Genomic and Biodegradation Potential of Bacillus altitudinis DG4 for Naphthalene Removal from Contaminated Environments

doi:10.21203/rs.3.rs-6271957/v1

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Genomic and Biodegradation Potential of Bacillus altitudinis DG4 for Naphthalene Removal from Contaminated Environments

https://doi.org/10.21203/rs.3.rs-6271957/v1

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Background: Polycyclic aromatic hydrocarbon contamination presents significant environmental challenges, requiring effective bioremediation solutions.

Methods: Bacillus altitudinisDG4, isolated from the Damanganga River in Vapi, India, was investigated for its genomic characteristics and naphthalene degradation capabilities using whole genome sequencing and Gas Chromatography-Mass Spectrometry (GC-MS) analysis.

Results: Genome sequencing revealed a 3,831,796 base pair genome with 4,120 protein-coding sequences. Genome annotation identified genes involved in diverse metabolic pathways including xenobiotic degradation. Pan-genome analysis of 122 B. altitudinisgenomes revealed 2,403 core genes, 1,588 accessory genes, and 40 unique genes in strain DG4. Naphthalene degradation experiments showed that strain DG4 exhibited the highest degradation efficiency (90.0%) among five isolates tested, despite a moderate growth rate. GC-MS analysis confirmed the metabolism of naphthalene and formation of key intermediates, including 1-naphthalenol, 1,8-naphthalic anhydride, 1-acenaphthanone, and benzoic acid, suggesting a specific degradation pathway.

Conclusion: The presence of genes associated with degradation pathways and the experimental validation of naphthalene degradation highlight the strong bioremoval potential of B. altitudinis DG4 for naphthalene-contaminated environments. These findings contribute to understanding microbial degradation mechanisms and support the development of effective bioremediation strategies for polycyclic aromatic hydrocarbon pollution.

Bacillus altitudinis DG4

genome sequencing

Damanganga River

naphthalene degradation

environmental cleanup

Polycyclic aromatic hydrocarbons (PAHs), including naphthalene, are ubiquitous environmental pollutants that pose a significant risk to ecosystems and human health. These compounds are known for their toxicity, with many PAHs being acutely toxic, mutagenic, or carcinogenic [1]. Among the 16 priority compounds of special concern owing to their toxicological effects, the US Environmental Protection Agency has listed naphthalene, along with pyrene and phenanthrene [2]. PAHs can contaminate various environmental matrices, including freshwater ecosystems, sediments, and aquifers [3] Their presence in surface waters, particularly urbanized rivers and drains, has been documented to vary significantly across locations and seasons [4]. Interestingly, the environmental fate of PAHs can differ under both aerobic and anaerobic conditions. Although many PAHs are known to biodegrade under aerobic conditions, most contaminated sediments are anaerobic, which can limit their degradation [5]. Recent advances in understanding the anaerobic degradation of PAHs, including naphthalene, have opened new avenues for assessing their fate in anoxic environments [3]. Bioremediation, including surfactant-mediated biodegradation, has emerged as a promising approach for eliminating PAHs from contaminated sites [6]. However, the environmental impact of PAHs remains a significant concern, particularly in areas with high urban emissions and industrial activity [4]. Continued research and monitoring of PAHs in various environmental matrices are crucial for developing effective strategies to mitigate their impact on ecosystems and human health.

Microbial degradation has emerged as a significant approach for pollution control, offering an eco-friendly and effective solution for environmental contamination caused by xenobiotics and other pollutants. Microorganisms possess unique metabolic capabilities, diverse enzymes, and various degradation pathways that enable them to transform contaminants into non-toxic forms, thereby reducing environmental pollution [7]. Microbial degradation is particularly important for addressing the growing concern regarding microplastic pollution in aquatic environments. Microorganisms form biofilms on the surfaces of pollutants, creating a region known as the plastisphere, where they interact and produce acids and enzymes for microplastic degradation [8]. This natural process has a significant impact on the reduction of plastic waste in the environment. Microbial degradation is also crucial in the bioremediation of various pollutants, including hexachlorocyclohexane (HCH) residues in the soil. Different bacterial and fungal strains have been reported to degrade HCH isomers via complex metabolic pathways, including reductive dechlorination, hydrolysis, and ring cleavage [9]. Similarly, microbial enzymatic degradation has shown promise for breaking down biodegradable plastics, offering a solution that does not result in waste accumulation [10]. The significance of microbial degradation extends to the treatment of toxic organic compounds such as cyanide, where enhanced biological degradation has been demonstrated both in laboratory and field conditions [11]. Additionally, microbial degradation plays a crucial role in the breakdown of pesticides, such as glyphosate, offering an eco-friendly method for their removal from the environment [12]. Its effectiveness, coupled with its environment-friendly nature, makes it a promising approach for sustainable pollution management and ecosystem restoration.

