Assessing hydrocarbon degradation capacity of Isoptericola peretonis sp. nov. and related species: a comparative study

Frontiers in Microbiology 01 frontiersin.org Assessing hydrocarbon degradation capacity of Isoptericola peretonis sp. nov. and related species: a comparative study ÀngelaVidal-Verdú 1*, AdrielLatorre-Pérez 2, JavierPascual 2, RuthMañes-Collado 1, AitanaNevot-Terraes 1 and ManuelPorcar 1,2* 1 Institute for Integrative Systems Biology I2SysBio (Universitat de València-CSIC), Paterna, Spain, 2 Darwin Bioprospecting Excellence SL. Parc Científic Universitat de València, Paterna, Spain Since the beginning of their production and use, fossil fuels have affected ecosystems, causing significant damage to their biodiversity. Bacterial bioremediation can provide solutions to this environmental problem. In this study, the new species Isoptericola peretonis sp. nov. 4D.3 T has been characterized and compared to other closely related species in terms of hydrocarbon degradation and biosurfactant production by invitro and in silico analyses. Biosurfactants play an important role in microbial hydrocarbon degradation by emulsifying hydrocarbons and making them accessible to the microbial degradation machinery. The tests performed showed positive results to a greater or lesser degree for all strains. In the synthesis of biosurfactants, all the strains tested showed biosurfactant activity in three complementary assays (CTAB, hemolysis and E24%) and rhamnolipid synthesis genes have been predicted in silico in the majority of Isoptericola strains. Regarding hydrocarbon degradation, all the Isoptericola strains analyzed presented putative genes responsible for the aerobic and anaerobic degradation of aromatic and alkane hydrocarbons. Overall, our results highlight the metabolic diversity and the biochemical robustness of the Isoptericola genus which is proposed to beof interest in the field of hydrocarbon bioremediation. KEYWORDS Isoptericola, new species, hydrocarbon degradation, biosurfactants, bioremediation Introduction Hydrocarbons, derived primarily from fossil fuels, are one of the major pollutants on Earth and are ubiquitous due to industrial activities, transportation, and accidental spills. Given their hydrophobic nature, these are very recalcitrant compounds that persist into ecosystems and considerably pollute soils, aquifers, and marine environments (Daher Hazaimeh and Ahmed, 2021; Lien etal., 2014; Wang etal., 2021). Therefore, microbial communities from polluted sites are shaped by the prolonged exposure to hydrocarbons. On top of that, not only natural environments are affected, which have been the main focus for studies on hydrocarbon pollution (Acosta-González etal., 2015; Cabral etal., 2021; Jimoh etal., 2022), but also urban artificial environments such as gas stations, fuel tanks and car tank components that have been shown to harbor adapted microbial communities to the presence of fuels (Bücker etal., 2014; da Fonseca etal., 2019; Lima etal., 2019; Vidal-Verdú etal., 2022). OPEN ACCESS EDITED BY Fu-Li Li, Chinese Academy of Sciences (CAS), China REVIEWED BY Balaram Mohapatra, Gujarat Biotechnology University, India Priscilla Zwiercheczewski De Oliveira, University of Mons, Belgium *CORRESPONDENCE Àngela Vidal-Verdú [email protected] Manuel Porcar [email protected] RECEIVED 26 July 2024 ACCEPTED 22 January 2025 PUBLISHED 05 February 2025 CITATION Vidal-Verdú À, Latorre-Pérez A, Pascual J, Mañes-Collado R, Nevot-Terraes A and Porcar M (2025) Assessing hydrocarbon degradation capacity of Isoptericola peretonis sp. nov. and related species: a comparative study. Front. Microbiol. 16:1471121. doi: 10.3389/fmicb.2025.1471121 COPYRIGHT © 2025 Vidal-Verdú, Latorre-Pérez, Pascual, Mañes-Collado, Nevot-Terraes and Porcar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 05 February 2025 DOI 10.3389/fmicb.2025.1471121 Vidal-Verdú et al. 10.3389/fmicb.2025.1471121 Frontiers in Microbiology 02 frontiersin.org In this context, bioremediation and bioprospecting arise together to address this critical issue. On the one hand, bioremediation, understood as the intervention aimed at alleviating pollution by exploitation of biological activities either by natural attenuation, bio-stimulation or bio-augmentation (de Lorenzo, 2008), is a cost-effective, environmentally friendly and minimally invasive technique when applied in situ compared to traditional ex situ contaminant removal techniques (Orellana etal., 2017; Perelo, 2010). On the other hand, hydrocarbondegrading microorganisms occur naturally in ecosystems, constituting less than 1% of the total microbial communities under normal conditions. When there is an increase in the presence of petroleum hydrocarbons, they can account for up to 10% of the total microbial communities (Atlas, 1995). Hence, bioprospecting of oil-polluted sites is a directed strategy aimed at discovering hydrocarbon-degrading microorganisms or activities for bioremediation. In addition, surface-active molecules (biosurfactants) play a key role by emulsifying and thereby enhancing the bioavailability of highly hydrophobic molecules. This process is critical for microbial access and subsequent degradation of hydrocarbons, highlighting the