Flavobacterium supellecticarium sp. nov., isolated from an abandoned construction timber

Abstract

A bacterial strain CC-CTC003T was isolated from a synthetic wooden board. Cells of strain CC-CTC003T were Gram-stained-negative, rod-shaped, motile by gliding and formed yellow colonies. Optimal growth occurred at 25 ºС, pH 7 and in the presence of 1 % NaCl. The phylogenetic analyses based on 16S rRNA genes revealed that strain CC-CTC003T belonged to the genus Flavobacterium and was most closely related to Flavobacterium cerinum (95.3 % sequence identity), Flavobacterium maris (94.9 % sequence identity), Flavobacterium qiangtangense (94.8 %) and Flavobacterium subsaxonicum (94.7 %) and had less than 94.7 % sequence similarity to other members of the genus. Average nucleotide identity (ANI) values between strain CC-CTC003T and the type strains of other closely related species were 70.1–74.1 %. The digital DNA–DNA hybridization (dDDH) with F. cerinum was 19.4 %. Strain CC-CTC003T contained C15:0, iso-C15:0, iso-C15:0 3-OH, iso-C17:0 3-OH, summed feature 3 (C16:1 ω6c / C16:1 ω7c) and summed feature 9 (C16:0 10-methyl / iso-C17:1 ω9c) as the predominant fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, four uncharacterized aminophospholipids, two aminolipids and one unidentified glycolipid. The major polyamine was sym-homospermidine and contained MK-6 as major isoprenoid quinone. The DNA G+C content of the genomic DNA was 39.2 mol%. On the basis of the phylogenetic inference and phenotypic data, strain CC-CTC003T should be classified as a novel species, for which the name Flavobacterium supellecticarium sp. nov. is proposed. The type strain is CC-CTC003T (=BCRC 81146T=JCM 32838T).

Full text

3731
Flavobacterium supellecticarium sp. nov., isolated from an
abandoned constructiontimber
Shih- YaoLin1, Wen- MingChen2, Guan- HuaHuang1, AsifHameed1, Chun- TseChang1, Chia- FangTsai1 and Chiu-
ChungYoung1,3,*
TAXONOMIC DESCRIPTION
Lin etal., Int. J. Syst. Evol. Microbiol. 2020;70:3731–3739
DOI 10.1099/ijsem.0.004227
Author aliations: 1Department of Soil and Environmental Sciences, College of Agriculture and Natural Resources, National Chung Hsing University,
Taichung City, Taiwan, ROC; 2Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung University of Science and Technology,
Kaohsiung City, Taiwan, ROC; 3Innovation and Development Center of Sustainable Agriculture, National Chung Hsing University, Taichung, Taiwan, ROC.
*Correspondence: Chiu- Chung Young, ccyoung@ mail. nchu. edu. tw
Keywords: average nucleotide identity; digital DNA–DNA hybridization; Flavobacterium supellecticarium; Flavobacterium pallidum; Flavobacterium
qiangtangense.
Abbreviations: AL, aminophospholipid; ANI, average nucleotide identity; APL, aminophospholipid; BCRC, Bioresource Collection and Research
Center; dDDH, digital DNA–DNA hybridization; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen; FAME, fatty acid methyl esters;
GL, glycolipid; JCM, Japan Collection of Microorganisms; KCTC, Korean Collection for Type Cultures; MA, marine agar; MIDI, Microbial Identification
System; MK-6, Menaquinone 6; NA, nutrient agar; NB, nutrient broth; ND, no data available; PE, phosphatidylethanolamine; PGAP, Prokaryotic Genome
Annotation Pipeline; PL, phospholipid; R2A, Reasoner’s 2A agar; tr, trace; TSA, tryptic soy agar; UBCG, upto- date bacterial core gene; w, weak positive
reaction.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and the draft genome of Flavobacterium supellecticarium strain CC- CTC003T are
MH028244 and SSNZ00000000, respectively.