These findings highlight the potential of various microorganisms for naphthalene bioremediation under different environmental conditions. Bacillus species have shown promising potential for the degradation of polycyclic aromatic hydrocarbons (PAHs), including naphthalene. Although several studies have investigated the naphthalene degradation capabilities of various Bacillus strains, there is limited genomic information specifically on Bacillus altitudinis for PAH degradation [13], [14].

Recent studies have highlighted the diverse microbial capabilities of naphthalene, which is a key environmental pollutant. For instance, Pseudomonas sp. strain SA3 demonstrated high efficiency, achieving 98.74% degradation within 96 h under optimized conditions [15]. In contrast, Pseudomonas sp. LBKURCC149 showed a moderate degradation of 13.95% after seven days of glucose supplementation [16]. Environmental microcosms exhibited mineralization half-lives ranging from 2.4 to 4.4 weeks, reflecting variability in natural ecosystems [17]. Additionally, Coelastrella saipanensis achieved 100% degradation within 12 days, whereas Bacillus thermoleovorans Hamburg 2 effectively degraded naphthalene under thermophilic conditions [18], [19].

Interestingly, some Bacillus strains have demonstrated a high naphthalene tolerance and degradation efficiency. For instance, Bacillus cereus 28BN was reported to degrade 72 ± 4% of naphthalene after 20 days of incubation [20]. Similarly, Bacillus licheniformis JUG GS2 and Bacillus sonorensis JUG (RS2(3)) showed naphthalene degradations of 73% and 52%, respectively, at a concentration of 100 mg/L [14]. These findings highlight the potential of Bacillus species for field applications in PAH remediation. Although genomic insights into the role of B. altitudinis strains in PAH degradation are limited, recent studies have provided valuable information on other aspects of this species. For example, genome sequencing of B. altitudinis GLB197 revealed the presence of a non-ribosomal peptide synthetase (NRPS) gene cluster, which may contribute to its biocontrol properties [21]. Additionally, proteomic studies have identified diverse lysine modifications in B. altitudinis under salt stress, providing insights into its adaptation mechanisms [22]. These genomic and proteomic approaches can be extended to study B. altitudinis strains with PAH degradation capabilities, potentially addressing the research gap and identifying strains with high naphthalene tolerance for field applications.

2.1. Sample Collection

Water samples were collected from the Damanganga River, Vapi City, India (geolocation: 20.373680, 72.878786), as depicted in Fig. 1. Sampling was conducted at three different locations along the river to account for potential variations in the bacterial distribution. At each location, water samples were collected at a depth of approximately 15 cm using sterile glass bottles (500 mL). The aseptic techniques were strictly followed during the sampling process to prevent contamination. Figure 1 shows the map of the sampling location (A) and a representative image of the Damanganga River sampling site (B) where Bacillus altitudinis DG4 was isolated.

2.2. Isolation and Purification of Bacillus altitudinis

The Bushnell Hass (BH) medium was used for enrichment and isolation. The composition of BH medium (per liter) was as follows: 0.2 g MgSO₄.7H₂O, 0.02 g CaCl₂. 2H₂O, 1.0 g KH₂PO₄, 1.0 g K₂HPO₄, 1.0 g NH₄NO₃, 0.05 g FeCl₃ and trace elements (1 ml/l). For the preparation of solid culture media, 15 g agar per liter was added to the above media and spilled into plates. Subsequently, 200 mg/l of naphthalene (Mol. wt. 178.2, HiMedia with ≥ 98% purity) were dissolved antecedently in 0.1mL acetone and added to the medium. After perfect evaporation of acetone, 5 g of soil or 5 ml of water samples were added to the BH medium, and the flasks were placed in a shaker incubator (120rpm, Patel Scientific, India) at 37°C for 7days. Then 5 ml aliquots were transferred to fresh medium. After a series of two passages, inoculants from the flask were streaked, and phenotypically, various colonies purified on BH-agar medium. Phenotypically, various colonies prepared from the plates were transferred to fresh media containing (200 mg/L) naphthalene to delete agar-consuming bacteria. Finally, the isolates showing significant growth on naphthalene were stored in stock media with glycerol at -20°C.

