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Microscopy: advances in scientific research and education (A. Mndez-Vilas, Ed.

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Microbial biotechnologies to preserve and restore stone monuments


C. Ercole*, P. Cacchio and M. Del Gallo
Department of Life, Health and Environmental Sciences, University of LAquila, Coppito 67010, Italy
*
Corresponding author: claudia.ercole@univaq.it
The role of microbes as decaying promoters of stone surfaces and of environmental factors they control has been
established since long time. On the contrary, the positive effects of microbial interventions on the stone surface
preservation and restoration has deserved, so far, much less attention. The present work describes calcite precipitation by
bacterial strains isolated from carbonate rich environments. Also, this study investigates the potential relationship between
extracellular polymeric materials (EPM) - such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) isolated
from calcifying bacteria -and calcium carbonate crystals precipitation. The ability to form CaCO3 crystals, the extent of the
precipitation, and the type of crystals formed are influenced by temperature as well as by microbial cellsgrowth. Shapes
and dimensions of the crystals depend on both bacterial strain and its growth conditions. Further researches are in progress
directly aiming to develop biotechnological processes by which stone decaying surfaces are restored by EPM or by
isolated protein produced from them.
Keywords: calcite precipitation; historic monuent preservation; EPS e CPS; calcifying bacteria

1. Introduction
Monumental stone decay is a consequence of interactions between material, and environmental factors and living
organisms.
Throughout history, mankind has used the most beautiful, durable and strongest stones, such as limestone, for the
construction of civil structures and historic monuments. Limestones are mainly composed of calcium carbonate.
Calcium carbonate is one of the most common and widespread minerals present on the earth. Its highly porous and
hydrophilic nature makes it highly susceptible to damage by weathering and atmospheric condition (such as
temperature, humidity, pollutants and acid rain). The high porosity allows penetration of water along with corrosive
ions, acids and salts inside the porosity of the stone and cause severe damage to them [1].
Besides, persistent environmental and industrial pollutants often may be deposited on limestone monuments and
stone works and as a result accelerate their deterioration. Besides stone surfaces supports the growth of some
characteristic group of microorganisms which cause deterioration in different ways [2]. Over the last few decades
numerous works of art, which have survived in good condition for centuries or ever millennia, have been degraded and,
in some cases, in an irreversible way.
Historic stone structures are often large and located outdoors; for this reason they pose particular conservation
challenges that require collaborative efforts between conservators and scientists [3].
Consequently the immediate need to develop preventive and remedial methodologies is necessary to safeguard these
cultural heritage monuments and stone works of art at their original location. In recent years several physical,
mechanical and chemical methods [1, 4-6] have been devised and used for cleaning and restoration purposes but none
of the tested treatment methods have proved to be satisfactory for the preservation consolidation and waterproofing of
deteriorated monuments and works of art [7-8].
Some of these methods have yielded rather poor results and accelerated alterations in the treated stones in terms of
texture, physical strength and aesthetic appearance [1]. Conventional treatments, both organic and inorganic, employed
for the consolidation of ornamental stones have several disadvantages: long-term incompatibility from the substrate and
the cement used for consolidation, plugging of pores induced by the new cement, all in all leading, in some cases, to the
acceleration of stone alteration [8, 9]. The observed incompatibilities have encouraged for search and development of
new conservation treatments for protection and consolidation of ornamental stone, being biological methods apparently
more suitable than traditional ones [10]. In particular, attention has been drawn to bioconservation using bacterial
carbonatogenesis (i.e. bacterially induced calcium carbonate precipitation) as a new eco-friendly conservation
treatment, particularly suitable for carbonate stones [8].

2. Biomineralization
Biomineralization is the process by which organisms form minerals, by creating physical and chemical conditions
necessary for mineral formation and growth.
Microorganisms are active in a wide range of mineralization processes and have been involved in the deposition of
minerals throughout the history of the Earth [11, 12]. Some aspects of biomineralization overlap with some aspects of
geomicrobiology and of other scientific disciplines.

