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Isolation of five Rubrobacter strains from

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Article in The Science of Nature November 2008
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Naturwissenschaften (2009) 96:7179

DOI 10.1007/s00114-008-0452-2

ORIGINAL PAPER

Isolation of five Rubrobacter strains from biodeteriorated

monuments
L. Laiz & A. Z. Miller & V. Jurado & E. Akatova &
S. Sanchez-Moral & J. M. Gonzalez & A. Dionsio &
M. F. Macedo & C. Saiz-Jimenez

Received: 11 March 2008 / Revised: 7 September 2008 / Accepted: 14 September 2008 / Published online: 25 October 2008
# Springer-Verlag 2008

Abstract In the last few years, the microbial colonisation

of mural paintings in ancient monuments has been attracting the attention of microbiologists and conservators. The
genus Rubrobacter is commonly found in biodeteriorated
monuments, where it has been reported to cause rosy
discolouration. However, to date, only three species of this
genus have been isolated, all from thermophilic environments. In this paper, we studied three monuments: the
Servilia and Postumio tombs in the Roman Necropolis of
Carmona (Spain), and Vilar de Frades church (Portugal), in
search of Rubrobacter strains. In all cases, biodeterioration
and the formation of efflorescences were observed, and five
Rubrobacter strains were isolated. These isolates showed
different physiology and migration in denaturing gradient
gel electrophoresis, suggesting they might represent new
species within this genus. The isolates reproduced some
L. Laiz : V. Jurado : E. Akatova : J. M. Gonzalez :
C. Saiz-Jimenez (*)
Instituto de Recursos Naturales y Agrobiologia, CSIC,
Apartado 1052,
41080 Seville, Spain
e-mail: saiz@irnase.csic.es
A. Z. Miller : M. F. Macedo
Departamento de Conservao e Restauro,
Faculdade de Cincias e Tecnologia,
Universidade Nova de Lisboa,
Lisbon, Portugal
S. Sanchez-Moral
Museo Nacional de Ciencias Naturales, CSIC,
Madrid, Spain
A. Dionsio
Laboratrio de Mineralogia e Petrologia,
Departamento de Engenharia de Minas e Georrecursos,
Instituto Superior Tcnico,
Lisbon, Portugal

biodeterioration processes in the laboratory and revealed

their biomediation in crystal formation.
Keywords Rubrobacter . Efflorescences . Biodeterioration .
Mural paintings . Struvite

Introduction
In the last decade, the topic of microbial colonisation and
biodeterioration of mural paintings in ancient monuments or
of plaster walls in churches has been attracting the attention of
microbiologists and conservators (Rlleke et al. 1998; Ciferri
1999; Gurtner et al. 2000; Piar et al. 2001). Many different
phenomena can be observedthe most commonly reported
include efflorescence formation, detachment, colour changes,
overgrowth of green photosynthetic biofilms, etc.
In the initial stages of colonisation, growth of microorganisms on a mural painting causes only aesthetic damage,
since there is little or no alteration of the painted surface.
Later, cells and hyphae penetrate the painted layer, and
chemical attack results in pitting, detachment, cracking, and
loss of the paint. This damage is added to by microbial
metabolites, which often modify the original colour, sometimes producing red or pink pigmentation (SchabereiterGurtner et al. 2001; Tiano and Tomaselli 2004; Realini et al.
2005; Imperi et al. 2007).
Schabereiter-Gurtner et al. (2001) investigated the wall
paintings of two historical buildings in Austria and
Germany, where a correlation between Rubrobacter-related
bacteria and the phenomenon of rosy discolouration of
masonry and lime wall paintings was found. Similar data
were obtained by Ortega-Morales et al. (2004) and Imperi
et al. (2007). However, those authors failed to isolate any
Rubrobacter strain.

72

Naturwissenschaften (2009) 96:7179

In this paper, we report the isolation of five Rubrobacter

strains from biodeteriorated monuments, using different
culture media. Some physiological characteristics and the
involvement of the strains in biodeterioration processes are
described.

