Geomicrobium sediminis sp. nov., a novel bacterium isolated from a sediment sample collected from the South China Sea, and emended description of the genus Geomicrobium

ORIGINAL PAPER

Geomicrobium sediminis sp. nov., a novel bacterium isolatedfrom a sediment sample collected from the South China Sea,and emended description of the genus Geomicrobium

Zi-Jun Xiong • Yong-Guang Zhang •

Dao-Feng Zhang • Bing-Bing Liu • Li Li •

Xiao-Mei Zhang • Li-Hua Xu • Wen-Jun Li

Received: 15 June 2013 / Accepted: 16 September 2013 / Published online: 24 September 2013

� Springer Science+Business Media Dordrecht 2013

Abstract A novel bacterium, designated YIM

M13075T, was isolated from a sediment sample

collected from the South China Sea. Growth occurred

from 4 to 45 �C (optimum 28 �C), pH 6.0–11.0

(optimum pH 8.0). The strain formed yellow-cream

colonies after 5 days incubation on TSA modified with

5 % NaCl medium at 28 �C. Cells were Gram-

positive, short rods and motile. Phylogenetic analyses

based on 16S rRNA gene sequences indicated that

strain YIM M13075T was affiliated with the genus

Geomicrobium (93.5 %). The strain YIM M13075T

contained meso-diaminopimelic acid in the cell wall.

The major polar lipids were diphosphatidylglycerol

and phosphatidylglycerol. The major fatty acids were

iso-C15:0 and anteiso-C15:0. The predominant men-

aquinones were MK-7 and MK-6. The genomic DNA

G?C content was 42.7 mol%. On the basis of the

morphological and chemotaxonomic characteristics as

well as genotypic data, strain YIM M13075T repre-

sents a novel species in the genus Geomicrobium, for

which the name Geomicrobium sediminis sp. nov. is

proposed. The type strain is YIM M13075T (=DSM

25540T =JCM 18144T =CCTCC AB 2013245T). An

emended description of the genus Geomicrobium is

also proposed in the light of the new data.

Keywords Geomicrobium sediminis sp. nov. �South China Sea � Polyphasic taxonomy

Introduction

A number of haloalkaliphilic taxa that belong to the

order Bacillales, such as Alkalibacillus, Gracilibacil-

lus, Halobacillus and Sinobaca, had been isolated

from various saline environments (Fritze 1996; Jeon

et al. 2005; Romano et al. 2005; Spring et al. 1996;

Zi-Jun Xiong and Yong-Guang Zhang contributed equally to

this work.

Electronic supplementary material The online version ofthis article (doi:10.1007/s10482-013-0039-6) contains supple-mentary material, which is available to authorized users.

Z.-J. Xiong � Y.-G. Zhang � D.-F. Zhang �X.-M. Zhang � L.-H. Xu � W.-J. Li (&)

Key Laboratory of Microbial Diversity in Southwest

China, Ministry of Education and Laboratory for

Conservation and Utilization of Bio-Resources,

Yunnan Institute of Microbiology,

Yunnan University, Kunming 650091,

People’s Republic of China

e-mail: [email protected]; [email protected]

Z.-J. Xiong � Y.-G. Zhang � L. Li � W.-J. Li

Key Laboratory of Biogeography and Bioresource in Arid

Land, CAS, Xinjiang Institute of Ecology and Geography,

Chinese Academy of Sciences, Ur}umqi 830011, People’s

Republic of China

B.-B. Liu

College of Life Sciences, North East Agricultural

University, Harbin 150030, People’s Republic of China

123

Antonie van Leeuwenhoek (2013) 104:1177–1183

DOI 10.1007/s10482-013-0039-6

http://dx.doi.org/10.1007/s10482-013-0039-6

Wainø et al. 1999; Li et al. 2006). In recent years,

however, some haloalkaliphilic strains such as Alka-

libacillus silvisoli (Usami et al. 2007), Geomicrobium

halophilum (Echigo et al. 2010) and Halalkalibacillus

halophilus (Echigo et al. 2007) had been isolated from

ordinary field soils in Japan.

