the water displacement method. The composition of fermentation gas was analysed by a gas chromatograph (PerkinElmer, USA) equipped with a Thermal Conductivity Detector (TCD) as described earlier (Lanjekar et al. 2023 ). The supernatant (2 ml) was withdrawn to measure activities of cellulases (avicelase, CMCase, and β-glucosidase), xylanase, and esterases (acetyl esterase, ρ-coumaroyl esterase, and feruloyl esterase) in a microplate-based assay (Mansour et al. 2016 ; Dagar et al. 2018 ). The reaction conditions for each enzyme activity are summarised in Table S1 . The concentration of released monomeric units was calculated using spectrophotometric (A 595 for glucose & xylose, and A 412 for ρ-nitrophenol), and HPLC (for ρ-coumaric acid and ferulic acid) based methods. The heat-denatured culture supernatants were used as the control, and all the enzymatic activities were calculated in International Units (IU). One IU was defined as the amount of enzyme that released 1.0 µmol of a monomeric unit/ml/h. All the experiments were conducted in triplicate. Additionally, the concentrations of ethanol, total volatile fatty acids (VFAs) and non-VFAs were also estimated as described earlier (Lanjekar et al. 2023 ). Results and Discussion Assessment of environmental factors of hot springs Various abiotic environmental factors have been found to impact the structures and diversity of microbial communities in hot springs. These factors include pH, temperature, mineralisation, and geological history, among others (Saghatelyan et al. 2021 ; Li et al. 2023 ). Although pH and temperature are considered critical factors, minerals and sediment composition also play a significant role in shaping microbial community assembly in hot springs (Li et al. 2021 ). Studies have shown that bacterial diversity in samples from springs with similar chemistries tends to overlap more, suggesting that chemistry, including mineral content, plays a crucial role in determining microbial community composition and diversity (Mathur et al. 2007 ). These findings indicate the complex interplay between environmental factors such as minerals, pH, temperature, and chemistry in influencing microbial diversity and community structures in hot springs. The hot springs studied here showed a range of surface temperatures from 42 to 85 ℃, with the exception of Unkeshwar and Shahada, which measured at ≤ 30 ℃. Such low temperatures of these particular hot springs may be due to their distant sub-surface hot water source. Additionally, we noted slight variations in pH levels amongst all the hot springs, with a range of 7.7 to 9.1, indicating their alkaline nature. The details of the geographical location and water quality analysis of the samples collected are mentioned in Fig. 1 and Table S2. Our assessment of conductivity and anion composition revealed significant differences among the hot springs. Still, we did not observe any correlation between the temperature gradient of the hot spring and environmental factors (Chiriac et al. 2017 ). (Place Fig. 1 Here) Based on the World Health Organisation's (WHO) guidelines from 2017, the levels of chloride, nitrate, and sulphate were within the recommended limits at all hot springs except for Chopada and Unhavare hot springs, where chloride levels exceeded 250 mg/Kg (WHO 2017 ). Interestingly, phosphate was only present in Unkeshwar hot spring, while Tural hot spring contained solely chloride. All other hot spring sites contained chloride and sulphate, with the exception of Shahada hot spring, which had chloride and nitrate. However, the nitrate level at Shahada hot spring was found to be higher than the WHO-approved limit (Table S2). It's worth noting that the environmental factors that impact the microbial diversity of hot springs largely remain unresolved (MEYER-DOMBARD et al. 2005; Chiriac et al. 2017 ). Enrichments, isolation and cryopreservation Cellulose and xylan (a major constituent of hemicellulose) are key components of plant biomass, and lignin is a complex aromatic polymer that is highly abundant in lignocellulosic materials. These substrates are crucial as they mimic the components found in lignocellulosic materials, enabling the selective enrichment of bacteria capable of breaking down these complex compounds (Wagner and Wiegel 2008 ). Studies have shown that thermophilic anaerobic bacteria isolated using cellulose and xylan-containing media exhibit efficient degradation of these substrates, with versatility in lignocellulose degradation (Sizova et al. 2011 ; Jia et al. 2016 ). Along with cellulose and hemicellulose, crude lignocellulosic substrates like rice straw contain soluble content, viz. , pectin, which poses a challenge in isolating efficient lignocellulose-degrading strains (Chimphango et al. 2020 ). NDF thus offers a distinct advantage as a carbon source over crude agricultural biomass, due to its lack of soluble components. NDF was included in the enrichment medium to enrich the growth of efficient lignocellulose-degrading thermophilic anaerobic bacteria, which can be used for various biotechnological applications. Most of these industrial applications, including anaerobic digestion, generally function optimally at neutral pH. Hence, a similar pH range was selected for the enrichment process. Out of a total of 160 enrichments, 29 were found to have positive results based on turbidity, gas, and hydrogen production (Table S3). Most of the enrichments showed positive results on xylan-based medium at temperatures of 55°C, 70°C, and 85°C, indicating the presence of a xylanolytic thermophilic anaerobic community in the samples (Fig. 2 ). Many of the enrichments were also positive on cellulose- and NDF-based media, indicating the presence of cellulolytic and lignocellulolytic communities. (Place Fig. 2 Here) Notably, cellulose-based enrichments from Chopada and Tural hot springs showed complete substrate degradation at 55°C within seven days of incubation, indicating efficient cellulose hydrolysis. Furthermore, enrichments from Unhavare showed growth at all high temperatures on both cellulose- and xylan-based media. Similar observations were made for enrichments from Tural hot spring, but only at 55°C and 70°C. These results indicate the presence of thermophilic cellulolytic and xylanolytic anaerobic bacteria at various temperature ranges in these hot springs. It is important to note that none of the enrichments at 40°C were found to be positive except for one on xylan of Aravali, highlighting the absence of mesophilic lignocellulolytic anaerobic bacteria. Also, none of the enrichments from Jakrem and Unkeshwar hot springs showed growth, indicating the absence of anaerobic lignocellulolytic bacteria in these samples. Additionally, none of the lignin-based enrichments showed growth at all the tested temperatures, signifying the limitation of lignin degradation at such elevated temperatures and low levels of oxygen in hot springs. Lignin degradation is generally more favourable under aerobic conditions compared to anaerobic conditions. The degradation and solubilisation of lignin-related compounds, especially high-molecular-weight lignin, are facilitated through oxidative reactions under aerobic conditions (Geib et al. 2008 ). Studies have shown that lignin deconstruction is primarily described in aerobic systems, with biological lignin deconstruction being less prevalent in anaerobic environments (Ko et al. 2009 ; Weng et al. 2021 ; Lankiewicz et al. 2023 ). Overall, 9 cellulose-, 14 xylan-, and 6 NDF-based enrichments were further processed for the isolation of lignocellulolytic thermophilic anaerobic bacteria (Table S3). The isolation procedures from these enrichments led to the growth of morphologically diverse colonies of anaerobic bacteria on roll bottles. Over 300 colonies were picked into their respective liquid medium and incubated at respective temperatures. Of the total colonies picked, the most diverse and the largest number of colonies were observed at 55 ℃, and the roll tubes at 70 ℃ and 85 ℃ yielded only a single type and fewer colonies in all the samples. Based on gas and hydrogen production, 83 isolates were established, of which 41 were cellulolytic (Avicel, 25 at 55 ℃; CMC, 16 at 55 ℃) and 42 were xylanolytic (8 at 40 ℃, 29 at 55 ℃, 4 at 70 ℃ and 1 at 85 ℃). The isolates were sub-cultured several times before confirming their purity based on phase-contrast microscopy and Gram staining. The majority of the cultures were Gram-negative in character with long or short or chain-forming rods (Figure S1 ). All the pure cultures were cryopreserved in 5 ml vials at -80°C and in 2 ml cryo-vials at -196°C, in duplicates (Hivarkar et al. 2023 ). Molecular identification and phylogenetic analysis The in-silico analysis using HaeIII restriction enzyme generated different ribotypes, thus differentiating bacteria at the genus level. The PCR amplification of isolated DNA generated PCR products of c.a. 1400 bp for all isolates. The actual restriction digestion of PCR products of eighty-three isolates using HaeIII produced nineteen types of restriction patterns (Figure S2), which helped document the diversity and minimise the number of sequencing reactions. Twenty-seven isolates were chosen for sequencing and identification based on differences in geographical locations and growing conditions. The identification results established these isolates as members of nineteen different species under thirteen genera: Bacillus , Caldibacillus , Caldicoprobacter , Caloramator , Clostridium , Lacrimispora , Limnochorda , Pseudoclostridium , Seramator , Sporanaerobium , Tepidimicrobium , Thermoanaerobacter and Thermoanaerobacterium . All the strains were members of families Bacillaceae , Caldicoprobacteraceae , Caloramatoraceae , Clostridiaceae , Dysgonomonadaceae , Lachnospiraceae , Limnochordaceae , and Thermoanaerobacteraceae , and Tissierellaceae of phylum Bacillota (synonym Firmicutes ) (Table 1 ). Table 1 Cultivation and identification details of pure isolates of thermophilic anaerobic bacteria obtained from different hot springs across India. Name of hot spring Location Incubation temp. No. of isolates Substrate Representative strain Closest phylogenetic affiliate and GenBank accession number % sequence coverage % similarity Strain designation GenBank accession number Chumathang Ladakh 55°C 2 Xylan XLC5596-1 OR960429 Caldibacillus thermoamylovorans (NR_117028) 100 99.71 70°C 2 XLC7096-1 OR960431 Caldibacillus thermoamylovorans (NR_117028) 100 99.71 Chopada North Maharashtra 55°C 1 CMC CUC P 55106-1 OR960433 Pseudoclostridium thermosuccinogenes (NR_119284) 100 99.67 1 Xylan XUC P 55106-1 OR960434 Limnochorda pilosa (NR_136767) 98 94.82 70°C 1 XUC B 70106-2 OR960435 Caldibacillus thermoamylovorans (NR_117028) 100 99.71 Rajwadi West Maharashtra 55°C 1 Xylan XMR5567-3 OR960436 Clostridium thermobutyricum (LT626257) 99 99.86 Tural West Maharashtra 55°C 12 Avicel AMT5567-1 OR960437 Thermoanaerobacterium aotearoense (NR_026296) 100 98.66 1 AMT5567-11 OR960439 Caloramator viterbensis (NR_025044) 100 99.63 1 AMT5567-15 OR960440 Thermoanaerobacterium butyriciformans (NR_178863) 99 98.80 2 CMC CMT5567-10 OR960441 Thermoanaerobacterium aotearoense (NR_026296) 97 98.58 5 CMT5567-9 OR960444 Tepidimicrobium ferriphilum (NR_117380) 100 99.77 1 CMT5567-11 OR960446 Pseudoclostridium thermosuccinogenes (NR_119284) 100 99.74 3 CMT5567-13 OR960447 Thermoanaerobacterium thermostercoris (NR_122103) 99 98.67 10 Xylan XMT5567-1 OR960448 Thermoanaerobacterium thermostercoris (NR_122103) 99 99.49 1 XMT5567-6 OR960451 Caloramator coolhaasii (NR_024955) 100 99.56 Unhavare West Maharashtra 55°C 11 Avicel AMU5567-5 OR960452 Caloramator proteoclasticus (LM999757) 100 99.71 1 CMC CMU5567-1 OR960456 Caldicoprobacter guelmensis (NR_109614) 99 95.51 1 CMU5567-4 OR960457 Caldicoprobacter faecalis (NR_117173) 100 94.17 1 CMU5567-6 OR960458 Caldibacillus thermoamylovorans (NR_117028) 100 99.71 1 CMU5567-8 OR960459 Caloramator proteoclasticus (LM999757) 100 99.71 14 Xylan XMU5567-13 OR960460 Caloramator coolhaasii (NR_024955) 100 99.50 70°C 1 Xylan XMU7067-6 OR960464 Thermoanaerobacter wiegelii (NR_029301) 97 99.41 85°C 1 Xylan XMU8567-5 OR960465 Bacillus thermozeamaize (NR_137401) 100 99.66 Aravali West Maharashtra 40°C 1 Xylan XHS19111 MF806589 Lacrimispora celerecrescens (NR_026100) 100 99.70 5 XHS29132 MF806588 Lacrimispora indolis (AB971794) 99 99.78 1 XHS1971 KX553978 Sporanaerobium hydrogeniformans (NR_189186) 100 100 1 XHS2771 KX553979 Seramator thermalis (MK170161) 97 99.66 (Place Table 1 Here) From the hot spring of Chumathang in the Ladakh region (northern India), only Caldibacillus thermoamylovorans strains were obtained at 55 ℃ and 70 ℃. A related strain of this organism was also found in the hot spring of Chopada (central India) at 70 ℃ and the hot spring of Unhavare (western India) at 55 ℃ from the Maharashtra region. All these strains obtained from different geographic locations were isolated from a xylan-based enrichment. Along with this strain, Pseudoclostridium thermosuccinogenes and a putatively novel genus were also isolated at 55 ℃ from the Chopada hot spring. Like Caldibacillus thermoamylovorans , a related strain of Pseudoclostridium thermosuccinogenes was also found in the Tural hot spring (western India) from the Maharashtra region. Both these strains were isolated from a CMC-based enrichment at 55 ℃. A maximum number of isolates were obtained from Tural hot springs, c.a. 36, followed by Unhavare hot springs, c.a. 31, both from the same geographical region (western India). Tural hot spring was dominated by species from the genus Thermoanaerobacterium ( T . aotearoense , T. butyriciformans and T. thermostercoris ), Tepidimicrobium ferriphilum followed by single strains of Caloramator viterbensis and Caloramator coolhaasii . We also found a novel species belonging to the genus Thermoanaerobacterium . All these cultures from the Tural hot spring were isolated at 55 ℃ and from avicel-, CMC- and xylan-based enrichments. Unlike the Tural hot spring, the Unhavare hot spring was mostly dominated by the species from genus Caloramator ( C. proteoclasticus and C. coolhaasii ), and two strains of a new genus at 55 ℃, followed by each strain of Thermoanaerobacter wiegelii (at 70 ℃) and Bacillus thermozeamaize (at 85 ℃). Further, from the Aravali hot spring (western India), we isolated thermo-tolerant strains of Lacrimispora indolis , Lacrimispora celerecrescens and Seramator thermalis from xylan-based enrichments. Along with these cultures, we also isolated a novel genus within the family Lachnospiraceae , which we established as Sporanaerobium hydrogeniformans (Hivarkar et al. 2023 ). From the same geographical region, the Rajwadi hot spring (western India) had the least diversity, where we could only find a single strain of Clostridium thermobutyricum . This can be attributed to the high anthropogenic activity observed in the hot spring during sample collection, which may have increased the oxygen levels in the hot spring, limiting the population of anaerobic microorganisms in them (Lindstrom et al. 2002 ). Nonetheless, hot springs are characterised by low biodiversity due to their extreme conditions in terms of temperature and chemical characteristics (Saxena et al. 2017 ; Chiriac et al. 2017 ; Narsing Rao et al. 2021 ). The relationship between the bacteria isolated in this study using 16S rRNA gene-based phylogenetic analysis was also analysed, which revealed the grouping of all the cultures in nine clusters representing nine different families of bacteria (Fig. 3 ). It was observed that all strains of the genus Thermoanaerobacterium and Caloramator were highly related among themselves despite having different origins. Similarly, four identical strains of Caldibacillus thermoamylovorans (Chumathang, Chopada and Unhavare) and two strains of Pseudoclostridium thermosuccinogenes (Chopada and Tural) showed close grouping. We also report two putative novel genera within the family Caldicoprobacteraceae (strains CMU5567-1 and CMU5567-4) and Limnochordaceae (strain XUCP55106-1) and one putative novel species belonging to the genus Thermoanaerobacterium (strain CMT5567-10). Isolation of such novel strains of anaerobic bacteria emphasises that, despite the traditionally low biodiversity associated with hot springs, they offer untapped sources for novel anaerobic microorganisms. The exploration of microbial diversity in hot springs continues to provide new insights into the richness of microbial life in these extreme environments, paving the way for further discoveries and advancements in microbiology (Li et al. 2023 ). (Place Fig. 3 Here) Lignocellulolytic activities, substrate utilisation profile and fermentation product analysis Thermophilic anaerobic bacteria found in oxygen-limited and high-temperature conditions exhibit an extraordinary ability for growth across diverse thermal spectra that align with their ecological niches and physiological characteristics. Although many thermophiles prefer neutral pH conditions, they are adapted to thrive in various environmental conditions, showcasing their ecological versatility (Arbab et al. 2022 ). This variable growth capability is further supported by findings that highlight the robust enzymatic systems employed by thermophiles, enabling the breakdown of complex organic substrates, indicating their potential utility in biotechnological applications, such as biofuel production from plant biomass (Bashir et al. 2019 ; Paredes-Barrada et al. 2024 ). Several research efforts have documented the enzyme production capabilities of thermophilic bacteria isolated from hot spring ecosystems. For instance, studies focused on the enzymatic activities of these bacteria have reported significant production of hydrolytic enzymes, such as cellulases and xylanases, which facilitate fiber degradation (Bala and Singh 2019 ; Ajeje et al. 2021 ; Singh et al. 2022 ; Castañeda-Barreto et al. 2024 ). Here, several thermophilic anaerobic strains isolated from Indian hot springs were evaluated for their lignocellulolytic enzyme activities, substrate utilisation profiles, and fermentation end-products. These isolates represent a range of taxonomic groups and functional traits, enabling us to document strain-specific variations in their ability to deconstruct lignocellulosic biomass and convert it into biofuels and other metabolites under thermophilic conditions. Bacillus The isolated Bacillus thermozeamaize strain XMU8567-5 was cultured from Unhavare hot spring, which was the only culture obtained at 85 ℃ from this study. The first strain of Bacillus thermozeamaize , sporulating aerobic bacilli, was isolated from a corn steep liquor-based fermenter (Mak 2003 ). But the strain XMU8567-5 was obtained under strict anaerobic conditions at elevated temperatures, which suggests that the sporulating nature of this organism might have aided in the survival of this strain under such extreme environmental conditions. The 16S rRNA gene-based phylogenetic analysis using GenBank database revealed presence of similar strains or clones in the compost, hot spring, industrial and household wastes, manure, and soil from China, Denmark, Finland, Ireland, South Korea, UK, and USA (Figure S3). The occurrence of such related strains in varying environments rich in lignocellulosic biomass indicates the species’ ability to degrade complex substrates. In this study, the strain XMU8567-5 was enriched in a xylan-based medium, which indicated its xylanolytic potential. However, the strain could not grow under the tested conditions on rice straw, wheat straw and other simple sugar-containing media. This may be due to its inability to degrade crude lignocellulosic biomass under obligate anaerobic conditions. Nonetheless, anaerobic strains of Bacillus spp. are known to harbour lignocellulolytic activity (Chukwuma et al. 2021 ; Thakur et al. 2021 ). Caldibacillus The characteristic features of Caldibacillus thermoamylovorans strains obtained from Chumathang (strain XLC5596-1) and Unhavare (strain CMU5567-6) were compared based on their lignocellulolytic activities and fermentation production analysis. The type strain of Caldibacillus thermoamylovorans (homotypic synonym, Bacillus thermoamylovorans ), a non-sporulating, amylolytic, facultative anaerobic bacillus, was first isolated from palm wine, a tropical alcoholic beverage (COMBET-BLANC et al. 1995). Like our strains XLC5596-1 and CMU5567-6, similar strains and clones of this organism were also detected in the hot springs across China, Indonesia, Nigeria, South Korea and Turkey. This organism is also widely present in faeces of buffalo, cow, goat, human, mice and pig, bioreactor and compost from Germany, Japan, Russia, South Africa, Sweden, Taiwan, and UK (Figure S4). Studies have shown that Caldibacillus thermoamylovorans positively influences microbial ecosystems by reducing lag phases and enhancing the growth of other microorganisms during hydrogen-producing bioprocesses (Cabrol et al. 2017 ). Its capacity to generate thermostable enzymes, such as lipases, medium-chain-length polyhydroxyalkanoate (mcl-PHA), etc., further emphasises the diverse lignocellulolytic potential of this species (Deive et al. 2012 ; Choonut et al. 2020 ). The strains of Caldibacillus thermoamylovorans showed a vast difference in lignocellulolytic activities, with strain XLC5596-1 producing 9.29 ± 1.0 IU β-glucosidase, whereas strain CMU5567-6 produced 4.32 ± 1.8 IU and 99.14 ± 6.2 IU acetyl and ρ-coumaroyl esterase, respectively, on rice straw as substrate. On wheat straw, both the strains showed moderate xylanase activity, while strain XLC5596-1 showed 8.53 ± 0.8 IU β-glucosidase and 35.54 ± 8.4 IU acetyl esterase activities (Figs. 4 & 5 ). Similar differences between the strains were also observed for the ρ-coumaroyl and feruloyl esterases, where rice straw stimulated the esterase enzyme production in them (Fig. 5 ). These results indicate strain and substrate-level variations in the enzyme activities of these isolates. The strains produced major quantities of ethanol followed by acetic-, lactic- and