Arch Microbiol
DOI 10.1007/s00203-015-1180-6
ORIGINAL PAPER
Paenibacillus puernese sp. nov., a β‑glucosidase‑producing
bacterium isolated from Pu’er tea
Dan‑Dan Wang1 · Yeon‑Ju Kim1 · Van‑An Hoang1 · Ngoc‑Lan Nguyen1 ·
Priyanka Singh1 · Chao Wang1 · Deok Chun‑Yang1,2 
Received: 4 September 2015 / Revised: 11 December 2015 / Accepted: 17 December 2015
© Springer-Verlag Berlin Heidelberg 2015
Abstract  A Gram-staining-positive, endospore-forming,
aerobic, rod-shaped bacterium, designated as DCY97T, was
isolated from ripened Pu’er tea and was identified by using
a polyphasic approach. 16S rRNA gene sequence analysis
showed that strain DCY97T was closely related to Paeniba-
cillus dongdonensis KUDC0114T (98.0 %), Paenibacillus
oceanisediminis L10T (97.7 %), and Paenibacillus barci-
nonensis BP-23T (97.2 %). The phenotypic and chemotaxo-
nomic characteristics of strain DCY97T matched with the
characteristics of members belonging to the genus Paeniba-
cillus. The major identified polar lipids included phosphati-
dylglycerol, phosphatidylethanolamine, and diphosphati-
dylglycerol. The predominant quinone was MK-7. The
major fatty acids were anteiso-C15:0 (35.1 %), anteiso-C16:0
(19.0 %), and iso-C16:0 (13.9 %). The peptidoglycan cell
wall was composed of meso-diaminopimelic acids, alanine,
and d-glutamic acid. The genomic DNA G + C content
Communicated by Erko Stackebrandt.
The GenBank/EMBL/DDBJ accession number for the DCY97T
is KM403403.
Electronic supplementary material  The online version of this
article (doi:10.1007/s00203-015-1180-6) contains supplementary
material, which is available to authorized users.
* Yeon‑Ju Kim
yeonjukim@khu.ac.kr
* Deok Chun‑Yang
deokchunyang@yahoo.co.kr
1 microorganisms commonly isolated from various sources,
Department of Oriental Medicine Biotechnology
and Ginseng Bank, College of Life Science, Kyung Hee
University, Yongin 446‑701, Republic of Korea
2
Graduate School of Biotechnology, College of Life Science,
Kyung Hee University, Yongin  446‑701, Republic of Korea
was determined to be 46.7 mol%. The DNA–DNA related-
ness between strain DCY97T and Paenibacillus dongdon-
ensis KCTC 33221T, Paenibacillus oceanisediminis KACC
16023T, Paenibacillus barcinonensis KCTC 13019T were
27, 19, and 10 %, respectively. Based on the genotypic,
phenotypic, and chemotaxonomic characteristics, strain
DCY97T is considered as a novel species of the genus
Paenibacillus, for which the name Paenibacillus puernese
sp. nov. is proposed. The type strain is DCY97T (=KCTC
33596T = JCM 140369T).
Keywords  Paenibacillus puernese · Fermented Pu’er
tea · Biotransformation
Introduction
The genus Paenibacillus, a novel genus of the family Paeni-
bacillaceae, was first proposed by Ash et al. (in 1993). Based
on the phylogenetic analysis of 16S rRNA gene sequence,
the genus Paenibacillus was separated from the genus Bacil-
lus as rRNA group 3 bacilli (Ash et al. 1993) and latter was
emended by Shida et al. (1997) and Behrendt et al. (2010).
At the time of writing, 166 species of Paenibacillus have
been described (http://www.bacterio.net/c/panebacillus.
html). The genus Paenibacillus is commonly characterized
by rod-shaped, Gram-stain-positive, endospore-forming bac-
teria with the DNA G + C content of 39 to 62.6 mol% (Saha
et al. 2005; Vaz-Moreira et al. 2007; Sukweenadhi et al.
2014). Members of the genus Paenibacillus are widespread
including air (Rivas et al. 2005), alkaline soil (Yoon et al.
