Clostridium aromaproducens sp. nov., isolated from pit mud used for producing Wuliangye baijiu

doi:10.21203/rs.3.rs-2228679/v1

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Clostridium aromaproducens sp. nov., isolated from pit mud used for producing Wuliangye baijiu

https://doi.org/10.21203/rs.3.rs-2228679/v1

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published 16 May, 2023

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A strictly anaerobic, gram-positive bacterium, designated as WLY-B-L2^T, was isolated from pit mud of a fermentation pit located at Wuliangye 501# baijiu workshop in Yibin (Sichuan province, PR China). The strain has straight or somewhat rod-shaped cells, which are 0.5–0.7 µm wide and 1.7–3.1 µm long, arranged singly or in pairs. The strain can produce butyric acid and utilize D-galacturonic acid, methyl pyruvate, L-lactamine, L-alanyl-L-glutamine, L-alanyl-L-histidine, glycerol, pyruvate, L-alanyl-L-threonine, L-methionine, L-phenylalanine, L-valine + L- aspartic acid, L-serine, L-valine, and thymidine as carbon sources. Major cellular fatty acids are C_{16: 0} (24.6%), anteiso-C_{15: 0} (16.5%), and iso-C_{15: 0} (14.1%). Based on 16S rRNA gene sequence, WLY-B-L2^T is most closely related to Clostridium luticellarii FW431^T; they exhibit 97.42% 16S rRNA similarity. Additionally, the digital DNA-DNA hybridization (dDDH) value between them is 28.10%. WLY-B-L2^T has a G + C content of 34.16 mol%. Based on the evidence presented here, WLY-B-L2^T (CICC 25133^T = JCM 35127^T) is proposed as the type strain of a novel species, designated as Clostridium aromatiproducens sp. nov.

Wuliangye baijiu

Clostridium aromaproducens sp. nov.

fermentation pit

novel species.

Baijiu is a traditional alcoholic beverage made in China. It is one of the oldest distilled liquors in the world. Its popularity is increasing around the world owing to its unique taste and flavor [1, 2]. According to the aroma characteristics, baijiu is classified into strong-aroma-type, light-aroma-type, sauce-aroma-type, and rice-aroma-type. Wuliangye is the typical representative of a strong-aroma-type baijiu (SATB) [3]. The SATB is fermented in a mud pit, which is one of the crucial factors influencing its liquor quality and flavor [4, 5].

The SATB is batch-brewed in a fermentation pit made of mud. The brewing process involves spontaneous solid-phase fermentation for 70–90 days. During the fermentation, the fermentation pit is in anaerobic condition and contain high ethanol concentrations reaching 6.2–8.0 mg/g [6, 7]. The brewing process leads to accumulation of diverse anaerobic and facultatively aerobic microbes [7, 8]. These adaptive micro-organisms can produce many low-molecular-mass organic acids, which are esterified to various esters or other volatile compounds and these compounds determine the quality and aromatic style of the liquor [9]. Here we report the isolation and characterization of a strictly anaerobic, Gram-positive bacterium, designated WLY-B-L2^T, isolated from a fermentation pit mud. The ecological features of the environment are anaerobic, acidic, and moist.

The genus Clostridium belongs to domain Bacteria, phylum Firmicutes, class Clostridia and family Clostridiaceae. The genus Clostridium typically contains fermentative bacteria. Clostridium species can metabolize complex carbohydrates and their fermentation products include short-chain fatty acids, ethanol, and other carbohydrate derivatives. Bacteria belonging to the genus Clostridium are the major functional bacteria that provide short-chain fatty acids for ester formation in Chinese baijiu brewing [10]. According to the report [11], the proportion of Clostridium in pit mud is 0.04–1.43% based on high-throughput sequencing. It was determined by culture-dependent approaches that the Clostridium in the pit mud are C. celerecrescens, C. cochlearium, C. carboxidivorans, C. sporogenes, C. sartagoforme, C. thermopalmarium, C. aurantibutyricum and C. butyricum [12].