Moreover, many novel species were isolated from

South China Sea during the course of investigation of

bacterial diversity, such as Streptomyces glycovorans,

Streptomyces xishensis and Streptomyces abyssalis

(Xu et al. 2012), Sinomicrobium oceani (Xu et al.

2013), Bacillus sediminis (You et al. 2013), among

others. A novel species represented by strain YIM

M13075T was also isolated from marine sediment of

South China Sea, which was moderately alkali-

resisting halotolerant. This discovery further revealed

the bacterial diversity of South China Sea.

The genus Geomicrobium was first proposed by

Echigo et al. in (2010), and the haloalkaliphilic G.

halophilum BH1T was the only species of the genus.

Highest sequence similarities of YIM M13075T were

found with G. halophilum BH1T (93.5 %), followed

by Oceanobacillus profundus CL-MP28T (92.3 %)

and Bacillus patagoniensis PAT 05T (92.3 %). After

polyphasic taxonomic research, we propose that this

novel strain represents a novel species in the genus

Geomicrobium and an emended description of the

genus Geomicrobium is also presented.

Materials and methods

Strains and culture conditions

Strain YIM M13075T was isolated from a marine

sediment sample collected from the South China Sea

(113�29.704 E, 18�32.409 N) at a depth of 1,468 m.

Sediment samples (1 g) were added to 9 ml sterile

distilled water and mixed by vortexing. A 10-fold

dilution of this soil suspension was prepared in

sterilized distilled water and 0.1 ml was spread on a

agar medium (raffinose 5 g, histidine 1 g, KNO3 1 g,

CaCl2 2 g, K2HPO4 1 g, MgSO4�7H2O 1 g, NaCl

35 g, agar 15.0 g; distilled water 1 l; adjusted to pH

7.0). Then, the plates were incubated at 28 �C for two

weeks. The isolated strain was routinely cultivated on

marine agar 2216 at 28 �C and stored as aqueous

glycerol suspensions (20 %, v/v) at -80 �C. Biomass

for chemical and molecular studies was obtained by

cultivation on tryptic soy agar (TSA) modified with

5 % NaCl plates for 7 days at 28 �C.

Morphological, physiological and biochemical

characteristics

Gram staining was carried out by using the standard

gram stain and motility was tested by the hanging-drop

technique (Skerman 1967) using phase-contrast micros-

copy (Olympus). Microscopic observation of strain

YIM M13075T grown on TSA modified with 5 % NaCl

medium for 5 days at 28 �C was made by light

microscopy (BH 2; Olympus). Cells negatively stained

with 2 % uranyl acetate were used for the detection of

the presence of flagella using a JEM-2100 transmission

electron microscope (Vreeland et al. 1980). Growth was

tested at 4, 10, 15, 20, 28, 30, 35, 40, 45 and 50 �C on

TSA modified with 5 % NaCl medium by incubating

the cultures for 14 days. Growth characteristics were

determined under the following conditions with liquid

medium tryptic soy broth (TSB) modified with 5 %

NaCl as basal medium: The ability of the strain to grow

at different pHs (pH 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13,

using the buffer system described by Xu et al. 2005) and

NaCl concentrations [0, 1, 2, 3, 4, 6, 7, 8, 9 and

10–30 % (w/v) at 5 % (w/v) intervals, pH 8] was

examined at 28 �C for 14 days. Anaerobic cultivation

was performed on TSA modified with 5 % NaCl using

the Oxoid AnaeroGen system (Miller et al. 1995).