formic-acids, and hydrogen gas in traces. Among these end-products, ethanol and acetic acids were produced in higher quantities by strain CMU5567-6, while strain XLC5596-1 produced high amounts of lactic and formic acids. Differences were also found between the strains with respect to substrate profile, where strain XLC5596-1 did not grow under the tested conditions on any of the six monomeric sugars. The strain CMU5567-6 could only ferment hexose and not pentose sugars, where it produced similar end-products as were obtained from crude lignocellulosic substrate rice and wheat straws. Interestingly, the strain CMU6567-6 produced butyric acid and traces of succinic acid from rhamnose sugar instead of lactic acid (Fig. 6 ). These results indicate that the substrate influences the metabolic pathways in the Caldibacillus thermoamylovorans strains (Yue et al. 2021 ). Moreover, considering their high temperature survival and production of lignocellulolytic enzymes at 55 ℃, Caldibacillus thermoamylovorans strains can be considered a potential candidate to produce lactic acid from crude lignocellulosic substrates. Caldicoprobacteraceae Members of the family Caldicoprobacteraceae , specifically the genus Caldicoprobacter , are of industrial importance due to their ability to produce industrially significant enzymes and biochemical compounds. They are all strictly anaerobic thermophilic heterotrophic bacteria utilising sugars but not proteinaceous compounds and are mostly found in faeces of herbivores and terrestrial hot springs (Bouanane-Darenfed et al. 2014 ). Similarly, we found related species and clones of strain CMU5567-1 and strain CMU5567-4, both isolated from Unhavare hot spring, mostly from hot spring and sheep faeces, with the exception of some clones being detected in compost, reactors, sludge and soil (Figure S5). Moreover, the only genus Caldicoprobacter within this family has been classified to play an important role in lignocellulosic biomass bioconversion by producing extracellular xylanase and cellulase enzymes (Jensen et al. 2021 ; Soares et al. 2022 ). Such findings suggest that our strains CMU5567-1 and CMU5567-4, which belong to a putatively new genus within the family Caldicoprobacteraceae , could also demonstrate fibrolytic activities. However, in this study, only strain CMU5567-1 could hydrolyse and utilise rice straw, wheat straw and monomeric sugars, due to the specific requirement of C/N ratio in some members of this family (Fernandez-Bayo et al. 2020 ). The Caldicoprobacteraceae bacterium strain CMU5567-1 showed high feruloyl esterase activity of 564.7 ± 46.8 IU and 132 ± 15.4 IU on wheat and rice straw, respectively, at 55℃. The culture also produced comparatively higher xylanases enzyme (12.39 ± 2.95 IU) on wheat straw, with the exception of acetyl esterase enzyme, which was produced in higher titer on rice straw (Figs. 4 & 5 ). The fermentation end-products of the strain were ethanol, hydrogen, acetic- and formic-acid, while it additionally produced trace amounts of succinic acid from wheat straw. This indicates that the culture prefers wheat straw over rice straw, as was evident by high enzyme titer and metabolic end-products, like other members of this family (Jensen et al. 2021 ). Furthermore, Caldicoprobacteraceae bacterium strain CMU5567-1 showed vast differences in the metabolic end-products utilising the six hexose and pentose sugars. Propionic acid was produced from pentoses like xylose and arabinose, while hexose sugars like rhamnose led to the production of butyric acid; all these end-products, alongside the other metabolites, viz. , ethanol and acetic acid. Hydrogen gas was not produced from glucose, xylose, mannose and galactose, whereas formic acid was produced comparatively in higher quantities from hexoses than pentoses (Fig. 6 ). We also found traces of lactic acid being produced by this isolate utilising the six monomeric sugars. The habitat of strain CMU5567-1, along with the production of high titres of lignocellulolytic enzymes and associated end-products, highlights its importance in bioconversion pathways, emphasising its role in the efficient utilisation of lignocellulosic biomass and supporting the placement of the strain in the family Caldicoprobacteraceae . Caloramator In this study, we obtained three species of genus Caloramator, i.e., Caloramator coolhaasii , Caloramator proteoclasticus and Caloramator viterbensis at 55 ℃. The two Caloramator coolhaasii strains were isolated from Tural and Unhavare hot springs, while the other two strains of Caloramator proteoclasticus were obtained from Unhavare hot spring, but on avicel and CMC-based medium, while Caloramator viterbensis was isolated from Tural hot spring and enriched only on avicel-based medium. These three species of Caloramator have been identified as organisms with significant lignocellulolytic potential (Ledbetter et al., 2007 ), and majority of the related members are detected in the lignocellulosic biomass-rich environments like hot springs and bioreactors across China, Colombia, Finland, Italy, Japan, Netherlands, Tibet and USA (Figure S6). Amongst Caloramator coolhaasii has been characterised as a glutamate-degrading anaerobe with distinct physiological characteristics (Plugge et al. 2000 ). Additionally, Caloramator proteoclasticus has been reported to produce propionate and butyrate (Ding et al. 2022 ). Caloramator viterbensis , a glycerol-fermenting anaerobe, utilises various substrates including sugars, amino acids, and starch, producing acetate, ethanol, and lactate as end products (Seyfried et al. 2002 ). The genus Caloramator , to which these species belong, has been recognised for its thermophilic and glutamate-degrading properties (Baena and Patel 2015 ). Furthermore, the lignocellulolytic enzymes produced by these organisms have been of interest for their potential in biomass deconstruction (Lee et al. 2018 ). In the genus Caloramator , all the species produced a significant amount of hydrogen ranging from 7.06 ± 0.36–11.94 ± 0.45 mM (from rice straw) and 10.19 ± 0.25–13.49 ± 0.45 mM (from wheat straw), except Caloramator coolhaasii strain XMT5567-6, which produced a meagre amount. High hydrogen production can be linked to the fibrolytic nature of the culture, but none of the cultures showed significant lignocellulolytic activities, except esterase activities. Interestingly, high feruloyl esterase activity was expressed by the cultures on rice straw (382.7 ± 0.5–492.72 ± 3.4 IU) than on wheat straw (11.74 ± 1.5–53.86 ± 18.1 IU) (Fig. 5 ). Xylanase enzyme was produced by both strains of Caloramator proteoclasticus on wheat straw, whereas the Caloramator coolhaasii strain XMU5567-13 produced xylanase enzyme on rice straw and β-glucosidase enzyme on wheat straw (Fig. 4 ). Caloramator coolhaasii strain XMT5567-6 and Caloramator viterbensis strain AMT5567-11 did not produce any of the cellulase or xylanase enzymes. Furthermore, Caloramator coolhaasii strain XMU5567-13 produced 79.14 ± 1.4 mM ethanol from wheat straw, which was highest amongst the cultures studied in this work (Fig. 6 ). Along with hydrogen and ethanol, these cultures produced acetic acid (all strains), lactic acid ( Caloramator coolhaasii strain XMT5567-6) and formic acid ( Caloramator coolhaasii strain XMU5567-13 and Caloramator proteoclasticus ). The differences in enzyme production and metabolite end-products were apparent at substrate-, strain- and species-levels among the members of the genus Caloramator . (Place Fig. 4 Here) Like crude lignocellulosic substrates, the fermentation end-products of Caloramator species from monomeric sugars were ethanol, hydrogen and acetic acid, while there was variation in the production of other metabolites like propionic-, butyric-, succinic- and formic acids. Formic acid was not produced by Caloramator viterbensis strain AMT5567-11, but it was the only member of the genus Caloramator which utilised all six sugars tested. We could observe strain- and substrate-level variations in the strains of Caloramator coolhaasii and Caloramator proteoclasticus . The Caloramator coolhaasii strains could not ferment arabinose and rhamnose sugar, and they additionally produced 25.31 ± 2.07–29.61 ± 2.21 mM propionic acid from xylose sugar. Amongst them, strain XMT5567-6 also produced succinic acid (1.37 ± 0.05 mM) from xylose and lactic acid was comparatively produced in higher amounts by strain XMT5567-13 from other sugars. Furthermore, both strains of Caloramator proteoclasticus could not utilise arabinose, while the strain CMU5567-8 was also not able to utilise xylose and rhamnose sugars. Caloramator proteoclasticus strain AMU5567-5 produced propionic acid (37.71 ± 3.42 mM) instead of acetic acid from xylose and it also produced butyric acid (21.85 ± 0.99 mM) from rhamnose (Fig. 6 ). Similarly, Caloramator viterbensis strain AMT5567-11 also produce propionic acid instead of acetic acid from pentose sugars xylose and arabinose. It also produced 5.93 ± 0.14 mM butyric acid and minor quantities of propionic acid from rhamnose sugar. These results indicate that the substrates direct the metabolic pathway in the members of genus Caloramator . To the best of our knowledge, this is the first study to document the lignocellulolytic potential of Caloramator coolhaasii , Caloramator proteoclasticus and Caloramator viterbensis species, and highlight their industrial importance in the production of bioenergy products. Further specific studies on Caloramator species’ lignocellulolytic capabilities would be beneficial to understand its role in biomass degradation. Clostridium Several studies have extensively investigated various species within the genus Clostridium for their lignocellulolytic properties. Clostridium thermocellum , also known as Hungateiclostridium thermocellum or Acetivibrio thermocellus , is the most profoundly studied lignocellulolytic bacterium known for its multienzyme complex, the cellulosome, which is of great potential value in lignocellulose biorefinery (Yan et al. 2022 ). Other important strains studied for industrial applications include Clostridium butyricum , Clostridium tyrobutyricum , and Clostridium thermobutyricum . These species have shown potential in lignocellulose biorefinery processes due to their enzymatic capabilities in degrading lignocellulosic biomass (Chu et al. 2021 ). Clostridium thermobutyricum especially has the potential for high butyrate titer and volumetric productivity (Wang et al. 2015 ). The first representative strain of Clostridium thermobutyricum was a thermotolerant strain isolated from the cellulolytic enrichment of horse manure (WIEGEL et al. 1989 ). On the contrary, we obtained Clostridium thermobutyricum strain XMR5567-3, a thermophilic strain, from the xylanolytic enrichment of sediments from the Rajwadi hot spring at 55 ℃. The 16S rRNA gene-based ecological distribution revealed presence of this species in diverse environments like biogas plant sludge, bioreactor, leachate sediment, and solvent factory sludge in the regions of Canada, China, Germany, Thailand and USA (Figure S7). Such habitats reveal that the organism survives harsh environments, majorly rich in acidic content. Moreover, the species is not well studied to identify its lignocellulolytic potential, and most of the research is focused on the butyric acid production from sugars (Wang et al. 2015 ). Here, we tried to determine the ability of Clostridium thermobutyricum strain XMR5567-3 to degrade crude lignocellulosic substrates, rice and wheat straw, and measure whether the liberated sugars from them can further be converted to high titres of butyric acid. Strain XMR5567-3, under the tested conditions at 55 ℃, secreted 118.72 ± 16.23 IU ρ-coumaroyl esterase and 483.10 ± 4.77 IU feruloyl esterase from rice straw and 48.55 ± 5.72 IU feruloyl esterase from wheat straw (Fig. 5 ). Secretion of only esterase enzymes may have led to the release of a very minor amount of sugars; hence, the culture produced a mere 1.17 ± 0.06 mM butyric acid from wheat straw (Fig. 6 ). Interestingly, rice straw substrate led to the metabolic shift in the organism, which produced 14.40 ± 2.77 mM ethanol instead of butyric acid. Also, the strain XMR5567-3 could not ferment any of the six sugars tested in this study due to the sequential transfer of the culture, which increased butyric acid concentrations and decreased the level of glucose tolerance in the inoculum used (Canganella et al. 2002 ). Moreover, production of esterase enzymes and ethanol contradicts the fact that Clostridium thermobutyricum strain only produces butyric acid from sugars. Hence, we propose that more studies should be focused on determining the metabolic pathways followed by this organism under different conditions to recognise its industrial potential. (Place Fig. 5 Here) Lacrimispora Clostridium celerecrescens and Clostridium indolis have been recently reclassified as Lacrimispora celerecrescens and Lacrimispora indolis (Haas and Blanchard 2020 ). Both these strains were not well studied until 2021, when Kobayashi et al. ( 2021 ) identified Lacrimispora indolis as a species with lignocellulolytic potential, providing genomic insights into its enzymatic systems. Here, we obtained several copies of Lacrimispora indolis strain XHS29132 and a single strain of Lacrimispora celerecrescens strain XHS19111 from the Aravali hot spring on a xylan enrichment at 42 ℃. To identify the potential of these strains to disintegrate lignocellulosic substrates, a 16S rRNA-based ecological distribution was carried out. The related cultured and non-cultured strains of Lacrimispora indolis were found either in the rumen of herbivores or rumen content augmented anaerobic digesters operated in China, Germany, India, Japan, and Tunisia (Figure S8). Conversely, the similar cultured and non-cultured strains of Lacrimispora celerecrescens showed diverse habitat preference, including faeces of mealworms, pig and wild-boar, harsh environments like coal seam gas bore wells, Uranium mining wastes, corroding gas pipeline, volcanic steam vents and petroleum/ pesticide contaminated soils, etc. (Figure S8). The presence of Lacrimispora indolis species in the lignocellulosic-rich environment and recent findings suggest that they may have lignocellulolytic potential. However, neither species could grow on complex or simple substrates under the tested conditions. Limnochordaceae We obtained another culture belonging to a putatively novel genus under the family Limnochordaceae , viz. , Limnochordaceae bacterium strain XUC P 55106-1. This family has been lately classified with the sole species, i.e., Limnochorda pilosa (Watanabe et al. 2015 ), hence to the best of our knowledge not many studies are available in literature highlighting its fiber degrading potential. Also, the type strain of Limnochorda pilosa is a moderately thermophilic, facultatively anaerobic, sporulating pleomorphic bacterium isolated from a brackish meromictic lake in Japan. While our strain Limnochordaceae bacterium strain XUC P 55106-1 is a thermophilic obligately anaerobic bacterium isolated from Unhavare hot spring with an in-situ temperature of > 70℃. The phylogenetic analysis of the 16S rRNA gene of strain XUC P 55106-1 showed that highly similar clones were detected in the swine manure and municipal waste-based composts (Figure S9). This suggested that the strain XUC P 55106-1 may have fibrolytic capabilities. This was verified when the strain when grown on rice straw and wheat straw at 55 ℃, showed 26.54 ± 5.1 IU and 13.5 ± 7.1 IU xylanase and acetyl esterase activities, respectively, along with β-glucosidase (35.76 ± 3.2 IU), ρ-coumaroyl esterase (238.08 ± 38.6 IU), feruloyl esterase (10.63 ± 4.8 IU) and moderate CMCase (0.23 ± 0.5 IU) activities (Figs. 4 & 5 ). The majority of the enzyme titers were higher when rice straw was used as a substrate, and it led to the production of 28.66 ± 0.25 mM acetic acid, 25.67 ± 0.09 mM formic acid, 20.02 ± 2.17 mM ethanol and 10.57 ± 1.09 mM hydrogen as by-products. Similar fermentation end-products were produced by the strain from wheat straw but comparatively in lower quantities as the enzyme secretions were also lower in those experimental bottles (Fig. 6 ). Considering the strain’s ability to produce a broad range of enzymes, it can be termed a fibrolytic organism that can utilise untreated rice straw efficiently at elevated temperatures. The strain XUC P 55106-1 was a hexose-fermenting bacterium as it did not grow in the xylose and arabinose-containing medium. Also, it did not produce hydrogen gas in any of the hexose-containing media. It produced higher amounts of ethanol followed by acetic- and formic-acid from glucose, mannose, rhamnose and galactose (Fig. 6 ). Interestingly, a metabolic shift was also observed in this bacterium as it additionally produced 20.26 ± 0.31 mM butyric acid from rhamnose sugar. These results indicate that Limnochordaceae bacterium strain XUC P 55106-1 can be termed as an industrially important strain to produce thermostable lignocellulolytic enzymes and can also be used in a single-pot consolidated bioprocessing to produce bioenergy products. (Place Fig. 6 Here) Pseudoclostridium The thermophilic Pseudoclostridium thermosuccinogenes strains are known to produce succinate from lignocellulosic-derived sugars, showcasing their potential as a platform organism for various industrial applications (Ganguly et al. 2022 ). Studies have shown that these strains are incapable of degrading cellulose, but they exhibit rapid growth on inulin and various monosaccharides, emphasising their unique metabolic pathways and enzymatic capabilities (Koendjbiharie et al. 2020 ). Our phylogenetic analysis showed the prevalence of this organism in cellulolytic enrichments and lignocellulose-rich compost, cow manure and soil (Figure S10), indicating the lignocellulose-degrading capability of these strains. Also, we obtained two strains of thermophilic Pseudoclostridium thermosuccinogenes , viz. , strain CMT5567-1 and strain CUC P 55106-1, from cellulolytic enrichments of Tural and Chopada hot springs, respectively, at 55 ℃. Surprisingly, only Pseudoclostridium thermosuccinogenes strain CUC P 55106-1 could produce cellulose-attacking avicelase (1.00 ± 0.8–1.57 ± 0.4 IU), CMCase (1.34 ± 0.2–2.44 ± 0.5 IU), and β-glucosidase (12.10 ± 0.9– 44.07 ± 2.8 IU) enzymes alongside feruloyl esterase and ρ-coumaroyl esterase. The other strain, CMT5567-1, did not produce any of the cellulolytic enzymes and only produced a high titer of feruloyl esterase and lower titers of xylanase, β-glucosidase and ρ-coumaroyl esterase enzymes (Figs. 4 & 5 ). Strain variation was also seen among these strains, such as the species of Caldibacillus and Caloramator . Though both strains showed significant growth on wheat straw over rice straw, the fermentation end products varied among the substrates. The strain CMT5567-1, like most strains of Pseudoclostridium thermosuccinogenes , produced 2.35 ± 0.10 to 2.93 ± 0.33 mM succinic acid along with ethanol, hydrogen and acetic acid. Whereas the other strain, CUCP55106-1, which was fibrolytic, produced 7.60 ± 0.64 to 8.00 ± 0.39 mM lactic acid instead of succinic acid, along with other similar end products (Fig. 6 ). Furthermore, neither strain could utilise any of the six monomeric sugars. Such variation in growth pattern and end product generation could be attributed to the CO 2 limitation, which decreases the NAD + /NADH ratio, ultimately leading to stress and an increase in lactic acid levels (Koendjbiharie et al. 2020 ). Seramator Seramator thermalis is a new genus and new species added recently to the family Dysgonomonadaceae (Liu et al. 2020b ). This moderately thermophilic type strain was isolated from a hot spring in China and had cellulose and xylan degrading ability. Similarly, we also obtained the Seramator thermalis strain XHS2771 from the Aravali hot spring, exhibiting the same characteristics. The occurrence of this organism across anaerobic digesters, municipal waste digesters, etc., explains its ability to degrade complex substrates like lignocellulosic biomass (Figure S11). However, the optimal growth temperature of around 45 ℃ limited the strain’s growth on the rice straw, wheat straw and other sugar containing media. Nonetheless, the strain’s ability to simultaneously degrade cellulose and xylan marks this species as a vital part of the anaerobic digestion process of agricultural wastes (Maus et al. 2020 ; Schroeder et al. 2022 ). Sporanaerobium In addition to the strain XHS2771, we also isolated a new species having cellulolytic and xylanolytic activities belonging to the family Lachnospiraceae from the same Aravali hot spring sample. We established the novelty of this strain and named it Sporanaerobium hydrogeniformans strain XHS1971 (Figure S12) (Hivarkar et al. 2023 ). It was isolated from xylan enrichment and exhibited good cellulase activity in addition to xylanase activity and produced significant quantities of biohydrogen. However, being a moderate thermophilic strain growing optimally at 45 ℃, its lignocellulolytic potential and substrate profile could not be determined at 55 ℃. The strain XHS1971 belonged to the family Lachnospiraceae , whose members are acknowledged as one of the most potent polysaccharide degraders owing to their ability to produce free or complex hydrolytic enzymes, it can be used for lignocellulosic biomass-derived biofuel production (Biddle et al. 2013 ). Tepidimicrobium Moderately thermophilic and Fe(III)-reducing Tepidimicrobium ferriphilum cultures are known to utilise proteinaceous compounds and not carbohydrates (Slobodkin et al. 2006 ). Yet, a thermophilic strain, viz. , Tepidimicrobium ferriphilum strain CMT5567-9, was obtained from a cellulose enrichment of the Tural hot spring sediment. This may be attributed to the presence of yeast extract and traces of Fe(III) salts present in the enrichment medium. But the strain could not grow under the tested conditions on rice straw, wheat straw and other simple sugar-containing media due to limiting