Material and methods

Studied sites Three different indoor sampling sites were
considered in this work. Two of them were the Servilia and
Postumio tombs in the Roman Necropolis of Carmona,
Spain (Fig. 1a). The Necropolis was discovered at the end of
the nineteenth century. It is one of the most significant burial
sites in southern Spain used during the first and second
centuries A.D. The Necropolis comprises a large number
(about 600) of underground tombs excavated in the rock, a
highly porous calcarenite, which is easily affected by
weathering and processes of microkarstification. A very
characteristic effect of these processes is the crystallisation of
abundant salts at specific sites as a consequence of
evaporation. In both tombs, efflorescences were observed
on the mural paintings due to wetting and drying processes.
A few papers on the deterioration of the tombs of this
necropolis have been published elsewhere (Ario and
Saiz-Jimenez, 1997; Ario et al. 1997; Piar et al. 2001;
Akatova et al. 2007). Samples were collected at selected
points under aseptic conditions into sterile tubes and stored
at 4C until processed.
The third site is in PortugalVilar de Frades church,
integrated in the Vilar de Frades Monastery, at Barcelos
(northern Portugal). The monastery was founded in 566 and
has undergone several architectural modifications. It was
reconstructed in the Romanesque style, using granite as the
main building material. The most important change was in
the sixteenth century, when the old Romanesque monastery
was substantially modified and the deteriorated church was
re-built in granite (Fig. 1b, c). In its present state, the interior
of the monastery is clearly affected by biodeterioration.
Inside the church, the massive occurrence of green biofilms
composed of cyanobacteria and microalgae was reported
(Miller and Macedo 2006). The church was sampled at 40,
80, and 120 cm above the ground, under conditions similar
to those reported for the tombs. Efflorescences were also
found on the granite walls inside the church.
Isolation and characterisation of microorganisms Solid
media were prepared in Petri dishes following standard
microbiological protocols. Media developed for the isolation
of halobacteria (DSMZ Media 372 and 1018) were inoculated
with samples collected from the Roman Necropolis of
Carmona. Bacteria collected from Vilar de Frades church
were isolated on DSMZ Medium 372 and Trypticase Soy

Fig. 1 a Postumio Tomb, Necropolis of Carmona, Spain. b and c

Vilar de Frades church, Portugal

Agar (TSA) supplemented with NaCl (3%) and MgSO47H2O

(2%). The composition of DSMZ media is described in
http://www.dsmz.de/media. Incubation was carried out at
28C for 30 days. Isolation followed standard microbiological procedures. Five strains of Rubrobacter spp. were
isolated. The isolates from the Necropolis of Carmona were
denominated CO5-S3 and CO5-S14, and those of Vilar de
Frades church VFA-S1, VFA-S4, and VFA-S5.
The methods for characterising the strains isolated in this
study were previously described by Jurado et al. (2005a,b).
Filter-sterilised carbon sources (see Table 1), ammonium
sulphate, and yeast extract were added to basal salts to
perform single-carbon-source assimilation tests (Chen et al.
2004). In addition, 3% NaCl (w/v) and 2% MgSO47H2O
(w/v) were added for VFA-S1, VFA-S4, VFA-S5, and CO5S3 strains. The same carbon sources were added to basal
salts medium with 15% NaCl (w/v) and 2% MgSO47H2O
(w/v) for the CO5-S14 strain.
Characterisation of microorganisms and microbial communities Molecular techniques were used for the detection of
microorganisms and microbial communities. DNA from

Naturwissenschaften (2009) 96:7179

73

Table 1 Phenotypic characteristics of Rubrobacter spp

Characteristic
Growth
5055C
TSA Na-Mg
TSA
Nitrate reduction
Decomposition or hydrolysis ofa
DNA
Gelatin
Acid produced from
D-Glucose
D-Melibiose
D-Fructose
Enzymatic activity
Alkaline phosphatase
Leucine arylamidase
Valine arylamidase
N-acetyl--glucosaminidase
Utilisation ofa,b
D-Arabinose
D-Fructose
D-Galactose
Glycerol
D-Mannitol
D-Mannose
Myo-Inositol
D-Xylose