Carbon source utilization was tested using GP2 micro-

plates from the Microlog system (Biolog), which

contained 95 substrates. Catalase activity was detected

by the production of bubbles after the addition of a drop

of 3 % (v/v) H2O2. Oxidase activity was determined by

the oxidation of tetramethyl-p-phenylenediamine. Tests

for hydrolysis of gelatin, starch and Tweens 20, 40, 60

and 80, utilization of urea and nitrate reduction were

performed as described by Gonzalez et al. (1978). Other

physiological and biochemical tests were performed by

using the API ZYM and API 50CH strips (bioMerieux)

according to the manufacturer’s instructions.

Chemotaxonomy

The isomer of diaminopimelic acid was analysed

according to the procedures developed by Hasegawa

et al. (1983). Menaquinones were extracted by using

the methods of Collins et al. (1977) and separated by

HPLC (Tamaoka et al. 1983). Cellular fatty acid

1178 Antonie van Leeuwenhoek (2013) 104:1177–1183

123

analysis was performed by using the Microbial

Identification System (Sherlock Version 6.1; MIDI

database: TSBA6). Biomass for fatty acid analysis was

obtained by cultivation on TSA at 28 �C for 3 days.

Polar lipids were extracted and analysed by two-

dimensional TLC according to Embley and Wait

(1994). The genomic DNA G?C content of strain

YIM M13075T was determined by using the HPLC

method of Mesbah et al. (1989).

Molecular analysis

Extraction of genomic DNA, PCR amplification and

sequencing of the 16S rRNA gene were carried out as

described by Li et al. (2007). The values for sequence

similarity among the closest strains were determined

using the EzTaxon-e server Database (http://eztaxon-e.

ezbiocloud.net/; Kim et al. 2012). Multiple alignments

with sequences of the most closely related bacteria

were carried out using the CLUSTAL_X 1.8 program

(Thompson et al. 1997). Phylogenetic trees were con-

structed by the neighbour-joining (Saitou and Nei

1987), maximum-parsimony (Fitch 1971) and maxi-

mum-likelihood (Felsenstein 1981) tree-making algo-

rithms by using the software packages MEGA version

5.05 (Tamura et al. 2011). The stability of relationships

was assessed by performing bootstrap analyses with

1,000 resamplings (Felsenstein 1985). Some related

genera in the order Bacillales were included in the

phylogenetic trees. Brevibacillus brevis ATCC 8246T

(AB271756) was used as an outgroup.

Results and discussion

Strain YIM M13075T formed yellow-cream colonies

after 5 days incubation on TSA modified with 5 %

NaCl medium at 28 �C. Cells of strain YIM M13075T

were Gram-positive, short rods, 0.2–0.5 9 0.9–2.9 lm

and motile on TSA modified with 5 % NaCl medium

(Fig. S1). Growth temperature range was 4–45 �C, and

the optimal growth temperature was 28 �C. Growth

occurred at pH 6.0–11.0, and optimally at pH 8.0. The

NaCl tolerance range was up to 25 % and optimally at

5 % NaCl. The results of the other physiological and

biochemical analyses are summarized in Table 1 and

the species description. Comparison with the closest

relative G. halophilum BH1T, it was observed that

strain YIM M13075T could grow at lower temperatures

between 4 and 15 �C. Growth pH range also differen-

tiate strain YIM M13075T from G. halophilum BH1T,

such as YIM M13075T could grow at pH 11.0, while the

strain G. halophilum BH1T could not tolerate such

extreme pH levels. Other various physiological and

biochemical characteristics also supported the distinc-

tiveness of strain YIM M13075T from G. halophilum

BH1T (Table 1).

