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

Experimental, Genomic, and Structural Evidence Supporting Putative PET‐Hydrolases in Thermophilic Bacteria Isolated From Hot Springs in Cajamarca, Peru

ABSTRACT Polyethylene terephthalate (PET) waste represents a major environmental challenge due to limited recycling solutions. Thermophilic bacteria from geothermal environments harbor diverse enzymatic machinery adapted to extreme conditions, offering promising biocatalysts for plastic degradation; however, biological resources from Peru and other South American countries remain scarce. We characterized four bacterial strains isolated from two geothermal sites in Cajamarca, Peru, screened for PET hydrolysis at 50°C. Whole‐genome sequencing using hybrid assembly achieved near‐complete circular genomes. GTDB‐Tk classification identified three species: Neobacillus thermocopriae (strain 19A), Bacillus licheniformis (strains 16P and BI2), and Brevibacillus agri (strain BI8). Quantitative assays revealed that strain 16P achieved the highest mass loss (0.598%), followed by strain BI8 (0.449%). ATR‐FTIR analysis of the incubated sheets showed a significant reduction of the ester carbonyl index in strains 16P, 19A, and BI8 relative to both non‐incubated PET and an abiotic control, whereas strain BI2 did not differ from the controls, indicating preferential modification of ester bonds at the sheet surface. Genome mining and structure‐based homology searches identified multiple candidate enzymes similar to validated PETases and carboxylesterases, including PETase46‐like homologs in strains BI8 and 16P and a terephthalate‐active carboxylesterase homolog in strain 16P. Molecular docking supported the conservation of catalytic geometry and substrate‐binding sites in these candidates. This work represents one of the first systematic genomic and structural characterizations of putative PET‐hydrolases in Peruvian geothermal bacteria, expanding knowledge of extremophile diversity and advancing thermostable enzymes for sustainable plastic waste management.

Isolation and Characterization of Thermophilic Microbial Enzymes Protease and Amylase from Hot Spring Madkot, Pithoragarh in Uttarakhand

Thermophilic environments such as hot springs are invaluable natural reservoirs of extremophilic microorganisms that produce thermostable enzymes with significant industrial utility. This study explored the thermophilic microbial community of the Madkot geothermal hotspot (Pithoragarh), Uttarakhand. Samples collected from high-temperature niches were subjected to enrichment and isolation under thermophilic conditions, followed by comprehensive biochemical profiling including indole, methyl red, Voges–Proskauer, phosphate solubilization, catalase, oxidase, urease and nitrate reduction tests to elucidate metabolic capabilities relevant to industrial processes. Selected thermophilic strains were molecularly identified by 16S rRNA gene sequencing and affiliated with the genera Bacillus and Pseudomonas. Both taxa demonstrated the capacity to produce industrially relevant enzymes under elevated temperature regimes: a Bacillus sp. isolate exhibited protease activity with a maximum specific activity of 23.05 U/ml, while a Pseudomonas sp. isolate synthesized amylase with a peak specific activity of 31.02 U/ml. The thermostability and activity profiles of these enzymes suggest suitability for applications that demand resilience to heat and harsh chemical conditions, including biofuel generation, high-temperature food processing, pharmaceutical enzyme formulations and bioremediation of thermally stressed or contaminated environments. Overall, the results underscore the rich, yet underexplored, thermophilic microbial diversity of Uttarakhand hot springs and emphasize their importance as a source of sustainable, heat-tolerant biocatalysts for industrial biotechnology. Future work will focus on enzyme purification, detailed thermostability profiling and cost effective pilot-scale production in relevant process conditions.

Production and Characterization of Poly(3-hydroxybutyrate) by Thermophilic Geobacillus sp. Strain SL28 Isolated from a Natural Hot Spring

