Isolation and Characterization of Marine Nonylphenol-DegradingBacteria and Description of Pseudomaricurvus alkylphenolicusgen. nov., sp. nov.

Hiroaki Iwaki • Makoto Fujioka • Yoshie Hasegawa

Received: 30 April 2013 / Accepted: 30 July 2013 / Published online: 20 September 2013

� Springer Science+Business Media New York 2013

Abstract Two novel aerobic p-n-nonylphenol-degrading

bacterial strains were isolated from seawater obtained from

the coastal region of Ogasawara Islands, Japan. The 16S

rRNA gene sequence analysis indicated that the strains are

affiliated with the order Alteromonadales within the class

Gammaproteobacteria. One isolate, strain KU41G2, is

most closely related to Maricurvus nonylphenolicus (99.2 %

similarity), and is tentatively identified as M. nonylpheno-

licus. The other isolate, strain KU41GT, is also most closely

related to M. nonylphenolicus; however, the 16S rRNA

gene sequence similarity was only 94.7 %. Cells of strain

KU41GT are Gram-negative rods with a single polar fla-

gellum. The predominant respiratory lipoquinone was ubi-

quinone-8, and the major cellular fatty acids were C17:1 x8c

(24.2 %); C15:0 iso 2-OH; and/or C16:1 x7c (16.3 %), C15:0

(10.3 %), C11:0 3-OH (9.5 %), C9:0 3-OH (6.7 %), C10:0

3-OH (6.4 %), and C18:1 x7c (5.5 %). The DNA G?C

content was 53.3 mol%. On the basis of physiological,

chemotaxonomic, and phylogenetic data, strain KU41GT is

suggested to represent a novel species of a new genus, for

which we propose the name Pseudomaricurvus alkylphe-

nolicus gen. nov., sp. nov. The type strain of P. alkylphe-

nolicus is KU41GT (=JCM 19135T = KCTC 32386T).

Introduction

Alkylphenols and alkylphenol polyethoxylates are widely

distributed as pollutants in the environment from a variety

of industries, and contamination of the marine environ-

ment with alkylphenols is of great public concern because

of their toxicity and endocrine-disrupting activity in

humans and marine organisms [1]. Many alkylphenol-

degrading microorganisms have been isolated and char-

acterized [2, 3], and the mechanism of alkylphenol deg-

radation has been studied extensively [4–6]. However,

these organisms have been isolated mainly from terrestrial

or freshwater sites. Recently, we reported the isolation

and characterization of a marine p-n-nonylphenol-

degrading bacterium Maricurvus nonylphenolicus strain

KU41ET [7]; however, information regarding alkylphe-

nol-degrading bacteria from marine environments is rel-

atively scarce. Here, we report on the isolation of two

novel aerobic p-n-nonylphenol-degrading bacteria. Com-

parative 16S rRNA gene sequence analysis indicated that

one of the isolates, strain KU41GT, forms an independent

branch within the order Alteromonadales. Therefore, we

aimed to determine the exact taxonomic position of strain

KU41GT by a polyphasic characterization that included

phenotypic and chemotaxonomic properties and a detailed

phylogenetic analysis based on the 16S rRNA gene

sequence.

Materials and Methods

Strain Isolation

Two p-n-nonylphenol-degrading bacterial strains desig-

nated KU41GT and KU41G2 were isolated from seawater

collected from the coastal region of Chichi-jima Island of

Ogasawara Islands, Japan in August 2011, as described by

Iwaki et al. [7]. The strains KU41GT and KU41G2 were

routinely cultured on Marine Agar 2216 (MA; Becton–

H. Iwaki (&) � M. Fujioka � Y. Hasegawa

Department of Life Science and Biotechnology, Kansai

University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan

e-mail: iwaki@kansai-u.ac.jp

123

Curr Microbiol (2014) 68:167–173

DOI 10.1007/s00284-013-0455-x

Dickinson) and stored at -80 �C in the commercial arti-

ficial seawater (Marine Art SF-1; Tomita Pharmaceutical)

and Marine broth 2216 (MB; Becton–Dickinson), respec-

tively, with 10 % (w/v) glycerol.