importance of harnessing bacterial biosurfactant production for enhanced bioremediation projects (Elumalai etal., 2021). Hydrocarbon-polluted environments, especially artificial urban niches, which have not been studied much to date, may constitute a biodiversity hotspot. This proved true in our previous bioprospecting study of the car tank lid in which the environmental strain 4D.3 T , tentatively identified as Isoptericola sp., was found to degrade diesel (Vidal-Verdú etal., 2022). To the extent of our knowledge, before our finding, only the strain I. chiayiensis 103-Na4 had been characterized in certain detail as biosurfactant producer and being able to degrade crude oil (Lee etal., 2018a), phenanthrene and pyrene (Lee etal., 2018b). Other studies had also pointed out the ability of other Isoptericola strains to play a role in hydrocarbon degradation (Ahmad etal., 2021; Al-Mailem etal., 2015; Polivtseva etal., 2020; Radwan etal., 2010). Along with this fuel catabolic potential, strains from this genus have also been described as possessing industrially relevant features such as cellulolytic (Bae etal., 2021), alginate lyase (Chen etal., 2018), and chitinase (Wu etal., 2011) enzymatic activities as well as antimicrobial (Girão etal., 2019; Kaushik etal., 2021) and plantgrowth promoting capabilities (Alghamdi et al., 2023) and polyhydroxybutyrate (PHB) synthesis (Krishnan etal., 2021). The genus Isoptericola was first described by Stackebrandt etal. (2004) who reclassified Cellulosimicrobium variable MX5 T , a cellulolytic and xylanolytic strain that differed substantially from the type species of the genus Cellulosimicrobium (Bakalidou etal., 2002), to I. variabilis. At the time of writing, this genus is composed of 14 species, 11 with validly published names, 1 and belongs to the family Promicromonosporaceae and the Actinomycetes class. The isolation environments of the members of this genus have been very diverse, including the hindgut of a termite (Bakalidou etal., 2002), a Roman catacomb (Groth etal., 2005), the root tissue of a cucumber (Kämpfer etal., 2016) and, mainly, soil samples (Bing etal., 2024; Huang etal., 2012; Kaur N. etal., 2014; Kumar etal., 2021; Tseng etal., 2011; Yoon etal., 2006) and saline sediments (Guan etal., 2013; Ming etal., 2020; OuYang etal., 2023; Wu etal., 2010; Zhang etal., 2005). 1 https://lpsn.dsmz.de/genus/isoptericola Therefore, wenow describe here, by a polyphasic approach, a new species of the genus Isoptericola isolated from an urban hydrocarbonpolluted environment, the car tank lid, for which the name Isoptericola peretonis sp. nov. is proposed. On top of that, wealso compare by both experimentally and genomic analysis, the new species and several species from the genus Isoptericola, on their ability to produce biosurfactants by in vitro assays as well as to degrade various hydrocarbons following in silico analyses, in order to characterize the bioremediation potential of this poorly known genus. This study reveals that species from the genus Isoptericola have outstanding potential as chassis for biotechnological applications, mainly on bioremediation, especially hydrocarbon remediation, since they show high physicochemical robustness and great metabolic diversity. Materials and methods Strain isolation Strain 4D.3 T was isolated during a previous study of the microbiota on a car tank lid and its ability to degrade diesel (Vidal-Verdú etal., 2022). Briefly, sampling was carried out in the parking areas of the Institute for Integrative Systems Biology (I2SysBio; Paterna, Spain). Strain 4D.3 T was isolated after an enrichment process using growth on a basal medium supplemented with diesel as the main carbon source as a selection pressure. The enriched culture consisted of the inoculation of 3 mL of Minimal Medium (Composition in g/L: 2 NaNO 3 , 1 K 2 HPO 4 , 0.5 MgSO 4 ·7H 2 O, 0.5 KCl, 0.5 sucrose) supplemented with diesel 10% (v/v) with the dust sampled from a car tank lid. Cultures were incubated at 30°C and 120 rpm for 1 week. Afterwards, 25 μL of the culture were transferred to new fresh media and this process was repeated weekly for 4 weeks. After the enrichment period, several dilutions of the culture were carried out in Phosphate Buffer Saline (PBS, composition in g/L: 8.0 NaCl, 0.2 KCl, 1.44 Na 2 HPO 4 , 0.24 KH 2 PO 4 ; adjusted to pH 7.4) and inoculated on Lysogeny Broth Agar (LB, composition in g/L: 10.0 tryptone, 10.0 NaCl, 5.0 yeast extract, 15.0 agar). Plates were incubated for 2 weeks at 30°C and isolated colonies were purified, presumptively identified by 16S rRNA gene sequencing and cryopreserved in 20% glycerol (v/v). 