Genomic and functional insights into the thermophilic strain Geobacillus sp. Geo 8.1: a source of thermostable xylanase for sustainable bioprocesses
Songül Yaşar Yıldız, Ilaria Finore, Bora Ceylan, Doğa Umaç, Ceyda Kasavi, Gennaro Finore, Antonio Federico, Francesco Nizzolino, Luigi Leone, Annarita Poli, Ebru Toksoy Öner
TL;DR
A heat-loving bacterium from a hot spring in Italy produces stable enzymes that could help in sustainable industrial processes.
Contribution
Discovery of a new thermophilic strain with a complete genomic profile and thermostable xylanase activity for bioprocessing.
Findings
The draft genome of Geobacillus sp. Geo 8.1 includes genes for carbohydrate, lipid, and protein metabolism.
Crude enzyme extracts showed high xylanase activity at 65°C, with xylose as the main product.
The strain has a complete D-xylose utilization pathway, linking hemicellulose breakdown to central metabolism.
Abstract
A thermophilic bacterium, designated Geobacillus sp. Geo 8.1, was isolated from a submarine hydrothermal spring of Ischia Island (Italy) and characterized through genomic and biochemical analyses to evaluate its biotechnological potential. The draft genome (3.41 Mbp; GC 52.5%) revealed 3,751 coding sequences, including complete pathways for carbohydrate, lipid, and protein metabolism, and enzymes involved in stress response and hydrocarbon degradation. Phylogenomic and digital DNA–DNA hybridization analyses placed Geo 8.1 within the Geobacillus thermoleovorans/Geobacillus kaustophilus cluster. Functional annotation highlighted diverse genes encoding thermostable hydrolases such as xylanases, β-xylosidases, lipases, proteases, and α-amylases, together with catalases and dehalogenases relevant to environmental and industrial applications. Crude enzyme extracts exhibited strong xylanase…
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Figure 4- —Istanbul Medeniyet University
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Taxonomy
TopicsBiofuel production and bioconversion · Polysaccharides and Plant Cell Walls · Advanced Cellulose Research Studies
Introduction
Thermophilic bacteria represent a diverse group of microorganisms capable of thriving at elevated temperatures, typically between 50 °C and 80 °C. These extremophiles have evolved specialized enzymes that remain active and stable under harsh industrial conditions, making them particularly attractive for biotechnological applications (Yasar Yildiz et al. 2014).
Within this group, the genus Geobacillus has emerged as a prolific source of thermostable enzymes, particularly xylanases. These Gram-positive, spore-forming bacteria are widely distributed in geothermal habitats and compost piles, where they play a pivotal role in decomposing plant biomass. Geobacillus species are recognized for their high catalytic activity, exceptional thermostability and broad pH tolerance, which make them ideal candidates for various applications such as biofuel production, pulp and paper bioleaching and improvement of food and feed quality. Significant species, including G. stearothermophilus, G. thermoleovorans and G. thermodenitrificans, have been studied extensively for their enzyme potential, with significant advances in their genomic and structural characteristics (Ajeje et al. 2021; Anand et al. 2013; Bhalla et al. 2014; Verma and Satyanarayana 2012).
The progress in genome sequencing and bioinformatics has significantly accelerated the discovery and optimization of industrial enzymes. Genomic studies provide tools to search for gene clusters, regulatory regions, and gene-encoded transport mechanisms controlling substrate consumption pathways in microbes. These aspects are applied in designing enzyme mixes in accordance with different industrial requirements (Brumm et al. 2015; Carbonaro et al. 2024).
In addition to the confirmation of known metabolism, these methods discover previously uncharacterized gene clusters, secondary metabolites, and mechanisms of environmental adaptation, thereby defining the complete biotechnological capability of microorganisms. These discoveries facilitate the delimitation of capabilities related to the production of industrial enzymes, antimicrobial agents, and bioplastic precursors, in addition to identifying essential metabolic functions such as carbon, nitrogen, sulfur, and phosphorus metabolism (Yıldız and Radchenkova 2025). Furthermore, genome-scale information defines the roadmap applicable to metabolic engineering and synthetic biology, thereby facilitating the redesign of gene overexpression and optimization of regulatory metabolic networks, in addition to increasing the efficiency of enzymes. Therefore, genome analysis forms the basis of comprehensive understanding and integration of new biocatalysts in bioprocesses (Yasar Yildiz et al. 2022; Yaşar Yıldız 2025; Yıldız and Radchenkova 2025; Yildiz et al. 2015).
Xylan, the principal hemicellulosic component of plant cell walls, is a complex polysaccharide predominantly composed of β-1,4-linked xylose residues. Its enzymatic hydrolysis is catalyzed by xylanases, which cleave the xylan backbone into xylooligosaccharides and xylose. This reaction is crucial for lignocellulosic biomass conversion, as it enhances cellulose accessibility for further breakdown (Mendonça et al. 2023). Thermostable xylanases from Geobacillus and other heat-adapted bacteria are therefore valuable assets in industrial biotechnology.
Thermophilic xylanases from Geobacillus species typically exhibit optimal activity between 50 °C and 90 °C, with many strains peaking around 70 °C (Finore et al. 2023; Verma and Satyanarayana 2012). This high-temperature preference accelerates reaction kinetics, improves substrate solubility, and reduces solution viscosity, enabling more efficient biomass hydrolysis. These traits are particularly advantageous in industrial processes that demand rapid conversion rates, lower enzyme dosages, and shorter processing times (Ajeje et al. 2021; Bhalla et al. 2014). Furthermore, many Geobacillus-derived xylanases retain activity over a broad pH range, a property beneficial for applications such as pulp bio-bleaching and food processing where operational conditions may vary from mildly acidic to alkaline (Anand et al. 2013; Marcolongo et al. 2015). Structural features including ionic bonds, hydrophobic interactions, and disulfide bridges confer resilience against thermal denaturation, while protein engineering strategies—such as domain fusion and site-directed mutagenesis—have further enhanced these traits (Kumar et al. 2018; Marcolongo et al. 2015).
Operating at elevated temperatures offers additional mechanistic advantages: reduced microbial contamination, prolonged enzyme stability, and compatibility with thermochemical pretreatments (Ajeje et al. 2021; Bhalla et al. 2014). When applied in synergy with cellulases, xylanases enhance lignocellulose saccharification by removing hemicellulosic barriers, thus increasing yields of fermentable sugars. In the pulp and paper industry, their selective hemicellulose degradation reduces the need for chlorine-based chemicals, improving both environmental sustainability and workplace safety (Marcolongo et al. 2015; Verma and Satyanarayana 2012).
Genome-enabled research has revealed conserved gene clusters and regulatory networks responsible for xylanase and β-xylosidase production. These modules encompass extracellular enzymes, specific transporters, and intracellular hydrolases, providing a systems-level perspective on xylan utilization (Yasar Yildiz et al. 2022).
Insights gained from genomic and functional analyses provide a direct framework for evaluating the industrial relevance of thermophilic enzymes, particularly xylanases produced by Geobacillus species. The industrial potential of Geobacillus xylanases spans multiple sectors. In food processing, they improve dough rheology, enhance juice clarity, and modulate dietary fiber content. In animal feed, they boost digestibility and generate prebiotic xylooligosaccharides that promote gut health (Kumar et al. 2018). In detergents and textiles, their stability under alkaline and high-temperature conditions supports bio-scouring and stain removal (Marcolongo et al. 2015).
Although the industrial value of these thermophilic xylanases is well established, large-scale production still presents several challenges. Continued optimization of fermentation strategies and exploration of efficient recombinant hosts remain active research areas. Meanwhile, the integration of metagenomic discovery, synthetic-biology tools, and AI-assisted enzyme design is rapidly broadening the diversity and applicability of novel biocatalysts for future sustainable processes (Atalah et al. 2019; Verma et al. 2019). Notably, cloning and expression of genes such as xyl267 from G. stearothermophilus and β-xylosidases from G. thermodenitrificans have demonstrated long enzyme half-lives and high product yields, underscoring their industrial relevance (Irfan et al. 2016; Bhalla et al. 2014).