In January 2019, WLY-B-L2 ^T was isolated from pit mud collected from the wall of a baijiu fermentation cellar (501#) located in Yibin Wuliangye Group Co., Ltd. (Yibin city, Sichuan Province, PR China; 28° 47′ N, 105° 36′ E). The mud sample was collected from the bottom of a fermentation pit. A total of 20 g pit mud was collected in a triangular flask containing 180 mL sterile water. The flask was evenly shaken and the sample was bathed at 80°C for 20 minutes. After gradient dilution in sterile water, the diluent was spread onto 54b medium agar plates. The ingredients of 54b medium are (per liter): 20 g glucose, 10 g yeast extract, 20 g calcium carbonate, and 20 g agar. The pH was adjusted to 7.0 before autoclaving. The plates were incubated for 5 days at 37 ℃ in an anaerobic system (Don Whitley Scientific M35) filled with a gas mixture of N₂/H₂/CO₂ (93:2:5). WLY-B-L2 ^T was isolated using gradient dilution and streak plate techniques. The strain was picked as a single colony and further purified by transferring to 54b agar plates three times. The purified strain was stored at -80℃ in tubes containing 20% glycerol solution.

After culturing for 5 days, WLY-B-L2^T genomic DNA was extracted using a DNA kit (Shenggong, Shanghai), following the manufacturer’s instructions, and purified as described by Lane [13]. The 16S rRNA gene sequence was amplified by using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3 ') [14]. The PCR product was inserted into the pEASY-T1 Cloning Vector and subsequently transformed in Escherichia coli DH5α competent cells (TianGen, Beijing, China) and sequenced using M13 forward primer and M13 reverse primer [15]. The sequence was 1436 bp long. A BLAST search of the 16S rRNA gene sequence against the GenBank database showed that WLY-B-L2^T was most closely related to Clostridium luticellarii FW431^T with 16S rRNA gene sequence similarities of 97.42%. Its 16S rRNA gene sequence similarity to other Clostridium type strains were lower than 98.65% [19]. The results indicated that strain WLY-B-L2^T is a member of the genus Clostridium. A phylogenetic tree based on the 16S rRNA gene sequence from strain WLY-B-L2^T and other closely related species, obtained from the EzTaxon server [16], was reconstructed using the neighbor-joining method [17] in MEGA version 7.0 [18] with bootstrap analysis of 1000 replications. Seventeen species were selected for the phylogenetic analysis. Distance algorithms, including interior-branch test and bootstrap method, were used for the phylogenetic tree construction to clarify the taxonomic status of the isolate. Phylogenetic analysis based on 16S rRNA gene showed that strain WLY-B-L2^T formed a distinct branch with C. luticellarii FW431^T in the neighbor-joining tree (Fig. 2).

According to the Ribosomal Database Project (RDP) (http://rdp.cme.msu.edu/classifier/classifier.jsp) analysis, WLY-B-L2^T belongs to Clostridium sensu stricto.

To support the results of the phylogenetic analysis and to get deeper taxonomic insights, the genome of WLY-B-L2^T was sequenced. Genome sequencing of WLY-B-L2^T was performed on the BGISEQ-500 platform [14] and the G + C content was determined from the whole sequence data [20]. The evolutionary relationships among bacteria can be studied by using orthologous genes of the core genome The inference of the corresponding phylogenetic tree offers deeper insights. Hence, a phylogenomic analysis was conducted based on the three core marker genes. The phylogenetic tree (Fig. 3) demonstrated a clear clustering of WLY-B-L2^T with the Clostridium sp.

At the Genome-to Genome Distance Calculation (GGDC) website (http://ggdc.dsmz.de/distcalc2.php), Genome sequence based digital DNA-DNA hybridization (dDDH) values were calculated using Formula 2, described by Meier-Kolthoff et al [20]. The digital DNA-DNA hybridization (dDDH) value between WLY-B-L2^T and Clostridium luticellarii FW431^T (NR_145907) was 28.10%. This value is below the threshold value of 70% dDDH species boundary, [21] thus revealing that WLY-B-L2^T does not represent any of these related species.

The genome sequence of WLY-B-L2^T was 3.88 Mb in size. The G + C content of the type strain was determined as 34.16 mol% while that of C. luticellarii FW431^T was 44.4 mol% [22].