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The most abundant cation in most known biominerals is calcium and the most abundant anions are carbonates.
Microbial metabolism is highly versatile and is characterized by the frequent release of metabolic products, such as
organic acids, sugars and enzymes. These products can change the physicochemical environment, (e.g. pH), they can
also catalyze redox reactions, which directly or indirectly change the redox state of metals, favouring mineral
deposition.
The type of mineral produced, however, is more dependent on the environmental conditions in which the microorganism is living than on the biological processes involved in its formation. Thus, the same bacterial species can
produce different minerals in different environmental conditions.
One of the most relevant and well-known examples of mineralization driven by bacteria is the precipitation of
CaCO3. This group of micro-organisms is known as carbonatogenic microorganisms or calcifying microbes, because of
their inherent capability of producing calcium carbonate [13].
2.1

The process of carbonatogenesis

In natural conditions, the precipitation of CaCO3 (carbonatogenesis) can be considered as the result of a series of
chemical (abiotic precipitation) and biochemical processes (biotic precipitation) (1). The equilibrium exists between
insoluble carbonate and soluble bicarbonate forms in the water. The depletion of CO2 from water favours the deposition
of carbonate.
(1) Ca++ + 2HCO3- <=> CaCO3 + CO2 + H2O
Abiotic chemical precipitation can occur due to a decrease in the partial CO2 pressure, shaking or stirring of water, an
increase in temperature, or a decrease in hydrostatic pressure.
Biotic precipitation is due to different bacterial species that precipitate carbonate in alkaline environments rich in
calcium ions. Bacteria from soils, freshwater and saline habitats have frequently been reported to be able to precipitate
calcium carbonate both in natural and in laboratory conditions. This capability has been related to the formation of
marine calcareous skeletons, carbonate sediments, and soil carbonate deposits [13-16]. According to Boquet et al. [17],
calcium carbonate precipitation is a widespread phenomenon in the bacterial world and under suitable conditions most
bacteria are able to precipitate CaCO3. Mechanisms proposed, occurring mostly in marine environments, are passive or
active. They include abiotic changes in seawater chemical conditions (pH, component concentrations, bicarbonate ion
production) and biotical changes driven by bacteria acting as crystallization nuclei. The primary role of bacteria in the
precipitation process has been ascribed to their ability to create an alkaline environment by physiological activities [1820]. Microorganisms involved in calcium carbonate are both autotrophic and heterotrophic (both in aerobiosis and in
anaerobiosis).
Though, the precise role of bacteria and their activities in carbonate crystallization remains unclear, they seem to fall
into three hypotheses:
1. As a first hypothesis, mineralization occurs as by-product of microbial metabolism involving either autotrophic or
heterotrophic pathways [15, 16-19]. Most bacteria are involved in autotrophic production: methanogenic archaea,
sulphurous or non-sulphurous, purple and green bacteria, and cyanobacteria [19]. All use CO2 as a carbon source to
produce organic matter and induce local CO2 depletion of the medium. In heterotrophic bacteria several pathways
from the nitrogen and sulphur cycle are involved in carbonate precipitation. In these processes, reaction, such as
enzymatic hydrolysis of urea or disassimilatory reduction of nitrate and sulphate, cause an increase in pH, which
shifts the carbonate-bicarbonate equilibrium towards the production of more carbonate ions and, ultimately, the
precipitation of calcium carbonate, if free calcium ions are present.
2. As a second hypothesis, carbonate nucleation takes place on the cell wall [16, 21]. Bacterial surfaces also play an
important role in calcium carbonate precipitation [22]. Bacterial surfaces can act as important sites for the
absorption of cations and constitute particularly favourable templates for heterogeneous nucleation and crystal
growth. Due to the presence of several negatively charged groups, at a neutral pH, positively charged metal ions (i.e.
calcium ions) could be bound on bacterial surfaces, favouring heterogeneous nucleation [18]. The first step is a
stoichiometric interaction of metal with reactive chemical groups, present on wall cellular: after complexation, these
sites nucleate the deposition of more metal as a chemical precipitate. Commonly, carbonate precipitates develop on
the external surface of bacterial cells by successive stratification and bacteria can be embedded in growing carbonate
crystals [19, 23]. The events on the cell surface can be summarized as follows:
Ca2+ + Cell Cell-Ca2+
Cell-Ca2+ + CO32- Cell-CaCO3
3. The third hypothesis involves role of extracellular macromolecules [24-26]. This last hypothesis involves
extracellular macromolecules such as extracellular polymeric materials (i.e. exopolysaccarides, EPS, and capsular
polysaccharides, CPS) produced by bacteria [27]. The extracellular polymeric macromolecules are a class of

