Genomic and structural insights into the high-efficiency poly(3-hydroxybutyrate) biodegradation in Terrabacter sp. AAH1

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.

Enhanced biological desilication by Bacillus mucilaginosus mutant: Multi-omics insights into extracellular polymeric substance overproduction and metabolic adaptations.

Recovering scandium from silicate-rich tailings remains a major challenge due to refractory mineral matrices. Although biological desilication using microbial secretomes offers an eco-friendly solution, its industrial application is limited by the poor understanding of the intracellular metabolic programs that govern high-efficiency desilication. Here, an N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced Bacillus mucilaginosus mutant, BM3, was evaluated using integrated whole-genome sequencing, transcriptomics, non-targeted metabolomics and mineral-interface measurements. BM3 achieved a 1.7-fold increase in desilication rate and subsequent chemical leaching of the bio-pretreated tailings substantially elevated scandium extraction from 21% (untreated) and 38% (wild-type-pretreated) to 53%. Spectroscopic analyses indicated that this modified secretome acted as a reactive interface, preferentially eroding recalcitrant pyroxene phases via carboxyl, amino, and hydroxyl ligand coordination. Multi-omics integration suggested that a coordinated metabolic reprogramming, including changes in phenylalanine metabolism, purine metabolism, and folate one-carbon metabolism, may underpin this phenotype. Overall, these findings reveal that the enhanced desilication by BM3 is associated with coordinated metabolic reprogramming and a more reactive EPS-mineral interface, providing an effective biological pretreatment for disrupting silicate matrices and improving scandium recovery from refractory tailings.

Acinetobacter guillouiae, a lipolytic strain isolated from sludge capable of partially depolymerising polyethylene terephthalate: genomic, proteomic, and biochemical insights

Acinetobacter guillouiae I-MWF was isolated by incubating amorphous polyethylene terephthalate (PET) film in sludge samples. The strain partially depolymerised PET powder with 11.3% crystallinity, as confirmed by FT-IR, HPLC-UV, and LC-MS analyses. Extracellular enzymes released terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET). Genomic analysis identified 18 putative extracellular hydrolases, including lipases and esterases, each with a conserved catalytic triad. Proteomic profiling revealed expression of two triacylglycerol lipases and two additional lipase-family proteins when the strain was cultivated with PET or a PET–Tween 80 mixture. These enzymes were cloned in Escherichia coli, but most formed insoluble, inactive inclusion bodies, and one was not expressed. Molecular modelling highlighted structural features likely to influence their catalytic interaction with PET. Although the strain partially depolymerised PET powder, it was unable to grow on PET, TPA, or ethylene glycol, indicating that PET depolymerisation occurs as a side activity rather than supporting growth. Instead, A. guillouiae displayed strong lipolytic activity and a clear preference for lipid-based substrates, achieving its highest growth with Tween 80. A lipid transporter was also expressed under these conditions, suggesting adaptation to hydrocarbon-rich environments. These findings indicate that A. guillouiae I-MWF can mediate partial PET depolymerisation without assimilating the resulting monomers, while preferentially growing on lipid-like substrates.

Characterization of a Novel GH3 β-Glucosidase with Alkali-Tolerant and Halophilic Properties from Pontixanthobacter luteolus SW-109.

Bioinformatics analysis of extracellular glycosyl hydrolases mined from the genome of Halophilic Meridianimaribacter sp. CL38.

Biological pre-treatment of lignocellulosic waste is crucial for efficient fermentation in second-generation biofuel production. Halophilic bacteria produce glycosyl hydrolases with enhanced stability for industrial application. This study aimed to characterise putative glycosyl hydrolases (cellulases and hemicellulases) encoded in the genome of the halophilic bacterium Meridianimaribacter sp. CL38 using bioinformatic approaches, in order to address the scarcity of structural information for this genus. Four putative glycosyl hydrolases (GH9, GH3, GH144 and GH16) were analysed, focusing on their functional domains and structural properties. Phylogenetic analysis indicated a close relationship with other members of the Flavobacteriaceae family. Structural analyses revealed significant predicted halophilic and thermostable properties, suggesting these putative enzymes may endure industrial conditions. This study provides insights into putative glycosyl hydrolase candidates for efficient lignocellulosic waste pre-treatment.

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.