We used Glimmer 3.02 software to annotate the genome of strain WLY-B-L2 ^T and predicted 3744 coding sequences. The KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Cluster of Orthologous Groups) and GO (Gene Ontology) databases were used for functional analysis of the predicted coding genes. Gene function analysis with KEGG showed majority of the genes were involved in carbohydrate metabolism (218 genes), amino acid metabolism (218), metabolism of cofactors and vitamins (154), energy metabolism (159), and membrane transport (87).

Cell morphology was observed under a light microscope (BH-2, Olympus, Japan) and by scanning electron microscopy (SU 8010, Hitachi, Japan) after incubation on Reinforced Clostridium Medium (RCM) [23] plates for 72 h [24] (Fig. 1). Gram staining was performed as described by Gerhardt et al. [25] in parallel with the KOH lysis method. In order to determine the optimal growth conditions, cultures of WLY-B-L2^T were inoculated in RCM for 7 days at following temperature: 4, 10, 15, 20, 25, 30, 37, 40, 45, and 50 ℃. The pH was varied between 3.0–10.0 at 1.0 pH unit intervals and NaCl concentration (w/v) was varied from 0–5% with increments of 1% and the growth was measured at 37℃. The different pH values were adjusted with sodium acetate buffer (pH 3.0–6.0), phosphate buffer (pH 6.0–8.0), Tris/HCl buffer (pH 8.0–9.0), and carbonate buffer (pH 9.0–10.0). Ethanol tolerance was measured at 0–10% ranges with 2% intervals. Growth at varied conditions such as temperature, pH, NaCl and ethanol was detected after 72 h, when the strain was in the exponential growth phase. The result showed that WLY-B-L2^T formed convex and round colonies with entire boundaries on RCM agar plates, like C. luticellarii FW 431^T. Colonies were yellow in color and had a diameter of 0.5–1.0 mm. The cells were mixed with 3% KOH (P816399-500g, Xilong Chemical) thoroughly on a glass slide (Cat No.7105, Mingzhu) and no gelling was observed within 5 to 60 s. The strain was strictly anaerobic, Gram-positive with straight or somewhat rod shaped cells which were 0.5–0.7 µm wide and 1.7–3.1 µm long, arranged singly or in pairs. It produced spores. The strain was able to grow at 15–45 ℃ and pH 5.0–8.0, with optimal growth at 37 ℃ and optimal pH 7.0. This is similar to C. luticellarii FW 431^T optimal growth temperature and pH. The two strains showed the same range of tolerance to NaCl. Optimal growth was at 1% (w/v) NaCl, and WLY-B-L2^T did not grow at NaCl concentration (w/v) greater than 4.0%. The two strains showed the same range of tolerance to ethanol as well and the ranges were 0–6%.

The Biolog Microbial Identification system (MicroStation, USA) was used to determine the dominant carbon sources that could be metabolized by WLY-B-L2^T and C. luticellarii FW 431^T. After 72 h of incubation in RCM broth, cells were centrifuged and washed twice with carbon-free Biolog buffer. The samples were suspended using the same buffer and adjusted to a turbidity of 65%. Then, a 100 ml aliquot of the cell suspension was added to each well of the Biolog AN MicroPlate panel as described by Ivanova et al. [26]. The AN plate was cultivated at 37 ℃ for 72 h in an anaerobic system filled with an anaerobic bag (Mitsubishi Gas Chemical Co. Inc., Japan). The results showed that, of the 95 tested carbon sources, the dominant compounds that could be metabolized by WLY-B-L2^T were D- galacturonic acid, methyl pyruvate, L-lactamine, L-alanyl-L-glutamine, L-alanyl-L-histidine, glycerol, pyruvate, L-alanyl-L-threonine, L-methionine, L-phenylalanine, L-valine + L- aspartic acid, L-serine, L-valine and thymidine. It was different in C. luticellarii FW431^T where the predominant carbon sources were D-galacturonic acid, DL-α-glycerophosphate, D-malate, methyl pyruvate, L-lactamine, D- fructose, etc. The other carbon sources that could be utilized by the two strains are mentioned in Table 1.

