Sporolactobacillus fermentans sp. nov., an obligately anaerobic and lactic acid bacterium isolated from pit mud

doi:10.21203/rs.3.rs-7975107/v1

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Sporolactobacillus fermentans sp. nov., an obligately anaerobic and lactic acid bacterium isolated from pit mud

https://doi.org/10.21203/rs.3.rs-7975107/v1

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A gram-positive, endospore-forming bacterial strain CQH2019^T was successfully isolated from pit mud samples of Chinese Nongxiang-flavor Baijiu. The cells were anaerobic, Gram-positive, rod-shaped, with 0.5–0.8 µm in width and 3–5 µm in length. The optimal growth conditions are 37°C, pH 5.5 and NaCl concentrations of 0.5%. This obligate chemoorganoheterotrophic strain utilized complex organic compounds, carbohydrates (L-arabinose, D-xylose, D-galactose, D-fructose, D-mannose, L-rhamnose, and D-tagatose), organic salts (potassium gluconate), glycosides (amygdalin, arbutin, salicin) and produced acetate, butyric and lactic acid as end products from RCM. The predominant cellular fatty acids (> 10%) of stain CQH2019^T were anteiso-C17:0 (29.95%), C16:0 (17.47%) and anteiso-C15:0 (14.42%). Phylogenetic analysis based on 16S rRNA gene sequences revealed the closest relatives were S. putidus QC81-06^T (95.74%), S. vineae SL153^T (95.74%), S. pectinivorans GD201205^T (95.38%) and S. shoreae BK92^T (95.19%). The size of the genome was 4.5 Mb and the DNA G + C content was 47.58%. The digital DNA-DNA hybridization (dDDH) values between CQH2019^T and these closely related strains were ranged from 19.3% to 26.8%, whereas the whole-genome average nucleotide identity (ANI) values were 69.26 − 82.78%. These polyphasic taxonomy analysis results indicate that strain CQH2019^T represents a novel species based on both criteria. Also based on polyphasic analysis, strain CQH2019^T represents a novel species within the genus Sporolactobacillus, for which the name Sporolactobacillus fermentans sp. nov. is proposed. The type strain is CQH2019^T (= MCCC 1A19398^T = KACC 23552^T).

Anaerobe

Pit mud

Polyphasic taxonomy analysis

Sporolactobacillus

The genus Sporolactobacillus was first described by Kitahara and Suzuki (1963) as Gram-positive, catalase-negative bacteria capable of endospore formation and lactic acid production (Kitahara et al., 1963). Current taxonomic classification recognizes 15 species and 2 subspecies within this genus (https://lpsn.dsmz.de/search?word=Sporolactobacillus), including S. inulinus (type species), S. kofuensis, S. lactosus, S. laevolacticus, S. nakayamae, S. terrae, S. vineae, S. putidus, S. spathodeae, S. pectinivorans, and S.mangiferae. These facultatively anaerobic bacteria are predominantly isolated from plant materials, soil ecosystems, and food fermentation systems(Chang et al., 2008; Fujita et al., 2010; Lan et al., 2016; Thamacharoensuk et al., 2015; Tolieng et al., 2017).

In the context of traditional Chinese Baijiu production, pit mud - a unique fermentation matrix in cuboid cellars (typical dimensions: 3.4 m × 1.8 m × 2.0 m) - serves as both microbial habitat and biochemical reactor for developing the characteristic Nongxiang-flavor profile (Qian et al., 2021; Zheng et al., 2020). In the cellar mud of different cellar ages (20–200 years), Bacillota (including Sporolactobacillus) play a key role in the synthesis of flavor compounds (Xu et al., 2017) (Liu et al., 2017). Notably, Sporolactobacillus spp. significantly influence organic acid metabolism, particularly acetic acid production, which ultimately leads to differences in the quality of liquor (Liu et al., 2019).

Herein, we report the isolation and characterization of strain CQH2019^T (MCCC 1A19399^T = KACC 23552^T) from aged pit mud. Polyphasic taxonomic analysis reveals this anaerobic isolate represents a novel species within the genus Sporolactobacillus.

Sampling and isolation of strains

The strain CQH2019^T was isolated from pit mud obtained from a Nongxiang-flavor Baijiu company in Sichuan Province, China. Pit mud samples were collected from the bottom of cellar and immediately transferred to an anaerobic bag with an oxygen indicator (Hopebio HBYY007, Qingdao, China) and transported to the laboratory for bacterial separation.