(+)

+
+
+
+

+
+
+
+

+
ND
+
+

+
+

+
+
+

+
+
+

+
+
+

+
+

+
+

+
+

+
+

+
+
+

+
+
+

+
+/

+
+/
+
+
+

+
+/
(+)
+
(+)
+
+
+

+
+
+
+
+
+
+
+

+
+
+
+
+

+
+

+
+
+

+
+
+

+
+
+

+
+
+

+
+

+
+
+

All strains are gram-positive, catalase-positive and oxidase-negative.

1 VFA-S1, 2 VFA-S4, 3 VFA-S5, 4 CO5-S3, 5 CO5-S14, 6 R. taiwanensis JCM 12932T , 7 R. xylanophilus DSMZ 9441T , 8 R. radiotolerans
DSMZ 5868T , negative, + positive, (+) weakly positive, +/ variable, ND not determined
a
Data obtained in this work for strains 1 to 5, and data from Chen et al. (2004) for strains 6 to 8
b
Results after 1 week of incubation

naturally colonising communities was extracted using the

Nucleospin Food DNA Extraction Kit (Macherey-Nagel,
Dren, Germany). Microbial communities were characterised by their molecular fingerprints obtained by DGGE
analysis. The 16S ribosomal RNA gene (16S rRNA) was
used for the identification of bacteria.
Amplification of DNA was carried out by PCR. Bacterial
16S rRNA genes were amplified using the primer pair 27F
(5-AGA GTT TGA TYM TGG CTC AG) and 1510R (5GGC TAC CTT GTT ACG ACT T; Gonzalez and SaizJimenez 2005; Echigo et al. 2005).
Total RNA was extracted using the RNAqueous4PCR kit
(Ambion Inc., Austin, USA). Reverse transcription was
carried out with the reverse transcriptase Thermoscript
(Invitrogen, Carlsbad, USA), using a 16S rRNA genespecific primer, 518R (5-ATT ACC GCG GCT GCT GG)
(Gonzalez and Saiz-Jimenez 2004), at an annealing temperature of 55C for 1 h.
Amplification protocols comprised one cycle of denaturation (95C for 2 min), followed by 35 cycles of
denaturation (15 s at 95C), annealing (55C for 15 s),

and elongation (72C for 2 min) and a terminal elongation

cycle (72C for 10 min). Amplifications were performed in
a BioRad iCycler iQ thermal cycler (BioRad, Hercules,
CA). PCR products were purified and sequenced by
SECUGEN Sequencing Services (CSIC, Madrid, Spain).
Sequence data were edited using the software Chromas,
version 1.45 (Technelysium, Tewantin, Australia). Homology
searches with those sequences were performed using the Blast
algorithm (Altschul et al. 1990) on the NCBI database (http://
www.ncbi.nlm.nih.org/blast/). Sequences were checked for
chimeric structures using CCODE (Gonzalez et al. 2005).
Crystal precipitation The precipitation of crystals was
tested in different media, as described elsewhere (Groth et
al. 2001). Positive results were obtained in the culture
medium TSA supplemented with NaCl (3%) and
MgSO4.7H2O (2%). Crystals were extracted from the solid
agar media with a small spatula, washed with distilled
water, and air-dried at 37C. The mineral composition of
the precipitates was characterised by powder X-ray diffraction (Philips-PW-1710 diffractometer) and scanning elec-