2005), rhizospheric soil (Cheong et al. 2005), fermented
Pu’er tea (Oh et al. 2008; Kim et al. 2009), ripened Pu’er tea
(Niu et al. 2015), and fresh water (Baik et al. 2011).
13

Pu’er tea is ripened tea leaves, which contains numer-
ous species belonging to the phylum: Proteobacteria, Fir-
micutes, Actinobacteria, Cyanobacteria, and Bacteroidetes
(Zhao et al. 2015). Nowadays, crude enzyme from many
microorganisms isolated from soil was used in the bio-
conversion of ginsenoside. However, these microorgan-
isms are not of food grade and restricted in the use of food
industry. Therefore, we attempted to isolate novel bacterial
strain from ripened Pu’er tea sample and check their abil-
ity for biotransformation of ginsenosides. In this study, we
reported a novel strain of the genus Paenibacillus, desig-
nated DCY97T, isolated from a ripened Pu’er tea sample
and exhibited biotransformation of ginsenoside Rb1 to gin-
senoside CK.
Materials and methods
Isolation and culture condition
Pu’er tea, which had been stored (post-fermented) since
2006 and packaged in 2013, was obtained from a manu-
facturer in Yunnan, China. We isolated the sample in
October 2013 by a serial dilution technique using sterile
saline [0.85 % (w/v) of NaCl]. One gram of sample was
suspended in 10 ml sterile water containing 0.85 % NaCl
and 0.8 % (w:v) peptone. Serial dilutions were prepared
up to 10−5 using the same solution. Subsequently, 100 μl
of 10−3 to 10−5 dilution was spread onto the Tryptic soy
agar (TSA, BD, USA), which was incubated at 30 °C for
5 days. Single colonies were purified by transferring to new
TSA plates. Among the isolates for 16S rRNA sequencing,
strain DCY97T was shown as a candidate for a novel strain
of the genus Paenibacillus. Therefore, we selected this
strain for further study. The isolate was routinely cultured
on TSA at 30 °C and preserved as a suspension in Tryptic
soy broth (TSB; BD, USA) with glycerol (25 %, w:v) at
−70 °C. Strain DCY97T has been deposited in the Korean
Collection for Type Cultures (as KCTC 33596T) and Japan
Collection of Microorganisms (as JCM 140369T). The type
strains Paenibacillus dongdonensis KCTC 33221T, Paeni-
bacillus oceanisediminis KACC 16023T, and Paenibacillus
barcinonensis KCTC 13019T were obtained from Korean
Collection for Type Cultures (KCTC) and Korean Agricul-
tural Culture Collection (KACC).
16S rRNA sequencing and phylogenetic construction
Genomic DNA extraction for 16S rRNA gene sequencing
was performed using a commercial genomic DNA extrac-
tion kit (Gene All Biotechnology, Republic of Korea).
The primer pairs 27F/1492R (Lane 1991) and 518F/800R
(Weisburg et al. 1991) were used to amplify the 16S rRNA
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Arch Microbiol
gene. The purified PCR product was sequenced by Geno-
tech (Daejeon, Republic of Korea) according to Kim et al.
(2005). The nearly full-length sequence (1462 bp) was
compiled using SeqMan software (DNASTAR). The 16S
rRNA gene sequence of strain DCY97T was compared
with those in NCBI database (http://blast.ncbi.nlm.nih.gov/
Blast.cgi) and EzTaxon-e server (http://eztaxon-e.ezbio-
cloud.net/) (Kim et al. 2012a, b) to determine pairwise sim-
ilarity. The multiple sequence alignments of DCY97T and
related type strains were performed by using the CLUSTAL
X program (Thompson et al. 1997). Phylogenetic trees
were then constructed using maximum likelihood (Felsen-
stein 1981) and maximum parsimony (Fitch 1972) as well
as the neighbor-joining method (Saitou and Nei 1987) with
the MEGA5 program. Bootstrap values were analyzed
based on 1000 replications (Felsenstein 1985).
Phenotypic characteristics analysis
Cell morphology and the presence of flagella were
detected by transmission electron microscopy (Carl Zeiss
LOE912AB) using cells grown on TSA for 1 day at 30 °C.
The type of Gram reaction status was determined by using
the Gram staining kit (bioMérieux, France). The occur-
rence of spores was checked by staining with malachite
green (Prescott and Harley 2001).