For enrichment cultivation, 5 g homogenized pit mud was inoculated into 50 mL reinforced clostridial medium (RCM; ATCC Medium 1524) containing (per liter): 10.0 g beef extract, 3.0 g yeast extract, 3.0 g sodium acetate, 10.0 g tryptone, 3.0 g glucose, 1.0 g soluble starch, and 0.15 g CaCl₂·2H₂O. The medium pH was adjusted to 6.8 ± 0.1 using 1 mol/L NaOH. Enrichment cultures were maintained in COY vinyl anaerobic chambers (Coy Laboratory Products, Model 1000) with 85% N₂, 10% H₂, 5% CO₂ atmosphere at 37.0°C ± 0.5°C for 15 days. Subsequent isolation employed on solid RCM agar (add 1.5% w/v agar) with three successive streak-plate purification under anaerobic conditions. The selected pure culture (strain CQH2019^T) with 25% (final v/v) glycerol was cryopreserved at -80°C.

Morphology

Use YCFA solid medium for the pure culture of strain CQH2019^T and perform morphological observation of the strain. YCFA medium contained: tryptone, 10 g; yeast extract, 2.5 g; sodium acetate, 3.5 g; NaCl, 4.5g; CaCl2·6H2O, 0.09g; KH2PO4, 0.45g; K2HPO4, 0.45g; MgSO4·7H2O, 0.09g; L-Cysteine Hydrochloride, 0.8g; Sodium propionate, 0.865g; Sodium butyrate, 0.11g; Agar powder, 17.5g. The pH was adjusted to pH 7.0 with 10M NaOH. In an anaerobic chamber at 37°C for 48 h for observation. Cell morphology of the isolated strain was examined using a light microscope (Leica DM500, Leica Microsystems, Germany) and transmission electron microscope(Hitachi HT7800, Hitachi High-Tech, Japan).

Physiology and chemotaxonomy

To determine the salinity, pH, and temperature tolerance of strain CQH2019^T, cultures were grown anaerobically in bottles containing 30 mL of Reinforced Clostridial Medium (RCM) broth. Salinity tolerance was assessed at NaCl concentrations ranging from 0 to 7% (w/v). The pH growth range was evaluated across pH 3.0–9.0 (adjusted at 0.5-unit intervals), and the temperature tolerance was tested from 4 to 50°C. All experiments were performed in triplicate biological replicates. Growth was monitored by measuring the optical density at 600 nm (OD₆₀₀) to determine the optimal conditions. To determine catalase activity, bacterial cells grown on RCM medium were exposed to a 3% hydrogen peroxide solution. Amylase activity was assessed by applying approximately 0.1 ml of Lugol's iodine solution around colonies grown on RCM agar plates containing 2% starch. Oxidase activity and nitrate reduction were identified using Oxyswab (bioMérieux, Marcy-l'Étoile, France) according to the manufacturer’s instructions.(Lan, Chen, Lin, Ye, Yan, Huang, Liu, Yang, 2016). Gram staining was conducted using a commercial kit (Hangzhou Tianhe Microbiology Reagent Co., Ltd.) following manufacturer instructions .

In RCM broth, after 7 days of anaerobic incubation at 37°C, the utilization of carbohydrates were determined according to the API 50CH (bioMérieux, Marcy-l'Étoile, France) instructions. and used HPLC (Shimadzu Corporation, Kyoto, Japan) to measure its fermentation end products(Duarte-Villanueva et al., 2025).The fatty acids were extracted following the instructions supplied with the MIDI / Hewlett Packard Microbial Identification System (MIDI Inc., Newark, DE, USA), and the subsequent separation and identification by Agilent 6850 gas chromatography (Agilent Technologies, Santa Clara, CA, USA) were performed (Athalye et al., 1985). The polar lipid composition was analyzed using two-dimensional thin-layer chromatography (TLC Merck, Germany)(Minnikin et al., 1979). The first-dimensional separation was performed with a chloroform-methanol-water solvent system (65:25:4, v/v), followed by second-dimensional development with chloroform-methanol-acetic acid-water (85:12:15:4, v/v). Total lipids were visualized by spraying with 10% (v/v) molybdatophosphoric acid solution. Isoprenoid quinones were extracted and analyzed according to the protocol established by Minnikin et al. (1984) (Minnikin et al., 1984). For quantification of short-chain fatty acids in fermentation products, including lactic acid, acetic acid, and butyric acid, identical analytical methodology was employed as described by Reyes et al. (2025) (Reyes et al., 2025).