Table 1

Phenotypic characteristics of strain WLY-B-L2^T and a closely related phylogenetic neighbor in the genus *Clostridium.* Strains: 1, WLY-B-L2^T; 2, *C. luticellarii* FW431^T. Data are from the present study, except where indicated. +, Positive; −, negative; +^w, weakly positive.
Characteristics	1	2
Growth temperature range (℃)	15–45	15–45
Optimal temperature (℃)	37	37
Growth pH range	5–8	5–8
Optimal pH	7	7
Growth NaCl concentration range (%)	0–3	0–3
Optimal NaCl concentration (%)	1	1
Growth ethanol range (%)	0–6	0–6
G + C content (mol%)	34.2	44.4
Major fatty acids	C_16:0, anteiso-C_15:0, iso-C_15:0, C_14:0	C_16:0, C_14:0, C_15:0, C_18:0
Organic acid(s) in fermentation	Butyric acid	Butyric acid
Dominant carbon sources (Biolog AN system)
D-Galacturonic acid	+	+
DL-α-Glycerophosphate	+^w	+
D-Malate	-	+
Methyl Pyruvate	+	+
L-Lactamine	+	+
D-Fructose	+^w	+
Poly-β-hydroxybutyric acid	-	+
L-Malate	-	+
L-Alanyl-L-Glutamine	+	-
L-Fucose	+^w	+^w
Glucose-6-Phosphates	-	+^w
3-Methyl-D-Glucose	-	+^w
L-Alanyl-L-Histidine	+	+
Glycyl-L-Proline	-	+^w
Dulcitol	-	+
Glycerol	+	+
Methyl-α-D-Galactoside	+^w	+
Isomaltulose	+^w	+^w
D-Glucitol	+^w	+
Turanose	+^w	+
D-Methyl lactate	+^w	+
Pyruvate	+	+
L-Alanyl-L-Glutamine	+	+^w
L-Alanyl-L-Threonine	+	-
L-Methionine	+	+
L-Phenylalanine	+	+
L- Valine + L- Aspartic Acid	+	+^w
Uridine	+^w	+^w
L-Serine	+	+
2′-Deoxyadenosine	+^w	-
L-Threonine	-	+
L-Valine	+	-
Thymidine	+	+^w

The analysis of the organic acids and the metabolites produced by the strains in the RCM cultures were determined using HPLC (Agelent Venusil ASB) according to the previously described protocol [27]. The results showed that butyric acid was the major organic acid (588 mg/L) produced during fermentation by WLY-B-L2^T. It could also produce acetic acid. Similar results were obtained for C. luticellarii FW431^T. For these experiments, the cells were incubated at 37°C for 72 h on RCM under anaerobic condition using a anaerobic atmosphere generation bag. The growth curve of WLY-B-L2^T is shown in Fig. 4.

The cells were grown on RCM at 37 ℃ for 72 h for fatty acid methyl ester (FAME) identification. Cellular fatty acids were extracted, methylated and analyzed using the Sherlock Microbial Identification System (MIDI, USA) according to the manufacturer’s instructions. The results (Table 2) showed that the major FAMEs in WLY-B-L2^T were C_{16: 0} (24.6%), anteiso-C_{15: 0} (16.5%), iso-C_{15: 0} (14.1%), and C_{14: 0} (8.3%). This profile is significantly different from that of C. luticellarii FW 431^T which predominantly comprised C_16:0 (37.0%), C_14:0 (10.0%), C_15:0 (7.4%), and C_18:0 (6.5%).

