Cellulomonas electrica sp. nov., isolated from an electrochemical enrichment culture

Abstract

A Gram-positive, facultatively anaerobic, rod-shaped actinomycete bacterium, strain NTE-D12^T was isolated from the filtrate (< 0.22 μm pore size) of an electrochemical enrichment culture. The strain grew in R2A medium at 15–40 °C (optimum 30 °C), pH 6–8 (optimum pH 6), and in the presence of 0–6% NaCl (optimum 0%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain NTE-D12^T is closely related to Cellulomonas algicola TKZ-21^T (97.9%), Cellulomonas biazotea DSM 20112^T (97.5%), and Cellulomonas fimi DSM 20113^T (97.5%). Genome-based phylogenetic analysis indicated that the closest relative is Cellulomonas citrea AO-9^T. Orthologous average nucleotide identity (OrthoANI) and digital DNA–DNA hybridization (dDDH) values between strain NTE-D12^T and related species ranged from 75.40–76.73% and 20.2–21.0%, respectively, clearly below the species delineation thresholds. In contrast, average amino acid identity (AAI) and percentage of conserved proteins (POCP) values ranged from 68.33–70.89% and 58.33–62.26%, respectively, indicating affiliation within the genus Cellulomonas. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol, aminoglycophospholipid, and glycophospholipid. The peptidoglycan type was A4β with L-Orn–D-Ser–D-Glu. The predominant cellular fatty acid was anteiso-C_15:0 (65.6%). The major quinone was menaquinone-10, distinguishing the strain from the other Cellulomonas species. The genomic DNA GC content was 73.1%. Based on phenotypic, phylogenetic, and chemotaxonomic characteristics, strain NTE-D12^T (= NBRC 115591^T = DSM 114595^T) represents a novel species of the genus Cellulomonas, for which the name Cellulomonas electrica sp. nov. is proposed.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10482-026-02356-0.

Introduction

Extracellular electron transfer (EET) is a respiratory system that facilitates direct electron transfer to conductive solids (Myers and Nealson 1988; Shi et al. 2016). While the core mechanisms and vital genes associated with EET have been detailed in Gram-negative bacteria, primarily focusing on the genera Shewanella and Geobacter (Lovley and Phillips 1988; Myers and Nealson 1988), a myriad of other bacteria exhibit EET capabilities, even in Gram-positive bacteria (Wrighton et al. 2011; Naradasu et al. 2019, 2020; Pankratova et al. 2019). Light et al. elucidated this mechanism specifically in Listeria monocytogenes (Light et al. 2018). Nevertheless, the number of Gram-positive bacteria identified with EET abilities pales in comparison to Gram-negative species. Thus, our understanding of EET mechanisms in gram-positive bacteria, beyond L. monocytogenes, remains limited. Uncovering novel Gram-positive EET-capable bacteria is vital to broadening our understanding of their underlying mechanisms.

The genus Cellulomonas, classified under the family Cellulomonadaceae and first described in 1923, comprises Gram-positive, aerobic bacteria (Bergey et al. 1923). Forty-one species have been recognized and isolated from diverse environments, such as termite gut, deep-sea and terrestrial environments (Zhang et al. 2013; Sun et al. 2018; Lee et al. 2020). Recent findings spotlight certain Cellulomonas ability to employ cellulose or glucose as electron donors and carbon electrodes as ultimate electron acceptors, hinting at their potential application in microbial fuel cells (Khawdas et al. 2019).

Cellulomonas sp. strain NTE-D12^T was isolated from an electrochemical enrichment of a soil-derived sample that had been passed through a 0.22 μm filter (Ihara et al. 2022). Intriguingly, the majority of microorganisms that pass through the 0.22 μm filter exhibit phylogenetic diversity (Naganuma et al. 2007; Nakai et al. 2013). In particular, the strain NTE-D12^T showcases an elevated current production capability and houses a distinct electron carrier activity gene cluster, setting it apart from related species in terms of its enhanced EET proficiency (Ihara et al. 2022).

Our study embarked on a comprehensive analysis of the phenotype, chemotaxonomy, and molecular phylogeny of strain NTE-D12^T. Our findings reveal several distinguishing traits compared to the relative strains. Notably, the Orthologous average nucleotide identity (orthoANI) and digital DNA–DNA hybridization (dDDH) analyses solidified the unique classification of NTE-D12^T. Consequently, we suggest that strain NTE-D12^T be recognized as a novel species within the genus Cellulomonas, for which the name Cellulomonas electrica sp. nov. is proposed.

