System. Appl. Microbiol.

24, 572–587 (2001)

© Urban & Fischer Verlag
http://www.urbanfischer.de/journals/sam

A Polyphasic Taxonomic Study of Thermophilic Bacilli from

Shallow, Marine Vents
TERESA L. MAUGERI1, CONCETTA GUGLIANDOLO1, DANIELA CACCAMO1, and ERKO STACKEBRANDT2
1
Dipartimento di Biologia Animale ed Ecologia Marina, Sez. Ecologia Microbica e Biotecnologie, Messina, Italy
2
DSMZ, Braunschweig, Germany

Received July 17, 2001

Summary
Eighty-seven thermophilic, aerobic, spore-forming bacteria were isolated from shallow, marine, thermal
vents of the Eolian Islands (Italy) and tested for a broad spectrum of phenotypic characteristics. A nu-
merical taxonomy study was performed on these isolates and 8 thermophilic Bacillus and Geobacillus
reference strains by 89 selected features. Results from cluster analysis showed the formation of nine clus-
ters. Most of the isolates (83%) fell into several phenetically well distinguished clusters, loosely related to
Geobacillus thermodenitrificans. The remaining isolates grouped together with different reference
strains. Eighteen isolates, representative of the different clusters, were selected for subsequent genotypic
characterisation, including partial 16S rDNA sequence analysis of 18 strains and almost complete 16S
rDNA sequences of 9 strains. Subsequent DNA/DNA reassociation studies and determination of the base
composition of DNA identified seven isolates as Geobacillus thermodenitrificans, two isolates as G. ther-
moleovorans and one isolate as Bacillus pallidus. Four isolates represented two novel species of Bacillus.
The remaining four represented novel Geobacillus species, one of which has recently been described as
Bacillus vulcani DSMZ 13174 T.

Key words: shallow hydrothermal vents – thermophilic bacilli – phenetic diversity – numerical analysis –
genotypic characterization – novel Geobacillus species – novel Bacillus species

Introduction
Thermophilic bacteria belonging to the genus Bacillus
are able to grow at or above 60 °C showing optimal
growth temperatures in the range ≥45– ≤70 °C. Most of
the high temperature bacilli have been isolated from hot
environments (hot springs, solfataras or geothermally
heated soils) or from man-made thermal systems (hot
water pipelines, heat exchangers, waste treatment plants,
burning coal refuse piles), while thermotolerant bacilli
may be isolated from both thermophilic and mesophilic
environments, including soil samples, composting vegeta-
tion, sewage digester, river-, lake- and sea-water. They
have been also isolated from shallow water marine hot
springs and from deep-sea hydrothermal vents (HJÖR-
LEIFSDOTTIR et al., 1989; MARTEINSSON et al., 1996).
The first publication on the characterisation of an aer-
obic spore-former able to grow at 70 °C, Bacillus ther-
mophilus, was carried out by MIQUEL in 1888. Since then
several reports on thermophilic Bacillus strains have been
published (GORDON and SMITH, 1949; SHARP et al., 1992;
WHITE et al., 1993).
On the basis of 16S rRNA sequence analysis of the
genus Bacillus it was shown that most thermophilic
species are members of Bacillus rRNA group 5 (ASH
et al., 1991; RAINEY et al., 1994), the majority of which
have recently been reclassified as Geobacillus (NAZINA
et al., 2001): Geobacillus stearothermophilus, G. ther-
moglucosidasius, G. thermodenitrificans (MANACHINI
et al., 2000), G. kaustophilus, G. thermocatenulatus and
G. thermoleovorans. The latter species embraces the in-
valid species “B. caldolyticus”, “B. caldotenax” and
“B. caldovelox” (SUNNA et al., 1997). Other members of
Bacillus rRNA group 5 are “B. thermoantarcticus”
(NICOLAUS et al., 1996) and B. vulcani, (CACCAMO et al.,
2000) as well as Saccharococcus thermophilus and S. cal-
doxylosilyticus (AHMAD et al., 2000).
The interest for these bacteria arises from their
biotechnological importance as sources of themostable
enzymes (proteases, amylases, pullulanases, glucose-iso-
merases, lipases, xylanases and DNA restriction endonu-
cleases) and products for industrial use, such exopolysac-

