Crassaminicella indica sp. nov., a novel thermophilic
anaerobic bacterium isolated from a deep-sea
hydrothermal vent

Ze-Xi Jiao, Xue-Gong Li, Huan-Huan Zhang, Jun Xu, Shi-Jie Bai, Jie Dai, Jin
Lin, Wei-Jia Zhang, Xiao-Qing Qi, Long-Fei Wu

► To cite this version:

Ze-Xi Jiao, Xue-Gong Li, Huan-Huan Zhang, Jun Xu, Shi-Jie Bai, et al.. Crassaminicella indica sp. nov., a novel thermophilic anaerobic bacterium isolated from a deep-sea hydrothermal vent. International Journal of Systematic and Evolutionary Microbiology, 2023, 73 (3), 10.1099/ijsem.0.005725 . hal-04131915

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*Crassaminicella indica* sp. nov., a novel thermophilic
anaerobic bacterium isolated from a deep-sea
hydrothermal vent

5 Ze-Xi Jiao¹,², Xue-Gong Li¹,³,⁴,⁵*, Huan-Huan Zhang⁶; Jun Xu⁷; Shi-Jie Bai⁴,⁸, Jie Dai⁹, Jin Lin¹,², Wei-Jia

6 Zhang¹,³,⁴,⁵, Xiao-Qing Qi¹,³,⁴, Long-Fei Wu³,⁹

7 ¹Laboratory of Deep-Sea Microbial Cell Biology, Institute of Deep-sea Science and Engineering,

8 Chinese Academy of Sciences, Sanya, China

9 ²University of Chinese Academy of Sciences, Beijing, China

10 ³International Associated Laboratory of Evolution and Development of Magnetotactic Multicellular

11 Organisms, CNRS-Marseille/ CAS- Sanya

12 ⁴CAS Key Laboratory for Experimental Study under Deep-sea Extreme Conditions, Institute of Deep-

13 sea Science and Engineering, Chinese Academy of Sciences, Sanya, China

14 ⁵Institution of Deep-sea Life Sciences, IDSSE-BGI, Hainan Deep-sea Technology Laboratory, Sanya,

15 Hainan, China

16 ⁶School of Oceanography, Shanghai Jiao Tong University, Shanghai, China

17 ⁷State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology,

18 Shanghai Jiao Tong University, Shanghai, China

19 ⁸Laboratory of Marine Viruses and Molecular Biology, Institute of Deep-sea Science and

20 Engineering, Chinese Academy of Sciences, Sanya, China

21 ⁹Aix Marseille Univ, CNRS, LCB, IMM, IM2B, Marseille, France

22 Correspondence: Xue-Gong Li: xuegongli@idsse.ac.cn

23 **Keywords: *Crassaminicella indica*; thermophile; anaerobe; Central Indian Ridge**

24 New tara: *Bacillota* and related organisms

25 Running title: *Crassaminicella indica* sp. nov.

26 The GenBank/EMBL/DDBJ accession number for the 16S rRNA sequence of strain 143-21T is

27 OP271781. The complete genome sequence of 143-21T has been deposited at GenBank/ENA/DDBJ

28 under the accession number CP078093.
30

ABSTRACT

A novel moderately thermophilic heterotrophic bacterium, designated strain 143-21T, was isolated
from a deep-sea hydrothermal chimney sample collected on the Central Indian Ridge at a depth of
2,440 m. Phylogenetic analysis indicated that strain 143-21T belongs to the genus *Crassaminicella*. It
was most closely related to *Crassaminicella thermophila* SY095T (96.79 % 16S rRNA gene sequence
similarity) and *Crassaminicella profunda* Ra1766HT (96.52 %). Genomic analysis showed that strain
143-21T share 79.79-84.45 % average nucleotide identity (ANI) and 23.50-29.20 % digital DNA-
DNA hybridization (dDDH) with the species of the genus *Crassaminicella*, respectively. Cells were
rod-shaped, non-motile, Gram-positive-staining. Terminal endospores were observed in stationary-
phase cells when strain 143-21T was grown on TRM medium. Strain 143-21T was able to grow at 30-
60 °C (optimum 50 °C), pH6.5-8.5 (optimum 7.0), and in NaCl concentration of 1.0-7.0 % (w/v)
/optimum 2.0 %, w/v). Strain 143-21T can utilize fructose, glucose, maltose, mannose, ribose, N-
acetyl-D-(+)-glucosamine and casamino acids, as well as amino acids including glutamate, lysine,
histidine, and cysteine. The main fermentation products from glucose were acetate (2.07 mM), H₂ and
CO₂. It did not reduce elemental sulfur, sulfate, thiosulfate, sulfite, fumarate, nitrate, nitrite and Fe
(III). The predominant cellular fatty acids were C₁₄:₀ (48.8 %), C₁₆:₀ (12.9 %), and summed Feature 3
contained C₁₆:₁ ω7c and/or C₁₆:₁ ω6c (10.2 %). The major polar lipids were diphosphatidylglycerol,
phosphatidylethanolamine, and phosphatidylglycerol, as well as two unidentified phospholipids, and
four unidentified aminolipid. No respiratory quinones were detected. Based on the phylogenetic
analysis and physiological characteristics, strain 143-21T is considered to represent a novel species of
the genus *Crassaminicella*, for which the name *Crassaminicella indica* sp. nov. is proposed. The type
strain is strain 143-21T (=DSM 114408T = MCCC 1K06400T).

