Oceanobacillus neutriphilus sp. nov., isolated from activated sludge in a bioreactor.

Abstract

A Gram-stain-positive, neutrophilic, rod-shaped bacterium, strain A1g(T), was isolated from activated sludge of a bioreactor and was subjected to a polyphasic taxonomic characterization. The isolate grew in the presence of 0-17.0 % (w/v) NaCl and at pH 6.0-9.0; optimum growth was observed in the presence of 3.0-5.0 % (w/v) NaCl and at pH 7.0. Strain A1g(T) was motile, formed cream-coloured colonies, was catalase- and oxidase-positive and was able to hydrolyse aesculin, Tween 40 and Tween 60. Chemotaxonomic analysis revealed menaquinone-7 as the predominant respiratory quinone and anteiso-C₁₅:₀, anteiso-C₁₇:₀, iso-C₁₆:₀ and iso-C₁₅:₀ as major fatty acids. The genomic DNA G+C content of strain A1g(T) was 36.3 mol%. Comparative 16S rRNA gene sequence analysis revealed that the new isolate belonged to the genus Oceanobacillus and exhibited closest phylogenetic affinity to the type strains of Oceanobacillus oncorhynchi subsp. incaldanensis (97.9 % similarity) and O. oncorhynchi subsp. oncorhynchi (97.5 %), but less than 97 % sequence similarity with respect to the type strains of other recognized Oceanobacillus species. Levels of DNA-DNA relatedness between strain A1g(T) and reference strains O. oncorhynchi subsp. incaldanensis DSM 16557(T), O. oncorhynchi subsp. oncorhynchi JCM 12661(T) and Oceanobacillus iheyensis DSM 14371(T) were 29, 45 and 38 %, respectively. On the basis of phenotypic and genotypic data, strain A1g(T) is considered to represent a novel species of the genus Oceanobacillus, for which the name Oceanobacillus neutriphilus sp. nov. is proposed. The type strain is A1g(T) (=CGMCC 1.7693(T) =JCM 15776(T)).

Full text

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Oceanobacillus neutriphilus sp. nov., isolated from
activated sludge in a bioreactor
Jun-Yi Yang,1,2Ying-Yi Huo,3Xue-Wei Xu,1,2Fan-Xu Meng,1,2Min Wu3
and Chun-Sheng Wang1,2
Correspondence
Chun-Sheng Wang
wangsio@sio.org.cn
1Laboratory of Marine Ecosystem and Biogeochemistry, State Oceanic Administration,
Hangzhou 310012, People’s Republic of China
2Second Institute of Oceanography, State Oceanic Administration, Hangzhou 310012,
People’s Republic of China
3College of Life Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China
A Gram-stain-positive, neutrophilic, rod-shaped bacterium, strain A1gT, was isolated from
activated sludge of a bioreactor and was subjected to a polyphasic taxonomic characterization.
The isolate grew in the presence of 0–17.0% (w/v) NaCl and at pH 6.0–9.0; optimum growth
was observed in the presence of 3.0–5.0% (w/v) NaCl and at pH 7.0. Strain A1gTwas motile,
formed cream-coloured colonies, was catalase- and oxidase-positive and was able to hydrolyse
aesculin, Tween 40 and Tween 60. Chemotaxonomic analysis revealed menaquinone-7 as the
predominant respiratory quinone and anteiso-C15:0, anteiso-C17:0, iso-C16:0and iso-C15:0as
major fatty acids. The genomic DNA G+C content of strain A1gTwas 36.3 mol%. Comparative
16S rRNA gene sequence analysis revealed that the new isolate belonged to the genus
Oceanobacillus and exhibited closest phylogenetic affinity to the type strains of Oceanobacillus
oncorhynchi subsp. incaldanensis (97.9% similarity) and O. oncorhynchi subsp. oncorhynchi
(97.5%), but less than 97% sequence similarity with respect to the type strains of other
recognized Oceanobacillus species. Levels of DNA–DNA relatedness between strain A1gTand
reference strains O. oncorhynchi subsp. incaldanensis DSM 16557T, O. oncorhynchi subsp.
oncorhynchi JCM 12661Tand Oceanobacillus iheyensis DSM 14371Twere 29, 45 and 38%,
respectively. On the basis of phenotypic and genotypic data, strain A1gTis considered to
represent a novel species of the genus Oceanobacillus, for which the name Oceanobacillus
neutriphilus sp. nov. is proposed. The type strain is A1gT(5CGMCC 1.7693T5JCM 15776T).