The new strain contained meso-diaminopimelic

acid (meso-DAP) as the diagnostic diamino acid in the

peptidoglycan. The menaquinones of strain YIM

M13075T were MK-7 and MK-6. Major fatty acids

([5 %) of strain YIM M13075T were saturated

branched-chain fatty acids: iso-C15:0 (54.6 %), ante-

iso-C15:0 (10.84 %), iso-C17:0 (7.22 %) and anteiso-

C17:0 (8.1 %). The polar lipid pattern consisted of

diphosphatidylglycerol (DPG), phosphatidylglycerol

(PG), unknown phosphoglycolipid (PGL), phosphati-

dylinositol (PI), phosphatidylinositol mannosides

(PIM), unknown phospholipids (PL) and unidentified

polar lipid (UL) as minor components (Fig. S2). The

DNA G?C content of strain YIM M13075T was

42.7 mol%. Comparison of the predominant mena-

quinone (MK-6) and the major cellular fatty acid (iso-

C17:0) with the closest relatives G. halophilum BH1T,

and close genera B. salarius BH 169T, Marinococcus

halophilus ATCC 27964T and Sinobaca qinghaiensis

YIM 70212T, it was observed that strain YIM M13075 T

Table 1 Differential phenotypic characteristics of strain YIM

M13075T and the related species Geomicrobium halophilum

BH1T

Characteristic 1 2

Colonial pigmentation Yellow-cream Brown

Cell morphology Short rods Bean-shaped

Motility ? -

Temperature 4–45 20–40

NaCl (%) 0–25 5–25

pH 6–11 6–10

Acid production from

Ribose ? -

Fermentation of

Glucose - ?

Aesculin ? -

G?C content (mol%) 42.7 45

Taxa: 1 YIM M13075T (data from this study); 2 G. halophilum

BH1T (data from Echigo et al. 2010). ? positive reaction;

- negative reaction

Antonie van Leeuwenhoek (2013) 104:1177–1183 1179

123

http://eztaxon-e.ezbiocloud.net/

http://eztaxon-e.ezbiocloud.net/

was consistent with genus Geomicrobium but different

from the close genera (Table 2).

Phylogenetic analysis of 16S rRNA gene sequence

(1,536 bp) showed that strain YIM M13075T was related

to the genus Geomicrobium (G. halophilum BH1T,

93.5 % similarity). The sequence similarities between

strain YIM M13075T and other genera of the order

Bacillales were below 93.0 %. Phylogenetic analysis

showed that strain YIM M13075T formed a distinct clade

with G. halophilum BH1T that was different from any

recognized genera or species of the order Bacillales

(Fig. 1). Maximum-likelihood and maximum-parsi-

mony method of phylogenetic reconstruction showed

relationships similar to those presented by neighbour-

joining method (Figs. S3, S4).

The chemotaxonomic characteristics of strain YIM

M13075T and the 16S rRNA gene sequence compar-

ison supported the classification of the isolate into the

genus Geomicrobium. However, differentiating char-

acteristics (Table 1) and phylogenetic analysis distin-

guished strain YIM M13075T from the only member

G. halophilum BH1T of the genus Geomicrobium.

Therefore, strain YIM M13075T is proposed to

represent a hitherto unrecognized species of the genus

Geomicrobium, for which the name G. sediminis sp.

nov. is proposed. An emended description of the genus

Table 2 Differential chemotaxonomic characteristics of strain YIM M13075T and the related genera in the order Bacillales

Characteristic 1 2 3 4 5

Predominant

quinone (s)

MK-7, MK-6 MK-7, MK-6 MK-7 MK-7 MK-5

Major polar

lipids

DPG, PG DPG, PG ND ND DPG, PG

Major fatty

acids

iso-C15:0, anteiso-C15:0,

iso-C17:0, anteiso-C17:0

iso-C15:0,

iso-C17:0,

iso-C18:0

iso-C15:0, anteiso-C15:0,

iso-C16:0, anteiso-C17:0

anteiso-C15:0,

iso-C16:0,C16:0,

anteiso-C17:0

anteiso-C15:0,

anteiso-C17:0

Taxa: 1 YIM M13075T (data from this study); 2 Geomicrobium halophilum BH1T (data from Echigo et al. 2010); 3 Bacillus salarius

BH 169T (data from Lim et al. 2006); 4 Marinococcus halophilus ATCC 27964T (data from Hao et al. 1984); 5 Sinobaca qinghaiensis