Materials and methods

Molecular analysis

The Quick-DNA HMW MagBead Kit (Zymo Research, Irvine, CA, USA) was used for genomic DNA extraction. We used universal primers, 27F (5′- AGAGT TTGAT CCTGG CTCAG -3′) and 1492R (5′- GGTTA CCTTG TTACG ACTT-3′), to amplify the near full-length of 16S rRNA gene by polymerase chain reaction (PCR). The PCR product was purified using the NucleoSpin® Gel and PCR Clean-up (Takara Bio, Kusatsu, Shiga, Japan). The amplified PCR products were directly sequenced. To construct the nearly full-length 16S rRNA gene, the obtained sequence data were compiled using BioEdit software (Hall 1999). The nearly full-length 16S rRNA gene (1,403 bp) was compared to existing sequences in the national center for biotechnology information rRNA/ITS database using the nucleotide basic local alignment search tool (BLASTN) program (Altschul et al. 1990).

Whole genome sequence, de novo assembly, and gene annotation were conducted in the previous study (accession no. AP026442) (Ihara et al. 2022). The genome completeness was evaluated using CheckM2 ver 1.0.2 (Chklovski et al. 2023). OrthoANI and dDDH values were compared between related species using EzBio Cloud server (Lee et al. 2016) and Genome-to-Genome Distance Calculator 3.0 (Auch et al. 2010), respectively. Average amino acid identity (AAI) and the percentage of conserved proteins (POCP) were calculated using CompareM v0.1.2 (https://github.com/dparks1134/CompareM) and the Nextflow-based POCP-nf pipeline (https://github.com/hoelzer/pocp) to assess genus-level novelty.

Sequence data of 16S rRNA genes and whole genomes of related type strains were retrieved from the GenBank database. For 16S rRNA gene analysis, sequences were aligned using the MUSCLE program (Edgar 2004). The alignment was trimmed to positions 83–1427 according to the Escherichia coli numbering system (Brosius et al. 1978). Phylogenetic trees based on 16S rRNA gene sequences were constructed using the neighbor-joining (NJ) (Saitou and Nei 1987) and maximum-likelihood (ML) (Felsenstein 1981) methods with the Jukes–Cantor substitution model and maximum-parsimony (MP) (Takahashi and Nei 2000) method with the Subtree-Pruning-Regrafting algorithm implemented in the mega11 program (Tamura et al. 2021). Tree topologies were evaluated by bootstrap analysis with 1,000 resamplings (Felsenstein 1985). For genome-based phylogenetic analysis, single-copy housekeeping genes were identified and aligned using GTDB-Tk v2.4.0 with the Bac120 marker set. The resulting alignments were trimmed using trimAl v1.5.0 with the automated trimming option (Capella-Gutierrez et al. 2009). A maximum-likelihood phylogenetic tree was then constructed using IQ-TREE2 v2.3.6 with the LG + F + G4 substitution model. Branch support was assessed using 1,000 ultrafast bootstrap replicates and 1,000 SH-like approximate likelihood ratio tests (Minh et al. 2020).

Physiological analysis

R2A medium was used to determine the growth ranges for temperature, pH, and NaCl concentration. Growth at different temperatures was assessed by incubating strain NTE-D12^T on R2A agar plates at 4, 10, 15, 20, 30, 37, 40, and 45 °C for 14 days. The pH range for growth was determined in buffered R2A liquid medium at 30 °C over a pH range of 4.0–10.0 for 14 days. The medium was buffered as follows: pH 4.0–5.0 with 0.1 M citric acid/0.1 M sodium citrate; pH 6.0–8.0 with 0.1 M KH₂PO₄/0.1 M NaOH; and pH 9.0–10.0 with 0.1 M NaHCO₃/0.1 M Na₂CO₃. Growth at different NaCl concentrations (0–7%, w/v) was examined in R2A liquid medium at 30 °C for 14 days. All cultures were incubated aerobically with shaking at 180 rpm. Anaerobic growth was tested in Gifu anaerobic medium after purging with N₂/CO₂ (80:20, v/v) gas for 1 h, followed by incubation for 5 days. All growth experiments were performed in triplicate. Enzymatic activities and carbon source utilization were evaluated using API ZYM and API 50CHL systems (bioMérieux, Marcy-l’Étoile, France), according to the manufacturer’s instructions. Cellulose degradation was assessed using CMC-Na agar [composition (g L⁻¹): 5.0 CMC-Na, 1.0 KNO₃, 0.5 MgSO₄·5H₂O, 0.01 FeSO₄·7H₂O, 1.0 tryptone, and 15.0 agar]. Bacterial suspensions (OD₆₀₀ = 0.5, 10 µL) were spotted onto the plates and incubated at 30 °C for 7 days. Plates were then stained with 0.1% (w/v) Congo red solution for 20 min and destained with 1 M NaCl, followed by three washes. Cellulose degradation was evaluated based on the formation of clear zones (Sun et al. 2018).