52

Deep-sea hydrothermal vent represent a typical chemosynthetic ecosystem, which are fueled by
geothermal chemical energy (1, 2). The chemical-rich hydrothermal fluid erupted from the vent and
rapidly mixed with surrounding cold seawater, resulting in steep thermal and chemical gradients.
Chemoautotrophic microorganisms can exploit these chemical disequilibria to produce energy for
carbon fixation and support the vent food chain (3). Vent ecosystem also contains a high fraction of
heterotrophic microorganisms, such as *Alkaliphilus pronyensis* (4), *Caminicella sporogenes* (5),
*Clostridium tepidiprofundi* (6), *Paramaledivibacter caminithermalis* (7), *Serpentinicella alkaliphila*,
and *Wukongibacter baidiensis* (7). Interestingly, microbial communities inhabiting deep-sea
hydrothermal vent varies with the chimney from the cradle to the grave, heterotrophic
microorganisms would become dominant after the hydrothermal activity has ceased (8). In addition,
numerous heterotrophic groups have been isolated from hydrothermal sediments and exhibited
hydrolysis activity of hexadecane or polycyclic aromatic hydrocarbons (9). These versatile
heterotrophic groups play a vital role in the deep-sea biogeochemical cycles.

Species in the genus *Crassaminicella*, originated in the deep-sea environment, are heterotrophic and
was first proposed by R. Lakhal et al (10), which can ferment carbohydrates and organic acids. The
genus *Crassaminicella* belongs to the family *Clostridiaceae* of the order *Eubacteriales* of the class
*Clostridia* in the phylum *Bacillota*. At the time of writing, this genus includes only two valid
published species (https://lpsn.dsmz.de/genus/crassaminicella), *Crassaminicella profunda* Ra1766HT
(10) isolated from the sediment of Guaymas Basin at a depth of 2,002 m, and *Crassaminicella
thermophila* SY095T (11) isolated from the chimney sample of the Southwest Indian Ridge at a depth
of 2,730 m. Genome analysis has revealed their diverse metabolic characteristics and adaptive
strategies in the deep-sea environment (12, 13). Here, we report the isolation and characterization of a
novel species of the genus *Crassaminicella*, strain 143-21T, originating from a deep-sea hydrothermal
76 vent in the Center Indian Ridge (CIR). The phenotypic and physiological features of novel species

77 will expand our knowledge about the genus *Crassaminicella*.

78

79

## Isolation and Ecology

80 During the TS10-3 cruise of the research vessel 'Tan Suo Yi Hao' in February 2019, the active

81 chimney fragments were collected at a depth of 2,440 m from the *Kali* hydrothermal field (25.32°S,

82 70.04°E) of CIR, by using the manned submersible 'Shen Hai Yong Shi'. The chimney surface was

83 covered by blind shrimps, few sea anemones attached to the rock near the root of the chimney. The

84 highest temperature of the hot vent fluid was 373 °C, which measured by a K-type thermocouple

85 probe inserted into the vent opening. The chimney fragments were obtained by the robotic arm of

86 manned submersible and placed in a closed biobox filled with sterile seawater. Once onboard, the

87 fragment was crushed under sterile conditions, and then stored anaerobically at 4 °C until culturing

88 attempts.