Thermostable xylanases from Geobacillus spp. exemplify the synergy between microbial adaptation and genome-guided innovation. Their exceptional biochemical robustness, cross-sector versatility, and amenability to engineering position them as key drivers in the next generation of sustainable, efficient, and cost-effective industrial processes. Ischia Island is the western most active volcanic complex of the Campania region area and belongs to the Phleagrean volcanic district of Southern Italy. Ischia is composed mainly of volcanic rocks and subordinately of sediments (Bucci et al. 2011). This island is well known for the presence of hydrothermal vents, and each year hosts many tourists coming from all over the world attracted from the therapeutical properties of thermal baths. The microbial diversity is still unexplored; therefore, the main aim of this study was to explore the thermophilic community of hot underground water collected in Ischia Island, as source of novel and biotechnologically interesting enzymes. For this, thermophiles isolated from water and sediment samples were characterized in terms of their pH and temperature dependent growth, salinity tolerance and morphology. A single cream/white-pigmented colony, designated as strain Geo 8.1, was selected for further study as it exhibited the fastest growth rate among the cultured microorganisms, forming colonies even at highest dilutions—indicating its robust physiological adaptability, and potential ecological dominance in its native hydrothermal environment.
Materials and methods
Sample collection
A mixture of water and sediment samples were collected in May 2021 from a submarine hydrothermal spring located off the coast of volcanic Ischia Island, Gulf of Naples, Italy (40°43’51’’N; 13°56’30’’E), at a depth of 105 m below sea level. The sampling site was characterized by hydrothermal activity, with the temperature of the venting water measured at approximately 70 °C at the time of collection. Samples were aseptically retrieved using sterile equipment to avoid contamination and were immediately transferred into sterile, airtight tubes. Subsequently, the collected samples were transported under refrigerated conditions and stored at 4 °C until further microbiological and physicochemical analyses were conducted in the laboratory.
One gram of sediment sample was used as an inoculum for enrichment in Medium A, which consisted of 0.2% peptone and 0.1% yeast extract, adjusted to pH 7.0. The culture was incubated at 60 °C for 48 h, after which microbial growth was observed. A volume of 100 µl from the enriched culture was then spread in duplicate onto solid Medium A, solidified with 1.8% (w/v) agar (Oxoid™). Following incubation at 60 °C for 48 h, distinct colonies that developed on the agar plates were isolated and purified using the serial dilution plating technique. Purification was further achieved by repeated streaking onto fresh Petri dishes containing the same solid medium. A cream/white-pigmented colony was designated as strain Geo 8.1.
For sub-culturing, the isolates were transferred into Tryptone Soy Broth (TSB; Oxoid) and incubated at 60 °C for 24 h. The cultures were then preserved as glycerol stocks (Oxoid) at − 20 °C for long-term storage (Strain Geo 8.1 is stored at CE-ICB collection, Extremophiles Collection at ICB CNR, Pozzuoli, Naples, Italy, under accession number CE-ICB 108).
To assess the purity of the isolates, cell morphology was examined using phase-contrast microscopy (Nikon Eclipse E400, Nikon Europe, Badhoevedorp, The Netherlands). In addition, colony homogeneity was evaluated under a stereomicroscope (M8, Leica Microsystems, Mannheim, Germany).
Growth conditions and morphological characterization
The optimal growth parameters of the isolate, its tolerance and preferences for salinity, pH, and temperature values, were evaluated in liquid Medium A under aerobic conditions. Salinity tolerance was tested by supplementing Medium A with different concentrations of NaCl (0%, 0.2%, 0.5%, 1%, 5%, 7.5%, and 10%, w/v). The pH range for growth was assessed by adjusting the medium to values between pH 6.0 and 9.0, while the temperature range was tested by incubating cultures at various temperatures between 60 °C and 80 °C.
Growth was monitored spectrophotometrically by measuring the turbidity of the cultures at λ540 nm. Morphological characterization of the strain was performed on cultures grown on Tryptone Soy Agar (TSA; Oxoid) incubated at 60 °C for 24 h. Cellular and colony morphologies were examined as above reported.
Biochemical characterization of the strain Geo 8.1
All biochemical assays were carried out under the strain’s optimal growth conditions, in terms of temperature, pH, and NaCl concentration. Gram staining was performed according to the method described by Halebian et al. (Halebian et al. 1981). Oxidase activity was evaluated by monitoring the oxidation of tetramethyl-p-phenylenediamine, while catalase activity was assessed based on bubble formation upon exposure to 3% (v/v) hydrogen peroxide, following standard procedures (Jurtshuk Jr and McQUITTY 1976).
Hydrolytic activities of various substrates were tested on solid medium A. Starch hydrolysis was assessed by flooding the plates with Lugol’s iodine after incubation on starch-supplemented medium (0.2% w/v). Xylan and cellulose degradation were evaluated by flooding the respective agar plates with 0.1% Congo red followed by 1 M NaCl; the solid media were supplemented with 1.0% (w/v) xylan or 0.5% (w/v) carboxymethyl cellulose (CM-cellulose), respectively (Yildiz 2024). Lipase activity was tested using solid medium A supplemented with 2.5% (w/v) olive oil and 0.001% rhodamine B, according to the protocol of Kouker and Jaeger (Kouker and Jaeger 1987). Casein hydrolysis was tested on solid medium A enriched with 5.0% (w/v) skim milk (Brown and Foster 1970).
Endospore formation was stimulated by supplementing liquid medium with 0.001% (w/v) MnCl₂·4 H₂O. Spore formation was assessed microscopically after 24 and 48 h of incubation. Antibiotic susceptibility was tested using Sensi-discs (6 mm, Oxoid) on solid medium A, and the results were evaluated based on inhibition zone diameters.
Phylogenetic assignment
Genomic DNA was isolated from the cultures using DNAzol (Molecular Research Centre, Inc. Cincinnati, OH, USA) according to the manufacturer’s instruction. PCR amplification and sequencing of the almost full-length 16 S rRNA gene was obtained from the genomic-DNAs amplification by using universal primers 8 F and 1517R of broad specificity in a polymerase chain reaction (PCR). The nucleotide sequence of 16 S rRNA gene was analyzed by EzTaxon-e server (https://www.ezbiocloud.net), and the values for pairwise 16 S rRNA gene sequence similarity among the closest species were determined using the EzTaxon-e server (Finore et al. 2020).
Genomic DNA extraction and whole-genome sequencing
25 mL of actively growing culture, coming from 12 h of incubation at 60 °C, was centrifuged, and the resulting cell pellet was used for genomic DNA extraction. Whole-genome sequencing was performed by MicrobesNG (Birmingham, UK) using Illumina short-read high-throughput sequencing platforms. Paired-end short-read sequencing libraries were prepared. Raw reads were subjected to quality control and adapter trimming before de novo genome assembly. Genome assembly was performed using standard assembly pipelines provided by MicrobesNG, based on SPAdes assembler, generating contigs that were subsequently used for downstream genome annotation and comparative analyses.
Genome annotation and functional characterization
Genome annotation and gene prediction were carried out using a multi-platform strategy combining automated pipelines with manual curation to maximize accuracy and functional depth. At first step, genome assemblies were uploaded to the RAST server (Rapid Annotations using Subsystems Technology; http://rast.nmpdr.org/), which provided preliminary predictions of coding sequences (CDSs), rRNA and tRNA genes, and subsystem-based functional assignments (Aziz et al. 2008). Results of RAST server were validated and refined by using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (https://www.bv-brc.org/) (Olson et al. 2023).
For orthology-based classification, protein-coding genes were clustered into Non-supervised Orthologous Groups (OGs) using the eggNOG database, with further classification according to COG (Clusters of Orthologous Groups) categories. Functional annotations were enhanced with Gene Ontology (GO) terms and protein domain data from SMART and PFAM databases.
To facilitate comparative genome analysis, all annotated assemblies were uploaded to the MicroScope Genome Annotation & Analysis Platform (MaGe) (https://mage.genoscope.cns.fr/microscope/home/index.php) (Vallenet et al. 2009). MaGe enabled visualization of genomic organization, identification of conserved and unique genes, and COG-based functional profiling of Geo 8.1.
Whole-genome-based taxonomic and phylogenomic analyses
To determine the taxonomic position and phylogenetic relatedness of the isolates, assembled genomes were submitted to the Type (Strain) Genome Server (TYGS) (https://tygs.dsmz.de), a dedicated platform for whole-genome-based taxonomic classification (Meier-Kolthoff and Göker 2019). Supporting nomenclature and synonymy were retrieved from the List of Prokaryotic names with Standing in Nomenclature (LPSN) (https://lpsn.dsmz.de) (Meier-Kolthoff et al. 2022).