Electrochemical measurements of strain NTE-D12^T were performed using a single-chamber three-electrode system as described previously (Naradasu et al. 2019), with a tin-doped indium oxide (ITO) glass working electrode, a platinum wire counter electrode, and an Ag/AgCl (sat. KCl) reference electrode. The electrolyte consisted of 4.0 mL defined medium (DM) [composition (g L⁻¹): 1.0 NH₄Cl, 0.8 MgCl₂·6H₂O, 0.1 CaCl₂·2H₂O, 0.5 KH₂PO₄, and 1.0 NaHCO₃]. Cells were cultured in R2A liquid medium for 30 h, harvested, washed twice with DM, and resuspended in DM to an OD₆₀₀ of 5.0, followed by purging with > 99.9% N₂ gas for 30 min. During the electrochemical measurements, a 0.5 mL aliquot of the cell suspension and 0.5 mL of either glucose solution (100 g L⁻¹) or sterile water were added to the reactor, resulting in a final OD₆₀₀ of 0.5 and a final glucose concentration of 10 g L⁻¹ (when added). Electrochemical measurements were performed by single-potential amperometry using an automatic polarization system (VMP3; Bio-Logic Science Instruments) at 30 °C, with the working electrode poised at + 0.2 V (vs. Ag/AgCl, sat. KCl).

Morphological characterization

Single colonies were obtained on R2A agar after incubation at 30 °C for 24 h. Colony morphology was examined after cultivation on R2A agar for 14 days at 30 °C. Gram staining was performed as described in Hucker method (Gerhardt et al. 1981). Cell morphology was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after aerobic cultivation in R2A medium for 30 h. SEM observations were carried out following previously described procedures(Ihara et al. 2022). The cells were stained with 2% phosphotungstic acid for TEM observation. TEM observation was outsourced to Tokai Electron Microscopy, Inc (Aichi, Japan).

Chemotaxonomic characterization

Cells for fatty acid analysis were grown aerobically at 28 °C for 4 days. Wet biomass was collected by centrifuge at 7,000 g and converted to fatty acid methyl esters (FAMEs) using a methylation kit (Nacalai Tesque, Japan). FAMEs were analyzed by GC/MS (8890 GC coupled with MSD 5977B; Agilent Technologies) using an HP-88 column (30 m × 0.25 mm, 0.20 μm) with helium as the carrier gas and an injector temperature of 250 °C. The oven temperature was programmed from 80 °C (2 min) to 130 °C at 10 °C min⁻¹ (15 min hold), then to 160 °C at 3 °C min⁻¹, and finally to 200 °C at 20 °C min⁻¹ (6 min hold). Detection was performed in scan mode, and fatty acids were identified based on retention times and mass spectra using FAME standards (Cayman Chemical). Cells for other chemotaxonomic analyses were grown aerobically at 30 °C for 30 h. Total lipids were extracted using the revised Bligh and Dyer method (Bligh 1959), and quinones were purified by solid-phase extraction (Sep-Pak Plus silica; Waters). Quinone profiles were analyzed using an ACQUITY UPLC H-Class system (Waters) equipped with a BEH C18 column (2.1 × 150 mm, 1.7 μm) under the following conditions: methanol/isopropanol (7:3, v/v) as the mobile phase, flow rate 0.3 mL min⁻¹, and column temperature 35 °C. Quinones were identified using authentic standards or estimated based on the equivalent number of isoprene units (ENIU) (Tamaoka et al. 1983). Polar lipids were analyzed by two-dimensional thin-layer chromatography with appropriate detection reagents (Minnikin et al. 1977; Yassin et al. 1993). Peptidoglycan was hydrolyzed with 4 N HCl at 100 °C for 4 h and analyzed by GC/MS as N-heptafluorobutyric acid isobutyl esters (Schumann 2011; Schumann et al. 2021).