89 The stored chimney debris (about 0.5 g) were transferred into Hungate tubes containing 10 ml of

90 anaerobic Thermococcales Rich Medium (TRM) with N₂ as the headspace gas, and then incubated at

91 50 °C without shaking. After incubation over one week, the medium becomes cloudy indicating the

92 growing of microorganisms. To obtain pure cultures, the enrichment culture was spread anaerobically

93 on plates of TRM solidified with agar (2 %, w/v), and grow at 50 °C for one week. One colony was

94 picked for repeated plate streaking, the purity of the isolate was confirmed by microscopic

95 observations and by repeated sequencing of the 16S rRNA gene. The purified strain was designated

96 143-21T and stored at -80 °C in TRM supplemented with 5 % (v/v) DMSO for long-term

97 preservation. The strain 143-21T can also grow well in the JCM 909 medium composed of NaCl,

98 NH₄Cl, KH₂PO₄, K₂HPO₄, L-cysteine-HCl, MgCl₂·6H₂O, NaHCO₃, yeast extract and glucose etc. The

99 anaerobic JCM 909 medium was prepared as described by Li et al. (11). Further investigations were

100 carried out using JCM 909 medium unless otherwise stated.

101

102

## 16S RNA phylogeny

103 The complete sequence of the 16S rRNA gene (1,527 bp) of strain 143-21T was retrieved from the

104 genomic sequence, and was then compared with available sequences in the NCBI GenBank

105 (nucleotide collection nr/nt database). 16S rRNA gene sequence analysis indicated that strain 143-21T

106 belongs to the family *Clostridiaceae* of the phylum *Bacillota*. The closest phylogenetic relative is

107 *Crassaminicella thermophila* SY095T (96.79 %), and *Crassaminicella profunda* Ra1766H<sup>T</sup> (96.52 %),

108 followed by *Inediibacterium massiliense* Mt12<sup>T</sup> (94.63 %), *Anaerophilus nitritogenes* MJB2<sup>T</sup>

109 (94.45 %), *Geosporobacter ferrireducens* IRF9<sup>T</sup> (94.00 %), *Marinisporobacter balticus* 59.4M<sup>T</sup>

110 (93.58 %), *Wukongibacter baidiensis* DY30321<sup>T</sup> (92.73 %), *Anaerosolibacter carboniphilus* IRF19<sup>T</sup>

111 (92.29 %), *Salimesophilobacter vulgaris* Zn2<sup>T</sup> (91.54 %). All the 16S rRNA gene sequences of the

112 closely related strains obtained from the GenBank database were aligned using sina aligner

113 (https://www.arb-silva.de/aligner/), phylogenetic analysis was performed by using the software

114 MEGA (version 11). A neighbor-joining phylogenetic tree of representative members was constructed

115 based on the 16S rRNA gene sequences. Bootstrap analysis was performed with 1000 replications.

116 Phylogenetic relationships showed that strain 143-21T belonged to the genus *Crassaminicella* and

117 formed a clade with *C. thermophila* SY095T and *C. profunda* Ra1766H<sup>T</sup> (Fig. 1). The phylogenetic
118

topology of these *Crassaminicella* species was also confirmed using the minimum-evolution (Fig.

119

S1A) and maximum-likelihood methods (Fig. S1B).

120

121

## Genome Features

122

For genomic DNA extraction, strain 143-21T was grown on the JCM 909 medium at 50 °C. Cells

123

were harvested in late exponential-phase by centrifugation (4500 g, 20 min). Genomic DNA was

124

extracted using a MagAttract DNA Kit (Qiagen, USA) according to the manufacturer's instructions.

125

The whole-genome sequencing was carried out by Nextomics Biosciences Co., Ltd. (Wuhan, China)

126

by using the PromethION and MGI-SEQ 2000 systems. More than 2.5 Gb of clean data were

127

generated for the genome assembly. Briefly, strain 143-21T genome contains a single circular

128

chromosome comprising 2,756,133 nucleotides with 31.10 % G+C content. A total of 2,427 protein-

129

coding sequences (CDS), 121 tRNA genes, and 33 rRNA genes including 5S, 16S, and 23S rRNAs

130

each containing 11 gene copies were identified (13). These 16S rRNA gene sequences show 99.94 %

131

similarity. The genome sequence of strain 143-21T was deposited at DDBJ/ENA/GenBank with an

132

accession number of CP078093. To date, three *Crassaminicella* genomes have been reported. Among

133

them, strain 143-21T and *C. thermophila* SY095T are moderate thermophiles with optimal growth of

134

50 °C, while *C. profunda* Ra1766H^T is mesophilic with optimal growth of 30 °C. Comparative

135

genome analysis showed that the abundance of COG category in strain 143-21T and *C. thermophila*

136

SY095T, such as Nucleotide transport and metabolism (F), Coenzyme transport and metabolism (H),

137

Translation, ribosomal structure and biogenesis (J), and Mobilome: prophages, transposons (X), are

138

higher than the *C. profunda* Ra1766H^T. These differences may reflect the genome characteristics of

139

moderate thermophile, which will help them to survive in the deep-sea hydrothermal vent.