004227 © 2020 The Authors
Abstract
A bacterial strain CC- CTC003T was isolated from a synthetic wooden board. Cells of strain CC- CTC003T were Gram- stain-
negative, rod- shaped, motile by gliding and formed yellow colonies. Optimal growth occurred at 25 °C, pH 7 and in the pres-
ence of 1 % NaCl. The phylogenetic analyses based on 16S rRNA genes revealed that strain CC- CTC003T belonged to the genus
Flavobacterium and was most closely related to Flavobacterium cerinum (95.3 % sequence identity), Flavobacterium maris (94.9 %
sequence identity), Flavobacterium qiangtangense (94.8 %) and Flavobacterium subsaxonicum (94.7 %) and had less than 94.7 %
sequence similarity to other members of the genus. Average nucleotide identity (ANI) values between strain CC- CTC003T and
the type strains of other closely related species were 70.1–74.1 %. The digital DNA–DNA hybridization (dDDH) with F. cerinum
was 19.4 %. Strain CC- CTC003T contained C15 : 0, iso- C15 : 0, iso- C15 : 0 3- OH, iso- C17 : 0 3- OH, summed feature 3 (C16 : 1 ω6c / C16 : 1 ω7c)
and summed feature 9 (C16 : 0 10- methyl / iso- C17 : 1 ω9c) as the predominant fatty acids. The polar lipid profile consisted of phos-
phatidylethanolamine, four uncharacterized aminophospholipids, two aminolipids and one unidentified glycolipid. The major
polyamine was sym- homospermidine and contained MK-6 as major isoprenoid quinone. The DNA G+C content of the genomic
DNA was 39.2 mol%. On the basis of the phylogenetic inference and phenotypic data, strain CC- CTC003T should be classified as
a novel species, for which the name Flavobacterium supellecticarium sp. nov. is proposed. The type strain is CC- CTC003T (=BCRC
81146T=JCM 32838T).
INTRODUCTION
e genus Flavobacterium, belonging to the family Flavobac-
teriaceae of the phylum Bacteroidetes, was rst described by
Bergey et al. [1] and subsequently emended by Bernardet et al.
[2], Dong et al. [3], Kang et al. [4] and Kuo et al. [5]. At the time
of writing, there are about 211 validly named members of the
genus (LPSN; www. bacterio. net/ avobacterium. html), with
Flavobacterium aquatile as the type species of the genus [1, 6]
( www. bacterio. net/ avobacterium. html). Cells of members of
the genus Flavobacterium are typically Gram- stain- negative,
non- spore- forming, yellow- pigmented and rod- shaped
bacteria that are non- motile or motile. Chemotaxonomically,
members of this genus contain C15 : 0, iso- C15 : 0, iso- C15 : 1 G, iso-
C15 : 0 3OH, summed feature 3 (C16 : 1 ω6c / C16 : 1 ω7c), iso- C16 : 0
3OH, iso- C17 : 1 ω9c and iso- C17 : 0 3OH as the predominant
fatty acids. Phosphatidylethanolamine is a major polar lipid
among members of this genus [3]. Menaquinone 6 (MK-6)
is the major respiratory quinone and sym- homospermidine
is the major polyamine [7] and DNA G+C contents within
30–52 mol% [8].
e present study describes extensive taxonomic investiga-
tion of a presumably novel bacterium (designated strain

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Lin etal., Int. J. Syst. Evol. Microbiol. 2020;70:3731–3739
CC- CTC003T) based on analyses of its 16S rRNA gene
sequencing (accession number: MH028244), whole- genome
sequencing (accession number: SSNA00000000), physi-
ological, biochemical and chemotaxonomic analyses [9, 10].
ISOLATION AND ECOLOGY
e construction timber was obtained from an abandoned
piece of furniture (synthetic wooden board) in the University
campus (National Chung Hsing University, 24°12′ N, 120°68′
E), Taichung City, Taiwan. Samples were stored in black
centrifuge tubes and processed for the analysis of bacterial
diversity. Bacteria were isolated using the standard serial
dilution- plating technique on nutrient agar (NA, Hi- Media)
and Reasoner’s 2A (R2A) agar (BD Difco) aer aerobic
incubation at 30 °C for 3 days. A light yellow- colour colony
(dened as strain CC- CTC003T) was selected, routinely sub-
cultured under the same conditions on NA and stored at
−80 °C in nutrient broth (NB, Hi- Media) supplemented with
30 % (v/v) glycerol for a long- term preservation.
PCR ASSAYS AND GENE PHYLOGENY
Bacterial genomic DNA was isolated by using UltraClean
Microbial Genomic DNA Isolation Kit (Qiagen) following
the manufacturer’s instructions and used for further study.
e 16S rRNA gene was amplied with bacterial universal
primers 8F and 1541R [11], the amplied gene fragments
were screened by 2.0 % (w/v) agarose gel electrophoresis. PCR
products were sequenced using the Bigdye terminator kit [12]
and ABI 3730 Genetic Analyzer (Applied Biosystems). e
DNA fragments were assembled using the Vector NTI 9.0
soware (IBI) and deposited in GenBank using Bankit.
Identication of phylogenetic neighbours and calculation of
pair- wise 16S rRNA gene sequence similarity were achieved
using the EzBioCloud server ( www. ezbiocloud. net/) [13]
and GenBank database. e collected sequence was aligned
through _ (1.83) program [14]. e phylogenetic
analysis was computed with the  X soware [15] by
using the Kimura two- parameter model. e phylogenetic
trees were reconstructed by using neighbour- joining [16],
maximum- likelihood [17] and maximum- parsimony [18]
methods based on the 16S rRNA sequence. Evolutionary
distance matrix was calculated using the correction of Jukes
and Cantor [19] and evaluated by bootstrap analyses [20] aer
1000 replications.
