Metabolic specialization and synergy in alkane degradation by a microbial consortium: an integrated genomic and geochemical approach

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Alkanes, being one of the most abundant and challenging fractions of petroleum in terms of biodegradation, demand microorganisms with high metabolic specialization and the incorporation of bacterial consortia, which possess high efficiency due to inter-species synergy. This study had as its objective to integrate genomic and geochemical analyses to unveil the potential and mechanisms of alkane degradation by nine bacterial strains ( Bacillus, Brevibacillus, Pseudomonas , and Stenotrophomonas ) isolated from a contaminated mangrove. The genomic sequencing and functional annotation revealed the presence of key genes and enzymes associated with alkane activation and oxidation, distributed among different bacterial genera. The geochemical analyses by GC-FID demonstrated the superiority of the complete consortium and the specificity of the strains, with highlight to Pseudomonas and Stenotrophomonas in the rapid degradation of the light and intermediate fractions, and Bacillus in the slower and more efficient degradation of the heavy fractions. Furthermore, the study evidenced the complexity of geochemical data analysis in multiphasic systems, where the negative variations were identified as methodological artifacts resulting from biofilm formation and a consequent heterogeneous sampling. The integration of genomic and geochemical results revealed itself to be a powerful tool to elucidate the microbial interactions and to understand the specific alkane degradation mechanisms of each genus in the bioremediation process. Bioremediation Microbial Consortium Petroleum Alkanes Functional Genomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Synopsis Unveiling metabolic synergy within microbial consortia provides mechanistic insights for designing efficient, sustainable bioremediation of alkane contamination. 1. Introduction Bioremediation has established itself, since the 1980s, as a sustainable and effective alternative for the treatment of environments contaminated by organic and inorganic pollutants. This approach gained notoriety after its application in the Exxon Valdez oil tanker disaster, which occurred in 1989, that evidenced the potential of microorganisms in the degradation of complex and persistent contaminants, such as crude oil (Baniasadi; Mousavi, 2018; Young, 2023). Since then, the increase in global demand for petroleum and its derivatives, estimated at up to 110 million barrels per day by 2045, according to OPEC, and the growing occurrence of spills during prospecting, transport, and refining have reinforced the need for ecological and economically viable strategies to mitigate the environmental impacts of these accidents (“Global oil products demand outlook 2050 | Statista”, 2025). Thus, bioremediation which utilizes the metabolic capacity of autochthonous microorganisms to degrade, detoxify, or transform petroleum compounds, represents an efficient, sustainable, and low-cost solution, especially when compared with traditional physical and chemical methods (Ueno et al., 2007 ; Verma & Jaiswal, 2016 ; Khan et al ., 2022). This process is based on complex enzymatic cascades, in which hydrocarbons are converted into less toxic intermediates and, finally, used as a source of carbon and energy (Amini; Giyahchi; Moghimi, 2024 ). Among the main constituents of petroleum, alkanes stand out for being a major fraction and for the structural variability that directly influences their biodegradability. Although short and medium-chain alkanes are generally more susceptible to microbial oxidation, long-chain ones present greater recalcitrance due to low solubility and enzymatic accessibility (Park; Park, 2018). The degradation of these compounds involves specific metabolic routes initiated by the action of monooxygenases and dehydrogenases, culminating in the conversion of alkanes into alcohols, aldehydes, and, subsequently, into fatty acids that integrate the β-oxidation cycle (Gao et al ., 2024; Wang et al ., 2021). The diversity of these routes is potentiated in microbial consortia, symbiotic systems formed by different bacterial species with complementary metabolic capacities. This cooperation increases degradation efficiency, broadens tolerance to pollutant toxicity, and favors the production of compounds such as biosurfactants, which reduce surface tension and facilitate access to hydrophobic hydrocarbons (Parus et al., 2023 ; Costa et al., 2007 ; Mahjoubi et al., 2023 ). Despite advances, important gaps still persist in the understanding of the molecular mechanisms that sustain alkane biodegradation, especially regarding the interaction between species in a consortium and the regulation of the enzymatic routes involved (Rawat; Rangarajan, 2019 ; Che; Men, 2019 ). In this sense, omics technologies, such as functional genomics, have shown themselves to be fundamental tools for the elucidation of metabolic pathways and the specific contribution of each microorganism within complex communities (Ahmed et al., 2023 ). In this way, the present study aimed to integrate genomic and geochemical analyses to investigate the alkane degrading potential in bacterial strains isolated from a mixed consortium originating from a petroleum-contaminated mangrove. Through the combination of functional analyses and experimental evaluation, it was sought to understand the metabolic routes involved in alkane degradation and to identify the main microorganisms and enzymes associated with this process, contributing to the development of more efficient strategies in the bioremediation of aliphatic hydrocarbons. 2. Metodologia 2.1 Biological Material The microorganisms used in this study are part of a microbial consortium composed of 36 organisms, isolated in the Baía de Todos os Santos and protected by the patent of Lima et al . (2021), with deposit number at the National Institute of Industrial Property (INPI) BR 10 2021 002341 4. For the present investigation, 9 bacterial strains were selected based on taxonomic diversity and preliminary metabolic profile. The taxonomic identifications were performed from the analysis of the 16S rRNA region, associated with classic microbiology techniques, and the respective genera are listed in Table 1. Table 1. Taxonomic identification of the bacterial strains selected for genomic analysis. 2.1.1 Experimental Configuration For this study, an assay was designed to analyze the degradation of hydrocarbons by the bacterial isolates with 3 distinct growth conditions: Standard condition: microorganisms cultivated in standard nutritive medium, destined for genomic analyses; Experimental condition: cultivation with crude oil as the only carbon source, simulating a contaminated environment; Negative control (blank): medium containing only the crude oil, without bacterial inoculation, to evaluate the abiotic degradation of the petroleum. 2.2 Genomic sequencing and data processing 2.2.1 Genome sequencing The genomic sequencing of the strains was performed by a third-party service at the University of Göttingen, Germany, using the Illumina Hi-Seq 2500 platform, with generation of 2 × 150 bp paired-end reads (500 bp insert). The quality control of the raw reads was performed with the FastQC software adopting a phred in the 30s range, followed by low-quality filtering, adapter removal and end trimming by means of the fastp tool (Chen et al ., 2018). The genome assembly was performed with Unicycler, and the evaluation of assembly metrics, such as N50, number of contigs and coverage, was conducted with QUAST (Gurevich et al ., 2013). 2.2.2 Genomic annotation The annotation of the bacterial genomes was performed in a combined manner, by means of the RASTtk (Rapid Annotation using Subsystem Technology) pipeline, accessed via the BV-BRC (Bacterial and Viral Bioinformatics Resource Center) platform, and by Prokka (rapid prokaryotic genome annotation). The integrated annotation was submitted to GhostKOALA for KO number attribution to the functional data, followed by the reconstruction of pathways by KEGG Mapper simultaneously with the comparison and validation of the PATRIC (Pathosystems Resource Integration Center) platform, allowing the integrated functional and taxonomic analysis between the sequenced strains with emphasis on the identification of genes associated with the degradation of complex organic compounds. Furthermore, the annotations were enriched with the integration of specialized databases, including KEGG (Kyoto Encyclopedia of Genes and Genomes), UniProt, InterProScan, Gene Ontology (GO), and BRENDA, with the objective of maximizing the accuracy in identifying gene functions and reconstructing metabolic pathways. 2.2.3 Comparative analysis From the annotated data, a functional analysis was performed, focused on the identification of genes and metabolic pathways associated with the biodegradation of aliphatic hydrocarbons. For this, the KEGG pathway corresponding to the fatty acid degradation pathway (map00071) was consulted. The comparative analysis between the strains was conducted based on the presence or absence of key genes for each pathway, variables, and number of repetitions, with a focus on enzymes catalyzing rate-limiting steps, such as monooxygenases (alkB, ladA, almA), cytochromes P450, and enzymes of the β-oxidation of fatty acids. The data were integrated by means of the visualization tools of the BV-BRC platform and organized into heatmaps and comparative tables with the objective of interpreting the functional organization and the biodegrading potential of the consortium. Furthermore, promoter region predictions for genes of interest were performed with the BPROM tool (Softberry), allowing to infer the potential for gene expression in an environmental context. 