16S rRNA gene sequencing The whole 16S rRNA gene from 4D.3 T was amplified by PCR using primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) and sequenced by Sanger sequencing with these two primers as well as the internal primers 1055F (5′- ATGGCTGTCGTCAGCT-3′) and 341R (5′-CTGCTGCCT CCCGTAGG-3′). The nearly complete 16S rRNA gene sequence (1,356 bp, accession number MZ562363) was analyzed by the EzBioCloud database tool 2 and the most closely related type strains were acquired from the DSMZ (Leibniz Institut, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany) as reference strains for the comparative study: Isoptericola cucumis DSM 2 https://www.ezbiocloud.net/ Vidal-Verdú et al. 10.3389/fmicb.2025.1471121 Frontiers in Microbiology 03 frontiersin.org 101603T; Isoptericola variabilis DSM 10177T; Isoptericola nanjingensis DSM 24300T; Krasilnikoviella flava DSM 21481T. Genome sequencing, assembly and annotation of I. peretonis sp. nov. 4D.3T, I. nanjingensis and I. variabilis strains Genomic DNA extraction of all strains was carried out from the recovered biomass of an overnight culture of each strain on the suggested culture medium by DSMZ (DSMZ medium 92: Tryptic Soy Agar) (TSA, composition in g/L: 15.0 Tryptone, 5 Soya peptone, 5 NaCl, 15 Agar) at 30°C and DNeasy Power Soil kit (Qiagen) was used by following manufacturer’s instructions. DNA concentration was measured by Qubit x1 dsDNA HS Assay Kit (Qubit 2.0 Fluorometer, Thermo Fisher, Waltham, UnitedStates). Genome sequencing was carried out with the NovaSeq 6000 system (Illumina), and the quality of sequence reads was evaluated with the FastQC tool (v. 0.11.5). SPAdes (v. 3.14.1) was utilized for genome assembly, and QUAST (v. 5.0.2) and CheckM (v. 1.1.3) were employed to calculate assembly statistics and evaluate completeness and contamination levels, respectively. Genome annotation was performed both with prokka (Seemann, 2014) and the RAST tool kit (RAStk) integrated in PATRIC v. 3.6.8. 3 TYGS (Meier-Kolthoff and Göker, 2019) was used to identify the most closely related type strains to 4D.3 T with publicly available genomes and to calculate the digital DNA–DNA hybridization (dDDH) index, while ANI was computed using FastANI (v. 1.33) (Jain etal., 2018). Phylogenetic and phylogenomic analysis Based on the 16S rRNA gene sequences, the phylogenetic reconstruction was obtained by maximum-likelihood (ML) (Felsenstein, 1981) and neighbor-joining (NJ) (Saitou and Nei, 1987) methods with the software MEGAX v.10.1.8. 4 Evolutionary models used were Tamura-Nei + gamma distribution (G) + invariant sites (I) for ML and Kimura two-parameter for NJ trees. Bootstrap analysis was performed with 500 replicates for the ML tree and 1,000 replicates for the NJ tree to assess branch pattern reliability (Felsenstein, 1985). The phylogenomic tree was reconstructed by using the UBCG (v.3.0) pipeline based on 92 housekeeping genes (Na etal., 2018) using the default options. Final trees were formatted with MEGAX. It must be noted that not all Isoptericola species had publicly available genomes at the time the analysis was carried out. Biochemical, physiological, and morphological characterization The comparative analysis of the phenotypic characteristics of 4D.3T was performed in parallel with four reference strains Isoptericola cucumis DSM 101603 T , Isoptericola nanjingensis DSM 24300 T , 3 https://www.patricbrc.org 4 http://www.megasoftware.net/ Isoptericola variabilis DSM 10177 T and Krasilnikoviella flava DSM 21481 T and was carried out after 4 days of growth on the suggested DSMZ medium 92 (TSA) at 30°C unless otherwise specified. All growth assays were conducted in duplicate. The cell morphology was examined by crystal violet stain and visualization under an optical microscope (DM2500 LED, Leica) and mycelium formation was tracked after 18 h, 48 h, and 72 h of incubation in TSA at 30°C and the same procedure for microscope visualization. At the same time, cell size was determined by measuring the minimum and maximum sizes of a random cell population (n = 25). The hanging-drop method was used to check the strain motility (Bernardet etal., 2002). Catalase activity was assessed by hydrogen peroxide 30% (v/v) and bubble formation was recorded as positive result. Oxidase activity was tested with the commercial Oxidase Test Stick (PanReac Applichem). Gram type test was conducted with KOH 3% (w/v) and the lack or appearance of viscosity was recorded as a Gram-negative or Gram-positive type, respectively (Halebian etal., 1981). The ability of all the strains to grow at different temperatures (4, 12, 16, 20, 24, 30, 37, 38, 40, 42, 45°C) and salt tolerance (NaCl concentrations 0–15% at intervals of 0.5%) was assessed in TSA medium after 4 days of incubation. Growth at different pH values (4.0 to 10.0 at intervals of 1.0 pH unit) in Tryptic Soy Broth (TSB, composition in g/L: 15.0 Tryptone, 5 Soya peptone, 5 NaCl) using specific buffers at 10 mM (MES for pH 4–6, HEPES for pH 7–8, and CHES for pH 9–10) was also tested after the same incubation time (Pascual etal., 2015). To assess the ability to grow under anaerobic and microaerophilic conditions, the BD GasPak EZ pouch system (Becton, Dickinson and Company) and the candle jar method were used, respectively. API 20NE and API ZYM system strips (bioMérieux) along with BIOLOG GEN III MicroPlates (BIOLOG) were used according to the manufacturer’s instructions to test the assimilation of C-sources and enzymatic activities. Analysis of cellular fatty acids for each strain was performed from a 24 h-old culture in TSB incubated at 30°C and was performed by the Spanish Type Culture Collection (CECT) by a gas chromatography system (model 6850, Agilent) and following the MIDI Microbial Identification System with the TSBA6 method (MIDI, 2008; Sasser, 1990). Biosurfactant synthesis