Sequence identity calculations of the 16S rRNA gene (1511
nt, accession number: MH028244) indicated that strain
CC- CTC003
T
appeared to belong to the genus Flavobacte-
rium, being most related to Flavobacterium cerinum 1E403T
(95.3 % similarity), Flavobacterium maris KMM 9535
T
(94.9 %
similarity), followed by Flavobacterium qiangtangense F3T
(94.8 %), Flavobacterium subsaxonicum WB 4.1-42T (94.7 %),
and lower sequence similarity to other species. e sequence
similarity of 16S rDNA was also related to some unculturable
colonies in GenBank, such as Flavobacterium sp. W29 (acces-
sion number: AY770969, 99.4 % similarity), Flavobacterium
sp. 7A6 (accession number: DQ298762, 96.5 % similarity),
Flavobacterium sp. 3A13 (accession number: DQ298760,
96.0 % similarity), Flavobacterium sp. Z1 (accession number:
CP012388, 95.3 % similarity). e results of these analyses
suggested that strain CC- CTC003T might belong to the
genus Flavobacterium, the lower sequence similarity values
suggested that strain CC- CTC003T possibly represents a
novel species since sequence similarity values less than
the threshold (<98.7 %) is considered as strong evidence at
species- level [9, 21].
e phylogenetic neighbourhood of strain CC- CTC003T
was constructed as evidenced through neighbour- joining,
maximum- likelihood and maximum- parsimony algorithms.
In the neighbour- joining tree based on nearly complete
16S rRNA gene sequence of strain CC- CTC003T and other
closely related Flavobacterium species, strain CC- CTC003T
formed a phylogenetic cluster with Flavobacterium pallidum
(93.8 %), and separated another clade with Flavobacterium
qiangtangense (94.6 %), Flavobacterium cerinum (95.3 %),
Flavobacterium subsaxonicum (94.5 %), Flavobacterium
aquatile (94.5 %, type species of the genus Flavobacterium),
Flavobacterium niveum (93.4 %), and Flavobacterium hauense
(94.2 %) within other Flavobacterium species (Fig.1). Regard-
less of multiple evolutionary comparisons, similar topology
was obtained in all phylogenetic trees, which indicated that
novel strain CC- CTC003
T
is most closely related to Flavobac-
terium species.
WHOLE GENOME SEQUENCE STUDIES
Genome features
To further investigate the taxonomic rank of strain
CC- CTC003T, a dra genome sequence was prepared by
Welgene Biotech Co., Ltd. (Taipei, Taiwan) using the MiSeq
sequencer platform (Illumina) following the manufacturer’s
protocols. e resulting reads were quality trimmed to the
Q20 condence level. e dra genome was assembled using
SPAdes (version 3.12.0) [22]. In this study, 30 contigs were
obtained from the dra genome of strain CC- CTC003T (acces-
sion number: SSNA00000000), with an average coverage of
438× and an N50 size of 974 477 bp. e estimated genome
size was 3.8 Mb, with an average G+C content of 39.2 %. Gene
prediction and annotation by the NCBI Prokaryotic Genome
Annotation Pipeline (PGAP) resulted in the identication
of 3413 protein encoding genes, three rRNA genes and 53
tRNA genes.
OrthoANI analysis and digital DNA–DNA
hybridization (dDDH)
Species identication by ribosomal MLST was performed
form the PubMLST. org website (https:// pubmlst. org/), and
the predicted taxonomy was within the genus Flavobacterium
with 100 % support. e estimated genome- sequence- based
digital DNA–DNA hybridization (dDDH) values were calcu-
lated as described by Meier- Koltho et al. [23]. In addition,
the average nucleotide identity (ANI) values were calculated
by OrthoANI analysis [24] between the genome of strain

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Flavobacterium hercynium DSM 18292T (MUGW01000033)
Flavobacterium chryseum CCM 8826T (MH100900)
Flavobacterium succinicans LMG 10402T (JATV01000001)
Flavobacterium chungbukense CS100T (HM627539)
Flavobacterium foetidum CJ42T (KY056226)
Flavobacterium frigoris DSM 15719T (jgi.1107976)
Flavobacterium tegetincola DSM 22377T (AUDN01000041)
Flavobacterium ovatum W201ET (KY352658)
Flavobacterium phocarum SE14T (KX911210)
Flavobacterium collinsense 4-T-2T (KM288594)
Flavobacterium buctense T7T (KP744923)
Flavobacterium aquatile LMG 4008T (JRHH01000003)