2.3 Geochemical experiment 2.3.1 Experimental setup The microorganisms used in the experiments were cultivated in Petri dishes for 48 hours. Then, they were transferred to 250 mL Erlenmeyer-type flasks containing liquid culture medium, with the objective of cultivating the isolates. After growth, the isolates were transferred to 25 mL of saline solution, and their concentrations were standardized using a microplate reader (Loccus). The microgeological experiment was set up in four 500 mL Erlenmeyer-type flasks containing: 300 mL of Bushnell-Haas Broth (BH) culture medium, poor in carbon; 9 mL of the standardized saline solution containing the bacterial strain in the experimental case; and 3 g of the carbon source. The crude oil used was supplied by the independent operator of onshore oil and gas exploration and production Petrorecôncavo S.A., Field: Norte Fazenda Caruaçu – Recôncavo Basin, Depth: 1064.0m, Density @ 20°C: 0.8371, °API @ 20°C: 37.36. It is a paraffinic oil from the Recôncavo Basin, Bahia - Brazil. The Erlenmeyer-type flasks were then incubated at 30°C for a period of 31 days, during which oil samples were collected at times 0h (T0), 16 days (T1), and 31 days (T2). The samples were dissolved in dichloromethane (DCM), filtered through an open column of activated sodium sulfate, and stored. 2.3.2 Analytical Methods The Total Petroleum Hydrocarbons were analyzed by GC/FID on an Agilent 7890B chromatograph, split method, whose usage specifications are described in table 2. The quantification of the compounds was performed by the external standard method, based on peak area, within the time interval of 2.5 to 40 minutes. The data were acquired and processed using the ChemStation software (Agilent Technologies), with the sensitivity and rejection parameters adjusted to guarantee the accuracy in the identification and quantification of peaks. Table 2. Parameters of the chromatographic analysis by GC/FID. Finally, a statistical analysis of the chromatographic data was performed using the degradation rate as the response variable. Normality was verified by means of the Shapiro-Wilk test, followed by analysis of variance (ANOVA). When statistically significant differences were observed, Tukey's test was applied as a post-hoc for the identification of multiple comparisons between groups. And all tests were conducted with a significance level of α = 0.05, using the R statistical software. 3. Results and discussion 3.1 Complete genome sequencing and comparative analysis Complete genomic sequencing was performed for the nine bacterial strains integrating the microbial consortium, generating high-quality data and adequate coverage for de novo assembly of the genomes (Table 3). The quality control and assembly steps were conducted using the FastQC, Fastp, and Unicycler tools, and evaluated by means of QUAST, as described in the methodology. Table 3. Quality parameters of the sequencing and assembly of the bacterial genomes. The assembled genomes presented sizes varying from 4.09 to 13.44 Mbp, with GC content between 38.2% and 67.5%, and total number of CDS between 3,950 and 12,400, values compatible with those expected for environmental bacteria with broad metabolic plasticity. The variation observed between the genera reflects the structural diversity of the consortium, suggesting the coexistence of microorganisms with different physiological and metabolic strategies. The number of contigs varied from 31 to 254, with the lowest values observed in strains of the Bacillus and Brevibacillus genera, which indicates greater continuity and assembly quality—reinforced by the N50 values. This structural heterogeneity is consistent with data from environmental bacterial genomes (Tully et al ., 2018; Parks et al ., 2020), and may be associated with the presence of mobile genetic elements and repetitive regions that hinder genomic closure in short-read sequencing (Orellana et al ., 2023). In general, the obtained assemblies were sufficiently robust for functional analyses and metabolic pathway reconstruction, allowing the identification of key genes associated with hydrocarbon degradation. The observed genomic diversity, reflected in the GC content and genetic size, reinforces the cooperative potential of the consortium in contaminated environments, since different physiological profiles favor adaptation to variable conditions and the functional division of catabolic routes (Pandolfo et al ., 2024; Rojas-Vargas et al ., 2024). 3.1.1 Alkane metabolization pathway The functional reconstruction of metabolic pathways revealed the broad representation of the fatty acid β-oxidation route (KEGG map00071) among the nine bacterial strains of the consortium. The mapping of EC numbers (Figure 1) indicated the complete presence of the main enzymes involved in the oxidative catabolism of alkanes, including acyl-CoA synthetase (EC 6.2.1.3), acyl-CoA dehydrogenase (EC 1.3.8.1), enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), and thiolase (EC 2.3.1.16). These enzymes participate in the sequential steps of oxidation and cleavage of fatty acids down to acetyl-CoA, evidencing the complete functioning of the downstream β-oxidation route. Furthermore, genes related to the initial activation of alkanes were identified, such as alkane 1-monooxygenase (EC 1.14.15.3), Cytochrome P450 (1.14.14.1), alcohol dehydrogenase (EC 1.1.1.1), and aldehyde dehydrogenase (EC 1.2.1.3), responsible for the conversion of alkanes into alcohols and subsequently into fatty acids. These results indicate a complete functional arrangement, spanning from the initial oxidation to the mineralization of the aliphatic compounds. (Guibert et al ., 2015; Wang et al ., 2022) The consortium demonstrated the presence of genes for different monooxygenases, including those specific for short chains, such as AlkB ( alkB1_2, alkM ), and the more versatile ones, such as Cytochrome P450 (102A3, 102A2, 102A5), both responsible for the terminal hydroxylation of linear alkanes. In addition to those of β-oxidation distributed among the different strains, suggesting interspecies metabolic cooperation and the existence of a biochemical strategy for the catabolism of aliphatic compounds present in petroleum. The lineages of the Pseudomonas and Stenotrophomonas genera (PD5, PD6, RC6) were highlighted, due to the presence of alkB, constantly used as a parameter for evaluating the alkane degradation potential in biodegrading organisms, while Cytochrome CYP102A5 is normally associated with the Bacillus genus (OH4 and RB4), as a versatile and promiscuous enzyme in its targets, capable of metabolizing non-natural substrates, including polyethylene film (Tarara; Ahuja; Mellies, 2025; Hidalgo et al ., 2020; Liu et al ., 2015; Vieto et al ., 2021). The redundancy of genes associated with alkane metabolism in multiple strains, combined with the presence of promoters and regulatory elements linked to oxidative stress response, suggests an evolutionary adaptation to the contaminated environment, in which continuous exposure to hydrocarbons favored the selection of specialized lineages (Brzeszcz; Kaszycki, 2018). This scenario reinforces the hypothesis of functional distribution of catabolic routes within the consortium, maximizing its degrading efficiency. 3.2 Total Petroleum Hydrocarbons For the comparative analysis of the individual degradative potential of the studied strains, previous results representative of the degradative capacity of the complete consortium under standardized conditions were used. In conjunction with the negative control, in which there was no inoculation, it was possible to infer that any contrast obtained between the samples results directly from microbial action. Furthermore, as all experimental conditions were standardized regarding the initial petroleum and inoculum concentration, a direct comparison between the treatments becomes viable (Fig. 2) The analysis of the microbial consortium revealed a high degradation rate throughout the experiment (T0–30), with a general average of 46%, reaching 52% just in the intermediate fraction in the first 15 days. This pattern reinforces the fundamental role of consortia in the synergistic degradation of hydrocarbons, especially in the initial phases of bioremediation, when there is greater nutrient availability and less accumulation of toxic metabolites (Yu et al ., 2022). In the last 15 days of the experiment, a reduction in the degradation rate for the intermediate fraction (29%) was observed, possibly as a consequence of the microbial decline phase and the limiting growth conditions in the system, such as metabolite accumulation, competition for nutrients, and a decrease in substrate bioavailability. In the case of the individual analyses, none of the isolated strains showed global performance equivalent to that of the complete consortium, an expected result, considering that crude oil is a complex mixture of hydrocarbons with different molecular weights, which restricts the action of isolated microorganisms (Li et al ., 2016). Even so, relevant specific behaviors were observed, especially when considering particular fractions of the degraded compounds. The strains PD5, RC6, and PD6, corresponding to the Pseudomonas and Stenotrophomonas genera, stood out as rapid degraders, with a bimodal profile, thanks to the presence of the alkane monooxygenase identified in the genomic analyses. This profile was characterized by high degradation rates for short-chain (C26) alkanes, contrasting with a sharp drop in efficiency for the intermediate range. Such behavior