# Phenylobacterium ferrooxidans sp. nov., isolated from a sub-surface geothermal aquifer in Iceland

Eva Pouder, Erwann Vince, Karen Jacquot, Maimouna Batoma Traoré,

Ashley Grosche, Maria Ludwig, Mohamed Jebbar, Loïs Maignien, Karine

Alain, Sophie Mieszkin

► To cite this version:

Eva Pouder, Erwann Vince, Karen Jacquot, Maimouna Batoma Traoré, Ashley Grosche, et al.. Phenylobacterium ferrooxidans sp. nov., isolated from a sub-surface geothermal aquifer in Iceland. Systematic and Applied Microbiology, 2024, 48 (1), pp.126578. 10.1016/j.syapm.2024.126578. hal-04854002

HAL Id: hal-04854002

https://hal.science/hal-04854002v1

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*Phenylobacterium ferrooxidans* sp. nov., isolated from a sub-surface geothermal aquifer in Iceland

Eva Pouder, Erwann Vince, Karen Jacquot, Maimouna batoma Traoré, Ashley Grosche, Maria Ludwig, Mohamed Jebbar, Loïs Maignien, Karine Alain, Sophie Mieszkin*

Univ Brest, CNRS, IFREMER, EMR 6002 BIOMEX, Unité Biologie et Écologie des Écosystèmes marins Profonds BEEP, F-29280 Plouzané, France

ARTICLE INFO

Keywords:

Subsurface geothermal aquifer

*Phenylobacterium* sp.

Cultivation

Genomics

Iron oxidation

Nitrate reduction

A B S T R A C T

A novel bacterial strain, HK31-G<sup>T</sup>, was isolated from a subsurface geothermal aquifer (Hellisheidi, SW-Iceland) and was characterized using a polyphasic taxonomic approach. Phylogenetic analysis of 16S rRNA gene along with phylogenomic position indicated that the novel strain belongs to the genus *Phenylobacterium*. Cells are motile Gram-negative thin rods. Physiological characterization showed that strain HK31-G<sup>T</sup> is a mesophilic bacterium able to grow from 10 to 30 °C, at pH values between 6 and 8 and at NaCl concentrations between 0 and 0.5 %. Optimal growth was observed without sodium chloride at 25 °C and pH 6. Strain HK31-G<sup>T</sup> is chemoorganoheterotroph and its major saturated fatty acids are C<sub>18:1</sub>ω7c, C<sub>16:1</sub>ω6c and C<sub>16:0</sub>, the predominant quinone is Q-10 and the major polar lipid is phosphatidylglycerol. The new strain also possesses the capacity to use ferrous iron (Fe(II)) as the sole energy source and can also be considered as a chemolithoautotrophic microorganism. The overall genome of strain HK31-G<sup>T</sup> was estimated to be 4.46 Mbp in size with a DNA G + C content of 67.95 %. Genes involved in iron metabolism were identified, but no genes typically involved in Fe(II)-oxidation were found. According to the overall genome relatedness indices (OGRI) values, six MAGs from groundwater have been assigned to the same species as the new strain HK31-G<sup>T</sup>. Furthermore, OGRI values between the genome of strain HK31-G<sup>T</sup> and the genomes of its closest relatives are below the species delineation threshold. Therefore, given the polyphasic approach used, strain HK31-G<sup>T</sup> represents a novel species of the genus *Phenylobacterium*, for which the name *Phenylobacterium ferrooxidans* sp. nov. is proposed. The type strain is HK31-G<sup>T</sup> (DSM 116432<sup>T</sup> = UBOCC-M-3429<sup>T</sup> = LMG 33376<sup>T</sup>).

Introduction

Carbon Capture Storage (CCS) technologies offer a way to sequester
anthropogenic CO2 emissions from the atmosphere in the subsurface,
lowering environmental consequences such as greenhouse-gas emissions
(Snæbjörnsdóttir et al., 2020). Through injections of CO2 dissolved in
water into deep basalts or peridotites rich in calcareous and ferromag-
nesian silicates, CO2 will react and precipitate as solid Ca-Mg-Fe-car-
bonate minerals over a two-year timespan (Snæbjörnsdóttir et al.,
2020). The carbonates formed will be stable for thousands of years. This
deep mineral carbon storage in basalt was developed in the framework
of the Carbfix project (https://www.carbfix.com) at the CarbFix-1 site
near the Hellisheidi geothermal power plant (SW-Iceland) (Matter et al.,
2011). Following the injection and dissolution of CO2 into the surface,
protons are released, resulting in acidic and oxidative conditions along
with an increase of inorganic carbon source promoting growth of

chemolithoautotrophic microbial communities (Mu and Moreau, 2015;
O'Mullan et al., 2015). At the CarbFix-1 site, it was shown that after CO2
was injected into basalt, microbial richness decreased but lithoauto-
trophic Fe(II)-oxidizing *Betaproteobacteria* and aromatic compound de-
graders became dominant (Trias et al., 2017).

During a study of bacterial diversity associated with CCS gas injec-
tion at the CarbFix-1 site, but on different wells from the study by Trias
et al. (2017) (Fig. S1), an attempt was also made to enrich and isolate
neutrophilic Fe(II)-oxidizing bacteria (FeOB). The bacterial strain HK31-
G<sup>T</sup> was finally isolated and appeared to belong to the genus *Phenyl-
obacterium*. At the time of writing, this genus encompasses 18 species
with validly published names, according to the List of Prokaryotic names
with Standing in Nomenclature (LPSN), from a variety of environments,
but mainly soil and water (Thomas et al., 2022). The genus *Phenyl-
obacterium* belongs to the class *Alphaproteobacteria* and the family *Cau-
lobacteraceae*, and the type species is *P. immobile* (Lingens et al., 1985).

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: Sophie.Mieszkin@univ-brest.fr (S. Mieszkin).</footnote>
To date, species of the genus *Phenylobacterium* are characterized as aerobic or facultatively anaerobic, Gram-negative, motile or non-motile, straight to slightly curved rods, coccobacilli or cocci, occurring either singly, in pairs or in short chains. The major respiratory quinone is Q-10 while C₁₈:₁ω₇c and/or C₁₈:₁ω₆c are the major fatty acids, and phosphatidylglycerol the main polar lipid. DNA G + C contents range from 64 to 72.3 % (Thomas et al., 2022).

In the present study, we performed a polyphasic taxonomic characterization of strain HK31-GT and provided phenotypic, chemotaxonomic, phylogenomic and genomic evidence that it meets criteria for the delineation of a new species of the genus *Phenylobacterium*. Interestingly, we also reported the capacity of this strain to oxidize ferrous iron (Fe(II)) under microaerophilic as well as anaerobic and neutrophilic conditions highlighting the versatility of its metabolism. This is in line with the changing environmental conditions that could be encountered during CCS gas injection.

## Materials and methods

*Isolation, ecology and deposit in public culture collections.*

Strain HK31-GT was isolated from a deep subsurface hydrothermal aquifer (well HK-31) at the Carbfix-1 injection site of an adjacent geothermal power plant at Hellisheidi (SW-Iceland), which is the third largest geothermal power plant in the world (64° 02′ 14" N, 21° 24′ 03" W) (Matter et al., 2011). The Carbfix-1 was used for pilot scale injections of CO₂ in 2012 where 175 tons of pure commercial CO₂ was injected, while industrial scale injection is today carried out at the Carbfix-2 injection site (Aradóttir et al., 2015). At the Carbfix-1 site, the CO₂ injection well (HN-2, located at a depth of 2000 m and into which CO₂ was injected at a depth between 400 and 800 m) is located approximately at 1750 m from the well HK-31, from which the new strain HK31-GT is originated (Fig. S1) (Aradóttir et al., 2011). Water, for CO₂ injection, was pumped from the upstream well HN-1, then re-injected with CO₂ into HN-2. Overall, the Carbfix-1 storage formation is composed of fresh basaltic lavas interbedded with hyaloclastites. The ground waters are located between 400 and 800 m depth and are characterized by poor dioxygen concentrations and by temperatures and pH ranging respectively from 18 to 33 °C and from 8.4 to 9.4 (Alfredsson et al., 2008; Aradóttir et al., 2011). Prior to sampling in July 2019, the well was pumped continuously for 24 h and water samples were collected using sterile 50 mL Falcon tubes and then stored at 4 °C. Before storage, the pH of the aqueous samples was measured and was established at 8.78. The temperature was recorded at 24.1 °C and the oxido-reduction potential (EH) measured was 268 uS/s.

In order to enrich and isolate neutrophilic Fe(II)-oxidizing bacteria (FeOB), semi-solid Fe(II)/O₂ gradient tubes under microaerophilic conditions were aseptically inoculated in the laboratory immediately after sample collection, as described elsewhere (Emerson and Floyd, 2005). After enrichment culture at 20 °C for 15 days, a serial dilution-to-extinction method in semi-solid Fe(II)/O₂ gradient tubes was applied for isolation at the same temperature. A co-culture was finally obtained and the two strains were then isolated by plating this co-culture on Rea-soner's 2 A (R2A) agar medium at 20 °C and pH 7.2 (Khan et al., 2018; Reasoner and Geldreich, 1985), which enabled distinct colonies to be isolated. The two strains were then purified by three successive quadrant streaks on R2A agar medium at 20 °C and pH 7.2 to obtain pure cultures. The first strain, HK31-GT, was affiliated to the *Phenylobacterium* genus while the second, HK31-P, was affiliated to the *Phreatobacter* genus. Strain HK31-GT was then grown routinely on R2A broth or agar adjusted to pH 7.2 over respectively, 3 or 5 days at 20 °C, under agitation (250 r.p.m). Its purity was routinely confirmed by microscopic observations (Olympus BX60 and CX40) and by sequencing its 16S rRNA gene. Stock cultures were stored at -80 °C in R2A broth medium supplemented with 5 % (v/v) dimethylsulfoxide (DMSO). *Phenylobacterium ferrooxidans* type strain HK31-GT (DSM 116432T; UBOCC-M-3429T; LMG 33376T) is

available in three public culture collections, namely Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; https://www.dsmz.de/, UBO Culture Collection (UBOCC; https://www.univ-brest.fr/ubocc) and Belgian Coordinated Collections of Microorganisms (BCCM; https://bccm.belspo.be/).