Flavobacterium aciduliphilum JJ013T (JN712178)
Flavobacterium chungnamense ARSA-103T (GU295971)
Flavobacterium koreense ARSA-42T (GU295967)
Flavobacterium supellecticarium CC-CTC003T
(MH028244)
Flavobacterium pallidum HYN0049T (CP029187)
Flavobacterium qiangtangense F3T (KF726983)
Flavobacterium cerinum 1E403T (MH209250)
Flavobacterium subsaxonicum WB 4.1-42T (JRLY01000044)
Flavobacterium hauense BX12T (JX066803)
Flavobacterium niveum TAPW14T (LT703450)
Flavobacterium cheniae NJ-26T (EF407880)
Flavobacterium cucumis DSM 18830T (jgi.1107703)
Flavobacterium piscinae ICH-30T (MF801369)
Flavobacterium orientale SP3T (KT985058)
Flavobacterium ponti GSW-R14T (GQ370387)
Flavobacterium maris KMM 9535T (AB973429)
Flavobacterium ahnfeltiae 10Alg 130T (KC247342)
Flavobacterium haoranii LQY-7T (GQ988780)
Flavobacterium urocaniciphilum DSM 27078T (jgi.1108040)
Flavobacterium sediminilitoris YSM-43T (MF595513)
Flavobacterium aquimarinum Dol 15-39T (KY612936)
Flavobacterium squillarum CMJ-5T (JQ029111)
Flavobacterium fontis DSM 25660T (jgi.1107739)
Flavobacterium lutivivi HQQT (KT215492)
Flavobacterium columnare IFO 15943T (AB078047)
Flavobacterium inkyongense IMCC27201T (KX025140)
Flavobacterium verecundum TTM-46T (LN849950)
Flavobacterium vireti THG-SM1T (KM576853)
Flavobacterium terrae DSM 18829T (jgi.1107701)
Flavobacterium suncheonense GH29-5T (JRLW01000007)
Flavobacterium pedocola UCM-R36T (KU052688)
Flavobacterium limnosediminis JC2902T (AVGG01000026)
Flavobacterium saliperosum S13T (AVFO01000001)
Flavobacterium cauense R2A-7T (AVBI01000023)
Myroides odoratus DSM 2801T (M58777)
100
100
100
66
100
99
100
99
70
100
61
99
98
99
99
99
85
58
82
55
91
66
73
55
82
67
74
73
0.010
Fig. 1. Phylogenetic analysis based on nearly complete 16S rRNA gene sequence of strain CC- CTC003T and other closely related species
of the genus Flavobacterium. Distances and clustering were calculated by using the neighbour- joining method with the software package
 version 6. The open circles indicate that the corresponding nodes were recovered with maximum- likelihood; the filled circles
indicate that the corresponding nodes were also recovered in the tree reconstructed based on the maximum- likelihood and maximum-
parsimony algorithms. Bootstrap values (>50 %) based on 1000 replications are listed as percentages at the branching points.

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Lin etal., Int. J. Syst. Evol. Microbiol. 2020;70:3731–3739
CC- CTC003T and the related type strains of genus Flavobac-
terium whose whole genome sequences are publicly available
were obtained from GenBank.
e results showed that the ANI values were 70.0–74.1 %
(n=13) compared to the type strains of Flavobacterium acidu-
liphilum, Flavobacterium aquatile, Flavobacterium cheniae,
Flavobacterium cucumis, Flavobacterium fontis, Flavobac-
terium frigoris, Flavobacterium glaciei, Flavobacterium
hercynium, Flavobacterium limnosediminis, Flavobacterium
pallidum, Flavobacterium subsaxonicum, Flavobacterium
succinicans and Flavobacterium tegetincola. e ANI value
between strain CC- CTC003T and Flavobacterium cerinum
was 62.3 %, subsequently, F. cerinum showed lower ANI
values 61.1–64.3 % to other Flavobacterium species (n=13).
e result showed that ANI values were lower than the cut- o
values (95–96 %) previously proposed for species delimitation
[9, 10], indicating that strain CC- CTC003T represents a new
species within the genus Flavobacterium.
Gene annotation and up-to-date bacterial core gene
(UBCG) analysis
Gene annotation was applied from the RAST Annotation
Server (http:// rast. theseed. org), the result was compared to
the most- related species. In order to investigate the relation-
ships among strain CC- CTC003T and related Flavobacterium
species, the genome of strain CC- CTC003
T
and the related
type strains of genus Flavobacterium (n=32) whose whole
genome sequences are publicly available were obtained from
GenBank. An up- to- date bacterial core gene set (UBCG) and
pipeline was utilized for phylogenetic tree reconstruction as
described by Na et al. [25].