proteinaceous substrates. A 16S rRNA gene-based phylogenetic analysis of this isolate showed the prevalence of such species in cow faeces, hot springs, and mesophilic and thermophilic anaerobic digesters (Figure S13). Such habitat preference directs that this species may possess unique metabolic pathways and enzymatic activities that contribute to its survival and function in anaerobic and thermophilic niches (Shikata et al. 2018 ). While specific properties of Tepidimicrobium ferriphilum may not be extensively documented, drawing from the broader characteristics of thermophilic anaerobes can provide a foundational understanding of their potential traits. Further research focusing directly on Tepidimicrobium ferriphilum would be valuable to elucidate its distinct properties and metabolic features in more detail. Thermoanaerobacter Thermoanaerobacter is a genus known for its thermophilic and anaerobic characteristics, with species capable of saccharolytic metabolism producing ethanol, acetic acid, lactic acid, alanine, CO 2 , and H 2 as end-products (Onyenwoke and Wiegel 2015 ; Michael Scully and Orlygsson 2019 ). Thermoanaerobacter wiegelii , a species within this genus, has been identified as an obligately anaerobic, thermophilic, and polysaccharolytic bacterium capable of fermenting various sugars to produce end products such as ethanol, acetic-, lactic-, propionic-acid, and H 2 (COOK et al. 1996 ). Genomic evaluations have highlighted the potential of Thermoanaerobacter spp., including Thermoanaerobacter wiegelii , for metabolic engineering to improve lignocellulosic biofuel production due to their high ethanol yields from hemicellulosic sugars (Brynjarsdottir et al. 2012 ; Verbeke et al. 2013 ). Studies have even indicated that Thermoanaerobacter wiegelii , along with other Thermoanaerobacter species, can enhance glycerol conversion when co-cultivated with methanogenic partners (Magalhães et al. 2020 ). Here, a hyperthermophilic culture of Thermoanaerobacter wiegelii , strain XMU7067-6, was obtained from a xylan enrichment of Unhavare hot spring sediment at 70 ℃. The strain’s ecological analysis based on 16S rRNA-based phylogeny showed that it mostly preferred hot environments like hot springs, hydrothermal vents and thermophilic digesters for dwelling (Figure S14). This directed us to look for its fibrolytic potential, so the strain XMU7067-6 was subjected to rice straw and wheat straw-based medium to evaluate the enzyme profile and subsequent fermentation products. The strain XMU7067-6 at 70 ℃ exhibited higher feruloyl esterase activity of 655.03 ± 3.8 IU on rice straw and merely 74.69 ± 7.4 IU activity on wheat straw (Fig. 5 ). Xylanase and β-glucosidase activities also differed on different straws, where they showed 11.18 ± 1.2 IU on rice straw and 15.99 ± 0.7 IU on wheat straw, respectively (Fig. 4 ). Production of this range of enzymes led to the metabolites like ethanol, hydrogen, acetic acid, and minor amounts of lactic and formic acids. End products were higher on wheat straw where strain XMU7067-6 produced 36.23 ± 1.33 mM ethanol, 14.05 ± 1.00 mM hydrogen and 7.17 ± 0.32 mM acetic acid with traces of lactic acid, i.e., 1.50 ± 0.13 mM (Fig. 6 ). These distinctive characteristics, along with its metabolic versatility and lignocellulolytic capabilities, position Thermoanaerobacter wiegelii strain XMU7067-6 as a valuable candidate for various biotechnological applications, particularly in the realm of biofuel production from wheat straw (Georgieva et al. 2008 ; Brynjarsdottir et al. 2012 ). Further, the strain XMU7067-6 co-utilised both hexose and pentose sugars to produce similar end-products. Fermenting hexose sugars, it produced 13.20 ± 1.37 to 18.76 ± 1.20 mM ethanol, 6.79 ± 0.47 to 6.98 ± 1.06 mM hydrogen, and 5.58 ± 0.26 to 7.38 ± 0.21 mM acetic acid; while 1.97 ± 0.07 mM lactic acid was produced only from glucose (Fig. 6 ). Interestingly, rhamnose sugar led to the generation of low ethanol and no hydrogen, but 19.62 ± 0.66 mM butyric acid, which was not reported in this species earlier. Moreover, the strain XMU7067-6 produced 10.08 ± 0.13 mM propionic acid and 3.00 ± 0.05 mM formic acid in addition to ethanol, hydrogen, acetic acid and lactic acid from arabinose sugar (Fig. 6 ). A similar end-product profile was also observed with xylose, indicating the shift in the metabolic pathway in Thermoanaerobacter wiegelii due to pentose sugars (Lin et al. 2011 ). These findings underscore the potential of utilising Thermoanaerobacter wiegelii and related species in biofuel production, leveraging their thermophilic, lignocellulolytic, and pentose and hexose co-utilisation properties. Understanding how Thermoanaerobacter wiegelii manages the metabolism of pentose sugars like xylose or arabinose alongside hexose sugars can provide crucial information for optimising biofuel production processes. However, the specific properties and applications of Thermoanaerobacter wiegelii would need to be directly studied to fully understand its capabilities and contributions to lignocellulosic biofuel production. Thermoanaerobacterium Thermoanaerobacterium species, recognised for their thermophilic nature, have garnered attention for their potential in biofuels production and various industrial applications due to their unique properties and capabilities. These species can directly utilise lignocellulosic materials, such as various paper substrates, to produce biofuels, showcasing a broad substrate range and high operating temperatures which enhance the efficiency of lignocellulose degradation and reduce the risk of microbial contamination (Popova et al. 2021 ; Wu et al. 2021 ; Dai et al. 2023 ). Amongst Thermoanaerobacterium species, Thermoanaerobacterium aotearoense , Thermoanaerobacterium butyriciformans , and Thermoanaerobacterium thermostercoris are anaerobic thermophilic bacteria with diverse metabolic properties, including lignocellulolytic capabilities. Thermoanaerobacterium aotearoense has been identified as a slightly acidophilic thermophile capable of forming elemental sulfur from thiosulfate and growing at acidic pH values at elevated temperatures (LIU et al. 1996). This species has been associated with bioethanol and biohydrogen production and the expression of thermostable enzymes like β-glucosidase for biomass conversion (Huang et al. 2015 ; Yang et al. 2015 ). Thermoanaerobacterium butyriciformans is another spore-forming anaerobic thermophilic bacterium known for its ability to degrade cellulose and hemicellulose to produce acetic acid, butyric acid, and alcohols (López et al. 2017 ). Thermoanaerobacterium thermostercoris , a non-sporulating member of this genus, has been studied for its hemicellulolytic activities, particularly xylanase production, highlighting its potential for biomass valorisation and lignocellulosic waste utilisation (Romano et al. 2010 ; Finore et al. 2021 ). Here, we characterise two strains each of Thermoanaerobacterium aotearoense (strains AMT5567-1 & CMT5567-10), Thermoanaerobacterium thermostercoris (strains CMT5567-13 & XMT5567-1) and one strain of Thermoanaerobacterium butyriciformans (strain AMT5567-15), which were obtained from avicel, CMC and xylan-based medium of Tural and Unhavare hot springs. The 16S rRNA gene-based phylogeny indicated that all these strains are closely related to each other and found in diverse locations like canned food, compost, oil reservoir fluids, and thermophilic reactors (Figure S15); so, most of the by-products and properties of them would overlap. Fibrolytic analysis revealed that Thermoanaerobacterium aotearoense strain CMT5567-10 showed majority of the tested lignocellulolytic activities, i.e. avicelase (3.61 ± 1.4 IU), CMCase (2.85 ± 0.6 IU), xylanase (17.86 ± 0.9 IU), β-glucosidase (38.24 ± 3.1 IU) and feruloyl esterase (613.04 ± 10.2 IU) on rice straw, producing a high titer of ethanol, i.e. 44.54 ± 4.94 mM and 7.77 ± 0.16 mM hydrogen (Figs. 4 & 5 ). Additionally, it also produced 19.33 ± 1.4 mM acetic acid and 13.94 ± 0.21 mM formic acid, which were comparatively higher than those of Thermoanaerobacterium aotearoense strain AMT5567-1 (Fig. 6 ). Equally, strain CMT5567-10 produced avicelase (1.16 ± 0.2 IU), xylanase (6.52 ± 1.1 IU), β-glucosidase (18.26 ± 0.6 IU), acetyl esterase (22.25 ± 9.1 IU), ρ-coumaroyl esterase (25.01 ± 1.4 IU) and feruloyl esterase (8.46 ± 3.7 IU) on wheat straw, producing 26.76 ± 2.83 mM ethanol and 9.40 ± 0.76 mM hydrogen. This culture thus may act as a potential candidate for single-pot co-production of hydrogen and ethanol from untreated agricultural wastes. Besides, the Thermoanaerobacterium aotearoense strain AMT5567-1 produced the highest ρ-coumaroyl esterase of 404.22 ± 24.7 IU from rice straw, indicating its substrate specificity (Fig. 5 ). Furthermore, Thermoanaerobacterium butyriciformans strain AMT5567-15 showed lower CMCase and xylanase activities, while 10.05 ± 0.7 IU β-glucosidase (on wheat straw) and 433.42 ± 35.07 IU feruloyl esterase (on rice straw) showcased substrate variability. The strain AMT5567-15 preferred wheat straw over rice straw as it produced a high concentration of ethanol, i.e. 30.74 ± 1.55 mM from wheat straw. No butyric acid was produced by this strain from crude lignocellulosic biomass. Among the Thermoanaerobacterium thermostercoris strains, strain XMT5567-1 showed fibrolytic properties as it produced comparatively higher avicelase, CMCase, xylanase, β-glucosidase, and esterase activities (Figs. 4 & 5 ). Still, the strain CMT5567-13 produced a higher titer of ethanol (45.22 ± 3.45 mM, rice straw), acetic acid (13.49 ± 0.56 mM, rice straw) and hydrogen (6.03 ± 0.31 mM, wheat straw). The fermentation end products of both the Thermoanaerobacterium thermostercoris strains were ethanol, acetic acid and hydrogen, while strain XMT5567-1 additionally produced a minor amount of lactic acid. Strain and substrate level variation was also evident among this group of organisms. These results explain why this group has garnered interest for biofuel production from lignocellulosic biomass. In terms of monomeric sugar utilisation, Thermoanaerobacterium butyriciformans strain AMT5567-15 was the only strain among this group to ferment all six monomeric sugars. It produced a higher amount of hydrogen, i.e. 20.55 ± 0.12 mM to 25.56 ± 0.15 mM, followed by 12.88 ± 0.24 mM to 23.02 ± 0.05 mM ethanol, 5.11 ± 0.38 mM to 11.61 ± 1.00 mM acetic acid and 4.91 ± 0.26 mM to 8.96 ± 0.06 mM butyric acid. Lactic acid in the range 7.84 ± 0.14 mM was also produced by this strain, which was the highest amongst this group. Surprisingly, 5.32 ± 0.05 mM and 18.69 ± 1.36 mM propionic acid were produced from xylose and arabinose, respectively; while 2.06 ± 0.18 mM succinic acid from rhamnose sugar was also produced by this species. Similarly, Thermoanaerobacterium aotearoense strains also had a similar metabolite profile of hydrogen, ethanol, acetic acid and butyric acid. Between them, strain AMT5567-1 produced a comparatively higher amount of hydrogen, whereas strain CMT5567-10 produced a higher amount of ethanol and lactic acid (Fig. 6 ). Interestingly, strain CMT5567-10 produced 8.52 ± 0.63 mM propionic acid from xylose sugar. Acetic acid and butyric acid production were equivalent between these strains, while they did not ferment arabinose sugar. Thermoanaerobacterium thermostercoris strains also utilised arabinose sugar, and they too produced ethanol, hydrogen, acetic acid and butyric acid with trace amounts of lactic acid as their metabolic end products. Among them, strain XMT5567-1 produced comparatively higher ethanol, i.e. 28.03 ± 0.07 mM to 41.99 ± 0.15 mM, while strain CMT5567-13 produced higher hydrogen, i.e. 21.99 ± 0.06 mM to 26.53 ± 0.55 mM. Lactic acid was produced from glucose (6.13 ± 1.34 mM) and mannose (5.21 ± 0.07 mM) by the strain CMT5567-13, whereas 1.48 ± 0.17 mM succinic acid was produced from rhamnose sugar. On the contrary, strain XMT5567-13 produced 17.11 ± 1.74 mM propionic acid in addition to other metabolites from xylose sugar. The strain level variation was also noticed during the substrate profiling of these isolates. In literature, Thermoanaerobacterium thermostercoris strains are reported to be higher hydrogen producers (Wu et al. 2021 ; Finore et al. 2021 ; Dai et al. 2023 ); however, we found strains of Thermoanaerobacterium aotearoense as high hydrogen producing culture followed by Thermoanaerobacterium butyriciformans . In summary, Thermoanaerobacterium species exhibit a wide range of industrially relevant properties, including efficient lignocellulolytic activities, the capability to ferment sugars to ethanol and hydrogen, and unique metabolic pathways for the degradation of cellulose and xylan. These attributes underscore their potential in biofuel production and other sustainable biotechnological applications. Conclusion This study highlights a comprehensive exploration of thermophilic anaerobic bacteria inhabiting Indian hot spring environments with a focus on their potential to degrade lignocellulosic biomass. Through systematic sample collection from ten hot springs across varied geothermal provinces, combined with a rigorous enrichment and isolation strategy, a diverse set of 83 bacterial strains was recovered and characterised. Phylogenetic analysis revealed their affiliation with 19 species across 13 genera, encompassing both known and potentially novel taxa. Functional assays demonstrated that many of these isolates possess significant lignocellulolytic activity, with several strains showing efficient degradation of complex plant polymers and the ability to produce key fermentation metabolites such as ethanol, hydrogen, and volatile fatty acids. Notable strain-level variability was observed in enzyme profiles, substrate preferences, and metabolic outputs, underscoring the functional diversity of this microbial community. Importantly, various strains of Caldibacillus thermoamylovorans , Thermoanaerobacter wiegelii , and Thermoanaerobacterium spp. emerged as promising candidates for bioenergy applications, especially in consolidated bioprocessing setups. These results confirm that Indian hot springs harbour phylogenetically diverse and metabolically versatile thermophilic anaerobic bacteria with strong potential for industrial use in lignocellulosic biomass valorisation. Declarations Acknowledgements The authors are thankful to Dr Karthick Balasubramanian, Agharkar Research Institute, Pune and Dr. Raymond A Duraiswami, Savitribai Phule Pune University, for sampling support. We duly acknowledge the help of Dr. Akshay Joshi extended during the study. We also appreciate the technical assistance from Dr. Bhushan Shigwan, Dr. Radhakrishnan Cheran & Dr. Vikram Lanjekar. We are grateful to the Director, Agharkar Research Institute, for providing the necessary infrastructure support. Author contributions SSH: Investigation, formal analysis, writing—original draft, visualisation, data curation, writing—review and editing; PKD: Conceptualisation, supervision, resources; SSD: Conceptualisation, supervision, resources, funding acquisition, writing—review and editing Funding This research was financially supported by the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India, with project number YSS/2015/000718 awarded to SSD and Junior Research Fellowship to SSH. Conflict of Interest: The authors declare that they do not have any conflicts of interest. References Ajeje SB, Hu Y, Song G, et al (2021) Thermostable Cellulases / Xylanases From Thermophilic and Hyperthermophilic Microorganisms: Current Perspective. Front Bioeng Biotechnol 9:. https://doi.org/10.3389/fbioe.2021.794304 Altschul SF, Gish W, Miller W, et al (1990) Basic local alignment search tool. 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07:28:18","extension":"png","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154052,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/46116582ad36479ca58fa347.png"},{"id":96079796,"identity":"8e72c305-0318-4da5-b3c1-16041bf3ab9d","added_by":"auto","created_at":"2025-11-17 11:22:23","extension":"xml","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":306710,"visible":true,"origin":"","legend":"","description":"","filename":"3a43c5cc3319418e9e62c90e9e42131d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/affd8f63daf165e1395dcec6.xml"},{"id":96079778,"identity":"e7f3c6cf-9cb3-4d72-b241-995eff51a3fb","added_by":"auto","created_at":"2025-11-17 11:22:22","extension":"html","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":323037,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/da610a6f5047dcfbb0aa0d2b.html"},{"id":96079749,"identity":"6f2a805c-36fa-4569-8ddc-8d510784dc5f","added_by":"auto","created_at":"2025-11-17 11:22:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":566989,"visible":true,"origin":"","legend":"\u003cp\u003eMap of India showing hot spring sampling locations with enlarged specific site photos and physiological data of the sampling site.\u003c/p\u003e","description":"","filename":"Figure1Samplingsitelocation.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/b9db5459a505090248677518.jpg"},{"id":96079756,"identity":"0e693baa-9e6b-4260-b2d5-b1b86ab67372","added_by":"auto","created_at":"2025-11-17 11:22:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":803496,"visible":true,"origin":"","legend":"\u003cp\u003eSpatial graph depicting the positive enrichments established at different culturing conditions in an increasing number.\u003c/p\u003e","description":"","filename":"Figure2Spatialgraphofpositiveenrichments.png","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/1654b4664cd697e188374c7d.png"},{"id":96079750,"identity":"02ca9e0b-89a1-4d26-adca-71eba3240d58","added_by":"auto","created_at":"2025-11-17 11:22:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4139280,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood-based phylogenetic tree based on 16S rRNA gene sequences showing the clustering and phylogenetic positions of the isolates obtained in this study with their closest phylogenetic neighbour. The tree was built using W-IQ-TREE with fast model selection via ModelFinder and ultrafast bootstrap approximation, as well as an approximate likelihood-ratio test for branches. Bootstrap values (≥52%) based on 1000 replicates are indicated at branching points. The GenBank accession number of each strain is listed in parentheses. Bar, 0.2 changes per position.\u003c/p\u003e","description":"","filename":"Figure3Phylogenetictreeallisolates.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/3099c655a86c9700fb86d98b.jpg"},{"id":96079753,"identity":"b13c4f97-121d-4fd7-a92e-ffb3b44e1b7c","added_by":"auto","created_at":"2025-11-17 11:22:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1779964,"visible":true,"origin":"","legend":"\u003cp\u003eBubble plot comparing the activities of cellulolytic (avicelase, CMCase, β-glucosidase) and xylanolytic (xylanase) enzymes produced by the bacterial isolates. Activities were measured using rice straw and wheat straw as substrates. The size of each bubble is proportional to the specific enzyme activities (IU).\u003c/p\u003e","description":"","filename":"Figure4CellulaseandXylanaseenzymesoftheisolates.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/7ee9dd767bb568b5109e8083.jpg"},{"id":96079758,"identity":"c3b14309-e187-45df-bfa2-913df94386c3","added_by":"auto","created_at":"2025-11-17 11:22:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1695379,"visible":true,"origin":"","legend":"\u003cp\u003eBubble plot comparing the activities of acetyl-, ρ-coumaroyl- and feruloyl-esterase enzymes produced by the bacterial isolates. Activities were measured using rice straw and wheat straw as substrates. The size of each bubble is proportional to the specific enzyme activities (IU).\u003c/p\u003e","description":"","filename":"Figure5Esteraseenzymesoftheisolates.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/ab9d9c167f5a9e931da8022d.jpg"},{"id":96079771,"identity":"d6f27b6c-bada-46ff-af66-eb8f2993420b","added_by":"auto","created_at":"2025-11-17 11:22:22","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6791468,"visible":true,"origin":"","legend":"\u003cp\u003eBubble plot illustrating the concentration (mM) of key metabolites produced by bacterial isolates (y-axis) grown on different carbon sources (x-axis), including rice straw, wheat straw and various monosaccharides. The panels show results for Ethanol, Hydrogen, Acetic Acid, Propionic Acid, Butyric Acid, Succinic Acid, Lactic Acid, and Formic Acid, with bubble size indicating the amount produced.\u003c/p\u003e","description":"","filename":"Figure6Metabolitesoftheisolates.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/dc3e74d1abfa695bb7b342c5.jpg"},{"id":96256343,"identity":"8d3c5507-8cf6-4353-8377-fe8f17c877af","added_by":"auto","created_at":"2025-11-19 07:50:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17195081,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/1876723e-7c92-477f-8c64-84b1899076bb.pdf"},{"id":96079776,"identity":"66e20dcb-6219-4490-b74a-ac01ec8c83a2","added_by":"auto","created_at":"2025-11-17 11:22:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19078627,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary03.11.25.docx","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/1926d9b158c924670c57a602.docx"},{"id":96079757,"identity":"b4a047e1-6e08-4c60-aa12-8fbbbbae7434","added_by":"auto","created_at":"2025-11-17 11:22:22","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5341474,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8018409/v1/d1d6d3ae90f4ff4716742083.