2.3. Naphthalene Biodegradation Experiment for Selection of the Efficient Bacterium

To select the most efficient naphthalene degradation, all five organisms (DG1, DG2, DG3, DG4, and DG5) were inoculated for seven days in 250 ml Erlenmeyer flasks containing 100 ml BH media containing 200ppm of concentration of naphthalene, the incubation temperature was 37°C, and agitation speed was maintained at 120 rpm. After the seventh day of incubation, naphthalene degradation efficiency was calculated as a percentage.

2.4. Extraction of Genomic DNA and Whole Genome Sequencing of Strain Bacillus altitudinis DG4

Genomic DNA (gDNA) was extracted and purified using a HiPurA® Bacterial Genomic DNA Purification Kit (HiMedia Biochemicals, Maharashtra, India) according to the manufacturer's instructions. The quality of the gDNA was determined by agarose gel electrophoresis and spectrophotometry.

DNA quality and concentration were assessed using a Qubit Fluorometer in conjunction with the Qubit dsDNA HS Assay (Thermo Fisher Scientific). Subsequently, paired-end genomic libraries for each isolate were constructed using the Nextera DNA Flex Library Preparation Kit (Illumina, San Diego, CA, United States). Sequencing was carried out using the MiSeq Reagent Kit v2 (2 × 250 base pairs [bp]) on the Illumina MiniSeq platform (Illumina, San Diego, CA, United States).

2.5. Genome Annotation Function and Prediction

2.5.1. Comprehensive Genome Analysis using BV-BRC

The assembled genome of Bacillus altitudinis DG4 was analyzed using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) platform (https://www.bv-brc.org/). BV-BRC provides a suite of tools for comprehensive genome analysis, including annotation, comparative genomics, and functional analyses[23].

Data Upload and Genome Annotation: The assembled genome of B. altitudinis DG4 was uploaded to the BV-BRC platform. Genome annotation was performed using the RAST (Rapid Annotations using Subsystems Technology) pipeline available on BV-BRC, which identifies coding sequences, ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and other genomic features.

Comparative Genomics: Comparative genomic analysis was conducted by selecting related Bacillus species from the BV-BRC database. The comparison involved identifying orthologous genes, synteny analysis, and phylogenetic analysis using tools such as the BV-BRC Genome Alignment Service and Phylogenetic Tree Services.

Functional Analysis: Functional annotation of the genome was performed using the BV-BRC Functional Annotation Service. This service assigns functional roles to genes based on subsystem categorization, allowing for the identification of pathways and metabolic capabilities.

2.5.2. Pan-Genome Analysis

The assembled genome of Bacillus altitudinis DG4 was analyzed within the context of the pan-genome of the species using the Integrated Prokaryotic Genome and Pan-genome Analysis (IPGA) service, version 1.09. The IPGA v1.09 tool facilitates comprehensive pan-genome analysis, including orthologous group clustering, gene presence/absence matrix construction, and core and accessory genome delineation[24].

For the comprehensive genome annotation of Bacillus altitudinis DG4, 122 assemblies were prepared and analyzed using Integrated Prokaryotic Genome Annotation (IPGA) tools. This approach allowed for the accurate identification and functional annotation of genes across different assemblies, providing a detailed insight into the genetic composition of Bacillus altitudinis DG4.

Analysis of the Bacillus altitudinis DG4 genome using IPGA tools and Panaroo revealed several key insights into the gene content and variability within this strain. These data highlight the presence or absence of various genes in different genomes.

3.1. Isolation and Morphological Characterization of Bacillus altitudinis DG4

The bacterial isolate obtained from the Damanganga River water samples displayed distinct colony morphologies on the nutrient agar plates. The colonies were medium to large (2–5 mm in diameter), circular with entire (smooth) edges, and exhibited raised or umbonate elevation. The surface appeared smooth and glistening with a moist or mucoid appearance. The colonies demonstrated a buttery or creamy texture with a soft and opaque consistency. The color ranged from cream to off-white or pale yellow with an opaque appearance and entire or smooth margins (Table 1).

Table 1

Morphological characteristics of Bacillus altitudinis DG4 colonies on nutrient agar.
Morphology	Characteristic
Size	Medium to large in size
Diameter	2–5 mm
Shape	Circular in shape with entire (smooth) edges
Elevation	Raised or umbonate elevation
Surface	Smooth and glistening, with a moist or mucoid appearance
Texture	Buttery or creamy
Consistency	Soft and opaque
Color	Cream, off-white or pale yellow
Opacity	Opaque
Margin	Entire or smooth

Biochemical tests were performed on Bacillus altitudinis DG4, which tested positive for catalase, methyl red, and citrate utilization, but negative for oxidase, indole production, and Voges-Proskauer tests. These results indicate that B. altitudinis DG4 possesses catalase activity, can utilize citrate as a sole carbon source, and follows the mixed acid fermentation pathway (Methyl Red positive), but does not produce oxidase, indole, or acetoin. Gram staining of the pure isolate confirmed its classification as a Gram-positive bacterium.