Phylogeny analysis of 16S RNA gene

Genomic DNA was extracted from pure culture of strain CQH2019^T using a commercial DNA extraction kit (Beijing SBS Genetech Co., Ltd). The nearly complete 16S rRNA gene sequence (1,458 bp) was amplified using universal primers 27F(5′-AGAGTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Ferdous, 2024). To ensure high accuracy of 16S rRNA gene, bidirectional Sanger sequencing was performed, and the final sequence was deposited in GenBank databases (accession number: OP925886). The 16S rRNA gene sequence was blasted using NCBI RefSeq database (https://www.ncbi.nlm.nih.gov/) and the EzBioCloud 16S database (https://www.ezbiocloud.net/), respectively. Related similar sequences were collected and phylogenetic trees were built by the neighbor-joining (NJ), minimum-evolution and maximum-likelihood (ML) algorithms using MEGA 7(Kimura, 1980; Kumar et al., 2016).

Genome Features

The whole-genome sequencing was conducted by Shanghai Meiji biomedical technology Co., Ltd (Shanghai, PR China) using Hiseq4000(Illumina)and Sequel II༈PacBio༉system according to the manufacturer’s protocols. The complete genome was assembled using SOAPdenovo2༈http://soap.genomics.org.cn/༉(Luo et al., 2012). Chromosomal completeness was confirmed by BUSCO (https://busco.ezlab.org/) with score of 99.2%. The genome sequences were annotated through the NCBI Prokaryotic Genome Annotation Pipeline (NCBI-PGAP) and deposited in the DDBJ, ENA, and GenBank databases under the INSDC framework (https://www.insdc.org/). The whole-genome sequencing project has been assigned with the BioProject accession number PRJNA908277. Transfer RNA (tRNA) genes were identified using tRNAscan-SE v1.21 and ARAGORN v1.2.41(Chan et al., 2019; Laslett et al., 2004), while ribosomal RNA (rRNA) genes were predicted with RNAmmer v1.2(Lagesen et al., 2007). The complete genome sequence was deposited in the NCBI GenBank database.

The digital DNA-DNA hybridization (dDDH) values were determined using Formula 2 of the Genome-to-Genome Calculator (http://ggdc.dsmz.de/ggdc.php) (Meier-Kolthoff et al., 2013). The ANI values between the genomes of strain CQH2019^T and the close strains were calculated using the ANI calculator tool from EzBioCloud (Yoon et al., 2017) and amino acid identity (AAI) values were calculated by the AAI calculator (http://enve-omics.ce.gatech.edu/aai/) (Rodriguezr et al., 2016).

The taxonomic position of the strain was further confirmed by whole-genome phylogeny. A phylogenomic tree was constructed using the Up-to-date Bacterial Core Gene (UBCG) method, which leverages a set of 92 core genes (Na et al., 2018).

Phenotypic and chemotypic characteristics of strain CQH2019^T

The discrepancies between strain CQH2019^T and related Sporolactobacillus species are presented in Table 1. Strain CQH2019^T was determined to be Gram-positive and anaerobic, exhibiting oval rod-shaped cells (0.5–0.8 µm in width × 3–5 µm in length) with terminal endospores and enlarged sporangia (Fig. S1). Colonies appeared round, flat and white. The strain grew within temperature (25–45°C), pH (4.0–7.0), and NaCl concentration (0–2% w/v), with optimal growth at 37°C, pH 5.5 and NaCl concentrations of 0–2%.

Using the API CH50 system, strain CQH2019^T was able to ferment L-arabinose, D-xylose, D-galactose, D-fructose, D-mannose, L-rhamnose, amygdalin, arbutin, salicin, D-tagatose, and potassium gluconate. Unlike other members of Sporolactobacillus, strain CQH2019^T showed the ability to utilize salicin and amygdalin. After 72 h of growth in RCM, the strain produced 8.14 g/L lactic acid, 7.04 g/L butyric acid, and 5.68 g/L acetic acid (Fig. 1).

