A novel polyhydroxyalkanoate-storing bacterium Thauera carbonocopians sp. nov. isolated from a sequencing batch reactor fed with volatile fatty acids

Abstract

A Gram-negative bacterial strain, namely Sel9^T, was isolated from a sequencing batch reactor for the selection of a polyhydroxyalkanoate (PHA)-storing microbial biomass, fed with volatile fatty acids. 16S rRNA gene sequence and core genome analyses performed with maximum likelihood method evidenced that Sel9^T belongs to the genus Thauera with the highest phylogenetic relatedness with Thauera butanivorans DSM 2080^T (98.99%) and Thauera linaloolentis DSM 12138^T (98.49%). Digital DNA–DNA hybridization and Average Nucleotide Identity (OrthoANI) values between strain Sel9^T and the closest taxon, T. butanivorans DSM 2080^T, were 53.20 and 93.71%, respectively, which were below the cut-off values for species delineation. The predominant cell fatty acids were summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:0 and summed feature 8 (C18:1 ω6c/C18:1 ω7c). Phosphatidylethanolamine and phosphatidylglycerol were the main polar lipids in the cell. Genome mining detected nine biosynthetic gene clusters, including ectoine, pyrroloquinoline quinone (PQQ)-redox and a genus-rare nonribosomal peptide synthetase (NRPS) gene cluster, plus the acyclic terpene utilization pathway predicting growth on linalool. The combination of phylogenetic, chemotaxonomic and phenotypic features led to consider strain Sel9^T as a representative of a novel species within the genus Thauera. Therefore, given its remarkable ability to store carbon sources, for the type strain Sel9^T (=LMG 33225^T =BAC RE RB 2381^T =VUCC 376^T) the name of Thauera carbonocopians sp. nov. is here proposed. Eventually, this study represents the first comprehensive investigation of biosynthetic gene clusters and the comparative genomics analysis of PHA metabolism within the genus Thauera.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-37556-1.

Introduction

Described in 1993, the genus Thauera belongs to the family Zoogloeaceae, order Rhodocyclales, class Betaproteobacteria¹. Species of Thauera include aerobic Gram-negative, rod-shaped, oxidase and catalase positive bacteria, characterised by a chemoorganotrophic respiratory metabolism. As of May 2025, the List of Prokaryotic names with Standing in Nomenclature (LPSN) indicates there are 12 validly published species within the Thauera genus, mainly isolated from natural habitats such as sediments, saline lakes^2–5, contaminated waters⁶ biofilm of a microbial fuel cell⁷ or plants for the treatment of landfill leachate as well as sewage sludge^8,9.

It has been previously reported that the genus Thauera is able to produce polyhydroxyalkanoates (PHAs), linear polyesters stored as intracellular granular reserve of carbon and energy¹⁰. These molecules have been explored for a wide range of applications, such as production of biodegradable plastics, packaging, development of biomedical devices as well as animal feed preparations^11,12. One of the most interesting potential applications of PHA-accumulating bacterial strains is their use as feed additive in aquaculture, which represents one of the fastest growing food production sectors over recent decades¹³. As such, PHA supplementation either in crystalline form (extracted from bacterial biomass) or amorphous form (as granules inside the cells) is a promising application in fish and crustacean farming as it promotes growth and resistance towards pathogens, while reducing the use of antibiotics^14–16.

During an earlier study aimed at monitoring the PHA-storing microbial biomass in a sequencing batch reactor (SBR), a novel strain called Thauera sp. Sel9^T was isolated^17,18. Although Thauera spp. can represent more than 50% of the total bacterial biomass in mixed microbial cultures (MMCs)¹⁷, Sel9^T was the first strain belonging to this genus isolated from a SBR aimed at selecting PHA-storing microbial biomass under feeding with volatile fatty acids (VFAs)^17,18. Further studies have already shown that Sel9^T can accumulate PHAs by using different VFAs as carbon sources, reaching a concentration above 60% (w/w) when butyrate was supplied¹⁸. Therefore, the use of low-cost biobased VFAs derived from the acidogenic fermentation of agro-industrial waste as growth substrate for Sel9^T could be a suitable strategy to achieve an economically affordable accumulation of PHAs¹⁹.

On the other hand, to use a bacterial strain for feed, a rigorous pre-market safety assessment is required for the inclusion in the Qualified Presumption of Safety (QPS) list by the European Food Safety Authority²⁰. To achieve this status, the taxonomic identification of a microbial candidate is the primary and essential prerequisite before the safety assessment²¹.

