# Alkalicella caledoniensis gen. nov., sp. nov., a novel alkaliphilic anaerobic bacterium isolated from ‘La Crouen’ alkaline thermal spring, New Caledonia

Marianne Quéméneur, Gaël Erauso, Manon Bartoli, Céline Vandecasteele, Laura Wils, Lisa Gil, Christophe Monnin, Bernard Pelletier, Anne Postec

► To cite this version:

Marianne Quéméneur, Gaël Erauso, Manon Bartoli, Céline Vandecasteele, Laura Wils, et al.. Alkalicella caledoniensis gen. nov., sp. nov., a novel alkaliphilic anaerobic bacterium isolated from ‘La Crouen’ alkaline thermal spring, New Caledonia. International Journal of Systematic and Evolutionary Microbiology, 2021, 71 (5), pp.004810. 10.1099/ijsem.0.004810. hal-03231234

HAL Id: hal-03231234

https://amu.hal.science/hal-03231234

Submitted on 29 Apr 2022

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 *Alkalicella caledoniensis* gen. nov., sp. nov., a novel alkaliphilic anaerobic bacterium

2 isolated from ‘La Crouen’ alkaline thermal spring, New Caledonia

3

4 Marianne Quéméneur¹*, Gaël Erauso¹, Manon Bartoli¹, Céline Vandecasteele², Laura Wils¹,

5 Lisa Gil², Christophe Monnin³, Bernard Pelletier⁴ and Anne Postec¹

6

7 ¹ Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO, Marseille, France

8 ² INRAe, US 1426, GeT-PlaGe, Genotoul, Castanet-Tolosan, France

9 ³ GET UMR5563 (CNRS/UPS/IRD/CNES), Géosciences Environnement Toulouse, 14 Avenue

10 Edouard Belin, 31400 Toulouse, France

11 ⁴ Centre IRD de Nouméa, 101 Promenade Roger Laroque, BP A5 – 98848 Nouméa cedex,

12 Nouvelle-Calédonie.

13

14 *Corresponding author: Marianne Quéméneur

15 E-mail: marianne.quemeneur@ird.fr; Fax number: ++33 (0) 4 91 82 85 70.

16

17 Running title: *Alkalicella caledoniensis*

18

19 **Keywords:** *Firmicutes*, anaerobic, alkaliphilic, alkaline thermal spring, *Proteinivoraceae*,

20 *Alkalicella caledoniensis*

21

22 **Section/Category:** New taxa - *Firmicutes* (Class *Clostridia*, Order *Clostridiales*)

23 ** Repositories:** The GenBank accession number for the 16S rRNA gene sequence of strain

24 LB2T is KR349724, and the one for the whole genome sequence is CP058559.

25 The word count of the article is 3604.
26 **Abstract**

27

28 A novel anaerobic, alkaliphilic, mesophilic, Gram-stain-positive, endospore-forming bacterium
29 was isolated from an alkaline thermal spring (42°C, pH 9.0) in New Caledonia. This bacterium
30 designated strain LB2T grew at 25-50°C (optimum 37°C) and pH 8.2-10.8 (optimum 9.5).

31 Added NaCl was not required for growth (optimum 0-1%) but was tolerated up to 7%. Strain
32 LB2T utilized a limited range of substrates, such as peptone, pyruvate, yeast extract and xylose.

33 End products detected from pyruvate fermentation were acetate and formate. Both ferric citrate
34 and thiosulfate were used as electron acceptors. Elemental sulfur, nitrate, nitrite, fumarate,
35 sulfate, sulfite, and DMSO were not used as terminal electron acceptors. The two major cellular
36 fatty acids were iso-C15:0 and C16:0. The genome consists of a circular chromosome (3.7 Mb)
37 containing 3626 predicted protein-encoding genes with a G+C content of 36.2 mol%.

38 Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate is a
39 member of the family Proteinivoraceae, order Clostridiales within the phylum Firmicutes.

40 Strain LB2T was most closely related to the thermophilic Anaerobranca gottschalkii LBS3T
41 (93.2% 16S rRNA gene sequence identity). Genome-based analysis of average nucleotide
42 identity (ANI) and digital DNA-DNA hybridization (dDDH) of strain LB2T with A.
43 gottschalkii LBS3T showed respective values of 70.8% and 13.4%. Based on phylogenetic,
44 genomic, chemotaxonomic, and physiological properties, strain LB2T is proposed to represent
45 the first species of a novel genus, for which the name Alkalicella caledoniensis gen. nov., sp.
46 nov. is proposed (type strain LB2T = DSM 100588T = JCM 30958T).

47

48
Over the last twenty years, alkaliphilic bacteria have been extensively studied because they are sources of biomolecules and enzymes stable at extreme conditions, precious in biotechnology and bioremediation [1, 2, 3]. Alkaliphiles are microorganisms that optimally grow at pH values above 9 but cannot grow or slowly grow at near-neutral pH [3]. They inhabit various alkaline ecosystems (e.g., soda lakes, thermal springs, oceans, industrial or mining-impacted areas). Some of them can grow under multiple extreme conditions like a combination of high salt concentrations and high temperature [4]. Anaerobic alkaliphiles, especially members of the order *Clostridiales* (e.g., the genera *Alkaliphilus*, *Anaerobranca*, *Natronoincola*, *Serpentinicella*, or *Tindallia*), have been isolated from different sites around the world and include halo-, meso-, and thermo-alkaliphiles [4, 5].

In New Caledonia (Southwest Pacific), several thermal springs are located in the southeastern half of the main island [6] (see the map in [7]). Such springs are found down south in the Prony Bay area and the area close to the city of Canala [7]. A sampling campaign was conducted in November 2014 during which water and sediment samples were collected at one of the springs of the Canala region, namely the spring feeding the abandoned spa facility of ‘La Crouen’ (165° 53' 20.6" E 21° 32' 06.6" S). On-site measurements showed that its waters are characterized by high pH (9.0), low Eh value (-174 mV, ref H₂), low dissolved O₂ (0.71 mg/L), low conductivity (314 µS/cm), and moderate temperature (42°C) (data from our study). Deville and Prinzhofer [8] reported that the gas emitted at the springs is dinitrogen (N₂) with traces of methane (CH₄ <3%), but we did not observe degassing in the spring during our visit. Our dissolved gases analyses showed that N₂ is dominant, followed by O₂, but no CH₄ or hydrogen (H₂) was detected. Both the geological setting and geochemistry differ from those of the well-known hyperalkaline springs located in the peridotite nappe in the Prony Bay Hydrothermal Field (PBHF). These on-shore or submarine springs discharge a warm (<40°C), highly reduced, high-pH (up to 11.2) fluid enriched in N₂, H₂, and CH₄ [9], produced by the serpentinization process
74 (i.e., a hydrothermal alteration that transforms ferromagnesian minerals contained in
75 ultramafic rocks into serpentine minerals [10]). Prokaryotic communities of PBHF have been
76 studied [11-15] and several anaerobic *Clostridiales* strains have been isolated and described as
77 new taxa [15-21]. On the contrary, microbial communities of 'La Crouen' spring had not been
78 studied previously. Here we report the isolation and characterization of a novel bacterium
79 isolated from 'La Crouen' spring, which original features allow the proposition of a novel
80 species within a novel genus belonging to the family *Proteinivoraceae*, phylum *Firmicutes*.