Flavobacterium cauense R2A-7T
Flavobacterium saliperosum CGMCC_1.3801T
Flavobacterium limnosediminis JC2902T
Flavobacterium enshiense DK69T
Flavobacterium suncheonense DSM_17707T
Flavobacterium supellecticarium CC-CTC003T
Flavobacterium subsaxonicum DSM 21790T
Flavobacterium arcticum SM1502T
Flavobacterium pallidum HYN0049T
Flavobacterium cerinum 1E403
T
Flavobacterium fontis DSM 25660T
Flavobacterium lacus CGMCC 1.12504T
Flavobacterium swingsii DSM 21789T
Flavobacterium psychrophilum ATCC 49418T
Flavobacterium tegetincola DSM 22377T
Flavobacterium succinicans DSM 4002T
Flavobacterium psychrolimnae LMG 22018T
Flavobacterium weaverense DSM 19727T
Flavobacterium glycines Gm-149T
Flavobacterium chungangense LMG 26729T
Flavobacterium saccharophilum DSM 1811T
Flavobacterium hercynium DSM 18292T
Flavobacterium croceum DSM 17960T
Flavobacterium aciduliphilum DSM 25663T
Flavobacterium aquatile LMG 4008T
Flavobacterium urocaniciphilum DSM 27078T
Flavobacterium terrigena DSM 17934T
Flavobacterium gelidilacus DSM 15343T
Flavobacterium cucumis DSM 18830T
Flavobacterium columnare ATCC 23463T
Flavobacterium terrae DSM 18829T
Myroides odoratus DSM 2801T
92
46
92
92
52
92
67
9
92
92
92
88
81
61
67
64
32
92
86
87
87
73
92
92
38
20
22
15
4
0.50
Fig. 2. Phylogenetic tree inferred using UBCGs (concatenated alignment of 92 core genes). Gene support indices (GSIs) values are given
at branching points. Bar, 0.05 substitution per position.

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Lin etal., Int. J. Syst. Evol. Microbiol. 2020;70:3731–3739
e phylogenetic tree based on the coding sequences of 92
protein clusters showed that strain CC- CTC003T formed
a phylogenetic cluster with Flavobacterium suncheonense,
Flavobacterium enshiense, Flavobacterium limnosediminis,
Flavobacterium saliperosum, Flavobacterium cauense, and
formed a distinct phylogenetic lineage with Flavobacterium
subsaxonicum, Flavobacterium pallidum and Flavobacterium
cerinum, which supported that strain CC- CTC003T should be
assigned to the genus Flavobacterium (Fig.2).
Physiology, phenotypic characterization and
chemotaxonomy
For taxonomic purposes, type strains of Flavobacterium
aquatile BCRC 80432T (type species of the genus Flavobacte-
rium), Flavobacterium maris KCTC 42093T, Flavobacterium
qiangtangense JCM 19739T, Flavobacterium subsaxonicum
DSM 21790
T
were purchased from Bioresource Collection
and Research Centre (BCRC), Korean Collection for Type
Cultures (KCTC), Japan Collection of Microorganisms (JCM)
and Deutsche Sammlung von Mikroorganismen und Zellkul-
turen (DSMZ), respectively. For direct comparative analysis,
strain CC- CTC003T and reference strains were analysed
under the same conditions on NA at 30 °C, unless specied
otherwise.
Colony morphology and the morphology of cells were investi-
gated using the colonies/cells grown on NA for 2 days at 30 °C.
e cell morphology and presence of agellum were observed
by transmission electron microscopy (JEOL JEM-1400) aer
cells had been negatively stained with 0.2 % uranyl acetate
(Fig.3). Catalase activity was determined by assessing bubble
production by cells in 3 % (v/v) H2O2 and oxidase activity
was determined by using 1 % (w/v) N,N,N,N,-tetramethyl-
1,4- phenylenediamine reagent (bioMérieux). e presence
of exirubin- type pigments was investigated using a 20 %
(w/v) KOH solution and the presence of capsule (extracellular
glycan) was evaluated by the Congo red test, according to
the minimal standards for describing novel taxa in the family
Flavobacteriaceae [26].
Growth of the strain CC- CTC003T was also tested on tryptic
soy agar (TSA, Difco), marine agar (MA, Difco) and Reason-
er’s 2A (R2A) agar (BD Difco) to determine its morphological
characteristics. Gram- staining reaction was investigated using
standard methods [27], and bacterial motility was observed
by light microscopy (model A3000, Zeiss). Anaerobic growth
was tested on NA supplemented with potassium nitrate
(0.1 %, w/v) using an anaerobic chamber (COY) lled with
H2:CO2:N2 (5 : 19 : 76) gas. Hydrolysis of gelatin, skimmed
milk, starch (1 %), Tweens 20, 40, 60 and 80 (1 %) and DNase
test (Hi- Media) were determined as described previously
using nutrient agar as basal media [28, 29]. H2S production
was checked on both 3 g l–1 Na2S2O3 and 0.1 g l–1 cysteine.