The temperature range for growth was tested in TSB
and TSA at 4, 10, 20, 28, 30, 34, 35, 37, and 45 °C. Tol-
erance for salinity was evaluated in modified TSB with
ingredients following formula of commercial products
(TSB, BD, USA) except for NaCl that was supplemented
with 0–15.0 % (w/v) NaCl, at 1.0 % intervals at 30 °C. This
experiment was carried out in triplicates. Growth of strain
DCY97T was determined over a pH range of 4–11 in TSB
(BD, USA) by adjusting the pH with citric acid–Na2HPO4
(for pH 4–6), NaHPO4–Na2HPO4 (for pH 7–8), glycine–
NaOH (for pH 9–10), and Na2HPO4–NaOH (for pH 11).
Growth on different media including R2A, TSA, Nutrient
Agar (NA, MB cell), Luria–Bertani (LB, MB cell) agar,
Potato Dextrose agar (PDA, MB cell), and MacConkey
Agar (Difco) was also tested at 30 °C for 7 days. Catalase
activity was determined by the production of oxygen bub-
bles in the presence of a 3 % (v/v) H2O2 solution. Oxidase
activity was tested using 1 % (w/v) N,N,N,N-tetramethyl-
p-phenylenediamine reagent (bioMérieux) according to the
manufacturer’s instructions. Nitrate reduction was tested in
nitrate broth containing 0.2 % KNO3 (Skerman 1967). Tri-
ple sugar iron agar was used to determine H2S production.
Indole production was analyzed using Kovács’s reagent in
1 % tryptone broth (Skerman 1967). Antibiotic susceptibil-
ity was tested on Mueller–Hinton (Difco) agar at 30 °C for
48 h using the disk diffusion method (Bauer et al. 1966).
The inhibition zones were interpreted according to the
Arch Microbiol
manufacturer’s instructions. The antibiotic disks (antibi-
otics per disk) used were as follows: penicillin G (10 U),
erythromycin (15 μg), cefazolin (30 μg), oleandomycin
(15 μg), ceftazidime (30 μg), vancomycin (30 μg), tetracy-
cline (30 μg), novobiocin (30 μg), carbenicillin (100 μg),
rifampicin (5 μg), and neomycin (30 μg).
Based on the pairwise 16S rRNA gene sequence similar-
ities of strain DCY97T, the closely related strains, P. dong-
donensis KCTC 33221T, P. barcinonensis KCTC 13019T,
and P. oceanisediminis KACC 16023T, were selected for
comparative study. Hydrolysis of esculin, starch, casein,
gelatin, DNA, Tween 20, and Tween 80 was also analyzed
as previously described by Cowan and Steel (1974). Addi-
tional biochemical characteristics were analyzed by using
API 50CHB, API 20NE, and ZYM according to the manu-
facturer’s instructions (bioMérieux).
Determination of DNA G + C content
The genomic DNA of strain DCY97T was extracted using an
Exgene TM Cell SV mini-kit (Gene All Biotechnology, Repub-
lic of Korea) according to the manufacturer’s instructions, puri-
fied by the method of Moore and Dowhan (1995) and degraded
enzymatically into nucleosides (nuclease P1 and alkaline phos-
phatase; Sigma) (Nguyen et al. 2015; Mesbah et al. 1989).
DNA–DNA relatedness
DNA–DNA hybridization experiments were performed
fluorometrically using photobiotin-labeled DNA probes
and microdilution wells as reported by Ezaki et al. (1989).
Hybridization was conducted in five replicates for each
sample, and the optimal hybridization temperature was
43 °C. The highest and lowest values obtained from each
sample were excluded, and the mean of the remaining three
values was used for determining the DNA–DNA related-
ness value.
Chemotaxonomic analysis
For the analysis of fatty acid, strain DCY97T and the three
related strains were grown on TSA at 30 °C for 24 h. The
cellular fatty acids were extracted, methylated, saponified,
and analyzed according to the standard protocol described
by the Sherlock Microbial Identification System (MIDI)
(Sasser 1990). Extracts were analyzed by gas chromato-
graph HP 6890 using the TSBA library, version 6.1.