81 For culturing experiments, a sterile glass bottle was filled with sediment slurry sample,
82 then hermetically sealed to preserve anoxic conditions, and stored at 4°C. In the laboratory,
83 anaerobic enrichment cultures were carried out at 37°C using 0.5 mL of the slurry sample
84 (corresponding to 10% inoculum) in Hungate tubes containing 5 mL of nutrient-rich LB (Luria-
85 Bertani) medium (i.e., 10g/L Tryptone, 5 g/L yeast extract, and 5 g/L NaCl), supplemented with
86 25 mM Tris-Base. Before inoculation, the culture medium was adjusted to pH 9.5, boiled for 5
87 min, and then cooled to room temperature under a flow of O₂-free N₂ gas. The medium was
dispensed into Hungate tubes, degassed under a flow of N₂, and subsequently autoclaved (45
88 min, 120°C). The following sterile solutions were injected in each tube: 0.1 mL of 2%
89 Na₂S.9H₂O (reducing agent) and 0.5 mL of 8% Na₂CO₃ (to adjust and buffer the final pH to
90 9.5). The Hungate technique for anaerobic cultivation was used throughout this study [22].
91 Temperature, pH, and NaCl concentration ranges (respectively tested: 20-70°C, 6.0-12.0, 0-
92 10%) for growth of the isolate were determined in duplicate using Hungate tubes containing
93 LB medium, supplemented with 25 mM Tris-Base (except for testing the optimal pH for
94 growth, which was determined using different buffer solutions: 0 mM Tris-Base for pH 6.0-
95 7.0, 25 mM Tris-Base for pH 7.0-9.0, or 20 mM CAPS for pH 9.0-12.0). The pH of the culture
96 media was adjusted with anaerobic sterile stock solutions of 8% Na₂CO₃ or 1M NaOH. The pH
97 was always checked both after inoculation and at the end of incubation. Water baths were used
98
for incubation at temperatures up to 70°C. For studies of NaCl requirements, NaCl was weighed and added directly into the tubes before culture medium addition. Each test culture was sub-cultured at least once under the same experimental conditions before determining growth rates.

A range of inorganic and organic substrates was tested as substrate for the isolated growth in the basal medium containing (per liter of distilled water): 0.3 g KH₂PO₄, 0.3 g K₂HPO₄, 1 g NH₄Cl, 1 g NaCl, 0.1 g KCl, 0.5 g cysteine.HCl, and 10 mL of trace elements solution [23].

The pH was adjusted to 9.5 with a 1M NaOH solution. The following compounds were tested as potential substrates (at a final concentration of 20 mM for simple substrates and 2g/L for complex substrates): adenine, adenosine 5'-diphosphate, albumin, arabinose, casamino-acids, casein, cellobiose, fructose, galactose, gelatin, glucose, glycerol, guanine, lactate, lactose, mannose, peptone, pyruvate, ribose, starch, sucrose, tryptone, uracil, xylose, yeast extract, H₂/CO₂ (80/20, v/v) and H₂/CO₂ (80/20, v/v) in the presence of acetate (2 mM) as carbon source. Elemental sulfur (1% w/v), sulfate (20 mM), thiosulfate (20 mM), sulfite (2 mM), nitrate (10 mM), nitrite (2 mM), fumarate (20 mM), FeIII-citrate (10 mM), and DMSO (10 mM) were tested as terminal electron acceptors using pyruvate as electron donor. Sensitivity to antibiotics (ampicillin, bacitracin, chloramphenicol, cycloserine, kanamycin, metronidazole, novobiocin, penicillin, rifampicin, streptomycin, tetracycline, vancomycin) was tested by adding them separately to the LB medium (supplemented with 25 mM Tris-Base) at a final concentration of 25 µg/mL or 50 µg/mL. Each test and control, carried out in parallel, was run in duplicate Hungate tubes. Controls were (i) inoculated tubes without tested compounds and (ii) uninoculated tubes with tested compounds. Bacterial growth was monitored by measuring the turbidity increase at 600 nm by inserting Hungate tubes into the tube holder of a spectrophotometer (Cary 50, Varian). H₂S production was measured photometrically as colloidal CuS, as described by Cord-Ruwisch [24]. End-products of metabolism were measured
123

by high-performance liquid chromatography (HPLC) and gas chromatography (GC) after 1 day
of incubation at 37°C [19].

124

125

Cellular morphology and purity of the strain and the presence of spores were assessed by
observation using a phase-contrast microscope (Optiphot; Nikon). For transmission electron
microscopy (using a MET FEI Tecnai G2), exponentially grown cells were negatively stained
with sodium phosphotungstate (to observe flagella) or fixed with glutaraldehyde and osmium
tetraoxide, epon-embedded and stained with uranyl acetate and lead citrate (to watch the cell
wall), as described by Quéméneur *et al.* [25].

126

127

128

129

130

131

Cellular fatty acids were determined in the late exponential growth phase (after 24-hour incubation at 37°C using LB-Tris medium at pH 9.5) by the Identification Service of DSMZ with modified Miller extraction protocol [26] using the MIDI Microbial Identification system (version 4.0, MIS operating manual March 2001) [27].

132

133

134

135

Near-complete 16S rRNA genes were amplified from genomic DNA extracted as described
by Quéméneur et al. [28], using the primers 27F and 1492R [29], then sequenced by GATC-
Biotech (Konstanz, Germany). The 16S rRNA gene sequence was deposited and aligned with
available sequences from NCBI database using the MUSCLE program [30] implemented in
MEGA7 software [31]. Evolutionary analyses were conducted from 16 nucleotide sequences
using three different methods: maximum-likelihood (ML) [32], maximum-parsimony (MP)
[33], and neighbor-joining (NJ) [34].