0723-2020/01/24/04-572 $ 15.00/0

charides or compatible solutes (HARWOOD, 1989; SHARP
et al., 1992).
This paper is the first report on the isolation and char-
acterisation of thermophilic bacilli from different shal-
low, marine thermal vents located around the Eolian Is-
lands (Italy). We describe here the biodiversity of the iso-
lates by numerical analysis of their phenotypic features in
comparison with 8 thermophilic Bacillus and Geobacillus
reference strains. This approach has been followed by the
genotypic characterisation of 18 selected isolates.
Materials and Methods
Sampling sites
The Eolian Islands represent an arc of volcanic origin where
submarine hot waters and gases flow from the sea-floor at vari-
ous depths. The sites of the collection of samples in shallow,
submarine volcanic systems were located at different depths
around the Eolian Islands (Italy) (GUGLIANDOLO et al., 1999).
Water and sediment samples were collected at different shal-
low marine thermal vents during an oceanographic cruise
(September 1996) around Eolian Islands. Sampling was made by
SCUBA divers using sterile sampler (GUGLIANDOLO et al., 1999).
Sites depth, temperature and pH were immediately recorded by
a multiparameter probe.
Enrichment and isolation
All samples were immediately treated aboard ship. The en-
richment of aerobic, heterotrophic, thermophilic bacteria was
obtained using the following protocol. Water samples were fil-
tered through 0.45 mm membrane filters. Filters were inoculat-
ed into Bacto Marine Broth 2216 (Difco, Detroit, MI, USA) and
liquid Medium D (MD) (DEGRYSE et al., 1978) or placed onto
plates of Bacto Marine Agar 2216 (Difco) (MA) and MD agar.
The media were incubated at 65 °C for three days in aerobic
conditions. Isolation was made on the same media supplement-
ed with agar (2%). All colonies obtained on Petri dishes were
picked and purified by streaking on MA at least three times. Se-
rial dilutions of sediment samples were treated by the same
method.
Reference strains
The following thermophilic strains of Bacillus rRNA group 5
were included as reference strains: G. stearothermophilus DSM
22T, G. thermocatenulatus DSM 730T, G. thermodenitrificans
DSM 465T, G. thermoleovorans DSM 5366T, G. kaustophilus
DSM 7263 as well as three strains, which were recently pro-
posed as members of G. thermoleovorans (SUNNA et al., 1997),
namely, “B. caldolyticus” DSM 405, “B. caldotenax” DSM
406, and “B. caldovelox” DSM 411.
Phenotypic studies
All phenotypic tests were made in triplicate and repeated
when inconsistent results were observed. All growth tests, unless
otherwise specified, were performed at 55 °C.
Gram- and spore-staining, observation of motility, test of ox-
idase and catalase activity, were performed as described by SMIB-
ERT and KRIEG (1994).
Temperature and pH range for growth was determined fol-
lowing incubation of the strains for 3 days at 37, 45, 55, 60, 65,
70 and 75 °C and pH 5.5, 6.0, 7.0, 8.0 and 9.0 in Bacto Marine
Broth (Difco).
Halotolerance was tested after 3 days of incubation in Bacto
Nutrient Broth supplemented with 0, 2, 3, 5, 7 and 10% (w/v)
NaCl.
Biodiversity of thermophilic bacilli from Eolian Islands 573
Optimal growth was evaluated by measuring the increase in
turbidity at 600 nm with a spectrophotometer (Ultraspec 3000,
Amersham Pharmacia Biotech, Buckinghamshire, UK).
Biochemical characteristics were screened by test strips of
API system (bioMérieux, Marcy l’Etoile, FR). The first 11 tests
in the API 20 E and 49 tests in the API 50 CHB (acid production
from carbohydrates) were used. Constitutive enzyme production
was tested by the API ZYM according to SHARP et al. (1980). Ni-
trate reduction and carbohydrate assimilation were detected by
the API 20 NE. Strips were incubated at 55 °C in a plastic bag
onto a bed of wet cotton in order to minimise evaporation. Re-
sults were scored reading at 12 and 24h (LOGAN and BERKE-
LEY,1984).
Utilization of glucose (0.6% w/v), sucrose (0.6% w/v), carra-
genan (0.1 % w/v) as sole carbon source was tested adding the
carbohydrates to MD agar deprived of tryptone. Growth was
scored after 3 days of incubation at optimal temperature.
Starch, casein and tributyrin hydrolysis was tested according
to MARTEINSSON et al. (1996); xylan and dextran hydrolysis was
tested according to WHITE et al. (1993).
Lipolytic activity on Tween 20 and Tween 80 was tested on
Sierra agar modified (DEGRYSE et al., 1978).
To assess possible error, tests were performed in duplicate on
18 random strains (SNEATH and JOHNSON, 1972).
Cluster analysis
Results obtained were arranged in a binary data matrix and
analysed using the Sokal and Michener (SSM) and Jaccard (J)
similarity coefficients together with the unweighted pair group
method with arithmetic average algorithm (UPGMA) (SNEATH and
SOKAL, 1973). Cophenetic correlation values were determined
for the two dendrograms obtained using the NTSYS-PC © 1.70
program (1992 – Exeter Software, Applied Biostatistics Inc,
New York).
Genotypic studies
Randomly selected strains from different clusters were subse-
quently studied for genotypic characteristics.
Determination of DNA mol % G+C content
The DNA was isolated according to CASHION et al. (1977).
The mol% G+C was determined by HPLC as described by MES-
BAH et al. (1989).
16S rDNA sequence determination and analysis
Genomic DNA extraction, 16S rDNA amplification via PCR
and sequencing of the purified products were performed accord-
ing to RAINEY et al. (1996). Sequence reaction mixture was elec-
trophoresed by using a model 373A automated DNA sequencer
(Applied Biosystem, Foster City, California, USA). The 16S
rDNA sequences obtained were manually aligned with reference
sequences of Bacillus and Geobacillus spp. using the ae2 editor
(MAIDAK et al., 1999). Evolutionary distances were calculated by
the method of JUKES and CANTOR (1969). For partial sequences
dendrograms were generated by the distance analysis methods
of DE SOETE (1983) and SAITOU and NEI contained in the PHYLIP
package (FELSENSTEIN, 1993). Phylogenetic clustering of almost
complete sequences were done by neighbour-joining and maxi-
mum-likelihood methods (FELSENSTEIN, 1993). Bootstrap analy-
sis, following a Kimura-2 correction of similarity values
(FELSENSTEIN, 1988) by performing 100 resamplings, was used
to evaluate the tree topology of the maximum-likelihood den-
drograms.
DNA-DNA hybridisation
The DNA was isolated as described by CASHION et al. (1977).
DNA-DNA hybridisations were carried out according to DE LEY
574 T. L. MAUGERI et al.

et al. (1970), with the modifications proposed by HUB et al.
(1983), in a Gilford System model 2600 spectrophotometer
equipped with a Gilford model 2527-R thermoprogrammer and
plotter. The renaturation rates were determined by the Transfer
BAS program (JAHNKE, 1992).
Results and Discussion
Sampling and isolation
Eighty-seven strains, 54 from venting-water and 33
from sediment close to the vents, were isolated from col-
lected samples.
Phenotypic studies
All 87 strains were rod-shaped, Gram positive, spore
forming and grew aerobically at 55 °C, reaching the ex-
ponential growth phase within 18 h. Seventy- seven
strains were able to grow at 60 °C, 65 strains at 65 °C
and 10 strains at 70 °C. Salt was not required for growth
by 59 strains. All strains grew with 2–3% NaCl, 23
strains grew with 5% NaCl, six with 7% NaCl and only
one strain with 10% NaCl. Almost all strains grew at
neutral or slightly alkaline pH. All strains were positive
for gelatin hydrolysis and most strains were positive for
esculin hydrolysis. Most strains fermented ribose, fruc-
tose, maltose, keto-gluconate, and grew on sucrose

Table 1. Designation and source of the 87 isolates and eight reference strains assigned to clusters, or unclustered, defined at 68–80%
SSM/UPGMA level (in bold, strains selected for genotypic characterization).