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diverse Thermophilic Anaerobes from Indian Hot Springs Exhibit High Potential for Bioenergy Production from Lignocellulosic Biomass","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThermophilic anaerobic bacteria represent a distinct and ecologically significant group of microorganisms capable of thriving in extreme temperatures (45\u0026ndash;80\u0026deg;C) under oxygen-free conditions. Their significance lies primarily in their ability to break down complex organic materials, particularly lignocellulosic biomass such as rice straw, wheat straw, corn straw, and sugarcane bagasse, predominantly consisting of lignin, cellulose, and hemicellulose. Research indicates that cellulose and hemicellulose constitute approximately 50\u0026ndash;75% of this biomass, while lignin represents the remaining fraction (Rasool and Irfan \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kukreti et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Woźniak et al. \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Sugars released from the hydrolysis of these polymers can be biologically converted into biohydrogen, bioethanol, and other industrially relevant bio-based products (Svetlitchnyi et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mokhtarani et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThermophilic anaerobic bacteria are essential in decomposing organic matter and play a critical role in carbon cycling within geothermal ecosystems. Hot springs, characterised by high temperatures, diverse mineral compositions, and variable pH levels, create unique habitats that host specialised microbial communities adapted to these extreme conditions (Yabe et al. \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Oliverio et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). While much research has focused on mesophilic anaerobic bacteria, particularly those associated with the digestive systems of ruminants (e.g., \u003cem\u003eFibrobacter succinogenes\u003c/em\u003e, \u003cem\u003eRuminococcus flavefaciens\u003c/em\u003e, and others), the potential of thermophilic anaerobes remains largely underexplored (Awan et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mokhtarani et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Tjo et al. \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Hsin et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Prominent thermophilic anaerobic lignocellulose degraders include genera such as \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eCaldibacillus\u003c/em\u003e, \u003cem\u003eCaldicoprobacter\u003c/em\u003e, \u003cem\u003eCaldicellulosiruptor\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e and \u003cem\u003eThermoanaerobacter\u003c/em\u003e, recognised for their robust enzymatic profiles suitable for high-temperature industrial applications (Mhuantong et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Brunecky et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; de Souza et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn India, there is a rich array of geothermal activity with over 300 hot springs located across seven provinces, namely, the Himalayan, Northeast, Son-Narmada-Tapti (SONATA) lineament belt, the Sahara Valley, the West Coast, the Godavari and Mahanadi basins (Yadav et al. \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Each of these environments possesses unique physicochemical properties that support diverse microbial life forms. However, the microbial diversity within these hot springs, particularly thermophilic anaerobic lignocellulolytic bacteria, is understudied, largely because past investigations have centered on aerobic thermophiles or general microbial surveys (Saxena et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Verma et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Soy et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Priyadharshini et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sharma and Kumar \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Existing literature reveals gaps in understanding the metabolic capabilities of the anaerobes, which may present significant opportunities for industrial biomass conversion (Chukwuma et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLately, studies have been increasingly focused on exploring hot springs for novel anaerobic bacterial isolates, given their promising bioenergy applications. Strains belonging to genera such as \u003cem\u003eCaldicellulosiruptor\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e, and \u003cem\u003eThermoanaerobacter\u003c/em\u003e have shown significant potential due to their efficiency in lignocellulose degradation (Liu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Rodionov et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Orlygsson and Scully \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Le et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Hsin et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Building upon valuable prior contributions from Indian researchers who isolated key thermophilic anaerobes like cellulolytic \u003cem\u003eClostridium thermocellum\u003c/em\u003e and xylanolytic \u003cem\u003eThermoanaerobacter\u003c/em\u003e strains (Singh et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), a significant opportunity remains to explore the untapped microbial diversity within India's numerous, yet understudied, hot springs. Many of these unique geothermal niches have not been systematically investigated for their potential to harbour novel bacteria capable of efficient lignocellulose breakdown (Poddar and Das \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study aims to bridge this knowledge gap by systematically investigating previously unexplored Indian hot springs. Employing targeted anaerobic cultivation strategies, our objectives were to (1) isolate and identify novel thermophilic anaerobic bacteria possessing lignocellulolytic capabilities, (2) conduct comprehensive physiological characterisation, evaluating their substrate degradation profiles (cellulose, hemicellulose), fermentation end-products, key enzymatic activities, and optimal growth parameters, and (3) perform robust phylogenetic and genomic analyses to elucidate their taxonomic position, evolutionary relationships, and the genetic determinants underpinning their lignocellulolytic potential and adaptation to thermophilic conditions. By addressing these objectives, this work seeks to uncover novel microbial taxa from India's unique geothermal environments. We anticipate that the detailed characterisation of the isolates will reveal unique enzymatic machinery and metabolic pathways that are valuable for converting lignocellulosic biomass into sustainable bioenergy. Ultimately, this research is poised to contribute unique biocatalysts and robust microbial chassis, thereby advancing the development of agricultural biomass-based renewable energy solutions in India.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSample collection and environmental factors\u0026rsquo; characterisation\u003c/h2\u003e\u003cp\u003eSediment and water samples were collected from various hot springs across different geothermal provinces of India, located in Ladakh (North-Western Himalaya), Maharashtra (West Coast), and Meghalaya (North-Western Himalaya), using Whirl-Pak\u0026reg; High-Temperature bags. The pH, temperature and conductivity of the water were measured using a portable multiparameter (HANNA\u0026trade; Instruments, USA). An aliquot of the collected sample was filtered through a 0.2 \u0026micro;m syringe filter for the determination of ionic composition. All the samples were transported to the lab in an expanded polystyrene insulated box and immediately processed for setting up the enrichments.\u003c/p\u003e\u003cp\u003eThe filtered water sample aliquots were subjected to anion determination using an Ion Chromatograph (Metrohm Compact IC plus, Switzerland) equipped with a conductivity detector. Metrosep A Supp 5 (250/4.0) column was used with sodium carbonate and bicarbonate (1.5 mmol/L: 2 mmol/L) mobile phase at a 0.5 ml/min flow rate. Organic disturbance in the sample was suppressed by adding 10% (v/v) acetonitrile in the eluent. To reduce the background conductivity of the eluent and enhance the conductivity of the analytes, 50 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and 0.45% oxalic acid solution were passed through the Metrohm Suppressor Module (MSM). The temperature of the column was kept at ambient temperature. Multielement Ion Chromatography Anion Solution (89886, Supelco, Merck, USA) was used as an anion standard, which contained 10.0 mg/kg\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2% of F\u003csup\u003e\u0026minus;\u003c/sup\u003e, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, Br\u003csup\u003e\u0026minus;\u003c/sup\u003e, NO\u003csup\u003e3\u0026minus;\u003c/sup\u003e, PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e anions. For analysis, a 20 \u0026micro;l sample volume was used. The data acquisition and analysis were performed using MagIC Net IC 2.0 software.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEnrichment, isolation and cryopreservation\u003c/h3\u003e\n\u003cp\u003eThe collected samples were flushed with N\u003csub\u003e2\u003c/sub\u003e gas, mixed with sterile anaerobic diluent and transferred to a 125-ml sterile N\u003csub\u003e2\u003c/sub\u003e-flushed serum bottle. The anaerobic diluent consisted of (litre\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 150 ml each of solution 1 [0.3% K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e], solution 2 [0.3% KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.6% (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 0.6% NaCl, 0.06% MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO and 0.06% CaCl\u003csub\u003e2\u003c/sub\u003e], 1 ml resazurin (0.1%), 2 ml hemin (0.05%), 1 g L-cysteine-HCl and pH of the medium was buffered by addition of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e to 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 in the presence of N\u003csub\u003e2\u003c/sub\u003e:CO\u003csub\u003e2\u003c/sub\u003e (80:20) as the headspace gas (McSweeney et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). After adequate flushing, the bottles were sealed to maintain the positive gas pressure. For enrichments, basal salt medium with four different substrates, i.e., Neutral Detergent Fibre (NDF) (Wu et al. \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), cellulose mixture [microcrystalline cellulose (avicel) and carboxymethyl cellulose (CMC)], xylan, and lignin (471003, Sigma-Aldrich, USA) was used at a final concentration of 1% (w/v). The basal salt medium consisted of (litre\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 150 ml solution 1, 150 ml solution 2, 0.5 g yeast extract, 1 ml resazurin (0.1%), 1 ml 10x Pfennig trace element solution (McSweeney et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), 2 ml hemin (0.05%), 0.5 g L-cysteine-HCl and pH of the medium was buffered by addition of NaHCO\u003csub\u003e3\u003c/sub\u003e to 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 in the presence of N\u003csub\u003e2\u003c/sub\u003e:CO\u003csub\u003e2\u003c/sub\u003e as the headspace gas. After sterilising the medium, a 10% v/v sample was inoculated into the medium and incubated at varying temperatures of 40\u0026thinsp;\u0026minus;\u0026thinsp;85\u0026deg;C with an interval of 15\u0026deg;C. Enrichments were monitored routinely for gas production and turbidity, and positive enrichments were sub-cultured every 7\u0026thinsp;\u0026minus;\u0026thinsp;10 days. In terms of individual gas production, the enrichments showing\u0026thinsp;\u0026ge;\u0026thinsp;10% hydrogen gas production were selected for regular sub-culturing.\u003c/p\u003e\u003cp\u003ePositive enrichments were sub-cultured at least 3\u0026thinsp;\u0026minus;\u0026thinsp;4 times before carrying out isolations. The serum roll bottle method (Hungate \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Bryant \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1972\u003c/span\u003e) was used to isolate pure cultures of thermophilic anaerobic bacteria from different dilutions. For this, enrichments were serially diluted up to 10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003e using the diluent medium. Briefly, 0.1 ml inoculum was added to 125-ml glass serum bottles containing 10 ml culture medium (pH 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2), which comprised of similar basal salt medium but supplemented with 0.5 g trypticase and agar (2%) or Gelrite (0.8%) as the gelling agent. The substrate used for isolation was avicel, CMC and xylan at a final concentration of 0.2% (w/v). The serum bottles were further incubated at their respective temperatures till bacterial colonies were observed. After incubation, the morphologically distinct colonies were picked under anaerobic conditions and inoculated into the fresh liquid culture medium containing simple sugars, \u003cem\u003eviz.\u003c/em\u003e, glucose or xylose (1%, w/v), instead of a complex substrate to facilitate the better growth of isolates.\u003c/p\u003e\u003cp\u003eThe cultures were examined for purity, cell shape, size, motility, etc., using a phase-contrast microscope (Nikon Eclipse 80i, Japan) and Gram staining using a differential interference contrast (DIC) microscope (Olympus BX53, Japan) equipped with a digital camera (Olympus DP 73, Japan). Mixed cultures were purified by repeated serum roll bottle method till pure cultures were obtained. Pure cultures thus obtained were cryopreserved in 15% glycerol at \u0026minus;\u0026thinsp;80\u0026deg;C and \u0026minus;\u0026thinsp;196\u0026deg;C (liquid nitrogen) in duplicates.\u003c/p\u003e\n\u003ch3\u003eMolecular identification, phylogenetic analysis and ecological distribution\u003c/h3\u003e\n\u003cp\u003eThe genomic DNA of the cultures were extracted using the Bacterial Genomic DNA Isolation Kit (RKN15-250D, Chromous Biotech, India) following the manufacturer\u0026rsquo;s instructions. The genomic DNA thus obtained were quality checked by gel electrophoresis, quantified by UV spectrophotometry (NanoDrop 2000 Spectrophotometer, Thermo Scientific, USA) and amplified using 27F and 1492R universal bacterial primers (Hivarkar et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The amplified PCR products were subjected to restriction digestion using \u003cem\u003eHaeIII\u003c/em\u003e restriction enzyme (R0108, New England Biolabs, USA) as per the manufacturer's protocol. The isolates showing different restriction patterns or having the same patterns but differing in sampling locations and growth conditions were outsourced for sequencing at 1st BASE, Singapore. The obtained 16S rRNA gene sequence of the isolates was deposited in the NCBI GenBank database and compared with sequences of other type strains using the BLASTn program (Altschul et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Benson et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) to confirm their identities.\u003c/p\u003e\u003cp\u003eFor phylogenetic analyses, the 16S rRNA gene sequences of type strains of nearest matches were downloaded from the NCBI GenBank database. All the obtained sequences were aligned using the MAFFT (Katoh et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) with default settings. The aligned sequences were used to construct a maximum-likelihood-based phylogenetic tree in W-IQ-TREE (Minh et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) using ModelFinder (Kalyaanamoorthy et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) to estimate substitution models and tested by 1000 bootstrap replications.\u003c/p\u003e\u003cp\u003eTo determine the occurrence of our strains in other environmental habitats, the 16S rRNA gene sequences of our strains were queried with the GenBank dataset. All the closely related cultured and uncultured sequences were downloaded, and their phylogenetic positions were inferred using a maximum-likelihood-based phylogenetic tree, as described earlier. Next, we documented the ecological distribution of the nearest clades from the GenBank dataset.\u003c/p\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cem\u003eLignocellulolytic activities, substrate utilisation profile and fermentation product analysis\u003c/em\u003e\u003c/div\u003e\u003cp\u003eBased on the phylogenetic analysis, phylogenetically distinct thermophilic cultures were selected for the determination of lignocellulolytic activities, substrate utilisation profile and fermentation product analysis. Representative strains were selected based on their isolation location, substrate specificity, temperature and hydrogen production ability to study the strain-level variation. For all the characterisation experiments, strains were revived from the glycerol stock in basal salt medium with glucose to minimise the strain generation error. To determine the lignocellulolytic activities, the selected strains (OD\u003csub\u003e600\u003c/sub\u003e 0.5; 1 ml) were inoculated in 9 ml of basal salt medium (pH 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1) containing 10 g/L rice straw or wheat straw as the sole carbon source and incubated at respective temperatures. After three days of incubation, the total produced gas was measured by the water displacement method. The composition of fermentation gas was analysed by a gas chromatograph (PerkinElmer, USA) equipped with a Thermal Conductivity Detector (TCD) as described earlier (Lanjekar et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The supernatant (2 ml) was withdrawn to measure activities of cellulases (avicelase, CMCase, and β-glucosidase), xylanase, and esterases (acetyl esterase, ρ-coumaroyl esterase, and feruloyl esterase) in a microplate-based assay (Mansour et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Dagar et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The reaction conditions for each enzyme activity are summarised in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The concentration of released monomeric units was calculated using spectrophotometric (A\u003csub\u003e595\u003c/sub\u003e for glucose \u0026amp; xylose, and A\u003csub\u003e412\u003c/sub\u003e for ρ-nitrophenol), and HPLC (for ρ-coumaric acid and ferulic acid) based methods. The heat-denatured culture supernatants were used as the control, and all the enzymatic activities were calculated in International Units (IU). One IU was defined as the amount of enzyme that released 1.0 \u0026micro;mol of a monomeric unit/ml/h. All the experiments were conducted in triplicate. Additionally, the concentrations of ethanol, total volatile fatty acids (VFAs) and non-VFAs were also estimated as described earlier (Lanjekar et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAssessment of environmental factors of hot springs\u003c/h2\u003e\u003cp\u003eVarious abiotic environmental factors have been found to impact the structures and diversity of microbial communities in hot springs. These factors include pH, temperature, mineralisation, and geological history, among others (Saghatelyan et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although pH and temperature are considered critical factors, minerals and sediment composition also play a significant role in shaping microbial community assembly in hot springs (Li et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Studies have shown that bacterial diversity in samples from springs with similar chemistries tends to overlap more, suggesting that chemistry, including mineral content, plays a crucial role in determining microbial community composition and diversity (Mathur et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These findings indicate the complex interplay between environmental factors such as minerals, pH, temperature, and chemistry in influencing microbial diversity and community structures in hot springs.\u003c/p\u003e\u003cp\u003eThe hot springs studied here showed a range of surface temperatures from 42 to 85 ℃, with the exception of Unkeshwar and Shahada, which measured at \u0026le;\u0026thinsp;30 ℃. Such low temperatures of these particular hot springs may be due to their distant sub-surface hot water source. Additionally, we noted slight variations in pH levels amongst all the hot springs, with a range of 7.7 to 9.1, indicating their alkaline nature. The details of the geographical location and water quality analysis of the samples collected are mentioned in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table S2. Our assessment of conductivity and anion composition revealed significant differences among the hot springs. Still, we did not observe any correlation between the temperature gradient of the hot spring and environmental factors (Chiriac et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e(Place Fig. 1 Here)\u003c/h3\u003e\n\u003cp\u003eBased on the World Health Organisation's (WHO) guidelines from 2017, the levels of chloride, nitrate, and sulphate were within the recommended limits at all hot springs except for Chopada and Unhavare hot springs, where chloride levels exceeded 250 mg/Kg (WHO \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Interestingly, phosphate was only present in Unkeshwar hot spring, while Tural hot spring contained solely chloride. All other hot spring sites contained chloride and sulphate, with the exception of Shahada hot spring, which had chloride and nitrate. However, the nitrate level at Shahada hot spring was found to be higher than the WHO-approved limit (Table S2). It's worth noting that the environmental factors that impact the microbial diversity of hot springs largely remain unresolved (MEYER-DOMBARD et al. 2005; Chiriac et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eEnrichments, isolation and cryopreservation\u003c/h3\u003e\n\u003cp\u003eCellulose and xylan (a major constituent of hemicellulose) are key components of plant biomass, and lignin is a complex aromatic polymer that is highly abundant in lignocellulosic materials. These substrates are crucial as they mimic the components found in lignocellulosic materials, enabling the selective enrichment of bacteria capable of breaking down these complex compounds (Wagner and Wiegel \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Studies have shown that thermophilic anaerobic bacteria isolated using cellulose and xylan-containing media exhibit efficient degradation of these substrates, with versatility in lignocellulose degradation (Sizova et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Jia et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Along with cellulose and hemicellulose, crude lignocellulosic substrates like rice straw contain soluble content, \u003cem\u003eviz.