Growth at dierent temperatures was monitored in NB
at 4, 15, 20, 25, 30, 37 and 40 °C with a 48 h incubation
period (4 and 15 °C with 72 h). e requirement for NaCl
was determined using NB containing 0–10 % NaCl (in 1 %
increments). Cell growth at various pH (pH 4.0–10.0, in
intervals of 1.0 pH unit) was examined using the following
buer systems: 0.1 M citric acid/0.1 M trisodium citrate for
pH 4.0–5.0; 0.2 M Na2HPO4/0.2 M NaH2PO4 for pH 6.0–8.0
and 0.1 M NaHCO3/0.1 M Na2CO3 for pH 9.0–10.0. Oxida-
tion of carbohydrates was determined by using Biolog GNII
MicroPlate system (Biolog). Nitrate reduction, indole produc-
tion, activities of β- galactosidase and urease, hydrolysis of
aesculin and gelatin, assimilation of dierent substrates were
tested with API 20NE strips (bioMérieux). API 20NE and
API ZYM strips (bioMérieux) were used to determine the
physiological and biochemical properties. All experiments
conducted using commercial kits were performed according
to the manufacturer’s instructions. Strain CC- CTC003T
showed several distinct physiological and biochemical
characteristics that distinguished itself from other members
of the genus Flavobacterium. e detailed results from the
phenotypic and biochemical analyses are provided in Table1
and in the species description.
Fatty acid profile
For the investigation of the chemotaxonomic characteristics,
cell biomass of strain CC- CTC003T and reference strains
were harvested at a similar physiological age with similar
growth kinetics. For analysis of cellular fatty acids, strains
were grown on R2A medium (pH 7.0) at 30 °C for 48 h (same
conditions as in our previous studies). Cells were harvested in
the mid- exponential growth phase and subjected to saponi-
cation, methylation and extraction [30]. Fatty acid methyl
esters (FAME) were extracted and analysed according to the
standard protocol [31] of the Microbial Identication System
(MIDI) [32]. Identication and comparison were made by
using the Aerobe (RTSBA6) database of the MIDI System
(Sherlock version 6.0).
e predominant fatty acids (>5 %) in strain CC- CTC003
T
were C15 : 0 (7.8 %), iso- C15 : 0 (15.3 %), iso- C15 : 0 3- OH (5.8 %),
iso- C17 : 0 3- OH (13.6 %), C16 : 1 ω6c / C16 : 1 ω7c (15.2 %) and
Fig. 3. Transmission electron micrograph of negatively stained cells of
strain CC- CTC003T after growth in nutrient broth at 30 °C for 48 h. Bar
0.5 µm.

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Lin etal., Int. J. Syst. Evol. Microbiol. 2020;70:3731–3739
C16 : 0 10- methyl / iso- C17 : 1 ω9c (7.1 %). e composition was
similar among strains CC- CTC003T and reference species
of the genus Flavobacterium that contained C15 : 0, iso- C15 : 0,
iso- C15 : 0 3- OH, summed feature 3 (C16 : 1 ω6c / C16 : 1 ω7c) and
iso- C17 : 0 3OH as the predominant fatty acids. Nonetheless, the
higher composition of C
15 : 0
, iso- C
13 : 0
, iso- C
14 : 0
, anteiso- C
15 : 0
,
iso- C16 : 0, summed feature 3 (C16 : 1 ω6c / C16 : 1 ω7c); and lower
composition of iso- C
15 : 0
, iso- C
17 : 0
3- OH, summed feature 9
(C16 : 0 10- methyl / iso- C17 : 1 ω9c) was able to be distinguishing
strain CC- CTC003T from F. mar is KCTC 42093T and other
related tested species (Table2).
Polyamine profile and respiratory system
Polyamines were extracted as described by Scherer and
Kneifel [33], and analysed by HPLC. e dansyl derivatives
were separated by using a Hitachi L-2130 equipped with a
Hitachi L-2200 autosampler, Hitachi L-2485 uorescence
detector (excitation at 360 nm and emission at 520 nm), and
a reverse- phase C18 column (Phenomenex Synergi Fusion-
RP80, 250×4.60 mm, 4 µm particle size). e isoprenoid
quinones of strain CC- CTC003T were extracted and puri-
ed by the methods according to the method of Minnikin et
al. [34] and analysed by HPLC as described by Collins [35].
e only respiratory quinone detected was menaquinone 6
(MK-6) and the major polyamine is sym- homospermidine.
Two-dimensional TLC for polar lipids
For analysis of polar lipids, cells of strain CC- CTC003T were
cultivated in NA for 3 days at 30 °C. Polar lipids were extracted
and analysed by two- dimensional TLC [34], and total lipids
proles were stained with 10 % ethanolic molybdatophos-
phoric acid; aminolipids were detected with a 0.2 % (w/v)
solution of ninhydrin in butanol; phospholipids were detected
with Dittmer and Lester’s Zinzadze reagent; glycolipids were
detected with α- naphthol spray reagent.