Table 1
Phenotypic properties of strain CQH2019^T and type strains of related species: 1, strain CQH2019^T; 2, *S. putidus* QC81-06^T; 3, *S. vineae* SL153^T; 4, *S. pectinivorans* GD201205^T; 5, *S. shoreae* BK92^T.
Characteristics	1	2	3	4	5
Isolation source	Pit mud	spoiled juice	soil	spoiled jelly	tree barks
Colony color	White	White	ivory-white	White	White
Temperature (℃)
Ranges	25–45	30–45	20–45	20–40	25–40
Optimum	37	35	37	37	30
NaCl (%, w/v)
Ranges	0–2	0–2	0–7	0–2	1–2
Optimum	0.5	-	-	1	1
pH
Ranges	4–7	3–6	4–9	4–7	4.5-8.0
Optimum	5.5	4.5-5	5	6	6
Catalase	+	-	-	-	-
Acid production from:
Amygdalin	w	-	-	-	-
Galactose	+	+	-	-	+
Maltose	-	+	w	-	-
Mannitol	-	-	+	-	-
Methyl a-D-glucoside	-	-	+	-	-
Raffinose	-	-	-	+	-
Salicin	+	-	-	-	-
Sorbitol	-	-	+	-	-
Sorbose	-	-	+	-	-
Sucrose	-	+	W	+	+
D-Tagatose	+	+	-	-	+
Trehalose	-	+	W	-	-
Turanose	-	+	-	+	-

As shown in Table S1, the major fatty acids (≥ 10% of total content) in strain CQH2019^T were anteiso-C17:0 (29.95%), C16:0 (17.47%), and anteiso-C15:0 (14.42%). Additionally, five fatty acids (iso-C15:0, C16:1ω9c, iso-C16:0, iso-C17:0 and C18:0) each accounted for more than 0.5% of the total fatty acid composition. In strain CQH2019^T, anteiso-C17:0 (29.95%) represented the predominant fatty acid, a feature shared with other spore-forming Lactobacillus species. Notably, the C16:1ω9c content (5%) in this strain was significantly higher than that in related species.

The polar lipid profile (Fig.S2) revealed the presence of phosphatidylethanolamine, phosphatidylcholine, four unidentified aminolipids, and four unidentified glycolipids.

Phylogenetic analysis of 16S RNA gene

Phylogenetic analysis positioned strain CQH2019^T within the genus Sporolactobacillus. Comparative 16S rDNA sequence analysis showed 95.19–95.74% similarity to established type strains; the highest similarity (95.74%) was observed with S. putidus QC81-06^T and S. vineae SL153^T, followed by S. pectinivorans GD201205^T (95.38%) and S. shoreae BK92^T (95.19%). Phylogenetic trees were consistently clustered stain CQH2019^T within Sporolactobacillus species. Notably, it formed a distinct clade with S. pectinivorans GD201205^T and S. vineae SL153^T (Fig. 2), a topology strongly supported by both minimum-evolution and maximum-likelihood analyses (Figs. S3 and S4).

Genome features

The genome of Sporolactobacillus strain CQH2019^T comprises a single circular chromosome of 4,513,405 bp, with no plasmids or additional genetic elements detected. It is larger than the genomes of four closely related Sporolactobacillus species, which range from 2.97 to 3.91 Mb. The genome contains 4,250 annotated genes, including 4,180 coding sequences (CDSs), 3,517 protein-coding genes, 7 rRNA operons, and 63 tRNA genes. Its genomic DNA displays a characteristic G + C content of 47.58%. Comparative genomic analyses with type strains S. putidus QC81-06^T, S. vineae SL153^T, S. pectinivorans GD201205^T, and S. shoreae BK92^T yielded average nucleotide identity (ANI) values of 71–83% (Table S2), falling below the established prokaryotic species threshold (95–96%)(Richter et al., 2009). Digital DNA-DNA hybridization (dDDH) values further confirmed taxonomic distinction, with pairwise comparisons showing 26.8% (S. putidus QC81-06^T), 19.9% (S. pectinivorans GD201205^T), 19.9% (S. vineae SL153^T), and 19.3% (S. shoreae BK92^T) – all significantly below the 70% species delineation benchmark(Meier-Kolthoff et al., 2022; Rodriguezr, Konstantinidis, 2016).