\u003c/em\u003e, pectin, which poses a challenge in isolating efficient lignocellulose-degrading strains (Chimphango et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). NDF thus offers a distinct advantage as a carbon source over crude agricultural biomass, due to its lack of soluble components. NDF was included in the enrichment medium to enrich the growth of efficient lignocellulose-degrading thermophilic anaerobic bacteria, which can be used for various biotechnological applications. Most of these industrial applications, including anaerobic digestion, generally function optimally at neutral pH. Hence, a similar pH range was selected for the enrichment process.\u003c/p\u003e\u003cp\u003eOut of a total of 160 enrichments, 29 were found to have positive results based on turbidity, gas, and hydrogen production (Table S3). Most of the enrichments showed positive results on xylan-based medium at temperatures of 55\u0026deg;C, 70\u0026deg;C, and 85\u0026deg;C, indicating the presence of a xylanolytic thermophilic anaerobic community in the samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Many of the enrichments were also positive on cellulose- and NDF-based media, indicating the presence of cellulolytic and lignocellulolytic communities.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e(Place Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e Here)\u003c/h2\u003e\u003cp\u003eNotably, cellulose-based enrichments from Chopada and Tural hot springs showed complete substrate degradation at 55\u0026deg;C within seven days of incubation, indicating efficient cellulose hydrolysis. Furthermore, enrichments from Unhavare showed growth at all high temperatures on both cellulose- and xylan-based media. Similar observations were made for enrichments from Tural hot spring, but only at 55\u0026deg;C and 70\u0026deg;C. These results indicate the presence of thermophilic cellulolytic and xylanolytic anaerobic bacteria at various temperature ranges in these hot springs. It is important to note that none of the enrichments at 40\u0026deg;C were found to be positive except for one on xylan of Aravali, highlighting the absence of mesophilic lignocellulolytic anaerobic bacteria. Also, none of the enrichments from Jakrem and Unkeshwar hot springs showed growth, indicating the absence of anaerobic lignocellulolytic bacteria in these samples.\u003c/p\u003e\u003cp\u003eAdditionally, none of the lignin-based enrichments showed growth at all the tested temperatures, signifying the limitation of lignin degradation at such elevated temperatures and low levels of oxygen in hot springs. Lignin degradation is generally more favourable under aerobic conditions compared to anaerobic conditions. The degradation and solubilisation of lignin-related compounds, especially high-molecular-weight lignin, are facilitated through oxidative reactions under aerobic conditions (Geib et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Studies have shown that lignin deconstruction is primarily described in aerobic systems, with biological lignin deconstruction being less prevalent in anaerobic environments (Ko et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Weng et al. \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lankiewicz et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Overall, 9 cellulose-, 14 xylan-, and 6 NDF-based enrichments were further processed for the isolation of lignocellulolytic thermophilic anaerobic bacteria (Table S3).\u003c/p\u003e\u003cp\u003eThe isolation procedures from these enrichments led to the growth of morphologically diverse colonies of anaerobic bacteria on roll bottles. Over 300 colonies were picked into their respective liquid medium and incubated at respective temperatures. Of the total colonies picked, the most diverse and the largest number of colonies were observed at 55 ℃, and the roll tubes at 70 ℃ and 85 ℃ yielded only a single type and fewer colonies in all the samples. Based on gas and hydrogen production, 83 isolates were established, of which 41 were cellulolytic (Avicel, 25 at 55 ℃; CMC, 16 at 55 ℃) and 42 were xylanolytic (8 at 40 ℃, 29 at 55 ℃, 4 at 70 ℃ and 1 at 85 ℃).\u003c/p\u003e\u003cp\u003eThe isolates were sub-cultured several times before confirming their purity based on phase-contrast microscopy and Gram staining. The majority of the cultures were Gram-negative in character with long or short or chain-forming rods (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All the pure cultures were cryopreserved in 5 ml vials at -80\u0026deg;C and in 2 ml cryo-vials at -196\u0026deg;C, in duplicates (Hivarkar et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMolecular identification and phylogenetic analysis\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003ein-silico\u003c/em\u003e analysis using \u003cem\u003eHaeIII\u003c/em\u003e restriction enzyme generated different ribotypes, thus differentiating bacteria at the genus level. The PCR amplification of isolated DNA generated PCR products of c.a. 1400 bp for all isolates. The actual restriction digestion of PCR products of eighty-three isolates using \u003cem\u003eHaeIII\u003c/em\u003e produced nineteen types of restriction patterns (Figure S2), which helped document the diversity and minimise the number of sequencing reactions. Twenty-seven isolates were chosen for sequencing and identification based on differences in geographical locations and growing conditions.\u003c/p\u003e\u003cp\u003eThe identification results established these isolates as members of nineteen different species under thirteen genera: \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eCaldibacillus\u003c/em\u003e, \u003cem\u003eCaldicoprobacter\u003c/em\u003e, \u003cem\u003eCaloramator\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e, \u003cem\u003eLacrimispora\u003c/em\u003e, \u003cem\u003eLimnochorda\u003c/em\u003e, \u003cem\u003ePseudoclostridium\u003c/em\u003e, \u003cem\u003eSeramator\u003c/em\u003e, \u003cem\u003eSporanaerobium\u003c/em\u003e, \u003cem\u003eTepidimicrobium\u003c/em\u003e, \u003cem\u003eThermoanaerobacter\u003c/em\u003e and \u003cem\u003eThermoanaerobacterium\u003c/em\u003e. All the strains were members of families \u003cem\u003eBacillaceae\u003c/em\u003e, \u003cem\u003eCaldicoprobacteraceae\u003c/em\u003e, \u003cem\u003eCaloramatoraceae\u003c/em\u003e, \u003cem\u003eClostridiaceae\u003c/em\u003e, \u003cem\u003eDysgonomonadaceae\u003c/em\u003e, \u003cem\u003eLachnospiraceae\u003c/em\u003e, \u003cem\u003eLimnochordaceae\u003c/em\u003e, and \u003cem\u003eThermoanaerobacteraceae\u003c/em\u003e, and \u003cem\u003eTissierellaceae\u003c/em\u003e of phylum \u003cem\u003eBacillota\u003c/em\u003e (synonym \u003cem\u003eFirmicutes\u003c/em\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCultivation and identification details of pure isolates of thermophilic anaerobic bacteria obtained from different hot springs across India.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eName of hot spring\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLocation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eIncubation temp.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNo. of isolates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSubstrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eRepresentative strain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eClosest phylogenetic affiliate and GenBank accession number\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e% sequence coverage\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e% similarity\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStrain designation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eGenBank accession number\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eChumathang\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLadakh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eXylan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXLC5596-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960429\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_117028)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e70\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXLC7096-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960431\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_117028)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eChopada\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eNorth Maharashtra\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e55\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCMC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCUC\u003csub\u003eP\u003c/sub\u003e55106-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960433\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003ePseudoclostridium thermosuccinogenes\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_119284)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eXylan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXUC\u003csub\u003eP\u003c/sub\u003e55106-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960434\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eLimnochorda pilosa\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_136767)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e94.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e70\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXUC\u003csub\u003eB\u003c/sub\u003e70106-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960435\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_117028)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRajwadi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWest Maharashtra\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eXylan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXMR5567-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960436\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eClostridium thermobutyricum\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(LT626257)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e\u003cp\u003eTural\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"8\" rowspan=\"9\"\u003e\u003cp\u003eWest Maharashtra\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"8\" rowspan=\"9\"\u003e\u003cp\u003e55\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAvicel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAMT5567-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960437\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eThermoanaerobacterium aotearoense\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_026296)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e98.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAMT5567-11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960439\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eCaloramator viterbensis\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_025044)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAMT5567-15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960440\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eThermoanaerobacterium butyriciformans\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_178863)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e98.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eCMC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCMT5567-10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960441\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eThermoanaerobacterium aotearoense\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_026296)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e98.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCMT5567-9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960444\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eTepidimicrobium ferriphilum\u003c/em\u003e (NR_117380)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCMT5567-11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960446\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003ePseudoclostridium thermosuccinogenes\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_119284)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.74\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCMT5567-13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960447\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eThermoanaerobacterium thermostercoris\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_122103)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e98.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eXylan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXMT5567-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960448\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eThermoanaerobacterium thermostercoris\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_122103)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXMT5567-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960451\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eCaloramator coolhaasii\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_024955)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eUnhavare\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eWest Maharashtra\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003e55\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAvicel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAMU5567-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960452\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eCaloramator proteoclasticus\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(LM999757)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eCMC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCMU5567-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960456\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eCaldicoprobacter guelmensis\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_109614)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e95.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCMU5567-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960457\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eCaldicoprobacter faecalis\u003c/em\u003e (NR_117173)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e94.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCMU5567-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960458\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_117028)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCMU5567-8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960459\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eCaloramator proteoclasticus\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(LM999757)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eXylan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXMU5567-13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960460\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eCaloramator coolhaasii\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_024955)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e70\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eXylan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXMU7067-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960464\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_029301)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eXylan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXMU8567-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOR960465\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eBacillus thermozeamaize\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_137401)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eAravali\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eWest Maharashtra\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e40\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eXylan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXHS19111\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMF806589\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eLacrimispora celerecrescens\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_026100)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXHS29132\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMF806588\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eLacrimispora indolis\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(AB971794)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXHS1971\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eKX553978\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eSporanaerobium hydrogeniformans\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(NR_189186)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eXHS2771\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eKX553979\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eSeramator thermalis\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(MK170161)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e99.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e(Place Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e Here)\u003c/h2\u003e\u003cp\u003eFrom the hot spring of Chumathang in the Ladakh region (northern India), only \u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e strains were obtained at 55 ℃ and 70 ℃. A related strain of this organism was also found in the hot spring of Chopada (central India) at 70 ℃ and the hot spring of Unhavare (western India) at 55 ℃ from the Maharashtra region. All these strains obtained from different geographic locations were isolated from a xylan-based enrichment. Along with this strain, \u003cem\u003ePseudoclostridium thermosuccinogenes\u003c/em\u003e and a putatively novel genus were also isolated at 55 ℃ from the Chopada hot spring. Like \u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e, a related strain of \u003cem\u003ePseudoclostridium thermosuccinogenes\u003c/em\u003e was also found in the Tural hot spring (western India) from the Maharashtra region. Both these strains were isolated from a CMC-based enrichment at 55 ℃. A maximum number of isolates were obtained from Tural hot springs, c.a. 36, followed by Unhavare hot springs, c.a. 31, both from the same geographical region (western India). Tural hot spring was dominated by species from the genus \u003cem\u003eThermoanaerobacterium\u003c/em\u003e (\u003cem\u003eT\u003c/em\u003e. \u003cem\u003eaotearoense\u003c/em\u003e, \u003cem\u003eT. butyriciformans\u003c/em\u003e and \u003cem\u003eT. thermostercoris\u003c/em\u003e), \u003cem\u003eTepidimicrobium ferriphilum\u003c/em\u003e followed by single strains of \u003cem\u003eCaloramator viterbensis\u003c/em\u003e and \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e. We also found a novel species belonging to the genus \u003cem\u003eThermoanaerobacterium\u003c/em\u003e. All these cultures from the Tural hot spring were isolated at 55 ℃ and from avicel-, CMC- and xylan-based enrichments. Unlike the Tural hot spring, the Unhavare hot spring was mostly dominated by the species from genus \u003cem\u003eCaloramator\u003c/em\u003e (\u003cem\u003eC. proteoclasticus\u003c/em\u003e and \u003cem\u003eC. coolhaasii\u003c/em\u003e), and two strains of a new genus at 55 ℃, followed by each strain of \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e (at 70 ℃) and \u003cem\u003eBacillus thermozeamaize\u003c/em\u003e (at 85 ℃).