Table 1. Distinguished characteristics of selected Flavobacterium species
Strain: 1, CC- CTC003T; 2, Flavobacterium aquatile BCRC 80432T (type species of the genus Flavobacterium); 3, Flavobacterium subsaxonicum DSM 21790T;
4, Flavobacterium qiangtangense JCM 19739T; 5, Flavobacterium maris KCTC 42093T; 6, Flavobacterium cerinum KCTC 62960T [36]. ＋, Positive reaction;
－, negative reaction; , no data available; w, weak positive reaction.
Characteristic 1 2 3 4 5 6
Growth temperature (℃)* 15–40 10–37 2–38 4–30 5–36 4–37
pH for growth* 6.0–9.0 6.5–8 7.0–7.6 5.5–9.5  6.0–9.0
NaCl concentration for growth (%, w/v)* 0–3 0–0.5 0–2 0–3 0–3 0–2
Hydrolysis of gelatin ＋ ＋ ＋ － － ＋
Nitrate reduction ＋－＋－ － －
Utilization of carbohydrates (Biolog GN2):
α- - Glucose ＋ ＋ ＋ － － ＋
Citric acid, Succinamic acid ＋－＋－ － －
- Galactose, - Gluconic acid ＋－＋－＋－
- Glucose-6- phosphate ＋－ － － － －
- Rhamnose, Sucrose, ,- Lactic acid ＋ ＋ － － ＋－
Enzymatic activities (API ZYM):
Cystine arylamidase ＋ ＋ － － － －
Lipase (C14) ＋－ － － － －
Leucine arylamidase ＋－ － － ＋ ＋
Valine arylamidase ＋ ＋ － － － ＋
API 20NE reactions:
Adipic acid (assimilation) ＋－ － － － 
β- Glucosidase (hydrolysis) ＋＋ ＋ －
Malic acid (assimilation) ＋－ － － ＋
DNA G+C content (mol%)* 39.2 32.2 43.3 35.5 28.8 36.8
*Data in columns 2–5 was collected from [37–40].

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Lin etal., Int. J. Syst. Evol. Microbiol. 2020;70:3731–3739
e complex polar lipid proles of strain CC- CTC003T
contained glycolipid (GL), phosphatidylethanolamine (PE),
two unknown aminophospholipid (AL1-2), four uncharacter-
ized aminophospholipids (APL1-4) and one unknown phos-
pholipid (PL) (Fig.4). e possession of PE as the common
major polar lipid is consistent with previous descriptions of
species of Flavobacterium [3, 4].
In summary, strain CC- CTC003
T
shared highest (95.3 %)
16S rRNA gene sequence similarity with Flavobacterium
cerinum, ANI calculations gave OrthoANI similarity range
of 70.0–74.1 % compared to related species from which
genomic data were able to be obtained. e combination
of genetic, phylogenetic, phenotypic and chemotaxonomic
characteristics validated strain CC- CTC003T constitute a
novel species of the genus Flavobacterium, for which the
name Flavobacterium supellecticarium sp. nov. is proposed.
DESCRIPTION OF Flavobacterium
supellecticarium SP. NOV.
( su. pel. lec. ti. ca' ri. um. L. neut. adj. supellecticarium relating
to furniture)
Cells are Gram- stain- negative, rod- shaped, 1.6–1.9 µm in
length and 0.5–0.8 µm in diameter, and motile by gliding.
Colonies are yellow in colour, circular and convex after
2 days of incubation on nutrient agar (NA); colony size
is about 2–3 mm. Growth occurs at 15–40 °C (optimal
30 °), pH 6.0–10.0 (optimal 7.0) and tolerates 3 %
(w/v) NaCl concentration (optimal 1 %). Flexirubin- type
Fig. 4. Two- dimensional thin layer chromatographs showing the polar
lipids of strain CC- CTC003T. Total polar lipids were stained with 10 %
ethanolic molybdatophosphoric acid. Abbreviation: AL1-2, unknown
aminophospholipids; APL1-4, aminophospholipids; GL, glycolipid; PE,
phosphatidylethanolamine; PL, unknown phospholipid.
Table 2. Cellular fatty acid composition of Flavobacterium
supellecticarium CC- CTC003T and phylogenetically closely related
Flavobacterium species
Strains: 1, CC- CTC003T; 2, Flavobacterium aquatile BCRC 80432T (type
species of the genus Flavobacterium); 3, Flavobacterium subsaxonicum
DSM 21790T; 4, Flavobacterium qiangtangense JCM 19739T; 5,
Flavobacterium maris KCTC 42093T; 6, Flavobacterium cerinum KCTC
62960T [36]. The major composition (>5.0 %) was emphasized as bold
type. －, Not detected; , trace (<1.0 %).