74

tron microscopy (Philips XL-20 SEM), using a Philips

Dx4i analytical microanalyser (EDAX).
Inoculation of rocks The strains CO5-S3 and VFA-S1 were
used for rock inoculation and reproduction of biodeterioration
processes in the laboratory. A limestone (Campaspero) and a
sandstone (Villamayor) were sliced into squares of 33
0.5 cm, sterilised in an autoclave, and inoculated with a
suspension of each bacterium in fresh culture medium TSA
with 3% NaCl and 2% MgSO47H2O, and incubated at 37C.
The rock probes were left to dry out three times (for periods of
57 days each) and, thereafter, wetted with new media. At the
end of the third dryness period, each probe was re-wetted with
medium for 48 h and processed for dehydrogenase activity.
Dehydrogenase activity The method of Warscheid et al.
(1990) was used for assessing microbial activity and
colonisation. This is based on the reduction of colourless
2,3,5-triphenyltetrazolium to red 2,3,5-triphenylformazan
by the active bacteria as the result of dehydrogenase
activity. After incubation, the probes were examined under
a binocular microscope.

Results
The two tombs of the Necropolis of Carmona and the Vilar
de Frades church presented evident biodeterioration processes. The areas of efflorescence in the Necropolis tombs
and in the church were sampled for determination of the
microbial communities using molecular tools and for
isolation of Rubrobacter strains.
A Rubrobacter strain (C05-S3) was isolated in a
sampling carried out in May 2005, 8 months after cleaning
and restoration of the Tomb of Servilia. Another Rubrobacter strain (C05-S14) was isolated from efflorescences
collected in the Tomb of Postumio, at a few hundred metres
from the former tomb, in this case shortly (two weeks) after
restoration, also in May 2005. In July 2006, three more
Rubrobacter strains (VFA-S1, VFA-S4 and VFA-S5) were
isolated from the altar of Vilar de Frades church.
Table 1 shows the phenotypic characteristics of the
Rubrobacter isolates and those of the three described type
strains. A major difference between our isolates and the
type strains was the absence of growth at 5055C and in a
TSA medium. Our strains required the presence of sodium
and magnesium as supplement in a TSA medium, denoting
a moderate halophilic behaviour. In addition, our isolates
did not decompose or hydrolyse DNA and gelatin, and did
not produce acid from sugars. The enzymatic activity of
valine arylamidase present in the type strains was also
absent in our isolates.

Naturwissenschaften (2009) 96:7179

Sequence comparison of strains C05-S3, C05-S14 and

VFA-S1 revealed their high similarity to each other but
only a 94% similarity to Rubrobacter radiotolerans 16S
rRNA gene. The percentage of similarity between the 16S
rRNA gene from strains C05-S3 and VFA-S1 was 99.3%.
However, DGGE mobility observed in our five strains was
different to those of the three type strains (Fig. 2),
suggesting that our isolates might correspond to a different
species.
In the Postumio tomb, 7.1% of 28 clones processed
corresponded to Rubrobacter spp. However, when the
study was focused on RNA (Gonzalez et al. 2006), the
percentage increased to 9.1%, indicating that the Rubrobacter spp. were metabolically active in the community. In
the Servilia tomb, of 96 clones of RNA, 8.3% corresponded
to Rubrobacter spp. Furthermore, the sequence of clones
CAR-D5 and CAR-A7 corresponded to the isolated strain
C05-S3, indicating that a metabolically active Rubrobacter
strain was also successfully isolated.
Figure 3 shows the phylogenetic tree of the Rubrobacter
type species, of our strains, and of the sequences obtained
by Schabereiter-Gurtner et al. (2001) and Imperi et al.
(2007). The strains isolated in this work and the sequences
obtained from rosy discolouration form a separate cluster
from the three Rubrobacter type strains.
The Rubrobacter isolates were tested for the production
of salt crystals, as described by Groth et al. (2001). All
strains except CO5-S14 were able to induce crystal
formation in TSA culture medium supplemented with NaCl
and MgSO4.7H2O (Fig. 4). XRD revealed that the crystals
were struvite, a magnesium ammonium phosphate,
(MgNH4PO46H2O), as shown in Fig. 5.
Two rocks were selected for reproduction of biodeterioration processes in the laboratory. Campaspero is a compact,
somewhat porous limestone (density 2.3 g/cm3, open porosity
14%), classified as a fossiliferous micrite. Villamayor is an
arkosic sandstone, the cementing material composed of
palygorskite, smectite and small amounts of clay minerals
and oxides, with a density of 1.8 g/cm3 and an open porosity
of 35%.
Inoculation of rocks with two Rubrobacter strains (CO5S3 and VFA-S1) resulted in the formation of a biofilm on the
limestone. The limestone presented a thin (0.1 mm) bacterial
film formed by an accumulation of cells which retracted
when dried (Fig. 6a). Bacterial cells were also observed in
the rock fissures (Fig. 6b). In the sandstone, in addition to
biofilm formation on the surface (Fig. 6c), there was evident
cell penetration into the mineral matrix, reaching up to 1 mm
as revealed by the dehydrogenase activity test. This was due
to the higher porosity of the sandstone matrix, with wide
holes and masses of palygorskite fibres, as observed in the
SEM micrographs (Fig. 6d). Scattered struvite crystals were
observed on the Rubrobacter biofilm (Fig. 6e and f). When