YIM 70212T (data from Li et al. 2006)

YIM M13075T (KF040368)Geomicrobium halophilum BH1T (AB449106)

Bacillus salaries BH 169T (AY667494)Marinococcus halophilus ATCC 27964 T (X90835)

Sinobaca qinghaiensis YIM 70212T (DQ168584)Bacillus saliphilus 6AGT (AJ493660)

Bacillus agaradhaerens PN-105T (X76445)Bacillus akibai 1139T (AB043858)

Bacillus oshimensis K11T(AB188090)Halolactibacillus halophilus M2-2T (AB196783)

Gracilibacillus halotolerans NNT (AF036922)Oceanobacillus profundus CL-MP28T (DQ386635)

Virgibacillus halophilus 5B73CT (AB243851)Virgibacillus pantothenticus B0018T (D16275)

Brevibacillus brevis ATCC 8246T (AB271756)

72*

98*

91*

100*

100*

99*

91

67*

69*

77*

52*

0.01

Fig. 1 Neighbour-joining tree based on 16S rRNA gene

sequences showing the relationships between strain YIM

M13075T and related genera of the order Bacillales. Bootstrap

values ([50 %) based on 1,000 replicates are shown at the

branch nodes. Asterisks indicate that the corresponding branches

were also recovered in trees generated with the maximum-

parsimony and maximum-likelihood methods. Brevibacillus

brevis ATCC 8246T was used as the outgroup. Bar, 0.01

substitutions per nucleotide position


123

Geomicrobium is also proposed in the light of the new

data.

Emended description of Geomicrobium Echigo

et al. (2010)

The emended description is based on data from Echigo

et al. (2010) and this study. Cells are Gram-positive,

non-spore-forming, motile or non-motile, short rods,

alkaliphilic, mesophilic and halophilic. Catalase- and

oxidase-positive. Cell walls contain A1c, meso-dia-

minopimelic acid-type murein. The major polar lipids

are diphosphatidylglycerol and phosphatidylglycerol.

The predominant isoprenoid quinones are MK-7 and

MK-6. The major fatty acids are iso-C15:0 and anteiso-

C15:0. The genomic DNA G?C contents are about

42–45 mol%. The type species is G. halophilum.

Description of Geomicrobium sediminis sp. nov.

Geomicrobium sediminis (se.di’ mi.nis. L. gen. n.

sediminis of a sediment).

Grows well in saliferous complex media, such as

trypticase soy agar modified with 5 % NaCl and marine

agar 2216 media. The colonies are cream-yellow. Cells

are Gram-positive, non-spore-forming, motile, short

rods, 0.2–0.5 9 0.9–2.9 lm and have flagellum.