Fatty acid analysis was performed by Dojin-Glocal Co., Ltd. (Kumamoto, Japan), quinone analysis by Techno Suruga Lab Co., Ltd. (Shizuoka, Japan), and polar lipid and peptidoglycan analyses by DSMZ Services (Braunschweig, Germany).

Results and discussion

16S rRNA gene and genome-based phylogenetic analysis

A nearly full-length 16S rRNA gene sequence (1,403 bp) was obtained. Based on the maximum-likelihood (ML) phylogenetic tree, strain NTE-D12^T clustered within the genus Cellulomonas (Fig. 1). The three closest relatives were Cellulomonas algicola TKZ-21^T, Cellulomonas biazotea DSM 20112^T and Cellulomonas fimi 20113^T, showing 16S rRNA gene sequence similarities of 97.9%, 97.5%, and 97.5%, respectively. The neighbor-joining (NJ) and maximum-parsimony (MP) trees also supported these three species as the closest relatives (Figs. S1 and S2). However, the bootstrap support for the clustering between strain NTE-D12^T and C. algicola was below 50% in these 16S rRNA gene trees, indicating low confidence in this relationship. In contrast, genome-based phylogenomic analysis identified Cellulomonas citrea Ao-9^T as the closest relative (Fig. 2). The phylogenomic tree was strongly supported, with ultrafast bootstrap and SH-aLRT values of 96.7% and 97%, respectively, indicating high reliability. Compared with the 16S rRNA gene-based phylogeny, the genome-based phylogeny provided a more robust inference of the evolutionary relationship.

Fig. 1.

Phylogenetic tree derived from 16S rRNA gene sequences between strain NTE-D12^T and the type strains of related Cellulomonas species. The tree was constructed using the Maximum-likelihood method using the Jukes-Cantor model. The black circular dot indicates that corresponding nodes were also recovered in trees generated with the maximum-parsimony method using the Subtree-Pruning-Regrafting algorithm and neighbor-joining method using the Jukes-Cantor model. Numbers at branch points indicate bootstrap percentages (only values ≥ 50% are shown). Bar, 0.01 nucleotide substitution position

Fig. 2.

Maximum-likelihood phylogenomic tree of strain NTE-D12^T and related Cellulomonas type strains. The tree was constructed using IQ-TREE2 v2.3.6 with the LG + F + G4 substitution model based on single-copy housekeeping genes identified and aligned with the GTDB-Tk v2.4.0 with the Bac120 gene marker set. Branch support values indicate ultrafast bootstrap (1,000 replicates) and SH-aLRT (1,000 tests). Nodes with ≥ 95% bootstrap and ≥ 80% SH-aLRT support were considered reliable (Minh et al. 2013). Bar, 0.1 nucleotide substitution position

Genome-based species and genus delineation

The complete genome of strain NTE-D12^T consists of one circular chromosome (3.68 Mbp) and one circular plasmid (5,386 bp) with 99.97% completeness evaluated using CheckM2. The whole genome contains 6 rRNA, 54 tRNA, 1 tmRNA and 3,333 CDSs. The genome has 73.1% G + C content.

OrthoANI and dDDH values between strain NTE-D12^T and its related type strains were less than 76.63% and 21.0%, respectively (Table 1). These values are well below the accepted species delineation thresholds of 95–96% for OrthoANI and 70% for dDDH (Rosselló-Mora and Amann 2001; Goris et al. 2007). In contrast, AAI and POCP values between strain NTE-D12^T and related type strains were below 70.89% and 62.26%, respectively. These values fall within or above the proposed genus-level thresholds (AAI: 65–72%; POCP: 50%) (Qin et al. 2014; Konstantinidis et al. 2017). Collectively, these results indicate that strain NTE-D12^T represents a novel species but does not constitute a novel genus.

Table 1.