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polymers implicated in various processes such as calcium carbonate precipitation [24, 26]. They are made from
organic substances such as polysaccharides, proteins, nucleic acid, lipids and uronic acids [28]. Researchers [29]
suggested that specific proteins present in biological extracellular polymeric materials may cause the formation of
different and polymorph CaCO3 crystals. Aims of our researches are both to investigate the role of microbes in the
large scale geo-biochemical deposition of carbonates and to develop basic knowledge and processes for
biotechnological exploitation of calcite deposition by bacteria.
Our research was aimed at investigating in vitro bacterial precipitation of calcium carbonate in different synthetic
media and at different incubation time.
In this chapter the potential relationship between extracellular polymeric materials (EPM), such as
exopolysaccharides (EPS) and capsular polysaccharides (CPS) isolated from calcifying bacteria and calcium carbonate
crystals precipitated are also discussed.

3. Experimental
3.1 Isolation of calcifying bacteria
Bacterial strains were isolated from speleothems (Fig.1) in Stiffes Cave, a limestone cave located near LAquila
(central Italy) that is open to the public.
Fig. 1 Speleothems from Stiffe cave from which the calcifying bacteria have been
isolated.

A small sample from the calcareous speleothems were ground to a powder


using a sterile pestle and mortar and then suspended in saline solution. Serial
dilutions were spread on B-4medium (B-4M) [17] and incubated at 32C for 23 weeks to allow the growth of heterotrophic bacteria. Individual colonies were
selected and purified by repeated subculture on solid B-4M. Each isolate was
periodically examined by optical microscopy for the presence of crystals. Pure
calcifying bacteria were isolated and identified by cyto-morphological,
physiological and biochemical tests utilizing both API and Biolog system.
3.2

Precipitation of calcium carbonate by micro-organisms

We tested the ability of the selected microbial strains to precipitate calcium carbonate on both liquid and solid B-4M.
B-4M medium plates were inoculated and incubated at 4, 22 and 32C in aerobic conditions. All experiments were
carried out in triplicate. Plates were examined under a light microscope every day for 30 days (Leitz Biomed) for the
presence of crystals.
From calcifying bacteria (two strains, Nocardia calcarea (Fig 2A) Bacillus firmus (Fig 2B), were chosen. We chose
these bacteria both for the amount and for the rapidity by which they produce crystals (1 -2 days). Both strains produce
mucoid colonies that precipitate calcite crystals coated with thick mucilage. We looked for cell structures of bacteria
involved in calcite crystal formation.

Fig. 2

B
B

Nocardia calcarea (A) and Bacillus firmus(B) Gram-stained observed at the optical microscope (magnification1000x).

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3.3

Staining of bacterial glycocalyx

Many bacteria may be surrounded by a polysaccharide containing outer layer termed glycocalyx. When this layer is
tightly bound and remains attached to cells, it is referred to as a capsule.
To visualize outer cellular structure as the glycocalyx (i.e capsule) produced by the microorganisms we utilize dyes
as follow: microorganisms grown for days 7 on solid B4-M were studied under a phase-contrast microscope after
staining with Manevalsmethod [congo red solution (0.1% at pH 5.2) and Manevals stain (10 % FeCl3 - 5 % phenol Acid fuchsin - Acetic acid)]. The presence of glycocalyx cells was also determined by India ink.
3.4 Extracellular polymeric materials extraction from bacteria
Extracellular polymeric materials, exopolysaccharides (EPS) and capsular polysaccharides (CPS) produced by
microorganisms may be tightly bound to the cell (cell attached or capsular) or loosely adherent to cells (slime type, free,
or released) or in the form of free dissolved material (27, 30).
We isolate outer structures (EPS and CPS) from B. firmus and N. calcarea to check on their influence in calcite
precipitation
B. firmus and N. calcarea were inoculated in different synthetic media with or without calcium ions: (a) B-4M
containg calcium as calcium acetate (2.8 g/L), (b) B4Mmod contains sodium acetate (2.3 g/L) instead of calcium acetate
present in B-4M.
Individual colonies were transferred to B4-M or B4Mmod liquid media and then incubated for 24 hat 30C (starter
cultures). Cultures were inoculated in flasks containing B-4M or B4-M mod liquid media and incubated for different
times (7, 14 or 21 days) at 30C. After incubation, bacterial cultures were processed for extraction of extracellular
polymeric materials (CPS and EPS). For extracting CPS and EPS bound to cells the combination of both physical and
chemical methods are utilized. Physical methods involve the techniques of separation with physical forces, for example,
centrifugation, dialysis and filtration. Chemical methods utilize chemical reagents, like phosphate buffered saline,
protease inhibitors, ethanol, thioglicolic acid, Hepes-TritonX [25].
3.5