suggests that these strains possess multiple enzymatic systems, with distinct specificities for different chain lengths. (Passler; Ditchkoff; Walz, 2016; Li; Pan; Ma, 2019) On the other hand, strains OH4 and RB4, belonging to the Bacillus genus, demonstrated a clear behavior of specialization in medium/long chains and late degradation (Fig. 4). With highlight to OH4 which initially presented low degradation of short and medium-chain alkanes, possibly associated with its slower metabolism and the absence of the AlkB enzyme. In contrast, it registered a continuous and later degradation of the heavy fractions, reaching 67% in the last 15 days of the experiment. This pattern of preferential and delayed consumption of complex hydrocarbons is typical of biosurfactant-producing microorganisms, such as surfactin, where the bacterium first produces and secretes the biosurfactants to emulsify the waxy alkanes, making them accessible to its degrading enzymes. (Dai et al ., 2020; Lima et al ., 202; Thirumurugan et al ., 2023). It is important to highlight that the apparent absence of degradation, or even the observed negative rates, represented in white to facilitate the visualization of effective degradation, of some intermediate-chain alkanes do not represent the biological production of these compounds. This phenomenon, also known as floating degradation, anomalous or negative values, is caused due to a methodological artifact resulting from the intense biofilm formation at the oil-water interface. (LI et al ., 2024) With the development of this biofilm, the system becomes heterogeneous, and the residual oil can be sequestered in biomass aggregates. Thus, the collection with a spatula at time 2 resulted in unrepresentative and over-concentrated samples in these aggregates, producing artificially higher values than those from the previous period. In this way, the negative rates reflect sampling limitations in a complex multiphasic system, and not necessarily a metabolic inefficiency for those fractions, a factor evidenced by the complex genomic structure presented by the strains for their degradation. (Johnsen; Wick; Harms, 2004; Neu et al ., 2019; Joannis; Delia; Riba, 1998) 4. Conclusion The study successfully demonstrated that the high alkane degradation efficiency of the target microbial consortium in paraffinic oil is occasioned by functional synergy and by an ecological niche partitioning among its members. The integration of genomic and geochemical data allowed to confirm the consortium's catabolic potential and to assign specific roles to the most representative genera, as Pseudomonas and Stenotrophomonas acted as primary and rapid degraders, while Bacillus specialized in the slower and later degradation of the long-chain recalcitrant compounds, probably mediated by biosurfactant production. The geochemical results corroborated the genomic inferences and evidenced the need for a critical interpretation in multiphasic systems, since anomalous or negative degradation values reflect methodological artifacts resulting from biofilm formation and sampling heterogeneity, and not a metabolic limitation. Together, the findings reinforce that the application of microbial consortia with functional diversity constitutes a robust and adaptable strategy for the bioremediation of complex contaminants, such as petroleum alkanes. Declarations Author Contribution P.G.B.S.N. conceptualized the study, performed data curation, conducted formal analysis, carried out the investigation, wrote the original draft, and reviewed and edited the final manuscript. A.A.M.N. contributed to the study conceptualization, performed data curation, and conducted formal analysis of specific data. L.A.O. assisted with data analysis, prepared figures, and reviewed and edited the final manuscript. D.F.L. contributed to the conceptualization, provided supervision, and critically reviewed and edited the manuscript. A.F.S.Q. and O.M.C.O. provided overall supervision, acquired funding, and contributed resources for the project. All authors reviewed and approved the final manuscript. Acknowledgement The authors gratefully acknowledge the support of Shell Brasil through the project "Research in Geomicrobiology: Microbial Biotechnology Applied to Petroleum Bioremediation and Recovery of Degraded Areas at the Institute of Geosciências of the Federal University of Bahia (UFBA) – GEOQPETROL – GEOMICRO" – ANP project 20720-9, and recognize the strategic importance of the support granted by the National Agency of Petroleum, Natural Gas and Biofuels (ANP), through the regulation of the R&D investment clause. To the laboratories LEPETRO – Excellence in Petroleum Geochemistry, Energy and Environment – and IBTEC – Institute of Biotechnology. This study was financed, in part, by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Brazil [Financing Code 001]. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References AHMED, M. et al . Optimizing Synergistic Metabolism in Xenobiotic Biodegradation: Engineering a Pseudomonas putida Based Microbial Consortium. American Journal of Life Science and Innovation , 3(1), 1–7, 27 dez. 2023. http://dx.doi.org/10.54536/ajlsi.v3i1.2236 AMINI, Fatemeh; GIYAHCHI, Minoo; MOGHIMI, Hamid. Bioremediation of Petroleum Contamination by Microorganisms: Role of Microbial Communities and Applications. In: Royal Society of Chemistry , 2024. p. 136–170. DOI: 10.1039/bk9781837673131-00136. BANIASADI, Mahsa; MOUSAVI, Seyyed Mohammad. A Comprehensive Review on the Bioremediation of Oil Spills. In: Springer, Singapore , 2018. p. 223–254. DOI: 10.1007/978-981-13-1840-5_10. BRZESZCZ, Joanna; KASZYCKI, Paweł. Aerobic bacteria degrading both n-alkanes and aromatic hydrocarbons: an undervalued strategy for metabolic diversity and flexibility. Biodegradation , v. 29, n. 4, p. 359–407, 2018. DOI: 10.1007/S10532-018-9837-X. C. JOANNIS; DELIA, M. L.; RIBA, J. P. Comparison of four methods for quantification of biofilms in biphasic cultures. Biotechnology Techniques , v. 12, n. 10, p. 777–782, 1 jan. 1998. 10.1023/A:1008835811731 CHE, Shun; MEN, Yujie. Synthetic microbial consortia for biosynthesis and biodegradation: promises and challenges. Journal of Industrial Microbiology & Biotechnology , v. 46, n. 9, p. 1343–1358, 2019. DOI: 10.1007/S10295-019-02211-4. COSTA, C. D. S. et al. Produção de biossurfactante por microrganismos isolados de áreas contaminadas com petróleo. Revista Brasileira de Biociências , v. 5, supl. 1, p. 69–71, 2007. DAI, X. et al . Heavy Oil Biodegradation by Mixed Bacterial Consortium of Biosurfactant-Producing and Heavy Oil-Degrading Bacteria. Polish Journal of Environmental Studies , v. 30, n. 1, p. 71–80, 3 set. 2020. https://doi.org/10.15244/pjoes/120769. GAO, Y. et al . Response Characteristics of the Community Structure and Metabolic Genes of Oil-Recovery Bacteria after Targeted Activation of Petroleum Hydrocarbon-Degrading Bacteria in Low-Permeability Oil Reservoirs. ACS Omega , v. 9, n. 31, p. 33448–33458, 27 jul. 2024. 10.1021/acsomega.3c10334. GUIBERT, L. M. et al . Diverse Bacterial Groups Contribute to the Alkane Degradation Potential of Chronically Polluted Subantarctic Coastal Sediments. Microbial Ecology , v. 71, n. 1, p. 100–112, 7 nov. 2015. 10.1007/S00248-015-0698-0. GUREVICH, Alexey et al . QUAST: quality assessment tool for genome assemblies. Bioinformatics , v. 29, n. 8, p. 1072–1075, 2013. DOI: https://doi.org/10.1093/bioinformatics/btt086. HIDALGO, K. J. et al . AlkB monooxygenases as key enzymes in alkane degradation: distribution and diversity in hydrocarbon-contaminated environments. Journal of Applied Microbiology , v. 129, n. 6, p. 1440–1455, 2020. DOI: https://doi.org/10.1111/jam.14667. JOHNSEN, A. R.; WICK, L. Y.; HARMS, H. Principles of microbial PAH-degradation in soil. Environmental Pollution , v. 133, n. 1, p. 71–84, jan. 2005. https://doi-org.ez10.periodicos.capes.gov.br/10.1016/j.envpol.2004.04.015 KHAN, Saimah; TRIPATHI, Ayush Kumar; SRIVASTAVA, Rohan; et al . Bioremediation of Petroleum Contamination: A Short Review. Ecological Questions , 33(2), 1–18, 2022. http://dx.doi.org/10.12775/EQ.2022.012 LI, L. et al . Dynamic responses in Pseudomonas stutzeri M3 bioaugmentation of crude-oil-contaminated soil: Hydrocarbons, microbial community structures, and functional genes. Journal of environmental chemical engineering , v. 12, n. 1, p. 111863–111863, 4 jan. 2024. https://doi.org/10.1016/j.jece.2023.111863 LI, Y. P.; PAN, J. C.; MA, Y. L. Elucidation of multiple alkane hydroxylase systems in biodegradation of crude oil n ‐alkane pollution by Pseudomonas aeruginosa DN1. Journal of Applied Microbiology , v. 128, n. 1, p. 151–160, 1 out. 2019. https://doi.org/10.1111/jam.14470. LI, xinfei; ZHAO, lin; ADAM, Mohamed. Biodegradation of marine crude oil pollution using a salt-tolerant bacterial consortium isolated from Bohai Bay, China. Marine Pollution Bulletin, v. 105, n. 1, p. 43–50, 2016. https://doi.org/10.1016/j.marpolbul.2016.02.073. LIMA, F. T. S. et al . Consórcio microbiano com capacidade para degradação de petróleo. Pedido de patente BR 10 2021 002341 4. Instituto Nacional da Propriedade Industrial – INPI, 2021. LIMA, S. D. et al . Isolation and characterization of hydrocarbon-degrading bacteria from gas station leaking-contaminated groundwater in the Southern Amazon, Brazil. figshare , 26 mar. 2021. 10.6084/m9.figshare.14322077. LIU, C. et al . Diversity and distribution of alkB genes in bacteria capable of degrading medium- and long-chain n-alkanes in oil-contaminated environments. International Biodeterioration & Biodegradation , v. 94, p. 116–122, 2015. DOI: https://doi.org/10.1016/j.ibiod.2014.07.016. MAHJOUBI, M. et al . Bacterial consortia as a promising strategy for enhanced petroleum hydrocarbons degradation. Environmental Science and Pollution Research , v. 30, p. 53973–53992, 2023. DOI: https://doi.org/10.1007/s11356-023-27138-5. NEU, L. et al . Small-Scale Heterogeneity in Drinking Water Biofilms . Frontiers in Microbiology , v. 10, 29 out. 2019. https://doi.org/10.3389/fmicb.2019.02446 ORELLANA, L. H. et al . Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies. Research Square , 2023. DOI: https://doi.org/10.21203/rs.3.rs-2357036/v1. PANDOLFO, Emiliana et al . Metagenomic analyses of a consortium for the bioremediation of hydrocarbons polluted soils. AMB Express , v. 14, n. 1, 2024. DOI: 10.1186/s13568-024-01764-7. PARK, C.; PARK, W. Survival and Energy Producing Strategies of Alkane Degraders Under Extreme Conditions and Their Biotechnological Potential. Frontiers in Microbiology , v. 9, 25 maio 2018. https://doi.org/10.3389/fmicb.2018.01081. PARKS, D. H. et al . A complete domain-to-species taxonomy for Bacteria and Archaea. Nature Biotechnology , v. 38, p. 1079–1086, 2020. DOI: https://doi.org/10.1038/s41587-020-0501-8. PARUS, A. et al . Basic principles for biosurfactant-assisted (bio)remediation of soils contaminated by heavy metals and petroleum hydrocarbons – A critical evaluation of the performance of rhamnolipids. Journal of Hazardous Materials , v. 443, p. 130171, 2023. DOI: https://doi.org/10.1016/j.jhazmat.2022.130171. PASSLER, T.; DITCHKOFF, S. S.; WALZ, P. H. Bovine Viral Diarrhea Virus (BVDV) in White-Tailed Deer (Odocoileus virginianus). Frontiers in Microbiology , v. 7, 20 jun. 2016. https://doi.org/10.3389/fmicb.2016.00945. RAWAT, Madhu; RANGARAJAN, Srishti. Omics approaches for elucidating molecular mechanisms of microbial bioremediation. In: Academic Press , 2019. p. 191–203. DOI: 10.1016/B978-0-12-818307-6.00011-1. ROJAS-VARGAS, Jorge et al . A comparative genomic study of a hydrocarbon-degrading marine bacterial consortium. PLOS ONE , v. 19, n. 8, p. e0303363, 2024. DOI: 10.1371/journal.pone.0303363. Shifu Chen, Yanqing Zhou, Yaru Chen, Jia Gu; fastp: an ultra-fast all-in-one FASTQ preprocessor, Bioinformatics , Volume 34, Issue 17, 1 September 2018, Pages i884–i890, https://doi.org/10.1093/bioinformatics/bty560. STATISTA. Global oil products demand outlook 2050. Statista, 2025. DOI: https://www.statista.com/statistics/282774/global-product-demand-outlook-worldwide/. TARARA, M.; AHUJA, S.; MELLIES, J. L. A Cytochrome P450 Facilitates Polyethylene Metabolism in a Microbial Community. International Journal of Molecular Sciences, v. 26, n. 18, p. 8775–8775, 9 set. 2025. https://doi.org/10.3390/ijms26188775 THIRUMURUGAN, D. et al . Impact of biosurfactant produced by Bacillus spp. on biodegradation efficiency of crude oil and anthracene. Chemosphere , 344, 140340, 1 dez. 2023. https://doi.org/10.1016/j.chemosphere.2023.140340 TULLY, B. J. et al . The reconstruction of 2,631 draft metagenome-assembled genomes from the global oceans. Scientific Data , v. 5, p. 170203, 2018. DOI: https://doi.org/10.1038/sdata.2017.203. UENO, A. et al . Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation. World Journal of Microbiology and Biotechnology , v. 23, n. 12, p. 1739–1745, 2007. DOI: https://doi.org/10.1007/s11274-007-9423-6. VERMA, Jay P.; JAISWAL, Durgesh K. Book Review: Advances in Biodegradation and Bioremediation of Industrial Waste. Frontiers in Microbiology , 6, 2016. https://doi.org/10.3389/fmicb.2015.01555 VIETO, Sofía et al . The potential of Pseudomonas for bioremediation of oxyanions. Environmental Microbiology Reports , v. 13, n. 6, p. 773–789, 2021. DOI: 10.1111/1758-2229.12999. WANG, Q. et al . Community-wide distribution of generalist catabolic genes in hydrocarbon-degrading microbial consortia. Frontiers in Microbiology , v. 14, p. 1183407, 2023. DOI: https://doi.org/10.3389/fmicb.2023.1183407. WANG, S. et al . A novel alkane monooxygenase (alkB) clade revealed by massive genomic survey and its dissemination association with IS elements. PeerJ , v. 10, p. e14147–e14147, 28 set. 2022. 10.7717/peerj.14147. YOUNG, John K. Bioremediation of Oil Spill Cleanup: Case Studies of Two Major Oil Spills. Sustainable Environment , p. 117–133, 2023. DOI: 10.1007/978-981-19-8464-8_7. YU, T. et al . Microbial community succession during crude oil-degrading bacterial enrichment cultivation and construction of a degrading consortium. Frontiers in Microbiology , 13, 4 nov. 2022. https://doi.org/10.3389/fmicb.2022.1044448 Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.png Highlights.pdf Table123.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 Feb, 2026 Reviews received at journal 01 Feb, 2026 Reviews received at journal 01 Feb, 2026 Reviews received at journal 14 Jan, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviewers agreed at journal 11 Jan, 2026 Reviewers agreed at journal 11 Jan, 2026 Reviewers invited by journal 08 Jan, 2026 Editor assigned by journal 31 Dec, 2025 Submission checks completed at journal 31 Dec, 2025 First submitted to journal 30 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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2","display":"","copyAsset":false,"role":"figure","size":114565,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation rate of total petroleum hydrocarbons in the medium inoculated with complete consortium under standardized conditions.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8481249/v1/8ed88ef6511e48966cb8e0da.png"},{"id":100367660,"identity":"b3a6ca79-79aa-4353-8b08-32964bc02ee8","added_by":"auto","created_at":"2026-01-16 07:57:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":201664,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation rate of total petroleum hydrocarbons in the medium inoculated with PD5, RC6 and PD6.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8481249/v1/8630fc01eadb4aa53c594d46.png"},{"id":100367950,"identity":"be2867e7-ad84-4177-b54d-8745098da877","added_by":"auto","created_at":"2026-01-16 07:57:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":131839,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation rate of total petroleum hydrocarbons in the medium inoculated with OH4 and RB4.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8481249/v1/65d0b91d732e0023be5384b6.png"},{"id":100422014,"identity":"2780ef69-0118-4cde-a30c-9d3e737a5e19","added_by":"auto","created_at":"2026-01-16 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within microbial consortia provides mechanistic insights for designing efficient, sustainable bioremediation of alkane contamination.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eBioremediation has established itself, since the 1980s, as a sustainable and effective alternative for the treatment of environments contaminated by organic and inorganic pollutants. This approach gained notoriety after its application in the Exxon Valdez oil tanker disaster, which occurred in 1989, that evidenced the potential of microorganisms in the degradation of complex and persistent contaminants, such as crude oil (Baniasadi; Mousavi, 2018; Young, 2023).\u003c/p\u003e \u003cp\u003eSince then, the increase in global demand for petroleum and its derivatives, estimated at up to 110\u0026nbsp;million barrels per day by 2045, according to OPEC, and the growing occurrence of spills during prospecting, transport, and refining have reinforced the need for ecological and economically viable strategies to mitigate the environmental impacts of these accidents (\u0026ldquo;Global oil products demand outlook 2050 | Statista\u0026rdquo;, 2025).\u003c/p\u003e \u003cp\u003eThus, bioremediation which utilizes the metabolic capacity of autochthonous microorganisms to degrade, detoxify, or transform petroleum compounds, represents an efficient, sustainable, and low-cost solution, especially when compared with traditional physical and chemical methods (Ueno et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Verma \u0026amp; Jaiswal, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Khan \u003cem\u003eet al\u003c/em\u003e., 2022). This process is based on complex enzymatic cascades, in which hydrocarbons are converted into less toxic intermediates and, finally, used as a source of carbon and energy (Amini; Giyahchi; Moghimi, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the main constituents of petroleum, alkanes stand out for being a major fraction and for the structural variability that directly influences their biodegradability. Although short and medium-chain alkanes are generally more susceptible to microbial oxidation, long-chain ones present greater recalcitrance due to low solubility and enzymatic accessibility (Park; Park, 2018). The degradation of these compounds involves specific metabolic routes initiated by the action of monooxygenases and dehydrogenases, culminating in the conversion of alkanes into alcohols, aldehydes, and, subsequently, into fatty acids that integrate the β-oxidation cycle (Gao \u003cem\u003eet al\u003c/em\u003e., 2024; Wang \u003cem\u003eet al\u003c/em\u003e., 2021).