Genome comparison between strain NTE-D12^T and closely related Cellulomonas species

Taxon (Accession No.)	orthoANI value (%)	dDDH (d0; %)	dDDH (d4; %)	dDDH (d6; %)	AAI (%)	POCP (%)
Cellulomonas citrea AO-9T (GCA_009829685.1)	76.19	19	20.2	18.4	70.39	62.26
Cellulomonas endophytica SYSUP0004T (GCA_004123805.1)	75.40	15.9	20.5	16.9	68.33	59.07
Cellulomonas marina NBRC 110784T (GCA_016862715.1)	75.52	17	20.5	15.9	69.07	60.20
Cellulomonas algicola TKZ-21T (GCA_003851725.1)	76.73	17.7	21	17.5	70.68	58.33
Cellulomonas biazotea DSM 20112T (GCF 004306155.1)	76.51	18.5	20.6	18.1	70.89	59.47
Cellulomonas fimi DSM 20113T (GCA_000212695.1)	76.39	19.2	20.4	18.6	70.75	61.61

Values are presented as differences relative to strain NTE-D12^T. OrthoANI was calculated using the EZBioCloud server(https://www.ezbiocloud.net/tools/ani), and dDDH values were estimated using GGDC 3.0 (https://ggdc.dsmz.de/distcalc2.php). AAI was calculated using CompareM v0.1.2 (https://github.com/dparks1134/CompareM), and POCP was determined using the Nextflow-based POCP-nf pipeline (https://github.com/hoelzer/pocp)

Physiological and morphological features

Growth of strain NTE-D12^T occurred at 20–40 °C, at pH 6–7, and in the presence of 0–2% NaCl for 7 days of incubation. Growth was also observed at 15 °C, pH 8, and 3–6% NaCl after 14 days. In summary, this strain grew at 15–40 °C, pH 6–8, and in the presence of 0–6% NaCl. Strain NTE-D12^T was able to utilize D-arabinose, which is not utilized by most related species, whereas it did not utilize amygdalin, which is utilized by most related species (Table 2). The cellulose degradation activity was weaker than that of related species (Fig. S3). Colonies grown on R2A agar plate were circular, undulating, and lemon-yellow in color (Fig. S4). During electrochemical measurements, strain NTE-D12^T exhibited a gradual increase in current upon glucose addition compared to the no-glucose control, reaching a maximum of 238 nA cm⁻² at 112 h (Fig. S5). This indicates that the strain can generate current using glucose as an electron donor.

Table 2.

Differential characteristics between strain NTE-D12^T and closely phylogenetic relative species

Characteristics	1	2	3	4	5	6	7
Growth at:
6% NaCl	+	−	−	−	−	−	−
pH 9.0	−	+	+	−	+	+	−
Cell size
(length × width, μm)	0.9–1.8 × 0.4 − 0.6	2.0–2.4 × 0.5–0.8	N/A	N/A	0.5–0.8 × 0.4–0.5	N/A	N/A
Margin of colony	undulate	entire	N/A	N/A	curled	entire	entire
Major fatty acid*	anteiso-C15:0	anteiso-C15:0	anteiso-C15:0	anteiso-C15:0	anteiso-C15:0	anteiso-C15:0	anteiso-C15:0
(> 10%)		C:16:0	anteiso-C15:1 A	C:16:0	iso-C15:0	iso-C16:0	C:16:0
		C14:0	C:16:0			iso-C15:0	iso-C16:0
						iso-C14:0
Major menaquinone	MK-10	MK-9 (H4)	MK-9 (H4)	MK-9 (H4)	MK-9 (H4)	MK-9 (H4)	MK-9 (H4)
Carbon source utilization:
D-Arabinose	+	−	N/A	−	−	−	−
L-Rhamnose	−	−	+	+	+	+	+
D-Mannitol	+	−	−	+	−	+	−
Methyl-α-D-glucopyranoside	−	+	N/A	N/A	+	−	−
Amygdalin	−	+	N/A	N/A	+	+	+
D-Melibiose	−	−	+	−	+	+	+
D-Melezitose	−	−	N/A	N/A	+	+	+
D-Raffinose	−	−	+	−	+	+	+
Enzymatic activities:
Valine allyl amidase	+	−	+	N/A	−	−	−
Cystine allyl amidase	+	−	+	N/A	−	−	−
α-Galactosidase	+	−	+	N/A	−	−	−

Strain: 1, NTE-D12^T; 2, C.citrea AO-9^T; 3, C.endophytica SYSUP0004^T; 4, C.marina FXJ8.089^T; 5, C.algicola TKZ-21^T; 6, C.biazotea DSM 20112^T; 7, C.fimi DSM 20113^T