*Morphological, physiological and metabolic features.*

Colony morphology of strain HK31-GT was observed on R2A agar adjusted to pH 6 for 5 days at 25 °C. Cell morphology and motility were determined by light microscopy (Olympus BX60 and CX40) and transmission electron microscopy (TEM, Jeol JEM 1400). Motility was also confirmed by using mannitol-motility-nitrate (MMN) agar medium (composed of 10 g·L⁻¹ tryptic hydrolysate of casein, 1 g potassium nitrate, 7.5 g·L⁻¹ mannitol, 40 mg·L⁻¹ phenol red and 3.5 g·L⁻¹ agar), which was also used to evidence mannitol fermentation and nitrate reductase activity. Gram-staining was determined using standard procedures and confirmed with a KOH (3 %) test. Catalase and cytochrome oxidase activities were respectively evaluated using H₂O₂ and strips of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (Bio-Rad).

Physiological characterization of the novel strain HK31-GT was carried out aerobically, in triplicates, on R2A (agar or broth) adjusted to pH 6 at 25 °C and under agitation (250 r.p.m.). Determination of the temperature range for growth and salt tolerance were respectively tested over the range 5–55 °C, at 5 °C intervals and 0–5 % NaCl (w/v), at 0.5 % intervals, both for 12 days on R2A agar at pH 6. The pH range for growth was tested from pH 3 to 10 (at 25 °C), with increments of 1 unit in R2A broth for 12 days. Cells were routinely enumerated by direct cell counting using a modified Thoma chamber (Preciss; surface: 0.0025 mm², depth: 10 µm). The following buffers (each at 20 mM, Sigma-Aldrich) were used to adjust the required pH: pH 4 and 5 with HOMO-PIPES buffer, pH 6 with MES buffer, pH 7 with PIPES buffer, pH 8 with HEPES buffer, and pH 9 and 10 with CAPSO buffer. For pH 3, no buffer was used. The growth kinetics of HK31-GT under optimal conditions was then studied in triplicate, on R2A broth adjusted to pH 6 at 25 °C with no NaCl salt, under agitation (250 r.p.m.) for 13 days. Cell growth was monitored by direct cell counting, using a modified Thoma chamber, in order to determine the growth rate and doubling time of the strain under optimal culture conditions.

Metabolic features were estimated for the novel isolate HK31-GT and closely related strains previously described and available in public culture collections: *P. haematophilum* LMG 11050T (=DSM 21793T = CCUG 26751T), *P. conjunctum* FWC 21T (LMG 24262T) (Abraham et al., 2008), and *P. aquaticum* W2-3-4T (KACC 18306T) (Jo et al., 2016). Utilization of organic substrates as the sole carbon source was investigated using the mineral basis of R2A medium containing 0.3 g·L⁻¹ dipotassium phosphate and 0.05 g·L⁻¹ magnesium sulfate and adjusted to pH 6. Each substrate (xylose, glycerol, malonate, hydroxybutyrate, lactate, succinate, aspartate, acetate, glutamate, propionate, proline, alanine and phenylalanine) was supplied at a final concentration of 20 mM. Each strain was cultivated at its own optimal growth parameters (T°, pH and NaCl). Before inoculation, cells, at the end of their exponential growth phase, were harvested and washed three times with distilled water. The bacterial suspension was then adjusted to obtain a McFarland index value of 1. A medium consisting of R2A mineral base adjusted to pH 6, with no carbon source, was used as negative control for each carbon utilization bioassay and a positive control was performed using R2A broth adjusted to pH 6. Additional tests were carried out to determine: (i) mannitol fermentation and presence of nitrate reductase (MMN agar medium); (ii) fermentative pathways (mixed acids or butane-2,3-diol pathways; Clark and Lubs liquid medium); (iii) glucose and lactose fermentation, gas and H₂S production (Kligler-Hajna agar medium); and (iv) citrate utilization (Simmons citrate agar medium). For these assays, cell washes and preparation of bacterial suspensions from cultures of HK31-GT under optimal conditions on R2A medium were performed as described above and elsewhere (Mieszkin et al., 2021). Oxidative and
fermentative utilization of carbohydrates as well as enzymatic activities for the strain HK31-G<sup>T</sup> and closely related strains were also evaluated by using the API®20NE kit, API®20E kit and API®ZYM kit (BioMérieux) according to the manufacturer's instructions with slight modifications (McFarland index = 1; 3 days of incubation at 20 °C).

### Chemotaxonomic analyses

Characterization of respiratory quinones, polar lipids and fatty acids of cells of strain HK31-G<sup>T</sup> was carried out by the identification Service of the DSMZ (Braunschweig, Germany) as described by Tindall (1990a, 1990b) and Kuykendall et al. (1988). To carry out these analyses, cells were grown in R2A broth, pH 6 at 25 °C under agitation (250 r.p.m) for 5 days and were then harvested by centrifugation (800 g; 10 min) at the end of their exponential phase of growth.

### Iron oxidation metabolism

To evaluate the ability of strain HK31-G<sup>T</sup> to oxidize iron under microaerophilic and anaerobic conditions (with nitrate as the terminal electron acceptor), its growth was evaluated using a modified DSMZ 730 medium (Emerson and Merrill Floyd, 2005), adapted to non-marine strains. The mineral basis was composed of NaCl (0.50 g·L<sup>-1</sup>), MgCl<sub>2</sub>·6H<sub>2</sub>O (0.50 g·L<sup>-1</sup>), CaCl<sub>2</sub>·2H<sub>2</sub>O (0.10 g·L<sup>-1</sup>), KCl (0.34 g·L<sup>-1</sup>), K<sub>2</sub>HPO<sub>4</sub> (0.14 g·L<sup>-1</sup>), NH<sub>4</sub>Cl (0.24 g·L<sup>-1</sup>) and FeCl<sub>2</sub>·4H<sub>2</sub>O at final concentration of 20 mM, added as the sole electron donor. The medium was then adjusted to pH 6.5 and placed under an anaerobic atmosphere (N<sub>2</sub> (100%; 1 bar)) before autoclaving. Solutions of Na<sub>2</sub>S (0.30 g·L<sup>-1</sup>) under N<sub>2</sub> (100%) and Na<sub>2</sub>CO<sub>3</sub> (1.50 g·L<sup>-1</sup>) under N<sub>2</sub>/CO<sub>2</sub> (80/20) have been prepared and autoclaved separately and then added to the medium after sterilization. In addition, autoclaved solutions of vitamins (1 mL; DSMZ 141, 10×) and trace elements (10 mL, DSMZ 141, 1×) were also added to the medium that was finally distributed (10 mL) in 50 mL penicillin flasks. Flasks were placed under N<sub>2</sub>/CO<sub>2</sub> (80/20) atmosphere. To be under microaerophilic conditions, 4 mL of 0.22 µm filtered ambient air were injected into the flask to obtain a 2% final dioxygen concentration, while to be under anaerobic conditions, 200 µL of KNO<sub>3</sub> solution (1 mol·L<sup>-1</sup>) were added to obtain a final concentration of 20 mmol·L<sup>-1</sup>. Before inoculations that were performed in triplicate, cells of strain HK31-G<sup>T</sup> were previously grown in R2A broth, were washed three times with the mineral basis described above, in order to remove all traces of carbon source. Then, 250 µL of the washed cells suspension were used to inoculate the Fe(II)-oxidizing culture medium. Incubations were performed at 25 °C without agitation for 5 and 6 days (end of exponential growth phase) under microaerophilic and anaerobic conditions, respectively. Negative controls for each condition were added to the experiment (culture medium without inoculation of cell suspension). For the third consecutive cultures in these conditions, cell growth was estimated by direct cell count using epifluorescence microscopy (Olympus BX60) at T0 day and T5 (microaerophilic condition) or T6 (anaerobic condition) days of incubation. For each condition, the experiments were repeated twice and in an independent way. Each time, similar trends were obtained.

In parallel to each cell count, Fe(II) concentrations were estimated spectrophotometrically using the Ferrozine method with slight modifications (Rouxel et al., 2018; Viollier et al., 2000). At each measuring point, 150 µL of HCl 0.2% was added to 150 µL of bacterial culture to stop the Fe(II)-oxidation kinetics. To determine the Fe(II) concentrations, 100 µL of HCl at 6 mol·L<sup>-1</sup>, were added, and then supplemented with the ferrozine solution (100 µL; 10 mmol·L<sup>-1</sup>) and the analytical buffer (ammonium acetate (200 µL; 10 mol·L<sup>-1</sup>). The absorbance of the mixture was then measured spectrophotometrically at 562 nm. In order to link the OD<sub>562nm</sub> obtained for the cultures with Fe(II) concentrations, a standard curve (OD<sub>562nm</sub> versus Fe(II) concentrations (µmol·L<sup>-1</sup>)) was made using the culture medium (from 0 to 100 µmol·L<sup>-1</sup>), as previously described (Fig. S2). To evaluate if the Fe(II) concentrations were

significantly different between T0 and T5 or T6, the Mann-Whitney test (package rstatix version 0.7.2) was applied using RStudio (Version 4.2.1).

Transmission electron microscopy (TEM, Jeol JEM 1400) was finally performed to visualize cell-mineral interactions under both conditions.

### Phylogenetic analysis

The complete double-strand 16S rRNA coding gene sequence of strain HK31-G<sup>T</sup> was generated from an isolated colony, as described elsewhere (Alain et al., 2002). Pairwise 16S rRNA gene sequence similarity, of strains having validated published names was calculated using a global alignment algorithm implemented at the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/) (Yoon et al., 2017). Phylogenetic trees were reconstructed using the neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods using the software Seaview version 5.0.5 (Gouy et al., 2010). The evolutionary distances for the NJ, MP and ML methods were respectively calculated using the Kimura two-parameters, the Dnapars and the GTR models (Kimura, 1983; Saitou and Nei, 1987; Dereeper et al., 2008; Gouy et al., 2010; Guindon et al., 2010). The robustness of the inferred topology was assessed by bootstrap analyses based on 1000 replications.