The determination of closely related type strains was carried out in two steps:
- MASH Analysis: Genomes were compared against all type strain genomes in the TYGS database using the MASH algorithm (Ondov et al. 2016), which calculates intergenomic similarity based on k-mer profiles. The ten strains with the lowest MASH distances were selected.
- 16 S rRNA-Based Analysis: 16 S rRNA sequences were extracted using RNAmmer (Lagesen et al. 2007) and BLASTed against the TYGS 16 S rRNA dataset (Camacho et al. 2009). The top 50 hits were used to calculate precise intergenomic distances via Genome BLAST Distance Phylogeny (GBDP), using the ‘coverage’ algorithm and the d5 distance formula (Meier-Kolthoff et al. 2013). The ten closest genomes were selected based on these distances.
All pairwise comparisons among the genomes were conducted using GBDP with the ‘trimming’ algorithm and the d5 formula. For each comparison, 100 distance replicates were generated. Digital DNA–DNA hybridization (dDDH) values and their confidence intervals were computed using GGDC 4.0, following the recommended parameters (Meier-Kolthoff et al. 2013, 2022).
Phylogenetic relationships were inferred using FASTME 2.1.6.1, applying the balanced minimum evolution algorithm and SPR postprocessing (Lefort et al. 2015). Branch support was estimated via 100 pseudo-bootstrap replicates. The resulting phylogenetic tree was midpoint-rooted (Farris 1972) and visualized using PhyD3 (Kreft et al. 2017).
Species-level clusters were defined using a 70% dDDH threshold around the 13 type strains (Meier-Kolthoff and Göker 2019), while subspecies-level clusters were identified based on a 79% dDDH threshold (Meier-Kolthoff et al. 2014).
Whole-genome phylogenomic clustering of Geobacillus strains
For deeper insights into the genomic relationships between members of the Geobacillus group, the study involved phylogenomic analysis on the complete genome sequences of 13 different strains. The set of 13 strains was selected to represent the closest phylogenetic relatives of strain Geo 8.1 within the genus Geobacillus, including type strains of related species with available high-quality genome assemblies. This selection enabled a balanced comparison at both species and strain levels, providing sufficient resolution to assess genomic relatedness and evolutionary patterns within the G. thermoleovorans / G. kaustophilus species cluster.
Pairwise genomic similarity between the strains was calculated with the Mash algorithm, which uses MinHash sketching to efficiently estimate genome-wide distances between strains based on mutation events. These distances are known to be strongly correlated with Average Nucleotide Identity values, which are frequently used to evaluate the difference between genomes and define species boundaries (Konstantinidis and Tiedje 2005).
The Pairwise Mash distances were calculated and gathered to form a distance matrix, which was employed to reconstruct the evolutionary tree with the neighbor-joining method. The neighbor-joining method computes a tree topology that optimizes the total branch length, adjusting any negative values to ensure the accuracy of the resulting trees (Ondov et al. 2016).
To detect genome clusters at the level of species, the graph-based community detection method was employed via the MicroScope Genome Clustering (MICGC) framework. The genomes were modeled to represent the graph with edges defined by the Mash similarity values. For better resolution at the level of species, the edges with values corresponding to ANI cutoffs of less than 94% were eliminated, thereby distinguishing different genomic communities. Only high-quality genome assemblies with completeness higher than 90% and contamination lower than 5% were considered for analysis, as defined by evaluation with the CheckM tool (Vallenet et al. 2020).
Community identification was done using the Louvain modularity optimization algorithm (Blondel et al. 2008). The parameter optimization (k-mer length = 18, sketch size = 5000, resolution = 2) maximized sensitivity and specificity in community identification to provide phylogenetically relevant genomic communities to define species and identify evolutionary patterns in the Geobacillus genus.
Pan-genome and core-genome analysis
To assess the genomic diversity, gene content variation, and functional landscape of the Geobacillus genus, a pan/core genome analysis was performed. Whole-genome sequences of Geo 8.1 strain were compared with those of other 12 Geobacillus species and Anoxybacillus geothermalis using the MicroScope Microbial Genome Annotation and Analysis Platform (https://www.genoscope.cns.fr/agc/microscope/home/index.php). The comparative pan/core genome analysis was conducted using a dataset comprising 12 phylogenetically related Geobacillus strains and one representative Anoxybacillus geothermalis genome included as an outgroup. The Geobacillus strains were selected based on phylogenetic proximity to strain Geo 8.1 and the availability of high-quality, publicly accessible genome assemblies, ensuring consistent annotation and reliable pan/core genome inference. Inclusion of A. geothermalis as an outgroup enabled contextualization of core and accessory genome boundaries and provided an external reference for genus-level genomic differentiation.
The platform enabled consistent gene annotation and comprehensive comparative analysis across all genomes in the dataset. Homologous genes were identified using permissive thresholds—minimum 50% amino acid identity and at least 80% alignment coverage—to capture both highly conserved and moderately divergent gene sequences. This inclusive approach allowed for robust identification of shared core genes and accessory genes, highlighting functional traits unique to certain strains or subgroups (Deb 2022).
Through this comparative framework, insights were gained into evolutionary adaptation, functional redundancy, and diversification within Geobacillus. The analysis revealed patterns of gene conservation and loss, as well as subsystem-level variation. Additionally, annotations obtained from the RAST server were employed to refine comparisons among Geobacillus strains, facilitating the detection of genes potentially linked to ecological adaptation or industrial applications.
Xylanase production of the strain Geo 8.1
To investigate the xylanolytic potential of the strain Geo 8.1, cultures were grown in medium A, both with and without supplementation of 0.1% (w/v) birchwood xylan. After 24 h of incubation, cells were harvested by centrifugation at 10,000 rpm for 30 min. Xylanase activity was measured in extracellular crude enzyme preparations obtained from culture supernatants after ammonium sulfate precipitation and dialysis. The supernatants were subjected to ammonium sulfate precipitation at 80% saturation, followed by centrifugation at 10,000 × g for 1 h at 4 °C. The resulting pellets were dissolved in 50 mM phosphate buffer (pH 7.0) and dialyzed overnight against the same buffer (Yildiz 2024).
Protein concentration in the crude enzyme preparations was determined using the Bradford assay (Bradford 1976). Endo-xylanase activity was measured using 1% (w/v) birchwood xylan as substrate. The reaction mixture, comprising 0.4 ml of appropriately diluted enzyme solution and 0.1 ml of substrate, was incubated at 65 °C for 20 min under magnetic stirring in sealed glass vessels. Reactions were terminated by heating at 100 °C for 2 min. Xylanase activity was quantified via the dinitrosalicylic acid (DNS) method (Bernfeld 1955), and activity was expressed in nanomoles of xylose-equivalent reducing sugars released per minute under the assay conditions. All enzymatic assays were performed in triplicate, and the reported values represent mean values.
Hydrolysis products were further analyzed using Thin Layer Chromatography (TLC) with a solvent system consisting of n-butanol, acetic acid, and water (6:2:2, v/v/v). This allowed for the resolution and identification of xylan degradation products ranging from xylose to xylohexaose.
Results
Morphological, physiological and biochemical characteristics of strain Geo 8.1
Strain Geo 8.1 was isolated from a geothermal spring environment characterized by elevated temperature conditions. Environmental samples were collected and subjected to selective cultivation under thermophilic conditions to enrich heat-tolerant microorganisms. Following incubation at elevated temperatures, distinct colonies exhibiting robust growth were selected and repeatedly subcultured to obtain a pure isolate. This approach enabled the recovery of a stable thermophilic strain suitable for subsequent phenotypic and genomic characterization.
The bacterial isolate, referred to as strain Geo 8.1, was determined to be the most rapidly growing strain out of the subset of microorganisms that were cultured in medium A. Notably, it was the only isolate that was able to form visible colonies in the highest dilutions, indicating not only a high concentration in the sample, but also a strong biological capability to survive in the lab environment.
Strain Geo 8.1 showed growth in a relatively wide temperature range between 60 °C and 75 °C, with the optimal temperature being 65 °C, signifying that it was thermophilic in nature. The capability to grow in such high temperatures gives a possible indication that it may be harboring thermostable enzymes and stress resistance mechanisms that can be useful in biotechnological innovations. The strain showed the need for 0.2% NaCl concentration to grow well and was able to grow in a culture medium with pH 7.0, making it neutrophilic and halotolerant.