\u003c/p\u003e\u003cp\u003eFurther, from the Aravali hot spring (western India), we isolated thermo-tolerant strains of \u003cem\u003eLacrimispora indolis\u003c/em\u003e, \u003cem\u003eLacrimispora celerecrescens\u003c/em\u003e and \u003cem\u003eSeramator thermalis\u003c/em\u003e from xylan-based enrichments. Along with these cultures, we also isolated a novel genus within the family \u003cem\u003eLachnospiraceae\u003c/em\u003e, which we established as \u003cem\u003eSporanaerobium hydrogeniformans\u003c/em\u003e (Hivarkar et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). From the same geographical region, the Rajwadi hot spring (western India) had the least diversity, where we could only find a single strain of \u003cem\u003eClostridium thermobutyricum\u003c/em\u003e. This can be attributed to the high anthropogenic activity observed in the hot spring during sample collection, which may have increased the oxygen levels in the hot spring, limiting the population of anaerobic microorganisms in them (Lindstrom et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Nonetheless, hot springs are characterised by low biodiversity due to their extreme conditions in terms of temperature and chemical characteristics (Saxena et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chiriac et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Narsing Rao et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe relationship between the bacteria isolated in this study using 16S rRNA gene-based phylogenetic analysis was also analysed, which revealed the grouping of all the cultures in nine clusters representing nine different families of bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It was observed that all strains of the genus \u003cem\u003eThermoanaerobacterium\u003c/em\u003e and \u003cem\u003eCaloramator\u003c/em\u003e were highly related among themselves despite having different origins. Similarly, four identical strains of \u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e (Chumathang, Chopada and Unhavare) and two strains of \u003cem\u003ePseudoclostridium thermosuccinogenes\u003c/em\u003e (Chopada and Tural) showed close grouping. We also report two putative novel genera within the family \u003cem\u003eCaldicoprobacteraceae\u003c/em\u003e (strains CMU5567-1 and CMU5567-4) and \u003cem\u003eLimnochordaceae\u003c/em\u003e (strain XUCP55106-1) and one putative novel species belonging to the genus \u003cem\u003eThermoanaerobacterium\u003c/em\u003e (strain CMT5567-10). Isolation of such novel strains of anaerobic bacteria emphasises that, despite the traditionally low biodiversity associated with hot springs, they offer untapped sources for novel anaerobic microorganisms. The exploration of microbial diversity in hot springs continues to provide new insights into the richness of microbial life in these extreme environments, paving the way for further discoveries and advancements in microbiology (Li et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e(Place Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e Here)\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e\u003cem\u003eLignocellulolytic activities, substrate utilisation profile and fermentation product analysis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThermophilic anaerobic bacteria found in oxygen-limited and high-temperature conditions exhibit an extraordinary ability for growth across diverse thermal spectra that align with their ecological niches and physiological characteristics. Although many thermophiles prefer neutral pH conditions, they are adapted to thrive in various environmental conditions, showcasing their ecological versatility (Arbab et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This variable growth capability is further supported by findings that highlight the robust enzymatic systems employed by thermophiles, enabling the breakdown of complex organic substrates, indicating their potential utility in biotechnological applications, such as biofuel production from plant biomass (Bashir et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Paredes-Barrada et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Several research efforts have documented the enzyme production capabilities of thermophilic bacteria isolated from hot spring ecosystems. For instance, studies focused on the enzymatic activities of these bacteria have reported significant production of hydrolytic enzymes, such as cellulases and xylanases, which facilitate fiber degradation (Bala and Singh \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ajeje et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Casta\u0026ntilde;eda-Barreto et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHere, several thermophilic anaerobic strains isolated from Indian hot springs were evaluated for their lignocellulolytic enzyme activities, substrate utilisation profiles, and fermentation end-products. These isolates represent a range of taxonomic groups and functional traits, enabling us to document strain-specific variations in their ability to deconstruct lignocellulosic biomass and convert it into biofuels and other metabolites under thermophilic conditions.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eBacillus\u003c/h2\u003e\u003cp\u003eThe isolated \u003cem\u003eBacillus thermozeamaize\u003c/em\u003e strain XMU8567-5 was cultured from Unhavare hot spring, which was the only culture obtained at 85 ℃ from this study. The first strain of \u003cem\u003eBacillus thermozeamaize\u003c/em\u003e, sporulating aerobic bacilli, was isolated from a corn steep liquor-based fermenter (Mak \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). But the strain XMU8567-5 was obtained under strict anaerobic conditions at elevated temperatures, which suggests that the sporulating nature of this organism might have aided in the survival of this strain under such extreme environmental conditions. The 16S rRNA gene-based phylogenetic analysis using GenBank database revealed presence of similar strains or clones in the compost, hot spring, industrial and household wastes, manure, and soil from China, Denmark, Finland, Ireland, South Korea, UK, and USA (Figure S3). The occurrence of such related strains in varying environments rich in lignocellulosic biomass indicates the species\u0026rsquo; ability to degrade complex substrates. In this study, the strain XMU8567-5 was enriched in a xylan-based medium, which indicated its xylanolytic potential. However, the strain could not grow under the tested conditions on rice straw, wheat straw and other simple sugar-containing media. This may be due to its inability to degrade crude lignocellulosic biomass under obligate anaerobic conditions. Nonetheless, anaerobic strains of \u003cem\u003eBacillus\u003c/em\u003e spp. are known to harbour lignocellulolytic activity (Chukwuma et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Thakur et al. \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCaldibacillus\u003c/h2\u003e\u003cp\u003eThe characteristic features of Caldibacillus thermoamylovorans strains obtained from Chumathang (strain XLC5596-1) and Unhavare (strain CMU5567-6) were compared based on their lignocellulolytic activities and fermentation production analysis. The type strain of \u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e (homotypic synonym, \u003cem\u003eBacillus thermoamylovorans\u003c/em\u003e), a non-sporulating, amylolytic, facultative anaerobic bacillus, was first isolated from palm wine, a tropical alcoholic beverage (COMBET-BLANC et al. 1995). Like our strains XLC5596-1 and CMU5567-6, similar strains and clones of this organism were also detected in the hot springs across China, Indonesia, Nigeria, South Korea and Turkey. This organism is also widely present in faeces of buffalo, cow, goat, human, mice and pig, bioreactor and compost from Germany, Japan, Russia, South Africa, Sweden, Taiwan, and UK (Figure S4). Studies have shown that \u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e positively influences microbial ecosystems by reducing lag phases and enhancing the growth of other microorganisms during hydrogen-producing bioprocesses (Cabrol et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Its capacity to generate thermostable enzymes, such as lipases, medium-chain-length polyhydroxyalkanoate (mcl-PHA), etc., further emphasises the diverse lignocellulolytic potential of this species (Deive et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Choonut et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe strains of \u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e showed a vast difference in lignocellulolytic activities, with strain XLC5596-1 producing 9.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 IU β-glucosidase, whereas strain CMU5567-6 produced 4.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 IU and 99.14\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2 IU acetyl and ρ-coumaroyl esterase, respectively, on rice straw as substrate. On wheat straw, both the strains showed moderate xylanase activity, while strain XLC5596-1 showed 8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 IU β-glucosidase and 35.54\u0026thinsp;\u0026plusmn;\u0026thinsp;8.4 IU acetyl esterase activities (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similar differences between the strains were also observed for the ρ-coumaroyl and feruloyl esterases, where rice straw stimulated the esterase enzyme production in them (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results indicate strain and substrate-level variations in the enzyme activities of these isolates. The strains produced major quantities of ethanol followed by acetic-, lactic- and formic-acids, and hydrogen gas in traces. Among these end-products, ethanol and acetic acids were produced in higher quantities by strain CMU5567-6, while strain XLC5596-1 produced high amounts of lactic and formic acids.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDifferences were also found between the strains with respect to substrate profile, where strain XLC5596-1 did not grow under the tested conditions on any of the six monomeric sugars. The strain CMU5567-6 could only ferment hexose and not pentose sugars, where it produced similar end-products as were obtained from crude lignocellulosic substrate rice and wheat straws. Interestingly, the strain CMU6567-6 produced butyric acid and traces of succinic acid from rhamnose sugar instead of lactic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results indicate that the substrate influences the metabolic pathways in the \u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e strains (Yue et al. \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, considering their high temperature survival and production of lignocellulolytic enzymes at 55 ℃, \u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e strains can be considered a potential candidate to produce lactic acid from crude lignocellulosic substrates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCaldicoprobacteraceae\u003c/h2\u003e\u003cp\u003eMembers of the family \u003cem\u003eCaldicoprobacteraceae\u003c/em\u003e, specifically the genus \u003cem\u003eCaldicoprobacter\u003c/em\u003e, are of industrial importance due to their ability to produce industrially significant enzymes and biochemical compounds. They are all strictly anaerobic thermophilic heterotrophic bacteria utilising sugars but not proteinaceous compounds and are mostly found in faeces of herbivores and terrestrial hot springs (Bouanane-Darenfed et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Similarly, we found related species and clones of strain CMU5567-1 and strain CMU5567-4, both isolated from Unhavare hot spring, mostly from hot spring and sheep faeces, with the exception of some clones being detected in compost, reactors, sludge and soil (Figure S5). Moreover, the only genus \u003cem\u003eCaldicoprobacter\u003c/em\u003e within this family has been classified to play an important role in lignocellulosic biomass bioconversion by producing extracellular xylanase and cellulase enzymes (Jensen et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Soares et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Such findings suggest that our strains CMU5567-1 and CMU5567-4, which belong to a putatively new genus within the family \u003cem\u003eCaldicoprobacteraceae\u003c/em\u003e, could also demonstrate fibrolytic activities. However, in this study, only strain CMU5567-1 could hydrolyse and utilise rice straw, wheat straw and monomeric sugars, due to the specific requirement of C/N ratio in some members of this family (Fernandez-Bayo et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eCaldicoprobacteraceae\u003c/em\u003e bacterium strain CMU5567-1 showed high feruloyl esterase activity of 564.7\u0026thinsp;\u0026plusmn;\u0026thinsp;46.8 IU and 132\u0026thinsp;\u0026plusmn;\u0026thinsp;15.4 IU on wheat and rice straw, respectively, at 55℃. The culture also produced comparatively higher xylanases enzyme (12.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95 IU) on wheat straw, with the exception of acetyl esterase enzyme, which was produced in higher titer on rice straw (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The fermentation end-products of the strain were ethanol, hydrogen, acetic- and formic-acid, while it additionally produced trace amounts of succinic acid from wheat straw. This indicates that the culture prefers wheat straw over rice straw, as was evident by high enzyme titer and metabolic end-products, like other members of this family (Jensen et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, \u003cem\u003eCaldicoprobacteraceae\u003c/em\u003e bacterium strain CMU5567-1 showed vast differences in the metabolic end-products utilising the six hexose and pentose sugars. Propionic acid was produced from pentoses like xylose and arabinose, while hexose sugars like rhamnose led to the production of butyric acid; all these end-products, alongside the other metabolites, \u003cem\u003eviz.\u003c/em\u003e, ethanol and acetic acid. Hydrogen gas was not produced from glucose, xylose, mannose and galactose, whereas formic acid was produced comparatively in higher quantities from hexoses than pentoses (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). We also found traces of lactic acid being produced by this isolate utilising the six monomeric sugars. The habitat of strain CMU5567-1, along with the production of high titres of lignocellulolytic enzymes and associated end-products, highlights its importance in bioconversion pathways, emphasising its role in the efficient utilisation of lignocellulosic biomass and supporting the placement of the strain in the family \u003cem\u003eCaldicoprobacteraceae\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eCaloramator\u003c/h2\u003e\u003cp\u003eIn this study, we obtained three species of genus Caloramator, i.e., \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e, \u003cem\u003eCaloramator proteoclasticus\u003c/em\u003e and \u003cem\u003eCaloramator viterbensis\u003c/em\u003e at 55 ℃. The two \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e strains were isolated from Tural and Unhavare hot springs, while the other two strains of \u003cem\u003eCaloramator proteoclasticus\u003c/em\u003e were obtained from Unhavare hot spring, but on avicel and CMC-based medium, while \u003cem\u003eCaloramator viterbensis\u003c/em\u003e was isolated from Tural hot spring and enriched only on avicel-based medium. These three species of \u003cem\u003eCaloramator\u003c/em\u003e have been identified as organisms with significant lignocellulolytic potential (Ledbetter et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and majority of the related members are detected in the lignocellulosic biomass-rich environments like hot springs and bioreactors across China, Colombia, Finland, Italy, Japan, Netherlands, Tibet and USA (Figure S6). Amongst \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e has been characterised as a glutamate-degrading anaerobe with distinct physiological characteristics (Plugge et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Additionally, \u003cem\u003eCaloramator proteoclasticus\u003c/em\u003e has been reported to produce propionate and butyrate (Ding et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eCaloramator viterbensis\u003c/em\u003e, a glycerol-fermenting anaerobe, utilises various substrates including sugars, amino acids, and starch, producing acetate, ethanol, and lactate as end products (Seyfried et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The genus \u003cem\u003eCaloramator\u003c/em\u003e, to which these species belong, has been recognised for its thermophilic and glutamate-degrading properties (Baena and Patel \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, the lignocellulolytic enzymes produced by these organisms have been of interest for their potential in biomass deconstruction (Lee et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the genus \u003cem\u003eCaloramator\u003c/em\u003e, all the species produced a significant amount of hydrogen ranging from 7.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u0026ndash;11.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 mM (from rice straw) and 10.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u0026ndash;13.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 mM (from wheat straw), except \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e strain XMT5567-6, which produced a meagre amount. High hydrogen production can be linked to the fibrolytic nature of the culture, but none of the cultures showed significant lignocellulolytic activities, except esterase activities. Interestingly, high feruloyl esterase activity was expressed by the cultures on rice straw (382.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026ndash;492.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 IU) than on wheat straw (11.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u0026ndash;53.86\u0026thinsp;\u0026plusmn;\u0026thinsp;18.1 IU) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Xylanase enzyme was produced by both strains of \u003cem\u003eCaloramator proteoclasticus\u003c/em\u003e on wheat straw, whereas the \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e strain XMU5567-13 produced xylanase enzyme on rice straw and β-glucosidase enzyme on wheat straw (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e strain XMT5567-6 and \u003cem\u003eCaloramator viterbensis\u003c/em\u003e strain AMT5567-11 did not produce any of the cellulase or xylanase enzymes. Furthermore, \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e strain XMU5567-13 produced 79.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 mM ethanol from wheat straw, which was highest amongst the cultures studied in this work (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Along with hydrogen and ethanol, these cultures produced acetic acid (all strains), lactic acid (\u003cem\u003eCaloramator coolhaasii\u003c/em\u003e strain XMT5567-6) and formic acid (\u003cem\u003eCaloramator coolhaasii\u003c/em\u003e strain XMU5567-13 and \u003cem\u003eCaloramator proteoclasticus\u003c/em\u003e). The differences in enzyme production and metabolite end-products were apparent at substrate-, strain- and species-levels among the members of the genus \u003cem\u003eCaloramator\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e(Place Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e Here)\u003c/h2\u003e\u003cp\u003eLike crude lignocellulosic substrates, the fermentation end-products of \u003cem\u003eCaloramator\u003c/em\u003e species from monomeric sugars were ethanol, hydrogen and acetic acid, while there was variation in the production of other metabolites like propionic-, butyric-, succinic- and formic acids. Formic acid was not produced by \u003cem\u003eCaloramator viterbensis\u003c/em\u003e strain AMT5567-11, but it was the only member of the genus \u003cem\u003eCaloramator\u003c/em\u003e which utilised all six sugars tested. We could observe strain- and substrate-level variations in the strains of \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e and \u003cem\u003eCaloramator proteoclasticus\u003c/em\u003e. The \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e strains could not ferment arabinose and rhamnose sugar, and they additionally produced 25.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u0026ndash;29.61\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21 mM propionic acid from xylose sugar. Amongst them, strain XMT5567-6 also produced succinic acid (1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mM) from xylose and lactic acid was comparatively produced in higher amounts by strain XMT5567-13 from other sugars. Furthermore, both strains of \u003cem\u003eCaloramator proteoclasticus\u003c/em\u003e could not utilise arabinose, while the strain CMU5567-8 was also not able to utilise xylose and rhamnose sugars. \u003cem\u003eCaloramator proteoclasticus\u003c/em\u003e strain AMU5567-5 produced propionic acid (37.71\u0026thinsp;\u0026plusmn;\u0026thinsp;3.42 mM) instead of acetic acid from xylose and it also produced butyric acid (21.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99 mM) from rhamnose (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Similarly, \u003cem\u003eCaloramator viterbensis\u003c/em\u003e strain AMT5567-11 also produce propionic acid instead of acetic acid from pentose sugars xylose and arabinose. It also produced 5.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 mM butyric acid and minor quantities of propionic acid from rhamnose sugar. These results indicate that the substrates direct the metabolic pathway in the members of genus \u003cem\u003eCaloramator\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eTo the best of our knowledge, this is the first study to document the lignocellulolytic potential of \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e, \u003cem\u003eCaloramator proteoclasticus\u003c/em\u003e and \u003cem\u003eCaloramator viterbensis\u003c/em\u003e species, and highlight their industrial importance in the production of bioenergy products. Further specific studies on \u003cem\u003eCaloramator\u003c/em\u003e species\u0026rsquo; lignocellulolytic capabilities would be beneficial to understand its role in biomass degradation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eClostridium\u003c/h2\u003e\u003cp\u003eSeveral studies have extensively investigated various species within the genus \u003cem\u003eClostridium\u003c/em\u003e for their lignocellulolytic properties. \u003cem\u003eClostridium thermocellum\u003c/em\u003e, also known as \u003cem\u003eHungateiclostridium thermocellum\u003c/em\u003e or \u003cem\u003eAcetivibrio thermocellus\u003c/em\u003e, is the most profoundly studied lignocellulolytic bacterium known for its multienzyme complex, the cellulosome, which is of great potential value in lignocellulose biorefinery (Yan et al. \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Other important strains studied for industrial applications include \u003cem\u003eClostridium butyricum\u003c/em\u003e, \u003cem\u003eClostridium tyrobutyricum\u003c/em\u003e, and \u003cem\u003eClostridium thermobutyricum\u003c/em\u003e. These species have shown potential in lignocellulose biorefinery processes due to their enzymatic capabilities in degrading lignocellulosic biomass (Chu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eClostridium thermobutyricum\u003c/em\u003e especially has the potential for high butyrate titer and volumetric productivity (Wang et al. \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The first representative strain of \u003cem\u003eClostridium thermobutyricum\u003c/em\u003e was a thermotolerant strain isolated from the cellulolytic enrichment of horse manure (WIEGEL et al. \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). On the contrary, we obtained \u003cem\u003eClostridium thermobutyricum\u003c/em\u003e strain XMR5567-3, a thermophilic strain, from the xylanolytic enrichment of sediments from the Rajwadi hot spring at 55 ℃. The 16S rRNA gene-based ecological distribution revealed presence of this species in diverse environments like biogas plant sludge, bioreactor, leachate sediment, and solvent factory sludge in the regions of Canada, China, Germany, Thailand and USA (Figure S7). Such habitats reveal that the organism survives harsh environments, majorly rich in acidic content. Moreover, the species is not well studied to identify its lignocellulolytic potential, and most of the research is focused on the butyric acid production from sugars (Wang et al. \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Here, we tried to determine the ability of \u003cem\u003eClostridium thermobutyricum\u003c/em\u003e strain XMR5567-3 to degrade crude lignocellulosic substrates, rice and wheat straw, and measure whether the liberated sugars from them can further be converted to high titres of butyric acid.