### Genome sequencing, assembly and annotation

Genomic DNA of the novel strain HK31-G<sup>T</sup> was extracted according to a standard PCI (Phenol-Chloroform-Isoamyl alcohol 25:24:1) protocol (Charbonnier et al., 1995). Whole genome sequencing was performed by the Fasteris company (Plan-les-Ouates, Switzerland) using the Illumina MiSeq technology (2 × 150 bp paired reads; Micro Nano V2 chemistry). Reads quality control and genome assembly were performed on Galaxy France (https://usegalaxy.fr/) using, respectively, FastQC (Galaxy v0.73) (Andrews, 2010) and SPAdes assemblers (Galaxy Version 3.15.4) (Gangiredla et al., 2021). Quality of the genome assembly was then obtained with Quast (Galaxy Version 5.2.0) (Gurevich et al., 2013). Genome completeness and potential redundancy were estimated with CheckM (Parks et al., 2015) on the MicroScope Microbial Genome Annotation and Analysis Platform (MaGe; https://mage.genoscope.cns.fr) (Vallenet et al., 2020). The digital DNA-DNA hybridization (dDDH) scores, the average nucleotide identity scores (ANI; OrthoANI values) and the average amino acid identity (AAI) scores between the genome of strain HK31-G<sup>T</sup> and genomes of closely related type strains were respectively obtained using the genome-to-genome distance calculator (GGDC 2.1), using formula 2 (Meier-Kolthoff et al., 2013; Yoon et al., 2017), the ANI calculator tool from the EzBioCloud web server (https://www.ezbiocloud.net/tools/ani) as well as the FastANI tool on Galaxy France platform (Galaxy Version 1.3), and finally the EzAAI suite of workflows (Kim et al., 2021), respectively. Genome assembly of the new strain HK31-G<sup>T</sup> was annotated via the MaGe platform using KEGG and BioCyc databases but also using Prokka (Seemann, 2014) (Galaxy Version 1.14.6) on the Galaxy platform (Wee and Yap, 2021) and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016). In addition, EggNOG database was used to classify coding DNA sequences (CDS) to clusters of orthologous groups (COG) (Hernández-Plaza et al., 2023). To identify the closest Metagenomes Assembled Genomes (MAGs) to the genome of strain HK31-G<sup>T</sup>, the ANI scores between the genome of HK31-G<sup>T</sup> and the 262 sequences of available MAGs affiliated to *Phenylbacterium* sp. on NCBI database, were calculated using FastANI tool (Jain et al., 2018).

### Phylogenomic tree

The phylogenomic analysis focused on 20 reference genomes representatives of the *Brevundimonas*, *Caulobacter* and *Phenylbacterium* genera and phylogenetically closely related to strain HK31-G<sup>T</sup> (*Brevundimonas nasdae* Au29<sup>T</sup> (GCF_019395145.1), *Brevundimonas*
vancanneyttii NCTC9239 (GCF_901421975.1), *Caulobacter mirabilis* FWC 38T (GCF_002749615.1), *Caulobacter henricii* CB4 (GCF_001414055.1), *Caulobacter rhizosphaerae* KCTC 52215T (GCF_010977555.1), *Caulobacter soli* Ji-3-8T (GCF_011045195.1) *Caulobacter hibisci* KACC 18849T (GCF_016135805.1), *Caulobacter flavus* RHGG3T (GCF_003722335.1), *Caulobacter segnis* ATCC 21756T (GCF_000092285.1), *Caulobacter vibrioides* CB15T (GCF_000006905.1), *Phenylobacterium aquaticum* KACC 18306T (GCF_022695515.1), *Phenylobacterium haematophilum* DSM 21793T (GCF_014196295.1), *Phenylobacterium glauciei* 20 BVR1T (GCF_016772415.1), *Phenylobacterium parvum* HYN0004T (GCF_003150835.1), *Phenylobacterium immobile* ATCC 35973T (GCF_001375595.1), *Phenylobacterium hankyongense* HKS-05T (GCF_003254505.1), *Phenylobacterium deserti* YIM 73061T (GCF_003254705.1), *Phenylobacterium soli* LX32T (GCF_003254475.1), *Phenylobacterium kunshanense* BUT-10T (GCF_003254525.1) and *Phenylobacterium zucineum* HLK1T (GCF_000017265.1), the target genome HK31-GT and its six closely related publicly available MAGs from groundwater (classified as the species *Phenylobacterium* sp030693625 on the Genome Taxonomy Database (GTDB, https://gtdb.ecogenomic.org), with accession numbers GCA_030693625, GCA_030645635, GCA_030704785, GCA_030696765, GCA_030683775 and GCA_030652015). The phylogenomic tree was constructed with the Anvi'o software (version 7.1) following the workflow published in 2017 (https://merenlab.org/2017/06/07/phylogenomics/) (Eren et al., 2021). Briefly, genomes were recovered from NCBI database and an Anvi'o contigs database was generated for each of them using the *anvi-gen-contigs-database* command. To compare genomes, the *anvi-get-sequences-for-hmms-hits* command was used to annotate single copy core genes listed in the *Bacteria* 71 HMMs profile collection, as well as ribosomal RNAs profiles ('Ribosomal RNA_16S' (3 models), 'Ribosomal RNA_23S' (2 models), and 'Ribosomal RNA_5S' (5 models)). To build the tree, amino acid sequences were extracted, aligned and concatenated using the *anvi-get-sequences-for-gene-cluster* command. Then, to get the newick-formatted phylogeny artifact for these genomes and MAGs, the *anvi-gen-phylogenomic-tree* command was used with FastTree and the maximum-likelihood method (Price et al., 2009). The phylogenomic tree obtained was visualized using the *anvi'o* interactive interface thanks to the *anvi-interactive* command. The tree was rooted using the genome *Sphingomonas paucimobilis* ZJSH1 (GCA_016919545.1) as an outgroup. Finally, the single-copy-core genes corresponding to each of the genomes from the phylogenomic tree were used to calculate ANI values between each of them using the FastANI tool on Galaxy France platform (Galaxy Version 1.3) (Jain et al., 2018). Genes involved in iron metabolism were identified in the genome of strain HK31-GT and those of its closely related MAGs using FeGenie in command line (version 1.2) according to Garber et al. (2020). Sequences homologous to the *cyc2* gene were also subjected to an HMM profile search using the HMMER 3.4 software package (Eddy, 1992).

## Environmental distribution

To highlight the distribution of the novel strain HK31-GT across diverse ecosystems, the Sandpiper resource (v0.3.0) was used for the interrogation of public shotgun metagenome datasets through the use of SingleM pipeline (Woodcroft et al., 2024). To do so, the MAG of the species *Phenylobacterium* sp030693625 (same species as HK31-GT) was implemented on the Sandpiper web-interface (https://sandpiper.qut.edu.au/taxonomy/s_Phenylobacterium%20sp030693625) to obtain its relative abundance in public metagenomes.

## Results and discussion

### Morphological and physiological properties

Under optimal growth conditions, colonies of strain HK31-GT are circular with regular edges and smooth surface and with a light creamish

color. Cells are Gram-negative thin rods dividing by binary fission. They occur mainly singly (Fig. 1a) but can also form aggregates. Cell sizes range from 0.36 to 0.71 µm wide (mean 0.53 µm; *n* = 42) and from 1.00 to 3.59 µm long (mean 1.87 µm; *n* = 42). For many cells observed by TEM, refragent intracellular granules, which could be polyhydroxyalkanoates (PHA) storage granules, were also observed (Fig. 1b). Strain HK31-GT is motile, as confirmed by observations on MMN agar medium and the observation of a single polar flagellum per cell by TEM (Fig. 1c, d). Interestingly, some cells of strain HK31-GT harbor a stalk (Fig. 1a). Such cellular appendices were only previously observed for *P. conjunctum* FWC 21T cells. However, contrary to the study of Abraham et al. (2008), no 'rosette' of prosthecate cells was observed. Like all closely related type strains, HK31-GT is positive for oxidase activity but negative for catalase. (Table 1)

Growth was observed from 10 to 30 °C with optimal growth between 25 and 30 °C which is consistent with the optimal growth temperature of the other related type strains (Table 1). With respect to salt tolerance, the strain grew only at 0.5 % NaCl and showed optimal growth without sodium chloride. Only *P. haematophilum* LMG 11050T and *P. conjunctum* FWC 21T expressed greater salt resistance with growth up to 2 % NaCl (Table 1). Concerning the pH, growth of strain HK31-GT was observed from pH 6 to 8 with optimal growth at pH 6. This narrow pH tolerance was also observed for the closest type strains having all an optimum close to neutrality (Table 1). All these results are consistent with the environment where the strain was isolated. Indeed, at the time of sampling, a temperature of 24.1 °C and a pH of 8.78 were recorded, meaning that strain HK31-GT is adapted to these environmental conditions. Under optimal growth conditions on R2A broth, the growth rate and the generation time of strain HK31-GT are, respectively, 0.031 h⁻¹ and 32 h 32 min (Fig. S3).

The novel isolate is chemoorganoheterotroph and grows by aerobic respiration. It catabolizes L-alanine, L-proline, phenylalanine, malonate and to a lesser extent, (D,L)-β-Hydroxybutyrate. *P. haematophilum* LMG 11050T has a similar substrate-utilization range, except that it does not use malonate and (D,L)-β-hydroxybutyrate. The other closely related strains are not capable of using the substrates used by HK31-GT excepted *P. aquaticum* W2-3-4T that is also able to catabolize malonate as the sole carbon source (Table 1). Regarding the enzymatic activities that can differentiate the new strain HK31-GT from the other closely related strains, the α- and β-glucosidase as well as the α-mannosidase are only active for strain HK31-GT as shown by the API®Zym kit. It should be noted that β-galactosidase activity was also demonstrated for *P. haematophilum* LMG 11050T when using API®20E kit with PNPG as substrate. As with *P. glauciei* 20VBR1T and *P. aquaticum* W2-3-4T, the strain HK31-GT was able to use nitrate as a terminal electron acceptor, revealing nitrate reductase activity (Table 1). This activity was also experimentally demonstrated with the MMN medium for strain HK31-GT and *P. aquaticum* W2-3-4T.

### Chemotaxonomic characteristics

The major respiratory quinone of strain HK31-GT is Q-10 (98.79 %) as described for other members of the family *Caulobacteraceae* (Thomas et al., 2022). In addition, a low amount of Q-9 (1.21 %) was also detected. The polar lipids profile indicated the presence of phosphatidylglycerol (PG), which is in line with other species of *Phenylobacterium*, and by two unknown glycolipids (GL), an unknown glycophospholipid (GPL), two unknown phospholipids (PL) and four unidentified polar lipids (L) (Fig. S4). This profile is slightly different from that of the closest species *P. glauciei* 20VBR1T and *P. aquaticum* W2-3-4T. Indeed, in addition to PG, *P. glauciei* 20VBR1T was characterized by an unknown PG, an unknown GL and four unknown lipids, while *P. aquaticum* W2-3-4T was characterized by an unknown PL, four unknown GL and three unknown lipids (Thomas et al., 2022; Jo et al., 2016). This latter profile was similar to *P. conjunctum* FWC 21T (LMG 24262T) but quite different from that of *P. haematophilum* LMG 11050T (Abraham et al., 2008). Polar
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Transmission electron microscopy microphotographs of cells of strain HK31-G<sup>T</sup> grown on R2A at 25 °C for 3 days. (a) Some cells harbor a cellular appendage, so-called prostheca or stalk (indicated by black arrow). (b) Intracellular granules. (c) A cell with its polar flagellum. (d) Enlargement of image (c).</figcaption></figure>

lipids can therefore be used as biomarkers, in addition to other criteria,
to distinguish *Phenylobacterium* species (Abraham et al., 2008).