These observations are consistent with the known attributes of Bacillus species, which frequently demonstrate noticeable morphological traits that facilitate identification [25].

Existing knowledge regarding Bacillus altitudinis indicates its significance in various biotechnological and environmental applications. Studies have established that members of this genus possess diverse metabolic capabilities that are useful for industrial microbiology and bioremediation [26].

In comparison to other Bacillus species, B. altitudinis DG4 exhibited unique biochemical characteristics, notably catalase positivity and oxidase negativity. This finding aligns with the general classifications of the genus, where catalase activity is a common trait among Bacillus strains. Conversely, the absence of indole production in B. altitudinis DG4 presents a notable differentiation from some other Bacillus species, which are typically indole-positive [27]. These biochemical traits are important for further understanding metabolic pathways and ecological adaptations.

B. altitudinis DG4 possesses catalase activity and can utilize citrate as the sole carbon source, reinforcing its classification within the Bacillus genus. Such abilities suggest its potential biotechnological applications, particularly for degrading environmental pollutants or serving as a biocontrol agent in agriculture contexts [28].

3.2. Naphthalene Degradation by Bacillus altitudinis DG4

Among the five isolates, strain DG5 demonstrated in Table 2 the highest growth rate with an OD₆₀₀ value of 0.6070, indicating robust proliferation in the presence of naphthalene. However, the highest naphthalene degradation efficiency was observed for strain DG4, which removed 90.0% of the initial naphthalene concentration, despite exhibiting a moderate growth rate (OD₆₀₀ = 0.4241). This suggests that strain DG4 possesses highly efficient naphthalene-degrading enzymatic machinery.

Table 2

Growth rates and naphthalene degradation efficiencies of bacterial isolates after 7 days of incubation
No.	Organism	The growth rate (OD at λ600)	Napthelene degradation efficiency (%)
1	DG1	0.4092	80%
2	DG2	0.3476	79%
3	DG3	0.4343	82%
4	DG4	0.4241	90%
5	DG5	0.607	85%

Strain DG3 showed the second-highest degradation efficiency (82.0%) with a growth rate (OD₆₀₀ = 0.4343) comparable to DG4. Strain DG5, despite having the highest growth rate, achieved a naphthalene degradation efficiency of 85.0%, ranking third among the isolates. Strains DG1 and DG2 exhibited relatively lower degradation efficiencies of 80.0% and 79.0%, respectively, with DG2 showing the lowest growth rate (OD₆₀₀ = 0.3476) among all isolates.

Supporting this, the data presented in this study confirm that strain DG4 operates efficiently, even with a moderate growth profile, focusing its metabolic resources on the degradation of naphthalene [29]. These findings pave the way for further investigations focused on the practical application of this strain in the bioremediation of naphthalene-contaminated environments, although there remains a limitation due to the absence of in situ field trials to corroborate laboratory results under natural environmental conditions [25].

3.3. Genome Assembly and Annotation of Bacillus altitudinis DG4

Whole-genome sequencing of B. altitudinis DG4 yielded a high-quality genome assembly. The assembled genome consisted of 19 contigs with a total length of 3,831,796 base pairs (bp) and an average G + C content of 41.03%. The genome assembly displayed a Contig L50 of 2 and a Contig N50 of 987,862 bp, indicating robust assembly quality (Table 3). Plasmids were not identified within the genome. The complete genome sequence of Bacillus altitudinis strain DG4 is available in the European Nucleotide Archive (ENA) under project accession number PRJEB76590.0.

Table 3

Assembly statistics of Bacillus altitudinis DG4 genome
Assembly Parameter	Value
Contigs	19
GC Content	41.03%
Plasmids	0
Contig L50	2
Genome Length	3,831,796 bp
Contig N50	987,862
Chromosomes	0

Genome annotation using the RAST toolkit (RASTtk) assigned a unique genome identifier (293387.487) to B. altitudinis DG4. The taxonomic classification placed the organism in the superkingdom Bacteria, phylum Bacillota, class Bacilli, order Bacillales, family Bacillaceae, genus Bacillus, and genus Bacillus altitudinis.