assays To perform a screening of the genus Isoptericola on the ability to produce biosurfactants, three complementary assays previously described in the literature (Lee etal., 2018a; Walter etal., 2010) were carried out with several species of the genus. Specifically, the CTAB assay, the emulsification index E 24 % and the hemolytic activity assay were carried out and are explained in more detail in the following paragraphs. Eight strains were tested, including the aforementioned closer strains used for the biochemical and physiological characterization Isoptericola peretonis sp. nov. 4D.3 T , Isoptericola cucumis DSM 101603 T , Isoptericola nanjingensis DSM 24300 T , Isoptericola variabilis DSM 10177 T and Krasilnikoviella flava DSM 21481 T as well as Isoptericola chiayiensis DSM 27643T, Isoptericola halotolerans DMS 16376T, Isoptericola jiangsuensis DMS 21863 T and Isoptericola hypogeus DSM 16849 T also purchased from the DSMZ (Leibniz Institut, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany). Vidal-Verdú et al. 10.3389/fmicb.2025.1471121 Frontiers in Microbiology 04 frontiersin.org CTAB assay This method allows the detection of anionic biosurfactants throughout the formation of a dark precipitate when the biosurfactant reacts with the cetyltrimethylammonium bromide (CTAB) (Ref. 219374, Merck KGaA, Darmstadt, Germany) (Lee etal., 2018a). The assay was conducted in duplicate. Bushnell Haas medium plates (Composition in g/L: 0.4 MgSO 4 ·7H 2 O, 0.020 CaCl 2 , 1.0 KH 2 PO 4 , 1.0K2HPO4, 1.0 NH4NO3, 0.05 FeCl3, 15.0 Agar) supplemented with glucose 2% (w/v), CTAB (0.5 mg/mL) and methylene blue (0.2 mg/ mL) were prepared. Precultures of all the strains were grown in 5 mL of in TSB at 30°C and agitation during 24 h, 48 h and 72 h. After each incubation time, cultures were divided in two volumes and the cellfree supernatant was obtained by centrifugation of one of the volumes (2.5 mL) of the culture at 10,000 rpm for 15 min and 1 mL of each supernatant was transferred to a new tube. A drop assay was carried out by inoculating a drop of 10 μL of each cell-free supernatant and the containing-cell culture in CTAB agar plates. As positive control, a set of dilutions of the anionic surfactant SDS of 10, 5, 1, 0.5, and 0.1% (v/v) were used as well as for the non-ionic surfactant Tween 80 10% (v/v) as negative control (non-ionic surfactant), were added in drops of 10 μL. Plates were incubated for 24 h and the production of a dark precipitate was semi-quantitatively measured by the following rule: −, no dark precipitate appeared; +, a light dark precipitate appeared; ++, a moderate precipitate appeared; +++ a strong dark precipitate appeared; ++++ a very strong dark precipitate appeared. Emulsification index E24% The emulsification ability of all the strains tested was measured by modifying the emulsification index E 24 % assay described by Cooper and Goldenberg (1987). The assay was conducted in duplicate. Each strain was inoculated in 30 mL TSB cultures that were incubated for 48 h at 30°C and 180 rpm. Afterwards, half of the volume was centrifuged at 10,000 rpm for 15 min to get the cell-free supernatant. The assay was set in parallel with the cell-free supernatant and the cell-containing culture, using either mineral oil or diesel as hydrocarbons. Therefore, 6 mL of the cultures or cell-free supernatants were mixed with 3 mL of the selected hydrocarbon in an assay tube. Then, they were mixed at a maximum speed by agitation in a vortex for 2 min and were allowed to stand for 24 h in vertical position at RT. The same procedure was followed for negative controls (non-inoculated TSB) and positive controls (Tween 80 2:1 (v/v) and SDS 10% (v/v) solutions), in which these solutions were used instead of the bacterial cultures. Afterwards, to calculate the E 24 % index, the height of the emulsified layer (mm) and the total height of the liquid column (mm) were measured and determined by the equation: ( ) ( ) Height of emulsified layer mm E24% 100 Totalheight of liquid column mm = × Hemolytic activity assay Biosurfactants have hemolytic activity. Therefore, when a biosurfactant-producing strain is grown on Blood Agar plates (BA, Ref: 0931, Condalab, Spain) a halo due to hemolysis of erythrocytes is expected (Sharma etal., 2015). The assay was conducted in duplicate. TSB cultures of 5 mL were inoculated separately with each strain and were incubated at 30°C and agitation for 48 h. After the incubation time, the Optical Density 600 nm (OD 600 ) was adjusted to 1 and 10 μL drops were inoculated in BA. Also, 10 μL drops of the anionic surfactant SDS at different concentrations (10, 5, 1, 0.5, and 0.1%, v/v) as well as the non-ionic surfactant Tween 80 10% (v/v) were used as positive control. Results were measured semi-quantitatively by measuring the halo of hemolysis produced by each strain at different incubation times (24 h, 72 h, and 10 days) at 30°C following the rule: −, no hemolysis; +, incomplete hemolysis; ++, complete hemolysis with a diameter of lysis <0.5 cm; +++, complete hemolysis with a diameter of lysis >0.5 cm. Genome analysis for hydrocarbon degradation, rhamnolipid production and biosafety of Isoptericola strains At the time of performing this work, the genome of the 