136

137

138

139

140

141

142

Whole-genome analysis was performed from high molecular weight DNA of the isolate,
extracted using a phenol-chloroform method, then sequenced at the GeT-PlaGe platform
(INRAe Toulouse, France) by combining long reads (Oxford Nanopore) and short-reads
(Illumina MiSeq) technologies, as described by Quéméneur *et al.* [25] with some modifications
because it was sequenced by multiplex sequencing according to the manufacturer's instructions
"1D Native barcoding genomic DNA" (EXP-NBD104 and SQK-LSK109)". Base-calling was

143

144

145

146

147
carried out by MinKNOW-Live-Base-calling software version 3.6.0 (Oxford Nanopore Technologies, Oxford, UK). Read demultiplexing and adapter trimming were performed using `qcat v1.1.0` (https://github.com/nanoporetech/qcat) and genome assembly with Unicycler v0.4.6 [35]. The genome sequence was deposited in the NCBI database and annotated using the MaGe (Microscope Genome Annotation) [36] and the Integrated Microbial Genomes (IMG) systems [37]. The value of average nucleotide identity (ANI) was calculated using OrthoANI with `usearch` (http://www.ezbiocloud.net/tools/ani). Digital (or *in silico*) DNA-DNA hybridization (dDDH) was calculated using the Genome-to-Genome Distance Calculator (GGDC) 2.1 (http://ggdc.dsmz.de/) [38]. A whole genome-based taxonomic analysis was performed using the bioinformatics platform Type (Strain) Genome Server (TYGS) (https://tygs.dsmz.de) [39]. In brief, the TYGS analysis included a determination of the closest type strain genomes by (i) comparison against all type strain genomes available in the TYGS database *via* the MASH algorithm [40] and (ii) the closely related type strains determined *via* the 16S rDNA gene sequences. For the phylogenomic inference, all pairwise comparisons among the genomes were conducted using Genome BLAST Distance Phylogeny (GBDP) and accurate intergenomic distances inferred under the algorithm 'trimming' and distance formula d5. The resulting intergenomic distances were used to infer a balanced minimum evolution tree with branch support *via* FASTME 2.1.6.1 [41]. Branch support was inferred from 100 pseudo-bootstrap replicates each. The trees were rooted at the midpoint [42].

The G+C content of the DNA was also determined at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) using HPLC, as described by Mesbah et al. [43].

The enrichment culture of the 'La Crouen' slurry sample showed significant growth on LB-Tris-Base medium after two days of incubation at 37°C and at pH 9.5. This enrichment was then subcultured several times under the same growth conditions before isolation. The Roll-
tube method described originally by Hungate [22] was used for strain isolation. The culture was serially diluted tenth in roll tubes containing molten 1.6% agar medium. Single colonies were transferred into the liquid medium and immediately serially diluted tenth in roll tubes a second time. The colonies grown in roll tubes were white and circular (1–2 mm in diameter after one-week incubation at 30°C). After the third round of roll tubes isolation, a single colony was finally transferred into the liquid medium, resulting in the strain designated LB2T that was further physiologically and metabolically characterized.

Cells of strain LB2T stain Gram-positive are motile and endospore-forming bacterium (Fig. 1A). Endospores are terminal and oval (0.8–1 x 1.0–1.5 µm). Rods are approximately 6.0–10.0 µm long and 0.5–0.6 µm wide. They occur singly or in pairs and possessed peritrichous flagella (Fig. 1B). They are pleiomorphic, i.e., rod-shaped during the exponential growth phase (Fig. S1A), they turn coccoid in the stationary phase (after 24h, Fig. S1B).

Strain LB2T is mesophilic and grows only under anaerobic conditions, ranging from 25°C to 50°C (Table 1). The optimum temperature for growth is 37°C. The pH range for growth is 8.2–10.8, with an optimum at an initial pH of 9.5; the final pH is 9.2. NaCl is not required for growth, and the NaCl range for growth is 0–7% (optimum at 0–1%) but is tolerated up to 7%, indicating that strain LB2T is halotolerant and adapted to the low-salinity and high-pH fluid emitted by the alkaline spring of ‘La Crouen’ (New Caledonia).

Peptone, pyruvate, xylose, and yeast extract were the only substrates used for growth (Table 1). Yeast extract was degraded to acetate, formate, H₂, and propionate. Growth on yeast extract was improved by the addition of thiosulfate used as a terminal electron acceptor, reduced to sulfide. End products detected from pyruvate fermentation were acetate and formate. Both ferric citrate and thiosulfate were used as electron acceptors using pyruvate at pH 9.5. Elemental sulfur, sulfate, nitrate, nitrite, fumarate, and DMSO were not used as terminal electron
acceptors, using pyruvate as an electron donor. The maximal growth rate found was 0.07 h⁻¹ using pyruvate and thiosulfate as electron donor and acceptor, respectively.

Strain LB2T was not sensitive to the presence of antibiotics cycloserine and novobiocin added at 25 µg/mL and 50 µg/mL in the culture medium. It also grew in the presence of 25 µg/mL of chloramphenicol and kanamycin, but it was sensitive to these antibiotics at 50 µg/mL. Ampicillin, bacitracin, metronidazole, penicillin, rifampicin, streptomycin, tetracycline, and vancomycin at 25 µg/mL completely inhibited cell growth.

Phylogenetic analysis based on the almost full-length 16S rRNA gene sequence (1245 nt) indicated that strain LB2T is a member of the family *Proteinivoraceae* [44], within the order *Clostridiales*, phylum *Firmicutes* (Fig. 2). It forms a distinct branch among alkaliphilic clostridia of the family *Proteinivoraceae*, including the genera *Anaerobranca* and *Proteinivorax* (91.2–93.2% 16S rRNA gene sequence identity) (Fig. 2). The phylogenetic position of strain LB2T was confirmed by using three different methods: maximum-likelihood (ML, Fig 2), maximum-parsimony (MP, Fig S2A), and neighbor-joining (NJ, Fig S2B). The closest relatives of strain LB2T are type species: *Anaerobranca gottschalkii* LBS3T (93.2% identity) isolated from a hot lake inlet (Lake Bogoria, Kenya) [45], *Anaerobranca horikoshii* JWIYL-13S<sup>T</sup> (92.6% identity) isolated from Yellowstone National Park (USA) [46], *Anaerobranca californiensis* PAOHA-1<sup>T</sup> (91.7% identity) isolated from alkaline hot spring sediment (Mono Lake, CA, USA) [47], *Anaerobranca zavarzinii* JW/VK-KS5Y<sup>T</sup> (92.2% identity) isolated from a small shallow geothermally heated pool (Kamchatka, Russia) [48], *Proteinivorax hydrogeniformans* Z-710<sup>T</sup> (91.8% identity) and *Proteinivorax tanatarense* Z-910<sup>T</sup> (91.5% identity) isolated from a decaying algal bloom in the alkaline lake Tanatar (Russia) [44, 49].