For quinone analysis, cells were grown in TSB with aer-
ation at 160 rpm for 1 day at 30 °C, pelleted by centrifuga-
tion, and freeze-dried for quinone analysis. The cells were
extracted as described by Collins (1985).
Polar lipids of strain DCY97T and P. dongdonen-
sis KCTC 33221T were extracted and analyzed using
freeze-dried cell as described by Minnikin et al. (1984).
The peptidoglycans of strain DCY97T and P. dongdon-
ensis KCTC 33221T were hydrolyzed with 6 N HCl and
analyzed accordingly as described by Schleifer (1985).
The hydrolyzed peptidoglycans were analyzed by spot-
ting the samples on one-dimensional TLC plates (Merck
KGaA 20 × 20 cm). The TLC solvent [methanol/pyridine/
HCl (12 N)/water = (32/4/1/7, by vol)] was prepared 1 day
before running TLC as described by Schumann (2011).
Biotransformation of ginsenoside
Strain DCY97T was cultured in TSB, and 0.4 mg L−1 of
ginsenoside Rb1 was then added to the broth. The reaction
mixture was incubated in a shaking incubator (150 rpm) at
30 °C for 4 days (Kim et al. 2012a, b). Ginsenosides were
extracted by water-saturated n-butanol solution, followed
by TLC and HPLC analysis (Quan et al. 2011a, b).
Results and discussion
On the basis of 16S rRNA gene sequence similarity, the
closest type strains were P. dongdonensis KCTC 33221T
(98.0 %), P. oceanisediminis KACC 16023T (97.7 %), and
P. barcinonensis KCTC 13019T (97.2 %). The neighbor-
joining phylogenetic tree clearly showed the affiliation of
strain DCY97T within the genus Paenibacillus (Fig. 1),
which was also supported by the maximum likelihood tree
(Supplementary Fig. S1).
The biochemical and physiological characteristics of
strain DCY97T are shown in Table 1 and in the species
description. Strain DCY97T was susceptible to penicillin
G (P10, 10 units), vancomycin (VA30, 30 μg), neomycin
(N30, 30 μg), erythromycin (E15, 15 μg), rifampicin (RD5,
5 μg), tetracycline (TE30, 30 μg), cefazolin (KZ30, 30 μg),
carbenicillin (CAR100, 100 μg), and oleandomycin (OL5,
15 μg), but resistant to ceftazidime (CAZ30, 30 μg) and lin-
comycin (MY15, 15 μg). Compared to the closest strain P.
dongdonensis KCTC 33221T, strain DCY97T was positive
in fermentation of glucose, trypsin, naphthol-AS-BI-phos-
phohydrolase, N-Acetyl-β-glucosaminidase, glycerol, and
d-turanose.
The DNA G + C content of strain DCY97T was
46.7 mol%, which is consistent with those of other species
of the genus Paenibacillus, the DNA G + C contents of
which range from 39 to 62.6 mol% (Saha et al. 2005; Vaz-
Moreira et al. 2007; Sukweenadhi et al. 2014). The DNA–
DNA relatedness between strain DCY97T and P. dongdon-
ensis KCTC 33221T was 27 % and was below 20 % with
the other type strains. These values were significantly lower
than the threshold value of 70 % which is recommended
for species delineation (Wayne et al. 1987). Thus, strain
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 Arch Microbiol

Fig. 1  Neighbor-joining phylo-
genetic tree based on 16S rRNA
gene sequences showing the
relationships of strain DCY97T
with other Paenibacillus spe-
cies. Bootstrap values >50 %
based on 1000 replications are
shown at branching points.
Filled circles indicate that the
corresponding nodes were also
recovered in the tree generated
with the maximum parsimony
algorithm. Bacillus subtilis
DSM 10T (AJ276351) was used
as an outgroup. Bar 0.01 substi-
tutions per nucleotide position

DCY97T was clearly indicated to be a new species of the
genus Paenibacillus.