The genus Oceanobacillus was proposed by Lu et al. (2001,
2002) with the description of Oceanobacillus iheyensis as
the type species; the description of the genus was later
emended by Yumoto et al. (2005) and Lee et al. (2006). The
genus Oceanobacillus comprises aerobic, Gram-positive,
motile, rod-shaped bacteria that are characterized chemo-
taxonomically by the presence of menaquinone-7 as the
major isoprenoid quinone and anteiso-C15:0 as the
predominant cellular fatty acid (Lee et al., 2006).
At the time of writing, the genus comprises seven
recognized species, including two subspecies. Members of
the genus have been isolated from diverse sources such as
deep-sea sediment (O. iheyensis Lu et al. 2002; O. profundus
Kim et al. 2007), mural paintings [O. picturae (Heyrman
et al. 2003) Lee et al. 2006], freshwater fish (O. oncorhynchi
subsp. oncorhynchi Yumoto et al. 2005), algae (O.
oncorhynchi subsp. incaldanensis Romano et al. 2006), an
insect (O. chironomi Raats and Halpern 2007), activated
sludge (O. caeni Nam et al. 2008) and food (O. kapialis
Namwong et al. 2009). The type strains of O. oncorhynchi
subsp. oncorhynchi and O. oncorhynchi subsp. incaldanensis
share high 16S rRNA gene sequence similarity (99.5%) but
can be differentiated on the basis of phenotypic traits. The
former is sporulating, facultatively aerobic and obligately
alkaliphilic (pH 9–10), whereas the latter is non-sporulat-
ing, obligately aerobic and alkalitolerant (pH 6.5–9.5)
(Romano et al., 2006).
The aim of the present study was to determine the exact
taxonomic position of a novel Oceanobacillus-like strain by
using a polyphasic approach that included analysis of
phenotypic properties and phylogenetic analysis based on
16S rRNA gene sequences and DNA–DNA relatedness data.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain A1gTis EU709018.
Detailed fatty acid profiles of strain A1gTand related type strains and
maximum-parsimonyand maximum-likelihood
sequence-based phylogenetic trees are available as supplementary
material with the online version of this paper.
16SrRNA gene
International Journal of Systematic and Evolutionary Microbiology (2010), 60, 2409–2414
DOI 10.1099/ijs.0.016295-0
016295G2010 IUMSPrinted in Great Britain 2409

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Strain A1gTwas isolated from activated sludge of a
sequential batch reactor treating salt-containing wastewater
taken from a preserved Szechuan pickle factory. The
reactor had been operational for 9 months at the time
when the sludge was sampled. The average in situ tem-
perature was 30 uC and the pH was approximately 7.4. The
sludge sample was suspended in sterile wastewater and
vortexed for 15 min. A portion of the suspension was
spread directly on sterile wastewater agar plates, which
contained (per litre wastewater) 1.0 g glucose, 1.0 g
peptone (BD) and 15.0 g agar (pH 7.0). The plates were
incubated at 30 uC for several days. Single colonies on the
plates were picked out and strain A1gTwas obtained by
repeated restreaking. This isolate was routinely cultured on
marine agar 2216 (MA; BD) and was maintained as a
glycerol suspension (30% v/v) at 280 uC.
The optimal conditions for growth were determined in
PY broth (Lu et al., 2001) with different NaCl con-
centrations (0, 0.5, 1, 3, 5, 7.5, 10, 12.5, 15, 16, 17, 18, 19,
20, 22.5 and 25%, w/v). The pH range for growth was
determinedby adding MES
(pH 6.5–7.0), Tricine (pH 7.5–8.5), CAPSO (pH 9.0–
10.0) or CAPS (pH 10.5) to PY broth supplemented with
3% (w/v) NaCl. The temperature range for growth was
determined in PY broth (pH 7.0) at 4, 10, 15, 20, 25, 30,
35, 37, 40, 42, 45 and 48 uC. Cell morphology and
motility were examined by optical microscopy (Olympus
BX40) and electron microscopy (Hitachi H-7650 and
JEOL JEM-1230).
(pH 5.0–6.0), PIPES
Oxidase activity was determined based on oxidation of 1%
p-aminodimethylaniline oxalate and catalase activity was
determined based on bubble production in 3% (v/v) H2O2
solution (Dong & Cai, 2001). Biochemical characteristics
were determined according to the methods described by
Dong & Cai (2001) and Romano et al. (2006). Single
carbon source assimilation tests were performed by using
medium 2 supplemented with 3% (w/v) NaCl (Romano
et al., 2006). The corresponding filter-sterilized sugar
(0.2%), alcohol (0.2%), organic acid (0.1%) or amino
acid (0.1%) was added to liquid medium. Acid production
was tested by using MOF medium supplemented with
1.0% sugars or alcohols (Leifson, 1963; Xu et al., 2008).