140

The phylogenetic tree based on the 120 concatenated bacterial marker proteins was constructed (14),

141

and the topology indicated that strain 143-21T formed a distinct branch with *C. profunda* Ra1766H^T

142

and *C. thermophila* SY095T (Fig. S2). Average nucleotide identity (ANI) was calculated by online

143

analysis sites EZBioCloud (https://www.ezbiocloud.net/tools/ani) (15). The DNA-DNA hybridization (dDDH) between two genomes was calculated using GGDC 2.0 (http://ggdc.dsmz.de/ggdc.php#) (16).

145

The average amino acid identity (AAI) between strain 143-21T and its closest phylogenetic relative

146

was determined using MiGA (17). Strain 143-21T show 84.45 % and 79.79 % ANI value with *C.*

147

*profunda* Ra1766H^T and *C. thermophila* SY095^T respectively (Table S1), which are significantly

148

lower than the proposed threshold values of 95–96 % (18). The dDDH values between the genomes of

149

strain 143-21T and two reference genomes were significantly lower than 70 % cutoff value previously

150

proposed for species delimitation (19), which are 29.20 % and 23.50 % for *C. profunda* Ra1766H^T

151

and *C. thermophila* SY095T, respectively (Table S1). In addition, the AAI values between strain 143-

152

21T and the related species (81.50 % and 66.78 % for *C. profunda* Ra1766H^T and *C. thermophila*

153

SY095T, respectively) are also in the range of standard criteria (65 %<AAI < 95 %) for different

154

species of the same genus (20). Taken together, these results confirmed that strain 143-21T is a

155

distinct species of the genus *Crassaminicella*.

156

157

## Physiology and Chemotaxonomy

158

Strain 143-21T was grown on the JCM 909 medium at 50 °C. The morphology of cells from late

159

exponential-phase was observed by phase-contrast microscopy (BX53 with a DP80 digital camera;

160

Olympus, Tokyo, Japan) and transmission electron microscopy (JEM-1230; JEOL Ltd., Tokyo,
Japan). Cells were straight rod-shaped, with a length of 7.1 ± 1.5 µm and a width of 1.1 ± 0.3 µm. The rods were non-motile without flagella (Fig. 2). Cells were stained using Gram Stain Kit (Beijing Solarbio Science and Technology) and the results showed that cells were Gram-stain-positive. Terminal endospores were observed in stationary-phase cells when the strain 143-21T was grown on TRM medium.

Physiological characterization of strain 143-21T was carried out in the JCM 909 medium. 8 ml anaerobic JCM 909 medium was dispensed into the serum bottle with a volume of 11 ml with N₂ as the headspace gas. The prepared medium was inoculated with cells from late exponential-phase and the final cell number is approximately 10⁶ cells ml⁻¹. All experiments were carried out anaerobically and all conditions were tested in triplicate unless stated otherwise. Growth was monitored by measuring optical density at 600 nm as described by Dai et al. (21). Growth rates were calculated as described by Li et al. (11) and shown in Fig. S3. The temperature range for growth was determined at 20–75 °C with 5 °C intervals. Strain 143-21T grew over the range from 25 to 60 °C, the optimal growth temperature is 50 °C. No growth was observed at 20 °C and 65 °C after 48 h of incubation. The pH range for growth was tested at 50 °C from 5.0–9.0 at intervals of 0.5 pH units. The different pH values were adjusted at room temperature (about 28 °C) with 5 M HCl or 5 M NaOH. For different pH ranges, the JCM 909 medium was modified with following buffers (each at a concentration of 10 mM): MES [2-(*N*-morpholino) ethanesulfonic acid] for pH 5.0–6.0; PIPES (piperazine-*N*, *N*⁹-bis-2-ethane sulfonic acid) for pH 6.5–7.0; HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] for pH 7.5–8.5; and no buffer for pH 9.0. After autoclave, media with various pH values were adjusted to required values with sterilized stock solutions of 5 M HCl or 5 M NaOH. The pH growth range for strain 143-21T was 6.5–8.5, and the optimal pH for growth was pH 7.0. No growth was observed at pH 6.0 or pH 9.0. To determine the NaCl tolerance, strain 143-21T was grown at 50 °C and pH 7.0. JCM 909 medium with gradients of 0–8 % NaCl (w/v) concentration was prepared at 0.5 % intervals. Strain 143-21T grew in NaCl concentrations of 1.0–7.0 % (w/v), with optimum growth at 2.0 % (w/v).