12 publicly available genomes of the strains Isoptericola peretonis sp. nov. 4D.3T, Isoptericola jiangsuensis DMS 21863T, Isoptericola dokdonensis DS-3T, Isoptericola chiayiensis KCTC 19740 T , Isoptericola sediminis JC619 T , Isoptericola halotolerans KCTC 19046 T , Isoptericola croceus q2 T , Isoptericola cucumis CCM 8653 T , Isoptericola luteus NEAU Y5 T , Krasilnikoviella flava DSM 21481 T , Isoptericola nanjingensis DSM 24300 T and Isoptericola variabilis MX5 T were analyzed by bioinformatic tools for hydrocarbon degrading and rhamnolipid production activities as well as to determine some biosafety traits. Genes related to hydrocarbon degradation were annotated with CANT-HYD (Khot etal., 2022). Only hits with an e-value lower than 10−6 were considered positive. In the case that an ORF was annotated as different genes, only the annotation with a lower e-value was considered. BLASTp (v. 2.0.9+) was used to determine the presence of genes rhlA, rhlB and rhlC which are responsible of rhamnolipid production in bacteria (e-value threshold = 10 −6 ). The potential to produce surfactin was evaluated with both BLASTp (genes sfrAA, sfrAB and sfrAC) and antiSMASH (v. 7.0) (Blin etal., 2023). Genes were downloaded from UniProt along with other sequences that had at least 50% similarity to the reference sequences. Several bioinformatic tools were applied to assess the biosafety of the strains. Specifically, ABRicate 5 was used to find possible antimicrobial resistance (AMR) genes or virulence factors. This tool searches the proteins predicted from the genome of interest against a set of different databases: NCBI AMRFinderPlus (Feldgarden etal., 2019), CARD (Jia etal., 2017), ARG-ANNOT (Gupta etal., 2014), Resfinder (Zankari etal., 2012), MEGARES (Doster etal., 2020), EcOH (Ingle etal., 2016), PlasmidFinder (Carattoli etal., 2014), Ecoli_ VF and VFDB (Chen etal., 2016). Additionally, RGI-CARD6 (Alcock etal., 2022) and abriTAMR7 were also employed to detect potential AMR genes. Finally, Bakta was used to identify biogenic amines (Schwengers etal., 2021). 5 https://github.com/tseemann/abricate 6 https://github.com/arpcard/rgi 7 https://github.com/MDU-PHL/abritamr Vidal-Verdú et al. 10.3389/fmicb.2025.1471121 Frontiers in Microbiology 05 frontiersin.org Results Biochemical, physiological and morphological analysis The strain 4D.3 T is a Gram-positive, facultatively anaerobic and non-motile bacterium that formed primary mycelium with irregular and aggregated morphology from cocci to v-shaped rods varying in size (0.8–1.0 μm in diameter and 1–3 μm long) in the first 48 h. Homogenous cocci shape and absence of mycelium was observed after 72 h of incubation (Supplementary Figure S1). The colonies of 4D.3T after 72 h, were circular and raised with 2–3 mm in diameter and appeared opaque-orange, shiny and smooth with regular edges. The strain grew well under aerobic conditions and showed less growth under microaerophilic and anaerobic conditions. Strain 4D.3 T displayed a mesophilic growth, being able to grow between 12 and 42°C (optimum 30°C). It was able to grow well up to 8% of NaCl and weakly 10% (optimum 0–5%). It showed optimum growth between pH values 6–7 but it was able to grow up to pH 10. Strain 4D.3T tested catalase positive and oxidase negative. In carbon source assimilation tests using API 20 NE strips, 4D.3T assimilated d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glucosamine, d-maltose and potassium gluconate. In the BIOLOG GENIII assay, strain 4D.3 T was found to oxidize 46 out of the 71 tested carbon sources. Among them, d-sorbitol, 3-methyl glucose, d-fucose, N-acetyl-β-d-mannosamine, N-acetyld-galactosamine and l-serine were exclusively assimilated by 4D.3 T when compared with the reference strains I. cucumis DSM 101603 T , I. nanjingensis DSM 24300 T , I. variabilis DSM 10177 T and Krasilnikoviella flava DSM 21481T (Supplementary Table S1). Strain 4D.3 T was positive in various enzymatic activities, such as alkaline phosphatase, esterases (C4 and C8), leucine arylamidase, valine arylamidase, cystine arylamidase, naphtol-AS-BIphosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase (API ZYM), aesculin hydrolysis (API 20NE) and gelatinase. Conversely, strain 4D.3 T exhibited negative responses for lipase (C14), trypsin, α-chymotrypsin, acid phosphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase, urease, fermentation of glucose, arginine dihydrolysis and indole production. Reduction of nitrates to nitrites tested positive whereas reduction of nitrates to nitrogen tested negative. Table1 shows differential phenotypic characters between strain 4D.3T and its phylogenetically closest species. In terms of the fatty acid composition, only saturated fatty acids were detected at high concentrations as well as hydroxylated fatty acids in trace amounts. The major fatty acid of strain 4D.3 T was anteiso-C15:0 making up59.44% of the total composition, followed by iso-C 15:0 accounting for 29.02%. Other less abundant (<5%) fatty acids were anteiso-C 17:0 , isoC16:0 , C 16:0 , and iso-C 14:0 . This fatty acid pattern was very similar to that of the reference strains (Table2) as well other members of the genus Isoptericola (Stackebrandt and Schumann, 