Susceptibility to antibiotics was determined on agar plates
by using antibiotic discs with the following compounds
(amounts in mg unless otherwise stated): amoxicillin (10),
ampicillin (10), carbenicillin (100), cefotaxime (30),
cefoxitin (30), chloramphenicol (30), erythromycin (15),
kanamycin (30), neomycin (30), nitrofurantoin (300),
novobiocin (30), nystatin (100), penicillin (10), polymyxin
B (300 IU), rifampicin (5), streptomycin (10) and tetra-
cycline (30). Additional enzyme activities and biochemical
characteristics were determined by using API 20E, API 20
NE, API 50 CH and API ZYM kits as recommended by the
manufacturer (bioMe ´rieux). O. iheyensis DSM 14371T, O.
oncorhynchi subsp. oncorhynchi JCM 12661Tand O.
oncorhynchi subsp. incaldanensis DSM 16557Twere used
as controls in these tests.
Fatty acid methyl esters were obtained from cells grown on
MA for 2 days at 35 uC and were analysed by using GC/MS
(Kuykendall et al., 1988). Isoprenoid quinones were
analysed as described by Komagata & Suzuki (1987) by
using reversed-phase HPLC. Cell-wall peptidoglycan was
prepared and hydrolysed according to the methods given
by Kawamoto et al. (1981) and the amino acid composi-
tion was analysed with an automatic amino acid analyser
(Hitachi L-8900). Genomic DNA was obtained by using
the method described by Marmur (1961). Purified DNA
was hydrolysed with P1 nuclease and the nucleotides were
dephosphorylated with calf intestine alkaline phosphatase;
the G+C content of the resulting deoxyribonucleosides
was determined by reversed-phase HPLC and was calcu-
lated from the ratio of deoxyguanosine to thymidine
(Mesbah & Whitman, 1989).
The 16S rRNA gene was amplified and analysed as
described by Xu et al. (2007). PCR products were cloned
into pMD 19-T vector (TaKaRa) and then sequenced. An
almost-complete 16S rRNA gene sequence of strain A1gT
(1484 nt) was obtained and was compared with closely
related sequences of reference organisms from the EzTaxon
service (Chun et al., 2007). Sequence data were aligned
with CLUSTAL W 1.8 (Thompson et al., 1994). Phylogenetic
trees were constructed by using the neighbour-joining
(Saitou & Nei, 1987) and maximum-parsimony (Fitch,
1971) methods with the MEGA 4 program package (Tamura
et al., 2007) and by using the maximum-likelihood method
(Felsenstein, 1981) with the TreePuzzle 5.2 program.
Evolutionary distances were calculated according to the
algorithm of Kimura’s two-parameter model (Kimura,
1980) for the neighbour-joining method. Bootstrap
analysis was used to evaluate the tree topology by means
of 1000 resamplings.
Cells of strain A1gTwere Gram-stain-positive, sporulating
rods that were motile by means of polar flagella (Fig. 1).
The NaCl concentration, pH and temperature ranges for
growth in PY broth were 0–17% (w/v), pH 6.0–9.0 and
10–45 uC. The cell-wall diamino acid was meso-diamino-
pimelic acid. The isoprenoid quinone of strain A1gTwas
MK-7 and the DNA G+C content was 36.3 mol%. These
chemotaxonomic characteristics were in accordance with
those given for the genus Oceanobacillus (Yumoto et al.,
2005; Lee et al., 2006). Detailed results are given in the
species description below and in Table 1.
16S rRNA gene sequence comparisons showed that strain
A1gTshouldbeplacedwithinthegenusOceanobacillus,being
related most closely to the type strains of O. oncorhynchi
subsp. incaldanensis (97.9% similarity), O. oncorhynchi
subsp. oncorhynchi (97.5%) and O. iheyensis (96.3%); levels
of16SrRNA genesequencesimilarity withrespecttothetype
strains of other recognized Oceanobacillus species were 94.2–
95.3%. Phylogenetic analysis based on 16S rRNA gene
sequences showed that strain A1gThad closest affinity with
the type strains of O. oncorhynchi and O. iheyensis with high
levels of bootstrap support (Fig. 2). The topologies of the
J.-Y. Yang and others
2410International Journal of Systematic and Evolutionary Microbiology 60

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phylogenetic trees built by using the maximum-parsimony
and maximum-likelihood methods also supported the
notion that strain A1gTformed a stable clade with the type
strains of O. oncorhynchi and O. iheyensis (Supplementary
Fig. S1 in IJSEM Online).