\u003c/p\u003e \u003cp\u003eThe diversity of these routes is potentiated in microbial consortia, symbiotic systems formed by different bacterial species with complementary metabolic capacities. This cooperation increases degradation efficiency, broadens tolerance to pollutant toxicity, and favors the production of compounds such as biosurfactants, which reduce surface tension and facilitate access to hydrophobic hydrocarbons (Parus et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Costa et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Mahjoubi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite advances, important gaps still persist in the understanding of the molecular mechanisms that sustain alkane biodegradation, especially regarding the interaction between species in a consortium and the regulation of the enzymatic routes involved (Rawat; Rangarajan, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Che; Men, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this sense, omics technologies, such as functional genomics, have shown themselves to be fundamental tools for the elucidation of metabolic pathways and the specific contribution of each microorganism within complex communities (Ahmed et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this way, the present study aimed to integrate genomic and geochemical analyses to investigate the alkane degrading potential in bacterial strains isolated from a mixed consortium originating from a petroleum-contaminated mangrove. Through the combination of functional analyses and experimental evaluation, it was sought to understand the metabolic routes involved in alkane degradation and to identify the main microorganisms and enzymes associated with this process, contributing to the development of more efficient strategies in the bioremediation of aliphatic hydrocarbons.\u003c/p\u003e"},{"header":"2. Metodologia","content":"\u003ch2\u003e2.1 Biological Material\u003c/h2\u003e\n\u003cp\u003eThe microorganisms used in this study are part of a microbial consortium composed of 36 organisms, isolated in the Ba\u0026iacute;a de Todos os Santos and protected by the patent of Lima \u003cem\u003eet al\u003c/em\u003e. (2021), with deposit number at the National Institute of Industrial Property (INPI) BR 10 2021 002341 4. For the present investigation, 9 bacterial strains were selected based on taxonomic diversity and preliminary metabolic profile. The taxonomic identifications were performed from the analysis of the 16S rRNA region, associated with classic microbiology techniques, and the respective genera are listed in Table 1.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Taxonomic identification of the bacterial strains selected for genomic analysis.\u003c/h4\u003e\n\u003ch2\u003e2.1.1 Experimental Configuration\u003c/h2\u003e\n\u003cp\u003eFor this study, an assay was designed to analyze the degradation of hydrocarbons by the bacterial isolates with 3 distinct growth conditions:\u003c/p\u003e\n\u003cul class=\"decimal_type\"\u003e\n \u003cli\u003eStandard condition: microorganisms cultivated in standard nutritive medium, destined for genomic analyses;\u003c/li\u003e\n \u003cli\u003eExperimental condition: cultivation with crude oil as the only carbon source, simulating a contaminated environment;\u003c/li\u003e\n \u003cli\u003eNegative control (blank): medium containing only the crude oil, without bacterial inoculation, to evaluate the abiotic degradation of the petroleum.\u003c/li\u003e\n\u003c/ul\u003e\n\u003ch2\u003e2.2 Genomic sequencing and data processing\u003c/h2\u003e\n\u003ch3\u003e2.2.1 Genome sequencing\u003c/h3\u003e\n\u003cp\u003eThe genomic sequencing of the strains was performed by a third-party service at the University of G\u0026ouml;ttingen, Germany, using the Illumina Hi-Seq 2500 platform, with generation of 2 \u0026times; 150 bp paired-end reads (500 bp insert). The quality control of the raw reads was performed with the FastQC software adopting a phred in the 30s range, followed by low-quality filtering, adapter removal and end trimming by means of the fastp tool (Chen \u003cem\u003eet al\u003c/em\u003e., 2018). The genome assembly was performed with Unicycler, and the evaluation of assembly metrics, such as N50, number of contigs and coverage, was conducted with QUAST (Gurevich \u003cem\u003eet al\u003c/em\u003e., 2013).\u003c/p\u003e\n\u003ch3\u003e2.2.2 Genomic annotation\u003c/h3\u003e\n\u003cp\u003eThe annotation of the bacterial genomes was performed in a combined manner, by means of the RASTtk (Rapid Annotation using Subsystem Technology) pipeline, accessed via the BV-BRC (Bacterial and Viral Bioinformatics Resource Center) platform, and by Prokka (rapid prokaryotic genome annotation). The integrated annotation was submitted to GhostKOALA for KO number attribution to the functional data, followed by the reconstruction of pathways by KEGG Mapper simultaneously with the comparison and validation of the PATRIC (Pathosystems Resource Integration Center) platform, allowing the integrated functional and taxonomic analysis between the sequenced strains with emphasis on the identification of genes associated with the degradation of complex organic compounds. Furthermore, the annotations were enriched with the integration of specialized databases, including KEGG (Kyoto Encyclopedia of Genes and Genomes), UniProt, InterProScan, Gene Ontology (GO), and BRENDA, with the objective of maximizing the accuracy in identifying gene functions and reconstructing metabolic pathways.\u003c/p\u003e\n\u003ch3\u003e2.2.3 Comparative analysis\u003c/h3\u003e\n\u003cp\u003eFrom the annotated data, a functional analysis was performed, focused on the identification of genes and metabolic pathways associated with the biodegradation of aliphatic hydrocarbons. For this, the KEGG pathway corresponding to the fatty acid degradation pathway (map00071) was consulted. The comparative analysis between the strains was conducted based on the presence or absence of key genes for each pathway, variables, and number of repetitions, with a focus on enzymes catalyzing rate-limiting steps, such as monooxygenases (alkB, ladA, almA), cytochromes P450, and enzymes of the \u0026beta;-oxidation of fatty acids. The data were integrated by means of the visualization tools of the BV-BRC platform and organized into heatmaps and comparative tables with the objective of interpreting the functional organization and the biodegrading potential of the consortium. Furthermore, promoter region predictions for genes of interest were performed with the BPROM tool (Softberry), allowing to infer the potential for gene expression in an environmental context.\u003c/p\u003e\n\u003ch3\u003e2.3 Geochemical experiment\u003c/h3\u003e\n\u003ch4\u003e2.3.1 Experimental setup\u003c/h4\u003e\n\u003cp\u003eThe microorganisms used in the experiments were cultivated in Petri dishes for 48 hours. Then, they were transferred to 250 mL Erlenmeyer-type flasks containing liquid culture medium, with the objective of cultivating the isolates. After growth, the isolates were transferred to 25 mL of saline solution, and their concentrations were standardized using a microplate reader (Loccus). The microgeological experiment was set up in four 500 mL Erlenmeyer-type flasks containing: 300 mL of Bushnell-Haas Broth (BH) culture medium, poor in carbon; 9 mL of the standardized saline solution containing the bacterial strain in the experimental case; and 3 g of the carbon source. The crude oil used was supplied by the independent operator of onshore oil and gas exploration and production Petrorec\u0026ocirc;ncavo S.A., Field: Norte Fazenda Carua\u0026ccedil;u \u0026ndash; Rec\u0026ocirc;ncavo Basin, Depth: 1064.0m, Density @ 20\u0026deg;C: 0.8371, \u0026deg;API @ 20\u0026deg;C: 37.36. It is a paraffinic oil from the Rec\u0026ocirc;ncavo Basin, Bahia - Brazil. The Erlenmeyer-type flasks were then incubated at 30\u0026deg;C for a period of 31 days, during which oil samples were collected at times 0h (T0), 16 days (T1), and 31 days (T2). The samples were dissolved in dichloromethane (DCM), filtered through an open column of activated sodium sulfate, and stored.\u003c/p\u003e\n\u003ch3\u003e2.3.2 Analytical Methods\u003c/h3\u003e\n\u003cp\u003eThe Total Petroleum Hydrocarbons were analyzed by GC/FID on an Agilent 7890B chromatograph, split method, whose usage specifications are described in table 2. The quantification of the compounds was performed by the external standard method, based on peak area, within the time interval of 2.5 to 40 minutes. The data were acquired and processed using the ChemStation software (Agilent Technologies), with the sensitivity and rejection parameters adjusted to guarantee the accuracy in the identification and quantification of peaks.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Parameters of the chromatographic analysis by GC/FID.\u003c/h4\u003e\n\u003cp\u003eFinally, a statistical analysis of the chromatographic data was performed using the degradation rate as the response variable. Normality was verified by means of the Shapiro-Wilk test, followed by analysis of variance (ANOVA). When statistically significant differences were observed, Tukey\u0026apos;s test was applied as a post-hoc for the identification of multiple comparisons between groups. And all tests were conducted with a significance level of \u0026alpha; = 0.05, using the R statistical software.\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003ch2\u003e3.1 Complete genome sequencing and comparative analysis\u003c/h2\u003e\n\u003cp\u003eComplete genomic sequencing was performed for the nine bacterial strains integrating the microbial consortium, generating high-quality data and adequate coverage for de novo assembly of the genomes (Table 3). The quality control and assembly steps were conducted using the FastQC, Fastp, and Unicycler tools, and evaluated by means of QUAST, as described in the methodology.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eQuality parameters of the sequencing and assembly of the bacterial genomes.\u003c/h4\u003e\n\u003cp\u003eThe assembled genomes presented sizes varying from 4.09 to 13.44 Mbp, with GC content between 38.2% and 67.5%, and total number of CDS between 3,950 and 12,400, values compatible with those expected for environmental bacteria with broad metabolic plasticity. The variation observed between the genera reflects the structural diversity of the consortium, suggesting the coexistence of microorganisms with different physiological and metabolic strategies.