In the present study, an unambiguous taxonomic identification of the strain Thauera sp. Sel9^T is provided by relying on a polyphasic approach including 16S rRNA gene sequence, digital DNA–DNA hybridization (dDDH; Formula 2, d0, d4 and d6), Average Nucleotide Identity (ANI; OrthoANI, ANIb and ANIm), core genome, and comparative genome analyses – such as OGRI metrics, gene clusters analysis - combined with phenotypic characterization such as polar lipids and fatty acids along with cell quinone identification. Eventually, this study represents the first comprehensive investigation of either biosynthetic gene clusters (BGCs) or a comparative genomics analysis of PHA metabolism within the genus Thauera, significantly expanding our understanding of its genetic and metabolic diversity, as well as highlighting potential biotechnological applications.

Materials and methods

Sampling and isolation procedures

The strain Sel9^T was collected in 2018 from a selection sequencing batch reactor (S-SBR) located in Carbonera, Treviso, Italy (45.67445° N, 12.29164° E). The S-SBR was optimized for the selection of polyhydroxyalkanoate (PHA)-storing biomass and was initially fed with acetic and propionic acids in a 70:30 ratio from day 0 to day 106. From day 107 to day 145, the feeding regimen was switched to a mixture of volatile fatty acids (VFAs) ranging from C-2 to C-5 acids derived from cellulosic primary sludge (CPS) fermentation liquid¹⁷.

Samples from the S-SBR were transported to the laboratory at 4 °C, diluted in physiological saline solution (0.9% NaCl), and plated on Tryptone Soy Agar (TSA; Oxoid, Basingstoke, UK). Single colonies were isolated by repeated streaking until pure cultures were obtained. The isolated strain, designated Sel9^T, was screened for its capability to produce PHAs according to the protocol described by Andreolli et al.¹⁸. Routine cultivation was carried out in Tryptone Soy Broth (TSB; Oxoid, Basingstoke, UK) or TSA, and the strain was stored at –80 °C in 20% glycerol. The strain Sel9^T was deposited in Belgian Coordinated Collection of Microorganisms (BCCM-LMG), Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna (IZLER) and Verona University Culture Collection – Department of Biotechnology (VUCC-DBT).

Morphological and physiological characterization

(i) Colony morphology was observed on TSA after incubation for 3 days at 27 °C. (ii) Gram staining was performed following the method of Smibert and Krieg²², and spore staining was conducted using malachite green as the primary dye. (iii) Cell morphology was observed by transmission electron microscope (TEM). Cells grown in TSB for 48 h at 27 °C (150 rpm) were further transferred to modified Brunner medium supplemented with acetate (1 g COD L⁻¹; C:N ratio of 80) as described by Andreolli et al.¹⁸ and harvested for observation using a Hitachi H-7650 TEM operating at 120 kV. (iv) Growth was assessed on TSA at temperatures ranging from 4 °C to 45 °C over a 5-day incubation period. Salt tolerance was evaluated by culturing on TSA supplemented with 0–5% (w/v) NaCl and incubated at 27 °C for 5 days. The optimal pH for growth was determined within the range 5–12 by adjusting the pH of the growth medium and observing growth patterns. Anaerobic growth was assessed in TSB and TSA, with and without 0.1% (w/v) KNO₃. Agar plates were placed in an anaerobic chamber (Oxoid, UK), and inoculated tubes containing 5 mL of medium were overlaid with 2 mL of sterilized mineral oil and incubated at 27 °C for 5 days. (v) Nitrate reduction was tested in TSB supplemented with 0.1% KNO₃ by detecting nitrite with naphthylamine/sulfanilic acid reagents and residual nitrate with zinc powder²³. (vi) Oxidase activity was determined by streaking biomass on filter paper soaked with 1% (w/v) N,N,N′,N′-tetramethyl-p-phenylenediamine and observing a colour change to purple²⁴. (vii) Catalase activity was detected by adding 3% (v/v) H₂O₂ solution to the biomass and observing bubble formation. (viii) Motility was assessed on semi-solid medium containing 0.4% (w/v) agar²⁵.

Identification of carbon sources used for growth

Utilization of different substrates was tested using Biolog Phenotypic MicroArray plates PM1 and PM2A (Biolog, USA) with modified minimal Brunner medium¹⁸. The plates were incubated for 11 days at 27 °C with shaking at 200 rpm. Colorimetric changes indicating substrate utilization were measured using a BioTek Synergy™ Neo2 Hybrid Multi-Mode Reader (BioTek Instruments, USA) at wavelengths of 590 nm and 750 nm. Data were exported using BioTek Gen5 Software and analysed in R Statistical Software (v4.2.3)²⁶. The opm package (v1.3.77)²⁷ was used with a discretization approach applying a 0.15 cut-off value to classify carbon sources as positive or negative.

Acid production from carbohydrates was evaluated using the API 50CH fermentation kit (bioMérieux, Marcy-l’Étoile, France) according to the manufacturer’s instructions. The analysis was performed in triplicate.