The genome-based phylogenetic tree of 92 core genes was constructed to determine the phylogenetic position of strain CQH2019^T within the genus Sporolactobacillus and with more closely relationship with S. putidus QC81-06^T (Fig. 3).

Taken together, the results of genetic analyses, including phylogenetic tree analysis of 16S rRNA gene, dDDH, ANIb and AAI values, as well as multilocus sequence analysis provide compelling evidence that strain CQH2019^T represents a novel species within the genus Sporolactobacillus.

Description of Sporolactobaclllus fermentans sp. nov.

Sporolactobacillus fermentans (fer. men´tans; L. part. adj. fermentans, fermenting) is a Gram-positive, anaerobic species. The cells are slightly curved rods (0.5–0.8 × 3.0–8.0 µm) bearing oval endospores and sparse flagella. Colonies grown on YCFA agar for 7 days appeared as opaque white, regular round structures (0.3–0.5 cm diameter) with rough surfaces that were easily lifted. Growth occurred at 25–45°C (optimum 37°C), pH 4.0–7.0 (optimum pH 5.5), and NaCl concentrations of 0–2% (w/v) (optimum NaCl 0.5%). Biochemical characterization revealed catalase, oxidase, amylase production, and nitrate reduction were all negative. Acid production was observed from L-arabinose, D-xylose, D-galactose, D-fructose, D-mannose, L-rhamnose, amygdalin, arbutin, salicin, and D-tagatose. The predominant fatty acids were anteiso-C15:0, C16:0, and anteiso-C17:0, with phosphatidyl ethanolamine and phosphatidylcholine identified as major polar lipids.

While most Sporolactobacillus strains characteristically contain menaquinone-7 (MK-7) as their primary respiratory quinone, strain CQH2019^T strikingly lacked this essential isoprenoid component. The genomic profile of this type strain reveals a total size of 4,513,505 bp with a G + C content of 47.58%. The strain is formally designated as CQH2019^T (= MCCC 1A19398^T = KACC 23552^T), serving as the type strain for taxonomic reference.

1.4 Repositories:

The GenBank accession numbers of for the 16S rRNA gene sequence and the draft genome of strain CQH2019^T are OP925886 and JARAJZ010000000, respectively. Supplementary materials are available with the online Supplementary Materials.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Ethical approval

Not applicable.

Funding

This work was supported by Sichuan Science and Technology Program 2024NSFSC2064.

Author Contribution

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Xun Liu, Caiyu Zeng . The first draft of the manuscript was written by Guangbin Ye,Xiang Zeng and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

No datasets were generated or analysed during the current study.