DNA–DNA hybridization experiments were performed by
the thermal denaturation and renaturation method of De
Ley et al. (1970) as modified by Huß et al. (1983), by using
a Beckman DU 800 spectrophotometer. Levels of DNA–
DNA relatedness between strain A1gTand O. oncorhynchi
subsp. incaldanensis DSM 16557T, O. oncorhynchi subsp.
oncorhynchi JCM 12661Tand O. iheyensis DSM 14371T
were 29, 45 and 38%, respectively, significantly below the
value of 70% which is considered to be the threshold for
the delineation of species (Wayne et al., 1987).
The major fatty acids of strain A1gTwere anteiso-C15:0
(37.7%), anteiso-C17:0(18.9%), iso-C16:0(15.8%) and
iso-C15:0(6.8%). This profile was different from those
of O. iheyensis DSM 14371Tand O. oncorhynchi subsp.
incaldanensis DSM 16557T(Supplementary Table S1). The
iso-C14:0content of strain A1gT(3.8%) was lower than
that of O. oncorhynchi subsp. incaldanensis DSM 16557T
(17.5%) and O. iheyensis DSM 14371T(17.0%). Addi-
tionally, strain A1gTcould be differentiated from recog-
nized Oceanobacillus species on the basis of several
phenotypic characteristics, such as cultural conditions,
nitrate reduction, hydrolysis of substrates, acid production
from sugars or alcohols, susceptibility to antibiotics and
enzyme activities (Table 1).
On the basis of the genotypic and phenotypic data
presented in this study, strain A1gTshould be assigned to
a novel species within the genus Oceanobacillus, for which
the name Oceanobacillus neutriphilus sp. nov. is proposed.
Description of Oceanobacillus neutriphilus
sp. nov.
Oceanobacillus neutriphilus (neu.tri.phi9lus. L. adj. neuter
-tra -trum neither, used to refer to neutral pH; Gr. adj.
philos loving; N.L. masc. adj. neutriphilus preferring neutral
pH).
Cells are Gram-positive-staining, aerobic rods, 0.7–1.2 mm
wide and 1.5–2.5 mm long with rounded ends, that are
motile by means of polar flagella and produce ellipsoidal
spores in a central position. Colonies on MA are 1–2 mm
in diameter, of low convexity, smooth, circular with regular
borders and cream-coloured after 48 h. Growth occurs in
PY medium at NaCl concentrations of 0–17.0% (w/v) with
optimum growth at 3.0–5.0%. The pH and temperature
ranges for growth are 6.0–9.0 and 10–45 uC (optimum
growth at pH 7.0 and 37 uC). No growth is detected below
pH 5.5 or above pH 9.5. Positive for oxidase and catalase.
Hydrolyses aesculin, Tween 40 and Tween 60, but not
casein, DNA, gelatin, starch, Tween 80 or tyrosine. Nitrate
is not reduced to nitrite. H2S is not produced from
thiosulfate. Positive for o-nitrophenyl-b-D-galactopyrano-
sidase. Negative for arginine dihydrolase, indole produc-
tion, lysinedecarboxylase,
tryptophan deaminase and urease. Utilizes D-fructose, D-
glucose, maltose, mannitol, D-mannose and sucrose as sole
carbon and energy sources, but not acetate, L-alanine, L-
arabinose, citrate, L-cysteine, ethanol, formate, fumarate,
glutamate,
L-glutamine, glycine,
lactate, lactose, malate, malonate,
inositol, L-ornithine, propionate, pyruvate, raffinose, L-
serine, L-sorbitol, L-sorbose, starch, succinate or L-valine.
Acid is produced from D-glucose, maltose, D-mannose and
sucrose, but not from L-arabinose, ethanol, myo-inositol,
lactose, raffinose, L-sorbitol or L-sorbose. Susceptible to (mg
per disc unless indicated otherwise) amoxicillin (10),
ampicillin (10), carbenicillin (100), cefotaxime (30),
chloramphenicol (30), erythromycin (15), kanamycin
(30), neomycin (30), nitrofurantoin (300), novobiocin
(30) and rifampicin (5), but not to cefoxitin (30), nystatin
ornithine decarboxylase,
L-histidine, isoleucine,
L-methionine, myo-
Fig. 1. Transmission electron micrographs of cells of strain A1gT.
(a) Exponentially growing cell, showing polar flagella. Bar, 0.5 mm.
(b) Ultrathin section showing outer membrane of the cell. PG,
Peptidoglycan; OL, outer layer. Bar, 0.2 mm.