Analysis of fatty acids, respiratory quinones, and polar lipids

Analysis of the cellular fatty acid profile, respiratory quinones, and polar lipids was performed by Leibniz-Institute DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Fatty acids were extracted from biomass grown on TSB at 27 °C for 48 h (150 rpm) and analysed using the MIDI Sherlock Microbial Identification System with the TSBA6 database. All tests were performed independently in triplicate.

16S rRNA gene sequencing and phylogeny

Genomic DNA was extracted from a 4 mL liquid culture grown in TSB and incubated for 48 h at 27 °C (200 rpm) using the Wizard® SV Genomic DNA Purification System kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The 16S rRNA gene was amplified using primers fD1 (5’-AGAGTTTGATCCTGGCTCAG-3’) and rP2 (5’-ACGGCTACCTTGTTACGACTT3’) to obtain a 1392 bp fragment²⁸. The amplified gene was cloned into the pGEM-T® Easy Vector System (Promega) and sequenced by Eurofins Genomics (Ebersberg, Germany).

Sequences of 16S rRNA from Thauera and Pseudothauera strains, along with Stutzerimonas stutzeri 17588^T as an outgroup⁹, were retrieved from LPSN and aligned using MUSCLE (v5.1.0)²⁹. The gaps in the multiple sequence alignments were removed and the phylogenetic analysis was conducted using the maximum-likelihood approach implemented in IQ-TREE (v2.1.4-beta)³⁰ with the GTR+F+G4 substitution model and a maximum of 10,000 ultrafast bootstrap replicates³¹. Phylogenetic trees were visualized using the Interactive Tree of Life (iTOL: v6.8.1) tool³². The 16S rRNA gene sequence of Thauera sp. Sel9^T was deposited in GenBank under accession number OP279920.

Genome sequencing and taxonomic characterization

Genomic DNA was extracted, sequenced and assembled as previously described¹⁸. The assembled genome was submitted to GenBank under accession number JANUXN000000000.

Average Nucleotide Identity (ANI) was computed using three distinct algorithms to ensure robustness: ANI based on BLAST+ (ANIb) and MUMmer (ANIm) were calculated using pyani v0.2.13³³. Additionally, OrthoANI values were computed using OrthoANI algorithm³⁴. To assess genomic structural signatures, Tetranucleotide frequency correlation coefficients (TETRA) were calculated using pyani. For proteome-level comparisons, Average Amino Acid Identity (AAI) was determined using FastAAI v0.1.20³⁵.

Digital DNA-DNA hybridization (dDDH) values were calculated using the Genome-to-Genome Distance Calculator 3.0 (http://ggdc.dsmz.de/ggdc.php; Formula 2) and Type (Strain) Genome Server (https://tygs.dsmz.de/; d0, d4 and d6 DDH)³⁶. Genomic comparisons were performed using MASH on genomic nucleotide FASTA files of Thauera sp. Sel9^T and Thauera butanivorans DSM 2080^T obtained from NCBI RefSeq.

Core genome analysis

Core genome analysis was performed using strains from Thauera spp. and Pseudothauera spp. Genomic data were retrieved and processed using NCBI Datasets command-line tools (v13.22.0)³⁷. Genome quality was assessed using CheckM (v1.2.2)³⁸ with precomputed lineage-specific marker genes for the Thauera genus. Genomes were evaluated and excluded based on quality control criteria as reported by the Genome Taxonomy Database³⁹: CheckM completeness estimate >80%, CheckM contamination estimate <10%, Quality Score defined as completeness - 5 ⨉ contamination >50, contain <1000 contigs, had an N50 >5 kbp, and contained <100,000 ambiguous bases. In total, 170 genome assemblies were processed of which 81 passed our quality control criteria (Table S1).

The core genome, defined as single-copy orthologs present in all genomes, was identified using OrthoFinder (v2.5.4)⁴⁰ with DIAMOND (v2.0.11)⁴¹ for all-vs-all sequence searches and MUSCLE (v5.1.0)²⁹ for multiple sequence alignment of protein sequences predicted by Prodigal (v2.6.3)⁴². Multiple sequence alignments were concatenated, and gaps were removed using Python (v3.8.10)⁴³ and Biopython (v1.83)⁴⁴. Phylogenetic analysis was conducted using the maximum-likelihood approach implemented in IQ-TREE (v2.1.4-beta)³⁰ with the LG+F+G4 amino acid substitution model and a maximum of 10,000 ultrafast bootstrap replicates³¹. Phylogenetic trees were visualized using the Interactive Tree Of Life (iTOL; v6.8.1) tool³².

Comparative genomics and biosynthetic gene cluster analysis

A comprehensive analysis of biosynthetic gene clusters (BGCs) was conducted on the genomes used for core genome analysis. AntiSMASH (v6.1.1)⁴⁵ was employed to identify and categorize BGCs across the Thauera and Pseudothauera genera. The correlation between the number of contigs in each genome and the predicted number of BGCs was assessed using Pearson correlation analysis performed in R Statistical Software (v4.2.3)²⁶.