2015). Genomic, phylogenetic and phylogenomic analysis of Isoptericola 4D.3T sp. nov. The 16S rRNA gene sequence of strain 4D.3 T obtained by Sanger sequencing was almost complete (1,356 bp, MZ562363) and 100% identity was obtained when compared with the 16S rRNA gene sequence extracted from its genome (1,527 bp), as expected. According to the EzBioCloud database, the closest type strains of 4D.3T were Isoptericola nanjingensis H17T (99.8% 16S rRNA gene sequence similarity), Isoptericola cucumis AP-38 T (99.5%) and Isoptericola variabilis MX5 T (99.4%). Other type strains of the TABLE1 Differential phenotypic characteristics of strain 4D.3T and the other closely related reference strains: 1, Isoptericola peretonis sp. nov. 4D.3T; 2, Isoptericola cucumis DSM 101603T; 3, Isoptericola variabilis DSM 10177T; 4, Isoptericola nanjingensis DSM 24300T; 5, Krasilnikoviella flava DSM 21481T. Characteristic 1 2 3 4 5 Isolation source Car tank lid Cucumber root tissue Termite hindgut Soil Baltic sea sediment Growth at/in Temperature range (°C) (optimal) 12–42 (30) 12–40 (30) 12–42 (30) 12–38 (30) 12–30 (30) NaCl tolerance (%, w/v) (optimal) 0–10 (0–5) 0–11 (0–7) 0–10 (0–5) 0–10 (0–5) 0–9 (0–5) pH (optimal) 6–10 (6–7) 7–10 (8) 5–9 (6–9) 5–10 (6–9) 6–9 (6) Carbon source utilization (API 20NE) Malic acid −+− − − Enzymatic activity (API ZYM) Valine arylamidase w −w w w Cystine arylamidase w −w w w Trypsin − − − w− α-galactosidase +−+ w w N-acetyl-β-glucosaminidase −w− − − α-mannosidase −+−w w Data for all strains were obtained in the present study. +, positive; w, weak; −, negative. All strains are positive for alkaline phosphatase, esterase lipase (C4), esterase lipase (C8), leucine arylamidase, naphtol-AS-BI-phosphohydrolase, β-galactosidase, α-glucosidase, β-glucosidase, reduction of nitrates to nitrites, aesculin hydrolysis, gelatinase and the assimilation of d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glucosamine, d-maltose and potassium gluconate. All strains are negative for lipase (C14), α-chymotripsin, acid phosphatase, β-glucuronidase, α-fucosidase, reduction of nitrates to nitrogen, indole production, fermentation of glucose, arginine dihydrolysis, urease and the assimilation of capric acid, adipic acid, trisodium citrate, and phenylacetic acid. Vidal-Verdú et al. 10.3389/fmicb.2025.1471121 Frontiers in Microbiology 06 frontiersin.org genus Isoptericola showed sequence similarity values of less than 99.0%. In both the NJ and ML phylogenetic trees based on the 16S rRNA gene, strain 4D.3 T forms a monophyletic group with the type strain of the species I. cucumis, with I. nanjingensis and I. variabilis occurring as external species to this group. However, this group is not very stable considering the bootstrap values presented at the nodes (<75%). Interestingly, in both phylogenetic trees, ML and NJ, the two species of the genus Krasilnikoviella, K. flava, and K. muralis, are placed within the genus Isoptericola, appearing as a paraphyletic group. This result suggests that the members of the genus Krasilnikoviella should bereclassified as species of the genus Isoptericola (ML: Figure1; NJ: Supplementary Figure S2). The draft genome of the strain 4D.3T has a length of 4,474,848 pb and consisted of 20 contigs, with an N50 value of 867,559 bp and a genomic DNA G + C content of 74%. The genomic G + C content of strain 4D.3 T was within the range described for the genus Isoptericola, namely 70.0–74.1% (Stackebrandt and Schumann, 2015). The predicted annotation identified 4,139 coding sequences (CDSs), out of which 2,797 were predicted as proteins with functional assignments. Additionally, 49 tRNAs and 3 rRNAs were predicted. The genome completeness was 100% and the contamination level was 0.58%, indicating that the draft genome had high-quality for further analyses (Riesco and Trujillo, 2024). For a more precise determination of the phylogenetic position of 4D.3 T within the Isoptericola genus, a phylogenomic tree was constructed using genomic sequences (Figure2). Strain 4D.3T formed a well-defined monophyletic group with the type strains of the species I. nanjingensis, I. variabilis and K. flava. This clustering is stable, as proved by the high bootstrap values (100%) and the genetic support index (71 out of a total of 92 genes). The inclusion of the species K. flava into the genus Isoptericola suggests that this species should bereclassified as a member of the genus Isoptericola. However, as the genome of the type species of the genus Krasilnikoviella, namely K. muralis, was not publicly available, we could not confirm this hypothesis. The genomic index ANIb between strain 4D.3 T and its closest relatives was calculated to be88.22% (K. flava), 87.91% (I. nanjingensis) and 87.71% (I. variabilis). For the dDDH index, the values were 31.5, 30.1 and 29.8%, respectively (Table3). As these values were lower than the thresholds for the delimitation of novel bacterial species, i.e., 95% for ANIb (Richter and Rosselló-Móra, 2009) and 70% for dDDH (Meier-Kolthoff etal., 2013), they further supported the classification of I. peretonis sp. nov. 4D.3 T as a novel species of the genus Isoptericola (Chun and Rainey, 2014; Riesco and Trujillo, 2024). Biosurfactants production by several species