The predominant cellular fatty acids (>10 % of the total fatty acids) of strain HK31-G<sup>T</sup> were C<sub>18:1</sub>ω7c (also in summed feature eight comprising C<sub>18:1</sub>ω7c and/or C<sub>18:1</sub>ω6c; 44.54 %), followed by C<sub>16:1</sub>ω6c (also in summed feature three comprising C<sub>16:1</sub>ω6c and/or C<sub>16:1</sub>ω7c; 19.30 %) and the saturated fatty acid C<sub>16:0</sub> (11.62 %) (Table 2). Cells of strain HK31-G<sup>T</sup> and its closely related genera are characterized by high amounts (>10.2 %) of summed feature eight and C<sub>16:0</sub> as already observed for members of the genus *Phenylobacterium* (Jo et al., 2016; Stackebrandt, 2006). Summed feature three seems more abundant and in similar amounts in cells of strain HK31-G<sup>T</sup> and *P. glaciei* 20VBR1<sup>T</sup> (19.30 % and 19.18 %, respectively) compared to the closely related species (ranging from 1.88 % to 8.9 %). Conversely, the methyl ester fatty acid C<sub>18:1</sub>ω5c11-methyl, present in similar amount in cells of strain HK31-G<sup>T</sup> and *P. glaciei* 20VBR1<sup>T</sup> (2.54 % and 6.55 %, respectively), was less abundant compared to the closely related genera (ranging from 10.8 % to 28 %). Some minor quantitative differences can also be observed between the new strain HK31-G<sup>T</sup> and its closest relatives. Indeed, low amount of summed features 1 (comprising C<sub>13:0</sub>-OH and/or isoC<sub>15:1</sub>; 0.26 %) and 9 (comprising C<sub>17:1</sub>isoω9c and/or C<sub>16:0</sub>-methyl; 0.41 %) and isoC<sub>15:0</sub> (0.32 %) were only detected in cells of strain HK31-G<sup>T</sup> (Table 2). Overall, these whole-cell fatty acid profiles demonstrate that strain HK31-G<sup>T</sup> shares typical characteristics of species of the genus *Phenylobacterium* but with a fatty acid profile highly similar to that of *P. glaciei* 20VBR1<sup>T</sup>.

Iron oxidation metabolism

After three successive transfers in a medium targeting growth by iron
oxidation, cell density and Fe(II) concentrations were determined

immediately after inoculation in all cultures and after 5 and 6 days of
incubation under microaerophilic and anaerobic (with nitrate as the
terminal electron acceptor) conditions, respectively. For each condition,
a cell growth greater than 0.5 log unit in terms of number of cells/mL
was obtained for the new strain HK31-G'. Indeed, under microaerophilic
and anaerobic conditions, an increase of cells number by 2.2-fold and
2.3-fold was evidenced, respectively (Fig. 2). As abiotic Fe(II)-oxidation
occurs under the microaerophilic conditions, it was not possible to ev-
idence a significant biotic Fe(II)-oxidation by cells of strain HK31-G', in
the presence of low oxygen concentration by using the ferrozine
bioassay (P > 0.05; data not shown), despite the cellular growth, in
comparison with control. Conversely, under anaerobic condition, Fe(II)
concentrations were significantly lower between T0 (21.45 µmol.L⁻¹ ±
1.57 µmol.L⁻¹) and T6 days (17.22 µmol.L⁻¹ ± 1.39 µmol.L⁻¹) (P <
0.001) when the cellular growth of HK31-G' occurred demonstrating the
production of energy by the oxidation of Fe(II), whereas no significant
difference was observed for negative controls between T0 and T6 days
(P > 0.05). This cellular growth could be compared to the one reported
by Laufer-Meiser et al. (2024) for three strains of *Hydrogenovibrio* sp.
isolated from deep-sea hydrothermal vents and capable of Fe(II)-
oxidation, although no known oxidation pathway has been evidenced.
In addition, potential cell-mineral interactions necessary to the extra-
cellular oxidation of Fe, were also observed by TEM (Fig. S5). Overall,
cells of strain HK31-G' are capable of oxidizing Fe as sole energy source
under both microaerophilic and anaerobic conditions. To our knowl-
edge, *Phenylobacterium* species are not known to have a secondary
metabolism based on Fe(II)-oxidation. This shift in metabolism is
consistent with the environment in which the strain was isolated.
Indeed, following an injection of CO₂, environmental conditions become
favorable to the development of chemolithoautotrophic microbial
communities (Mu and Moreau, 2015; O'Mullan et al., 2015) and Fe(II)-
<caption>**Table 1**</caption>

Differential physiological characteristics of strain HK31-G<sup>T</sup> and related type strains of the genus *Phenylobacterium*. 1. HK31-G<sup>T</sup>, 2. *P. glaciei* 20VBR1<sup>T</sup> (DSM 111428<sup>T</sup>) (Thomas et al., 2022), 3. *P. aquaticum* W2-3-4<sup>T</sup> (KACC 18306<sup>T</sup>) (Jo et al., 2016), 4. *P. haematophilum* LMG 11050<sup>T</sup> (Abraham et al., 2008), 5. *P. conjunctum* FWC 21<sup>T</sup> (LMG 24262<sup>T</sup>) (Abraham et al., 2008).

<table><thead><tr><th>Characteristics</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th></tr></thead><tbody><tr><td>Colony color</td><td>Light creamish</td><td>Light creamish</td><td>Light brown</td><td>Light creamish</td><td>Light brown</td></tr><tr><td>Cell size (µm)</td><td>0.4-0.7 × 1.0-3.6</td><td>0.4-0.5 × 1.0-2.0</td><td>0.4-0.6 × 2.5-4.0</td><td>0.3-0.4 × 0.9-2.5</td><td>0.5-0.7 × 1.2-1.6</td></tr><tr><td>Mobility</td><td>+</td><td>—</td><td>—</td><td>+</td><td>—</td></tr><tr><td>Prostheca</td><td>+</td><td>—</td><td>—</td><td>—</td><td>+</td></tr><tr><td>Temperature range (optimum) (°C)</td><td>10-30 (25-30)</td><td>10-35 (20)</td><td>18-40 (25-30)</td><td>10-40 (37)</td><td>20-40 (25-30)</td></tr><tr><td>pH growth range (optimum)</td><td>6-8 (6)</td><td>6.5-8 (7)</td><td>6.0-8.0 (7)</td><td>6.0-8.5</td><td>5.5-8</td></tr><tr><td>NaCl tolerance (optimum) (%, w/v)</td><td>0-0.5 (0)</td><td>0-2.5 (0.5)</td><td>0-0.5</td><td>0-2</td><td>0-2</td></tr><tr><td>Nitrate reduction to nitrite</td><td>+</td><td>+</td><td>+</td><td>—</td><td>—</td></tr><tr><td>Carbon sources utilization:</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>L-Alanine</td><td>+</td><td>—</td><td>—</td><td>w</td><td>—</td></tr><tr><td>L-Proline</td><td>+</td><td>—</td><td>—</td><td>+</td><td>—</td></tr><tr><td>Phenylalanine</td><td>+</td><td>—</td><td>—</td><td>w</td><td>—</td></tr><tr><td>(D,L)-β-Hydroxybutyrate</td><td>w</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Malonate</td><td>+</td><td>—</td><td>+</td><td>—</td><td>—</td></tr><tr><td>Mannitol (MMN)</td><td>—</td><td>—</td><td>+</td><td>—</td><td>—</td></tr><tr><td>Glucose (Kligler-Hajna)</td><td>—</td><td>—</td><td>—</td><td>+</td><td>—</td></tr><tr><td>API® 20NE and 20E results</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Lysine decarboxylase</td><td>—</td><td>NA</td><td>+</td><td>—</td><td>—</td></tr><tr><td>Ornithine decarboxylase</td><td>—</td><td>NA</td><td>+</td><td>—</td><td>—</td></tr><tr><td>Urease</td><td>—</td><td>—</td><td>+</td><td>—</td><td>+</td></tr><tr><td>Tryptophan deaminase</td><td>+</td><td>NA</td><td>+</td><td>—</td><td>—</td></tr><tr><td>Gelatinase</td><td>+</td><td>NA</td><td>+</td><td>—</td><td>—</td></tr><tr><td>Arginine dihydrolase</td><td>—</td><td>—</td><td>+</td><td>—</td><td>+</td></tr><tr><td>β-Glucosidase (aesculin hydrolysis)</td><td>+</td><td>NA</td><td>—</td><td>+</td><td>—</td></tr><tr><td>Gelatin hydrolysis</td><td>—</td><td>—</td><td>+</td><td>—</td><td>+</td></tr><tr><td>β-Galactosidase (PNPG)</td><td>+</td><td>NA</td><td>—</td><td>+</td><td>—</td></tr><tr><td>API® ZYM results</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Valine arylamidase</td><td>—</td><td>+</td><td>—</td><td>+</td><td>+</td></tr><tr><td>α-Galactosidase</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>β-Galactosidase</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>α-Glucosidase</td><td>—</td><td>+</td><td>—</td><td>+</td><td>+</td></tr><tr><td>β-Glucosidase</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td></tr><tr><td>N-acetyl-β-glucosaminidase</td><td>+</td><td>—</td><td>—</td><td>+</td><td>+</td></tr><tr><td>α-mannosidase</td><td>+</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>DNA G + C content (mol%)</td><td>67.95</td><td>67.86</td><td>68.87</td><td>67.9</td><td>67.0</td></tr><tr><td>Isolation source</td><td>Deep subsurface geothermal aquifer</td><td>Glacier snout ice</td><td>Reservoir of water purifier</td><td>Human blood</td><td>Water biofilm</td></tr></tbody></table>

All data concerning nitrate reduction to nitrite, carbon sources utilization and API®20 NE, 20 E and ZYM come from this study. Otherwise, for other characteristics concerning related type strains, data come from the references cited above. Note that for *P. glaciei* 20VBR1<sup>T</sup>, the strain was not available from the two culture collections where it was deposited and the results presented here were taken from (Thomas et al., 2022).