Predicted protein sequences (Prodigal v2.6.3) were re-annotated using InterProScan (v.5.71.102)⁴⁶. The resulting domain assignments were filtered against a manually curated list that marks key genes and operons of interest through InterPro signatures, including: PHA metabolism, beta-oxidation of aromatic compounds, components of soluble butane monooxygenase (sBMO) complex, and acyclic terpene utilization (Atu) pathway. Ambiguous or missing calls were resolved by BLASTp (v2.16)⁴⁷ searches (E-value ≤ 1⨉10⁻³⁰, 70% query coverage, and 60% similarity) against corresponding UniProtKB sequences.

Results and discussion

Isolation and identification of strain Sel9^T

The bacterial strain Sel9^T was isolated from a sequencing batch reactor (SBR) optimized for the selection of polyhydroxyalkanoate (PHA)-storing biomass.

The phylogenetic tree based on 16S rRNA gene sequences of both the strain Sel9^T and all species belonging to the genera Thauera and Pseudothauera is reported in Fig. 1. Sequence analysis indicated that the strain Sel9^T is closely related to members of the genus Thauera and has the highest similarity values with Thauera butanivorans DSM 2080^T (98.99%) and Thauera linaloolentis DSM 12138^T (98.49%). All other type strains showed 16S rRNA gene sequence similarities well below the cut-off value of 98.7% considered the threshold for discriminating two species, including the type species of Thauera genus (Thauera selenatis ATCC 55363^T)^6,48.

Fig. 1.

Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences of strain Thauera sp. Sel9^T and related species. Stutzerimonas stutzeri CGMCC 1.1803^T was used as outgroup. Sequences were aligned and gaps were removed prior to phylogenetic analysis. The tree was constructed with GTR+F+G4 substitution model and bootstrap support assessed by 10,000 ultrafast bootstrap replicates. Bootstrap values are indicated at the branching points. The tree is rooted at midpoint and visualized on the iTOL platform. Branch lengths are in expected nucleotide substitutions per site (GTR+F+G4); scale bar = 0.1 substitutions/site.

The capability of Thauera spp. to synthetize PHAs in SBR fed with VFAs has been already reported elsewhere¹⁷. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis performed in the SBR where Sel9^T was isolated, revealed bands with sequence similarity ranging from 96% to 98% to Thauera butanivorans DSM 2080^T17. Moreover, fluorescence in situ hybridization (FISH) analysis demonstrated an increase in the abundance of Thauera spp. from 3.0 ± 0.02% to 58.2 ± 11.1% of the total bacterial population after 119 days¹⁷, underscoring the pivotal role of this genus in PHA production from VFAs within the reactor system. A complete overview of genetic pathways involved in PHAs biosynthesis within the Thauera genus will be discussed further.

Morphological and physiological features of strain Sel9^T

After cultivation on TSA for 72 h at 27 °C, colonies of Sel9^T appeared small, circular, convex, entire, smooth, and light brown. Cells are Gram staining negative, motile rods measuring 1.2–3.1 µm in length and 0.3–0.7 µm in width (Fig. 2). The cell size was similar to T. selenatis ATCC 55363^T and other related Thauera spp. (Table 1). No spores were observed after examination under the microscope, consistent with other members of the genus^{2–9,49–54}.

Fig. 2.

Transmission electron microscopy image showing Sel9^T containing the granules of PHAs. The image magnification was 18,000X¹⁸.

Table 1.

Comparison of strain Sel9^T and closely related species of the genus Thauera. The percentage of fatty acid composition is reported.

	Thauera sp. Sel9T [This study]	T. butanivorans DSM 2080T52	T. linaloolentis DSM 12138T49	T. propionica KCTC 52820T4	T. selenatis ATCC 55363T6,7
Cell Morphology	Rods	Rods	Rods	Rods	Rods
Cell size (µm)	1.2–3.1 x 0.3–0.7	1.1–2.4 x 0.6–0.8	1.4–2.7 x 0.5–0.8	1.6–2.2 x 0.6–0.8	1.4 x 0.56
Mobility	+	+	+	+	+
Nitrate reduction	+	+	+	+	+
Optimum temperature for growth (°C)	27–30	n.d.	32	37	25–30
Metabolism	Facultative anaerobe	Facultative anaerobe	Facultative anaerobe	Facultative anaerobe	Facultative anaerobe
G+C (%)	66.0	67.3	65.6	65.9	66
Fatty acid (%)
Summed feature 3*	45.3	43.6	47.1	50.6	43.8
C16.0	27.5	27.6	28.5	26.6	21.7
Summed feature 8*	14.6	14.8	12.9	7.0	14.9
C12:0	5.4	4.6	2.9	7.0	7.1
C10:0 3-OH	5.2	4.7	5.7	7.6	5.5

^*Summed feature 3 contains 16:1 ω7c and/or 16:1 ω6c; Summed feature 8 contains 18:1 ω7c and/or 18:1 ω6c. n.d.: no determined.