Naturwissenschaften (2009) 96:7179

Fig. 2 Details from the 3055% denaturing concentration region of

the ethidium bromide-stained gel, showing the 16S rDNA fingerprint
derived from the DNA extraction of isolates and Rubrobacter spp.
type strains. Lane A is a standard reference marker which includes
four strains: Pseudomonas sp., Escherichia coli, Paenibacillus sp.,
and Streptomyces sp. Lane B strain VFA-S1. Lane C strain VFA-S4.
Lane D strain VFA-S5. Lane E Rubrobacter radiotolerans
DSMZ5868T. Lane F Rubrobacter xylanophilus DSMZ9941T. Lane
G Rubrobacter taiwanensis JCM12932T. Lane H strain C05-S3. Lane
I strain C05-S14

75

et al. 2003a,b, 2005a,b; Lee et al. 2006, Smerda et al.

2006). In September 2004, the mural paintings were
consolidated with Primal AC-33 (acrylate copolymers)
and treated with benzalkonium chloride, but instead of
eliminating the microbial community, the treatment produced a complex DGGE pattern where bacteria were more
abundant (Akatova et al. 2007) and included Rubrobacter
sequences. The Postumio tomb was consolidated with Estel
1000 (ethyl silicate) and treated with benzalkonium
chloride.
At the Necropolis of Carmona sampling sites, in addition to
Rubrobacter, sequences from Sphingomonas, Phyllobacterium,
Ralstonia, Pseudomonas, Stenotrophomonas, Shigella and
Arthrobacter monumenti were retrieved. Interestingly, A.
monumenti, together with A. parietis and A. tumbae, were
isolated from the Tomb of Servilia by Heyrman et al. (2005b)
and described as new species.
In Vilar de Frades church, Rubrobacter was associated
with efflorescences and cyanobacterial biofilms composed
of Cyanidium which accounted for the majority of DGGE
bands.
Schabereiter-Gurtner et al. (2001) analysed the bacterial
colonisation and rosy discolouration of masonry and lime wall
paintings. Some 70% of sequences obtained from three rosy
biofilms were related to sequences of the genus Rubrobacter.
Moreover, the majority of these Rubrobacter-related seR. radiotolerans, DSM 5868, X98372
99

71

CO5-S3 strain, EU512989

56

AJ298577

82
92

dried, the biofilm separated from the rock surface and the
detachment removed mineral grains, producing a mechanical
deterioration (Fig. 6a). In addition, the continuity of the
biofilm was interrupted by emerging masses of crystals in a
process similar to the formation of efflorescences. The
penetration of Rubrobacter cells into the porous mineral
matrix, the formation of crystals in intimate contact with the
Rubrobacter film, and the detachment of mineral grains upon
biofilm retraction are well-known biodeterioration processes.