Microscopic observations showed that Geo 8.1 cells were rod-shaped, Gram-positive, and able to form endospores, which was an identifying feature associated with increased resistance to stress factors such as heat, drying, and starvation. On solid media A, the strain was observed to form large cream-white colonies with irregular margins, possibly indicating filamentous growth.
Biochemically, strain Geo 8.1 was observed to be oxidase-positive and catalase-negative, which reveal the unique characteristics of the strain concerning its respiratory metabolism in an oxygen-rich atmosphere. The enzymatic assay on agar media revealed that the strain was able to hydrolyze starch and xylan, meaning that it has extracellular amylase and xylanase enzymes, respectively, which play major roles in carbohydrate biodegradation processes. Lack of casein and carboxymethylcellulose (CMC) hydrolysis enzyme expression was observed, but the strain showed positive results for lipase activity, which indicated that it has the capability to degrade lipids, demonstrated by the strain’s capability to hydrolyze olive oil in the presence of rhodamine B.
Antibiotic susceptibility testing revealed that strain Geo 8.1 was sensitive to a broad spectrum of antibiotics, including Neomycin (30 µg), Penicillin G (2 µg and 10 µg), Chloramphenicol (50 µg), Fusidic Acid (10 µg), Erythromycin (5 µg and 30 µg), Tetracycline (30 µg and 50 µg), Vancomycin (30 µg), Lincomycin (15 µg), Kanamycin (30 µg), Novobiocin (30 µg), and Bacitracin (10 µg). This susceptibility profile suggests that the strain does not harbor common antibiotic resistance genes, at least under the tested conditions. Conversely, resistance to Nystatin (100 U), an antifungal agent targeting membrane sterols, was observed, which is consistent with the bacterial nature of the strain, as bacteria typically lack the sterol-rich membranes that Nystatin targets.
Taken together, these phenotypic and biochemical characteristics suggest that strain Geo 8.1 is a thermophilic, moderately halotolerant, aerobic, endospore-forming bacterium with enzymatic capabilities of potential biotechnological relevance, particularly in the degradation of complex carbohydrates and lipids under high-temperature conditions.
General genomic properties of the strain Geo 8.1
Based on its 16 S rRNA gene sequence data, strain Geo 8.1 belonged to the family Bacillaceae and genus Geobacillus, showing the highest similarity to Geobacillus kaustophilus NBRC 102,445^T^ (99.78%) and Geobacillus thermoleovorans KCTC 3570^T^ (99.78%). It is classified within the superkingdom Bacteria, kingdom Bacillati, phylum Bacillota, class Bacilli, and order Bacillales.
The genome length of Geobacillus Geo 8.1 was estimated to be approximately 3.41 Mbp, with a GC content of 52.5%. The genome assembly consists of 120 contigs, with an N50 value of 185,376 bp and an L50 value of 7. Quality assessment using CheckM indicated 100% completeness and 0.7% contamination. Both coarse (99.6) and fine (99) consistency values were high, confirming that the genome is of “good” quality (Table 1).
Table 1. General genomic properties of Geobacillus strain geo 8.1Taxonomy InfoAnnotation StatisticsTaxon ID129,337Number of tRNA genes93SuperkingdomBacteriaNumber of rRNA genes14KingdomBacillatiNumber of CDS (count)3751PhylumBacillotaCDS Ratio10.995.563ClassBacilliHypothetical CDS935OrderBacillalesHypothetical CDS Ratio0.37563315FamilyBacillaceaePLFAM CDS3502GenusGeobacillusPLFAM CDS Ratio0.9336177Genome StatisticsGenome QualityContigs120Coarse Consistency99.6Genome Length (bp)3,411,376Fine Consistency99GC Content (%)52.486.916CheckM Completeness (%)100Contig L507CheckM Contamination (%)0.7Contig N50185,376Genome QualityGood Annotation of the whole genome sequence revealed a total of 93 tRNA, 14 rRNA genes, 75 crispr repeat and 71 crispr spacer (Online Resource 1; Table S1, Table S2, Table S3). In addition, 3,751 coding sequences (CDS) were identified, of which 935 encode hypothetical proteins, representing approximately 24.9% of the total CDS (Online Resource 1; Table S5). Coding sequences assigned to any subsystem made up 47% of the total coding sequences (Online Resource 2; Table S6). Within family-specific classifications, 3,502 CDS were assigned to PLfam and 3,522 CDS to Pgfam (Table 1). Furthermore, the gene annotation analysis resulted in the presence of complete glycolysis, gluconeogenesis, Entner-Doudoroff, pentose phosphate pathways, TCA cycle, ethanol fermentation and genes encoding enzymes required for xylan degradation.
A circular map of the draft genome of Geobacillus strain Geo 8.1 was constructed to visualize its genomic organization. The map illustrates coding sequences on both forward and reverse strands, non-coding features, antimicrobial resistance genes, virulence factors, transporters, drug targets, as well as the distribution of GC content and GC skew across the genome (Fig. 1).
Fig. 1. The Circular Genome View Geobacillus strain Geo 8.1. The tracks on the viewer are displayed as concentric rings, from outermost to innermost: Position, Contigs/Chromosomes, CDS-forward, CDS-reverse, Non-CDS Features, GC Content, and GC Skew
Functional annotation assigned 2,815 proteins with known functions. Among these, 1,177 proteins were classified into subsystems, 723 into metabolic pathways, and 934 were associated with enzymatic activities (EC numbers). Additionally, 788 proteins were annotated with Gene Ontology (GO) terms, while 3,502 proteins were placed in PLfam and 3,522 proteins in PGfam families. A total of 935 hypothetical proteins were detected, whereas no proteins were linked to FIGfam classifications (Table 2).
Table 2. Protein features identified in Geobacillus strain geo 8.1Protein FeaturesProteins with functional assignments2816Proteins with Subsystem assignments1177Proteins with Pathway assignments723Proteins with PATRIC genus-specific family (PLfam) assignments3502Proteins with PATRIC cross-genus family (PGfam) assignments3522Proteins with GO assignments788Proteins with FIGfam assignments0Proteins with EC number assignments934Hypothetical proteins935
Several characteristic genes were identified in the genome of Geobacillus strain Geo 8.1. Thirty-one transporter genes were annotated based on the TCDB database. Furthermore, three metal resistance genes were detected according to the BacMet database. The TTD database identified four potential drug targets. Antibiotic resistance genes were found in both PATRIC (28 genes) and CARD (2 genes) databases. These features highlight the strain’s potential for environmental adaptation and its possible biotechnological applications (Table 3).
Table 3. Specialty genes detected in Geobacillus strain geo 8.1Specialty GenesSourceGenesTransporterTCDB31Metal ResistanceBacMet3Drug TargetTTD4Antibiotic ResistancePATRIC28Antibiotic ResistanceCARD2
The COG functional classification of coding sequences is summarized in Table 4. The largest proportion of genes (30.51%) was assigned to the category of unknown function. Genes related to information storage and processing accounted for 17.6% of the genome, mainly represented by replication, recombination and repair (6.52%), transcription (5.88%), and translation, ribosomal structure and biogenesis (5.13%).
Table 4. Number of genes associated with the general cluster of orthologous group (COG) functional categoriesCOG CategoryCOG Category DescriptionCDS%Cellular process and signalingDCell cycle control, cell division, chromosome partitioning551.59%MCell wall/membrane/envelope biogenesis1464.21%NCell motility772.22%OPost-translational modification, protein turnover, chaperones912.62%TSignal transduction mechanisms1323.81%UIntracellular trafficking, secretion, and vesicular transport521.50%VDefense mechanisms250.72%Information storage and processingBChromatin structure and dynamics10.03%JTranslation, ribosomal structure and biogenesis1785.13%KTranscription2045.88%LReplication, recombination and repair2266.52%MetabolismCEnergy production and conversion2035.85%EAmino acid transport and metabolism2848.19%FNucleotide transport and metabolism1243.58%GCarbohydrate transport and metabolism1855.33%HCoenzyme transport and metabolism1514.35%ILipid transport and metabolism1103.17%PInorganic ion transport and metabolism2065.94%QSecondary metabolites biosynthesis, transport and catabolism661.90%Poorly CharacterizedSFunction unknown105830.51%
Metabolism-related functions constituted a significant portion, with the highest representation in amino acid transport and metabolism (8.19%), followed by inorganic ion transport and metabolism (5.94%), energy production and conversion (5.85%), and carbohydrate transport and metabolism (5.33%). Genes involved in lipids, nucleotides, and coenzymes metabolism were also identified.