16S rRNA Gene Sequencing and Phylogenetic Analysis

The 16S rRNA genes were amplified and sequenced as

described by Iwaki et al. [8]. Alignment was carried out

using the CLUSTAL W tool in MEGA version 5.1 [9].

Phylogenetic trees were generated using the neighbor-

joining [10], maximum-parsimony [11], and maximum-

likelihood [12] methods in MEGA version 5.1. The

distance matrix was produced based on Kimura’s 2-parameter

model [13], and the topologies of the resultant trees were

evaluated with bootstrap analysis [14] of 1,000 replicates.

Sequence similarity values were calculated using GEN-

ETYX-MAC version 16 (Genetyx Corporation).

Morphological, Physiological, and Biochemical

Characterization

The cell morphology of strain KU41GT was examined

under a transmission electron microscope (H-7600, Hit-

achi) at 100 kV. Motility was examined on a semisolid MB

medium [15]. Gram staining was performed using a Favor-

G kit (Nissui), and the cells were observed under a light

microscope (BX50F4, Olympus). Catalase and oxidase

tests were performed as described by Barrow and Feltham

[16]. Growth was tested at 25 �C on MA, unless otherwise

stated. Salinity requirements were tested using modified

MA, which had the same composition as MA, except for

the NaCl concentration, i.e., it was supplemented with

0–4 % (w/v) NaCl. The pH range for growth was deter-

mined on MA, and the pH was adjusted to 5.0–10.0. Sus-

ceptibility to antibiotics was determined using the diffu-

sion method with antibiotic disks (Becton–Dickinson), as

Pseudoteredinibacter isoporae SW-11T (FJ347760)Eionea nigra 17X/A02/237T (AY576771)

KU41G2 (AB817936)Maricurvus nonylphenolicus KU41ET (AB626730)Pseudomaricurvus alkylphenolicus KU41GT (AB809161)

Saccharophagus degradans 2-40T (AF055269)Teredinibacter turnerae T7902T (AY028398)

Gilvimarinus chinensis QM42T (DQ822530)Simiduia agarivorans SA1T (EF617350)

Marinimicrobium koreense M9T (AY839869)Marinimicrobium agarilyticum M18T (AY839870)

Melitea salexigens 5IX/A01/131T (AY576729)Spongiibacter marinus HAL40bT (AM117932)

Haliea salexigens 3X/A02/235T (AY576769)Dasania marina KOPRI 20902T (AY771747)

Porticoccus litoralis IMCC 2115T (EF468719)Microbulbifer maritimus TF-17T (AY377986)

Microbulbifer agarilyticus JAMB A3T (AB158515)Microbulbifer hydrolyticus DSM 11525T (AJ608704)

Zooshikella ganghwensis JC2044T (AY130994)Endozoicomonas elysicola MKT110T (AB196667)

Umboniibacter marinipuniceus KMM 3891T (AB467279)Spongiispira norvegica Gp 4 7.1T (AM117931)

Oceanospirillum beijerinckii IFO 15445T (AB006760)Oceanospirillum linum ATCC 11336T (M22365)Neptuniibacter caesariensis MED92T (AY136116)

Amphritea balenae JAMM 1525T (AB330883)Neptunomonas japonica JAMM 0745T (AB288092)

Pseudomonas aeruginosa ATCC 10145T (AF094713)Cellvibrio japonicus NCIMB 10462T (AF452103)

Cellvibrio mixtus ACM 2601T (AF448515)Cellvibrio fulvus NCIMB 8634T (AF448514)

Escherichia coli ATCC 11775T (X80725)

0.01

100

99

99

89

99

100

99

99

100

100

100

97

77Fig. 1 Phylogenetic

relationships between strain

KU41GT and other members of

the class Gammaproteobacteria.

The tree was constructed using

the neighbor-joining algorithm.

Numbers at nodes are bootstrap

percentages based on 1,000

replications; only values[70 %

are shown. Filled circles

indicate that the corresponding

nodes were also recovered in

trees generated with the

maximum-likelihood and

maximum-parsimony

algorithms. Open circles

indicate that the corresponding

nodes were also recovered in the

tree generated with the

maximum-likelihood algorithm.