Thauera sp. Sel9^T was able to grow in a range from 12 to 38 °C with an optimum around 27–30 °C and tolerated up to 3% NaCl, with optimal growth at pH 7. These results are similar to those reported for most species belonging to genus Thauera ^{2–9,49–54}. The strain exhibited facultative anaerobic growth, reducing nitrate to nitrite when using NO₃⁻ as an electron acceptor. In fact, nitrate reduction and anaerobic growth by utilizing NO₃^- as an electron acceptor are common characteristics among the members of genus Thauera^{2,3,5,8,49–52}. Catalase and oxidase activities were positive, and the strain was motile, aligning with other Thauera species^{2–9,49–54}. The results and a comparison to its most closely related species and T. selenatis are summarized in Table 1.

Carbon sources used for growth

Biolog assays revealed that Thauera sp. Sel9^T utilized a range of substrates as sole carbon and energy sources, particularly acids and amino acids (Fig. S1). These include succinic acid, L-aspartic acid, L-proline, D-alanine, glycerol, L-lactic acid, L-glutamic acid, D,L-malic acid, tween 20, acetic acid, L-asparagine, alpha-keto-butyric acid, alpha-hydroxy-butyric acid, fumaric acid, propionic acid, glyoxylic acid, L-serine, L-alanine, D-malic acid, pyruvic acid, ethanolamine, butyric acid, caproic acid, beta-hydroxy-butyric acid and succinamic acid. The results confirm the capability of Sel9^T to grow on VFAs such as acetic, propionic, butyric¹⁸ and also caproic acid. Interestingly, among these 25 substrates, 18 compounds are acids (72%) and four are amino acids (16%). The strain did not ferment sugars, consistent with the metabolic profiles of other Thauera species^7,18,49. Previously studies evidenced that T. butanivorans DSM 2080^T, T. aromatica DSM 6984^T and T. linaloolentis DSM 12138^T can grow under aerobic conditions with butyrate^49,51,52. Strains DSM 2080^T and DSM 6984^T can use acetate, propionate, succinate and fumarate under aerobic conditions (no data are available for T. linaloolentis DSM 12138^T)^51,52. Comparative analysis showed distinctive carbon utilization patterns compared to closely related species. T. terpenica DSM 12139^T utilized D-serine, D-malic acid, beta-hydroxy-butyric acid, acetic and propionic acid⁴⁹; T. humireducens DSM 100429^T, T. aminoaromatica DSM 14742^T T. selenatis ATCC 55363^T, T. chlorobenzoica DSM 18012^T grew with acetate, butyrate and pyruvate^3,6,7,50. Eventually, the results here obtained confirm the low affinity of Thauera strains to use sugars as carbon substrates.

The inability to produce acid from carbohydrates, as evidenced by the API 50CH fermentation kit, further differentiates Sel9^T from some related species. For instance, T. linaloolentis DSM 12138^T showed activity only toward D-arabinose, whereas T. terpenica DSM 12139^T fermented mannitol and (weakly) inositol and sorbitol⁴⁹ (no data are available for T. butanivorans DSM 2080^T).

Profiles of constitutive fatty acids, respiratory quinones, and polar lipids

The major respiratory quinone in Thauera sp. Sel9^T was ubiquinone-8 (Q-8, 93.7%) followed by ubiquinone-7 (Q-7, 6.3%). Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) were the predominant polar lipids. The fatty acid profile was dominated by summed feature 3 (C16:1 ω6c/C16:1 ω7c, 45.31%), C16:0 (27.54%), and summed feature 8 (C18:1 ω6c/C18:1 ω7c, 14.65%). This lipid composition previously reported for closely related species and T. selenatis is reported in Table 1. Among them, T. propionica KCTC 52820ᵀ (grown on TSA at 30 °C) showed a fatty acid profile consistent with that of strain Sel9ᵀ⁴.

Genome sequencing and taxonomic characterization

The draft genome of Thauera sp. Sel9^T was assembled into 49 contigs totalling 4,524,829 bp, with an N50 value of 187.4 kb. The genome contains 4,152 predicted coding sequences, 51 tRNA genes, and one complete rRNA operon. A total of 14 pseudogenes were found. The G+C content is 66.0%, consistent with values reported for other Thauera species (63.6–70.6 mol%; as determined by HPLC, in silico or thermal denaturation method)^{2–9,49–54}.