Athalye, M., Noble, W. C., & Minnikin, D. E.(1985). Analysis of cellular fatty acids by gas chromatography as a tool in the identification of medically important coryneform bacteria. J Appl Bacteriol, 58(5): 507–512.http://dx.doi.org/10.1111/j.1365-2672.1985.tb01491.x
Chan, P. P., & Lowe, T. M.(2019). tRNAscan-SE: Searching for tRNA Genes in Genomic Sequences. Methods Mol Biol, 1962: 1–14.http://dx.doi.org/10.1007/978-1-4939-9173-0_1
Chang, Y. H., Jung, M. Y., Park, I. S., & Oh, H. M.(2008). Sporolactobacillus vineae sp. nov., a spore-forming lactic acid bacterium isolated from vineyard soil. Int J Syst Evol Microbiol, 58(Pt 10): 2316–2320.http://dx.doi.org/10.1099/ijs.0.65608-0
Duarte-Villanueva, M. C., Garner, M., Thompson, J., Diab, H., & Calle, A.(2025). Comparative analysis between organic acids and acid-producing bacteria to inhibit Salmonella serovars. Microb Pathog, 207: 107941.http://dx.doi.org/10.1016/j.micpath.2025.107941
Ferdous, R.(2024). Natural field diagnosis and molecular confirmation of fungal and bacterial watermelon pathogens in Bangladesh: A case study from the Natore and Sylhet districts. PLoS One, 19(11): e0307245.http://dx.doi.org/10.1371/journal.pone.0307245
Fujita, R., Mochida, K., Kato, Y., & Goto, K.(2010). Sporolactobacillus putidus sp. nov., an endospore-forming lactic acid bacterium isolated from spoiled orange juice. Int J Syst Evol Microbiol, 60(Pt 7): 1499–1503.http://dx.doi.org/10.1099/ijs.0.002048-0
Kimura, M.(1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16(2): 111–120.http://dx.doi.org/10.1007/BF01731581
Kitahara, K., Suzuki, J. J. J. o. G., & Microbiology, A.(1963). SPOROLACTOBACILLUS NOV. SUBGEN. 9: 59–71
Kumar, S., Stecher, G., & Tamura, K.(2016). MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol, 33(7): 1870–1874.http://dx.doi.org/10.1093/molbev/msw054
Lagesen, K., Hallin, P., Rødland, E. A., Staerfeldt, H. H., Rognes, T., & Ussery, D. W.(2007). RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res, 35(9): 3100–3108.http://dx.doi.org/10.1093/nar/gkm160
Lan, Q. X., Chen, J., Lin, L., Ye, X. L., Yan, Q. Y., Huang, J. F., Liu, C. C., & Yang, G. W.(2016). Sporolactobacillus pectinivorans sp. nov., an anaerobic bacterium isolated from spoiled jelly. Int J Syst Evol Microbiol, 66(11): 4323–4328.http://dx.doi.org/10.1099/ijsem.0.001351
Laslett, D., & Canback, B.(2004). ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res, 32(1): 11–16.http://dx.doi.org/10.1093/nar/gkh152
Liu, M. K., Tang, Y. M., Guo, X. J., Zhao, K., Tian, X. H., Liu, Y., Yao, W. C., Deng, B., Ren, D. Q., & Zhang, X. P.(2017). Deep sequencing reveals high bacterial diversity and phylogenetic novelty in pit mud from Luzhou Laojiao cellars for Chinese strong-flavor Baijiu. Food Res Int, 102: 68–76.http://dx.doi.org/10.1016/j.foodres.2017.09.075
Liu, M. K., Tang, Y. M., Zhao, K., Liu, Y., Guo, X. J., Tian, X. H., Ren, D. Q., & Yao, W. C.(2019). Contrasting bacterial community structure in artificial pit mud-starter cultures of different qualities: a complex biological mixture for Chinese strong-flavor Baijiu production. 3 Biotech, 9(3): 89.http://dx.doi.org/10.1007/s13205-019-1622-y
Luo, R., Liu, B., Xie, Y., Li, Z., Huang, W., Yuan, J., He, G., Chen, Y., Pan, Q., Liu, Y., Tang, J., Wu, G., Zhang, H., Shi, Y., Liu, Y., Yu, C., Wang, B., Lu, Y., Han, C., Cheung, D. W., Yiu, S. M., Peng, S., Xiaoqian, Z., Liu, G., Liao, X., Li, Y., Yang, H., Wang, J., Lam, T. W., & Wang, J.(2012). SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience, 1(1): 18.http://dx.doi.org/10.1186/2047-217x-1-18
Meier-Kolthoff, J. P., Auch, A. F., Klenk, H. P., & Göker, M.(2013). Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics, 14: 60.http://dx.doi.org/10.1186/1471-2105-14-60
Meier-Kolthoff, J. P., Carbasse, J. S., Peinado-Olarte, R. L., & Göker, M.(2022). TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acids Res, 50(D1): D801-d807.http://dx.doi.org/10.1093/nar/gkab902
Minnikin, D. E., Collins, M. D., & Goodfellow, M.(1979). Fatty Acid and Polar Lipid Composition in the Classification of Cellulomonas, Oerskovia and Related Taxa. Journal of Applied Bacteriology, 47(1): 87–95.http://dx.doi.org/https://doi.org/10.1111/j.1365-2672.1979.tb01172.x
Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A., & Parlett, J. H.(1984). An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. Journal of Microbiological Methods, 2(5): 233–241.http://dx.doi.org/https://doi.org/10.1016/0167-7012(84)90018-6
Na, S. I., Kim, Y. O., Yoon, S. H., Ha, S. M., Baek, I., & Chun, J.(2018). UBCG: Up-to-date bacterial core gene set and pipeline for phylogenomic tree reconstruction. J Microbiol, 56(4): 280–285.http://dx.doi.org/10.1007/s12275-018-8014-6
Qian, W., Lu, Z. M., Chai, L. J., Zhang, X. J., Li, Q., Wang, S. T., Shen, C. H., Shi, J. S., & Xu, Z. H.(2021). Cooperation within the microbial consortia of fermented grains and pit mud drives organic acid synthesis in strong-flavor Baijiu production. Food Res Int, 147: 110449.http://dx.doi.org/10.1016/j.foodres.2021.110449
Reyes, L., Bourgeois, C., Renard, G. G., Jame, P., Saupin, X., Nikitine, C., & Vilcocq, L.(2025). Analysis of aliphatic and phenolic compounds present in industrial black liquors using HPLC-DAD and IC-MS/MS methods. J Chromatogr B Analyt Technol Biomed Life Sci, 1253: 124442.http://dx.doi.org/10.1016/j.jchromb.2024.124442
Richter, M., & Rosselló-Móra, R.(2009). Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A, 106(45): 19126–19131.http://dx.doi.org/10.1073/pnas.0906412106
Rodriguezr, L. M., & Konstantinidis, K. T.(2016). The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes.
Thamacharoensuk, T., Kitahara, M., Ohkuma, M., Thongchul, N., & Tanasupawat, S.(2015). Sporolactobacillus shoreae sp. nov. and Sporolactobacillus spathodeae sp. nov., two spore-forming lactic acid bacteria isolated from tree barks in Thailand. Int J Syst Evol Microbiol, 65(Pt 4): 1220–1226.http://dx.doi.org/10.1099/ijs.0.000084
Tolieng, V., Prasirtsak, B., Miyashita, M., Shibata, C., Tanaka, N., Thongchul, N., & Tanasupawat, S.(2017). Sporolactobacillus shoreicorticis sp.nov., a lactic acid-producing bacterium isolated from tree bark. Int J Syst Evol Microbiol, 67(7): 2363–2369.http://dx.doi.org/10.1099/ijsem.0.001959
Xu, Y., Sun, B., Fan, G., Teng, C., Xiong, K., Zhu, Y., Li, J., & Li, X.(2017). The brewing process and microbial diversity of strong flavour Chinese spirits: a review. 123(1): 5–12.http://dx.doi.org/https://doi.org/10.1002/jib.404
Yoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, H., & Chun, J.(2017). Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol, 67(5): 1613–1617.http://dx.doi.org/10.1099/ijsem.0.001755
Zheng, Y., Hu, X., Jia, Z., Bodelier, P. L. E., Guo, Z., Zhang, Y., Li, F., & He, P.(2020). Co-occurrence patterns among prokaryotes across an age gradient in pit mud of Chinese strong-flavor liquor. Can J Microbiol, 66(9): 495–504.http://dx.doi.org/10.1139/cjm-2020-0012