The 16S rRNA gene sequence of strain LB2T was closely related (>97% identity) to few environmental sequences retrieved in August 2020 by a NCBI web BLAST search [50] and
originated from low-salt alkaline ecosystems: anaerobic fermentation reactor with waste activated sludge under mesophilic condition (pH 9.0; GU455162) [51] and surface rock/sediment of Lonar Crater Lake (pH 8.9-10.0; JQ738949) [52]. 16S rRNA genes affiliated to the family Proteinivoraceae (formerly known as the family Anaerobrancaceae) were also detected in the subsurface hyperalkaline fluids of the Semail Ophiolite (Oman) [53] and other oligotrophic, rock-hosted ecosystems, such as deep crystalline aquifer of Outokumpu (Finland) [54]. Still, their potential functional roles in the subsurface remain unclear.

The complete genome sequence of strain LB2T is a unique circular chromosome of 3.7 Mb long with a 36.2 mol% G+C content (Table 2), close to the G+C content measured by HPLC (36.4 mol%, Table 2). The G+C content of strain LB2T is higher than that of its closest Anaerobranca relatives (A. californiensis, 30.0-33.4 mol%; A. gottschalkii, 30.0-32.9 mol%; A. horikoshii, 32.0-34.0 mol%; A. zavarzinii, 32.5 mol%), and P. tanatarense (31.9-32.2 mol%) within the family Proteinivoraceae. The genome size of strain LB2T is much larger than that of its closest phylogenetic neighbor for which genomes are available, A. californiensis (2.0 Mb) and A. gottschalkii (2.3 Mb), but is similar to those of the mesophilic and alkaliphilic genus Alkaliphilus (>3 Mb). The ANI and dDDH values between LB2T and A. californiensis PAOHA-1T were 70.61% and 13.50%. The ANI and dDDH values between LB2T and A. gottschalkii LBS3T were 70.78% and 13.40% (Supplementary Table S1), i.e., far below the species boundaries of 95~96% and 70%, respectively, recommended by Chun *et al.* [55]. The LB2T strain genome comprises a single chromosome and 3,777 genes, including 3,626 protein-encoding genes and 94 RNA encoding genes. Amino acids, carbohydrates, and inorganic ion transport and metabolisms represented 7.4%, 5.7%, and 4.9% of the function-predicted genes. The distribution of genes into COGs functional categories is shown in Supplementary Table S2. In a phylogenomic analysis (Fig S3), strain LB2 clustered with other alkaliphilic Firmicutes.
246

It positions in a branch containing members of the genera *Alkalibacterium* (class *Bacilli*) and *Serpentinicella* (class *Clostridia*) distantly related to the *Anaerobranca* species.

247

248

The predominant cellular fatty acids (>5% of total fatty acids) were iso-C15:0 (15.3%), C16:0 (10.2%), C14:0 (8.0%), cyclo-C17:0 (6.9%), iso-C17:1 (8.8%), and C18:0 (6.1%). The proportion of iso-C15:0 and C16:0 was lower than that of *A. gottschalkii* and *P. tanatarense*, while the iso-C15:0 content was higher than that of *A. horikoshii* (Supplementary Table S3).

249

250

251

252

Table 1 summarizes the physiological and biochemical differences between strain LB2<sup>T</sup> and the type species of all genera of the family *Proteinivoraceae*, including *A. horikoshii* and *P. tanatarense*. Strain LB2<sup>T</sup> shares similar physiological and biochemical properties with this latter (e.g., proteinaceous substrate utilization and high-pH optimal growth ranging between 8.5 and 9.5) but differs by growing optimally higher pH (up to 10.8) compared to other *Proteinivoraceae* members. Unlike *Anaerobranca* and *Proteinivorax* genera, strain LB2<sup>T</sup> displays optimal growth in the absence of NaCl addition, similarly to another alkaliphilic anaerobe of the family *Clostridiaceae* *Serpentinicella alkaliphila* 3b<sup>T</sup>, isolated from the serpentinizing Prony Bay Hydrothermal Field of New Caledonia [20]. In contrast to the alkalithermophilic *Anaerobranca* spp., displaying optimal growth temperatures between 50-60°C, strain LB2<sup>T</sup> was mesophilic (the optimum temperature at 37°C) similarly to *P. tanatarense* and *P. hydrogeniformans* that grew optimally at 32-38°C and 30-32°C, respectively [44, 49]. Like other *Proteinivoraceae* members, strain LB2<sup>T</sup> utilizes proteinaceous substrates for growth (i.e., yeast extract). It can also use pyruvate as the sole carbon source, similarly to *A. californiensis* (or *A. zavarzinii* that co-metabolizes pyruvate with yeast extract). As reported for *A. zavarzinii*, both formate and acetate were the major end products detected. It suggests that pyruvate is mainly metabolized *via* pyruvate:formate lyase and not *via* the pyruvate:ferredoxin oxidoreductase as commonly reported in *Firmicutes*. In the latter case, acetate plus H<sub>2</sub> would be the major metabolic end-products. Genomic data supported this

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270
hypothesis. The gene encoding the formate lyase, also known as formate C-acetyltransferase (EC 2.3.1.54), is found in the genome of strain LB2T and not the gene of the pyruvate: ferredoxin oxidoreductase (PFOR, EC 1.2.7.1). The ability of strain LB2T to use both ferric citrate and thiosulfate as electron acceptors were also shared with *A. californiensis*. Sugars (i.e., xylose) was used by strain LB2T, similarly to *A. gottschalkii*, as well as *A. californiensis*, *A. zavarzinii*, *P. hydrogeniformans* and *P. tanatarense* ferment some sugars, but in the presence of tryptone or yeast extract.