All the strains were positive for oxidase activity but negative for catalase. For carbon sources utilization, all the strains were negative for xylose, glycerol, aspartate, acetate, lactate, succinate, glutamate, propionate and glucose through butanediol pathway. None are able of citrate fermentation. In API®20 NE and API®20 E, all the strains were negative for β-galactosidase and arginine dihydrolase, assimilation of d-glucose, L-arabinose, D-mannose, N-acetyl-glucosamine, D-maltose, citrate, gluconate, caprate, adipate, malate and phenyl-acetate, production of H₂S, indole and acetoin and acid production from d-glucose, d-mannitol, inositol, D-sorbitol, L-rhamnose, D-saccharose, D-melibiose, amygdalin and L-arabinose. In API®ZYM kits, all the strains were positive for alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for lipase, cystine arylamidase, chymotrypsine, β-glucuronidase and α-fucosidase.

+, positive; —, negative; w, weakly; w, weak; NA, Not Available.

oxidizing bacteria (Trias et al., 2017).

## Phylogeneticaffiliation

Comparative analysis of 16S rRNA gene sequences of HK31-G<sup>T</sup> (1372 bp) and closely related type strains with validated published names, indicated that its closest relatives were *P. glaciei* 20VBR1<sup>T</sup> (99.9 %), *P. aquaticum* W2-3-4<sup>T</sup> (97.52 %), *P. haematophilum* LMG 11050<sup>T</sup> (97.37 %) and *P. parvum* HYN0004<sup>T</sup> (96.94 %). In this case, the 16S rRNA gene marker did not allow strain HK31-G<sup>T</sup> to be differentiated from its closest relative *P. glaciei* 20VBR1<sup>T</sup>. Indeed, this phylogenetic marker is highly conserved within many bacterial genera and could be almost 100 % identical between strains of different species belonging to the same genus (Rosselló-Mora and Amann, 2001). The phylogenetic trees based on ML, MP and NJ algorithms using the sequence of the gene coding for the 16S rRNA of strains belonging to the genera *Phenylobacterium*, *Brevundimonas*, *Caulobacter*, *Aquiducibacter*, and *Terricaulis*, confirmed that strain HK31-G<sup>T</sup> clusters within the genus *Phenylobacterium*, and forms a clade (MP, ML and NJ algorithms) with *P. glaciei*

20VBR1<sup>T</sup>, *P. aquaticum* W2-3-4<sup>T</sup>, *P. haematophilum* LMG 11050<sup>T</sup> and *P. conjunctum* FWC 21<sup>T</sup> (Fig. S6).

The marker nucleotides of the 16S rRNA gene of the order *Caulobacterales* and in particular of the genus *Phenylobacterium*, described in Abraham et al. (2008) were used to determine the nucleotide signature of strain HK31-G<sup>T</sup> in relation to the type strains of the closest species, *P. glaciei* 20VBR1<sup>T</sup>, *P. aquaticum* W2-3-4<sup>T</sup>, *P. haematophilum* LMG 11050<sup>T</sup>, *P. conjunctum* FWC 21<sup>T</sup>, *P. parvum* HYN0004<sup>T</sup> and *P. koreense* Slu-01<sup>T</sup> (Table S1). We confirmed that the new strain and all compared species were missing nucleotides at positions 71–88, 183–190, 206–211 and 452–476 (Escherichia coli str. K12 subtr. MG1655 numbering), as expected for the order *Caulobacterales*. Strains HK31-G<sup>T</sup> and *P. glaciei* 20VBR1<sup>T</sup> have an A at position 1265 and a T at position 1270, which is different from the closest type strains (1265-T and 1270-A) (Table S1). It has been previously shown that *P. glaciei* 20VBR1<sup>T</sup> has the same nucleotide signature as *P. falsum* AC-49<sup>T</sup> and *P. panacis* DCY 109<sup>T</sup> (Thomas et al., 2022). These two nucleotides (1265-T and 1270-A) have been used so far to differentiate the genera *Phenylobacterium* and *Caulobacter* from the genera *Brevundimonas* and *Asticcacaulis*, where they are absent
<table><caption>**Table 2**
Comparison of whole-cell fatty acid profiles (% of the total) of strain HK31-G<sup>T</sup> with type strains of closely related species.</caption><thead><tr><th>Fatty acids<sup>a</sup> (%)</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th></tr></thead><tbody><tr><td><strong>Saturated</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>C<sub>10</sub>: 0</td><td>-</td><td>-</td><td>2.1</td><td>-</td><td>-</td></tr><tr><td>C<sub>11</sub>: 0</td><td>0.18</td><td>-</td><td>0.69</td><td>TR</td><td>0.8</td></tr><tr><td>C<sub>12</sub>: 0</td><td>-</td><td>-</td><td>0.3</td><td>2.3</td><td>-</td></tr><tr><td>C<sub>13</sub>: 0</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C<sub>14</sub>: 0</td><td>0.44</td><td>0.48</td><td>0.2</td><td>TR</td><td>0.5</td></tr><tr><td>C<sub>15</sub>: 0</td><td>TR</td><td>6.77</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C<sub>16</sub>: 0</td><td><strong>11.62</strong></td><td><strong>10.2</strong></td><td><strong>30.3</strong></td><td><strong>17.4</strong></td><td><strong>20.5</strong></td></tr><tr><td>C<sub>17</sub>: 0</td><td>4.87</td><td>4.0</td><td><strong>10.67</strong></td><td>7.6</td><td>6.9</td></tr><tr><td>C<sub>18</sub>: 0</td><td>0.76</td><td>-</td><td>3.7</td><td>TR</td><td>0.5</td></tr><tr><td>C<sub>20</sub>: 0</td><td>0.68</td><td>-</td><td>-</td><td>1.3</td><td>-</td></tr><tr><td><strong>Unsaturated</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>C<sub>12</sub>: 1ω7c</td><td>TR</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C<sub>16</sub>: 1ω11c</td><td>0.91</td><td>0.5</td><td>1.3</td><td>1.6</td><td>4.0</td></tr><tr><td>C<sub>16</sub>:1ω7c</td><td><strong>19.30</strong></td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C<sub>17</sub>: 1ω6c</td><td>6.86</td><td>4.20</td><td>1.27</td><td>2.0</td><td>1.7</td></tr><tr><td>C<sub>17</sub>: 1ω8c</td><td>1.76</td><td>1.56</td><td>1.09</td><td>0.9</td><td>0.8</td></tr><tr><td>C<sub>18</sub>: 1ω7c</td><td><strong>44.54</strong></td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><strong>Methyl ester</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>C<sub>18</sub>: 1ω5c 11-methyl</td><td>-</td><td>-</td><td>-</td><td>-</td><td>2.0</td></tr><tr><td>C<sub>18</sub>: 1ω7c 11-methyl</td><td>2.54</td><td>6.55</td><td><strong>21.7</strong></td><td><strong>28.0</strong></td><td><strong>10.8</strong></td></tr><tr><td><strong>Branched-chain fatty acid</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Iso-C<sub>15</sub>: 0</td><td>0.32</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Iso-C<sub>17</sub>: 0</td><td>0.96</td><td>0.5</td><td>0.75</td><td>TR</td><td>0.5</td></tr><tr><td>Anteiso-C<sub>15</sub>: 0</td><td>0.27</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><strong>Hydroxyl fatty acids</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>C<sub>12</sub>:0 3-OH</td><td>0.68</td><td>0.79</td><td>0.85</td><td>0.8</td><td>1.0</td></tr><tr><td>C<sub>12</sub>:1 3-OH</td><td>2.64</td><td>2.0</td><td>1.7</td><td>1.9</td><td>2.1</td></tr><tr><td><strong>Cyclo fatty acids</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>C<sub>17</sub>:0 cyclo ω8c</td><td>-</td><td>-</td><td>0.57</td><td>2.7</td><td>-</td></tr><tr><td><strong>Summed features</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1 (C<sub>13</sub>:0 3-OH and/or iso-C<sub>15</sub>:1H)</td><td>0.26</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>3 (C<sub>16</sub>:1ω6c and/or C<sub>16</sub>:1 ω7c)</td><td><strong>19.30</strong></td><td><strong>19.18</strong></td><td>1.88</td><td>5.9</td><td>8.9</td></tr><tr><td>8 (C<sub>18</sub>:1ω7c and/or C<sub>18</sub>:1 ω6c)</td><td><strong>44.54</strong></td><td><strong>40.25</strong></td><td><strong>16.86</strong></td><td><strong>25.4</strong></td><td><strong>38.8</strong></td></tr><tr><td>9 (C<sub>17</sub>:1ω8c ω9c and/or C<sub>16</sub>:0-methyl)</td><td>0.41</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

Taxa: 1. HK31-G<sup>T</sup>, 2. *P. glauciei* 20VBR1<sup>T</sup> (DSM 111428<sup>T</sup>) (Thomas et al., 2022), 3. *P. aquaticum* W2-3.4<sup>T</sup> (KACC 18306<sup>T</sup>) (Jo et al., 2016), 4. *P. haematophilum* LMG 11050<sup>T</sup> (Abraham et al., 2008), 5. *P. conjunctum* FWC 21<sup>T</sup> (LMG 24262<sup>T</sup>) (Abraham et al., 2008).

Values are percentages of the fatty acids that were assigned to fatty acids in the peak-naming table of the MIS database (MIDI, Microbial ID, Newark, DE 19711 U.S.A.). The nomenclature is as follows: the first number indicates the number of carbon atoms in the molecule; ‘OH’ and ‘cyclo’ indicate hydroxy or cyclic fatty acids; the second number following the colon indicates the number of double bonds present. The position of the double bond is indicated by the carbon atom position starting from the methyl (ω) end of the molecule. c, cis isomer. Major fatty acids (>10 % of the total fatty acids) are indicated in bold.

–, Not detected.

TR, trace amount.

As all strains were not grown under exactly the same conditions or in the same media, the fatty acid percentages must be compared with caution from one strain to another.

according to Abraham et al. (2008).