Genome annotation revealed 4,120 protein-coding sequences (CDS), 66 transfer RNA (tRNA) genes, and 3 ribosomal RNA (rRNA) genes (Table 4). Among the protein-coding sequences, 1,017 were annotated as hypothetical proteins and 3,103 were assigned functional annotations. Of these, 942 proteins received Enzyme Commission (EC) number assignments, 787 had Gene Ontology (GO) assignments, and 695 were mapped to KEGG pathways. Additionally, 3,819 proteins were classified into PATRIC genus-specific protein families (PLFams), and 3,924 proteins were assigned to PATRIC cross-genus protein families (PGFams).

Table 4

Annotated genome features of Bacillus altitudinis DG4
Feature Type	Count
CDS	4,120
tRNA	66
Repeat Regions	5
rRNA	3
Partial CDS	0
Miscellaneous RNA	0
Hypothetical proteins	1,017
Proteins with functional assignments	3,103
Proteins with EC number assignments	942
Proteins with GO assignments	787
Proteins with Pathway assignments	695
Proteins with PATRIC genus-specific family (PLfam) assignments	3,819
Proteins with PATRIC cross-genus family (PGfam) assignments	3,924

3.4. Subsystem Analysis and Specialty Genes

The complete genomic features of B. altitudinis DG4 was elucidated and is preseanted as a circular genome map in Fig. 2, whereby showing the arrangement of coding sequences, RNA genes, and other genomic features. Subsystem analysis of the B. altitudinis DG4 genome revealed a distribution of functional categories across the genome (Fig. 3). The analysis identified various functional subsystems including those related to metabolism, cellular processes, and stress responses.

Genome analysis also identified several specialty genes, including those homologous to known transporters, virulence factors, drug targets, and antibiotic resistance genes (Table 5). Specifically, 63 genes showed homology to transporters in the Transporter Classification Database (TCDB), 41 genes were associated with antibiotic resistance in the PATRIC database, and 21 genes were identified as potential drug targets in DrugBank.

Table 5

Specialty genes identified in the Bacillus altitudinis DG4 genome.
Specialty Category	Source	Genes
Antibiotic Resistance	CARD	2
Antibiotic Resistance	NDARO	2
Antibiotic Resistance	PATRIC	41
Drug Target	DrugBank	21
Drug Target	TTD	1
Transporter	TCDB	63
Virulence Factor	PATRIC_VF	2
Virulence Factor	Victors	2

Previous studies have demonstrated the significance of Bacillus altitudinis in adapting to high-altitude environments, hinting at the genetic underpinnings that facilitate resilience. The role of specific genetic factors, including metabolic pathways involved in stress tolerance, underscores the ecological versatility [27].

Compared with other Bacillus species, B. altitudinis DG4 possesses a distinctive set of transporters and antibiotic resistance genes. Specifically, our analysis identified a significant number of genes associated with these traits, indicating functional niches that may not be present in closely related species. However, comprehensive comparative genomic analyses of specific reference genes are necessary to establish these distinctions [30].

Our detailed analysis revealed that 63 genes showed homology to transporters documented in the Transporter Classification Database (TCDB), aligning with metabolic and transport functionalities essential for nutrient acquisition. Additionally, 41 genes associated with antibiotic resistance were identified in the PATRIC database, whereas 21 genes designated as potential drug targets were identified in DrugBank. These findings provide substantial evidence for the enhanced functional capacity of B. altitudinis DG4 [31], [32].

This study provides novel insights into the genetic landscape of B. altitudinis DG4, particularly concerning its specialized genes and functional subsystems that contribute to its environmental adaptability and potential uses in biotechnology [33].

3.5. Antimicrobial Resistance Gene Analysis

Genome annotation identified several genes associated with antimicrobial resistance (Table 6). These included genes encoding antibiotic inactivation enzymes (CatA6 family, FosB), antibiotic target protection proteins (BcrC), antibiotic target replacement proteins (fabL), and efflux pumps conferring antibiotic resistance (BceA, BceB, EbrA, and EbrB).