Within cellular processes and signaling, the most abundant categories included cell wall/membrane/envelope biogenesis (4.21%) and signal transduction mechanisms (3.81%). Additional categories such as cell motility (2.22%), defense mechanisms (0.72%), and intracellular trafficking (1.50%) were also represented.
Genome-based comparative and phylogenomic analysis of strain Geo 8.1
The genomic features of strain Geo 8.1 and representative Geobacillus species are summarized in Table 5, while the phylogenomic relationships inferred by TYGS are shown in Fig. 2. Geo 8.1 has a genome of 3,411,632 bp with a G + C content of 52.49% and 3,751 predicted coding sequences (CDSs). These values were most comparable to G. thermoleovorans KCTC 3570 (3,499,317 bp; 52.28% G + C; 3,298 CDSs) and G. kaustophilus NBRC 102,445 (3,446,385 bp; 51.99% G + C; 3,459 CDSs). By contrast, more distantly related genomes such as G. stearothermophilus ATCC 12,980 displayed smaller genome sizes (2,630,157 bp) and lower CDS counts (2,552), whereas G. proteiniphilus 1017 contained the highest number of CDSs (3,894). Notably, lower G + C contents were recorded in G. thermodenitrificans DSM 465 (49.05%) and G. thermodenitrificans subsp. calidus DSM 22,629 (48.94%). The outgroup genome Anoxybacillus geothermalis ATCC BAA2555 was clearly distinct, with a genome size of 6,975,976 bp, a lower G + C content (46.84%), and 7,055 proteins.
Table 5. Species and subspecies clustersTYGS IDPreferred NameStrainBase PairsPercent G + CNumber of proteinsBioproject AccessionBiosample AccessionAssembly Accession12,126 Geobacillus proteiniphilus 10173,548,45351,763894PRJNA353982SAMN06043560GCA_00190802512,880 Geobacillus zalihae NBRC 101,8423,528,23051,93519PRJDB415SAMD0045737GCA_00154413519,576 Geobacillus jurassicus NBRC 107,8293,447,57352,223470PRJDB428SAMD0045741GCA_00154431523,115 Geobacillus thermocatenulatus BGSC 93A13,563,80051,773350PRJNA224116SAMN06768585GCF_0022176553312 Geobacillus stearothermophilus ATCC 12,9802,630,15753,072552PRJNA212538SAMN03291237GCA_0012778053315 Geobacillus thermoleovorans KCTC 35703,499,31752,283298PRJNA310809SAMN04455789GCA_0016109554099 Geobacillus kaustophilus NBRC 102,4453,446,38551,993459PRJDB414SAMD00000360GCA_000739955U555179Geobacillus sp.Geo 8.13,411,63252,493751PRJNA1366803SAMN533246973317 Geobacillus subterraneus KCTC 39223,474,42652,23161PRJNA310054SAMN04445793GCA_0016186853639 Geobacillus icigianus G1w13,457,81052,033146PRJNA246135SAMN02850065GCA_0007500054270 Geobacillus thermodenitrificans DSM 4653,400,54649,053347PRJNA224955SAMN02386948GCA_0004965759482Geobacillus thermodenitrificans subsp. calidusDSM 22,6293,405,33348,943466PRJNA583236SAMN13173386GCA_0359845156255 Anoxybacillus geothermalis ATCC BAA25556,975,97646,847055PRJNA260743SAMN03025781GCA_000948315
Fig. 2. Phylogenetic tree inferred with FastME 2.1.6.1 (Lefort et al. 2015) using GBDP distances calculated from whole-genome comparisons. Branch lengths are scaled according to the GBDP distance formula d5, and the scale bar indicates substitutions per site. Numbers above branches represent GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 58.0%. The tree was rooted at the midpoint (Farris 1972). The coloured annotations on the right summarize genome-based metadata, including species and subspecies clusters defined by TYGS (dDDH thresholds of 70% and 79%, respectively), genomic G + C content (%), GBDP delta statistics, genome size (bp), predicted protein count, and SSU rRNA length (bp). The “User strain?” column marks the strain analysed in this study (Geobacillus sp. Geo 8.1), whereas the “Type species?” column indicates the type strains included in the analysis. For quantitative colour-coded annotations, darker shades indicate higher values
The TYGS-based phylogenomic tree (Fig. 2) confirmed the close relationship of strain Geo 8.1 with G. thermoleovorans and G. kaustophilus. These species formed a well-supported cluster, distinct from other Geobacillus representatives such as G. stearothermophilus and G. thermocatenulatus. Although G. zalihae clusters phylogenetically close to strain Geo 8.1 in the whole-genome tree (Fig. 2), its genomic similarity values remain below species delineation thresholds, and therefore it was not considered a primary reference for species-level assignment in this study.
Pairwise genome comparison values are provided in Table 6. Geo 8.1 displayed the highest digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values with G. thermoleovorans and G. kaustophilus. The dDDH values with these species exceeded the 70% threshold, and ANI values were above 95–96%, supporting species-level relatedness. Based on ANI and dDDH values exceeding species delineation thresholds, strain Geo 8.1 is not proposed as a novel species but represents a strain belonging to the G. thermoleovorans / G. kaustophilus species cluster. In contrast, other Geobacillus members such as G. stearothermophilus and G. thermocatenulatus showed dDDH values well below 50% and ANI values in the range of 85–90%, clearly separating them from Geo 8.1. G. thermodenitrificans DSM 465 and its subspecies calidus DSM 22,629 showed even lower similarities (< 40% dDDH; ANI < 85%), while Anoxybacillus geothermalis ATCC BAA2555 represented the most divergent taxon, with ANI values around 70% and negligible dDDH relatedness.
Table 6. Pairwise comparisons of user genomes vs. type strain genomesQuery StrainSubject StraindDDH (d0, %)C.I.(d0, %)dDDH (d4, %)C.I.(d4, %)dDDH(d6, %)C.I.(d6, %)G + C Content Difference (%)Geobacillus sp. Geo 8.1Geobacillus thermoleovorans KCTC 357083,9[80,1–87,7]91[88,8–92,8]88[85,0–90,4]0,21Geobacillus sp. Geo 8.1Geobacillus kaustophilus NBRC 102,44578,6[74,6–82,1]84,9[82,1–87,2]82,6[79,2–85,4]0,5Geobacillus sp. Geo 8.1Geobacillus proteiniphilus 101769,8[65,9–73,5]64,2[61,3–67,0]71[67,5–74,2]0,72Geobacillus sp. Geo 8.1Geobacillus zalihae NBRC 101,84271,3[67,4–75,0]62,9[60,0–65,7]72[68,5–75,2]0,59Geobacillus sp. Geo 8.1Geobacillus thermocatenulatus BGSC 93A171,5[67,6–75,2]54,1[51,4–56,8]69,9[66,4–73,1]0,71Geobacillus sp. Geo 8.1Geobacillus jurassicus NBRC 107,82966,8[63,0–70,5]41,9[39,4–44,4]61,9[58,6–65,1]0,27Geobacillus sp. Geo 8.1Geobacillus stearothermophilus ATCC 12,98058,5[54,8–62,0]38,8[36,3–41,3]54,1[51,0–57,2]0,58Geobacillus sp. Geo 8.1Anoxybacillus geothermalis ATCC BAA255530,1[26,7–33,7]37[34,6–39,5]29,9[27,0–33,0]5,65Geobacillus sp. Geo 8.1Geobacillus subterraneus KCTC 392253,4[50,0–56,9]31,3[28,9–33,8]47,3[44,2–50,3]0,29Geobacillus sp. Geo 8.1Geobacillus icigianus G1w143,4[40,0–46,8]30,5[28,2–33,0]39,5[36,5–42,5]0,46Geobacillus sp. Geo 8.1Geobacillus thermodenitrificans subsp. calidus DSM 22,62941,1[37,7–44,5]26,7[24,4–29,2]36,5[33,5–39,5]3,55Geobacillus sp. Geo 8.1Geobacillus thermodenitrificans DSM 46540,2[36,9–43,7]26,5[24,1–29,0]35,8[32,8–38,8]3,44
Together, the genomic features (Table 5), phylogenomic placement (Fig. 2), and similarity indices (Table 6) consistently support that strain Geo 8.1 belongs to the G. thermoleovorans/G. kaustophilus species cluster.