For the analysis of the utilization of sole carbon sources, the basal 909 medium was prepared as described by Lakhal *et al.* (10). The tested substrates were added to basal 909 medium with a final concentration of 20 mM. Then, the prepared medium with various substrates were filtered through sterilized 0.22 µm Millipore filters (Merck Millipore). These media were inoculated with cells from late exponential-phase and the final cell number is approximately 10⁶ cells ml⁻¹. The basal 909 medium and normal JCM 909 medium were also inoculated and set as the negative and positive control, respectively. The following substrates were tested: fructose, glucose, maltose, mannose, ribose, *N*-acetyl-D-(+)-glucosamine, cellobiose, galactose, glycerol, lactose, mannitol, raffinose, rhamnose, sorbose, sucrose, xylose, tryptone, acetate, butyrate, formate, lactate, pyruvate, succinate, ethanolamine, casamino acids, glutamate, lysine, histidine, cysteine, glutamine, methionine, serine, alanine, arginine, asparagine, aspartate, glycine, isoleucine, leucine, phenylalanine, proline, tyrosine, valine, tryptophan, and threonine. Strain 143-21T was found to be able to utilize fructose, glucose, maltose, mannose, ribose, and *N*-acetyl-D-(+)-glucosamine. Other single carbon sources were not used under the tested condition. Strain 143-21T was able to utilize casamino acids, glutamate, lysine, histidine, and cysteine. Other amino acids including glutamine, methionine, serine, alanine, arginine, asparagine, aspartate, glycine, isoleucine, leucine, phenylalanine, proline, tyrosine, valine, tryptophan, and threonine were not utilized. The fermentation products of glucose were analyzed as described by Li *et al.* (11). The main products of glucose fermentation were acetate (2.07 mM), H₂ and CO₂. The ability of strain 143-21T to use electron acceptors was tested by adding sulfur (1 %, w/v), sulfate (20 mM), thiosulfate (20 mM), sulfite (2 mM), fumarate (20 mM), nitrate (10 mM), nitrite (2 mM) and Fe (III) (13 mM) to the JCM 909 medium, respectively. None were used as terminal electron acceptors.
208 The cellular fatty acid profiles of strain 143-21T and reference strains were determined. Cells grown
209 on JCM 909 medium under optimal conditions were collected in late exponential-phase by
210 centrifugation (4500 g, 20 min). The optimal conditions is 50 °C, pH 7.0, NaCl 2.0 % (w/v) for strain
211 143-21T, 50 °C, pH 7.0, NaCl 2.5 % (w/v) for C. thermophila SY095T, and 30 °C, pH 7.0, NaCl 2.5 %
212 (w/v) for C. profunda Ra1776H T. Fatty acids were prepared by the Marine Culture Collection of
213 China (MCCC) according to the instructions of the Microbial Identification System (MIDI), and were
214 then analyzed by gas chromatography (model 6850; Agilent Technologies) and identified by the
215 TSBA 6.0 database (22). The primary cellular fatty acids of strain 143-21T were C14:0 (48.8 %), C16:0
216 (12.9 %), and summed Feature 3 contained C16:1 ω7c and/or C16:1 ω6c (10.2 %). The dominant fatty
217 acid was C14:0 (48.8 %), which was same as C. thermophila SY095T (C14:0, 60.5 %) and C. profunda
218 Ra1776H T (C14:0, 44.9 %). However, strain 143-21T contains higher proportion of C16:0 and C18:0 than
219 C. thermophila SY095T and C. profunda Ra1776H T (Table S2). The polar lipids were extracted using
220 a chloroform/methanol system and analyzed using one-& two-dimensional TLC. Strain 143-21T show
221 a polar lipid composition of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), and
222 phosphatidylglycerol (PG), two unidentified phospholipids (PL1, PL2), and four unidentified
223 aminolipids (AL1, AL2, AL3, AL4) (Fig. S4). No respiratory quinones were detected which was
224 consistent with the results of the reference strain.