Table 6

Antimicrobial resistance genes identified in Bacillus altitudinis DG4.
AMR Mechanism	Genes
Antibiotic inactivation enzyme	CatA6 family, FosB
Antibiotic target in susceptible species	Alr, Ddl, dxr, EF-G, EF-Tu, folA, Dfr, folP, gyrA, gyrB, inhA, fabI, Iso-tRNA, kasA, MurA, rho, rpoB, rpoC, S10p, S12p
Antibiotic target protection protein	BcrC
Antibiotic target replacement protein	fabL
Efflux pump conferring antibiotic resistance	BceA, BceB, EbrA, EbrB
Gene conferring resistance via absence	gidB
Protein altering cell wall charge conferring antibiotic resistance	GdpD, MprF, PgsA
Regulator modulating expression of antibiotic resistance genes	BceR, BceS, LiaF, LiaR, LiaS

3.6. Comparative Genome Analysis

This analysis further confirmed the taxonomic classification of the isolate and demonstrated its close relationship with other members of the Bacillus pumilus group, while showing clear separation from other Bacillus species such as B. anthracis and B. psychrosaccharolyticus. As shown in Fig. 4, this analysis positioned B. altitudinis DG4 within the Bacillus altitudinis clade, highlighted in red in the phylogenetic tree. Phylogenetic analysis based on the PATRIC global protein families (PGFams) positioned B. altitudinis DG4 within the Bacillus altitudinis clade (Fig. 5). This analysis further confirmed the taxonomic classification of these isolates.

3.7. Pan-Genome Analysis

The pan-genome analysis of B. altitudinis DG4, conducted using the Integrated Prokaryotic Genome and Pan-genome Analysis (IPGA) service with 122 B. altitudinis genomes, revealed a comprehensive view of the gene content and variability within this strain (Fig. 6). The analysis identified 2,403 core genes that were present across all genomes, indicating their essential roles in basic cellular functions and survival. Additionally, 1,588 accessory genes were identified, which were present only in a subset of the genomes, suggesting their involvement in adaptive functions. Notably, 40 unique genes were found to be specific to B. altitudinis DG4, potentially indicating recent acquisitions through horizontal gene transfer or unique adaptations.

Analysis of accessory genes in B. altitudinis DG4 revealed genes involved in diverse metabolic pathways, such as accA_1 (propanoate metabolism), accC_2 (biotin carboxylase), and acsA_1 (acetyl-coenzyme A synthetase). Notably, genes associated with degradation pathways, including acyP (1,4-dichlorobenzene degradation) and dhaT (naphthalene and anthracene degradation), were identified, suggesting their potential xenobiotic degradation capabilities. Additionally, genes for various transport systems (btuD_1 and btuD_6) and transcriptional regulators (gabR_1 and rhaS_4) were present, indicating complex gene regulation mechanisms and nutrient acquisition systems (Fig. 6).

Functional Analysis successfully identified key accessory genes, including accA_1, accC_2, and acsA_1, which are implicated in a range of metabolic pathways. Understanding these genes is vital because accessory genes play a significant role in bacterial adaptability and survival in various environments [34]. This insight underscores the potential application of B. altitudinis DG4 in bioremediation, where environmental pollutants pose challenges to health and ecosystems [35].

Previous studies have established that accessory genes contribute to the metabolic diversity of bacteria, enabling them to exploit a broader spectrum of substrates. In comparison to other Bacillus species, B. altitudinis DG4 has shown a unique set of accessory genes connected to xenobiotic degradation, specifically acyP and dhaT. These genes may provide a strain with enhanced capacities for utilizing and degrading environmental pollutants, contradicting earlier findings that suggested limited xenobiotic degradation abilities within Bacillus species [36], [37]. The data presented in this study indicate promising metabolic versatility in B. altitudinis DG4, opening avenues for its utilization in bioremediation strategies.

3.8. Analysis of Residual Naphthalene with GC-MS by B. altitudinis DG4 (continued)

Gas Chromatography-Mass Spectrometry (GC-MS) analysis of naphthalene degradation by B. altitudinis DG4 confirmed the metabolism of naphthalene and formation of intermediate metabolites. The major metabolites were 1-naphthalenol (18.21 min), 1,8-naphthalic anhydride (19.48 min), 1-acenaphthanone (20.31 min), and benzoic acid (7.70 min) (Fig. 7). The detection of these metabolites suggests that B. altitudinis DG4 employs a specific naphthalene degradation pathway, likely involving initial methylation followed by carboxylation.

The degradation of naphthalene by B. altitudinis DG4 requires a detailed understanding of the metabolic byproducts generated during the process, especially using techniques such as Gas Chromatography-Mass Spectrometry (GC-MS). The findings from our GC-MS analysis identified several key metabolites, including 1-naphthalenol and benzoic acid, indicating the significant metabolic activity of naphthalene by B. altitudinis DG4. This aligns with existing literature suggesting naphthalene's metabolic pathways in various bacterial strains, particularly that certain metabolites can be produced during the degradation of naphthalene by microbes [38], [39].