A high biodiversity has been reported in the mural

paintings of the Tomb of Servilia. Previous works resulted
in the isolation and description of a considerable number of
bacteria, mainly novel species of Arthrobacter, Bacillus,
Oceanobacillus, Paenibacillus and Virgibacillus (Heyrman

VFA-S1 strain, EU512991

CO5-S14 strain, EU512990
VFA-S4 strain, EU512992
VFA-S5 strain, EU512993
AJ298576

86

90

86

AM746681
AM746694

83

AJ298575
AM746684
81

68

Discussion

AJ298579

AM746685

R. taiwanensis, AF465803
R. xylanophilus, DSM 9941, CP000386

0.01

Fig. 3 Phylogenetic tree based on the 16S rRNA gene sequences

from Rubrobacter species, the isolates obtained during this study, and
related sequences. Accession numbers of the sequences represented in
this dendrogram are provided, as well as the bootstrap values at each
branching point. AJ sequences are from Herberstein, Austria and AM
sequences are from Matera, Italy. Bar represents changes per
nucleotide

76

Fig. 4 Rubrobacter VFA-S1 and struvite crystals at 20 days of

incubation. Medium TSA supplemented with NaCl (3%) and
MgSO47H2O (2%)

quences shared more than 95% similarity, indicating that the

corresponding bacteria were members of a single genus. The
low-sequence similarity (about 93%) to Rubrobacter species
revealed that they might represent new species within the
genus Rubrobacter. Our five strains showed 98% similarity to
the sequence of the Hb-clones present in the wall paintings of
the Castle of Herberstein (Austria).
Our strains also showed 98% similarity to the sequences of
the clones obtained by Imperi et al. (2007). Those authors
analysed the rosy discolouration following bacterial colonisation on Byzantine frescoes in the Crypt of the Original Sin

Fig. 5 X-ray diffraction pattern

of struvite crystals. Standard
struvite diffraction pattern is
marked as single lines

Naturwissenschaften (2009) 96:7179

(Matera, Italy) and provided evidence of a causal relationship

between heavy contamination by Rubrobacter-related
bacterioruberin-producing bacteria and rosy discolouration
of ancient wall paintings.
On an exterior, partially shaded, wall of the Anexo
building (a Mayan monument in Uxmal, Mexico,
characterised by high illumination and low water content), a thin, reddish biofilm community, containing
almost exclusively organisms related to Rubrobacter
xylanophilus and Rubrobacter radiotolerans was found
(Ortega-Morales et al. 2004).
Schabereiter-Gurtner et al. (2001) suggested that a
possible resistance to desiccation might be a selective
advantage for Rubrobacter growth on masonry and wall
paintings. For Imperi et al. (2007), the humidity decrease
and temperature increase resulting from local climatic
changes, combined with enhanced daylight irradiation,
may have promoted the outgrowth of xerotolerant heterotrophic bacteria, primarily Rubrobacter spp., at the expense
of the pre-existing microbial community.
Furthermore, it is known that the DNA of Rubrobacter
species is frequently cloned from desert soils where these
organisms may be very abundant (Rainey et al. 1999;
Holmes et al. 2000; Gundlapally and Garcia-Pichel 2006;
Kuhlman et al. 2006) and survive for long periods to grow
during sporadic rainy periods.
It should be mentioned that the underground chapel of
Castle Herberstein, where Rubrobacter-related sequences
were found, was used, in former times, for salt and food
storage. This, and the presence of efflorescences on the