\u003c/p\u003e\u003cp\u003eStrain XMR5567-3, under the tested conditions at 55 ℃, secreted 118.72\u0026thinsp;\u0026plusmn;\u0026thinsp;16.23 IU ρ-coumaroyl esterase and 483.10\u0026thinsp;\u0026plusmn;\u0026thinsp;4.77 IU feruloyl esterase from rice straw and 48.55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.72 IU feruloyl esterase from wheat straw (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Secretion of only esterase enzymes may have led to the release of a very minor amount of sugars; hence, the culture produced a mere 1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mM butyric acid from wheat straw (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, rice straw substrate led to the metabolic shift in the organism, which produced 14.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.77 mM ethanol instead of butyric acid. Also, the strain XMR5567-3 could not ferment any of the six sugars tested in this study due to the sequential transfer of the culture, which increased butyric acid concentrations and decreased the level of glucose tolerance in the inoculum used (Canganella et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Moreover, production of esterase enzymes and ethanol contradicts the fact that \u003cem\u003eClostridium thermobutyricum\u003c/em\u003e strain only produces butyric acid from sugars. Hence, we propose that more studies should be focused on determining the metabolic pathways followed by this organism under different conditions to recognise its industrial potential.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e(Place Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e Here)\u003c/h2\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eLacrimispora\u003c/h2\u003e\u003cp\u003e\u003cem\u003eClostridium celerecrescens\u003c/em\u003e and \u003cem\u003eClostridium indolis\u003c/em\u003e have been recently reclassified as \u003cem\u003eLacrimispora celerecrescens\u003c/em\u003e and \u003cem\u003eLacrimispora indolis\u003c/em\u003e (Haas and Blanchard \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Both these strains were not well studied until 2021, when Kobayashi et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) identified \u003cem\u003eLacrimispora indolis\u003c/em\u003e as a species with lignocellulolytic potential, providing genomic insights into its enzymatic systems. Here, we obtained several copies of \u003cem\u003eLacrimispora indolis\u003c/em\u003e strain XHS29132 and a single strain of \u003cem\u003eLacrimispora celerecrescens\u003c/em\u003e strain XHS19111 from the Aravali hot spring on a xylan enrichment at 42 ℃. To identify the potential of these strains to disintegrate lignocellulosic substrates, a 16S rRNA-based ecological distribution was carried out. The related cultured and non-cultured strains of \u003cem\u003eLacrimispora indolis\u003c/em\u003e were found either in the rumen of herbivores or rumen content augmented anaerobic digesters operated in China, Germany, India, Japan, and Tunisia (Figure S8). Conversely, the similar cultured and non-cultured strains of \u003cem\u003eLacrimispora celerecrescens\u003c/em\u003e showed diverse habitat preference, including faeces of mealworms, pig and wild-boar, harsh environments like coal seam gas bore wells, Uranium mining wastes, corroding gas pipeline, volcanic steam vents and petroleum/ pesticide contaminated soils, etc. (Figure S8). The presence of \u003cem\u003eLacrimispora indolis\u003c/em\u003e species in the lignocellulosic-rich environment and recent findings suggest that they may have lignocellulolytic potential. However, neither species could grow on complex or simple substrates under the tested conditions.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eLimnochordaceae\u003c/h2\u003e\u003cp\u003eWe obtained another culture belonging to a putatively novel genus under the family \u003cem\u003eLimnochordaceae\u003c/em\u003e, \u003cem\u003eviz.\u003c/em\u003e, \u003cem\u003eLimnochordaceae\u003c/em\u003e bacterium strain XUC\u003csub\u003eP\u003c/sub\u003e55106-1. This family has been lately classified with the sole species, i.e., \u003cem\u003eLimnochorda pilosa\u003c/em\u003e (Watanabe et al. \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), hence to the best of our knowledge not many studies are available in literature highlighting its fiber degrading potential. Also, the type strain of \u003cem\u003eLimnochorda pilosa\u003c/em\u003e is a moderately thermophilic, facultatively anaerobic, sporulating pleomorphic bacterium isolated from a brackish meromictic lake in Japan. While our strain \u003cem\u003eLimnochordaceae\u003c/em\u003e bacterium strain XUC\u003csub\u003eP\u003c/sub\u003e55106-1 is a thermophilic obligately anaerobic bacterium isolated from Unhavare hot spring with an \u003cem\u003ein-situ\u003c/em\u003e temperature of \u0026gt;\u0026thinsp;70℃.\u003c/p\u003e\u003cp\u003eThe phylogenetic analysis of the 16S rRNA gene of strain XUC\u003csub\u003eP\u003c/sub\u003e55106-1 showed that highly similar clones were detected in the swine manure and municipal waste-based composts (Figure S9). This suggested that the strain XUC\u003csub\u003eP\u003c/sub\u003e55106-1 may have fibrolytic capabilities. This was verified when the strain when grown on rice straw and wheat straw at 55 ℃, showed 26.54\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 IU and 13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1 IU xylanase and acetyl esterase activities, respectively, along with β-glucosidase (35.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 IU), ρ-coumaroyl esterase (238.08\u0026thinsp;\u0026plusmn;\u0026thinsp;38.6 IU), feruloyl esterase (10.63\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8 IU) and moderate CMCase (0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 IU) activities (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The majority of the enzyme titers were higher when rice straw was used as a substrate, and it led to the production of 28.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mM acetic acid, 25.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mM formic acid, 20.02\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17 mM ethanol and 10.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09 mM hydrogen as by-products. Similar fermentation end-products were produced by the strain from wheat straw but comparatively in lower quantities as the enzyme secretions were also lower in those experimental bottles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Considering the strain\u0026rsquo;s ability to produce a broad range of enzymes, it can be termed a fibrolytic organism that can utilise untreated rice straw efficiently at elevated temperatures.\u003c/p\u003e\u003cp\u003eThe strain XUC\u003csub\u003eP\u003c/sub\u003e55106-1 was a hexose-fermenting bacterium as it did not grow in the xylose and arabinose-containing medium. Also, it did not produce hydrogen gas in any of the hexose-containing media. It produced higher amounts of ethanol followed by acetic- and formic-acid from glucose, mannose, rhamnose and galactose (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, a metabolic shift was also observed in this bacterium as it additionally produced 20.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 mM butyric acid from rhamnose sugar. These results indicate that \u003cem\u003eLimnochordaceae\u003c/em\u003e bacterium strain XUC\u003csub\u003eP\u003c/sub\u003e55106-1 can be termed as an industrially important strain to produce thermostable lignocellulolytic enzymes and can also be used in a single-pot consolidated bioprocessing to produce bioenergy products.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e(Place Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e Here)\u003c/h2\u003e\u003cdiv id=\"Sec26\" class=\"Section4\"\u003e\u003ch2\u003ePseudoclostridium\u003c/h2\u003e\u003cp\u003eThe thermophilic \u003cem\u003ePseudoclostridium thermosuccinogenes\u003c/em\u003e strains are known to produce succinate from lignocellulosic-derived sugars, showcasing their potential as a platform organism for various industrial applications (Ganguly et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Studies have shown that these strains are incapable of degrading cellulose, but they exhibit rapid growth on inulin and various monosaccharides, emphasising their unique metabolic pathways and enzymatic capabilities (Koendjbiharie et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our phylogenetic analysis showed the prevalence of this organism in cellulolytic enrichments and lignocellulose-rich compost, cow manure and soil (Figure S10), indicating the lignocellulose-degrading capability of these strains. Also, we obtained two strains of thermophilic \u003cem\u003ePseudoclostridium thermosuccinogenes\u003c/em\u003e, \u003cem\u003eviz.\u003c/em\u003e, strain CMT5567-1 and strain CUC\u003csub\u003eP\u003c/sub\u003e55106-1, from cellulolytic enrichments of Tural and Chopada hot springs, respectively, at 55 ℃.\u003c/p\u003e\u003cp\u003eSurprisingly, only \u003cem\u003ePseudoclostridium thermosuccinogenes\u003c/em\u003e strain CUC\u003csub\u003eP\u003c/sub\u003e55106-1 could produce cellulose-attacking avicelase (1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u0026ndash;1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 IU), CMCase (1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026ndash;2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 IU), and β-glucosidase (12.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u0026ndash; 44.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 IU) enzymes alongside feruloyl esterase and ρ-coumaroyl esterase. The other strain, CMT5567-1, did not produce any of the cellulolytic enzymes and only produced a high titer of feruloyl esterase and lower titers of xylanase, β-glucosidase and ρ-coumaroyl esterase enzymes (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Strain variation was also seen among these strains, such as the species of \u003cem\u003eCaldibacillus\u003c/em\u003e and \u003cem\u003eCaloramator\u003c/em\u003e. Though both strains showed significant growth on wheat straw over rice straw, the fermentation end products varied among the substrates. The strain CMT5567-1, like most strains of \u003cem\u003ePseudoclostridium thermosuccinogenes\u003c/em\u003e, produced 2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 to 2.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 mM succinic acid along with ethanol, hydrogen and acetic acid. Whereas the other strain, CUCP55106-1, which was fibrolytic, produced 7.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64 to 8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 mM lactic acid instead of succinic acid, along with other similar end products (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Furthermore, neither strain could utilise any of the six monomeric sugars. Such variation in growth pattern and end product generation could be attributed to the CO\u003csub\u003e2\u003c/sub\u003e limitation, which decreases the NAD\u003csup\u003e+\u003c/sup\u003e/NADH ratio, ultimately leading to stress and an increase in lactic acid levels (Koendjbiharie et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eSeramator\u003c/h2\u003e\u003cp\u003e\u003cem\u003eSeramator thermalis\u003c/em\u003e is a new genus and new species added recently to the family \u003cem\u003eDysgonomonadaceae\u003c/em\u003e (Liu et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). This moderately thermophilic type strain was isolated from a hot spring in China and had cellulose and xylan degrading ability. Similarly, we also obtained the \u003cem\u003eSeramator thermalis\u003c/em\u003e strain XHS2771 from the Aravali hot spring, exhibiting the same characteristics. The occurrence of this organism across anaerobic digesters, municipal waste digesters, etc., explains its ability to degrade complex substrates like lignocellulosic biomass (Figure S11). However, the optimal growth temperature of around 45 ℃ limited the strain\u0026rsquo;s growth on the rice straw, wheat straw and other sugar containing media. Nonetheless, the strain\u0026rsquo;s ability to simultaneously degrade cellulose and xylan marks this species as a vital part of the anaerobic digestion process of agricultural wastes (Maus et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Schroeder et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eSporanaerobium\u003c/h2\u003e\u003cp\u003eIn addition to the strain XHS2771, we also isolated a new species having cellulolytic and xylanolytic activities belonging to the family \u003cem\u003eLachnospiraceae\u003c/em\u003e from the same Aravali hot spring sample. We established the novelty of this strain and named it \u003cem\u003eSporanaerobium hydrogeniformans\u003c/em\u003e strain XHS1971 (Figure S12) (Hivarkar et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It was isolated from xylan enrichment and exhibited good cellulase activity in addition to xylanase activity and produced significant quantities of biohydrogen. However, being a moderate thermophilic strain growing optimally at 45 ℃, its lignocellulolytic potential and substrate profile could not be determined at 55 ℃. The strain XHS1971 belonged to the family \u003cem\u003eLachnospiraceae\u003c/em\u003e, whose members are acknowledged as one of the most potent polysaccharide degraders owing to their ability to produce free or complex hydrolytic enzymes, it can be used for lignocellulosic biomass-derived biofuel production (Biddle et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eTepidimicrobium\u003c/h2\u003e\u003cp\u003eModerately thermophilic and Fe(III)-reducing \u003cem\u003eTepidimicrobium ferriphilum\u003c/em\u003e cultures are known to utilise proteinaceous compounds and not carbohydrates (Slobodkin et al. \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Yet, a thermophilic strain, \u003cem\u003eviz.\u003c/em\u003e, \u003cem\u003eTepidimicrobium ferriphilum\u003c/em\u003e strain CMT5567-9, was obtained from a cellulose enrichment of the Tural hot spring sediment. This may be attributed to the presence of yeast extract and traces of Fe(III) salts present in the enrichment medium. But the strain could not grow under the tested conditions on rice straw, wheat straw and other simple sugar-containing media due to limiting proteinaceous substrates. A 16S rRNA gene-based phylogenetic analysis of this isolate showed the prevalence of such species in cow faeces, hot springs, and mesophilic and thermophilic anaerobic digesters (Figure S13). Such habitat preference directs that this species may possess unique metabolic pathways and enzymatic activities that contribute to its survival and function in anaerobic and thermophilic niches (Shikata et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). While specific properties of \u003cem\u003eTepidimicrobium ferriphilum\u003c/em\u003e may not be extensively documented, drawing from the broader characteristics of thermophilic anaerobes can provide a foundational understanding of their potential traits. Further research focusing directly on \u003cem\u003eTepidimicrobium ferriphilum\u003c/em\u003e would be valuable to elucidate its distinct properties and metabolic features in more detail.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThermoanaerobacter\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eThermoanaerobacter\u003c/em\u003e is a genus known for its thermophilic and anaerobic characteristics, with species capable of saccharolytic metabolism producing ethanol, acetic acid, lactic acid, alanine, CO\u003csub\u003e2\u003c/sub\u003e, and H\u003csub\u003e2\u003c/sub\u003e as end-products (Onyenwoke and Wiegel \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Michael Scully and Orlygsson \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e, a species within this genus, has been identified as an obligately anaerobic, thermophilic, and polysaccharolytic bacterium capable of fermenting various sugars to produce end products such as ethanol, acetic-, lactic-, propionic-acid, and H\u003csub\u003e2\u003c/sub\u003e (COOK et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Genomic evaluations have highlighted the potential of \u003cem\u003eThermoanaerobacter\u003c/em\u003e spp., including \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e, for metabolic engineering to improve lignocellulosic biofuel production due to their high ethanol yields from hemicellulosic sugars (Brynjarsdottir et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Verbeke et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Studies have even indicated that \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e, along with other \u003cem\u003eThermoanaerobacter\u003c/em\u003e species, can enhance glycerol conversion when co-cultivated with methanogenic partners (Magalh\u0026atilde;es et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHere, a hyperthermophilic culture of \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e, strain XMU7067-6, was obtained from a xylan enrichment of Unhavare hot spring sediment at 70 ℃. The strain\u0026rsquo;s ecological analysis based on 16S rRNA-based phylogeny showed that it mostly preferred hot environments like hot springs, hydrothermal vents and thermophilic digesters for dwelling (Figure S14). This directed us to look for its fibrolytic potential, so the strain XMU7067-6 was subjected to rice straw and wheat straw-based medium to evaluate the enzyme profile and subsequent fermentation products. The strain XMU7067-6 at 70 ℃ exhibited higher feruloyl esterase activity of 655.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8 IU on rice straw and merely 74.69\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4 IU activity on wheat straw (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Xylanase and β-glucosidase activities also differed on different straws, where they showed 11.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 IU on rice straw and 15.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 IU on wheat straw, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Production of this range of enzymes led to the metabolites like ethanol, hydrogen, acetic acid, and minor amounts of lactic and formic acids. End products were higher on wheat straw where strain XMU7067-6 produced 36.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33 mM ethanol, 14.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 mM hydrogen and 7.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 mM acetic acid with traces of lactic acid, i.e., 1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These distinctive characteristics, along with its metabolic versatility and lignocellulolytic capabilities, position \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e strain XMU7067-6 as a valuable candidate for various biotechnological applications, particularly in the realm of biofuel production from wheat straw (Georgieva et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Brynjarsdottir et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurther, the strain XMU7067-6 co-utilised both hexose and pentose sugars to produce similar end-products. Fermenting hexose sugars, it produced 13.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37 to 18.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20 mM ethanol, 6.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 to 6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06 mM hydrogen, and 5.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 to 7.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mM acetic acid; while 1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mM lactic acid was produced only from glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, rhamnose sugar led to the generation of low ethanol and no hydrogen, but 19.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66 mM butyric acid, which was not reported in this species earlier. Moreover, the strain XMU7067-6 produced 10.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mM propionic acid and 3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mM formic acid in addition to ethanol, hydrogen, acetic acid and lactic acid from arabinose sugar (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). A similar end-product profile was also observed with xylose, indicating the shift in the metabolic pathway in \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e due to pentose sugars (Lin et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These findings underscore the potential of utilising \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e and related species in biofuel production, leveraging their thermophilic, lignocellulolytic, and pentose and hexose co-utilisation properties. Understanding how \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e manages the metabolism of pentose sugars like xylose or arabinose alongside hexose sugars can provide crucial information for optimising biofuel production processes. However, the specific properties and applications of \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e would need to be directly studied to fully understand its capabilities and contributions to lignocellulosic biofuel production.