from the genus Isoptericola In order to elucidate the ability to produce biosurfactants by the majority of the species of the genus Isoptericola, complementary invitro assays to check biosurfactant synthesis of strains close to the new Isoptericola strain 4D.3 T were performed, and results are summarized in Figure3. Eight strains belonging to the Isoptericola genus as well as the K. flava 21481T strain were included in the CTAB, Hemolytic and E 24 % assays as described in Materials and Methods section. Overall, all the strains tested showed able to produce biosurfactants and the increase in the incubation time correlated to a higher biosurfactant production. At the same time, the CTAB and the E24% assays were performed with the cell-free supernatants and cellcontaining cultures, to elucidate whether biosurfactants are secreted or remain embedded to the cell surface. Regarding the CTAB assay, which reveals the production of anionic biosurfactants, such as rhamnolipids, by the formation of a dark precipitate, it showed that all strains tested were positive in anionic biosurfactants synthesis. Furthermore, supernatants tested negative for anionic biosurfactants production and only cultures containing cells presented the dark precipitate. I. hypogeus DSM 16849T showed the most intense precipitate when the preculture was incubated 72 h at 30°C. I. peretonis sp. nov. 4D.3 T , I. varibilis DSM 10177T, I. cucumis DSM 101603T as well as I. chiayiensis DSM 27643T displayed the highest hemolytic activity after 10 days of incubation on blood-agar plates. In this case I. jiangjuensis DSM 21863 T differed from the rest of the strains by showing a very low hemolysis ability. TABLE2 Cellular fatty acid profile (%) of strain 4D.3T and its closest type strains. 1 2 3 4 5 Saturated iso-C14:0 1.09 2.94 2.37 1.28 tr C14:0 tr 1.76 1.52 1.33 1.44 iso-C15:0 29.02 17.75 25.14 25.56 22.73 anteiso-C15:0 59.44 64.44 54.17 59.17 59.88 iso-C16:0 2.38 3.61 7.16 4.07 3.79 C16:0 1.15 1.54 1.66 1.93 2.20 iso-C17:0 tr tr tr tr 1.14 anteiso-C17:0 4.86 7.09 6.86 5.99 8.01 Hydroxylated C12:0 2-OH tr tr tr – – Strains: 1, Isoptericola peretonis sp. nov. 4D.3T; 2, Isoptericola cucumis DSM 101603T; 3, Isoptericola variabilis DSM 10177T; 4, Isoptericola nanjingensis DSM 24300T; 5, Krasilnikoviella flava DSM 21481T. tr, trace (<1.0%); −, not detected. Vidal-Verdú et al. 10.3389/fmicb.2025.1471121 Frontiers in Microbiology 07 frontiersin.org FIGURE1 Maximum likelihood phylogenetic tree showing the taxonomic position of I. peretonis sp. nov. 4D.3T and other related members of the families Promicromonosporaceae and Cellulomonadaceae. The evolutionary model used was Tamura-Nei + gamma distribution (G) + invariant sites (I) and bootstrap values based on 500 replicates are shown at nodes (only values >50% are shown). As outgroup, Brevibacterium linens DSM 20425T (X77451) was used. Bar 0.050 fixed nucleotide substitutions per site. FIGURE2 Phylogenomic tree based on a multiple alignment of 92 housekeeping gene sequences using the UBCG (v.3.0) pipeline (Na etal., 2018) showing taxonomic relationships between I. peretonis sp. nov. 4D.3T and other related species in the family Promicromonosporaceae. Bootstrap analysis was performed using 100 replicates. Gene support indices (maximum value; 92 genes) and percentage bootstrap values (maximum value; 100%) are shown at branch points. Bar indicates 0.01 substitutions per nucleotide position. Xylanimonas cellulosilytica DSM 15894T (GCF_000024965.1) was used as an outgroup. Vidal-Verdú et al. 10.3389/fmicb.2025.1471121 Frontiers in Microbiology 08 frontiersin.org FIGURE3 Emulsification activity by Isoptericola peretonis sp. nov. 4D.3T and related species. Heatmap of three complementary assays to detect biosurfactants synthesis. CTAB assay: Detection of anionic surfactants by qualitative measurement of the appearance of a dark precipitate. In a blue gradient, the appearance of the dark precipitate is shown from dark blue (very strong precipitate = presence of anionic biosurfactant) to white (no precipitate = absence of anionic biosurfactant). Hemolytic assay: Semi-quantitative measurement of halo formation in Blood Agar plates due to hemolysis caused by the lysis of erythrocytes after strains growth. In a red gradient, the hemolytic activity from dark red (complete hemolysis with a lysis diameter > 0.5 cm) to white (no hemolysis). Emulsification index E24% assay: Quantitative measurement of the emulsification ability of a liquid culture of each strain (TSB, 48 h incubation, 30°C and 180 rpm) on diesel and mineral oil. Cell-containing culture and cell-free supernatant were tested. In a brown gradient, the E24% from dark brown (100% = complete emulsification) to white (0% = absence of emulsification). In all the assays positive controls displayed the maximum value and negative controls displayed the minimum value of precipitation/hemolysis/emulsification. In addition, the E 24 % assay, widely used to test biosurfactant production in strains of interest, was carried out using diesel and mineral oil as the organic phase that was mixed with the cell-free supernatant as well as the cell-containing culture. As shown in Figure3, I. chiayiensis DSM 27643 T was the only one giving a complete