Grows at 4–45 �C, in the presence of 0–25 % NaCl

and at pH 6–11. Optimum growth occurs at 28 �C, with

5 % NaCl and at pH 8.0. Activities of gelatinase,

catalase and oxidase are positive, while activities of

urease and tests of reduction of nitrate, hydrolysis of

starch, Tween 20, 40, 60, 80 are negative. Positive (API

ZYM) for C4 esterase, C8 lipase, leucine arylamidase,

valine arylamidase, a-chymotrypsin, naphthol-AS-BI-

phosphohydrolase. Negative (API ZYM) for alkaline

phosphatase, C14 lipase, cysteine arylamidase, trypsin,

acid phosphatase, a-galactosidase, b-galactosidase,

b-glucuronidase, a-glucosidase, b-glucosidase, N-

acetyl-b-glucosaminidase, a-mannosidase and b-fuco-

sidase. Positive (API 50CH) for glycerol, ribose,

mannitol, amygdalin, arbutin, esculin, sucrose, treha-

lose, starch, gentiobiose. Negative (API 50CH) for

erythritol, D-arabinose, L-arabinose, D-xylose, L-

xylose, Adonitol, b-methyl-D-xyloside, galactose, glu-

cose, fructose, mannose, sorbose, rhamnose, dulcitol,

inositol, sorbitol, a-methyl-D-mannoside, a-methyl-D-

glucoside, N-acetyl-D-glucosamine, salicin, cellobi-

ose, maltose, lactose, melibiose, Inulin, melezitose,

raffinose, glycogen, xylitol, D- turanose, lyxose, taga-

tose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluco-

nate, 2-keto-gluconate, 5-keto-gluconate. The

following carbon sources are utilized (Biolog GP2

system): propionic acid, succinic acid, L-alanine, L-

pyroglutamic acid, adenosine. And while the following

carbon sources are not utilized (Biolog GP2 system): a-

cyclodextrin, Tween 40, amygdalin, sucrose, glycerol,

D-L-a-glycerol-phosphate, dextrin, glycogen, D-

fructose, a-D-glucose, maltose, D-mannose, L-serine,

3-methyl-D-glucose, D-psicose, D-trehalose, turanose,

D-lactic acid methyl ester, L-lactic acid, pyruvic acid

methyl ester, b-cyclodextrin, inulin, mannan, Tween

80, N-acetyl-D-glucosamine, N-acetyl-b-D-mannosa-

mine, L-arabinose, D-arabitol, arbutin, D-cellobiose, L-

fucose, D-galactose, D-galacturonic acid, gentiobiose,

m-inositol, a-D-lactose, lactulose, maltotriose, D-man-

nitol, D-melezitose, D-melibiose, a-methyl-D-galacto-

side, b-methyl-D-galactoside, a-methyl-D-glucoside,

b-methyl-D-glucoside, a-methyl-D-mannoside, palati-

nose, D-raffinose, L-rhamnose, D-ribose, salicin, sedoh-

eptulosan, D-sorbitol, stachyose, D-tagatose, xylitol, D-

xylose, acetic acid, a-hydroxybutyric acid, b-hydroxy-

butyric acid, c-hydroxybutyric acid, b-hydroxyphe-

nylacetic acid, a-ketoglutaric acid, a-ketovaleric acid,

lactamide, D-malic acid, L-malic acid, succinic acid

monomethyl ester, pyruvic acid, succinamic acid,

N-acetyl-L-glutamic acid, L-alaninamide, D-alanine,

L-alanylglycine, L-asparagine, L-glutamic acid, glycyl-

L-glutamic acid, putrescine, 2,3-butanediol, 2-deoxy-

adenosine, inosine, thymidine, uridine, adenosine-50-monophosphate, thymidine-50-monophosphate, uri-

dine-50- monophosphate, D-fructose-6-phosphate, a-

D-glucose-1-phosphate and a-D-glucose-6-phosphate.

The type strain contains meso-DAP as the diagnostic

diamino acid in the peptidoglycan. The polar lipids are

diphosphatidylglycerol, phosphatidylglycerol and

unknown phosphoglycolipid with phosphatidylinosi-

tol, phosphatidylinositol mannosides, unknown phos-

pholipids and unidentified polar lipid as minor

components. The major fatty acids are iso-C15:0 and

anteiso-C15:0. The predominant menaquinones are

MK-7 and MK-6. The genomic DNA G?C content

of the type strain is 42.7 mol%.

The type strain, YIM M13075T (=DSM

25540T =JCM 18144T =CCTCC AB 2013245T) was

isolated from a marine sediment sample collected from

the South China Sea (113�29.704E, 18�32.409 N). The

16S rRNA gene sequence of strain YIM M13075T have


123

been deposited in GenBank under the accession

number KF040368.

Acknowledgments This research was supported by the

National Basic Research Program of China (No.

2010CB833801). Y-G Zhang was supported by West Light

Foundation of The Chinese Academy of Sciences. W-J Li was

also supported by ‘Hundred Talents Program’ of the Chinese

Academy of Sciences.

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