Bar 0.01 substitutions per

nucleotide position

168 H. Iwaki et al.: Pseudomaricurvus alkylphenolicus gen. nov., sp. nov.

123

described by Iwaki et al. [7], and the cell suspension was

adjusted to a 2 McFarland standard. Nitrate reduction,

indole production, acid production from glucose, hydro-

lysis of esculin and gelatin, and the presence of arginine

dihydrolase, urease, and b-galactosidase were tested using

the API 20NE (bioMerieux), according to the manufac-

turer’s instructions, except that the cell suspensions were

prepared using Marine Art SF-1. The results were obtained

after 24 h of incubation at 25 �C. Hydrolysis of casein,

Tween 40, and Tween 80 were tested on MA, using the

substrate concentrations described by Cowan and Steel

[17]. Other enzyme activities were also assayed using API

ZYM (bioMerieux) at 37 �C, according to the manufac-

turer’s instructions. Utilization of various carbon sources

was tested using the commercial artificial seawater med-

ium, Daigo’s IMK-SP (Nihon Pharmaceutical), containing

0.1 % carbon source. Alkylphenol degradation was tested

using solidified Daigo’s IMK-SP, containing 0.1 % alkyl-

phenol; formation of a clear zone around the growing

colonies was considered a positive result.

DNA G?C Content, Isoprenoid Quinone, and Fatty

Acid Analysis

The G?C content of the genomic DNA was determined by

the method used by Katayama-Fujimura et al. [18], using a

high-performance liquid chromatograph (HPLC; Nihon

Waters). Isoprenoid quinones were extracted from freeze-

dried KU41GT cells grown in MB for 4 days at 25 �C

according to the method devised by Nishijima et al. [19]

and analyzed using HPLC (Nihon Waters). Cellular fatty

acids from cells grown on MA for 3 days at 25 �C were

extracted, methylated, and analyzed by gas chromatogra-

phy (Agilent Technologies 7890A GC System equipped

with a HP Ultra2 column) according to the standard pro-

tocol of the Sherlock Microbial Identification System

(MIDI) version 6.0, and identified using the TSBA40

method and TSBA40 library.

Results and Discussion

Phylogenetic Analysis

The nearly complete 1,458 bp 16S rRNA gene sequences

of strains KU41GT and KU41G2 were determined and

deposited in DNA Data Bank of Japan under accession

numbers AB809161 and AB817936, respectively. The 16S

rRNA gene sequence analysis indicated that strains

KU41GT and KU41G2 are phylogenetically affiliated with

the order Alteromonadales within the class Gammaprote-

obacteria. Strain KU41G2 is most closely related to M.

nonylphenolicus with 99.2 % similarity and is tentatively

identified as M. nonylphenolicus. Strain KU41GT is also

most closely related to M. nonylphenolicus; however, the

similarity was only 94.7 %, and the strain forms a distinct

lineage within the order Alteromonadales (Fig. 1). Strain

KU41GT was also found to be related to Pseudoteredi-

nibacter isoporae SW-11T (93.6 % similarity), Teredinib-

acter turnerae T7902T (92.7 %), Eionea nigra 17X/A02/

237T (92.5 %), and Saccharophagus degradans 2-40T

(92.0 %). Therefore, strain KU41GT should be classified as

a novel genus and species in the order Alteromonadales

considering the phylogenetic analyses.

Phenotypic Characteristics

The cells of strain KU41GT were Gram-negative, aerobic,

curved rods (1.5–2.0 lm in length and 0.7–1.0 lm in

width), and motile by a single polar flagellum (Fig. 2), as

with the members of the order Alteromonadales [20]. The

KU41GT cells formed colonies that were pale yellow,

circular, smooth, convex, 1.0–2.0 mm in diameter, and

with an entire margin after 3-days incubation on MA.