emulsification in both, diesel and mineral oil, followed by K. flava DSM 21481T that presented a 100% emulsification index in the cellcontaining cultures. On average, strains presented a higher E 24 % in diesel when cells were present, except for I. hypogeus DSM 16849 T which supernatant had more emulsification capacity. Regarding the new species I. peretonis sp. nov. 4D.3T, both the supernatant and the cell-containing culture showed a moderate-high E24% index. In silico analysis of hydrocarbon degradation and biosurfactant production by Isoptericola All the publicly available genomes of Isoptericola species, including K. flava, were analyzed to elucidate the ability of these microorganisms to degrade aromatic hydrocarbons and alkanes (Figure 4). All the studied species contained four genes involved in the aerobic degradation of alkanes: almA (Group I), cyp153, ladA (beta subunit), and ladB. almA (Group I) and cyp153 encode the enzymes flavin-binding monooxygenase and cytochrome P450 alkane hydroxylase, respectively, which are involved in the degradation of C20-C32 (almA) and C5-C13 (cyp153) alkanes. The degradation of C15-C36 alkanes would bemediated by the ladA (beta) and ladB genes, which encode longchain alkane monooxygenases and were found in multiple copies in the genomes. However, the similarity between copies was low. For example, in the case of ORFs assigned as ladA or ladB in the I. peretonis genome, the similarity between ORFs annotated as the same gene was less than <40% in all cases, according to BLASTp. All the species, except I. croceus and I. halotolerans, presented a 1-methylalkyl (alkyl) succinate synthase (gene assA). In contrast, ahyA, a gene that encodes a putative alkane C2 methylene hydroxylase, was present in all the species except for I. sediminis. Both enzymes are involved in the anaerobic degradation of alkanes. Regarding the aerobic degradation of aromatic compounds, all the genomes contained copies of dszC, which encodes the dibenzothiophene desulfurization enzyme C. I. jiangsuensis, I. dokdonensis, I. sediminis, and I. halotolerans were the only species lacking non-NdoB type naphthalene dioxygenase alpha. Finally, K. flava and I. nanjingensis were the only species with the potential ability to produce an ethylbenzene dehydrogenase, which catalyzes the anaerobic oxidation of ethylbenzene to (S)-1-phenylethanol (Rabus etal., 2005). The organization of genes involved in hydrocarbon degradation revealed no evidence of complex gene clusters. This was anticipated, TABLE3 Genome distance indexes for strain 4D.3T compared to other close type strains. 2 3 4 5 6 ANIb 1. 4D.3T84.44 87.71 87.91 88.22 83.86 dDDH 1. 4D.3T24.6 (22.2–27.0) 29.8 (27.4–32.3) 30.1 (27.7–32.6) 31.5 (29.1–34.1) 23.9 (21.6–26.4) Average Nucleotide Identity values (ANIb) and in silico digital DNA–DNA Hybridization (dDDH) values together with the confidence intervals in parenthesis are shown in %. 1, Isoptericola peretonis sp. nov. 4D.3T; 2, Isoptericola cucumis DSM 101603T; 3, Isoptericola variabilis DSM 10177T; 4, Isoptericola nanjingensis DSM 24300T; 5, Krasilnikoviella flava DSM 21481T; 6, Isoptericola luteus NEAU Y5T. Vidal-Verdú et al. 10.3389/fmicb.2025.1471121 Frontiers in Microbiology 09 frontiersin.org as many pathways for hydrocarbon degradation remain incomplete, with accessory functions still unidentified. Moreover, most genomes analyzed were draft assemblies rather than fully closed genomes, limiting the detection of such clusters. Therefore, further research is necessary to clarify this aspect. Nevertheless, simpler gene organization patterns were observed. For example, the almA and cyp153 genes appeared consecutively in all Isoptericola genomes, suggesting a linked inheritance, evolution and regulation of their functions in degrading C20-C32 (almA) and C5-C13 (cyp153) alkanes. A similar arrangement was identified for the ahyA gene. Several hits consistent with this gene were found adjacent to each other in different Isoptericola genomes, often separated by one or more genes. These hits likely represent distinct subunits of the same enzyme, as they were annotated by Prokka as “Nitrate reductase alpha subunit” and “Assimilatory nitrate reductase catalytic subunit.” In contrast, the ladA (beta subunit) and ladB genes exhibited a more complex organization. For instance, in I. peretonis sp. nov. 4D.3T, seven of the eight identified copies of these genes were scattered across different contigs. However, an interesting pattern was observed in I. variabilis, I. nanjingensis, and K. flava. In these species, a genomic region (20–30 FIGURE4 Hydrocarbon degradation capabilities of Isoptericola species, including K. flava, as deduced from the CANT-HYD annotation of each genome. Only genes with at least one hit in one genome are shown (e-value threshold < 10−6). The size of the point is proportional to the number of gene copies present in the genome. In cases where an ORF was annotated as multiple genes, only the hit with the lowest e-value was considered. Panels show genes implicated in aromatic and alkane hydrocarbon degradation in aerobic or anaerobic conditions. Genomes in the y axis follow the order as they appear in the phylogenomic tree (Figure2). “ana.”: anaerobic.