## Genome overview

Based on MaGe, the total genome size of strain HK31-G<sup>T</sup> was 4.46 Mbp for 100 % completion and 1.41 % redundancy (seven markers were duplicated) and consisted of 104 contigs. The N50 and L50 were respectively 89,925 bp and 19 contigs. The G + C content of the genomic DNA of strain HK31-G<sup>T</sup> was 67.95 %, which is consistent with the closely related strains of the genus *Phenylobacterium* (G + C contents ranging from 66.73 % to 68.87 %) (Table 3). Annotations with MaGe resulted in 4667 CDS, two rRNA operons and 48 tRNA genes (corresponding to the 20 essential amino acids) (Table 3; Fig. S7). Most of the CDS (82.65 %) were assigned to at least one COG category (Table S2). Among major processes, COGs categories related to metabolism were dominant

(27.06 % of the CDS) and included (>4 % of the CDS) (i) inorganic ion transport and metabolism (4.90 %), (ii) amino acid transport and metabolism (4.58 %), and (iii) energy production and conversion (4.51 %). Then, 17.05 % of the CDS were involved in cellular processes and signaling represented mainly by signal transduction mechanisms (4.36 %) and cell wall, membrane and envelope biogenesis (4.19 %). Finally, 13.41 % of the COGs were dedicated to information storage and processing with mechanisms such as transcription (5.07 %), replication, recombination and repair (4.45 %) or signal transduction (4.36 %).

## Phylogenomic tree and Overall Genome Relatedness Indices (OGRI)

Calculation of ANI scores between genome of strain HK31-G<sup>T</sup> and all the sequences of MAGs affiliated to *Phenylobacterium* sp. available on NCBI database allowed to identify six MAGs (accession numbers: GCA_030693625, GCA_030645635, GCA_030704785, GCA_030696765, GCA_030683775 and GCA_030652015) with a score above the species delineation threshold and affiliated to species *Phenylobacterium* sp030693625 (GTDB) (Table S3). OrthoANIu values confirmed these results for the six MAGs identified previously, with values ranging from 97.11 % to 98.12 % (Richter and Rossello-Mora, 2009). These MAGs were therefore used to build the phylogenomic tree. Similarly to the phylogenetic analysis based on the 16S rRNA gene, the phylogenomic tree identified a cluster containing genomes of strain HK31-G<sup>T</sup>, *P. glauciei* 20VBR1<sup>T</sup>, *P. aquaticum* W2-3.4<sup>T</sup> and *P. haematophilum* DSM 21793<sup>T</sup> and the six MAGs affiliated to *Phenylobacterium* sp030693625. The genome of strain HK31-G<sup>T</sup> was most closely related to that of the MAG with accession number GCA_030645635 (Fig. 3). With regard to the reference genomes of *Phenylobacterium* representative species, the genome of *P. glauciei* 20VBR1<sup>T</sup> was the most closely related to the genome of HK31-G<sup>T</sup>, even though they represent two different species (Fig. 3 and Fig. S8.). Digital DNA-DNA hybridization (dDDH) scores between the genome of strain HK31-G<sup>T</sup> and the most closely related genome of reference species ranged from 20.1 to 35.0 % and were well below the threshold for distinguishing two different species (Table 3) (Wayne, 1987; Stackebrandt, 2006). In the same way, OrthoANIu values were all below the generally accepted threshold of 95–96 % for species delineation with values ranging from 75.14 to 88.97 % (Richter and Rossello-Mora, 2009). In addition, ANI values, calculated with FastANI between the most closely related genomes of reference species ranged from 79.4 to 89.1 % and were also well below the threshold for species delineation. FastANI values calculated between the genome of HK31-G<sup>T</sup> and its six closely related MAGs ranged from 96.94 to 97.90 %, confirming that they represent the same species (Table S4 and Fig. 4, Table S5). In addition, AAI values calculated between the genome of HK31-G<sup>T</sup> and its most closely related genomes of reference species ranged from 69.14 to 89.48 % and were also well below the threshold for species delineation (Konstantinidis et al., 2017). Despite the lack of sufficient resolution of 16S rRNA coding gene sequence comparisons to delineate the strain HK31-G<sup>T</sup> as a new species, OGRI (dDDH, ANI and AAI values) were powerful enough to lead to the conclusion that strain HK31-G<sup>T</sup> represents a new species of the genus *Phenylobacterium*. Therefore, phylogenomic distances as well as OGRI values confirmed that strain HK31-G<sup>T</sup> and *P. glauciei* 20VBR1<sup>T</sup> represent two distinct species of the genus *Phenylobacterium*.

## Environmental distribution

To highlight the distribution of the novel strain HK31-G<sup>T</sup> across diverse ecosystems, the Sandpiper resource (v0.3.0) was used for the interrogation of public shotgun metagenome datasets through the use of SingleM pipeline (Woodcroft et al., 2024). To do so, the MAG of the species *Phenylobacterium* sp030693625 (same species as HK31-G<sup>T</sup>) was implemented on the Sandpiper.

Using the Sandpiper resource, the MAG of the species *Phenylobacterium* sp030693625 (same species as HK31-G<sup>T</sup>) yielded 92 matches
<figure><img src="image_4.png" /><figcaption>Fig. 2. Cell concentrations of strain HK31-GT and Fe(II) concentrations at T0 and T6 days in Fe(II)-oxidizing medium (cultures were performed in triplicate, negative controls in duplicate). Cellular concentrations are represented by full black bars. Fe(II) concentrations in the Fe(II)-oxidizing medium inoculated with cells of HK31-GT are represented by striped bars while Fe(II) concentrations in negative controls (without inoculation of cells) are represented by dotted bars. A Mann-Whitman test was applied to show any significant difference of Fe(II) concentrations between T0 and T6 (P < 0.001).</figcaption></figure>

<table><caption>**Table 3**
Genome statistics and Overall Genome Relatedness Indices (OGRI) between the genome of strain HK31-GT and the reference genomes of closely related species of the genus *Phenylobacterium*. Species: 1, HK31-GT (data from this study); 2, *P. glaciei* 20VBR1T; 3, *P. aquaticum* W2-3-4T; 4, *P. haematophilum* DSM 21793T; 5, *P. immobile* strain ET.</caption><thead><tr><th></th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th></tr></thead><tbody><tr><td>Number of contigs</td><td>104</td><td>5</td><td>90</td><td>21</td><td>6</td></tr><tr><td>Size (Mbp)</td><td>4.46</td><td>4.24</td><td>5.25</td><td>4.43</td><td>3.33</td></tr><tr><td>G + C content (%)</td><td>67.95</td><td>67.86</td><td>68.87</td><td>67.90</td><td>66.73</td></tr><tr><td>Number of CDS</td><td>4667</td><td>4257</td><td>5552</td><td>4502</td><td>3316</td></tr><tr><td>rRNA</td><td>6</td><td>3</td><td>3</td><td>3</td><td>3</td></tr><tr><td>tRNA</td><td>48</td><td>43</td><td>46</td><td>47</td><td>45</td></tr><tr><td>16S rDNA similarity (%)</td><td>100</td><td>99.9</td><td>97.52</td><td>97.37</td><td>96.06</td></tr><tr><td>dDDH (%)</td><td>100</td><td>35.0</td><td>22.1</td><td>21.1</td><td>20.1</td></tr><tr><td>OrthoANIu (%)</td><td>100</td><td>88.97</td><td>78.87</td><td>77.64</td><td>75.14</td></tr><tr><td>FastANI (%)</td><td>100</td><td>89.1</td><td>82.2</td><td>80.9</td><td>79.4</td></tr><tr><td>AAI (%)</td><td>100</td><td>89.48</td><td>74.73</td><td>73.85</td><td>69.14</td></tr></tbody></table>

among the metagenomes present in the database, highlighting a worldwide distribution of the new strain HK31-GT (Fig. S9; i.e. USA, China, Brazil, Antarctica and Europe (Iceland, Portugal, Germany, Poland, Denmark). These metagenomes were associated mainly with aquatic environments (i.e. wastewater (25 matching), groundwater (14 matching), activated sludge (14 matching samples), aquifer (7 matching), freshwater (7 matching samples) or mine drainage (2 matching samples)) (Fig. S9; Table. S6). Interestingly, the highest relative abundance of the species *Phenylobacterium* sp030693625 (2.27 %) came from a groundwater metagenome where pyrite (FeS₂) weathering bacteria in shale were studied. In addition, this species was also identified (relative abundance from 0.43 to 0.02 % and coverage from 56.27 to 0.72) in the raw reads of aquifer metagenomes obtained from the CarbFix-1 site for

wells HK12, HK26, and well HK31, from which the novel isolate HK31-GT was isolated (Table S6). These results were in contrast with those obtained for *P. glaciei* where only 11 matching metagenomes were obtained with only four in common with the strain HK31-GT (Table S7).

### Central metabolism and energy production pathways

Based on genomic predictions, strain HK31-GT encodes complete pathways for the biosynthesis of the 20 essential amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine). Complete pathways for organo-heterotrophic growth, namely the glycolysis, Entner-Doudoroff, TCA cycle pathways and the pentose phosphate (oxidative and non-oxidative branches) pathways have been identified, as well as the degradation pathways for several amino acids such as alanine, asparagine, glutamate, glutamine, L-cysteine, L-serine and taurine. The genome also encodes complete pathways for glycerol and acetoacetate degradation and for oxidation of C1 compounds such as methanol and formaldehyde. The putative methylotrophic growth of strain HK31-GT remains to be experimentally validated. Concerning the CO₂ carbon fixation pathway, all genes involved in the reverse oxidative TCA cycle (roTCA) are encoded in the genome of strain HK31-GT (Table S8). In this pathway, associated with high CO₂ concentrations, citrate synthase is used as a replacement for ATP-citrate lyase encoded in the canonical reductive TCA (rTCA) cycle and works in a reversible way to cleave citrate (Mall et al., 2018; Steffens et al., 2021). Although this reaction is thermodynamically unfavorable, it allows one less ATP molecule to be expended. The roTCA cycle lacks unique enzymes (i.e. ATP-citrate lyase or citryl-CoA synthetase large subunit (CCS) and citryl-CoA lyase (CCL) for rTCA or Rubisco for Calvin-Benson-Bassham (CBB)).
<figure><img src="image_5.png" /><figcaption>Fig. 3. Phylogenomic tree showing the phylogenomic position of strain HK31-G<sup>T</sup> with respect to *Phenylobacterium*, *Caulobacter* and *Brevundimonas* species having sequenced genomes, and the six closely related MAGs belonging to the species *Phenylobacterium* sp030693625 (GTDB). The genome sequence of *Sphingomonas paucimobilis* ZJSH1 was used as an outgroup. The tree was built using FastTree implemented in Anvi'o and using the Maximum-Likelihood method. Bootstrap values below 0.5 are not shown. Bar, 0.07 substitutions per position.</figcaption></figure>

cycle; these enzymes were not identified in the genome of HK31-G<sup>T</sup>)
making it cryptic for genomic analyses and probably leading to its un-
derestimation among autotrophic microorganisms (Mall et al., 2018;
Steffens et al., 2021). Thus, given that present results rely solely on
genomic analysis, additional biochemical or transcriptomic analyses are
required to confirm this hypothesis. Another pathway, the CO<sub>2</sub> fixation
into oxaloacetate (anaplerotic), was identified in the genome of HK31-
G<sup>T</sup>. This pathway can be found in some autotrophic bacteria and archaea
and involves two enzymes, the carbonic anhydrase and the phospho-
enolpyruvate carboxylase, both encoded in the genome of strain HK31-
G<sup>T</sup>, to convert CO<sub>2</sub> to hydrogen carbonate and then to oxaloacetate,
respectively (Patel et al., 2004).