Cluster (n°of strains) Strain Source or Site of isolate

a (13) A1-3, A2, S1, S1-1, T1-3,B3s5, B3-1,

B3-72, B3-73, B3-74, B2-71, B3s, B3-75 Porto di Levante, Vulcano Island
b (28) B1-1, B1-2, B2-1, B2-2, B2-3, B2-4, B2-5, Porto di Levante, Vulcano Island
B3, B3-5, B3-15, B3-22, B3-29, s1b-1a,
T2a, T3-3, B3-s1, B3-s2, T2-2, T2-3, T3-2
3s-2 La Roya, Vulcano Island
s5s-1, s5s-2, 12-2, S12-1a Inzolfata, Lipari Island
10-15, g10 Zurro, Stromboli
g11-2 Ginostra, Stromboli
c (2) S7s1, 7s La Calcara, Panarea
d (16) B2-70, B3-76,T1-1, T1-4, T2, T2-1, T2-4, Porto di Levante, Vulcano Island
T2-6, T3-1, T1, T3 Punta Conigliara, Vulcano Island
4-4, 4-5, 4-6 Inzolfata, Lipari Island
5-1, 5-2
e (4) 1b-2, 1as Porto di Levante, Vulcano Island
4-1, 4s-1 Punta Conigliara, Vulcano Island
f (2) A3, S3 Porto di Levante, Vulcano Island
g (6) A1, S2 Porto di Levante, Vulcano Island
5s, 5s-2 Inzolfata, Lipari Island
S7-5 La Calcara, Panarea
10-16 Zurro, Stromboli
G. thermodenitrificans (DSM 465T)
h (20) “B. caldotenax” (DSM 406)
“B. caldolyticus” (DSM 405)
T3-4 Porto di Levante, Vulcano Island
U4-4 Punta Conigliara, Vulcano Island
10-1 Zurro, Stromboli
12-1 Inzolfata, Lipari Island
“B. caldovelox” (DSM 411)
G. thermoleovorans (DSM 5366 T)
1b-4, 1a-1, 1a-2, 1a-3 Porto di Levante, Vulcano Island
3s-1, 3s, M3s La Roya, Vulcano Island
M4, M4-1, 4-2 Punta Conigliara
G. thermocatenulatus (DSM 730 T)
B. kaustophilus (DSM 7263 T)
i (2) 1bw Porto di Levante, Vulcano Island
G. stearothermophilus (DSM 22T)
Unclustered strain B3s-3 Porto di Levante, Vulcano Island