225 Overall, strain 143-21T show a close phylogenetic relationship with C. thermophila SY095T and C.
226 profunda Ra1776H T from the genus Crassaminicella. However, strain 143-21T could distinguished
227 with them by its optimal growth conditions (Table 1), physiological characterization (Table 1), cell
228 morphology, cellular fatty acids composition (Table S2), and genome relatedness. Therefore, we
229 suggested that strain 143-21T (=DSM 114408T = MCCC 1K06400T) represents a novel species of the
230 genus Crassaminicella, for which the name Crassaminicella indica sp. nov. is proposed.

231 **Description of Crassaminicella indica sp. nov.**

232 *Crassaminicella indica* (in'di.ca. L. fem. adj. *indica*, pertaining to the Indian Ocean, where the strain
233 was isolated).

234 Cells are Gram-stain-positive, strictly anaerobic, non-motile, rod-shaped without flagella, 1.1 ± 0.3
235 µm in width and 7.1 ± 1.5 µm in length. Spores occur with terminal swelling in nutritional restriction.
236 Cell growth range is as followed: temperature 25-60 °C (optimum 50 °C), pH 6.5-8.5 (optimum 7.0),
237 NaCl 1.0-7.0 % (w/v) (optimum 2.0 %, w/v). Able to utilize fructose, glucose, maltose, mannose,
238 ribose, N-acetyl-D-(+)-glucosamine, casamino acids, glutamate, lysine, histidine, cysteine. Cellobiose,
239 galactose, glycerol, lactose, mannitol, raffinose, rhamnose, sorbose, sucrose, xylose, tryptone, acetate,
240 butyrate, formate, lactate, pyruvate, succinate, ethanolamine, glutamine, methionine, serine, alanine,
241 arginine, asparagine, aspartate, glycine, isoleucine, leucine, phenylalanine, proline, tyrosine, valine,
242 tryptophan, and threonine are not utilized. Terminal electron acceptors such as elemental sulfur,
243 sulfate, thiosulfate, sulfite, fumarate, nitrate, nitrite, and Fe (III) are not utilized. The major
244 fermentation products from glucose are acetate, H₂ and CO₂. The main cellular fatty acids consist of
245 C₁₄:₀ and C₁₆:₀. The major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine,
246 phosphatidylglycerol, two unidentified phospholipids, and four unidentified aminolipid. No
247 respiratory quinones are detected.

248 The type strain, strain 143-21T (=DSM 114408T = MCCC 1K06400T), was isolated from deep-sea
249 hydrothermal vent chimney at a depth of 2,440 m in the Kali hydrothermal field of the Central Indian
250 Ridge. The DNA G+C content of the type strain is 31.1 %. The GenBank/EMBL/DDBJ accession
251 number for the 16S rRNA sequence of strain 143-21T is OP271781. The complete genome sequence
252 of 143-21T has been deposited at GenBank/ENA/DDBJ under the accession number CP078093.
# AUTHOR STATEMENTS

## 1.1 Authors and contributors

Xue-Gong Li designed the research outline. Ze-Xi Jiao and Xue-Gong Li drafted the manuscript. Ze-Xi Jiao, Huan-Huan Zhang, Shi-Jie Bai, Jie Dai, Jin Lin, and Xiao-Qing Qi performed the experiments. Xue-Gong Li, Wei-Jia Zhang and Long-Fei Wu collected the sample from the Central Indian Ridge. Wei-Jia Zhang, Jun Xu, and Long-Fei Wu revised the manuscript. All authors read and approved the final manuscript.

## 1.2 Conflicts of interest

The authors declare that there is no conflict of interest.

## 1.3 Funding information

This research was funded by grant ZDKJ2019011 and ZDKJ2021028 from the Major Scientific and Technological Projects of Hainan Province, grant 321RC616 from the Natural Science Foundation of Hainan Province, grant NSFC 41806174, 42176121 and 42076127 from the National Natural Science Foundation of China, grant DBM 2021 from INSB-CNRS. This work is also supported in part by the China Scholarship Council (CSC No. 202004910195).

## 1.4 Acknowledgements

We are very grateful to all the participants of the TS10-3 cruise of R/V "Tan Suo Yi Hao" and the captain and crew of the "Shen Hai Yong Shi" for their professional service during this cruise.