Naturwissenschaften (2009) 96:7179

77

Fig. 6 Growth of Rubrobacter

spp. on rocks. a Desiccated
biofilm of Rubrobacter strain
VFA-S1 on the surface of a
limestone. b Rubrobacter strain
VFA-S1 on a limestone fissure.
c Biofilm of Rubrobacter strain
CO5-S3 covering sandstone
surface. d Detail of palygorskite
fibres and cells of Rubrobacter
strain CO5-S3. e Three struvite
crystals mixed with Rubrobacter
strain CO5-S3 biofilm. f Details
of a struvite crystal and
Rubrobacter strain CO5-S3
biofilm

walls, might explain the presence of Rubrobacter. Similarly,

the underground church where the crypt in Matera was
located showed efflorescences on the paintings as a
consequence of a dry and hot season (Imperi et al. 2007).
The Necropolis of Carmona is usually subject to a regional
drought from June to October. In the studied tombs, air
temperature ranged from 32C in August to 6C in
December, while rock temperature ranged from 27C in
August to 9C in December. Relative humidity (RH) ranged
from 99100% in December and March to 17% in August.
These facts suggest that the finding of Rubrobacter strains in

the Necropolis of Carmona might be related with the niches

suffering periods of drought with efflorescence formation, as
also demonstrated in the laboratory. Rubrobacter could,
therefore, survive through the year and grow during rainy
periods. In Vilar de Frades church, only a few data were
recorded during the sampling time. In April, the air
temperature was 13C and the RH 87%, while in July the
values were 22C and 75%, respectively.
The authentic type strains of the genus Rubrobacter were
isolated from hot spring water samples (Suzuki et al. 1988;
Chen et al. 2004) and hot runoff of a carpet factory (Carreto

78

et al. 1996). Although Rubrobacter species have been

isolated only from thermophilic environments, it is unlikely
that further novel species will be restricted to such
environments. The finding of environmental sequences in
different soils, rock varnish, and monuments, as well as the
isolation of our five strains, suggests that Rubrobacter is a
widespread group present in a variety of habitats. The use
of appropriate culture media should result in the isolation of
more Rubrobacter strains.
The strains found outside thermophilic environments
have different physiological characteristics to those of the
type strains. The analysis of phenotypic and molecular
characteristics suggests that our isolates are different from
the three Rubrobacter type strains, and that some of them
might represent new species. However, for the proposal of a
new species, further taxonomic study is needed.
Some research has been done on Rubrobacter type
species with regard to radiation resistance, carbohydrate
metabolism, etc. (Chen et al. 2004). It is suggested that
Rubrobacter from desert, arid soils and monuments play a
role in the biogeochemical cycle of elements. To get a
better understanding of this matter we investigated the
production of minerals in culture media and in rock probes.
We found that Rubrobacter strains form struvite both in
culture media and on the rock surfaces. Similar euhedral
crystals of struvite were found in the cultures of an
unidentified strain isolated from the Grotta dei Cervi, Porto
Badisco, Italy (Groth et al. 2001). This was subsequently
identified as Bacillus weihenstephanensis, strain P2-14
(Laiz et al. 2003).
The ability to form struvite is not widespread among
bacteria. Species of the genera Pseudomonas, Flavobacterium,
Arthrobacter (Rivadeneyra et al. 1983, 1992), Myxococcus
(Ben Omar et al. 1996) and Chromohalobacter (Rivadeneyra
et al. 2006) have been reported to produce struvite. Recently,
Sanchez-Roman et al. (2007) found that moderately halophilic gamma-proteobacteria (Halomonas spp., Salinivibrio
costicola, Marinomonas communis and Marinobacter hydrocarbonoclasticus) precipitate struvite.
The study of the non-thermophilic isolates gave an
insight into the physiology of Rubrobacter present in
monuments. The data indicate that these Rubrobacter
strains play an active role in efflorescence niches and in
mineral precipitation, and contribute to biodeterioration
processes, as demonstrated by the weathering tests performed in the laboratory.
Acknowledgements EA, VJ and LL acknowledge financial support
from a Marie Curie Action MEST-CT2004-513915, an I3P (CSICESF) postdoctoral contract, and project 200740/011, respectively. This
research was funded by the Consejera de Innovacin, Ciencia y
Empresa, Junta de Andaluca, project P06-RNM-02318 and by CSICFCT, project 2007PT0041.

Naturwissenschaften (2009) 96:7179

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