Oceanobacillus neutriphilus sp. nov.
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Table 1. Differential characteristics between strain A1gTand the type strains of related members of
Oceanobacillus
Strains: 1, A1gT; 2, O. oncorhynchi subsp. incaldanensis DSM 16557T; 3, O. oncorhynchi subsp. oncorhynchi JCM
12661T; 4, O. iheyensis DSM 14371T. Data were obtained in the present study unless indicated. +, Positive;
2, negative; W, weakly positive.
Characteristic1234
Spore position*
NaCl range for growth (%, w/v)
NaCl optimum (%, w/v)
Growth temperature range (uC)
pH optimum
Nitrate reduction
o-Nitrophenyl-b-D-galactopyranosidase
Hydrolysis of:
Casein
Gelatin
Tween 60
Urea
Acid production from:
Arbutin
Cellobiose
Gentiobiose
Glycerol
D-Mannitol
Raffinose
Salicin
Sucrose
D-Tagatose
Trehalose
Sensitivity to:
Cefotaxime (30 mg)
Cefoxitin (30 mg)
Nitrofurantoin (300 mg)
Penicillin G (10 mg)
Streptomycin (10 mg)
API ZYM results
a-Chymotrypsin
a-Glucosidase
b-Glucosidase
DNA G+C content (mol%) (HPLC)
C NSTT
0–17.0
3.0–5.0
10–45
7.0
2
+
0–20.0
10.0
10–45
9.0
+
2
0–22.5
7.5–10.0
10–45
9.5
+
2
0–18.0
3.0–5.0
15–42
8.0–8.5
2
2
2
2
+
2
2
2
2
2
2
2
2
+
+
+
+
2
++
+
+
2
+
2
+
+
+
+
+
+
+
2
2
+
+
+
+
+
2
2
2
+
W
2
W
+
2
+
+
+
+
W
2
2
2
2
2
+
2
+
2
2
+
+
+
+
2
2
+
2
+
+
+
+
+
+
2
+
+
+
36.3
+
+
+
+
+
+
2
2
2
40.1aD38.5b
35.8c
*C, Central; NS, not sporulated; T, terminal.
DData from: a, Romano et al. (2006); b, Yumoto et al. (2005); c, Lu et al. (2001).
Fig. 2. Neighbour-joining tree based on 16S
rRNA gene sequences, showing the phyloge-
netic relationships between strain A1gTand
related taxa. Bootstrap values at nodes are
based on 1000 replicates; only values .50%
are shown. Filled circles indicate nodes recov-
ered with bootstrap values .50% in the
maximum-parsimony and maximum-likelihood
trees. Bar, 0.02 substitutions per nucleotide
position.
J.-Y. Yang and others
2412International Journal of Systematic and Evolutionary Microbiology 60

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(100), penicillin (10), polymyxin B (300 IU), streptomycin
(10) or tetracycline (30). The following constitutive enzyme
activities are detected in API ZYM tests: acid phosphatase,
alkaline phosphatase, a-chymotrypsin, esterase (C4), ester-
ase lipase (C8), a-glucosidase, b-glucosidase and naphthol-
AS-BI-phosphohydrolase.N-Acetyl-b-glucosaminidase,cystine
arylamidase, a-fucosidase, a-galactosidase, b-galactosidase,
b-glucuronidase, lipase (C14), leucine arylamidase, a-
mannosidase, trypsin and valine arylamidase activities are
not observed. Oxidative acid production in API 50 CH
tests is positive for N-acetylglucosamine, arbutin, aesculin,
D-fructose, D-glucose, maltose, D-mannitol, salicin, suc-
rose, D-tagatose and trehalose and weakly positive for
cellobiose and glycerol. Principal fatty acids (.5%) are
anteiso-C15:0, anteiso-C17:0, iso-C16:0and iso-C15:0. The
isoprenoid quinone is MK-7. The cell-wall diamino acid is
meso-diaminopimelic acid. The DNA G+C content of the
type strain is 36.3 mol%.
The type strain, A1gT(5CGMCC 1.7693T5JCM 15776T),
was isolated from activated sludge of a bioreactor treating
salt-containing wastewater.
Acknowledgements
This work was supported by grants from the Ministry of Science and
Technology of China (973 Program, 2004CB719604-3; 863 Program,
2007AA021305), the National Natural Science Foundation of China
(40806066), the China Ocean Mineral Resources R & D Association
(COMRA) Special Foundation (DYXM-115-01-3-01, DYXM-115-01-
3-02) and the Key Project of Zhejiang Science and Technology
(2006C13053).
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