The naphthalene degradation pathway, as illustrated in Fig. 8, was investigated through the identification of key metabolites using chromatographic and spectroscopic analysis. The primary intermediates detected at different retention times included 1-naphthalenol, 1,8-naphthalic anhydride, 1-acenaphthanone, and benzoic acid.

These intermediates suggested a sequential degradation pathway involving hydroxylation, oxidation, and ring cleavage. The presence of benzoic acid as a degradation product indicates the possibility of its further breakdown into simpler aliphatic acids, which can enter the tricarboxylic acid (TCA) cycle for complete mineralization.

The experimental findings confirmed the successful degradation of naphthalene through microbial and oxidative processes, with the formation of key intermediates leading to the mineralization of the parent compound. The identification of 1,8-naphthalic anhydride and 1-acenapthol suggests an alternative degradation pathway involving anhydride formation before ring cleavage.

These results align with those of previous studies on naphthalene biodegradation by Bordetella avium and Paraburkholderia aromaticivorans, which identified similar intermediates and confirmed the role of dioxygenase enzymes in initiating naphthalene catabolism [38]. Zeng et al. (2024) also supported this degradation pathway, demonstrating the role of radical oxidation in producing naphthalenic intermediates [21].

Elucidating this degradation pathway is important for bioremediation strategies that target environments contaminated by PAHs. Insights gained from the metabolic pathways of B. altitudinis DG4 could play a crucial role in developing effective bioremediation techniques tailored for naphthalene-contaminated sites, thereby enhancing the efficacy of environmental clean-up efforts [40]. Although our research contributes to the understanding of microbial degradation, caution is warranted when extending these findings to natural environments, as in vivo conditions can differ from laboratory settings [39].

4.1. Genomic Features and Metabolic Potential of B. altitudinis DG4

The genomic analysis of B. altitudinis DG4 provides significant insights into its metabolic capabilities and adaptability. With a genome size of 3.83 Mb and 4,120 protein-coding sequences, DG4 possesses a substantial genetic repertoire that supports diverse biochemical functions. The presence of 942 proteins with Enzyme Commission (EC) number assignments indicates a rich enzymatic profile capable of catalyzing various biochemical reactions. Additionally, the mapping of 695 proteins to KEGG pathways highlights the strain's metabolic versatility, including potential degradation pathways for xenobiotic compounds.

The identification of 40 unique genes specific to strain DG4, as revealed by pan-genome analysis of 122 B. altitudinis genomes, suggests unique adaptations that may contribute to its environmental niche specialization. Of particular significance is the presence of genes like dhaT, which is associated with naphthalene and anthracene degradation pathways. This genetic feature provides a molecular basis for the observed superior naphthalene degradation capability of strain DG4 compared to other isolates.

4.2. Naphthalene Degradation Efficiency and Metabolic Pathway

The degradation experiments demonstrated that B. altitudinis DG4 possesses remarkable naphthalene degradation efficiency (90%) despite exhibiting only moderate growth rates. This observation suggests that DG4 allocates a significant portion of its metabolic resources towards naphthalene catabolism rather than biomass production. Such metabolic prioritization may represent an adaptation to environments contaminated with polycyclic aromatic hydrocarbons.

The GC-MS analysis of metabolites formed during naphthalene degradation revealed a specific degradation pathway involving the formation of key intermediates including 1-naphthalenol, 1,8-naphthalic anhydride, 1-acenaphthanone, and benzoic acid. The detection of these metabolites indicates a pathway involving initial hydroxylation of naphthalene to form 1-naphthalenol, followed by oxidative reactions leading to ring cleavage and ultimately the formation of benzoic acid. The presence of benzoic acid is particularly significant as it represents a common intermediate that can be further metabolized through the β-ketoadipate pathway and eventually enter the TCA cycle for complete mineralization.

4.3. Comparative Analysis with Other Naphthalene-Degrading Bacteria

When compared to other naphthalene-degrading bacteria, B. altitudinis DG4 demonstrates competitive degradation efficiency. While some strains such as Pseudomonas sp. strain SA3 have shown higher degradation rates (98.74% within 96 hours), DG4's 90% efficiency after 7 days is comparable to or exceeds many other reported Bacillus strains, such as B. cereus 28BN (72% after 20 days) and B. licheniformis JUG GS2 (73% at 100 mg/L).

The genomic features of DG4, particularly the presence of specific degradation genes like dhaT, provide a genetic foundation for its enhanced degradation capabilities. This genetic basis, combined with experimental validation of its degradation efficiency and pathway elucidation, positions B. altitudinis DG4 as a promising candidate for bioremediation applications targeting naphthalene-contaminated environments.