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## FIGURES AND TABLES

**Fig. 1.** Neighbor-joining phylogenetic tree based on 16S rRNA sequences of strain 143-21T and its relatives. Bootstrap values above 50 % based on 1,000 replicates are shown at nodes. Filled circles indicate nodes also recovered with minimum evolution and maximum-likelihood methods. The respective GenBank accession numbers are given in parentheses. Bar, 2 nucleotide substitutions per 1,000 nucleotide positions.

**Fig. 2.** Transmission-electron microscopy image of a cell of strain 143-21T. Bar: 1 µm.

**Table. 1.** Differential phenotypic characteristics of strain 143-21T and its closely related species

Strains: 1, *Crassaminicella indica* 143-21T; 2, *Crassaminicella thermophila* SY095T; 3, *Crassaminicella profunda* Ra1766H. +, Positive; –, negative.

<table><thead><tr><th>Characteristics</th><th>1</th><th>2</th><th>3</th></tr></thead><tbody><tr><td>Geographical origin</td><td>Central Indian Ridge</td><td>Southwest Indian Ridge</td><td>Guaymas basin</td></tr><tr><td>Depth (m)</td><td>2,440</td><td>2,730</td><td>2,002</td></tr><tr><td>Motility</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Size (µm)</td><td>1.1 ± 0.3 × 7.1 ± 1.5</td><td>0.7 ± 0.1 × 6.1 ± 1.0</td><td>0.5–1.0 × 6.0–10.0</td></tr><tr><td>Presence of spores</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Gram-stain</td><td>+</td><td>+</td><td>+</td></tr><tr><td><strong>Growth conditions*#</strong></td><td></td><td></td><td></td></tr><tr><td>Temperature range (°C)</td><td>25–60 (50)</td><td>45–60 (50–55)</td><td>20–40 (30)</td></tr><tr><td>pH range</td><td>6.5–8.5 (7.0)</td><td>6.0–7.5 (7.0)</td><td>6.0–8.0 (7.0)</td></tr><tr><td>NaCl (%, w/v)</td><td>1.0–7.0 (2.0)</td><td>1.0–4.5 (2.5)</td><td>0.5–4.0 (2.5)</td></tr><tr><td><strong>Substrates used#</strong></td><td></td><td></td><td></td></tr><tr><td>Fructose</td><td>+</td><td>+</td><td>-</td></tr><tr><td>Maltose</td><td>+</td><td>+</td><td>-</td></tr></tbody></table>
<table><tr><td>Mannose</td><td>+</td><td>-</td><td>+</td></tr><tr><td>Ribose</td><td>+</td><td>-</td><td>-</td></tr><tr><td>Acetate</td><td>-</td><td>-</td><td>+</td></tr><tr><td>Pyruvate</td><td>-</td><td>-</td><td>+</td></tr><tr><td>N-acetyl-D-(+)-<br>glucosamine</td><td>+</td><td>+</td><td>-</td></tr><tr><td>DNA G+C content (%)</td><td>31.1</td><td>30.8</td><td>31.5</td></tr></table>

351 * Optimum values in parenthesis.

352 # Data are from this study.

353 Fig 1

<figure><img src="image_2.png" /></figure>

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<figure><img src="image_3.png" /></figure>

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359 Supplementary Material Files

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*Crassaminicella indica* sp. nov., a novel thermophilic
anaerobic bacterium isolated from a deep-sea
hydrothermal vent

Ze-Xi Jiao<sup>1,2</sup>, Xue-Gong Li<sup>1,3,4,5*</sup>, Huan-Huan Zhang<sup>6</sup>; Jun Xu<sup>7</sup>; Shi-Jie Bai<sup>4,8</sup>, Jie Dai<sup>9</sup>, Jin Lin<sup>1,2</sup>, Wei-Jia

Zhang<sup>1,3,4,5</sup>, Xiao-Qing Qi<sup>1,3,4</sup>, Long-Fei Wu<sup>3,9</sup>

<sup>1</sup>Laboratory of Deep-Sea Microbial Cell Biology, Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya, China

<sup>2</sup>University of Chinese Academy of Sciences, Beijing, China

<sup>3</sup>International Associated Laboratory of Evolution and Development of Magnetotactic Multicellular Organisms, CNRS-Marseille/CAS- Sanya
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372 CAS Key Laboratory for Experimental Study under Deep-sea Extreme Conditions, Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya, China

373
374 Institution of Deep-sea Life Sciences, IDSSE-BGI, Hainan Deep-sea Technology Laboratory, Sanya, Hainan, China

