Unraveling disparate roles of organisms, from plants to bacteria, and viruses on built cultural heritage

Abstract

The different organisms, ranging from plants to bacteria, and viruses that dwell on built cultural heritage can be passive or active participants in conservation processes. For the active participants, particular attention is generally given to organisms that play a positive role in bioprotection, bioprecipitation, bioconsolidation, bioremediation, biocleaning, and biological control and to those involved in providing ecosystem services, such as reducing temperature, pollution, and noise in urban areas. The organisms can also evolve or mutate in response to changes, becoming tolerant and resistant to biocidal treatments or acquiring certain capacities, such as water repellency or resistance to ultraviolet radiation. Our understanding of the capacities and roles of these active organisms is constantly evolving as bioprotection/biodeterioration, and biotreatment studies are conducted and new techniques for characterizing species are developed. This brief review article aims to shed light on interesting research that has been abandoned as well as on recent (some ongoing) studies opening up new scopes of research involving a wide variety of organisms and viruses, which are likely to receive more attention in the coming years.

Key points
• Organisms and viruses can be active or passive players in heritage conservation
• Biotreatment and ecosystem service studies involving organisms and viruses are shown
• Green deal, health, ecosystem services, and global change may shape future research

Full text

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https://doi.org/10.1007/s00253-023-12423-5
MINI-REVIEW
Unraveling disparate roles oforganisms, fromplants tobacteria,
andviruses onbuilt cultural heritage
PatriciaSanmartín1,2 · PilarBosch‑Roig3 · DomenicoPangallo4,5 · LuciaKraková4· MiguelSerrano6
Received: 19 December 2022 / Revised: 19 December 2022 / Accepted: 31 January 2023
© The Author(s) 2023
Abstract
The different organisms, ranging from plants to bacteria, and viruses that dwell on built cultural heritage can be passive or
active participants in conservation processes. For the active participants, particular attention is generally given to organisms
that play a positive role in bioprotection, bioprecipitation, bioconsolidation, bioremediation, biocleaning, and biological
control and to those involved in providing ecosystem services, such as reducing temperature, pollution, and noise in urban
areas. The organisms can also evolve or mutate in response to changes, becoming tolerant and resistant to biocidal treat-
ments or acquiring certain capacities, such as water repellency or resistance to ultraviolet radiation. Our understanding of
the capacities and roles of these active organisms is constantly evolving as bioprotection/biodeterioration, and biotreatment
studies are conducted and new techniques for characterizing species are developed. This brief review article aims to shed
light on interesting research that has been abandoned as well as on recent (some ongoing) studies opening up new scopes of
research involving a wide variety of organisms and viruses, which are likely to receive more attention in the coming years.
Key points
• Organisms and viruses can be active or passive players in heritage conservation
• Biotreatment and ecosystem service studies involving organisms and viruses are shown
• Green deal, health, ecosystem services, and global change may shape future research
Keywords Biocontrol agents (BCAs)· Biotreatment· Ecological factors· Species adaptation· Species role in ecosystems·
Stone-built heritage
Introduction
The study of organisms with an interacting role with cul-
tural heritage, especially those with high metabolic activ-
ity and resilient to changes in environmental conditions,
either by their growth form or their potential for genetic
and physiological adaption, has become a research hotspot
to advance fundamental knowledge to address the natu-
ral imbrication of different types of conservation prob-
lems that is usually present in built cultural heritage. It
has been suggested that future research should focus on
metabolically active microorganisms and biochemical
* Patricia Sanmartín
patricia.sanmartin@usc.es
* Miguel Serrano
miguel.serrano@usc.es
Pilar Bosch-Roig
mabosroi@upvnet.upv.es
Domenico Pangallo
domenico.pangallo@savba.sk
1 GEMAP (GI-1243), Departamento de Edafoloxía e Química
Agrícola, Facultade de Farmacia, Universidade de Santiago
de Compostela, 15782SantiagodeCompostela, Spain
2 CRETUS, Universidade de Santiago de Compostela,
SantiagodeCompostela, Spain
3 Instituto Universitario de Restauración del Patrimonio,
Dpto. Conservación y Restauración del Patrimonio,
Universitat Politècnica de València, 46022Valencia, Spain
4 Institute ofMolecular Biology, Slovak Academy ofSciences,
Dúbravská cesta 21, 84551Bratislava, Slovakia
5 Caravella, s.r.o., Tupolevova 2, 85101Bratislava, Slovakia
6 Department ofBotany, Faculty ofPharmacy, University
ofSantiago de Compostela, 15782SantiagodeCompostela,
Spain
/ Published online: 23 February 2023
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1 3
reactions (Liu etal. 2022a), such as the transformation