Genomic predictions allowed identifying genes involved in poly-
hydroxyalkanoates (PHAs) synthesis. Indeed, *phaB* (locus_tag (PGAP):
OCL97_08250), *phaC* (locus_tag (PGAP): OCL97_01260) and *phaR*
(locus_tag (PGAP): OCL97_08240), encoding respectively for an
acetoacetyl-CoA reductase, the poly(3-hydroxyalkanoate) polymerase
and the PHA synthesis repressor were identified. In addition, genes
encoding polyhydroxyalkanoic acid system family protein (locus_tag
(PGAP): OCL97_12980) and a PHA depolymerase (locus_tag (PGAP):
OCL97_14885) were also evidenced. The presence of these genes

suggests that it is the class IV PHA synthase operon, widespread in
bacteria belonging to the genus *Bacillus* (Tsuge et al., 2015), that is at
work in strain HK31-G<sup>T</sup>. These results, combined with the observations
of intracellular granules by TEM, reinforce the hypothesis that the new
strain produces PHAs storage granules to adapt to harsh environments
where the organic matter is scarce. With regard to energy metabolism,
genes encoding cytochrome *c* oxidase complex for aerobic respiration
(*ctaC* (OCL97_16890), *ctaD* (OCL97_16895), *ctaG* (OCL97_16910), and
*ctaE* (OCL97_16915)) are coded in the genome, confirming that the
enzyme is at work in strain HK31-G<sup>T</sup>, which has been demonstrated
experimentally. Enzymes needed to resist oxidative stress have also been
predicted in the genome of strain HK31-G<sup>T</sup>. Indeed, catalase
(OCL97_19865), catalase peroxidase (OCL97_00270), superoxide dis-
mutase (OCL97_11735), and glutathione peroxidase *gpo* (OCL97_19215)
were identified in its genome but they were not demonstrated experi-
mentally at work excepted for catalase.

Interestingly, the genome of strain HK31-G<sup>T</sup> encodes for the *cbb3*
cytochrome (OCL97_16190 for the subunit I; OCL97_16185 for the
subunit II; OCL97_16175 and OCL97_16180 for the subunit III and
OCL97_16155 for the *cbb3*-type cytochrome oxidase assembly protein
CcoS), which has a high affinity for dioxygen, so allows growth in
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Heatmap showing FastANI scores between genomes and MAGs. FastANI values below 77.5 % are not shown. The red limit indicates FastANI values above 96 % referenced in Table S5. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

microaerophilic environments (Pitcher and Watmough, 2004). In addition, subunits I and II of cytochrome *bd* ubiquinol oxidase were identified (OCL97_15410 for the subunit I; OCL97_15415 for the subunit II) confirming the ability of the new strain to grow under microaerophilic conditions as experimentally shown in the present study (Jünemann, 1997). Finally, the *aa3* cytochrome oxidase was detected; it could be involved in the electron transfer mechanism from the extracellular Fe(II) to the intracellular O₂, as proposed by Peng et al. (2022). Predictions by MicroCyc on MaGe revealed the presence of a complete dissimilatory nitrate reduction pathway in the genome of strain HK31-GT. Indeed, genes encoding for the A subunit of nitrate reductase (subunits alpha (narG (OLC97_05710)), beta (narH (OLC97_05715)), and gamma (narI (OLC97_05720)) subunits), for the chaperone protein (narJ (OLC97_05725)), and the nitrate transporter (25*ark* (OLC97_05700)), as well as for the NADH: ubiquinone oxidoreductase subunits (*nuoB*, *nuoH*, *nuoI*, *nuoJ*, *nuoK*, *nuoM* and *nuoN*) were identified (for locus-tags see Table S9). The missing NADH:ubiquinone oxidoreductase, and transporter subunits (A, E, F, G and L) were finally detected from the synteny

map around genes annotated as *nuo*. The comparison to the Uniprot database allows identifying the *nuoA*, *nuoE*, *nuoF*, *nuoG* and *nuoL* genes confirming their putative function. The presence of this complete nitrate reduction pathway (Fig. 5) indicates the ability of strain HK31-GT to respire nitrate, which is consistent with the anaerobic respiration demonstrated experimentally. This is in line with the strain's ability to change ecological niches and adapt easily.

Iron metabolism

Concerning iron metabolism, i.e., iron acquisition, storage and oxidation/reduction, several genes were identified by FeGenie and annotations were completed by PGAP (NCBI), Prokka and MaGe predictions. A total of 72 Fe-related genes were identified. Among them, 38 genes were identified by FeGenie (Table 4) and only 5 of them were found by all approaches (Table S10). A majority of them (56) were involved in iron assimilation, and included eight genes involved in iron (II)/(III) transport (including the Fe(II)-transporters FeoA and FeoB and
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Overview of the dissimilatory nitrate reduction pathway in strain HK31-G<sup>T</sup> operating under anaerobic conditions. Black arrows represent the proton translocation and red arrows represent the electron fluxes in protein complexes. Here, *Nar1* is used to oxidize the ubiquinol (UQH<sub>2</sub>) into ubiquinone (UQ<sup>+</sup>), then two electrons are transferred to *NarG* through *NarH*. The *bis*-molybdopterin guanine dinucleotide (MGD) allows the final nitrate reduction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<table><caption>**Table 4**

List of genes involved in iron transport, storage and redox cycling identified in the genome of strain HK31-G<sup>T</sup> and in the genomes of closely related species of *Phenylobacterium* using the FeGenie tool. Species: 1, HK31-G<sup>T</sup>; 1a, GCA_030645635; 1b, GCA_030652015; 1c, GCA_030683775; 1d, GCA_030693625; 1e, GCA_030696765; 1f, GCA_030704785; 2, *P. glauciei* 20VBR1<sup>T</sup>; 3, *P. aquaticum* W2-3-4<sup>T</sup>; 4, *P. haematophilum* DSM 21793<sup>T</sup>; 5, *P. immobile* strain E<sup>T</sup>.</caption><thead><tr><th></th><th></th><th>1</th><th>1a</th><th>1b</th><th>1c</th><th>1d</th><th>1e</th><th>1f</th><th>2</th><th>3</th><th>4</th><th>5</th></tr></thead><tbody><tr><td rowspan="7">Iron acquisition</td><td>Iron transport</td><td>4</td><td>0</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td><td>0</td><td>4</td><td>2</td><td>2</td></tr><tr><td>Heme transport</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td></tr><tr><td>Heme oxygenase</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Siderophore synthesis</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>3</td></tr><tr><td>Siderophore transport</td><td>9</td><td>7</td><td>8</td><td>12</td><td>8</td><td>10</td><td>12</td><td>0</td><td>18</td><td>17</td><td>11</td></tr><tr><td>Siderophore transport potential</td><td>9</td><td>6</td><td>6</td><td>6</td><td>6</td><td>6</td><td>10</td><td>0</td><td>6</td><td>6</td><td>8</td></tr><tr><td>Iron oxidation</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="3">Energetic metabolism</td><td>Possible iron oxidation and reduction</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Probable iron reduction</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Magnetosome formation</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="2">Other</td><td>Iron gene regulation</td><td>15</td><td>11</td><td>17</td><td>9</td><td>16</td><td>12</td><td>13</td><td>18</td><td>20</td><td>16</td><td>10</td></tr><tr><td>Iron storage</td><td>1</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>1</td><td>1</td><td>1</td><td>2</td></tr></tbody></table>

the Fe(II)-permease *EfeU*), two genes involved in transport by hemes (namely *HmuU* (identified as *FpvE*, involved in siderophore transport, by FeGenie) and *HmuV*), and 42 genes involved in transport by siderophores, with most of them encoding for *Ton-B* dependent receptor. Interestingly, *Ton-B* was upregulated in *Hydrogenovibrio* sp. when grown on Fe(II)-oxidation medium showing the role of siderophores to keep Fe in solution. In addition, siderophores could be potentially involved in the formation of Fe(III) mineral structures underlying their importance for Fe-oxidation metabolism (Laufer-Meiser et al., 2024). Then, 15 genes were involved in iron regulation, one in iron storage, and two genes might be involved in dissimilatory iron reduction (Table S10). The first iron reductase was only predicted with MaGe and was 69.3 % similar to the gene NX02_p1185 present in the genome of *Sphingomonas sanxani-genens* NX02<sup>T</sup> (Uniprot database). The second iron reductase was predicted with PGAP, and identified as a ferric reductase-like transmembrane domain-containing protein. This enzyme could be involved in Fe(III) respiration or in iron assimilation for metalloproteins.

The co-transcribed *cyc1* and *cyc2* genes, encoding the outer-membrane cytochrome c4 (or C<sub>552</sub>) (Appia-Ayme et al., 1998), and known to be involved in Fe(II)-oxidation (McAllister et al., 2020), were not identified in the HK31-G<sup>T</sup> genome, by any of the annotation tools used, suggesting, either that an alternative Fe(II)-oxidation pathway

exists, or that the genome is not sufficiently complete to identify all the genes it encodes. However, the *cyc2* gene was identified, by FeGenie, in the genome of *P. glauciei* 20VBR1<sup>T</sup>, the closest relative of strain HK31-G<sup>T</sup>, and in the genome of *P. hankyoungense* HKS-05<sup>T</sup>. In addition, for the genome of strain HK31-G<sup>T</sup>, the analysis performed with HMMER to identify other genes than *cyc2* that could be involved in Fe(II)-oxidation allowed to obtain interesting results for *cyc1* (corresponding to c-type cytochromes) and for *fox* genes (*foxA*, *B*, *E*, *Y* and *Z*) (Table S11). The *fox* genes are classified as two three-genes-operons, *foxABC* mainly found in Gram-positive Fe(II)-oxidizers and *foxEYZ* mainly found in Gram-negative Fe(II)-oxidizers and especially in neutrophilic or photosynthetic bacteria (Bathe and Norris, 2007; Croal et al., 2007). In the present study, the first operon was not completely detected (*foxC* is missing) and *foxA* and *foxB* HMM profiles corresponded respectively to a c-type cytochrome subunit 1 and a hypothetical protein. Using UniProt database, *foxA* was respectively encoded by *ctaD* in *P. glauciei* or by *coxA* in *Caulobacter vibrioides*, and *foxB* was identified as a c-type cytochrome subunit 2 encoded by *coxB* in *P. glauciei* or by *ctaC* in *Brevundimonas* sp. Interestingly, the *coxAB* genes encoding for an *aa3*-type cytochrome (Appia-Ayme et al., 1999), are present in the *rus*-operon found in Fe(II)-oxidizing bacteria and overexpressed in presence of Fe(II) (Liu et al., 2011). In addition, the second operon *foxEYZ* was identified by HMM
profiles but *foxE* HMM profile provided a low e-value and according to Uniprot database, encoded a carotenoid 1,2-hydratase which is not in accordance with Croal et al. (2007) that have demonstrated that *foxE* encodes for a cytochrome *c* oxidase (Table S11). Overall, these findings suggest the presence of genes other than *cyc2* that could be involved in Fe(II)-oxidation in HK31-G<sup>T</sup> strain. Further investigations and functional approaches will be required to confirm this hypothesis.