In conclusion, the strain LB2T differs from other species of the closest genera *Anaerobranca* and *Proteinivorax* (belonging to the family *Proteinivoraceae*) by several features: (i) it forms a distinct branch among the family *Proteinivoraceae* in the 16S rRNA gene phylogenetic trees, (ii) it is only distantly related to *Anaerobranca* spp. in phylogenomic analyses, (iii) it displays a larger genome size and G+C content than other *Proteinivoraceae* members, (iv) it grows optimally at higher pH (up to 10.8) than other *Proteinivoraceae* members, (v) it grows optimally in the absence of NaCl addition, (vi) it has a much lower optimal growth temperature (37°C) than the closest *Anaerobranca* members, (vii) it is the only one in the family *Proteinivoraceae* (including pleomorphic bacteria) occurring as rods or cocci depending on the growth phase, as observed for the actinobacterial genus *Arthrobacter* [56]. In contrast, cells of the genus *Anaerobranca* are rod-shaped, sometimes showing branching [46, 48]. Based on morphological, phenotypic, phylogenetic, and genomic, strain LB2T is proposed to represent a novel species of the new genus *Alkalicella*, *A. caledoniensis* gen. nov., sp. nov.

Description of *Alkalicella* gen. nov.

*Alkalicella* (Al.ka.li.cel'la. N.L. n. *alkali* from Arabic *al-qaliy* ashes of saltwort; L. fem. n. *cella* a storeroom, chamber, and in biology a cell; N.L. fem. n. *Alkalicella*, a cell living in an alkaline
296 environment).

297 Gram-stain-positive (terminal and oval) endospore-forming bacterium with anaerobic
298 chemoorganotrophic metabolism. Occurs as rods or cocci under different conditions. Able to
299 utilize sugars and proteinaceous substrates. Use ferric citrate and thiosulfate as electron
300 acceptors. Alkaliphilic and mesophilic. Displays optimal growth in the absence of NaCl
301 addition. Belongs to the family Proteinivoraceae, order Clostridiales, class Clostridia within
302 the phylum Firmicutes. Most closely related to Anaerobranca gottschalkii LBS3T (93.2% 16S
303 rRNA gene sequence identity). The G+C content of the genomic DNA is 36.2%. The type
304 species is Alkalicella caledoniensis, and the genus description is based on that of the type
305 species, currently the only species ascribed to this genus.

306

307 Description of *Alkalicella caledoniensis* sp. nov.

308

309 *Alkalicella caledoniensis* (ca.le.do.ni.en'sis. N.L. fem. adj. caledoniensis pertaining to New
310 Caledonia, where strain LB2<sup>T</sup> was first isolated).

311 Displays the following characteristics in addition to those listed in the genus description. Cells
312 are approximately 6.0–10.0 µm long and 0.5–0.6 µm wide, occurring singly or in pairs. Cells
313 are motile and possess multiple flagella. They are pleiomorphic depending on the growth phases
314 (from rod to cocci). Colonies are white and circular and about 1–2 mm in diameter after seven
315 days of incubation at 30°C. Growth occurs at 25–50°C (optimum 37°C), at pH 8.2–10.8
316 (optimum pH 9.5) with 0–7% NaCl (optimum 0–1%). Added NaCl was not required for growth.
317 Pyruvate, yeast extract, xylose, and peptone were used as electron donors. Still, adenine,
318 adenosine 5'-diphosphate, albumin, arabinose, casamino-acids, casein, cellobiose, fructose,
319 galactose, gelatin, glucose, glycerol, guanine, lactate, lactose, mannose, ribose, starch, sucrose,
320 tryptone, uracil, and H<sub>2</sub>/CO<sub>2</sub> were not. End products detected from pyruvate fermentation were
acetate and formate. Both ferric citrate and thiosulfate were used as electron acceptors using pyruvate and reduced to ferrous iron and sulfide. Yeast extract was degraded into acetate, formate, H₂, and propionate. Elemental sulfur, nitrate, nitrite, fumarate, sulfate, sulfite, and DMSO were not used as terminal electron acceptors. The predominant fatty acids were iso-C15:0 (15.3%), C16:0 (10.2%), C14:0 (8.0%), cyclo-C17:0 (6.9%), iso-C17:1 (8.8%), and C18:0 (6.1%). The GenBank accession number for the 16S rRNA gene sequence of strain LB2T is KR349724, and that for the whole genome sequence is CP058559.

The type strain LB2T (=DSM 100588T = JCM 30958T) was isolated from a sediment slurry of 'La Crouen' thermal spring near Canala, New Caledonia.

## Conflicts of interest

The authors declare that there are no conflicts of interest.

## Funding Information

This project was financially supported by the French Research Institute for Development (IRD), the INTERVIE CNRS INSU program, the H2NAT project funded by the Midi-Pyrénées Observatory (OMP, Toulouse) and ANR MICROPRONY (N°19-CE02-0020-02). We thank Céline Roques at the GeT core facility, Toulouse, France (http://get.genotoul.fr) for the genome sequencing of LB2 strain, supported by France génomique. The LABGeM (CEA/Genoscope & CNRS UMR8030) is acknowledged for support within the MicroScope annotation platform.
344 **Figures legends**

345

346 **Fig 1:** Phase-contrast micrographs of cells of strain LB2<sup>T</sup> with terminal and oval endospore

347 formation (A; bar 10 µm), and transmission electron micrograph showing cells of strain LB2<sup>T</sup>

348 with flagella (B; bar 1 µm).

349

350 **Fig. 2:** Maximum-likelihood (ML) phylogenetic tree based on 1245 aligned bp of 16S rRNA

351 gene sequences showing the position of strain LB2<sup>T</sup> among the other family members

352 *Proteinivoraceae* within the order *Clostridiales*. *Clostridium butyricum* (M59085) was used as

353 an outgroup (not shown). Bootstrap values (based on 1000 replicates) are shown at branch

354 nodes. Accession numbers are indicated in parentheses. Bar: 0.02 substitutions per 100

355 nucleotides.

356
357 References

358

359

360

361

1. Preiss L, Hicks DB, Suzuki S, Meier T, Krulwich TA. Alkaliphilic bacteria with impact on industrial applications, concepts of early life forms, and bioenergetics of ATP synthesis. *Front Bioeng Biotech* 2015;3:75.