Study on Extracellular polymeric materials

Biomineralization Experiments. Extracellular polymeric materials (CPS or EPS) extracted from bacterial cells were
inoculated with a supersaturated bicarbonate solution to follow calcite crystallization. Tetracycline and ampicillin were
then added. The flasks were then incubated at room temperature (20-25C) under non shaking conditions.
Protein isolation. Proteins present on extracellular polymeric materials were isolated by electrophoresis. Sodium
Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is a technique for separating proteins, based on
their ability to move within an electrical field, which is a function of the length of their polypeptide chains or of their
molecular weight. CPS or EPS samples were loaded onto the gel and run at 200 V. The gels were then stained with
either Coomassie Brillant Blue or silver (Silver Stain Kit, Bio-Rad) to visualize the proteins.
3.6

SEM analysis

Morfology and size of the crystals deposited from both living bacterial cells and extracellular polymeric materials
isolated from them were studied by scanning electron microscopy (SEM- Philips XL30CP). SEM samples were
prepared as follows:
(1) Agar blocks of bacterial cultures grown on B-4M solid medium were dried (at 22 or 32C), and those richest in
crystallites were fixed into adhesive tape.
(2) Crystals deposited from bacterial cells on liquid medium were collected breaking the flasks and fixing into the
adhesive tape glass pieces containing crystals and microorganisms into an adhesive tape.
(3) Extracellular polymeric materials (CPS and EPS) incubated on calcifying solution were dried at 37C on glass
coverslip. The morphologies of the calcium carbonate crystals were characterized by SEM after being sputter-coated
with a thin layer of gold nanoparticles.

4. Results and Discussion


The calcareous fragments from Stiffe cave contained between 1.0 x103 4 x109 bacteria per gram (cultivated on B4-M
solid medium). The amount isolated bacteria varied enormously between the different samples. Bacteria might have
been scarce in some areas due to their trivial origin or due to other reasons (e.g. contamination due to the presence of
cave-dwelling animals and visitors). The existence of millions or billions of cells per gram requires suitable local
conditions for growth. The samples that contained the most bacteria also tended to contain a higher amount of
calcifying strains; we found a direct relationship between the number of calcifying microorganisms present and the
thickness of the speleothem walls. In the karst cave 30 calcifying bacterial strains were isolated and identified. All of
these findings confirm the permanent nature of these bacterial communities. Fig. 3 shows the time required for the
single strain to start precipitation of calcium carbonate at different temperatures. At 32C all calcifying bacteria needed

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7 days of incubation to precipitate carbonates. At 22C all cave strains started to form crystals after 15 days of
incubation. Furthermore, all calcifying bacteria incubated at 4C needed 25 days of incubation to precipitate crystals.
Microscopic observation showed that high temperatures decrease the time necessary for the initiation of the
precipitation process, which in turn increases the precipitation rate.

Time (days)

15
7

32C
22C
4C

5
2
0

20

40
60
80
Bacterial strains (%)

100

Fig. 3 Relationship between the percentage of crystal-forming strains on solid medium (B4-M) and the number of days in culture
needed to form them.

4.1 Glycocalyx (bacterial capsule)


Outer cellular structure such as the glycocalyx and EPS matrix is known to contain several different major categories of
molecules whose roles and involvement in precipitation are still under study. Nocardia calcarea and Bacillus firmus
produced mucoid colonies on B4-M solid medium (Fig. 4A). Slimy colonies were observed after 3-4 days-incubation.
Negative staining with Maneval (4B) and with India ink (4C) revealed encapsulated cells.

Fig. 4 Colonies of Nocardia calcarea grown on solid medium (A). Phase-contrast microscopy (magnification 1600 x) of Bacillus
firmus cells stained by Maneval's method (B) and Nocardia calcarea cells stained with India ink (C). The capsules are visualized
around the cells; note that the capsules are seen as a clear halo around both the rod-shaped (B) and cocci-shaped cells (C).