Fatty acid 1 2 3 4 5 6
Straight chain:
C15 : 07.8 24.7 4.6 4.0 3.2 －
C16 : 0 1.8 1.1 6.9 3.7 1.0 5.7
C20 : 0 －－－1.1  －
Branched:
iso- C13 : 0 4.4   1.1  －
iso- C14 : 0 3.6 2.1  1.7  
iso- C15 : 0 15.3 11.6 23.6 11.6 37.1 13.4
anteiso- C15 : 0 3.1 4.5 4.0 3.5  4.4
iso- C15 : 1 G 3.3 5.4  3.4 2.9 5.3
iso- C16 : 0 4.4 4.3  4.4  5.2
iso- C16 : 1 h  2.0    2.2
iso- C17 : 1 ω9c－ －  － － 3.8
iso- C20 : 0 － －  1.1 － －
Unsaturated:
C15 : 1 ω5c － － 3.4 －1.0 －
C15 : 1 ω6c 10.2  1.0  1.6
C17 : 1 ω6c －2.8  － －
C17 : 1 ω8c  1.3  － －
C18 : 1 ω9c   1.0  
Hydroxy:
iso- C14 : 0 3- OH    1.2 － －
C15 : 0 2- OH 1.2  1.1 2.4 －1.7
C15 : 0 3- OH 2.3   1.8  －
iso- C15 : 0 3- OH 5.8 5.6 5.5 10.5 7.3 4.2
C16 : 0 3- OH 2.6 tr 7.4 5.4 1.2 3.4
iso- C16 : 0 3- OH 1.4 5.0 2.9 10.8  4.3
C17 : 0 2- OH  1.0 1.4 1.1  －
C17 : 0 3- OH  1.0    8.5
iso- C17 : 0 3- OH 13.6 4.9 9.0 9.1 21.3 －
Summed Feature 2 － － 1.4 1.3 － －
Summed Feature 3 15.2 3.0 15.3 9.9 1.0 24.3
Summed Feature 9 7.1 1.9 2.7  16.4 －
Summed feature 2 consists of C14 : 0 3OH / iso- C16 : 1 I.
Summed feature 3 consists of C16 : 1 ω6c / C16 : 1 ω7c.
Summed feature 9 consists of C16 : 0 10- methyl / iso- C17 : 1 ω9c.

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Lin etal., Int. J. Syst. Evol. Microbiol. 2020;70:3731–3739
pigments are absent and Congo red is not absorbed by
colonies. Positive for catalase, oxidase and proteinase
activities and utilization of dextrin, glycogen, Tween 40,
Tween 80, N- acetyl- - glucosamine, adonitol, i- erythritol,
- fucose, - galactose, gentiobiose, α- - glucose, lactulose,
- mannose, melibiose, - psicose, - rhamnose, - sorbitol,
sucrose, trehalose, turanose, xylitol, citric acid, formic
acid, - gluconic acid, ,- lactic acid, succinic acid and
- glucose-6- phosphate as carbon sources. Nitrate is
reduced to nitrite; indole and H2S are not produced;
assimilates adipic acid and malic acid. Alkaline phos-
phatase, acid phosphatase, esterase (C4), esterase lipase
(C8), lipase (C14), leucine arylamidase, valine arylami-
dase, cystine arylamidase, acid phosphatase and naphthol-
AS- BI- phosphohydrolase are present. The major cellular
fatty acid consists with C15 : 0, iso- C15 : 0, iso- C15 : 0 3- OH, iso-
C
17 : 0
3- OH and C
16 : 1
ω6c / C
16 : 1
ω7c. Polar lipids present
are phosphatidylethanolamine (PE), four uncharacterized
aminophospholipids (APL1-4), two aminolipids (AL1-2)
and one unidentified glycolipid (GL). The predominant
respiratory system is menaquinone 6 (MK-6) and the
major polyamine is sym- homospermidine. The BioProject
ID for strain CC- CTC003
T
is PRJNA531783 and whole
genome sequencing NCBI record is SSNZ00000000.
e type strain is CC- CTC003T (=BCRC 81146T=JCM
32838T) isolated from an abandoned furniture. e DNA
G+C content was determined to be 39.2 mol%.
Funding information
This work was financially supported by the Ministry of Science and
Technology (Taiwan) under Grant No. MOST 108–2634 F-005-002 and
by the ‘Innovation and Development Centre of Sustainable Agriculture’
from The Featured Areas Research Centre Program within the frame-
work of the Higher Education Sprout Project by the Ministry of Educa-
tion (MOE) in Taiwan.
Author contributions
S- Y. L., analysed most of the data and wrote the manuscript. Professor
W- M. C., provided the help of analysed the polar lipid profile. A. H.,
contributed to providing critical revisions to this article. G- H. H. and
C- F. T., re- constructed the UBCG analysis. C- T. C., was responsible for
collecting samples and isolating the novel microorganism. All authors
discussed the results and revised the manuscript.
Conflicts of interest
The authors declare that they have no conflict of interest.
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