Pan/Core genome analysis
The pan-genome analysis of strain Geo 8.1 in comparison with other Geobacillus and Anoxybacillus representatives is summarized in Table 7. Geo 8.1 contained 3,468 CDSs, of which 3,271 were retained after removing artefact families. Within the pan-genome, Geo 8.1 contributed 1,957 CDSs to the core genome shared among all analyzed strains and 1,314 variable CDSs, including 168 strain-specific genes. The proportion of core genes in Geo 8.1 accounted for 59.83% of its genome, while variable and strain-specific fractions represented 40.17% and 5.14%, respectively.
Table 7. Pan-genome statistics of strain geo 8.1 compared with representative Geobacillus and Anoxybacillus species. The table summarizes the total coding sequences (CDSs), core genome size, variable and strain-specific CDSs, and their relative percentagesOrganismCDSCDS (w/o artefact fam.)Pan CDSCore CDSVar CDSStrain specific CDSCore CDS (%)VarCDS (%)Strain specific CDS (%)Geobacillus stearothermophilus ATCC 12,9803150296229621919104330564.78735.21310.297Geobacillus thermodenitrificans subsp calidus DSM 22,6293450325932591967129218560.35639.6445.677Geobacillus subterraneus KCTC 39223486327132711974129725860.34939.6517.887Geobacillus thermodenitrificans DSM 4653448326832681966130221960.15939.8416.701Geobacillus sp. Geo 8.13468327132711957131416859.82940.1715.136Geobacillus thermoleovorans KCTC 35703572335133511974137720558.90841.0926.118Geobacillus thermocatenulatus BGSC 93A13615341034101965144529257.62542.3758.563Geobacillus jurassicus NBRC 107,8293619339833981955144336057.53442.46610.594Geobacillus zalihae NBRC 101,8423672346234621977148529357.10642.8948.463Geobacillus icigianus G1w13724351435141974154052456.17543.82514.912Geobacillus proteiniphilus 10173762356035601977158332355.53444.4669.073Anoxybacillus geothermalis ATCC BAA255571676768676837423026127555.2944.7118.839Geobacillus kaustophilus NBRC 102,4453894364136411986165539054.54545.45510.711
Across the dataset, the number of CDSs varied between 3,150 in G. stearothermophilus ATCC 12,980 and 3,894 in G. kaustophilus NBRC 102,445, while A. geothermalis ATCC BAA2555 encoded a much larger gene set (7,167 CDSs). The fraction of core genes showed a decreasing trend from G. stearothermophilus (64.79%) to A. geothermalis (55.29%) with G. kaustophilus exhibiting the lowest proportion (54.55%), due to its highly divergent genome. In contrast, the proportion of variable and strain-specific genes was highest in G. icigianus G1w1 (43.83% variable; 14.91% strain-specific), G. kaustophilus (45.46% variable; 10.71% strain-specific), and A. geothermalis (44.71% variable; 18.84% strain-specific), reflecting their genomic plasticity.
Notably, Geo 8.1 clustered within the group of Geobacillus thermoleovorans KCTC 3570 and G. thermodenitrificans strains in terms of pan-genome composition, showing a similar balance between core and variable fractions. These findings suggest that Geo 8.1 shares a conserved genomic backbone with these species while maintaining a moderate set of unique genes that may contribute to strain-specific adaptations.
Xylanase activity and analysis of hydrolytic products
To assess the xylanolytic potential of strain Geo 8.1, crude enzyme extracts were prepared from cultures grown in both standard medium A and medium A supplemented with xylan as an inducer. The total protein content in the enzymatic supernatants was determined to be 1.52 mg/mL and 2.60 mg/mL for the standard and xylan-enriched media, respectively. Xylanase activity assays revealed that the presence of xylan in the culture medium significantly enhanced enzyme production, reaching 184 U/mL in the crude extract, with a corresponding specific activity of approximately 71 U/mg protein. In contrast, the extract from cultures grown without xylan exhibited an 88% decrease in activity, indicating that xylan acts as a strong inducer of xylanase expression in Geo 8.1.
To elucidate the enzymatic mechanism and identify the hydrolytic products, birchwood xylan was incubated with the crude enzyme extract at 65 °C, and reaction products were analyzed by thin-layer chromatography (TLC). During the early phase of hydrolysis (20 min), a complex mixture of xylo-oligosaccharides larger than xylotriose was observed, suggesting endo-type cleavage of the xylan backbone. After prolonged incubation (24 h), xylose appeared as the major end product, suggesting the presence of β-xylosidase enzyme activity that completes the saccharification process (Fig. 3).
Fig. 3. Thin-layer chromatographic (TLC) analysis of hydrolytic products obtained from crude enzyme extracts of Geo 8.1 incubated with birchwood xylan at 65 °C. Lane 1: reaction products after 20 min; lane 2: reaction products after 24 h; lanes 3–8: xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexose standards, respectively. The results show the progressive conversion of xylan-derived oligosaccharides to xylose, indicating the presence of both endo-xylanase and β-xylosidase activities
Taken together, these findings indicate that strain Geo 8.1 harbors a complete xylanolytic system comprising at least two synergistic enzymes: (i) an endo-1,4-β-xylanase responsible for the initial cleavage of the xylan polymer into shorter oligosaccharides, and (ii) a β-xylosidase that further hydrolyzes these intermediates into xylose monomers. The combined action of these enzymes enables efficient xylan depolymerization under thermophilic conditions, highlighting the strain’s potential for the bioconversion and valorization of lignocellulosic substrates.
Additionally, genome-based pathway reconstruction revealed the complete D-xylose utilization pathway in strain Geo 8.1 (Fig. 4). Xylan is initially degraded to D-xylose by xylanases (EC 3.2.1.37; Geo8.1peg.712,719,730) and β-xylosidases (EC 3.2.1.8; Geo8.1.peg.713,714). D-xylose is then transported into the cell through an ATP-binding cassette (ABC) transporter system (substrate-binding component Geo8.1.peg.707; ATP-binding component Geo8.1.peg.709). Intracellularly, D-xylose is isomerized to D-xylulose (EC 5.3.1.5; Geo8.1.peg.731), phosphorylated to D-xylulose-5-phosphate (EC 2.7.1.17; Geo8.1.peg.732), and subsequently converted into intermediates such as D-ribulose-5-phosphate (EC 5.1.3.4; Geo8.1.peg.690) and D-glyceraldehyde-3-phosphate (EC 2.2.1.1; Geo8.1.peg.1453), linking xylan degradation to the pentose phosphate pathway and glycolysis. These genomic insights support the experimental data and demonstrate that strain Geo 8.1 possesses both extracellular and intracellular enzymatic machinery for complete xylan degradation and xylose metabolism. It should be emphasized that these findings are based on genome-based predictions and bioinformatic analyses. While they are highly indicative and provide valuable insights into the metabolic and biotechnological potential of strain Geo 8.1, experimental validation will be required to confirm these predicted functions.
Fig. 4. Proposed xylan degradation and D-xylose catabolic pathway in Geo 8.1. Enzymes and their corresponding EC numbers are indicated along the pathway, and Geo 8.1 locus tags are provided in parentheses. The scheme illustrates the enzymatic steps from extracellular xylan hydrolysis to intracellular D-xylose catabolism via the pentose phosphate pathway and glycolysis
Features of industrial and biotechnological interest
Genome annotation of strain Geo 8.1 revealed a diverse repertoire of enzymes associated with industrially relevant bioprocesses, emphasizing its potential as a multifunctional thermophilic biocatalyst. Several genes encode hydrolases and oxidoreductases that play key roles in bioremediation, biomass conversion, and industrial catalysis (Table 8).