Polyhydroxyalkanoates (PHAs) are biodegradable microbial polyesters that may reduce dependence on petroleum-derived plastics, but broader use remains constrained by production costs and tightly controlled cultivation. Thermophilic microorganisms have attracted increasing attention as promising PHA producers because they can be cultivated at elevated temperatures, reducing contamination risks and operational costs. In this study, a thermophilic Geobacillus sp. strain SL28 isolated from a Vietnamese hot spring was evaluated for its PHA-producing capability. Temperature, initial pH, carbon and nitrogen sources, C/N ratio, and cultivation time were examined. Intracellular inclusions were assessed by Sudan Black B and Nile Blue A staining and transmission electron microscopy, whereas the recovered polymer was characterized by FE-SEM, Fourier-transform infrared spectroscopy (FTIR), gas chromatography–mass spectrometry (GC–MS), 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy, capillary viscometry, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and tensile testing. Under the optimized cultivation conditions identified by one-factor experimentation (50 °C, pH 7.0, sucrose, peptone, C/N 25:1, and 72 h), SL28 reached a dry cell weight of 2.215 ± 0.16 g/L and a PHA concentration of 1.383 ± 0.04 g/L, equivalent to 62.57 ± 2.82% of DCW. Structural characterization confirmed that the recovered polymer was poly(3-hydroxybutyrate) (PHB). The purified PHB also exhibited favorable thermal stability, a relatively high molecular weight, and satisfactory mechanical properties. These results demonstrate the potential of thermophilic Geobacillus sp. SL28 as a promising candidate for PHB production.

THERMOHALOPHILIC BACTERIA PRODUCING THERMOSTABLE LIPASE FROM WAWOLESEA HOT SPRING, SOUTHEAST SULAWESI

This study aimed to isolate and characterize thermohalophilic bacteria producing lipase and to purify thermostable lipase from the Wawolesea hot spring in Southeast Sulawesi. A total of 24 isolates were obtained, all of which demonstrated lipase-producing capability. Three selected isolates (BT2.M3, CT2.M4, and DT1.M2) were identified as Gram-negative bacilli and showed lipase activities ranging from 0.17 to 21 U/mL, with the highest activity recorded for BT2.M3 at 50°C and pH 6. Purification through ammonium sulfate fractionation yielded the highest activity in the 60–80% saturation fraction (43 U/mL) at pH 6–7 and 50–60°C. Protein analysis showed the highest concentration in the 80% dialysis fraction (0.954 mg/mL). These findings highlight the potential of Wawolesea thermohalophilic bacteria as a valuable source of thermostable lipase for biotechnological applications.

Purification, biochemical characterization, and industrial potential of a thermostable lipase from Aeribacillus pallidus IO3 isolated from a geothermal source.

Lipases obtained from thermophilic microorganisms are attracting great interest in the industrial field due to their stability under high-temperature conditions and their wide range of industrial applications. In this study, thermophilic bacterial strains were isolated from water and mud samples collected from Erzurum Ilica Hot Springs (Turkey). An isolate producing a potent extracellular lipase was selected, named IO3, and identified by 16S rRNA sequencing. Sequencing analysis revealed that this isolate showed 99% similarity to Aeribacillus pallidus. The lipase enzyme purified from the isolate was obtained using two comparative strategies. The conventional multi-stage chromatographic approach, which includes ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography, provided 9.3-fold purification with a 3.18% recovery. However, the alternative Three-Phase Partitioning (TPP) system provided rapid, single-step recovery with a significantly higher yield of 38.79%, although the purification fold remained 0.65 under the tested conditions. The molecular weight of the purified A. pallidus IO3 lipase was determined to be approximately 33.88 kDa by SDS-PAGE. The enzyme showed optimum activity at pH 8.0 and 50 °C and maintained considerable thermal stability; the enzyme retained significant activity levels (45-69%) even at high temperatures such as 70 °C and 80 °C after an incubation period of 120 minutes. Among the tested metal ions, Fe2+, Fe³+, Cu2+, and Zn2+ enhanced enzyme activity, while the enzyme showed stability in the presence of Li+ and Cu2+ ions. Lipase activity also increased in the presence of surfactants, while chloroform enhanced enzyme activity by 22-338% depending on solvent concentration. The enzyme showed the highest activity toward p-nitrophenyl palmitate. The enzyme was completely inhibited by DTNB, IAA, and EDTA. In addition, the enzyme retained low activity in the presence of β-mercaptoethanol. The Km and Vmax values were calculated as 0.46 mM and 51.44 µmol·min-1·mg-1, respectively, using nonlinear regression analysis. Overall, these findings indicate that A. pallidus IO3 lipase is a thermostable and chemically tolerant enzyme with potential applicability in industrial biocatalysis and detergent-related processes.

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