\u003c/p\u003e\n\u003cp\u003eThe number of contigs varied from 31 to 254, with the lowest values observed in strains of the \u003cem\u003eBacillus\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBrevibacillus\u0026nbsp;\u003c/em\u003egenera, which indicates greater continuity and assembly quality\u0026mdash;reinforced by the N50 values. This structural heterogeneity is consistent with data from environmental bacterial genomes (Tully \u003cem\u003eet al\u003c/em\u003e., 2018; Parks \u003cem\u003eet al\u003c/em\u003e., 2020), and may be associated with the presence of mobile genetic elements and repetitive regions that hinder genomic closure in short-read sequencing (Orellana \u003cem\u003eet al\u003c/em\u003e., 2023).\u003c/p\u003e\n\u003cp\u003eIn general, the obtained assemblies were sufficiently robust for functional analyses and metabolic pathway reconstruction, allowing the identification of key genes associated with hydrocarbon degradation. The observed genomic diversity, reflected in the GC content and genetic size, reinforces the cooperative potential of the consortium in contaminated environments, since different physiological profiles favor adaptation to variable conditions and the functional division of catabolic routes (Pandolfo \u003cem\u003eet al\u003c/em\u003e., 2024; Rojas-Vargas \u003cem\u003eet al\u003c/em\u003e., 2024).\u003c/p\u003e\n\u003ch3\u003e3.1.1 Alkane metabolization pathway\u003c/h3\u003e\n\u003cp\u003eThe functional reconstruction of metabolic pathways revealed the broad representation of the fatty acid \u0026beta;-oxidation route (KEGG map00071) among the nine bacterial strains of the consortium. The mapping of EC numbers (Figure 1) indicated the complete presence of the main enzymes involved in the oxidative catabolism of alkanes, including acyl-CoA synthetase (EC 6.2.1.3), acyl-CoA dehydrogenase (EC 1.3.8.1), enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), and thiolase (EC 2.3.1.16). These enzymes participate in the sequential steps of oxidation and cleavage of fatty acids down to acetyl-CoA, evidencing the complete functioning of the downstream \u0026beta;-oxidation route.\u003c/p\u003e\n\u003cp\u003eFurthermore, genes related to the initial activation of alkanes were identified, such as alkane 1-monooxygenase (EC 1.14.15.3), Cytochrome P450 (1.14.14.1), alcohol dehydrogenase (EC 1.1.1.1), and aldehyde dehydrogenase (EC 1.2.1.3), responsible for the conversion of alkanes into alcohols and subsequently into fatty acids. These results indicate a complete functional arrangement, spanning from the initial oxidation to the mineralization of the aliphatic compounds. (Guibert \u003cem\u003eet al\u003c/em\u003e., 2015; Wang \u003cem\u003eet al\u003c/em\u003e., 2022)\u003c/p\u003e\n\u003cp\u003eThe consortium demonstrated the presence of genes for different monooxygenases, including those specific for short chains, such as AlkB (\u003cem\u003ealkB1_2, alkM\u003c/em\u003e), and the more versatile ones, such as Cytochrome P450 (102A3, 102A2, 102A5), both responsible for the terminal hydroxylation of linear alkanes. In addition to those of \u0026beta;-oxidation distributed among the different strains, suggesting interspecies metabolic cooperation and the existence of a biochemical strategy for the catabolism of aliphatic compounds present in petroleum. The lineages of the \u003cem\u003ePseudomonas\u0026nbsp;\u003c/em\u003eand \u003cem\u003eStenotrophomonas\u0026nbsp;\u003c/em\u003egenera (PD5, PD6, RC6) were highlighted, due to the presence of alkB, constantly used as a parameter for evaluating the alkane degradation potential in biodegrading organisms, while Cytochrome CYP102A5 is normally associated with the \u003cem\u003eBacillus\u0026nbsp;\u003c/em\u003egenus (OH4 and RB4), as a versatile and promiscuous enzyme in its targets, capable of metabolizing non-natural substrates, including polyethylene film (Tarara; Ahuja; Mellies, 2025; Hidalgo \u003cem\u003eet al\u003c/em\u003e., 2020; Liu \u003cem\u003eet al\u003c/em\u003e., 2015; Vieto \u003cem\u003eet al\u003c/em\u003e., 2021).\u003c/p\u003e\n\u003cp\u003eThe redundancy of genes associated with alkane metabolism in multiple strains, combined with the presence of promoters and regulatory elements linked to oxidative stress response, suggests an evolutionary adaptation to the contaminated environment, in which continuous exposure to hydrocarbons favored the selection of specialized lineages (Brzeszcz; Kaszycki, 2018). This scenario reinforces the hypothesis of functional distribution of catabolic routes within the consortium, maximizing its degrading efficiency.\u003c/p\u003e\n\u003ch2\u003e3.2 Total Petroleum Hydrocarbons\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eFor the comparative analysis of the individual degradative potential of the studied strains, previous results representative of the degradative capacity of the complete consortium under standardized conditions were used. In conjunction with the negative control, in which there was no inoculation, it was possible to infer that any contrast obtained between the samples results directly from microbial action. Furthermore, as all experimental conditions were standardized regarding the initial petroleum and inoculum concentration, a direct comparison between the treatments becomes viable (Fig. 2)\u003c/p\u003e\n\u003cp\u003eThe analysis of the microbial consortium revealed a high degradation rate throughout the experiment (T0\u0026ndash;30), with a general average of 46%, reaching 52% just in the intermediate fraction in the first 15 days. This pattern reinforces the fundamental role of consortia in the synergistic degradation of hydrocarbons, especially in the initial phases of bioremediation, when there is greater nutrient availability and less accumulation of toxic metabolites (Yu \u003cem\u003eet al\u003c/em\u003e., 2022). In the last 15 days of the experiment, a reduction in the degradation rate for the intermediate fraction (29%) was observed, possibly as a consequence of the microbial decline phase and the limiting growth conditions in the system, such as metabolite accumulation, competition for nutrients, and a decrease in substrate bioavailability.\u003c/p\u003e\n\u003cp\u003eIn the case of the individual analyses, none of the isolated strains showed global performance equivalent to that of the complete consortium, an expected result, considering that crude oil is a complex mixture of hydrocarbons with different molecular weights, which restricts the action of isolated microorganisms (Li \u003cem\u003eet al\u003c/em\u003e., 2016). Even so, relevant specific behaviors were observed, especially when considering particular fractions of the degraded compounds.\u003c/p\u003e\n\u003cp\u003eThe strains PD5, RC6, and PD6, corresponding to the Pseudomonas and Stenotrophomonas genera, stood out as rapid degraders, with a bimodal profile, thanks to the presence of the alkane monooxygenase identified in the genomic analyses. This profile was characterized by high degradation rates for short-chain (\u0026lt;C13) and long-chain (\u0026gt;C26) alkanes, contrasting with a sharp drop in efficiency for the intermediate range. Such behavior suggests that these strains possess multiple enzymatic systems, with distinct specificities for different chain lengths. (Passler; Ditchkoff; Walz, 2016; Li; Pan; Ma, 2019)\u003c/p\u003e\n\u003cp\u003eOn the other hand, strains OH4 and RB4, belonging to the Bacillus genus, demonstrated a clear behavior of specialization in medium/long chains and late degradation (Fig. 4). With highlight to OH4 which initially presented low degradation of short and medium-chain alkanes, possibly associated with its slower metabolism and the absence of the AlkB enzyme. In contrast, it registered a continuous and later degradation of the heavy fractions, reaching 67% in the last 15 days of the experiment. This pattern of preferential and delayed consumption of complex hydrocarbons is typical of biosurfactant-producing microorganisms, such as surfactin, where the bacterium first produces and secretes the biosurfactants to emulsify the waxy alkanes, making them accessible to its degrading enzymes. (Dai \u003cem\u003eet al\u003c/em\u003e., 2020; Lima \u003cem\u003eet al\u003c/em\u003e., 202; Thirumurugan \u003cem\u003eet al\u003c/em\u003e., 2023).\u003c/p\u003e\n\u003cp\u003eIt is important to highlight that the apparent absence of degradation, or even the observed negative rates, represented in white to facilitate the visualization of effective degradation, of some intermediate-chain alkanes do not represent the biological production of these compounds. This phenomenon, also known as floating degradation, anomalous or negative values, is caused due to a methodological artifact resulting from the intense biofilm formation at the oil-water interface. (LI \u003cem\u003eet al\u003c/em\u003e., 2024) With the development of this biofilm, the system becomes heterogeneous, and the residual oil can be sequestered in biomass aggregates. Thus, the collection with a spatula at time 2 resulted in unrepresentative and over-concentrated samples in these aggregates, producing artificially higher values than those from the previous period. In this way, the negative rates reflect sampling limitations in a complex multiphasic system, and not necessarily a metabolic inefficiency for those fractions, a factor evidenced by the complex genomic structure presented by the strains for their degradation. (Johnsen; Wick; Harms, 2004; Neu \u003cem\u003eet al\u003c/em\u003e., 2019; Joannis; Delia; Riba, 1998)\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe study successfully demonstrated that the high alkane degradation efficiency of the target microbial consortium in paraffinic oil is occasioned by functional synergy and by an ecological niche partitioning among its members. The integration of genomic and geochemical data allowed to confirm the consortium's catabolic potential and to assign specific roles to the most representative genera, as Pseudomonas and Stenotrophomonas acted as primary and rapid degraders, while Bacillus specialized in the slower and later degradation of the long-chain recalcitrant compounds, probably mediated by biosurfactant production.