362

363

2. Sarethy IP, Saxena Y, Kapoor A, Sharma M, Sharma SK *et al*. Alkaliphilic bacteria: applications in industrial biotechnology. *J Ind Microbiol Biotechnol* 2011;38(7),769.

364

365

3. Horikoshi K. Alkaliphiles: some applications of their products for biotechnology. *Microbiol Mol Biol Rev* 1999;63(4):735-750.

366

367

4. Kevbrin V. Isolation and cultivation of alkaliphiles. *Adv Biochem Eng Biotechnol* 2020;172:53-84.

368

369

5. Wiegel J. Anaerobic alkaliphiles and alkaliphilic poly-extremophiles. *Extremophiles Handbook* 2011;81-97.

370

371

372

6. Cox M E, Launay J, Paris JP. Geochemistry of low temperature geothermal systems in New Caledonia. In *Pacific Geothermal conference and 4th NZ Geothermal Workshop* 1982;453-459.

373

374

375

376

7. Maurizot P, Sevin B, Lesimple S, Collot J, Jeanpert J *et al*. Chapter 9: Mineral resources and prospectivity of non-ultramafic rocks of New-Caledonia. In: Mortimer, N. (ed) *New Caledonia: Geology, Geodynamic Evolution and Mineral Resources*. *Geological Society, London, Memoirs* 2020;51:215-245.

377

378

379

8. Deville E, Prinzhofer A. The origin of N₂-H₂-CH₄-rich natural gas seepages in ophiolitic context: a major and noble gases study of fluid seepages in New Caledonia. *Chemical Geol* 2016;440:139-147.
380

9. Monnin C, Chavagnac V, Boulart C, Ménez B, Gérard M *et al.* Fluid chemistry of the low temperature hyperalkaline hydrothermal system of the Prony Bay (New Caledonia). *Biogeosciences* 2014;11:5687–5706.

381

382

383

10. Pinti DL. S Serpentinization. *Proc Natl Acad Sci* 2015;101:12818–12823.

384

11. Quéméneur M, Bes M, Postec A, Mei N, Hamelin J *et al.* Spatial distribution of microbial communities in the shallow submarine alkaline hydrothermal field of the Prony Bay, New Caledonia. *Environ Microbiol Rep* 2014;6:665–674.

385

386

387

12. Postec A, Quéméneur M, Bes M, Mei N, Aissa FB *et al.* Microbial diversity in a submarine carbonate edifice from the serpentinizing hydrothermal system of the Prony Bay (New Caledonia) over a 6-year period. *Front Microbiol* 2015;6:857.

388

389

390

13. Pisapia C, Gérard E, Gérard M, Lecourt L, Pelletier B *et al.* Mineralizing filamentous bacteria from the Prony Bay Hydrothermal Field give new insights into the functioning of serpentinization-based subseafloor ecosystems. *Front Microbiol* 2017;8:57.

391

392

393

14. Frouin E, Bes M, Ollivier B, Quéméneur M, Postec A *et al.* Diversity of rare and abundant prokaryotic phylotypes in the Prony Hydrothermal Field and comparison with other serpentinite-hosted ecosystems. *Front Microbiol* 2018;9:102.

394

395

396

15. Mei N, Postec A, Monnin C, Pelletier B, Payri CE *et al.* Metagenomic and PCR-based diversity surveys of [FeFe]-hydrogenases combined with isolation of alkaliphilic hydrogen-producing bacteria from the serpentinite-hosted Prony Hydrothermal Field, New Caledonia. *Front Microbiol* 2016a;7:1301.

397

398

399

400

16. Ben Aissa F, Postec A, Erauso G, Payri C, Pelletier B *et al.* *Vallitalea pronyensis* sp. nov., isolated from a marine alkaline hydrothermal chimney. *Int J Syst Evol Microbiol* 2014;64:1160–1165.

401

402

403

17. Ben Aissa F, Postec A., Erauso G., Payri C, Pelletier B *et al.* Characterization of *Alkaliphilus hydrothermalis* sp. nov., a novel alkaliphilic anaerobic bacterium, isolated

404
405

from a carbonaceous chimney of the Prony hydrothermal field, New
Caledonia. *Extremophiles* 2015;19:183–188.

406

407

18. Bes M, Merrouch M, Joseph M, Quéméneur M, Payri C et al. *Acetoanaerobium pronyense* sp. nov., an anaerobic mesophilic bacterium isolated from the Prony alkaline hydrothermal Field, New Caledonia. *Int J Syst Evol Microbiol* 2015;65:2574–2580.

408

409

410

19. Mei N, Zergane N, Postec A, Erauso G, Ollier A et al. Fermentative hydrogen production by a new alkaliphilic *Clostridium* sp. (strain PROH2) isolated from a shallow submarine hydrothermal chimney in Prony Bay, New Caledonia. *Int J Hydrogen Energ* 2014;39:19465–19473.

411

412

413

414

20. Mei N, Postec A, Erauso G, Joseph M, Pelletier B et al. *Serpentinicella alkaliphila* gen. nov., sp. nov., a novel alkaliphilic anaerobic bacterium isolated from the serpentinite-hosted Prony hydrothermal field, New Caledonia. *Int J Syst Evol Microbiol* 2016b;66(11):4464–4470.

415

416

417

418

21. Postec A, Quéméneur M, Lecoeuvre A, Chabert N, Joseph M et al. *Alkaliphilus serpentinus* sp. nov. and *Alkaliphilus pronyensis* sp. nov., two novel anaerobic alkaliphilic species isolated from the serpentinite-hosted Prony Bay Hydrothermal Field (New Caledonia). *Syst Appl Microbiol* 2021;44:126175.

419

420

421

422

22. Hungate RE. A roll tube method for cultivation of strict anaerobes, In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, Academic Press, London. 1969;3b:117–132.

423

424

425

23. Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe RS. Methanogens: reevaluation of a unique biological group. *Microbiol Rev* 1979;43:260–296.

426

427

24. Cord-Ruwisch R. A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfate-reducing bacteria. *J Microbiol Methods* 1985;4(1):33-36.

428
429

25. Quéméneur M, Erauso G, Frouin E, Zeghal E, Vandecasteele C *et al*. Hydrostatic pressure helps to cultivate an original anaerobic bacterium from the Atlantis Massif subseafloor (IODP Expedition 357): *Petrocella atlantisensis* gen. nov. sp. nov. *Front Microbiol* 2019;10:1497.