(0.6%), carragenan, dextran and xylan. Almost all pos-
sessed phosphatase alkaline and acid, butyrate and capry-
late esterase, Tween 20 and Tween 80 lipase, casein and
tributyrin hydrolase.
All strains were negative for arginine dehydrolase, ly-
sine and ornithine decarboxylase, urease and H2S produc-
tion; caprate was not utilised; D-arabinose, L-xylose,
adonitol, rhamnose, dulcitol, inulin, xylitol, and fucose
were not fermented.
Error based on mismatches of results of the duplicate
strains was estimated at 5.5%.
Cluster analysis
The final n × t matrix included 95 strains (87 isolates
and 8 reference strains) and 89 selected tests. Data arising
from growth tests at different conditions and antibiotic
sensitivity were not used for the numerical analysis.
Cophenetic values were 0.81 and 0.73 for SSM and
SJ/UPGMA analysis, respectively. Given the relatively
high cophenetic correlation values, detailed results are
presented for the SSM /UPGMA analysis.
Two main groups (A and B), which were separated at
63% similarity level, emerged in the dendrogram result-
ing from cluster analysis (Fig. 1). Group A included most
of the isolates (72), linking with G. thermodenitrificans
DSM 465T at 65% similarity level. Group B included 15
isolates and the remaining 7 reference strains linked at
67% similarity level. Several strains (8/15) of this group
clustered around G. thermoleovorans DSM 5366 at 70%
similarity level.
Any aggregation at ≥ 68% similarity level on the dendro-
gram was considered as a different cluster. The rational for
this cut off value was based on the finding that strains affili-
ated to the species G. thermoleovorans by high DNA-DNA
reassociation similarities, namely “B. caldolyticus” DSM
405, “B. caldotenax” DSM 406, “B. caldovelox” DSM
411, G. kaustophilus DSM 7263 and G. thermocatenulatus
DSM 730 (SUNNA et al., 1997) were linked at 68% similari-
ty level (AUSTIN and PRIEST, 1986) in the present study.
Six multimembered (a, b, d, e, g, h) and three double-
membered (c, f, i) clusters of isolates were recovered at
≥ 68% SSM (Fig. 1). Only one strain in Group A remained
unclustered.
In Group A, the cluster a was defined at 78% SSM level
and comprised 13 isolates from site U1 (Vulcano). Most
of them were positive for lipolytic activity on Tween 20
and Tween 80 and for nitrate reduction. Eight strains hy-
drolysed starch.
Cluster b (SSM ≥ 78%) was the largest one, comprising
28 isolates from water and sediment. Most isolates were
thermotolerant and halotolerant, and were also able to
reduce nitrates.
Cluster c (SSM ≥ 88%) comprised two halophilic iso-
lates from site U7.
Cluster d (SSM ≥ 75%) comprised 16 isolates, showing
lipolytic activity on Tween 20. They were all unable to
ferment most carbohydrates.
Cluster e (SSM ≥ 80%) comprised four isolates more
inert than other strains in carbohydrates fermentation.
Biodiversity of thermophilic bacilli from Eolian Islands 575
They were able to hydrolyse starch and casein and only
two strains reduced nitrates.
Cluster f (SSM ≥ 78%) comprised two strains from site
U1, strictly thermophilic and able to hydrolyse starch.
Cluster g (SSM ≥ 70%) comprised six isolates. They dif-
fered from other isolates in Group A in their ability to
ferment glucose, fructose, mannose, mannitol, sorbitol,
sucrose and trehalose. Moreover, they were unable to hy-
drolyse casein and starch.
In Group B, cluster h (SSM ≥ 68%) comprised twenty
strains, six of which were reference strains (G. ther-
moleovorans, G. kaustophilus, G. thermocatenulatus,
“B. caldotenax”, “B. caldolyticus” and “B. caldovelox”).
Most strains assimilated mannose, N-acetyl-glucosamine
and maltose and were able to use citrate and ferment
fructose, maltose, mannose, sucrose, D-xylose, trehalose
and starch.
Cluster i comprised G. stearothermophilus and one
field strain, strictly thermophilic and halophilic, linked at
68% similarity level.
Table 1 lists the designation and source of the 87 iso-
lates and eight reference strains assigned to clusters or
constituting individual lineages on the basis of ≥ 68%
SSM/UPGMA similarity values.
Table 2 (a, b, c) shows physiological/biochemical char-
acteristics of the multi-membered clusters of isolates and
reference strains.
Genotypic studies
Eighteen representative strains were randomly selected
from the 87 previously analysed by cluster analysis. The
main phenotypic properties of these 18 isolates are
shown in Table 3 in comparison with those of three relat-
ed Geobacillus reference strains (data from literature).
DNA base composition
The mol% G + C content of DNA of the 18 isolates
ranged from 39 to 53%, as shown in Table 3.
Phylogenetic position
As the previous characterisation indicated a high vari-
ability of their phenotypic properties, partial 16S rDNA
sequences (position 5′ terminus to about position 500)
were generated for the 18 selected strains. Binary similar-
ity values indicated that some strains were phylogeneti-
cally very similar, some of which were closely related to
some Geobacillus and Bacillus reference strains. The
topology of the neighbour-joining tree indicated the pres-
ence of five strains groups (I–V) (Fig. 2), two of which (I,
II) included members of the Geobacillus/Saccharococcus
rRNA group 5 (ASH et al., 1991; RAINEY et al., 1994).
Group I, harbouring 10 isolates, was divided in two
subgroups. In subgroup Ia, sequences of isolates A1 and
S3 were identical while those of strains A2, B3, T2, B3s
and B3-1 were almost identical to the sequence of G.
thermodenitrificans. Subgroup Ib consisted of strains 4-2,
3s-1 and 3s-2, sharing identical sequences with that of G.
576 T. L. MAUGERI et al.

Fig. 1. Simplified dendrogram based on simple matching coefficient (SSM) showing phenotypic relationships among the 87 field
strains and 8 thermophilic Bacillus and Geobacillus reference strains (on the right, in bold, strains selected for genotypic characteri-
zation).

kaustophilus and its close relatives G. stearo-
thermophilus, G. thermoleovorans, “B. caldotenax”, “B.
caldolyticus”, and G. thermocatenulatus.
Group II showed strains 5-2, 10-1, and 1bw to form a
moderately related strains cluster adjacent to G. ther-
moglucosidasius and some undescribed thermophilic iso-
lates, Bacillus strain ICP 56 and Bacillus strain Ak1.
Group III contained strain 5s, closely related to B. pal-
lidus, “B. thermoalkalophilus” and Bacillus sp. DSM
2349, a strain of B. pallidus (WHITE et al., 1993).
 Biodiversity of thermophilic bacilli from Eolian Islands 577

Table 2a. Characteristics of clusters arising from numerical analysis.

Legend: Within columns, number of positive strains when different from all positive (+) or negative (–). Responses of strains within double-
membered clusters (c, f, i), or unclustered, are not showed except for the reference strains DSM 22 and DSM 465.
§
DSM 465T – G. thermodenitrificans; DSM 405 – “B. caldolyticus”; DSM 406 – “B. caldotenax”; DSM 411 – “B. caldovelox”; DSM 5366T –
G. thermoleovorans; DSM 730T – G. thermocatenulatus; DSM 7263T – G. kaustophilus; DSM 22T – G. stearothermophilus. Strain DSM 5366
is the type strain of the species G. thermoleovorans which embraces also strains DSM 405, 406, 411, 730, 7263 (SUNNA et al., 1997); results are
given separately for the 6 strains in order to show the phenotypic heterogeneity of this species. Data for all reference strains were obtained at
our laboratory.
* Characters not selected for numerical analysis.
578 T. L. MAUGERI et al.