\u003c/p\u003e \u003cp\u003eThe geochemical results corroborated the genomic inferences and evidenced the need for a critical interpretation in multiphasic systems, since anomalous or negative degradation values reflect methodological artifacts resulting from biofilm formation and sampling heterogeneity, and not a metabolic limitation. Together, the findings reinforce that the application of microbial consortia with functional diversity constitutes a robust and adaptable strategy for the bioremediation of complex contaminants, such as petroleum alkanes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.G.B.S.N. conceptualized the study, performed data curation, conducted formal analysis, carried out the investigation, wrote the original draft, and reviewed and edited the final manuscript. A.A.M.N. contributed to the study conceptualization, performed data curation, and conducted formal analysis of specific data. L.A.O. assisted with data analysis, prepared figures, and reviewed and edited the final manuscript. D.F.L. contributed to the conceptualization, provided supervision, and critically reviewed and edited the manuscript. A.F.S.Q. and O.M.C.O. provided overall supervision, acquired funding, and contributed resources for the project. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge the support of Shell Brasil through the project \"Research in Geomicrobiology: Microbial Biotechnology Applied to Petroleum Bioremediation and Recovery of Degraded Areas at the Institute of Geosci\u0026ecirc;ncias of the Federal University of Bahia (UFBA) \u0026ndash; GEOQPETROL \u0026ndash; GEOMICRO\" \u0026ndash; ANP project 20720-9, and recognize the strategic importance of the support granted by the National Agency of Petroleum, Natural Gas and Biofuels (ANP), through the regulation of the R\u0026amp;D investment clause. To the laboratories LEPETRO \u0026ndash; Excellence in Petroleum Geochemistry, Energy and Environment \u0026ndash; and IBTEC \u0026ndash; Institute of Biotechnology. This study was financed, in part, by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) \u0026ndash; Brazil [Financing Code 001].\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAHMED, M. \u003cem\u003eet al\u003c/em\u003e. Optimizing Synergistic Metabolism in Xenobiotic Biodegradation: Engineering a Pseudomonas putida Based Microbial Consortium. \u003cstrong\u003eAmerican Journal of Life Science and Innovation\u003c/strong\u003e, 3(1), 1\u0026ndash;7, 27 dez. 2023. http://dx.doi.org/10.54536/ajlsi.v3i1.2236\u003c/li\u003e\n\u003cli\u003eAMINI, Fatemeh; GIYAHCHI, Minoo; MOGHIMI, Hamid. Bioremediation of Petroleum Contamination by Microorganisms: Role of Microbial Communities and Applications. In: \u003cstrong\u003eRoyal Society of Chemistry\u003c/strong\u003e, 2024. p. 136\u0026ndash;170. DOI: 10.1039/bk9781837673131-00136.\u003c/li\u003e\n\u003cli\u003eBANIASADI, Mahsa; MOUSAVI, Seyyed Mohammad. A Comprehensive Review on the Bioremediation of Oil Spills. In: Springer, \u003cstrong\u003eSingapore\u003c/strong\u003e, 2018. p. 223\u0026ndash;254. DOI: 10.1007/978-981-13-1840-5_10.\u003c/li\u003e\n\u003cli\u003eBRZESZCZ, Joanna; KASZYCKI, Paweł. Aerobic bacteria degrading both n-alkanes and aromatic hydrocarbons: an undervalued strategy for metabolic diversity and flexibility. \u003cstrong\u003eBiodegradation\u003c/strong\u003e, v. 29, n. 4, p. 359\u0026ndash;407, 2018. DOI: 10.1007/S10532-018-9837-X.\u003c/li\u003e\n\u003cli\u003eC. JOANNIS; DELIA, M. L.; RIBA, J. P. Comparison of four methods for quantification of biofilms in biphasic cultures. \u003cstrong\u003eBiotechnology Techniques\u003c/strong\u003e, v. 12, n. 10, p. 777\u0026ndash;782, 1 jan. 1998. 10.1023/A:1008835811731\u003c/li\u003e\n\u003cli\u003eCHE, Shun; MEN, Yujie. Synthetic microbial consortia for biosynthesis and biodegradation: promises and challenges. \u003cstrong\u003eJournal of Industrial Microbiology \u0026amp; Biotechnology\u003c/strong\u003e, v. 46, n. 9, p. 1343\u0026ndash;1358, 2019. DOI: 10.1007/S10295-019-02211-4.\u003c/li\u003e\n\u003cli\u003eCOSTA, C. D. S. et al. Produ\u0026ccedil;\u0026atilde;o de biossurfactante por microrganismos isolados de \u0026aacute;reas contaminadas com petr\u0026oacute;leo. \u003cstrong\u003eRevista Brasileira de Bioci\u0026ecirc;ncias\u003c/strong\u003e, v. 5, supl. 1, p. 69\u0026ndash;71, 2007.\u003c/li\u003e\n\u003cli\u003eDAI, X. \u003cem\u003eet al\u003c/em\u003e. Heavy Oil Biodegradation by Mixed Bacterial Consortium of Biosurfactant-Producing and Heavy Oil-Degrading Bacteria. \u003cstrong\u003ePolish Journal of Environmental Studies\u003c/strong\u003e, v. 30, n. 1, p. 71\u0026ndash;80, 3 set. 2020. https://doi.org/10.15244/pjoes/120769.\u003c/li\u003e\n\u003cli\u003eGAO, Y. \u003cem\u003eet al\u003c/em\u003e. Response Characteristics of the Community Structure and Metabolic Genes of Oil-Recovery Bacteria after Targeted Activation of Petroleum Hydrocarbon-Degrading Bacteria in Low-Permeability Oil Reservoirs. \u003cstrong\u003eACS Omega\u003c/strong\u003e, v. 9, n. 31, p. 33448\u0026ndash;33458, 27 jul. 2024. 10.1021/acsomega.3c10334.\u003c/li\u003e\n\u003cli\u003eGUIBERT, L. M. \u003cem\u003eet al\u003c/em\u003e. Diverse Bacterial Groups Contribute to the Alkane Degradation Potential of Chronically Polluted Subantarctic Coastal Sediments. \u003cstrong\u003eMicrobial Ecology\u003c/strong\u003e, v. 71, n. 1, p. 100\u0026ndash;112, 7 nov. 2015. 10.1007/S00248-015-0698-0.\u003c/li\u003e\n\u003cli\u003eGUREVICH, Alexey \u003cem\u003eet al\u003c/em\u003e. QUAST: quality assessment tool for genome assemblies. \u003cstrong\u003eBioinformatics\u003c/strong\u003e, v. 29, n. 8, p. 1072\u0026ndash;1075, 2013. DOI: https://doi.org/10.1093/bioinformatics/btt086.\u003c/li\u003e\n\u003cli\u003eHIDALGO, K. J. \u003cem\u003eet al\u003c/em\u003e. AlkB monooxygenases as key enzymes in alkane degradation: distribution and diversity in hydrocarbon-contaminated environments. \u003cstrong\u003eJournal of Applied Microbiology\u003c/strong\u003e, v. 129, n. 6, p. 1440\u0026ndash;1455, 2020. DOI: https://doi.org/10.1111/jam.14667.\u003c/li\u003e\n\u003cli\u003eJOHNSEN, A. R.; WICK, L. Y.; HARMS, H. Principles of microbial PAH-degradation in soil. \u003cstrong\u003eEnvironmental Pollution\u003c/strong\u003e, v. 133, n. 1, p. 71\u0026ndash;84, jan. 2005. https://doi-org.ez10.periodicos.capes.gov.br/10.1016/j.envpol.2004.04.015\u003c/li\u003e\n\u003cli\u003eKHAN, Saimah; TRIPATHI, Ayush Kumar; SRIVASTAVA, Rohan; \u003cem\u003eet al\u003c/em\u003e. Bioremediation of Petroleum Contamination: A Short Review. \u003cstrong\u003eEcological Questions\u003c/strong\u003e, 33(2), 1\u0026ndash;18, 2022. http://dx.doi.org/10.12775/EQ.2022.012\u003c/li\u003e\n\u003cli\u003eLI, L. \u003cem\u003eet al\u003c/em\u003e. Dynamic responses in Pseudomonas stutzeri M3 bioaugmentation of crude-oil-contaminated soil: Hydrocarbons, microbial community structures, and functional genes. \u003cstrong\u003eJournal of environmental chemical engineering\u003c/strong\u003e, v. 12, n. 1, p. 111863\u0026ndash;111863, 4 jan. 2024. https://doi.org/10.1016/j.jece.2023.111863\u003c/li\u003e\n\u003cli\u003eLI, Y. P.; PAN, J. C.; MA, Y. L. Elucidation of multiple alkane hydroxylase systems in biodegradation of crude oil n ‐alkane pollution by Pseudomonas aeruginosa DN1. \u003cstrong\u003eJournal of Applied Microbiology\u003c/strong\u003e, v. 128, n. 1, p. 151\u0026ndash;160, 1 out. 2019. https://doi.org/10.1111/jam.14470.\u003c/li\u003e\n\u003cli\u003eLI, xinfei; ZHAO, lin; ADAM, Mohamed. Biodegradation of marine crude oil pollution using a salt-tolerant bacterial consortium isolated from Bohai Bay, China. \u003cstrong\u003eMarine Pollution\u003c/strong\u003e Bulletin, v. 105, n. 1, p. 43\u0026ndash;50, 2016. https://doi.org/10.1016/j.marpolbul.2016.02.073.\u003c/li\u003e\n\u003cli\u003eLIMA, F. T. S. \u003cem\u003eet al\u003c/em\u003e. Cons\u0026oacute;rcio microbiano com capacidade para degrada\u0026ccedil;\u0026atilde;o de petr\u0026oacute;leo. Pedido de patente BR 10 2021 002341 4. Instituto Nacional da Propriedade Industrial \u0026ndash; INPI, 2021. \u003c/li\u003e\n\u003cli\u003eLIMA, S. D. \u003cem\u003eet al\u003c/em\u003e. Isolation and characterization of hydrocarbon-degrading bacteria from gas station leaking-contaminated groundwater in the Southern Amazon, Brazil. \u003cstrong\u003efigshare\u003c/strong\u003e, 26 mar. 2021. 10.6084/m9.figshare.14322077.\u003c/li\u003e\n\u003cli\u003eLIU, C. \u003cem\u003eet al\u003c/em\u003e. Diversity and distribution of alkB genes in bacteria capable of degrading medium- and long-chain n-alkanes in oil-contaminated environments. \u003cstrong\u003eInternational Biodeterioration \u0026amp; Biodegradation\u003c/strong\u003e, v. 94, p. 116\u0026ndash;122, 2015. DOI: https://doi.org/10.1016/j.ibiod.2014.07.016.\u003c/li\u003e\n\u003cli\u003eMAHJOUBI, M. \u003cem\u003eet al\u003c/em\u003e. Bacterial consortia as a promising strategy for enhanced petroleum hydrocarbons degradation. \u003cstrong\u003eEnvironmental Science and Pollution Research\u003c/strong\u003e, v. 30, p. 53973\u0026ndash;53992, 2023. DOI: https://doi.org/10.1007/s11356-023-27138-5.