430

431

432

433

26. Kuykendall LD, Roy MA, O'Neill JJ, Devine TE. Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of *Bradyrhizobium japonicum*. *Int J Syst Bacteriol* 1988;38:358-361.

434

435

436

27. Sasser M. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. Newark, DE:MIDI Inc. 1990

437

438

28. Quéméneur M, Palvadeau A, Postec A, Monnin C, Chavagnac V *et al*. Endolithic microbial communities in carbonate precipitates from serpentinite-hosted hyperalkaline springs of the Voltri massif (Ligurian Alps, Northern Italy). *Environ Sci Pollut Res* 2015;22:13613-24.

439

440

441

442

29. Lane DJ. "Nucleic acid techniques in bacterial systematics," in *Nucleic Acid Techniques in Bacterial Systematics*, eds E. Stackebrandt and M. Goodfellow (Hoboken, NJ: Wiley) 1991;115-175.

443

444

445

30. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. *Nucleic Acids Res* 2004;32(5):1792-1797.

446

447

31. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. *Mol Biol Evol* 2016;33(7):1870-1874.

448

449

32. Felsenstein J. Evolutionary trees from DNA sequences: a maximum likelihood approach. *J Mol Evol* 1981;17:368-376.

450

451

33. Fitch WM. Toward defining the course of evolution: minimum change for a specified tree topology. *Syst Zool* 1971;20:406-416.

452
453

34. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. *Molec Biol Evol* 1987;4:406-425.

454

455

35. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. *PLoS Comput Biol* 2017;13:e1005595.

456

457

458

36. Vallenet D, Belda E, Calteau A, Cruveiller S, Engelen S et al. MicroScope - an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data. *Nucleic Acids Res* 2013;41:636-647.

459

460

461

37. Chen IMA, Chu K, Palaniappan K, Pillay M, Ratner A et al. IMG/M v. 5.0: an integrated data management and comparative analysis system for microbial genomes and microbiomes. *Nucleic Acids Res* 2019;47(D1):D666-D677.

462

463

464

38. Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. *BMC Bioinform* 2013;14:60.

465

466

467

39. Meier-Kolthoff JP, Göker M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. *Nat Commun* 2019;10:2182.

468

469

40. Ondov BD, Treangen TJ, Melsted P, Mallonee AB, Bergman NH et al. Mash: Fast genome and metagenome distance estimation using MinHash. *Genome Biol* 2016;17:1-14.

470

471

472

41. Lefort V, Desper R, Gascuel O. FastME 2.0: A comprehensive, accurate, and fast distance-based phylogeny inference program. *Mol Biol Evol* 2015;32:2798-2800.

473

474

42. Farris JS. Estimating phylogenetic trees from distance matrices. *Am Nat* 1972;106:645-667.

475
476

43. Mesbah M, Premachandran U, Whitman W. Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. *Int J Syst Bacteriol* 1989;39:159-167.

477

478

44. Kevbrin V, Boltyanskaya Y, Zhilina T, Kolganova T, Lavrentjeva E et al. *Proteinivorax tanatarense* gen. nov., sp. nov., an anaerobic, haloalkaliphilic, proteolytic bacterium isolated from a decaying algal bloom, and proposal of *Proteinivoraceae* fam. nov. *Extremophiles* 2013;17(5):747-756.

479

480

481

482

45. Prowe SG, Antranikian G. *Anaerobranca gottschalkii* sp. nov., a novel thermoalkaliphilic bacterium that grows anaerobically at high pH and temperature. *Int J Syst Evol Microbiol* 2001;51(2):457-465.

483

484

485

46. Engle M, Li Y, Woese C, Wiegel J. Isolation and characterization of a novel alkalitolerant thermophile, *Anaerobranca horikoshii* gen. nov., sp. nov. *Int J Syst Evol Microbiol* 1995;45(3):454-461.

486

487

488

47. Gorlenko V, Tsapin A, Namsaraev Z, Teal T, Tourova T et al. *Anaerobranca californiensis* sp. nov., an anaerobic, alkalithermophilic, fermentative bacterium isolated from a hot spring on Mono Lake. *Int J Syst Evol Microbiol* 2004;54(3):739-743.

489

490

491

492

48. Kevbrin V, Boltyanskaya Y, Garnova E, Wiegel J. *Anaerobranca zavarzinii* sp. nov., an anaerobic, alkalithermophilic bacterium isolated from Kamchatka thermal fields. *Int J Syst Evol Microbiol* 2008;58(6):1486-1491.

493

494

495

49. Boltyanskaya Y., Detkova E, Pimenov N, Kevbrin V. *Proteinivorax hydrogeniformans* sp. nov., an anaerobic, haloalkaliphilic bacterium fermenting proteinaceous compounds with high hydrogen production. *Antonie van Leeuwenhoek* 2018;111(2):275-284.

496

497

498

50. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. "Basic local alignment search tool." *J Mol Biol* 1990;215:403-410.

499

500
501 51. Zhang P, Chen Y, Zhou Q, Zheng X, Zhu X *et al*. Understanding short-chain fatty acids accumulation enhanced in waste activated sludge alkaline fermentation: kinetics and microbiology. *Environ Sci Technol* 2010;44(24):9343-9348.

504 52. Antony CP, Shimpi GG., Cockell CS, Patole MS, Shouche, Molecular characterization of prokaryotic communities associated with lonar crater basalts. *Geomicrobiol J* 2014;31(6):519-528.

507 53. Rempfert KR, Miller HM, Bompard N, Nothaft D, Matter JM *et al*. Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman. *Front Microbiol* 2017;8, 56.

510 54. Purkamo L, Bomberg M, Kietäväinen R, Salavirta H, Nyyssönen M *et al*. Microbial co-occurrence patterns in deep Precambrian bedrock fracture fluids. *Biogeosciences* 2016;13(10):3091-3108.

513 55. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR *et al*. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. *Int J Syst Evol Microbiol* 2018;68:461-466.