Table 8. Annotated enzymes of geo 8.1 with potential industrial and biotechnological relevance. Each enzyme is listed with its functional category, representative EC number, and corresponding coding sequence (CDS)EnzymeCDSBioremediation of chlorinated organic compounds2-haloalkanoic acid dehalogenase (EC 3.8.1.2)Geo8.1.peg.1101Hydrolase, haloacid dehalogenase-like familyGeo8.1.peg.520Fat hydrolysis, biodiesel production, detergent additive, food flavoringsBacterial patatin-like phospholipase domain containing proteinGeo8.1.peg.1215Lipase/Acylhydrolase with GDSL-like motifGeo8.1.peg.850LipaseGeo8.1.peg.592lipase/acylhydrolase family proteinGeo8.1.peg.729Lipase/Acylhydrolase with GDSL-like motifGeo8.1.peg.1593Uncharacterized lipase YqhOGeo8.1.peg.276Hydrogen peroxide removal, food preservation, textile bleachingCatalase-peroxidase KatG (EC 1.11.1.21)Geo8.1.peg.1657Manganese catalase (EC 1.11.1.6)Geo8.1.peg.1005Manganese catalase (EC 1.11.1.6) = > Spore coat protein CotJCGeo8.1.peg.106Molecular biology, diagnostic kitsAlkaline phosphatase (EC 3.1.3.1)Geo8.1.peg.1753, 3057Alkaline phosphatase like proteinGeo8.1.peg.2208, 3022Alkaline phosphatase synthesis transcriptional regulatory protein PhoPGeo8.1.peg.1786Protein hydrolysis, detergent formulation, leather processing, food technologyATP-dependent Clp protease ATP-binding subunit ClpXGeo8.1.peg.1694ATP-dependent Clp protease proteolytic subunit ClpP (EC 3.4.21.92)Geo8.1.peg.1032ATP-dependent Clp protease, ATP-binding subunit ClpCGeo8.1.peg.3212ATP-dependent Clp protease, ATP-binding subunit ClpEGeo8.1.peg.1912ATP-dependent hsl protease ATP-binding subunit HslUGeo8.1.peg.1324ATP-dependent protease La (EC 3.4.21.53) LonB Type IGeo8.1.peg.1693ATP-dependent protease La (EC 3.4.21.53) Type IGeo8.1.peg.1692ATP-dependent protease subunit HslV (EC 3.4.25.2)Geo8.1.peg.1323CAAX amino terminal protease family proteinGeo8.1.peg.2692Carboxyl-terminal proteaseGeo8.1.peg.1158Carboxyl-terminal protease (EC 3.4.21.102)Geo8.1.peg.1065Cell division-associated, ATP-dependent zinc metalloprotease FtsHGeo8.1.peg.3346ClpCP protease substrate adapter protein MecAGeo8.1.peg.102, 2051Transglutaminase-like enzymes, putative cysteine proteasesGeo8.1.peg.2765Zinc proteaseGeo8.1.peg.1400Intracellular proteaseGeo8.1.peg.2333Intramembrane protease RasP/YluC, implicated in cell division based on FtsL cleavageGeo8.1.peg.1366Lon-like protease with PDZ domainGeo8.1.peg.1216Membrane protease family protein BA0301Geo8.1.peg.2404ProteaseGeo8.1.peg.2246Protease IVGeo8.1.peg.1844Protease production regulatory protein Hpr (ScoC)Geo8.1.peg.2152Protease PrsWGeo8.1.peg.98Protease synthase and sporulation negative regulatory protein PAI 2Geo8.1.peg.2617Putative membrane protease YugPGeo8.1.peg.2219Putative serine proteaseGeo8.1.peg.2726Secreted protease metal-dependent proteaseGeo8.1.peg.781Serine protease AprXGeo8.1.peg.3037Serine protease, DegP/HtrA, do-like (EC 3.4.21.-)Geo8.1.peg.3293SOS-response repressor and protease LexA (EC 3.4.21.88)Geo8.1.peg.1448Sporulation-specific protease YabGGeo8.1.peg.3375Stage II sporulation protein related to metaloproteases (SpoIIQ)Geo8.1.peg.2804Uncharacterized membrane zinc metalloprotease YwhCGeo8.1.peg.3123Uncharacterized protease YrrNGeo8.1.peg.420Uncharacterized protease YrrOGeo8.1.peg.419Uncharacterized zinc protease YmfHGeo8.1.peg.1401Starch hydrolysis, high-fructose syrup production, bioethanol productionalpha-amylaseGeo8.1.peg.1166alpha-amylase (EC 3.2.1.1) @ Pullulanase (EC 3.2.1.41)Geo8.1.peg.1164
Enzymes involved in bioremediation of halogenated compounds, including 2-haloalkanoic acid dehalogenase (EC 3.8.1.2; Geo8.1.peg.1101) and a haloacid dehalogenase-like hydrolase (Geo8.1.peg.520), suggest the strain’s capacity to degrade chlorinated organics, making it a promising candidate for detoxification processes in contaminated environments (Stockbridge and Wackett 2024).
The genome also encodes multiple lipolytic enzymes—such as GDSL-type lipases (Geo8.1.peg.850, Geo8.1.peg.1593), patatin-like phospholipase (Geo8.1.peg.1215), and uncharacterized lipase YqhO (Geo8.1.peg.276)—that could serve in biodiesel production, detergent formulation, and food flavor enhancement. These thermostable lipases are particularly valuable for high-temperature industrial reactions, where conventional enzymes often lose activity (Hamdan et al. 2021).
Oxidative stress response enzymes, including catalase-peroxidase KatG (EC 1.11.1.21; Geo8.1.peg.1657) and manganese catalases (EC 1.11.1.6; Geo8.1.peg.1005, Geo8.1.peg.106), enable hydrogen peroxide detoxification and have potential uses in food preservation, environmental cleaning, and textile bleaching applications (Khan 2025).
Genes encoding alkaline phosphatases (EC 3.1.3.1; Geo8.1.peg.1753, Geo8.1.peg.3057) and their regulatory components (PhoP; Geo8.1.peg.1786) highlight the strain’s relevance for molecular biology and diagnostic reagent production, particularly under alkaline conditions compatible with thermophilic processes.
A large group of proteolytic enzymes was also identified, including ATP-dependent Clp, Hsl, and Lon proteases, serine proteases (AprX, DegP/HtrA), and several uncharacterized metalloproteases. These enzymes are associated with protein turnover, stress tolerance, and sporulation, but they also hold industrial potential in detergent formulations, leather processing, and food technology due to their thermostability and broad substrate range (Song et al. 2023).
Furthermore, Geo 8.1 possesses enzymes involved in polysaccharide degradation, such as α-amylase (EC 3.2.1.1; Geo8.1.peg.1166) and pullulanase (EC 3.2.1.41; Geo8.1.peg.1164), which are crucial for starch liquefaction, saccharification, and bioethanol production. The presence of both α-amylase and debranching pullulanase indicates a complete enzymatic toolkit for starch hydrolysis under thermophilic conditions, providing opportunities for biofuel and sweetener industries.
Overall, the enzymatic inventory of strain Geo 8.1 demonstrates a remarkable metabolic versatility, with catalytic activities spanning lipid, protein, and carbohydrate processing as well as oxidative and halogenated compound detoxification. This broad enzymatic capacity underscores its potential as a thermophilic biocatalyst for diverse biotechnological and industrial applications.
Discussion
The combined genomic and biochemical findings presented in this study reveal Geobacillus sp. Geo 8.1 as a robust thermophilic bacterium with exceptional enzymatic and metabolic versatility, reflecting the ecological and functional diversity of Geobacillus species inhabiting extreme geothermal environments. Isolated from a submarine hydrothermal vent of Ischia Island, this strain not only adapts to high temperature and moderate salinity but also exhibits a genome organization and enzymatic profile indicative of strong industrial potential. Its affiliation within the G. thermoleovorans/G. kaustophilus complex—supported by both high digital DNA–DNA hybridization and ANI values (dDDH > 70%, ANI > 95%)—places Geo 8.1 among well-characterized thermophiles known for producing thermostable biocatalysts (Brumm et al. 2015; Meier-Kolthoff and Göker 2019). Based on ANI and dDDH values exceeding species delineation thresholds, strain Geo 8.1 is not proposed as a novel species but represents a strain belonging to the G. thermoleovorans / G. kaustophilus species cluster.