4.4. Potential for Bioremediation Applications

The findings of this study highlight the potential of B. altitudinis DG4 for bioremediation of naphthalene-contaminated environments. The strain's high degradation efficiency, combined with its genomic adaptations for xenobiotic degradation, suggests it could be effective in environmental cleanup strategies. Additionally, the moderate growth rate of DG4 may be advantageous in field applications, as it implies a sustainable metabolism that prioritizes pollutant degradation over rapid biomass production, potentially leading to more efficient bioremediation processes.

Future in situ field trials would be essential to validate the effectiveness of this strain under natural environmental conditions and to develop optimized bioremediation strategies. Such studies would need to address factors such as bioavailability of naphthalene, competition with indigenous microorganisms, and environmental conditions that might affect degradation efficiency.

This study presents a comprehensive analysis of Bacillus altitudinis DG4, isolated from the Damanganga River in Vapi, India, revealing its remarkable potential for naphthalene bioremediation. Whole genome sequencing identified a 3.83 Mb genome with 4,120 protein-coding sequences, including genes associated with xenobiotic degradation pathways. Pan-genome analysis of 122 B. altitudinis genomes revealed 40 unique genes in strain DG4, potentially contributing to its specialized metabolic capabilities. Experimentally, DG4 demonstrated superior naphthalene degradation efficiency (90%) compared with other isolates, despite showing only moderate growth rates. GC-MS analysis confirmed the metabolism of naphthalene through the formation of key intermediates, including 1-naphthalenol, 1,8-naphthalic anhydride, 1-acenaphthanone, and benzoic acid, indicating a specific degradation pathway involving hydroxylation, oxidation, and ring cleavage. The presence of genes such as dhaT, specifically associated with naphthalene degradation, provides a genetic basis for the observed biodegradation capabilities. These findings highlight B. altitudinis DG4 as a promising candidate for bioremediation of naphthalene-contaminated environments, offering an eco-friendly approach to address PAH pollution. Future in situ field trials are essential to validate the effectiveness of this strain under natural environmental conditions and to develop optimized bioremediation strategies.

Author Contributions

Manoj Godhaniya conducted the laboratory experiments, performed the naphthalene degradation assays, and drafted the initial manuscript. Rajesh Patel designed the research methodology, supervised the genomic analysis, and contributed to manuscript revision. Komal Antaliya performed the bioinformatics analysis, including genome annotation and pan-genome analysis. Pravin Dudhagara conducted the GC-MS analysis, interpreted the degradation pathway data, and assisted with the metabolic analysis. Charmy Kothari coordinated research and finalized the manuscript. All authors reviewed and approved the final version of the manuscript for publication.

Funding Statement

No external funding was received for this research. This study was conducted using the institutional resources provided by the home departments of the authors.

Ethical Compliance

This study did not involve human participants, human data or tissue, or animals. All environmental sampling was conducted with appropriate permissions.

Data Access Statement

Research data supporting this publication, including genome sequence available in ENA Under project accession number - PRJEB76590

Conflict of Interest Declaration

The authors declare that they have no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript.

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Genomic and Biodegradation Potential of Bacillus altitudinis DG4 for Naphthalene Removal from Contaminated Environments

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Sample Collection

2.1. Sample Collection

2.2. Isolation and Purification of Bacillus altitudinis

2.3. Naphthalene Biodegradation Experiment for Selection of the Efficient Bacterium

2.4. Extraction of Genomic DNA and Whole Genome Sequencing of Strain Bacillus altitudinis DG4

2.5. Genome Annotation Function and Prediction

2.5.1. Comprehensive Genome Analysis using BV-BRC

2.5.2. Pan-Genome Analysis

3. Results

3.1. Isolation and Morphological Characterization of Bacillus altitudinis DG4

3.2. Naphthalene Degradation by Bacillus altitudinis DG4

3.3. Genome Assembly and Annotation of Bacillus altitudinis DG4

3.4. Subsystem Analysis and Specialty Genes

3.5. Antimicrobial Resistance Gene Analysis

3.6. Comparative Genome Analysis

3.7. Pan-Genome Analysis

3.8. Analysis of Residual Naphthalene with GC-MS by B. altitudinis DG4 (continued)

4. Discussion

4.1. Genomic Features and Metabolic Potential of B. altitudinis DG4

4.2. Naphthalene Degradation Efficiency and Metabolic Pathway

4.3. Comparative Analysis with Other Naphthalene-Degrading Bacteria

4.4. Potential for Bioremediation Applications

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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