1: Data obtained from the present study, 2: Data referred from Lee et al. 2020 (Lee et al. 2020), 3: Data referred from Li et al. 2020 (Li et al. 2020), 4: Data referred from Zhang et al. 2013 (Zhang et al. 2013), 5,6,7: Data referred from Yamamura et al. 2019 (Yamamura et al. 2019), + , positive; w, weakly positive; − , negative; N/A, no data available

^*Fatty acid profiles for strains 1, 2, and 5–7 were determined in this study; detailed data are provided in Table S1 (Supplementary file)

Cells observed to be Gram-positive using optical microscopy. Rod shaped, flagellated cells were observed using TEM, and the size was length × width = 0.9 to 1.8 × 0.4 to 0.6 μm (Fig. 3). As demonstrated in a previous study, isolated cells did not pass through a 0.22 μm filter again (Ihara et al. 2022). It is presumed that the NTE-D12^T may only attain a filter-passing size under specific conditions such as environmental stresses (Hood and MacDonell 1987).

Fig. 3.

Microscopic images of strain NTE-D12^T after incubation using R2A medium aerobically. A SEM images of the cells on the glass. B TEM image shows a flagellum from the cells

Chemotaxonomic characteristics

The cellular fatty acid profiles (> 1% of the total fatty acids) were anteiso-C_15:0 (65.6%), C_14:0 (9.4%), C_16:0 (8.7%), iso-C_16:0 (6.8%), iso-C_14:0 (5.7%), iso-C_15:0 (2.1%), and anteiso-C_17:0 (1.6%) (Table S1).

Most members of the genus Cellulomonas possess menaquinone (MK)-9(H₄) as the predominant quinone, although MK-8(H₄) has been reported in some species (Kang et al. 2007). However, the presence of MK-10 as the predominant quinone has not been reported in this genus. The major peak of strain NTE-D12^T appeared at 7.72 min, which is closer to the retention time of MK-10 standard (7.73 min) than to the estimated retention time of MK-9(H₄) (7.30 min) based on ENIU, and was therefore assigned to MK-10 (Fig. S6). Although the quinone composition deviates from typical Cellulomonas species, genome-based phylogenetic metrics (AAI, POCP, and phylogenomics) consistently support its placement within the genus, indicating that quinone variation alone does not warrant genus-level reclassification. Taken together, these results indicate that strain NTE-D12^T is the first Cellulomonas species possessing MK-10 as the predominant quinone.

The major polar lipids consisted of diphosphatidylglycerol (DPG), phosphatidylinositol (PI), aminoglycophospholipid (AGPL), and glycophospholipid (GPL). Some unidentified phospholipids, aminolipids and lipids were observed as minor polar lipids (Fig. S7). The peptidoglycan contained the amino acids alanine (Ala), glutamic acid (Glu), serine (Ser), and ornithine (Orn) in the following molar ratio: 2.5: 2.0: 0.9: 0.4, respectively. The analysis of enantiomers showed the presence of D-Ser, L-Orn, D-Ala/L-Ala and D-Glu. Based on the obtained data, the peptidoglycan was classified as type A4β with L-Orn–D-Ser–D-Glu, which is the same peptidoglycan type as in related species.

Taxonomic conclusions

We characterized strain NTE-D12^T, isolated from an electrochemical enrichment culture of a 0.22 μm pore-size filter filtrate, based on phylogenetic, phenotypic, and chemotaxonomic analyses. Strain NTE-D12^T was clearly separated from related type strains of Cellulomonas in both the 16S rRNA gene-based phylogenetic tree and the GTDB-based phylogenomic tree (Figs. 1 and 2).

The ANI and dDDH values between strain NTE-D12^T and related species were below 76.73% and 21.0%, respectively. In addition, the AAI and POCP values were consistent with assignment to the genus Cellulomonas, indicating that strain NTE-D12^T does not represent a novel genus. The major quinone was MK-10, which differs from the predominant MK-9(H₄) observed in related species.

Based on the results of the phylogenetic, phenotypic, and chemotaxonomic characterizations, strain NTE-D12^T represents a novel species within the genus Cellulomonas, for which the name Cellulomonas electrica is proposed.

Description of Cellulomonas electrica sp. nov.

Cellulomonas electrica (e.lec’tri.ca. L. neut. n. electrum, amber; N.L. fem. adj. electrica, pertaining to electricity).