No competing interests reported.

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25 Dec, 2025
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25 Dec, 2025
Reviewers agreed at journal
16 Dec, 2025
Reviews received at journal
08 Dec, 2025
Reviewers agreed at journal
18 Nov, 2025
Reviewers invited by journal
16 Nov, 2025
Editor assigned by journal
31 Oct, 2025
Submission checks completed at journal
31 Oct, 2025
First submitted to journal
28 Oct, 2025

You are reading this latest preprint version

Sporolactobacillus fermentans sp. nov., an obligately anaerobic and lactic acid bacterium isolated from pit mud

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Sampling and isolation of strains

Morphology

Physiology and chemotaxonomy

Phylogeny analysis of 16S RNA gene

Genome Features

Results and discussion

Phenotypic and chemotypic characteristics of strain CQH2019^T

Phylogenetic analysis of 16S RNA gene

Genome features

Declarations

1.4 Repositories:

Competing Interests

Ethical approval

Funding

Author Contribution

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

Sporolactobacillus fermentans sp. nov., an obligately anaerobic and lactic acid bacterium isolated from pit mud

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Sampling and isolation of strains

Morphology

Physiology and chemotaxonomy

Phylogeny analysis of 16S RNA gene

Genome Features

Results and discussion

Phenotypic and chemotypic characteristics of strain CQH2019T

Phylogenetic analysis of 16S RNA gene

Genome features

Declarations

1.4 Repositories:

Competing Interests

Ethical approval

Funding

Author Contribution

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

Phenotypic and chemotypic characteristics of strain CQH2019^T