## Conclusion

The new strain HK31-G<sup>T</sup>, belonging to the putative species *Phenylobacterium* sp030693625 according to GTDB database, is a mesophilic, neutrophilic and chemoheterotrophic bacterium able to grow by dioxygen and nitrate respiration using a wide variety of organic substrates (Table 5). It is also able to grow under chemolithoautotrophic conditions, by Fe(II)-oxidation, using nitrate and low dioxygen concentrations as terminal electron acceptors, and CO<sub>2</sub> as the sole carbon source. The roTCA cycle could be involved in autotrophic CO<sub>2</sub> fixation. However, genomic analyses are not sufficient to validate the use of this cycle by strain HK31-G<sup>T</sup> due to the lack of unique enzymes. The possible ways to validate the use of the roTCA cycle would be to conduct biochemical experiments (i.e. <sup>13</sup>C NMR analysis) or to perform transcriptomic analyses as the citrate synthase is overexpressed during the roTCA cycle. To our knowledge, this secondary metabolism based on Fe (II)-oxidation has not previously been demonstrated in this genus. Genomic predictions indicate the presence of genes encoding for c-type and cbb3-type cytochrome oxidase and cytochrome bd ubiquinol oxidase as well as genes encoding for the complete nitrate reduction pathway, which is congruent with the strain's ability to develop under oxic, microoxic, and anoxic conditions in the presence of nitrate. Despite that Fe(II)-oxidation metabolism has been proven experimentally, key genes involved in Fe(II)-oxidation pathway could not be identified. Growth observed under different nutrients and physico-chemical conditions, suggests that HK31-G<sup>T</sup> can adapt to different ecological niches as demonstrated by its worldwide environmental distribution. In this respect, strain HK31-G<sup>T</sup> is thought to produce polyhydroxyalkanoate storage granules (PHA), compounds that confer an ecological advantage since PHAs can help to cope with different stress conditions. The new strain HK31-G<sup>T</sup> exhibits also a large distribution across diverse environments and is present in several subsurface geothermal aquifers of the CarbFix-1 site as well as in an environment associated to Fe-rich minerals. Phylogenomic relatedness indices, as well as phenotypic, metabolic and chemotaxonomic differences between strain HK31-G<sup>T</sup> and the most closely related strain *P. glaciei* 20VBR1<sup>T</sup> confirm that strain HK31-G<sup>T</sup> is a novel species of the genus *Phenylobacterium* for which we suggest the name *Phenylobacterium ferrooxidans* HK31-G<sup>T</sup>.

## Fundings

This research was funded by the S4CE collaborative project (H2020-EU.3.3.2.- Low-cost, low-carbon energy supply –764,810), the French National Research Agency for the project IRON2MI under the reference ANR-22-CE01-0013-01 to S.M., by the Sino-French IRP 1211 Microsea to K.A. and by the UMR6197.

## CRediT authorship contribution statement

**Eva Poudier:** Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Conceptualization.
**Erwann Vince:** Writing – review & editing, Investigation.
**Karen Jacquot:** Writing – review & editing, Investigation.
**Maimouna batoma Traoré:** Writing – review & editing, Investigation.
**Ashley Grosche:** Writing – review & editing, Methodology, Investigation.
**Maria Ludwig:** Writing – review & editing, Investigation.
**Mohamed Jebbar:** Writing – review & editing, Resources, Funding acquisition.
**Loïs Maignien:** Writing – review & editing, Resources.

<table><caption>**Table 5**

Description of *Phenylobacterium ferrooxidans* sp. nov.</caption><thead><tr><th>Parameters</th><th>Phenylobacterium ferrooxidans sp. nov.</th></tr></thead><tbody><tr><td>Guiding Code for Nomenclature</td><td>ICNP</td></tr><tr><td>Nature of the type material</td><td>strain</td></tr><tr><td>Genus name</td><td>Phenylobacterium</td></tr><tr><td>Species name</td><td>Phenylobacterium ferrooxidans</td></tr><tr><td>Specific epithet</td><td>ferrooxidans</td></tr><tr><td>Species status</td><td>sp. nov.</td></tr><tr><td>Species etymology</td><td>fer.ro.ox'i. dans sp. nov. L neut. n. ferrum, iron; N.L. v. oxido, oxidize; N.L. part. Adj. ferrooxidans, iron-oxidizing</td></tr><tr><td>Designation of the Type Strain</td><td>HK31-G<sup>T</sup></td></tr><tr><td>Strain Collection Numbers</td><td>DSM 116432<sup>T</sup> = UBOCC-M-3429<sup>T</sup> = LMG 33376<sup>T</sup></td></tr><tr><td>Type Genome, MAG or SAG accession Nr. [INSDC databases]</td><td>JAOTJD000000000</td></tr><tr><td>Genome status</td><td>Complete</td></tr><tr><td>Genome size</td><td>4.46 Mbp</td></tr><tr><td>GC mol%</td><td>67.95 %</td></tr><tr><td>16S rRNA gene accession nr.</td><td>OR652334</td></tr><tr><td>Description of the new taxon and diagnostic traits</td><td>Phenylobacterium ferrooxidans members are motile, Gram-negative thin rods (0.4–0.7 × 1.0–3.6 µm in size). Colonies appear smooth, circular, light creamish, convex and 0.5–1 mm in diameter. The type strain HK31-G<sup>T</sup> is mesophilic and grows at circumneutral pH. Growth occurs at 10–30 °C (optimum, 25–30 °C), at pH 2–12 (optimum, 6) and with 0.5 % NaCl concentration (optimum, 0 % NaCl). They are oxidase-positive, catalase-negative, aerobic and chemoorganotrophic when cultivated on R2A but microaerophilic and anaerobic and chemolithoautotrophic when cultivated on Fe(II)-oxidation liquid medium. Nitrate is reduced to nitrite. Capable of oxidizing iron under microaerophilic and anaerobic conditions and using CO<sub>2</sub> as carbon source on Fe(II)-oxidation rich medium. The following carbon sources are used: L-alanine, L-proline, phenylalanine, β-hydroxybutyrate and malonate. The following carbon sources are used: L-alanine, L-proline, phenylalanine, β-hydroxybutyrate and malonate. On the contrary, unable to use: xylose, glycerol, aspartate, acetate, lactate, succinate, glutamate, propionate, D-glucose, L-arabinose, D-mannose, N-acetyl-glucosamine, D-maltose, citrate, gluconate, caprate, adipate, malate and phenylacetate, D-mannitol, inositol, D-sorbitol, L-rhamnose, D-saccharose, D-melibiose, amygdalin and L-arabinose. Cells produce β-glucosidase (aesculin hydrolysis), α-galactosidase, β-galactosidase (PNPG), N-acetyl-β-glucosaminidase, α-mannosidase, tryptophan deaminase, gelatinase, alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase but unable to produce lysine decarboxylase, ornithine decarboxylase, urease, arginine dihydrolase, valine arylamidase, β-Galactosidase, arginine dihydrolase, lipase, α-glucosidase, β-glucosidase, crystalline arylamidase, chymotrypsine, β-glucuronidase, and α-fucosidase.</td></tr><tr><td>Country of origin</td><td>Iceland</td></tr><tr><td>Region of origin</td><td>Carbfix-1 site of an adjacent geothermal power plant at Hellisheidi</td></tr><tr><td>Date of isolation</td><td>08/06/2021</td></tr><tr><td>Source of isolation</td><td>Deep subsurface geothermal aquifer at an adjacent geothermal power plant at Hellisheidi</td></tr><tr><td>Sampling date</td><td>18/07/2019</td></tr><tr><td>Latitude</td><td>64° 02' 14" N</td></tr><tr><td>Longitude</td><td>21° 24' 03" W</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 5 (continued)**</caption><thead><tr><th>Parameters</th><th>Phenylobacterium ferrooxidans sp. nov.</th></tr></thead><tbody><tr><td>Number of strains in study</td><td>1</td></tr><tr><td>Information related to the Nagoya Protocol</td><td>Not applicable</td></tr></tbody></table>

**Karine Alain:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Sophie Mieszkin:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationship that could have appeared to influence the work reported in this paper.

## Acknowledgments

We gratefully acknowledge financial support provided by the S4CE collaborative project, funded by the European Union's Horizon 2020 research and innovation program under grant agreement number 764810. AG was supported by a postdoctoral fellowship from the S4CE project. We are grateful to Claire Geslin (UBO, BEEP) and Philippe Elies (UBO, Plateforme d'imagerie et de mesures en microscopie (PIMM)) for their technical assistance in TEM imaging. We acknowledge Nadège Bienvenu (UBO, BEEP) for her help concerning the deposit of the strain at the UBOCC and Olivier Rougel for his guidelines concerning the ferrozine assay protocol. We are grateful to Lukáš V. F. Novák for the use of HMM profiles in the search for certain genes. We thank Ranime Safieddine and Léna Ailliot (UBO, BEEP) for their technical help concerning the culture of the bacterial strains. The LABGeM (CEA/Genoscope and CNRS UMR8030), the France Génomique and French Bioinformatics Institute national infrastructures (funded as part of Investissement d'Avenir program managed by Agence Nationale pour la Recherche, contracts ANR-10-INBS-09 and ANR-11-INBS-0013) are acknowledged for support within the MicroScope annotation platform.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.syapm.2024.126578.

## Data availability

Data will be made available on request.

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