The major fatty acid compositions of DCY97T and the
three closest references strains are shown in Table 2. The
major cellular fatty acids of strain DCY97T were anteiso-
C15:0 (35.1 %), anteiso-C16:0 (19.0 %), and iso-C16:0
(13.9 %), which are similar with the other type strains of
the genus Paenibacillus (Shida et al. 1997). The predomi-
nant menaquinone of strain DCY97T was MK-7, and the
major polar lipids were phosphatidylethanolamine (PE),
phosphatidylglycerol (PG), diphosphatidylglycerol (DPG),
and one unidentified aminolipid (AL2). However, com-
pared to the closest related strain P. dongdonensis KCTC
33221T, strain DCY97 contained phosphatidylglycerol
(PG) as major polar lipid (Supplementary Fig. S3). The
peptidoglycan cell wall of strain DCY97T and strain P.
dongdonensis KCTC 33221T was similar by including
meso-diaminopimelic acids, alanine, and d-glutamic acid.
Overall, chemotaxonomic characteristics of strain DCY97T
are consistent to typical characteristics of members of the
genus Paenibacillus (Nguyen et al. 2015).
The HPLC profiles of ginsenoside Rb1 biotransforma-
tion using strain DCY97T are shown in Supplementary Fig.
S4. The peak for ginsenoside Rb1 disappeared, and a new
peak appeared with a retention time consistent with gin-
senoside compound K (CK). Hence, it is concluded that
strain DCY97T transformed ginsenoside Rb1 into ginseno-
side CK. Ginsenoside Rb1 as the major saponin in ginseng
root (about 1–3 %) is easy to be acquired; however, its oral
absorption is poor. In this regard, the biological usage rate
of ginsenoside Rb1 is limited. To improve the pharmaceuti-
cal efficacy, the major ginsenoside needs to be transformed
into minor ginsenoside CK (Quan et al. 2011a, b). The
minor ginsenoside CK can induce tumor cell apoptosis,
inhibit tumor metastasis, and restrain tumor invasion (Oh
et al. 2004; Wakabayashi et al. 1997, 1998). We suggested
that strain DCY97T had the potential to be applied for the
preparation of compound K.
Overall, the physiological, biochemical, and genotypic
tests results delineated strain DCY97T from other members
of the genus Paenibacillus. Therefore, strain DCY97T was
classified as a representative of novel species of the genus
Paenibacillus, for which the name Paenibacillus puernese
sp. nov has been proposed.
Description of Paenibacillus puernese sp. nov
Paenibacillus puernese (puer.nése. N.L. masc. adj. puer-
nese of or belonging to Pu’er tea, from which sample the
type strain was isolated).
Cells are Gram-staining-positive, catalase-positive,
oxidase-negative, rod-shaped (0.9–1.0 μm in width, 2.5–
2.8 μm in length), endospore-forming, aerobic, and motile
by means of peritrichous flagella. Colonies grown on TSA
are cream-colored, circular, and slightly convex. Cells
grow well on TSA, R2A, LA, and NA, but not on MRS and
MacConkey Agar. Growth occurs at 15–40 °C (optimum,

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Arch Microbiol

Table 1  Differential characteristics of strain DCY97T and the related Table 2  Cellular fatty acid profile of strain DCY97T and the related
species of genus Paenibacillus species of genus Paenibacillus
Characteristics 1 2 3 4 Fatty acid 1 2 3 4

Assimilation of (API 20NE) Saturated

 Reduction of nitrate + + + −  C14:0 3.94 3.97 1.79 3.08
 Fermentation of glucose + − + +  C16:0 18.99 14.34 13.23 14.88
 Hydrolysis of gelatin + + − + Branched chain
 Mannitol + + + −  iso-C14:0 7.15 8.48 4.9 6.85
 N-Acetyl-glucosamine − + + −  iso-C15:0 6.18 5.08 2.55 6.09
 Malic acid + + − −  iso-C16:0 13.86 12.01 17.83 14.71
Enzyme activity (API ZYM)  iso-C17:0 2.52 1.94 1.7 2.65
 Esterase + + − +  Anteiso-C15:0 35.12 41.1 38.52 39.69