The results of OrthoANI and dDDH (Formula 2) analyses are summarized in Table 2 and indicated that Sel9^T shares highest similarity with Thauera butanivorans DSM 2080^T, with OrthoANI values of 93.71% (with an ANIb of 93.4% and an ANIm of 94.0%) and dDDH values of 53.2%, both below the thresholds for species demarcation (95–96% for ANI^48,55 and 70% for dDDH^55,56. TYGS analysis yielded dDDH values of 53.3, 53.2, and 53.8% (for d0, d4, and d6, respectively), further indicating that strain Sel9ᵀ does not correspond to any currently described species. Moreover, Sel9 and T. butanivorans DSM 2080^T share only 144/1000 genomic objects according to the Mash mutational distance analysis; the data points more strongly support compared to ANI for the genetic isolation between these two closely related strains.

Table 2.

Average nucleotide identity (OrthoANI) and in silico DNA–DNA hybridization (isDDH; Formula 2) values between Sel9^T and type strains of closely related Thauera species.

Species	ANI (%)	DDH (%)	References
T. butanivorans DSM 2080T	93.71	53.20 [50.6 - 55.9]	52
T. linaloolentis DSM 12138T	86.35	31.30 [28.9 - 33.8]	49
T. propionica KCTC 52820T	83.13	26.40 [24.1 - 28.9]	4
T. humireducens DSM 100429T	82.98	26.30 [24.0 - 28.8]	7
T. phenylacetica DSMZ 14743T	81.91	25.90 [23.6 - 28.4]	50
T. chlorobenzoica DSM 18012T	81.62	25.10 [22.8 - 27.6]	3
T. aminoaromatica DSM 14742T	81.58	25.20 [22.9 - 27.7]	50
T. aromatica DSM 6984T	81.34	24.90 [22.5 - 27.3]	51
T. selenatis ATCC 55363T	81.21	25.20 [22.9 - 27.7]	6
T. terpenica DSM 12139T	80.94	24.00 [21.7 - 26.5]	49
T. sinica KACC 19216T	80.45	24.10 [21.8 - 26.5]	9
“T. phenolivorans NCBR 112379T”	80.45	23.30 [21.0 - 25.7]	54
T. mechernichenis DSM 12266T	80.26	23.50 [21.2–26.0]	8
“T. sedimentorum KCTC 72546T”	78.46	22.40 [20.1 - 24.8]	2

These results support the proposal that Sel9^T represents a novel species within the Thauera genus. Moreover, additional OGRI metrics (such as TETRA and AAI) were calculated and reported in Tab. S2.

Core genome analysis

Core genome analysis was based on 43 core genes identified among 81 genome sequenced of Thauera, Pseudothauera strains and Stutzerimonas stutzeri ATCC 17588^T as outgroup (Fig. 3). This study represents the first comprehensive core genome analysis of strains of these genera. The tree highlighted the presence of two distinct clusters containing T. butanivorans DSM 2080ᵀ and Thauera sp. Sel9ᵀ. This evidence is consistent with the ANI and dDDH results, confirming the phylogenetic relatedness with T. butanivorans and also supporting the notion that Sel9ᵀ may represent a new species. The results evidenced that the species affiliation was not established for most of strains belonging of these genera. In fact, the presence of different unique clusters (e.g. single cluster with Thauera sp. UBA 6194 or Thauera sp. UWPOP THAU1) suggests the presence of additional Thauera species not yet identified.

Fig. 3.

Maximum Likelihood Phylogenetic Tree of the Core Genome for Thauera sp. Sel9^T and strains within the Thauera and Pseudothauera genera using Stutzerimonas stutzeri CGMCC 1.1803^T as an outgroup. The tree is constructed from 43 core genes identified among 81 strains. The tree is rooted using the outgroup and visualized on the iTOL platform. Species names are annotated with assembly accession number and isolation source. Branch lengths are in expected amino-acid substitutions per site; scale bar = 1 substitution/site (LG+F+G4). Bootstrap values < 60 are not shown.

Analysis of biosynthetic gene clusters (BGC)

Overall, 651 BGCs were identified using the antiSMASH tool across 80 genomes belonging to Thauera and Pseudothauera (Fig. 4). These BGCs were classified into five major categories: “Other” (276), ribosomally synthesized and post-translationally modified peptides (RiPP; 224), terpenes (76), nonribosomal peptide synthetases (NRPS; 58), and polyketide synthases (PKS; 17). Further classification into 23 subgroups, based on protocluster types defined by antiSMASH⁴³, revealed that terpene (94%), betalactone (91%), RiPP-like (85%), acyl amino acids (78%), ranthipeptide (68%), and hserlactone (68%) clusters were the most prevalent, representing core BGCs across Thauera species. An average of eight BGCs per genome was observed, with Thauera phenylacetica B4P^T=DSM 14743^T containing the highest number (14 BGCs). No significant correlation was found between the number of genomic contigs and the predicted number of BGCs (Fig. S2).

Fig. 4.