Table 2b.
 Biodiversity of thermophilic bacilli from Eolian Islands 579

Table 2 (c)
580 T. L. MAUGERI et al.
Group IV harboured strains T3 and 4-1, sharing iden-
tical partial 16S rDNA sequences, which were distantly
related to B. methanolicus.
Group V included strains 1as and 7s, sharing identical
partial 16S rDNA sequences, which were distantly related
to B. infernus and some temperate Bacillus species.
In order to determine more precisely the phylogenetic
position of those strains which were not identical to de-
scribed Geobacillus and Bacillus species, the almost com-
plete (1450 nucleotides) 16S rDNA sequence of nine
strains was determined: three from group I (strains A2,
Fig. 2. Phylogenetic tree showing
the relationships of the 18 selected
strains, representative of ther-
mophilic bacilli isolated from the
Eolian Islands, to described ther-
mophilic Bacillus and Geobacillus
species.
3s-1, 4-2), three from group II (strains 5-2, 10-1, 1bw),
one from group III (strains 5s), one from group IV (strain
4-1) and one from group V (strain 1as). The extension of
the information from about 500 nucleotides to 1450 nu-
cleotides did not significantly change the overall picture
of relatedness (not shown).
The similarity values obtained for almost complete se-
quences (Table 4) indicated that strain A2 (subgroup Ia)
was nearly identical (99.7%) to G. thermodenitrificans
and similar to G. stearothermophilus (98.4%) and mem-
bers of G. thermoleovorans cluster (98.4%) (MAIDAK et
Table 3. Main chemo-taxonomic and phenotypic properties of the 18 selected isolates in comparison with those of three Geobacillus
reference strain.

Refer- Strain G+C Cata- Oxi- Mini- Opti- Maxi- Growth Opti- Growth Opti-
ence name mol(%) lase dase mum mum mum range mum pH mum
Cluster* tempe- tempe- tempe- with salt salt range pH
rature rature rature (%) (%)

a A2 51.3 + + 37 65 70 0–3 2 5.5–9 8

a B3-1 51.4 – + 45 60 65 0–3 2 5.5–9 6
a B3s 51.9 + – 37 45 65 0–5 2 6–8 7
b B3 50.8 – + 37 60 65 0–10 2 5.5–9 7
b 3s-2 52.4 – + 37 65 65 0–3 2 7–9 8
c 7s 39 – – 37 55 65 2–3 2 7–9 7
d T3 40.8 – + 37 45 65 2–3 2 5.5–9 6
d 5-2 43.1 – + 45 60 70 0–5 2 6–8 8
d T2 51.0 + – 37 60 65 0–3 2 8–9 9
e 4-1 40.8 – + 37 55 65 2–3 2 7–9 8
e 1as 39 – + 37 55 65 2–3 2 6–9 7
f S3 51.2 + + 45 65 70 0–3 2 5.5–9 8
g A1 51.6 + + 37 45 60 2–3 2 5.5–9 7
g 5s 40 + – 37 65 70 0–3 2 6–8 7
DSM 465** 50.3 + + 45 55–65 70 0–5 0–2 5.5–8 6–8
h 10-1 42.7 – – 37 60 70 0–5 2 6–9 7
h 4-2 52.4 – – 37 55 65 0–3 2 5.5–9 8
h DSM 5366** 51–56 v v 35 55–70 78 0–2 0 6–8 6–7.5
h 3s-1 53 – + 37 60 72 0–3 2 5.5–9 6
i 1bw 45 – – 45 60 70 0–5 2 7–9 8
i DSM 22** 52.6 – – 45 60 75 0–3 0 6.5–8 7
Production of acetoin
Reference Cluster*

Citrate utilisation

Nitrate reduction
Acid from:***

Hydrolysis of:
Strain name

cellobiose

Tween 20
Tween 80
trehalose

mannitol
mannose

fructose
maltose
glucose
sucrose

esculin
gelatin
ribose

starch
casein

Species identification
a A2 G. thermodenitrificans
a B3-1 G. thermodenitrificans
a B3s G. thermodenitrificans
b B3 G. thermodenitrificans
b 3s-2 G. thermoleovorans
c 7s Bacillus sp.
d T3 Bacillus sp.
d 5-2 Geobacillus sp.
d T2 G. thermodenitrificans
e 4-1 Bacillus sp.
e 1as Bacillus sp.
f S3 G. thermodenitrificans
g A1 G. thermodenitrificans
g 5s B. pallidus
DSM 465** G. thermodenitrificans
h 10-1 Geobacillus sp.
h 4-2 G. thermoleovorans
h DSM5366** G. thermoleovorans
h 3s-1 Geobacillus sp.
i 1bw Geobacillus sp.
i DSM 22** G. stearothermophilus