Growth occurred at 15–30 �C (optimally at 25–30 �C), at

pH range of at least 7.0 to 10.0 (optimally at pH 8.0), and

with 2.0–3.0 % NaCl. The strain degraded p-n-nonylphe-

nol, p-n-octylphenol, and p-n-heptylphenol, and utilized

these substrates as sole sources of carbon and energy. The

strain did not utilize p-n-hexylphenol, p–t-octylphenol, or

p-nonylphenol (a mixture of branched-chain isomers). The

other phenotypic properties of strain KU41GT are stated in

the genus and species descriptions and those characteristics

that differentiate strain KU41GT from phylogenetically

related taxa are listed in Table 1.

Fig. 2 Transmission electron micrograph of KU41GT cell grown on

MA. Bar 0.5 lm

H. Iwaki et al.: Pseudomaricurvus alkylphenolicus gen. nov., sp. nov. 169

123

Table 1 Differential characteristics of strain KU41GT and closely related genera

Characteristic 1 2 3 4 5 6

Cell morphology Rods Curved rods Rods Rods Pleomorphic rods Pleomorphic

rods

Cell size (lm) 0.7–1.0 9 1.5–2.0 0.3–0.8 9 1.0–2.5 0.3–0.4 9 0.7–1.2 0.5 9 1.4 0.4–0.6 9 3.0–6.0 0.5 9 1.5–3.0

NaCl range for growth (%, w/v) 2–3 1–4 2–4 0.7–7 0.6–3.5 1–10

pH range for growth 7 to at least 10 7–8 7–8 5–9 6–10.5 4.5–10

Temperature range for growth

(�C)

15–30 20–35 10–45 10–44 20–35 4–37

Indole production - - ? NR - -

Oxidase ? ? ? - ? ?

Enzyme activities (API ZYM tests)

Esterase lipase (C8), valine

arylamidase

? ? ? - ? -

Trypsin, chymotrypsin - - ? - ? -

a-Galactosidase,

b-galactosidase

- - - - ? -

Esterase (C4) ? ? ? w - -

Lipase (C14) ? ? ? - - -

Leucine arylamidase ? ? ? - NR NR

Cystine arylamidase - - ? - - -

Acid phosphatase ? - ? - ? -

Carbon utilization of

N-acetyl-glucosamine,

glycerol, L-histidine, L-

leucine, L-phenylalanine, L-

serine, L-threonine

? - ? - NR NR

L-Asparagine, L-asparate - - ? - NR NR

D-Galactose, maltose ? - - - NR ?

Cellobiose, succinate - - ? - ? NR

D-Mannitol, citrate - - - ? NR NR

Propionate, L-alanine ? ? ? - NR NR

D-Fructose - - - w ? ?

D-Mannose - - ? - NR ?

L-Rhamnose ? - NR - NR NR

D-Xylose ? - - NR ? ?

Lactose - - - - NR ?

Sucrose ? - - - ? ?

Trehalose ? ? - - - NR

Acetate ? ? ? - ? NR

Pyruvate ? ? - - ? NR

L-Glutamate ? - ? - ? NR

L-Proline ? - ? ? NR NR

Hydrolysis of

Casein - ? - NR NR NR

Gelatin - ? ? NR - -

Esculin - ? ? NR NR NR

Susceptibility to

Penicillin G, Streptomycin ? ? ? NR NR -

Ampicillin ? ? - NR NR ?

Polymyxin B ? ? NR NR NR -

170 H. Iwaki et al.: Pseudomaricurvus alkylphenolicus gen. nov., sp. nov.

123

Chemotaxonomic Characteristics

The G?C content of the genomic DNA was 53.3 mol%,

and was in the range of values 36–54 mol%, which is

characteristic of members of the order Alteromonadales

[20]. The major lipoquinone was ubiquinone-8 (ubiqui-

none-8, 98.5 %; ubiquinone-7, 1.5 %), corresponding to

the members of the order Alteromonadales [20]. The major

cellular fatty acids of strain KU41GT were C17:1 x8c

(24.2 %), summed feature 3 (C15:0 iso 2-OH and/or C16:1

x7c, 16.3 %), C15:0 (10.3 %), C11:0 3-OH (9.5 %), C9:0

3-OH (6.7 %), C10:0 3-OH (6.4 %), and C18:1 x7c (5.5 %)

(Table 2). The fatty acid composition could differentiate

strain KU41GT from the other phylogenetically related

taxa: M. nonylphenolicus KU41ET [7], P. isoporae SW-11T

[21], T. turnerae T7902T [21], E. nigra 17X/A02/237T

[22], and S. degradans 2-40T [21]. These results indicate

that strain KU41GT probably represents an independent

genus of the order Alteromonadales within the class

Gammaproteobacteria.