Distribution of biosynthetic gene clusters (BGCs) among Thauera and Pseudothauera species. The heatmap illustrates the presence and prevalence of different BGC types predicted by antiSMASH across 80 genomes analysed in this study, highlighting core and strain-specific BGCs. The stacked bar plot summarizes the total number of BGCs per genome, color-coded according to major BGC categories (RiPP, Terpene, NRPS, PKS, and Other).

A total of nine BGCs were predicted in Sel9^T, including all core BGCs, along with additional clusters encoding redox-cofactor (46%), ectoine (35%), and NRPS (11%). The redox-cofactor BGC identified in Sel9^T includes core genes homologous to pqqC, pqqE, and pqqD, suggesting the capability of pyrroloquinoline quinone (PQQ) biosynthesis. PQQ is recognized as an important redox cofactor involved in various enzymatic reactions, thereby enhancing metabolic capabilities⁵⁷. Comparative genomic analysis indicated that this redox-cofactor BGC occurs in approximately half of the Thauera strains examined, implying that its presence is not a conserved essential function across the genus but rather a selective adaptation, potentially conferring advantages under specific environmental conditions, particularly those where PQQ-mediated metabolic processes could provide a competitive edge⁵⁷.

The ectoine-like BGC identified in Thauera sp. Sel9^T, showed 75% similarity to the characterized ectoine BGC from Streptomyces anulatus (MIBiG accession: BGC0000853). Ectoine BGCs are typically found in halophilic and osmotolerant bacteria⁵⁸. Consequently, the presence of this ectoine-like BGC in Sel9^T suggests a potential role in osmoprotection and enhanced salt-stress tolerance as described previously.

Thauera sp. Sel9^T also harbors a non-ribosomal peptide synthetase (NRPS) BGC, spanning approximately 46.22 kbp. This cluster comprises core synthetase genes (amino acid adenylation domain-containing protein and a non-ribosomal peptide synthetase), as well as genes associated with transport and modification functions. Notably, the presence of this NRPS cluster is uncommon within the Thauera genus, having been identified in only a few other genomes.

This study represents the first comprehensive investigation of BGCs within the genus Thauera. This comparative genomic approach further distinguished Sel9^T from its closely related type strains, T. butanivorans DSM 2080^T and T. linaloolentis DSM 12138^T. Specifically, T. butanivorans DSM 2080^T possesses an arylpolyene BGC (10%), an uncommon cluster within the genus that is absent in both Sel9^T and T. linaloolentis DSM12138^T. Conversely, Sel9^T uniquely harbors an NRPS BGC, not found in T. butanivornas and T. linaloolentis type strains. Furthermore, T. linaloolentis notably lacks the acyl amino acid BGC (present in 78% of Thauera species), underscoring the distinct biosynthetic capabilities among these closely related taxa. Besides BGCs, a further analysis of the genome of Sel9^T displayed the presence of two spermidine synthase (JANUXN01P0000221 and JANUXN01P0001151), a polyamine aminopropyl transferase (JANUXN01P0001850) and a carboxynorspermidine decarboxylase (JANUXN01P0001849) which are likely involved in the production of spermidine and norspermidine polyamines.

Targeted comparative genomics of PHA metabolism and specialized pathways

PHA metabolism fundamentally involves three key enzymes: acetyl-CoA acetyltransferase, acetoacetyl-CoA reductase, and PHA synthase. This biosynthetic pathway begins with the condensation of CoA-activated substrates, catalyzed by β-ketothiolases. In Thauera species, Beta-ketothiolase (BktB), rather than PhaA, predominantly mediates these reactions. The main functional distinction between PhaA and BktB lies in their substrate specificity. While PhaA typically catalyzes the condensation of two acetyl-CoA molecules, BktB shows broader substrate promiscuity, efficiently catalyzing condensation reactions involving acetyl-CoA with propionyl-CoA and butyryl-CoA^59–61.

Thauera species harbour class I PhaC synthase, responsible exclusively for synthesizing short-chain length polyhydroxyalkanoates(scl-PHAs)⁶². An additional general signature sequence for PhaC was commonly detected across the analysed species. Concerning PHA depolymerization, most Thauera genomes encode the esterase PhaZ (IPR010126), which initiates polymer breakdown through substrate-specific hydrolysis. Additionally certain Thauera genomes, including Sel9^T, harbour the bdhA gene. In contrast to PhaZs, the BdhA enzyme facilitates the integration of the depolymerization-derived monomers into central metabolism^63,64. Regulatory elements including phaD, phaP subfamily 1, phaR, and phaM were broadly distributed across the Thauera genomes, indicating highly conserved regulatory mechanisms within the genus.