Legend: * Clusters arising from numerical analysis of phenetic characters of isolates and reference strains. ** Type strain from
Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Data from literature. *** Data obtained by the API 50 CHB.
v – variable; w – weak.
582
Table 3a
Reference cluster
Strain
a A2
a B3-1
a B3s
b B3
b 3s-2
c 7s
d T3
d 5-2
d T2
e 4-1
e 1as
f S3
g A1
g 5s
h 10-1
h 4-2
h 3s-1
i 1bw
´
Reference cluster
Strain
a A2
a B3-1
a B3s
b B3
b 3s-2
c 7s
d T3
d 5-2
d T2
e 4-1
e 1as
f S3
g A1
g 5s
h 10-1
h 4-2
h 3s-1
i 1bw
T. L. MAUGERI et al.
Antibiotic sensitivity
Growth pH range
Chloramphenicol
Growth tempe-
Growth range
Nalidixic acid
Erythromycin
with salt (%)
Stteptomycin
Polymyxin B
Novobiocin
Kanamycin
Tetracyclin
rature (°C)
Bacitracin
Penicillin
Mobility
Catalase
Oxidase
– + + 37–70 0–3 5.5–9 + + + + + + + – + +
– – + 45–65 0–3 5.5–9 + + + + + + + – + +
– + – 37–65 0–5 6–8 + + + + + + + – + +
– – + 37–65 0–10 5.5–9 + + + + + + + – + +
+ – + 37–65 0–3 7–9 + + + + + + + – + +
– – – 37–65 2–3 7–9 + + + + + + + – + +
– – + 37–65 2–3 5.5–9 + + + + + + + – + +
+ – + 45–70 0–3 6–8 + + + + + + + + + +
– + – 37–65 0–3 8–9 + + + + + + + – + +
+ – + 37–65 2–3 7–9 + + + + + + + – + +
– – + 37–65 2–3 6–9 + + + + + + + – + +
– + + 45–70 0–3 5.5–9 + + + + + + + – + +
– + + 37–60 2–3 5.5–9 + + + + + + + – – –
+ + – 37–70 0–3 6–8 + + + + + + + – + +
+ – – 45–70 0–3 5.5–9 + + + + + + + – + +
+ – – 37–65 0–3 5.5–9 + + + + + + + – + +
+ – + 37–72 0–3 5.5–9 + + + + + + + – + +
+ – – 45–70 2–3 7–9 + + + + + + + – + +
API 20E API 20NE
orto-nitro-phenyl-galactosidase
assimilation of phenylacetate
assimilation of arabinose
assimilation of gluconate
assimilation of N-acetyl-
assimilation of mannitol
assimilation of mannose
ornithine decarboxylase
assimilation of maltose
assimilation of glucose
assimilation of adipate
tryptophan deaminase
assimilation of malate
assimilation of citrate
arginine dehydrolase
lysine decarboxylase
acetoin production
gelatin hydrolysis
citrate utilisation
indol production
H2S production
NO3 reduction
glucosammine
urease
– – – – – – – – – – + – – – – – – – + – – – –
– – – – – – – – – – + + – – + – + + + + + – +
– – – – + – – – – – + + + – + – + – + – – – –
+ – – – – – – – – – + + – – – – – – + – + – –
– – – – + – – – – + + – + – + – – – – – – – –
– – – – – – – – – – + – – – – – – – – – – – –
– – – – + – – – – – + + + – + + – + + + – – –
– – – – – – – – – + + – + – + + + + + + – – –
– – – – – – – – – + + + + – + + + + + + + – +
– – – – + – – – – + + – + – – + – + + + + + –
– – – – + – – – – + + + + – + – + + + – – – –
– – – – – – – – – – + – + + + + – + + – + – –
+ – – – – – – – – + + + + + + + + + + – + – –
– – – – – – – – – + + – + – + + – + + + + – –
+ – – – – – – – – – + + + – + + + + + – + – +
+ – – – + – – – – + + – + + + + + + – – – – –
– – – – – – – – – – + + + – + + + + + + + – +
– – – – + – – – – + + – – – – – – – – – – – –
Table 3b
Reference cluster
1. glycerol
Strain
a A2
a B3-1
a B3s
b B3
b 3s-2
c 7s
d T3
d 5-2
d T2
e 4-1
e 1as
f S3
g A1
g 5s
h 10-1
h 4-2
h 3s-1
i 1bw
Table 3c
Reference cluster
26. Salicin
Strain
a A2
a B3-1
a B3s
b B3
b 3s-2
c 7s
d T3
d 5-2
d T2
e 4-1
e 1as
f S3
g A1
g 5s
h 10-1
h 4-2
h 3s-1
i 1bw
API 50 CHB-carbohydrate fermentation
20. α-methyl-D-mannoside
21. α-methyl-D-glucoside
22. N-acetyl-glucosamine
9. β-methyl-D-xyloside
25. Esculin hydrolysis
3. D-arabinose
4. L-arabinose
13. D-mannose
23. Amygdalin
15. Rhamnose
12. D-fructose
10. Galactose
14. L-sorbose
2. erythritol
7. L- xylose
18. mannitol
6. D-xylose
8. Adonitol
16. Dulcitol
24. Arbutin
19. sorbitol
17. Inositol
11. glucose
5. Ribose
– – – – + + – – – – + – + – – – – + – – – + – – +
– – – – + – – – – – – + – – – – – – – – – – – – +
– – – – – – – – – – – – – – – – – – – – – – – – –
– – – – + – – – – – – + – – – – – – – – – – – – +
– – – – + – – – + – – + – + – – – – – – – – – – +
– – – – + – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – + – – – – – – – – – + – + – +
– – – – – – – – – – + – + – – – – – – – – – – – –
– – – – + + – – – – + + – – – – – – – – – – – + +
+ – – – – – – – – – – + – – – – – – – – – – – – +
– – – – + – – – – – – + – + – – – – – – – – – – +
– – – – + + – – – – + + + – – – – + – – – – – – +
– – – + – – – – – – + + + – – – – – – – + + + + +
+ – – – – – – – – – + + + – – – + + + – + – + + +
+ – – – + – – – – + + + + – – – – + – – – + – – +
– – – + + + – – – – + + + – – – – – – + + + + + +
+ – – – + + – – – + + + + – – – – + – – + – + + +
+ – – – + + – – – – + + + + – – – + – – – + – + +
API 50 CHB-carbohydrate fermentation
48. 2-ketogluconate
49. 5-ketogluconate
39. β-gentiobiose
35. D-raffinose
40. D-turanose
42. D-tagatose
34. Melezitose
27. Cellobiose
45. D-arabitol
47. Gluconate
46. L-arabitol
30. Melibiose
32. Trehalose
37. Glycogen
41. D-lyxose
43. D-fucose
44. L-fucose
28. Maltose
29. Lactose
31. Sucrose
38. Xylitol
36. Starch
33. Inulin
+ + – – + – + – – – + – – – – – – – – – – – – –
– – – – – – – – – – + – – – – – + – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – + – – – – – – – + – – – – – – +
– – – – – + – – – – – – – – – + + – – – – – + +
– – + – – + + – – – + – – – – + + – – – – – + +
– – + – + + + – – – – – – – – – – – – – – – – –
– – + – – – – – – – – – – – + – – – – – – – + –
– – – + – – + – – – + + – – – – + – – – – – – +
– – + – – + + – – – + – – – – – + – – – – – – +
– – + – – – – – – – + – – – – – – – – – – – – –
+ + – – – + + – – – – – – – – – + – – – – – – –
+ + + – – + + – – – – – – – – – – – + + – – + –
– – + – + + + – – – – – – – – – – – – – – – + +
+ + + + + + + – – + + – – + + – – – – – – – – +
+ + + + + + + – + + + + – + + – – – – – – + + +
+ + + – + + + – + + + + – – – – – – – – – + + +
584 T. L. MAUGERI et al.