Taxonomic Conclusions

As shown by the 16S rRNA gene sequence analysis, strain

KU41GT belongs to the order Alteromonadales within the

class Gammaproteobacteria and forms a distinct lineage

from the related genera. Furthermore, strain KU41GT can

be differentiated from closely related genera by the com-

bination of fatty acid composition, G?C content of the

DNA, and phenotypic characteristics. Considering the data

from the polyphasic study, we suggest that strain KU41GT

represents a novel species of a new genus, for which we

propose the name Pseudomaricurvus alkylphenolicus gen.

nov., sp. nov.

Description of Pseudomaricurvus gen. nov.

Pseudomaricurvus (Pseu’do.ma.ri.cur’vus. Gr. adj. pseudes,

false; N. L. masc. n. Maricurvus, a bacterial genus name;

N. L. masc. n. Pseudomaricurvus, false Maricurvus).

Cells are Gram-negative, aerobic rods, and motile by a

single polar flagellum. Sodium ions are required for their

growth. The predominant fatty acids are C17:1 x8c, summed

feature 3 (C15:0 iso 2-OH and/or C16:1 x7c), C15:0, C11:0

3-OH, C9:0 3-OH, C10:0 3-OH, C18:1 x7c. The predominant

respiratory quinone is Q-8. The type species is Pseudomar-

icurvus alkylphenolicus.

Description of Pseudomaricurvus alkylphenolicus sp.

nov.

Pseudomaricurvus alkylphenolicus (al.kyl.phe.no’li.cus N.

L. n. alkylphenolis, alkylphenol; L. suff. -icus -a -um suffix

used with the sense of belonging to; N. L. masc. adj. al-

kylphenolicus referring to the substrate alkylphenol that

can be utilized by the species).

The description is identical to that for the genus, with

the following additions. Cells are 1.5–2.0 lm in length and

0.7–1.0 lm in width. Colonies are pale yellow, circular,

smooth, convex, 1.0–2.0 mm in diameter, and with an

entire margin after 3-days incubation on MA. Oxidase- and

catalase-positive. Growth is observed between 15 and

30 �C, with optimum growth occurring at 25–30 �C, a pH

range from at least 7.0 to 10.0, with optimum growth

occurring at pH 8.0 and at NaCl concentrations of 2–3 %.

The cells are susceptible to ampicillin (10 lg), chloram-

phenicol (30 lg), gentamicin (10 lg), kanamycin (30 lg),

nalidixic acid (30 lg), novobiocin (30 lg), penicillin G

(10 U), polymyxin B (300 U), rifampicin (5 lg), strepto-

mycin (10 lg), and tetracycline (30 lg), but not to linco-

mycin (2 lg). In API ZYM system, cells are positive for

alkaline phosphatase, esterase (C4), esterase lipase (C8),

lipase (C14), leucine arylamidase, valine arylamidase, acid

phosphatase, and naphthol-AS-BI-phosphohydrolase, but

negative for all other enzymes. Hydrolysis of Tween 40 and

Tween 80 are positive. Hydrolysis of casein, esculin, and gelatin,

nitrate reduction, indole production, acid production from glu-

cose (fermentation), arginine dihydrolase, urease, and b-galac-

tosidase are negative. The cells utilize the following compounds

as sole carbon and energy sources: p-n-nonylphenol, p-n-octyl-

phenol, p-n-heptylphenol, N-acetyl-glucosamine, D-galactose, D-

glucose, L-rhamnose, D-xylose, D-maltose, sucrose, trehalose,

glycerol, acetate, caprate, n-hexanoate, propionate, pyruvate,

benzoate, 4-hydroxybenzoate, L-alanine, L-arginine, L-gluta-

mine, L-glutamate, L-histidine, L-isoleucine, L-leucine, L-lysine,

L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,

Table 1 continued

Characteristic 1 2 3 4 5 6

Major quinone Q-8 Q-8 Q-9 (79 %), Q-8

(21 %)