\u003c/li\u003e\n\u003cli\u003eNEU, L. \u003cem\u003eet al\u003c/em\u003e. Small-Scale Heterogeneity in Drinking Water Biofilms\u003cstrong\u003e. Frontiers in Microbiology\u003c/strong\u003e, v. 10, 29 out. 2019. https://doi.org/10.3389/fmicb.2019.02446\u003c/li\u003e\n\u003cli\u003eORELLANA, L. H. \u003cem\u003eet al\u003c/em\u003e. Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies. \u003cstrong\u003eResearch Square\u003c/strong\u003e, 2023. DOI: https://doi.org/10.21203/rs.3.rs-2357036/v1.\u003c/li\u003e\n\u003cli\u003ePANDOLFO, Emiliana \u003cem\u003eet al\u003c/em\u003e. Metagenomic analyses of a consortium for the bioremediation of hydrocarbons polluted soils. \u003cstrong\u003eAMB Express\u003c/strong\u003e, v. 14, n. 1, 2024. DOI: 10.1186/s13568-024-01764-7.\u003c/li\u003e\n\u003cli\u003ePARK, C.; PARK, W. Survival and Energy Producing Strategies of Alkane Degraders Under Extreme Conditions and Their Biotechnological Potential. \u003cstrong\u003eFrontiers in Microbiology\u003c/strong\u003e, v. 9, 25 maio 2018. https://doi.org/10.3389/fmicb.2018.01081.\u003c/li\u003e\n\u003cli\u003ePARKS, D. H. \u003cem\u003eet al\u003c/em\u003e. A complete domain-to-species taxonomy for Bacteria and Archaea. \u003cstrong\u003eNature Biotechnology\u003c/strong\u003e, v. 38, p. 1079\u0026ndash;1086, 2020. DOI: https://doi.org/10.1038/s41587-020-0501-8.\u003c/li\u003e\n\u003cli\u003ePARUS, A. \u003cem\u003eet al\u003c/em\u003e. Basic principles for biosurfactant-assisted (bio)remediation of soils contaminated by heavy metals and petroleum hydrocarbons \u0026ndash; A critical evaluation of the performance of rhamnolipids. \u003cstrong\u003eJournal of Hazardous Materials\u003c/strong\u003e, v. 443, p. 130171, 2023. DOI: https://doi.org/10.1016/j.jhazmat.2022.130171.\u003c/li\u003e\n\u003cli\u003ePASSLER, T.; DITCHKOFF, S. S.; WALZ, P. H. Bovine Viral Diarrhea Virus (BVDV) in White-Tailed Deer (Odocoileus virginianus). \u003cstrong\u003eFrontiers in Microbiology\u003c/strong\u003e, v. 7, 20 jun. 2016. https://doi.org/10.3389/fmicb.2016.00945.\u003c/li\u003e\n\u003cli\u003eRAWAT, Madhu; RANGARAJAN, Srishti. Omics approaches for elucidating molecular mechanisms of microbial bioremediation. In: \u003cstrong\u003eAcademic Press\u003c/strong\u003e, 2019. p. 191\u0026ndash;203. DOI: 10.1016/B978-0-12-818307-6.00011-1.\u003c/li\u003e\n\u003cli\u003eROJAS-VARGAS, Jorge \u003cem\u003eet al\u003c/em\u003e. A comparative genomic study of a hydrocarbon-degrading marine bacterial consortium. \u003cstrong\u003ePLOS ONE\u003c/strong\u003e, v. 19, n. 8, p. e0303363, 2024. DOI: 10.1371/journal.pone.0303363.\u003c/li\u003e\n\u003cli\u003eShifu Chen, Yanqing Zhou, Yaru Chen, Jia Gu; fastp: an ultra-fast all-in-one FASTQ preprocessor, \u003cstrong\u003eBioinformatics\u003c/strong\u003e, Volume 34, Issue 17, 1 September 2018, Pages i884\u0026ndash;i890, https://doi.org/10.1093/bioinformatics/bty560.\u003c/li\u003e\n\u003cli\u003eSTATISTA. Global oil products demand outlook 2050. Statista, 2025. DOI: https://www.statista.com/statistics/282774/global-product-demand-outlook-worldwide/.\u003c/li\u003e\n\u003cli\u003eTARARA, M.; AHUJA, S.; MELLIES, J. L. A Cytochrome P450 Facilitates Polyethylene Metabolism in a Microbial Community. International Journal of Molecular Sciences, v. 26, n. 18, p. 8775\u0026ndash;8775, 9 set. 2025. https://doi.org/10.3390/ijms26188775 \u003c/li\u003e\n\u003cli\u003eTHIRUMURUGAN, D. \u003cem\u003eet al\u003c/em\u003e. Impact of biosurfactant produced by Bacillus spp. on biodegradation efficiency of crude oil and anthracene. \u003cstrong\u003eChemosphere\u003c/strong\u003e, 344, 140340, 1 dez. 2023. https://doi.org/10.1016/j.chemosphere.2023.140340\u003c/li\u003e\n\u003cli\u003eTULLY, B. J. \u003cem\u003eet al\u003c/em\u003e. The reconstruction of 2,631 draft metagenome-assembled genomes from the global oceans. \u003cstrong\u003eScientific Data\u003c/strong\u003e, v. 5, p. 170203, 2018. DOI: https://doi.org/10.1038/sdata.2017.203.\u003c/li\u003e\n\u003cli\u003eUENO, A. \u003cem\u003eet al\u003c/em\u003e. Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation. World Journal of \u003cstrong\u003eMicrobiology and Biotechnology\u003c/strong\u003e, v. 23, n. 12, p. 1739\u0026ndash;1745, 2007. DOI: https://doi.org/10.1007/s11274-007-9423-6. \u003c/li\u003e\n\u003cli\u003eVERMA, Jay P.; JAISWAL, Durgesh K. Book Review: Advances in Biodegradation and Bioremediation of Industrial Waste. \u003cstrong\u003eFrontiers in Microbiology\u003c/strong\u003e, 6, 2016. https://doi.org/10.3389/fmicb.2015.01555\u003c/li\u003e\n\u003cli\u003eVIETO, Sof\u0026iacute;a \u003cem\u003eet al\u003c/em\u003e. The potential of Pseudomonas for bioremediation of oxyanions. \u003cstrong\u003eEnvironmental Microbiology Reports\u003c/strong\u003e, v. 13, n. 6, p. 773\u0026ndash;789, 2021. DOI: 10.1111/1758-2229.12999.\u003c/li\u003e\n\u003cli\u003eWANG, Q. \u003cem\u003eet al\u003c/em\u003e. Community-wide distribution of generalist catabolic genes in hydrocarbon-degrading microbial consortia. \u003cstrong\u003eFrontiers in Microbiology\u003c/strong\u003e, v. 14, p. 1183407, 2023. DOI: https://doi.org/10.3389/fmicb.2023.1183407.\u003c/li\u003e\n\u003cli\u003eWANG, S. \u003cem\u003eet al\u003c/em\u003e. A novel alkane monooxygenase (alkB) clade revealed by massive genomic survey and its dissemination association with IS elements. \u003cstrong\u003ePeerJ\u003c/strong\u003e, v. 10, p. e14147\u0026ndash;e14147, 28 set. 2022. 10.7717/peerj.14147.\u003c/li\u003e\n\u003cli\u003eYOUNG, John K. Bioremediation of Oil Spill Cleanup: Case Studies of Two Major Oil Spills. \u003cstrong\u003eSustainable Environment\u003c/strong\u003e, p. 117\u0026ndash;133, 2023. DOI: 10.1007/978-981-19-8464-8_7.\u003c/li\u003e\n\u003cli\u003eYU, T. \u003cem\u003eet al\u003c/em\u003e. Microbial community succession during crude oil-degrading bacterial enrichment cultivation and construction of a degrading consortium. \u003cstrong\u003eFrontiers in Microbiology\u003c/strong\u003e, 13, 4 nov. 2022. https://doi.org/10.3389/fmicb.2022.1044448\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Bioremediation, Microbial Consortium, Petroleum, Alkanes, Functional Genomics","lastPublishedDoi":"10.21203/rs.3.rs-8481249/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8481249/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe constant demand for petroleum and the recurrence of environmental accidents led to the development of sustainable strategies for the treatment of contaminated areas, such as bioremediation. Alkanes, being one of the most abundant and challenging fractions of petroleum in terms of biodegradation, demand microorganisms with high metabolic specialization and the incorporation of bacterial consortia, which possess high efficiency due to inter-species synergy. This study had as its objective to integrate genomic and geochemical analyses to unveil the potential and mechanisms of alkane degradation by nine bacterial strains (\u003cem\u003eBacillus, Brevibacillus, Pseudomonas\u003c/em\u003e, and \u003cem\u003eStenotrophomonas\u003c/em\u003e) isolated from a contaminated mangrove. The genomic sequencing and functional annotation revealed the presence of key genes and enzymes associated with alkane activation and oxidation, distributed among different bacterial genera. The geochemical analyses by GC-FID demonstrated the superiority of the complete consortium and the specificity of the strains, with highlight to \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eStenotrophomonas\u003c/em\u003e in the rapid degradation of the light and intermediate fractions, and \u003cem\u003eBacillus\u003c/em\u003e in the slower and more efficient degradation of the heavy fractions. Furthermore, the study evidenced the complexity of geochemical data analysis in multiphasic systems, where the negative variations were identified as methodological artifacts resulting from biofilm formation and a consequent heterogeneous sampling. The integration of genomic and geochemical results revealed itself to be a powerful tool to elucidate the microbial interactions and to understand the specific alkane degradation mechanisms of each genus in the bioremediation process.\u003c/p\u003e","manuscriptTitle":"Metabolic specialization and synergy in alkane degradation by a microbial consortium: an integrated genomic and geochemical approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 13:51:25","doi":"10.21203/rs.3.rs-8481249/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-03T02:31:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-01T18:18:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-01T16:39:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-14T11:49:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181908796650096796601673680853367041345","date":"2026-01-14T09:56:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289301802059461169608393851755422344191","date":"2026-01-12T01:46:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265859884274967257704924175796769318530","date":"2026-01-11T20:08:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-08T23:29:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-31T19:23:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-31T10:29:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Current Microbiology","date":"2025-12-30T12:14:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5df1be81-d267-46dd-8e31-cfbadfa5fe51","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-18T04:54:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 13:51:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8481249","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8481249","identity":"rs-8481249","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}