Table 3d

API ZYM Growth Hydrolysis of:

on:

18. N-acetyl-β-glucosaminidase
2. phosphatase alcaline

6. leucine arylamidase

8. cystine arylamidase
4. esterase lipase (C8)

7. valine arylamidase

12. phosphohydrolase
11. phosphatase acid

15. β-glucuronidase
13. α-galactosidase
14. β-galactosidase

19. α-mannosidase
10. chymotrypsin

16. α-glucosidase
17. β-glucosidase
3. esterase (C4)

20. α-fucosidase
Reference cluster

5. lipase (C14)

9. trypsin

carragenan

tributyrin

Tween 20
Tween 80
dextran
glucose
sucrose

starch
casein
Strain

a A2
a B3-1
a B3s
b B3
b 3s-2
c 7s
d T3
d 5-2
d T2
e 4-1
e 1as
f S3
g A1
g 5s
h 10-1
h 4-2
h 3s-1
i 1bw
xylan
+ + + + – – – – – + + + – – + – – – – – + + + + + + – – +
+ + + + + – – – – – + + – – – – – – – – + + + + + + + + –
+ + + – – – – – – + + + – – – – – – – – + + + + + + + + +
– + – – – – – – – + + – – – – – – – – – + + + + + + + – +
– – – – – – – – – + + – – – – – – – – + + + + – + + – + –
+ + + + + + + + + + + + – – – + – – – – – + – – + – – – –
+ + + – – – – – – + + – – – + + – – – – + + + + – + – –
+ + + – – – – – – + + + – – + – – – – + + + + + – – + – –
+ + + + + – – – – + + + – – – – – – – – + + + + + + + +
– – – – – – – – – + + – – – + – – – – + + + + – + + + – –
– – – – – – – – – + + – – – + – – – – + + + + + + + + – –
+ + + + – – – – – + + + – – + – – – – – + + + + + + + – +
+ + + + – – – – – + + – – – – – – – – + + + – – – – – + –
+ + + – – – – – – + – – – – – – – – – + + – + + + – – + –
+ + + – – – – – – + + + + – + – – – – + + + + + + + + + +
+ + + – – – – – – + + + + – + + – – – + + + + + + + + – –
– – – – – – – – – + + – – – – – – – – + + + + + + + – + –
+ + + + + + – – – + + + – – – – – – – + + + + – + + – + –

al., 1999). This cluster also contained strains of subgroup species within the genus Bacillus, strains 4-1 and 1as
Ib, i.e., strains 4-2 and 3s-1 similar at 99.7–99.9% and should be considered two novel Bacillus species.
99.6–99.7%, respectively (Table 4). Strains 5-2, 10-1, The representative of subgroup Ia, strain A2, showed
1bw, members of group II, shared 98.6–99.1% similarity 98.9% DNA similarity with G. thermodenitrificans DSM
among each other and 98–98.6% between strain ICP 56 465T (Table 4) and must be considered a strain of this
and strain Ak1 to G. thermoglucosidasius (Table 4). The species. Similarly, strain 4-2 (subgroup Ib) was highly re-
sequence of the group III strain 5s was identical to that of lated to members of the G. thermoleovorans group
strain DSM 2349 and almost identical to that of B. pal- (79–89.6%), while reassociation values between DNAs
lidus (Table 4). Isolates in group IV (strain 4-1) and of strain 3s-1 and these organisms ranged between
group V (strain 1as) showed similarity values lower than 51.4–60.7%; reassociation of strain 3s-1 and strain 4-2
96% among themselves and to any described species of was 62.6% (Table 4). The G. thermoleovorans group in-
Geobacillus and Bacillus. cludes G. kaustophilus, G. thermocatenulatus, “B. caldo-
lyticus”, “B. caldotenax”, “B. caldovelox” and G. ther-
moleovorans that share DNA/DNA similarity values
DNA/DNA hybridisation
>70% and should be considered as one species (SUNNA et
The degree of DNA-DNA similarity as measured by al., 1997). The three isolates of group II (strains 5-2, 10-1,
the DNA/DNA reassociation method was determined for 1bw) exhibited 48.5–60% DNA/DNA similarity one to
seven of the nine isolates that showed high 16S rDNA each other (Table 4), while the representative of group III,
similarity values with type strains of Geobacillus species strain 5s, should be considered a strain of B. pallidus
(Table 4). Strains 4-1 and 1as were not hybridised against (100%) (Table 4). Since the DNA/DNA hybridisation ex-
any reference strain because of the isolated phylogenetic periments yielded less than 70% similarity with the phy-
position, remote from any Bacillus reference strain (lower logenetically closest Geobacillus species (Table 4), strains
than 96%) (STACKEBRANDT and GOEBEL, 1994). Accord- 3s-1, 5-2, 10-1 and 1bw are considered to represent new
ing to their phylogenetic distance from all reference species (WAYNE et al., 1987). One of these, strain 3s-1,
 Biodiversity of thermophilic bacilli from Eolian Islands 585
Table 4. 16S rDNA similarity values (%) and DNA-DNA reassociation levels (%) for seven isolates and the closest reference strains within three (I, II, III) of the five groups of

*Range of 16S rDNA and DNA/DNA similarity values obtained after comparison of our isolates with G. thermoleovorans, G. kaustophilus, “B. caldolyticus”, “B. caldotenax”,
has recently been described as B. vulcani (CACCAMO et

100
13

al., 2000). This species has not yet been reclassified as a

–
member of Geobacillus (NAZINA et al., 2001) because the

99.3
100
publication of this species occurred during the printing
12
Group III

–
process of the latter genus.