Cells are found to be Gram-stain positive, facultatively anaerobic, rod-shaped 0.9–1.8 μm long and 0.4–0.6 μm wide. Colonies formed on R2A agar are circular, undulate, and lemon-yellow color. Growth occurs at 15–40 °C (optimum 30 °C), pH 6–8 (optimum pH 6), and in 0–6% (w/v) NaCl (optimum 0%). Weakly positive for degradation of cellulose. With the API 50ch system, The following compounds are found to be capable of utilizing as a carbon source: glycerol, D-arabinose, L-arabinose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetyl-glucosamine, arbution, esculin ferric citrate, salicin, D-cellobiose, D-maltose, D-sucrose, D-trehalose, starch, glycogen, gentiobiose, D-turanose, and D-lyxose as a solo carbonsource, but are not found to be capable of utilizing Erythritol, D-ribose, L-xylose, D-adonitol, methyl-β-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl-α-D-mannopyranoside, methyl-α-D-Glucopyranoside, amygdalin, D-lactose, D-melibiose, Inulin, D-melezitose, D-raffinose, xylitol, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-keto-gluconate, and 5-keto-gluconate. With the API-ZYM system, This strain is found to have enzymatic activity to metabolize the following substrates: esterase (C4), leucine allyl amidase, valine allyl amidase, cysteine allyl amidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase, but not metabolize alkaline phosphatase, esterase-lipase (C8), Lipase (C14), trypsin, α-chymotrypsin, β-glucuronidase, α-mannosidase, and α-frucosidase. The predominant cellular fatty acid is anteiso-C_15:0. The major menaquinone is MK-10. The major polar lipids are DPG, PI, AGPL, and GPL. The peptidoglycan type is A4β with L-Orn–D-Ser–D-Glu. The GC content of the type strain is 73.1 mol %.

The type strain is NTE-D12^T (= NBRC 115591^T = DSM 114595^T), isolated from an electrochemical enrichment of a culture derived from soil that had been passed through a 0.22 μm filter. The original soil was collected from Fukushima prefecture, Japan (37° 21′ N, 141° 01′ E).

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

EET

Extracellular electron transfer

orthoANI

Orthologous average nucleotide identity

dDDH

Digital DNA–DNA hybridization

AAI

Average amino acid identity

POCP

Percentage of conserved proteins

PCR

Polymerase chain reaction

BLASTN

Nucleotide basic local alignment search tool

MUSCLE

Multiple sequence comparison by log-expectation

NJ

Neighbor-joining

MP

Maximum-parsimony

ML

Maximum-likelihood

GTDB

Genome Taxonomy Database

SEM

Scanning electron microscopy

TEM

Transmission electron microscopy

FAMES

Fatty acid methyl esters

ENIU

Equivalent number of isoprene units

MK

Menaquinone

DPG

Diphosphatidylglycerol

PI

Phosphatidylinositol

AGPL

Aminoglycophospholipid

GPL

Glycophospholipid

Ala

Alanine

Glu

Glutamic acid

Ser

Serine

Orn

Ornithine

Author contributions

Conceptualization, S.I. and A.O.; Data Curation, S.I. and A.O.; Formal Analysis, S.I.; Funding Acquisition, S.I. and A.O.; Investigation, S.I.; Methodology, S.I.; Project Administration, A.O.; Resources, A.O.; Software, S.I.; Supervision, A.O.; Validation, S.I.; Visualization, S.I.; Writing—Original Draft, S.I.; Writing—Review & Editing, S.I. and A.O.

Funding

This work was financially supported by a Grant-in-Aid for Research from the Japan Society for Promotion of Science (JSPS) KAKENHI (Grant No. 20H05590 and 22H02265), Grant-in-Aid for JSPS Fellows (Grant No. 24KJ0484) and PRIME from the Japan Agency for Medical Research and Development (Grant No. 19gm6010002h0004). This work was also supported by Japan Science and Technology Agency (JST), PRESTO (Grant No. JPMJPR19H1), the establishment of university fellowships towards the creation of science technology innovation (Grant No. JPMJSP2124) and by the JAEA Nuclear Energy S&T and Human Resource Development Project through concentrating wisdom (Grant No. PJA20P20333393). I.S. was supported by a grant from JST and JSPS.

Data availability

The sequence data of Genbank/EMBL/DDBJ accession number AP026442 was used as a complete genome of strain NTE-D12^T. The 16S rRNA sequence data has been deposited to the DNA Data Bank of Japan (DDBJ) under accession number LC779660.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethical approval

Not applicable.