516 56. Luscombe BM, Gray TRG. Characteristics of *Arthrobacter* grown in continuous culture. *Microbiology* 1974;82(2):213-222.
<table><caption>**Table 1.** Differential characteristics between strain LB2T (obtained in this study) and data obtained from previous studies for all type species of all genera of the family *Proteinivoraceae*. Strains: 1, Strain LB2T; 2, *Anaerobranca horikoshii* JWIYL-13S<sup>T</sup> [46]; 3, *Anaerobranca gottschalkii* LBS3T [45]; 4, *Anaerobranca californiensis* PAOHA-1T [47]; 5, *Anaerobranca zavarzinii* JW/VK-KS5YT [48]; 6, *Proteinivorax tanatarense* Z-910T [44]; 7, *Proteinivorax hydrogeniformans* Z-710T [49].</caption><thead><tr><th>Characteristic</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th></tr></thead><tbody><tr><td>Origin</td><td>La Crouen spring, New Caledonia</td><td>Yellowstone National Park, USA</td><td>Lake Bogoria, Kenya</td><td>Mono Lake, USA</td><td>Kamchatka, Russia</td><td>Lake Tanatar VI, Russia</td><td>Lake Tanatar III, Russia</td></tr><tr><td>Cell size (µm)</td><td>0.5–0.6 x 6–10</td><td>0.5–0.6 x 11–20</td><td>0.3–0.5 x 3–5</td><td>0.26–0.31 x 2.4–5.0</td><td>0.36–0.45 x 3.6–7.7</td><td>0.5 x 1.6–4</td><td>0.5–0.6 x 2.0–4.5</td></tr><tr><td>Spore formation</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td></tr><tr><td>Motility</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+/-</td><td>+</td></tr><tr><td>Temperature for growth (°C)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Range</td><td>25–50</td><td>34–64</td><td>30–65</td><td>45–70</td><td>34–64</td><td>16–52</td><td>12–45</td></tr><tr><td>Optimum</td><td>37</td><td>57</td><td>50–55</td><td>58</td><td>54–60</td><td>32–38</td><td>30–32</td></tr><tr><td>pH for growth</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Range</td><td>8.2–10.8*</td><td>6.7–10.3</td><td>6.0–10.5</td><td>8.6–10.4</td><td>7.7–9.9</td><td>7.6–10.5</td><td>7.7–10.4</td></tr><tr><td>Optimum</td><td>9.5</td><td>8.5</td><td>9.5</td><td>9.0–9.5</td><td>8.5–9.0</td><td>8.8</td><td>8.3–8.9</td></tr><tr><td>NaCl for growth (%, w/v)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Range</td><td>0–7</td><td>ND</td><td>0–4</td><td>0–6</td><td>ND</td><td>0.4–3.6</td><td>0.7–3.0</td></tr><tr><td>Optimum</td><td>0*–1</td><td>ND</td><td>1</td><td>1–2.5</td><td>ND</td><td>2–3</td><td>1.0–1.4</td></tr><tr><td>DNA G+C content (mol%)<sup>1</sup></td><td>36.2<sup>1+</sup></td><td>34<sup>2</sup></td><td>30.9<sup>2</sup></td><td>30<sup>2</sup></td><td>32.5<sup>2</sup></td><td>32.2<sup>2</sup></td><td>33.9<sup>2</sup></td></tr><tr><td>Substrates</td><td>Peptone, pyruvate, xylose, yeast extract</td><td>Peptone, tryptone, yeast extract</td><td>Peptone, tryptone, yeast extract, mono- and polysaccharides</td><td>Peptone, soytone, malt extract, Casamino acids</td><td>Soytone and yeast extract</td><td>Albumine, casein, gelatin, peptone, soytone, tryptone, yeast extract</td><td>Peptone, soytone, tryptone, yeast extract</td></tr><tr><td>Fermentation products</td><td>From yeast extract: acetate and propionate; from pyruvate: acetate and formate</td><td>From yeast extract: acetate, H<sub>2</sub>, CO<sub>2</sub></td><td>From glucose or starch: Acetate, ethanol (traces)</td><td>ND</td><td>From YE: Formate, acetate, H<sub>2</sub> (major), propionate (minor)</td><td>From tryptone: acetate, formate, NH<sub>4</sub>, H<sub>2</sub>, propionate, succinate, n-butyrate, iso-butyrate, 2-methylbutyrate, iso-valerate</td><td>From tryptone: acetate, hydrogen, ammonium (major), formate, propionate, n-butyrate, iso-butyrate, iso-valerate and succinate (minor).</td></tr><tr><td>Electron acceptors</td><td>Ferric citrate, thiosulfate</td><td>Fumarate</td><td>Thiosulfate</td><td>Sulfur, polysulfide, thiosulfate, ferric citrate</td><td>Fumarate, thiosulfate, sulfur</td><td>Fumarate, DMSO, thiosulfate, sulfur</td><td>Fumarate, thiosulfate, sulfur</td></tr></tbody></table>

+, Positive; -, negative; ND, not data available.

¹ G+C content determined from genomic data.

² G+C content determined by using HPLC.

* Characteristics distinguishing the strain LB2T from others presented.
526 **Table 2.** Genome statistics of strain LB2<sup>T</sup>, *Anaerobranca californiensis* PAOHA-1<sup>T</sup> [47], and

527 *Anaerobranca gottschalkii* LBS3<sup>T</sup> [45].

<table><thead><tr><th>Genome Name /<br>Sample Name</th><th>LB2<sup>T</sup></th><th>Anaerobranca<br>californiensis<br>PAOHA-1<sup>T</sup></th><th>Anaerobranca<br>gottschalkii LBS3<sup>T</sup></th></tr></thead><tbody><tr><td>GenBank accession<br>number</td><td>CP058559</td><td>NZ_FRAI00000000</td><td>NZ_FOIF01000000</td></tr><tr><td>IMG Genome ID</td><td>2888992949</td><td>2585428191</td><td>2602042032</td></tr><tr><td>Genome Size (bp)</td><td>3,695,371</td><td>2,034,462</td><td>2,300,172</td></tr><tr><td>Contig count</td><td>NA</td><td>51</td><td>115</td></tr><tr><td>Scaffold count</td><td>1</td><td>NA</td><td>110</td></tr><tr><td>GC (%)</td><td>36.2</td><td>33.4</td><td>32.9</td></tr><tr><td>Gene Count</td><td>3,777</td><td>2,132</td><td>2,382</td></tr><tr><td>Protein count</td><td>3,626</td><td>2,006</td><td>2,246</td></tr><tr><td>rRNA Count</td><td>27</td><td>22</td><td>16</td></tr><tr><td>tRNA Count</td><td>62</td><td>52</td><td>55</td></tr><tr><td>Other RNA</td><td>5</td><td>5</td><td>4</td></tr></tbody></table>

NA, data not applicable.

528

529