Table 2

Comparative FAME profiles: Strains: 1, WLY-B-L2^T; 2, *C. luticellarii* FW431^T. −, negative. Data are from the present study.
FAME profiles	1	2
9:0 FAME	-	3.08
14:0 FAME	8.33	9.99
14:0 DMA	2.72	3.78
15:0 iso FAME	14.09	-
15:0 anteiso FAME	16.50	0.57
15:0 FAME	4.22	7.41
16:0 iso FAME	1.11	0.72
16:1 cis 9 FAME	1.97	-
16:0 FAME	24.61	37.03
16:0 DMA	1.73	0.54
17:0 iso FAME	3.04	-
17:0 anteiso FAME	4.19	0.88
17:0 cyc FAME	0.95	1.18
17:0 FAME	3.21	1.55
17:0 cyc DMA	2.58	4.45
18:2 cis 9,12 FAME	0.58	4.73
18:0 FAME	3.50	6.46
19 cyc 9,10/:1 FAME	-	3.04
19 cyc 11,12/:1FAME	1.59	4.00
19:0 cyc 9,10 DMA	0.26	5.52

Therefore, based on phenotypic, genotypic, and chemotaxonomic characteristics and phylogenetic analysis, the strain WLY-B-L2^T (CICC 25133^T = JCM 35127^T) is suggested to represent a novel species of the genus Clostridium, for which the name Clostridium aromatiproducens sp. nov. is proposed.

Clostridium aromatiproducens (a.ro.ma. pro.du'cens. N.L. n. aroma a fragrant scent; L. part. adj. producens producing; N.L. part. adj. aromatiproducens aroma-producing).

Cells are strictly anaerobic, Gram-positive, straight or rod-shaped, 0.5–0.7 µm wide and 1.7–3.1 µm long, arranged singly or in pairs. Colonies are circular, light yellow, convex and opaque with irregular edges on RCM plates after 72 h incubation. Growth occurs at 15–45 ℃ (optimum 37 ℃), at pH 5–8 (optimum pH 7), at NaCl concentrations of 0–3% (optimum 1%), and at ethanol concentrations of 0–6%. Produces butyric acid when cultured on RCM. Utilizes D-galacturonic acid, methyl pyruvate, L-lactamine, L-alanyl-L-glutamine, L-alanyl-L-histidine, glycerol, pyruvate, L-alanyl-L-threonine, L-methionine, L-phenylalanine, L-valine + L-aspartic acid, L-serine, L-valine and thymidine as the carbon sources. The predominant cellular fatty acids are C_{16: 0}, anteiso-C_{15: 0}, iso-C_{15: 0} and C_{14: 0}.

The type strain is Clostridium aromatiproducens WLY-B-L2^T (CICC 25133^T = JCM 35127^T), isolated from a mud cellar located at the Wuliangye 501^# Baijiu making workshop in Yibin region of Sichuan province, PR China. The G + C content of strain WLY-B-L2^T was 34.16 mol%. The GenBank accession number for the 16S rRNA gene sequence of the strain WLY-B-L2^T is LC631612 and the DDBJ/ENA/GenBank accession number for the draft genome is JAJJPB000000000.

The GenBank accession numbers for the 16S rRNA sequences of strain WLY-B-L2^T is LC631612. The whole genomes of strain WLY-B-L2^T has been deposited at GenBank under the accession number JAJJPB000000000.

SATB: strong- aroma-type baijiu; MIDI: Sherlock Microbial Identification System; HPLC: High Performance Liquid Chromatography; dDDH: digital DNA-DNA hybridization; PCR: Polymerase Chain Reaction; GGDC: Genome-to Genome Distance Calculation; RCM: Reinforced Clostridium Medium.

Authors contributors

Qingchun Luo: Writing-original draft (lead); Jia Zheng: Concept design, and reviewing (lead); Investigation (lead). Dong Zhao: Funding acquisition (equal); Resources (equal). Duotao Liu: Resources (equal).

Competing interest

Authors are required to disclose non-financial interests.

Ethic approval

This work did not include humans or animals.

Consent for publication

This work did not include details, images, or videos relating to an individual person.

Acknowledgements

The authors thank Dr. Chaolan Liu (Faculty of Antibiotics Research and Re-evaluation Key Laboratory of Sichuan Province, School of Pharmacy, Chengdu University) for the help to the identification of WLY-B-L2^T.and Dr. Lei Zai (China Center of Industrial Culture Collection, CICC) for the help to the nomenclature of WLY-B-L2^T.

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Clostridium aromaproducens sp. nov., isolated from pit mud used for producing Wuliangye baijiu

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