 Trypsin + − − +  Anteiso-C17:0 3.21 2.66 4.57 4.2
 Naphthol-AS-BI-phosphohydrolase + − + +  C16:1ω11c ND tr 1.99 3.48
 β-Glucuronidase − + + − Hydroxy
 N-Acetyl-β-glucosaminidase + − + −  C18:0 3OH 7.15 8.5 11.79 2.9
Acid production from (API 50CHB)  Summed feature 2 tr tr 1.13 tr
 Glycerol + − + +
Strains: 1. DCY97T  ; 2. P. dongdonensis KCTC 33221T; 3. P. oceani-
 d-Arabinose − − + + sediminis KACC 16023T; 4. P. barcinonensis KCTC 13019T. All type
 Methyl β-d-xylopyranoside + + + − strains were collected after 24-h growth on TSA medium (Difco) at
 Methyl α-d-glucopyranoside − + + + 30 °C for fatty acid analysis. Summed feature 2 contained C12:0 alde-
 Sucrose + + + − hyde. All data are obtained from this study; fatty acids amount less
than 1.0 % in all strains were not listed
 Starch + + + −
ND not detected, tr trace amount (<1.0 %)
 Glycogen + + + −
 d-Turanose + − + −
 Potassium gluconate − + + − methyl α-d-glucopyranoside, N-acetylglucosamine, inu-
lin, d-melezitose, xylitol, d-lyxose, d-tagatose, d-fucose,
Strains: 1. DCY97T  ; 2. P. dongdonensis KCTC 33221T; 3. P. oceani-
l-fucose, d-arabitol, l-arabitol, potassium gluconate, potas-
sediminis KACC 16023T; 4. P. barcinonensis KCTC 13019T. +, posi-
tive result; −negative result. All results were obtained in this study sium 2-ketogluconate, and potassium 5-ketogluconate.
Based on the 20NE system, strain DCY97T is positive for
the reduction of nitrates, formation of glucose, hydrolysis
30 °C), at pH 5–11 (optimum, pH 6), and at 0–7 % NaCl of esculin, gelatin, β-galactosidase, assimilation of glu-
(optimum, 1 %). Strain DCY97T is able to hydrolyze cose, mannose, mannitol, maltose, potassium gluconate,
starch, Tween 20, Tween 80, gelatin, casein, and aescu- and malic acid, but is negative for indole production, argi-
lin, but not DNA. Production of H2S and indole is nega- nine dihydrolase, urease, and assimilation of arabinose,
tive. Based on the API ZYM system, DCY97T is positive N-acetyl-glucosamine, capric acid, adipic acid, trisodium
for alkaline phosphatase, esterase, esterase lipase, leucine citrate, and phenylacetic acid (Supplementary Table. S1).
arylamidase, trypsin, α-chymotrypsin, acid phosphatase, The DNA G + C content is 46.7 mol%. The major cel-
naphthol-AS-BI-phosphohydrolase, α-galactosidase, lular fatty acids are anteiso-C15:0, anteiso-C16:0, and iso-
β-galactosidase, α-glucosidase, β-glucosidase, and C16:0. The predominant quinone is MK-7. The major polar
N-acetyl-β-glucosaminidase activity; however, it is nega- lipids are phosphatidylglycerol, phosphatidylethanolamine,
tive for lipase, valine arylamidase, cystine arylamidase, diphosphatidylglycerol, and one unidentified aminolipid.
β-glucuronidase, α-mannosidase, and α-fucosidase. Acid The peptidoglycan cell wall contains meso-diaminopimelic
is produced from glycerol, l-arabinose, d-ribose, d-xylose, acids, alanine, and d-glutamic acid as the major amino
methyl β-d-xylopyranoside, d-galactose, d-glucose, d-fruc- acids.
tose, d-mannose, d-mannitol, amygdalin, arbutin, escu- The type strain DCY97T (=KCTC 33596T  = JCM
lin, salicin, d-cellobiose, d-lactose, d-melibiose, sucrose, 140369T) was isolated from Pu’er tea.
d-trehalose, d-raffinose, starch, glycogen, gentiobiose, and
d-turanose; however, it is not produced from erythritol, Acknowledgments  This research was supported by Korea Institute
of Planning & Evaluation for Technology in Food, Agriculture, For-
d-arabinose, l-xylose, d-adonitol, l-sorbose, l-rhamnose,
estry & Fisheries (KIPET No. 313038-03-2-SB010).
dulcitol, inositol, D-sorbitol, methyl α-d-mannopyranoside,

13
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