Q-8 Q-8 Q-8

DNA G?C content (mol%) 53.3 48.6 51.6 47.5 49–51 45.8

Genera 1 strain KU41GT, 2 M. nonylphenolicus KU41ET [7], 3 P. isoporae SW-11T [21], 4 E. nigra 17X/A02/237T [3], 5 T. turnerae T7902T [3,

21, 23], 6 S. degradans 2-40T [3, 21, 24, 25], ? positive reaction, - negative reaction, w weakly positive reaction, NR not reported

H. Iwaki et al.: Pseudomaricurvus alkylphenolicus gen. nov., sp. nov. 171

123

and L-valine, but not the following compounds: L-arabinose, D-

arabitol, D-fructose, D-mannitol, D-mannose, D-ribose, D-sorbitol,

myo-inositol, cellobiose, lactose, adipate, citrate, gluconate,

formate, DL-malate, succinate, L-asparagine, L-asparate, L-cys-

teine, L-glycine, and L-methionine. The DNA G?C content is

53.3 mol%.

The type strain, KU41GT (=JCM 19135T = KCTC

32386T), was isolated from seawater obtained from the coastal

region of Chichi-jima Island of Ogasawara Islands, Japan.

Acknowledgments This research was financially supported in part

by the Kansai University Grant-in-Aid for progress of research in

graduate course, 2013.

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Table 2 Cellular fatty acid composition (%) of strain KU41GT and

closely related genera

Fatty acid 1 2 3 4 5 6

C9:0 4.1 1.0 – – – –

C10:0 2.2 6.4 3.5 – 3.2 –

C9:0 3-OH 6.7 –

C10:0 3-OH 6.4 9.4 3.2 6.7 1.6 8.3

C11:0 – – 2.5 – – 2.4

C11:0 3-OH 9.5 – 4.0 – – –

C12:0 – – 3.0 – 2.5 3.0

C12:0 anteiso – – – – – 8.4

C12:0 3-OH – – 2.4 – 4.7 –

C12:1 3-OH – – – – – 4.3

C13:0 anteiso – – – – – 4.1

C14:0 – 1.3 1.1 – 4.0 5.1

C15:0 10.3 1.8 – – – –

C15:1 x6c 2.4 – – – – –

C15:1 x8c 1.2 0.1 – – – –

C16:0 3.5 17.0 10.3 17.8 20.9 28.9

C16:0 3-OH – – – 1.0 – –

C17:0 2.4 1.8 5.1 1.3 1.3 17.3

C17:1 x6c 3.0 – 1.2 – – –

C17:1 x8c 24.2 5.6 13.4 – – –

C17:1 anteiso x9c – – 10.0 – – –

C18:0 – 1.6 – 6.8 4.2 5.1

C18:1 x6c – 3.0 13.1b – 36.7b 9.9b

C18:1 x7c 5.5 19.8 29.0

Summed feature 3a 16.3c 28.4c 22.3d 36.8c 18.1d 3.4d

Values are percentages of the total fatty acids; fatty acids that make up

\1 % of the total are not shown or indicated by ‘‘-‘‘

Genera 1 strain KU41GT, 2 M. nonylphenolicus KU41ET [7], 3 P.

isoporae SW-11T [21], 4 E. nigra 17X/A02/237T [3], 5 T. turnerae

T7902T [21], 6 S. degradans 2-40T [21]a, c, d Summed features are groups of two fatty acids that cannot be

separated by GLC using the MIDI system. Summed feature 3 com-

prises cC15:0 iso 2-OH and/or C16:1 x7c; dC16:1 x7c and/or C16:1 x6cb C18:1 x6c and/or C18:1 x7c

172 H. Iwaki et al.: Pseudomaricurvus alkylphenolicus gen. nov., sp. nov.

123

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nigra gen. nov., sp. nov., a gammaproteobacterium from the

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23. Distel DL, Morrill W, MacLaren-Toussaint N, Franks D,

Waterbury J (2002) Teredinibacter turnerae gen. nov., sp. nov., a

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123