375 School of Oceanography, Shanghai Jiao Tong University, Shanghai, China

376
377 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

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379 Laboratory of Marine Viruses and Molecular Biology, Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya, China

380 Aix Marseille Univ, CNRS, LCB, IMM, IM2B, Marseille, France

381
382 Correspondence: Xue-Gong Li: xuegongli@idsse.ac.cn

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<figure><img src="image_4.png" /></figure>

A

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<figure><img src="image_5.png" /></figure>

B

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Fig. S1. Minimum evolution (A) and maximum likelihood (B) phylogenetic trees based on 16S rRNA sequences of strain 143-21T and its closest relatives. Bootstrap values above 50 % based on 1,000 replicates are shown at nodes. The respective GenBank accession numbers are given in parentheses.
Bar, 2 nucleotide substitutions per 1,000 nucleotide positions.

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<figure><img src="image_6.png" /><figcaption>Fig. S2. Neighbor-joining phylogenetic tree of strain 143-21T and its closest relatives based on the concatenated 120 bacterial marker proteins using the MEGA version 11. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The respective GenBank accession numbers are given in parentheses. Bootstrap values above 50 % based on 1,000 replicates are shown at nodes. Bar, number of amino acid differences per site.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. S3. Effects of temperature (A), pH (B) and NaCl concentration (C) on the growth of strain 143-21T. Each value is the mean of three replicates, mean values ± SD, n=3.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Fig. S4. Polar lipids composition of strain 143-21T. PL: Phospholipid, PG: Phosphatidylglycerol, DPG: Diphosphatidylglycerol, PE: phosphatidylethanolamine, AL, aminolipid, F-first dimension of TLC; S- second dimension of TLC.</figcaption></figure>

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<table><caption>**Supplementary Table S1.** Whole genome-based comparisons between strain 143-21T and closely relatives.</caption><thead><tr><th>Strains</th><th>DDH (%)</th><th>ANI (%)</th><th>AAI (%)</th></tr></thead><tbody><tr><td><i>Crassaminicella profunda</i> Ra1766H<sup>T</sup></td><td>29.20</td><td>84.85</td><td>81.50</td></tr><tr><td><i>Crassaminicella thermophila</i> SY095<sup>T</sup></td><td>23.50</td><td>79.79</td><td>66.78</td></tr></tbody></table>

<caption>**Supplementary Table S2.** Fatty acid composition of strain 143-21T and its closely related strains.</caption>

Strain: 1, 143-21T; 2, *Crassaminicella thermophila* SY095T; 3, *Crassaminicella profunda* Ra1766H<sup>T</sup>.

Values are percentages of total fatty acids. Fatty acids that represented <1 % in all strains were omitted. TR, Traces (<1 %).

<table><thead><tr><th rowspan="2">Fatty acid*</th><th colspan="3">Composition (%) in:</th></tr><tr><th>1</th><th>2</th><th>3</th></tr></thead><tbody><tr><td>C<sub>12:0</sub></td><td>TR</td><td>1.0</td><td>TR</td></tr><tr><td>C<sub>14:0</sub></td><td>48.8</td><td>60.5</td><td>44.9</td></tr><tr><td>C<sub>16:1</sub>ω9c</td><td>3.4</td><td>3.4</td><td>9.0</td></tr><tr><td>C<sub>16:0</sub></td><td>12.9</td><td>7.6</td><td>7.0</td></tr><tr><td>C<sub>18:0</sub></td><td>3.8</td><td>TR</td><td>TR</td></tr><tr><td>Summed Feature 1</td><td>2.7</td><td>5.6</td><td>1.9</td></tr><tr><td>Summed Feature 3</td><td>10.2</td><td>14.0</td><td>29.6</td></tr><tr><td>Summed Feature 4</td><td>9.9</td><td>3.6</td><td>3.7</td></tr><tr><td>Summed Feature 8</td><td>3.25</td><td>TR</td><td>TR</td></tr></tbody></table>

*Summed Features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed Feature 1 contained iso-C<sub>15:1</sub> H and/or C<sub>13:0</sub> 3-OH; Summed Feature 3 contained C<sub>16:1</sub> ω7c and/or C<sub>16:1</sub> ω6c; Summed Feature 4 contained iso-C<sub>17:1</sub> I and/or anteiso-C<sub>17:1</sub> B; Summed Feature 8 contained C<sub>18:1</sub> ω7c and/or C<sub>18:1</sub> ω6c.