\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eThermoanaerobacterium\u003c/h2\u003e\u003cp\u003e\u003cem\u003eThermoanaerobacterium\u003c/em\u003e species, recognised for their thermophilic nature, have garnered attention for their potential in biofuels production and various industrial applications due to their unique properties and capabilities. These species can directly utilise lignocellulosic materials, such as various paper substrates, to produce biofuels, showcasing a broad substrate range and high operating temperatures which enhance the efficiency of lignocellulose degradation and reduce the risk of microbial contamination (Popova et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dai et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Amongst \u003cem\u003eThermoanaerobacterium\u003c/em\u003e species, \u003cem\u003eThermoanaerobacterium aotearoense\u003c/em\u003e, \u003cem\u003eThermoanaerobacterium butyriciformans\u003c/em\u003e, and \u003cem\u003eThermoanaerobacterium thermostercoris\u003c/em\u003e are anaerobic thermophilic bacteria with diverse metabolic properties, including lignocellulolytic capabilities. \u003cem\u003eThermoanaerobacterium aotearoense\u003c/em\u003e has been identified as a slightly acidophilic thermophile capable of forming elemental sulfur from thiosulfate and growing at acidic pH values at elevated temperatures (LIU et al. 1996). This species has been associated with bioethanol and biohydrogen production and the expression of thermostable enzymes like β-glucosidase for biomass conversion (Huang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eThermoanaerobacterium butyriciformans\u003c/em\u003e is another spore-forming anaerobic thermophilic bacterium known for its ability to degrade cellulose and hemicellulose to produce acetic acid, butyric acid, and alcohols (L\u0026oacute;pez et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eThermoanaerobacterium thermostercoris\u003c/em\u003e, a non-sporulating member of this genus, has been studied for its hemicellulolytic activities, particularly xylanase production, highlighting its potential for biomass valorisation and lignocellulosic waste utilisation (Romano et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Finore et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHere, we characterise two strains each of \u003cem\u003eThermoanaerobacterium aotearoense\u003c/em\u003e (strains AMT5567-1 \u0026amp; CMT5567-10), \u003cem\u003eThermoanaerobacterium thermostercoris\u003c/em\u003e (strains CMT5567-13 \u0026amp; XMT5567-1) and one strain of \u003cem\u003eThermoanaerobacterium butyriciformans\u003c/em\u003e (strain AMT5567-15), which were obtained from avicel, CMC and xylan-based medium of Tural and Unhavare hot springs. The 16S rRNA gene-based phylogeny indicated that all these strains are closely related to each other and found in diverse locations like canned food, compost, oil reservoir fluids, and thermophilic reactors (Figure S15); so, most of the by-products and properties of them would overlap.\u003c/p\u003e\u003cp\u003eFibrolytic analysis revealed that \u003cem\u003eThermoanaerobacterium aotearoense\u003c/em\u003e strain CMT5567-10 showed majority of the tested lignocellulolytic activities, i.e. avicelase (3.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 IU), CMCase (2.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 IU), xylanase (17.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 IU), β-glucosidase (38.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 IU) and feruloyl esterase (613.04\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2 IU) on rice straw, producing a high titer of ethanol, i.e. 44.54\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94 mM and 7.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 mM hydrogen (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Additionally, it also produced 19.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 mM acetic acid and 13.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mM formic acid, which were comparatively higher than those of \u003cem\u003eThermoanaerobacterium aotearoense\u003c/em\u003e strain AMT5567-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Equally, strain CMT5567-10 produced avicelase (1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 IU), xylanase (6.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 IU), β-glucosidase (18.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 IU), acetyl esterase (22.25\u0026thinsp;\u0026plusmn;\u0026thinsp;9.1 IU), ρ-coumaroyl esterase (25.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 IU) and feruloyl esterase (8.46\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 IU) on wheat straw, producing 26.76\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83 mM ethanol and 9.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 mM hydrogen. This culture thus may act as a potential candidate for single-pot co-production of hydrogen and ethanol from untreated agricultural wastes. Besides, the \u003cem\u003eThermoanaerobacterium aotearoense\u003c/em\u003e strain AMT5567-1 produced the highest ρ-coumaroyl esterase of 404.22\u0026thinsp;\u0026plusmn;\u0026thinsp;24.7 IU from rice straw, indicating its substrate specificity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, \u003cem\u003eThermoanaerobacterium butyriciformans\u003c/em\u003e strain AMT5567-15 showed lower CMCase and xylanase activities, while 10.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 IU β-glucosidase (on wheat straw) and 433.42\u0026thinsp;\u0026plusmn;\u0026thinsp;35.07 IU feruloyl esterase (on rice straw) showcased substrate variability. The strain AMT5567-15 preferred wheat straw over rice straw as it produced a high concentration of ethanol, i.e. 30.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55 mM from wheat straw. No butyric acid was produced by this strain from crude lignocellulosic biomass. Among the \u003cem\u003eThermoanaerobacterium thermostercoris\u003c/em\u003e strains, strain XMT5567-1 showed fibrolytic properties as it produced comparatively higher avicelase, CMCase, xylanase, β-glucosidase, and esterase activities (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Still, the strain CMT5567-13 produced a higher titer of ethanol (45.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45 mM, rice straw), acetic acid (13.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56 mM, rice straw) and hydrogen (6.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 mM, wheat straw). The fermentation end products of both the \u003cem\u003eThermoanaerobacterium thermostercoris\u003c/em\u003e strains were ethanol, acetic acid and hydrogen, while strain XMT5567-1 additionally produced a minor amount of lactic acid. Strain and substrate level variation was also evident among this group of organisms. These results explain why this group has garnered interest for biofuel production from lignocellulosic biomass.\u003c/p\u003e\u003cp\u003eIn terms of monomeric sugar utilisation, \u003cem\u003eThermoanaerobacterium butyriciformans\u003c/em\u003e strain AMT5567-15 was the only strain among this group to ferment all six monomeric sugars. It produced a higher amount of hydrogen, i.e. 20.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mM to 25.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 mM, followed by 12.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 mM to 23.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mM ethanol, 5.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 mM to 11.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 mM acetic acid and 4.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 mM to 8.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mM butyric acid. Lactic acid in the range 7.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 mM was also produced by this strain, which was the highest amongst this group. Surprisingly, 5.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mM and 18.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 mM propionic acid were produced from xylose and arabinose, respectively; while 2.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 mM succinic acid from rhamnose sugar was also produced by this species. Similarly, \u003cem\u003eThermoanaerobacterium aotearoense\u003c/em\u003e strains also had a similar metabolite profile of hydrogen, ethanol, acetic acid and butyric acid. Between them, strain AMT5567-1 produced a comparatively higher amount of hydrogen, whereas strain CMT5567-10 produced a higher amount of ethanol and lactic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, strain CMT5567-10 produced 8.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 mM propionic acid from xylose sugar. Acetic acid and butyric acid production were equivalent between these strains, while they did not ferment arabinose sugar. \u003cem\u003eThermoanaerobacterium thermostercoris\u003c/em\u003e strains also utilised arabinose sugar, and they too produced ethanol, hydrogen, acetic acid and butyric acid with trace amounts of lactic acid as their metabolic end products. Among them, strain XMT5567-1 produced comparatively higher ethanol, i.e. 28.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mM to 41.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 mM, while strain CMT5567-13 produced higher hydrogen, i.e. 21.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mM to 26.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 mM. Lactic acid was produced from glucose (6.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34 mM) and mannose (5.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mM) by the strain CMT5567-13, whereas 1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 mM succinic acid was produced from rhamnose sugar. On the contrary, strain XMT5567-13 produced 17.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74 mM propionic acid in addition to other metabolites from xylose sugar. The strain level variation was also noticed during the substrate profiling of these isolates. In literature, \u003cem\u003eThermoanaerobacterium thermostercoris\u003c/em\u003e strains are reported to be higher hydrogen producers (Wu et al. \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Finore et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dai et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e); however, we found strains of \u003cem\u003eThermoanaerobacterium aotearoense\u003c/em\u003e as high hydrogen producing culture followed by \u003cem\u003eThermoanaerobacterium butyriciformans\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn summary, \u003cem\u003eThermoanaerobacterium\u003c/em\u003e species exhibit a wide range of industrially relevant properties, including efficient lignocellulolytic activities, the capability to ferment sugars to ethanol and hydrogen, and unique metabolic pathways for the degradation of cellulose and xylan. These attributes underscore their potential in biofuel production and other sustainable biotechnological applications.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights a comprehensive exploration of thermophilic anaerobic bacteria inhabiting Indian hot spring environments with a focus on their potential to degrade lignocellulosic biomass. Through systematic sample collection from ten hot springs across varied geothermal provinces, combined with a rigorous enrichment and isolation strategy, a diverse set of 83 bacterial strains was recovered and characterised. Phylogenetic analysis revealed their affiliation with 19 species across 13 genera, encompassing both known and potentially novel taxa.\u003c/p\u003e\u003cp\u003eFunctional assays demonstrated that many of these isolates possess significant lignocellulolytic activity, with several strains showing efficient degradation of complex plant polymers and the ability to produce key fermentation metabolites such as ethanol, hydrogen, and volatile fatty acids. Notable strain-level variability was observed in enzyme profiles, substrate preferences, and metabolic outputs, underscoring the functional diversity of this microbial community. Importantly, various strains of \u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e, \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e, and \u003cem\u003eThermoanaerobacterium\u003c/em\u003e spp. emerged as promising candidates for bioenergy applications, especially in consolidated bioprocessing setups.\u003c/p\u003e\u003cp\u003eThese results confirm that Indian hot springs harbour phylogenetically diverse and metabolically versatile thermophilic anaerobic bacteria with strong potential for industrial use in lignocellulosic biomass valorisation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to Dr Karthick Balasubramanian, Agharkar Research Institute, Pune and Dr. Raymond A Duraiswami, Savitribai Phule Pune University, for sampling support. We duly acknowledge the help of Dr. Akshay Joshi extended during the study. We also appreciate the technical assistance from Dr. Bhushan Shigwan, Dr. Radhakrishnan Cheran \u0026amp; Dr. Vikram Lanjekar. We are grateful to the Director, Agharkar Research Institute, for providing the necessary infrastructure support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSSH: Investigation, formal analysis, writing\u0026mdash;original draft, visualisation, data curation, writing\u0026mdash;review and editing; PKD: Conceptualisation, supervision, resources; SSD: Conceptualisation, supervision, resources, funding acquisition, writing\u0026mdash;review and editing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India, with project number YSS/2015/000718 awarded to SSD and Junior Research Fellowship to SSH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that they do not have any conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAjeje SB, Hu Y, Song G, et al (2021) Thermostable Cellulases / Xylanases From Thermophilic and Hyperthermophilic Microorganisms: Current Perspective. 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Metab Eng 83:39\u0026ndash;51. https://doi.org/10.1016/j.ymben.2024.03.001 \u003c/li\u003e\n\u003cli\u003ePlugge CM, Zoetendal EG, Stams AJ (2000) \u003cem\u003eCaloramator coolhaasii\u003c/em\u003e sp. nov., a glutamate-degrading, moderately thermophilic anaerobe. Int J Syst Evol Microbiol 50:1155\u0026ndash;1162. https://doi.org/10.1099/00207713-50-3-1155 \u003c/li\u003e\n\u003cli\u003ePoddar A, Das SK (2018) Microbiological studies of hot springs in India: A review. Arch Microbiol 200:1\u0026ndash;18. https://doi.org/10.1007/s00203-017-1429-3 \u003c/li\u003e\n\u003cli\u003ePopova LI, Bahl H, Egorova MA, et al (2021) Isolation of Cellulose-Degrading \u003cem\u003eThermoanaerobacterium\u003c/em\u003e Strains from Thermophilic Methanogenic Microbial Communities. 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Microorganisms 9:1473. https://doi.org/10.3390/microorganisms9071473 \u003c/li\u003e\n\u003cli\u003eSaxena R, Dhakan DB, Mittal P, et al (2017) Metagenomic Analysis of Hot Springs in Central India Reveals Hydrocarbon Degrading Thermophiles and Pathways Essential for Survival in Extreme Environments. Front Microbiol 7:223920. https://doi.org/10.3389/fmicb.2016.02123 \u003c/li\u003e\n\u003cli\u003eSaxena S, Moharil MP, Jadhav P V., et al (2025) Transforming waste into wealth: Leveraging nanotechnology for recycling agricultural byproducts into value-added products. Plant Nano Biology 11:100127. https://doi.org/10.1016/J.PLANA.2024.100127 \u003c/li\u003e\n\u003cli\u003eSchroeder BG, Logro\u0026ntilde;o W, Rocha UN da, et al (2022) Enrichment of Anaerobic Microbial Communities from Midgut and Hindgut of Sun Beetle Larvae (\u003cem\u003ePachnoda marginata\u003c/em\u003e) on Wheat Straw: Effect of Inoculum Preparation. 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Enzyme Microb Technol 118:66\u0026ndash;75. https://doi.org/10.1016/j.enzmictec.2018.07.001 \u003c/li\u003e\n\u003cli\u003eSingh B, Bala A, Anu, et al (2022) Biochemical properties of cellulolytic and xylanolytic enzymes from \u003cem\u003eSporotrichum thermophile\u003c/em\u003e and their utility in bioethanol production using rice straw. Prep Biochem Biotechnol 52:197\u0026ndash;209. https://doi.org/10.1080/10826068.2021.1925911 \u003c/li\u003e\n\u003cli\u003eSingh N, Mathur AS, Tuli DK, et al (2017) Cellulosic ethanol production via consolidated bioprocessing by a novel thermophilic anaerobic bacterium isolated from a Himalayan hot spring. Biotechnol Biofuels 10:73. https://doi.org/10.1186/s13068-017-0756-6 \u003c/li\u003e\n\u003cli\u003eSingh N, Puri M, Tuli DK, et al (2018) Bioethanol production potential of a novel thermophilic isolate \u003cem\u003eThermoanaerobacter\u003c/em\u003e sp. DBT-IOC-X2 isolated from Chumathang hot spring. 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Springer Nature Switzerland, Cham, pp 1\u0026ndash;34\u003c/li\u003e\n\u003cli\u003eYan F, Dong S, Liu Y-J, et al (2022) Deciphering Cellodextrin and Glucose Uptake in \u003cem\u003eClostridium thermocellum\u003c/em\u003e. mBio 13:. https://doi.org/10.1128/mbio.01476-22 \u003c/li\u003e\n\u003cli\u003eYang F, Yang X, Li Z, et al (2015) Overexpression and characterization of a glucose-tolerant \u0026beta;-glucosidase from \u003cem\u003eThermoanaerobacterium aotearoense\u003c/em\u003e with high specific activity for cellobiose. Appl Microbiol Biotechnol 99:8903\u0026ndash;8915. https://doi.org/10.1007/s00253-015-6619-9\u003c/li\u003e\n\u003cli\u003eYue S, Mizoguchi T, Kohara T, et al (2021) Meta‐fermentation system with a mixed culture for the production of optically pure L‐lactic acid can be reconstructed using the minimum isolates with a simplified pH control strategy. Biotechnol J 16:2100277. https://doi.org/10.1002/biot.20210027 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacteria, Diversity, Ecology, Phylogeny, Taxonomy","lastPublishedDoi":"10.21203/rs.3.rs-8018409/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8018409/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThermophilic anaerobic bacteria are crucial for degrading lignocellulosic biomass and producing biofuels under high-temperature, oxygen-limited conditions, presenting industrial relevance. However, their diversity and function from Indian hot springs remain underexplored. In this study, water and sediment samples were collected from ten geographically distinct Indian hot springs (25\u0026ndash;85\u0026deg;C), and physicochemical parameters were measured to characterise environmental heterogeneity. Enrichments were performed using cellulose, xylan, neutral detergent fibre, and lignin, which demonstrated significant hydrogen production, primarily at thermophilic temperatures (55\u0026ndash;85\u0026deg;C). Using anaerobic roll bottle isolation and redundancy reduction by RFLP, 83 distinct strains were obtained. Phylogenetic analysis of the 16S rRNA gene identified 19 species across 13 genera and 9 bacterial families, including \u003cem\u003eCaldibacillus\u003c/em\u003e, \u003cem\u003eCaloramator\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e, \u003cem\u003eThermoanaerobacterium\u003c/em\u003e, and \u003cem\u003eSporanaerobium\u003c/em\u003e. Numerous strains exhibited notable cellulase, xylanase, and esterase activities on untreated rice and wheat straw. Distinct strain-level variations were noted in enzyme activities and metabolite profiles. Isolates produced high yields of ethanol, hydrogen, and volatile fatty acids, including acetic, butyric, and propionic acids. Notably, strains of \u003cem\u003eCaldibacillus thermoamylovorans\u003c/em\u003e, \u003cem\u003eThermoanaerobacter wiegelii\u003c/em\u003e, and \u003cem\u003eThermoanaerobacterium\u003c/em\u003e spp. showed promise for consolidated bioprocessing applications. This represents the first comprehensive systematic study of lignocellulolytic thermophilic anaerobes from Indian geothermal ecosystems, highlighting their ecological diversity and significant potential for bioenergy production from agricultural residues.\u003c/p\u003e","manuscriptTitle":"Diverse Thermophilic Anaerobes from Indian Hot Springs Exhibit High Potential for Bioenergy Production from Lignocellulosic Biomass","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 11:22:16","doi":"10.21203/rs.3.rs-8018409/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-20T07:14:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-19T13:47:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-15T20:04:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-13T11:08:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233088700338198551470632587845688994939","date":"2025-11-11T13:49:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-10T04:23:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247703827026579214464650934677396397364","date":"2025-11-08T04:51:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141174613531776858035938145614974682555","date":"2025-11-06T12:08:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334944743421601717388850597707861472329","date":"2025-11-06T06:40:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153381549641516060811935539933273912764","date":"2025-11-06T04:35:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290198510610470799051848779363281390143","date":"2025-11-06T04:33:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-06T03:59:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-04T07:36:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-03T13:50:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2025-11-03T11:06:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3d01d153-09cd-44fd-9872-b0e88f25c8f6","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2025-11-20T07:23:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-17 11:22:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8018409","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8018409","identity":"rs-8018409","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}