The two type strains most closely related to Sel9^T, belonging to T. butanivorans and T. linaloolentis, each possess unique genetic capabilities. T. linaloolentis species is uniquely equipped to degrade and utilize linalool as its sole carbon source via the acyclic terpene utilization (Atu) pathway⁶⁵. Comparative genomic analyses indicate a notably high genetic similarity between Sel9^T and T. linaloolentis for the atu pathway, strongly suggesting Sel9^T as only the second known Thauera species capable of utilizing linalool. Conversely, T. butanivorans DSM 2080^T exclusively includes the soluble butane monooxygenase (sBMO) gene cluster, which encodes the enzymes responsible for butane oxidation⁶⁶. The absence of the sBMO gene cluster in Sel9 argues against its inclusion within T. butanivorans, whose nomenclature reflects a key phenotypic hallmark of its type strain⁶⁷. A summary of the results is available in Fig. S3.

Proposal of  Thauera carbonocopians as a new species

Based on (a) ANI and dDDH values, respectively 93.71% and 53.20%, between the isolate Sel9^T and the type strains of most closely related species T. butanivorans, both below the threshold range for species demarcation^48,55,56, (b) the presence of a distinct cluster in core-genome tree containing Sel9, separated from T. butanivorans DSM 2080^T (c) specific BGC patterns and (d) the absence of soluble butane monooxygenase (sBMO) gene cluster compared to T. butanivorans DSM 2080^T, we propose the isolate Sel9^T as representative of a new species of the genus Thauera. The suggested name for this new species is Thauera carbonocopians sp. nov.. The type strain is Sel9^T (=LMG 33225^T =BAC RE RB 2381^T =VUCC 376^T).

Description of Thauera carbonocopians sp. nov.

Rationale for the Latin nomenclature of the species – Thauera carbonocopians meaning that it greedily accumulates carbonaceous reserves (carbono = Latin neologism, prefix for carbonaceous substrate; copians, present participle of the deponent intransitive Latin verb of the 1st conjugation copior -aris -ari = to stock up).

The type strain Sel9^T was isolated from a sequencing batch reactor (SBR) for the selection of a PHAs storing microbial biomass^17,18. The DNA G+C content of this isolate is 66%. Sel9^T cells are Gram-negative, motile, facultatively anaerobic, rod-shaped (1.2–3.1 µm long and 0.3–0.7 µm wide). After cultivation on TSA for 72 h at 27° C, colonies appear small (1–2 mm in diameter), circular, convex, entire, smooth, and light brown. Sel9^T is catalase and oxidase positive. It can grow up to 3% NaCl concentration and between 12 to 38 °C (optimum 27–30 °C). Growth is observed in anaerobic conditions in the presence of NO₃^- as an electron acceptor. Succinic acid, L-aspartic acid, L-proline, D-alanine, glycerol, L-lactic acid, L-glutamic acid, D,L-malic acid, tween 20, acetic acid, L-asparagine, alpha-keto-butyric acid, alpha-hydroxy-butyric acid, fumaric acid, propionic acid, glyoxylic acid, L-serine, L-alanine, D-malic acid, pyruvic acid, ethanolamine, butyric acid, caproic acid, beta-hydroxy-butyric acid and succinamic acid can be used as sole sources of carbon and energy. No acid production was observed from carbohydrates (relying on the API 50CH fermentation kit). Sel9^T can accumulate and store PHAs while growing on different VFAs (acetate, propionate, butyrate and valerate) which are used as sole sources of carbon and energy⁸. Ubiquinone-8 (Q-8) represents the predominant respiratory quinone while the main polar lipids in Sel9^T are phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Finally, the representative fatty acids detected in the strain Sel9^T are summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:0 and summed feature 8 (C18:1 ω6c/C18:1 ω7c).

The GenBank accession number for the 16S rRNA gene sequence is OP279920 and the accession number of the genome sequence is JANUXN000000000.

Author contributions

Conceptualization: M.A. and S.L.; methodology: M.A., M.J. and E.S.; validation: S.L.; investigation: M.A., M.J., E.S., and P.V.; data curation: M.A., M.J., and E.S.; writing—original draft preparation: M.A., M.J.; writing—review and editing: E.S., G.V. and S.L.; supervision: S.L.. All authors have read and agreed to the published version of the manuscript.

Funding

Part of this work was granted by the Interconnected Nord-Est Innovation Ecosystem (iNEST) and received funding from the European Union - Next-Generation EU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR) – MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.5 – D.D. 1058 June 23, 2022, ECS00000043) and by the SUS-MIRRI.IT project "Strengthening the MIRRI Italian Research Infrastructure (RI) for Sustainable Bioscience and Bioeconomy”, code n. IR0000005,. We acknowledge the contribution and support from the RI MIRRI-IT.

Data availability

The datasets analysed during the current study are available in the GenBank repository: the 16S rRNA gene and genome were deposited with Accession no. OP279920 and no. JANUXN000000000.

Declarations

Competing interests

The authors declare no competing interests.