The draft genome of Geo 8.1 (3.41 Mbp, 52.5% GC) exhibits a balanced representation of core metabolic modules and specialized functional systems. Approximately 47% of the coding sequences were assigned to subsystems encompassing glycolysis, the TCA cycle, pentose phosphate, and D-xylose catabolism, underscoring a well-integrated central metabolism typical of metabolically versatile thermophiles. Pan-genome comparison with 12 representative Geobacillus species revealed that Geo 8.1 shares 59.8% core genes while maintaining 168 strain-specific CDSs, suggesting moderate genomic plasticity and niche-specific adaptation. The pan/core genome analysis further provides insights into the genomic basis of adaptation of strain Geo 8.1 to thermophilic environments. Although Geo 8.1 shares a substantial core genome with other members of the Geobacillus genus, the presence of strain-specific and variable genes suggests functional diversification linked to environmental fitness. Notably, several genes retained in the accessory genome are associated with stress response, protein turnover, chaperone systems, and regulatory functions, which are commonly implicated in thermophilic adaptation. Such features are consistent with the selective pressures encountered in hydrothermal habitats, including elevated temperatures, fluctuating redox conditions, and nutrient limitation. Therefore, the comparative pan-genome framework not only highlights genomic conservation within the G. thermoleovorans / G. kaustophilus species cluster but also emphasizes strain-level adaptations that may contribute to the ecological resilience and biotechnological relevance of Geo 8.1.The presence of metal-resistance determinants, oxidative stress response genes, and numerous transporters (TCDB; BacMet databases) likely contributes to its survival in dynamic submarine environments characterized by steep redox and ionic gradients (Bucci et al. 2011). Such adaptive features parallel those observed in other geothermal isolates, including Parageobacillus thermantarcticus strain M1 (Yasar Yildiz et al. 2022) and Brevibacillus thermoruber strain 423 (Yildiz et al. 2015), highlighting convergent evolution toward metabolic resilience in extreme niches.
Comparative evaluation of genomic properties places Geobacillus sp. Geo 8.1 well within the genomic landscape of the genus. The genome size, GC content, and number of coding sequences are comparable to those reported for other thermophilic Geobacillus species, particularly members of the G. thermoleovorans / G. kaustophilus species complex. Likewise, the predicted functional repertoire of Geo 8.1 reflects hallmark features of the genus, including an abundance of genes related to carbohydrate metabolism, stress response, chaperone systems, and protein turnover, which are commonly associated with thermophilic lifestyles. Notably, the presence and diversity of genes involved in xylan degradation and associated carbohydrate-active enzymes highlight a functional specialization consistent with, yet distinct from, related Geobacillus strains. Together, these comparisons suggest that while Geo 8.1 conforms to the conserved genomic framework of the genus, strain-level variations in predicted functions may underpin its ecological adaptation and biotechnological potential.
One of the defining features of strain Geo 8.1 is its broad enzymatic repertoire spanning carbohydrate, lipid, and protein hydrolysis pathways. Functional annotation revealed genes encoding α-amylases, pullulanases, lipases, proteases, catalases, and dehalogenases—many of which are central to high-temperature biotransformations. The apparent discrepancy between phenotypic assays and genome annotation likely reflects condition-dependent gene expression. Although catalase and protease-encoding genes were identified in the genome, their expression may be inducible or regulated by specific environmental or stress-related conditions not tested in the plate assays. Moreover, several annotated proteases are intracellular or regulatory enzymes that do not necessarily confer detectable caseinolytic activity on agar plates. The coexistence of hydrolases and oxidative enzymes reflects a dual ecological role: nutrient recycling and detoxification of oxidized or halogenated compounds. The detection of haloacid dehalogenases (Geo8.1.peg.520, 1101) supports a capacity for biodegradation of chlorinated organics, in line with the natural geochemical composition of hydrothermal environments rich in halides (Stockbridge And Wackett 2024). Similar genomic signatures have been reported in Thermomonas hydrothermalis (Yaşar Yıldız 2025), underscoring a recurring pattern of environmental adaptation across thermophilic lineages.
The occurrence of multiple GDSL-type lipases (Geo8.1.peg.850, 1593) and patatin-like phospholipases (Geo8.1.peg.1215) implies potential applications in biodiesel synthesis, detergent formulation, and lipid modification—industrial domains that favor thermotolerant, solvent-resistant enzymes (Hamdan et al. 2021). In parallel, the identification of Clp, Hsl, and Lon proteases, as well as serine proteases (AprX, DegP/HtrA), indicates both stress-management functions and potential utility in detergents, leather processing, and food biotechnology (Song et al. 2023). The combination of thermostability, oxidative resilience, and catalytic diversity thus positions strain Geo 8.1 as a multi-enzyme producer suitable for integrated bioprocesses operating under harsh physicochemical conditions.
The xylanase activity of Geo 8.1 represents one of the most remarkable biochemical traits observed among Geobacillus spp. The crude extracellular enzyme preparation exhibited a xylanase activity of 184 U/mL at 65 °C and pH 7.0, corresponding to a specific activity of approximately 71 U/mg protein. This activity level is markedly higher than those previously reported for G. thermoleovorans (~ 2.1 U/mL; (Verma and Satyanarayana 2012) and Geobacillus sp. DUSELR13 (6–31 U/mL after optimization; (Bibra et al. 2018). Compared with the highly alkaline xylanases from G. thermodenitrificans A333 (pH 9.0; (Marcolongo et al. 2015) or G. thermoleovorans (pH 8.5; (Verma And Satyanarayana 2012), Geo 8.1’s neutral pH optimum broadens its usability in industrial applications that require compatibility with mild process conditions and mixed enzyme systems.
The 88% increase in activity upon xylan induction reflects a tightly regulated operon structure analogous to the xylan-responsive systems described in G. stearothermophilus T6 and G. thermodenitrificans TSAA1 (Anand et al. 2013). Genome mapping confirmed that endo-1,4-β-xylanase (Geo8.1.peg.712–730) and β-xylosidase (Geo8.1.peg.713–714) genes are clustered with an ABC-type D-xylose transporter and catabolic enzymes, forming a complete D-xylose utilization pathway (Geo8.1.peg.707–732). This genetic organization is consistent with the modular carbohydrate-active enzyme (CAZyme) architecture conserved across Geobacillus genomes (Brumm et al. 2015; Carbonaro et al. 2024). TLC analysis corroborated this genomic evidence, showing sequential degradation of birchwood xylan into xylo-oligosaccharides and ultimately xylose, confirming synergistic endo- and exo-activity as reported in other thermophilic systems (Bhalla et al. 2014; Mendonça et al. 2023). At this stage, the enzymatic characterization should be regarded as exploratory, aimed at demonstrating functional xylanolytic potential rather than providing full biochemical optimization. Further studies addressing temperature and pH profiles, enzyme stability, and kinetic parameters will be required to fully assess industrial applicability.
Thermostable xylanases are pivotal in biofuel production, pulp and paper biobleaching, and feed enhancement, as their high-temperature tolerance accelerates hydrolysis, reduces viscosity, and minimizes microbial contamination (Ajeje et al. 2021; Kumar et al. 2018). The strong activity of Geo 8.1 at 65 °C and neutral pH aligns well with operational parameters in enzymatic saccharification and paper pulping. Moreover, the co-existence of α-amylase and pullulanase genes (Geo8.1.peg.1164, 1166) enables dual carbohydrate hydrolysis suitable for starch liquefaction and bioethanol production. The encoded catalase-peroxidase (KatG) and manganese catalases further enhance process robustness by protecting enzymatic formulations from oxidative inactivation (Khan 2025).
From a systems biology perspective, Geo 8.1’s genome provides a modular chassis for synthetic biology and metabolic engineering efforts aimed at consolidated bioprocessing. The coupling of hemicellulose degradation with intracellular D-xylose assimilation enables the direct conversion of lignocellulosic feedstocks into fermentable sugars or bio-based chemicals. Heterologous expression of Geo 8.1 hydrolases or reconstruction of its xylanase cluster in mesophilic hosts could enhance enzyme yield and simplify downstream processing. Future work integrating transcriptomic, proteomic, and structural studies will be essential to elucidate the regulatory networks governing carbohydrate and lipid metabolism and to identify thermostability determinants for rational protein engineering.
Overall, Geobacillus sp. Geo 8.1 represents high-performing thermophilic biocatalyst combining strong xylanolytic activity, a wide enzymatic portfolio, and genomic features conducive to environmental resilience. Compared with previously characterized Geobacillus strains (Carbonaro et al. 2024; Marcolongo et al. 2015; Verma and Satyanarayana 2012), Geo 8.1 exhibits one of the most balanced profiles of catalytic efficiency, thermostability, and neutral-pH functionality reported so far. Its genomic configuration provides a blueprint for exploring novel thermozymes and engineering sustainable bioprocesses aimed at lignocellulose valorization, bioremediation, and green chemical synthesis.
Supplementary Information
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The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1Irfan M, Güler Hİ, Shah AA, Sal FA, Inan K, Beldüz A (2016) Cloning, purification and characterization of halotolerant xylanase from Geobacillus thermodenitrificans C 5. JMBFS 5(6). 10.15414/jmbfs.2016.5.6.523-529