4.2

Crystal precipitation

We checked for calcifying ability both living bacterial cells both on solid and liquid culture (figures 5 and 6) and
extracellular polymeric materials isolated from cells grown both in the presence or absence of calcium ions.
Scanning electron micrographs showed that both living bacterial cells grown both on solid and liquid medium and
their extracellular polymeric materials induced precipitates of calcite. Figure 5 shows calcite crystals precipitates from
living bacterial cells on solid medium. In particular, fig. 5a e fig. 5b show elliptical crystals precipitated from
Brevibacillus brevis. The size and the shape of the microbial rods observed on the surface or around the crystals fit with
the prints observed in the inner portion of the biolith (Fig. 5a). Figure 5c shows the grouped forms of floral shape
bioliths deposited by Bacillus brevis. In figure 5d crystals precipitated, from Nocardia calcarea, both on and around
the colonies are observed by optical microscope (magnification 100x).

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Fig. 5 Scanning electron micrographs showing bacterially deposited precipitates of calcite by Brevibacillus brevis (5a, 5b) and
Bacillus brevis (5c) grown on solid medium (B-4M), and optical microscope micrograph (5d) showing precipitates both on and
around bacterial colonies.

Fig. 6 Scanning electron micrographs showing calcite precipitatesdeposited by Bacillus megaterium and Bacillus firmus grown on
B4-M solid medium (6a and 6c) or on B-4M liquid media (6b and 6d).

In figure 6, the crystals deposited on solid and liquid media from living bacterial cells are compared. In particular, in
figure 6a Bacillus megaterium produced elliptical crystals on solid medium and deposited irregular elliptical crystals on
liquid medium.
Bacillus firmus precipitated hemispherical crystals when grown on solid medium (fig. 6c), and deposited irregular
hemispherical crystal on liquid medium. In the micrograph it can be seen that crystals were bound together by mucous
matrix and a relation between crystals deposited and mucous matrix can be observed.
SEM observation revealed that both morphology and sizes of the calcite crystals were correlated with the species
involved in the calcification process and with the growth conditions.
When extracellular polymeric materials (CPS and EPS) extracted from Bacillus firmus and Nocardia calcarea
grown on liquid medium (containing calcium ion) are able to form a crystalline mineral when incubated on calcifying
solution and crystals are very similar to that of crystals precipitated by living cells [25].

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Protein isolation: extracellular polymeric material (CPS and EPS) was extracted at the stationary growth phase (3
weeks) from bacterial cells grown on synthetic media containing or not calcium ions. In figures 7A and 7B profiles of
the proteins present in the CPS extracted from bacteria are reported. It is possible observed that the presence of calcium
ions in the medium increases proteins expression (lines 1-A and 1-B). Gel electrophoresis of N. calcarea and B. firmus
show that some proteins (highlighted in the frame) are overexpressed when calcium ions are present in the medium
when compared with cultures grown on calcium free medium.
A

Fig. 7 SDS-PAGE protein profiles obtained from the


CPS fractions extracted from Bacillus firmus and
Nocardia calcarea grown on B-4M (presence of
calcium ions) or B4-M mod (absence of calcium ions),
lines 1A and 1B (CPS B4M), lines 2A and 2B (CPS
B4Mmod) and weight standards. The gels of B. firmus
and N. calcarea were stained with silver stain to
visualize the proteins.

5. Concluding remarks
Biocalcification by bacteria is an emerging restoration technique that is still under development and requires further
research. However, the production of acid substances, the development of colored spots by microbial metabolism, or
bacterial survival within the carbonate crystal could have serious implications in the restoration techniques. It has
already been demonstrated that uncontrolled bacterial growth can damage stone. To overcome these problems,
development of stone treatment without viable cells seems a better biotechnological tool.
Our studies demonstrated that bacteria isolated from calcareous stalactites, which are able to mediate CaCO3
precipitation in vitro, play a role in the formation of carbonate speleothems. Among our isolates, there are strains that,
because of their rapid ability to precipitate CaCO3 rapidly and because their yields as calcite producers, can have
interesting application.
Our studies show that extracellular polymeric secretions influence calcium carbonate precipitation in a positive way.
In this cellular fraction some proteins are overexpressed when calcium ions are present in the cultural media. Study of
these proteins will help in cloning their genes thus facilitating identification of the functions. Further investigation
should be directed to the isolation and purification of the protein responsible of bioprecipitation. Further researchers are
in progress directly aiming to develop biotechnological processes by which stone decaying surfaces can be restored
utilizing proteins isolated from bacteria without living cells. This avoid that some bacterial metabolite, excreted by
living cells, can negatively influence the restoration.

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