99.7
99.5

and “B. caldovelox”, considered as representative of the same species, G. thermoleovorans, on the basis of DNA/DNA reassociation >70% (SUNNA et al., 1997).
11

–
Conclusions

94.7
94.6
94.7
59.8
48.5 Comparison of the isolates from shallow hydrothermal
10

– vents of Eolian Islands with reference strains of Bacillus

strains recognised into the phylogenetic tree (upper right triangle: DNA/DNA reassociation values; lower left triangle: 16S rDNA similarity values).

94.7
94.6
94.7
98.6
and Geobacillus by numerical analysis methods did not
60

allow their unambiguous identification at the species

9

–
Group II

level. The majority of strains (83%) grouped together

94.7
94.8
99.1
98.6

forming several clusters within Group A, related to G.

95
8

thermodenitrificans. Taxonomically, the clear separation

between G. thermodenitrificans (Group A) and the other
94.5–94.6
51.4–60.7

96.6–96.8
96.3–96.4
96.9–97.1

reference strains (Group B) agrees with previously pub-

79–89.6

lished data on thermophilic bacilli (WHITE et al., 1993;

72–73

94.4
40.4
61.4

94.4

MARTEINSSON et al., 1996; MANACHINI et al., 2000).

7

Comparison of metabolic properties of seven strains se-

–

lected from Group A, namely strains A1, A2, B3, B3-1,

99.7–99.0

B3s, S3, and T2, revealed that they resembled G. ther-

modenitrificans, but were phenetically heterogeneous in
94.4
62.6

94.5
94.4
96.6
96.3
96.9

many respects, such as hydrolysis of casein and gelatin,

6

and acid production from sucrose, trehalose, cellobiose,

mannitol, dulcitol, erythritol. Only after the examination
99.6–99.7

of DNA-DNA similarity and 16S rDNA gene sequence

similarity of representatives from each cluster, we were
41.2

99.6

94.4
96.8
99.6

94.6
94.4
97.1

able to determine the SSM level at which individual ge-

5

nomic species were separated one from each other pheno-

typically. Comparison of our data with the most recent
99.1–99.2

literature confirms the high phenotypic intraspecific di-

versity of thermophilic bacilli, as demonstrated by the
99.1

94.6
99.1

94.7
94.6
96.8
96.4
97.1

formation at 68% similarity level of a cluster with six dif-

4

ferent strains (G. thermoleovorans, G. kaustophilus, G.

Subgroup Ib

98.9–99.1

thermocatenulatus, “B. caldolyticus”, “B. caldotenax”,

“B. caldovelox”) representative of the same species G.
98.9
98.9
98.9

94.6
96.4
96.1
96.7

94.7
94.6

thermoleovorans, phenetically heterogeneous and geneti-

48
3

cally homogeneous. Phylogenetic analyses of partial 16S

–

rDNA sequences and DNA/DNA reassociation of one

98.2–98.3

representative of these seven isolates, strain A2, indicated

that all of them should be considered strains of G. ther-
98.9

98.3
98.4
98.4
98.2

93.9
95.7
96.3
96.7

94.1

modenitrificans, which dominated among the ther-

Subgroup Ia

94
2

mophilic bacilli isolated from Porto di Levante, at Vul-

cano Island. Also the metabolic properties of G. ther-
99.7
98.4
98.6
98.5
98.4
98.4
95.9
95.5
97.1

94.3
94.1

modenitrificans are thus much broader than those indi-

94
1

cated in the description of the species, containing pre-

dominantly isolates from soil (MANACHINI et al., 2000).
G. thermodenitrificans is more halotolerant than other
“B. thermoalkalophilus”

described thermophilic Geobacillus and Bacillus species

Geobacillus strain 10-1
Geobacillus strain 1bw
Geobacillus strain 3s-1
G. thermodenitrificans
G. stearothermophilus
Geobacillus strain A-2

Geobacillus strain 4-2

Geobacillus strain 5-2

(SHARP et al., 1992) and it is therefore not surprising that

G. thermocatenulatus

G. thermoleovorans*

Geobacillus strain 5s

the high salinity of thermal vents at Porto di Levante

favours the presence of strains of this species.
The polyphasic characterization allowed the recogni-
B. pallidus

tion of two new Bacillus and four new Geobacillus

species, one of which, strain 3s-1, has recently been de-
scribed as B. vulcani (CACCAMO et al., 2000). Strain 4-1
Strains

and strain T3 (related to B. methanolicus), as well as

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.

strains 7s and 1as (related to B. pallidus) represent new

586 T. L. MAUGERI et al.
Bacillus species, while strains 1bw, 10-1 and 5-2 (all re-
lated to G. thermoglucosidasius) represent novel
Geobacillus species. The description of these species is in
preparation (MAUGERI et al., unpublished).
Many of these new isolates exhibited characteristics
potentially useful for biotechnological exploitation. All of
them were heavy-metals resistant (MAUGERI et al., 1999)
and able to utilize hydrocarbons (GUGLIANDOLO et al.,
1998); strains 4-1, 3s-1, 5-2, T3, 1bw and 10-1 produced
exopolysaccharides.
The results lead to the conclusion that marine hydro-
thermal vents of Eolian Islands harbour different thermo-
philic organisms the taxonomic novelty and biotechno-
logical potential are worth pursuing.
Acknowledgements
We wish thank Dr. Franco Italiano from IGF (CNR), Palermo
(Italy), chief scientist of the cruise (1996) aboard the M/V Ura-
nia, for inviting us to participate in the expedition.
This study was partly funded by the European Commission
project MAS3-CT 95-0034, which was coordinated by Prof.
Daniel Prieur, University of Brest, France.
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Corresponding author:
TERESA L. MAUGERI, Dipartimento di Biologia Animale ed Ecolo-
gia Marina, Sez. Ecologia Microbica e Biotecnologie, Salita
Sperone, 31, 98166 Messina, Italy
Tel.: ++39-90-676 5523; Fax: ++39-90-393409;
e-mail: tmaugeri@unime.it