of metal compounds into metal oxalates (biopatination)
by fungi and the consolidation of biofilms by extracel-
lular polymeric substances (EPS) produced by bacteria
(Joseph 2021). Bacteria can also induce precipitation of
silicate, phosphate, sulfide, oxide, and above all, carbon-
ate minerals (bioconsolidation), resulting from the inter-
actions between metabolic by-products on substrate sur-
faces and the surrounding environment (Hoffmann etal.
2021). The precipitation of biominerals is promoted by
high pH, as well as by high concentrations of calcium and
dissolved inorganic carbon and the availability of nuclea-
tion sites (Timoncini etal. 2022). Previous studies have
also confirmed microbial-rock interactions involving CO2
uptake or release processes; e.g., the development of act-
inobacterial biofilms on cave walls promotes uptake of
CO2, dissolution of the rock, and production of calcite
crystals during periods of low humidity and/or CO2 levels
(Martín-Pozas etal. 2020). Methanotrophic bacteria con-
sume CH4 in caves, produce methanobactins (bioactive
compounds with high affinity for metal ions), and exhibit
antibiotic activity against Gram-positive bacteria (Martín-
Pozas etal. 2020). Although there has been an increase in
these types of studies on biotreatments in heritage field,
other research that produced promising results more than
10years ago was unfortunately abandoned, e.g., studies
on the use of phages as natural antagonists of bacteria and
their use in bioremediation to remove algal growth from
stone-built heritage (Klaassen 2005; May etal. 2009). The
reasons for abandonment could be related to the lack of
effectiveness of the treatment in the field, as well as the
difficulty for its implementation in relation to costs and
method of application.
Regarding the resilience of organisms and their ability
to compete, Alexander Fleming warned, shortly after he
discovered penicillin, of the problems that abusive use of
the compound could bring about and that bacteria could
become resistant to the antibiotic (Marshall and McMurry
2005). In a similar way, repeated application on the same
target area of heritage interest of non-lethal concentra-
tions of the biocide benzalkonium chloride (probably the
most widely used quaternary ammonium compound in cul-
tural heritage) over time (cleaning campaigns conducted
during the 1980s, 1990s, 2000s) has probably driven a
significant reduction in microbial sensitivity to biocides,
creating tolerant and resistant species (Pinna 2022). This
situation led to the search for novel biocidal solutions
being heightened, so that at the beginning of the 1990s,
there were about two dozen of antibacterial biocides on the
market, and by 2005, they were 1000 or more (Marshall
and McMurry 2005). The search also promoted a shift
towards green biocides, for achieving more energy-, time-,
and cost-efficient production and consumption processes,
as promising alternatives to their industrial counterparts
(see, e.g., Caldeira 2021).
Furthermore, many paradigms are changing rapidly; e.g.,
lithobionts are now considered to have a dual role in biode-
teriorative and bioprotective effects, which sometimes take
place simultaneously (Favero-Longo and Viles 2020, and
references therein). Climbing plants and chromatic altera-
tions on stone surfaces caused by biological growth were
previously considered to enhance the aesthetic value of his-
torical buildings and ruins (Martines 1983; Guillitte 1993).
However, in later times, particularly in the late 1980s and
1990s, the presence of surface-colonizing organisms tended
to be viewed as negative and associated with physical and
chemical degradation, fouling, soiling, and loss of identity of
the object. This led to the complete eradication of all organ-
isms on the surfaces of the built heritage being favored and
promoted the presence of “stone cities” (karst landscape),
rather than “green cities.” However, this situation was coun-
tered by bioprotection studies, understood as “the positive
ways in which organisms growing on the surfaces of rocks
and building materials protect the surface from other pro-
cesses of weathering and erosion” (Carter and Viles 2005;
Sanmartín etal. 2021). Bioprotection roles include umbrella
effects against rain droplets, wind abrasion, and solar radia-
tion; the development of resistant outer layers, which pre-
vent erosion or weathering due to pollutants; stabilization
caused by filamentous actinobacteria, fungi, and lichens;
and consolidation by secondary minerals, precipitates, and
EPS-chelates (Liu etal. 2022b). Some bioprotective organ-
isms are material binders and act as good substitutes for
cementitious materials.
Likewise, long-term protection and the permanence of
active organisms should be considered; e.g., live Pseu-
domonas stutzeri cells should be allowed to remain in treated
areas of stonework to continue their nitrate biocleaning func-
tion (Pavlović etal. 2022).
This mini-review covers some case studies involving cul-
tural heritage which have attempted to understand the active
roles of various viruses and organisms (Fig.1), ranging from
bacteria to vascular plants, shedding light on interesting
research which will probably receive more attention in the
coming years.
QAC‑tolerant andQAC‑resistant
microorganisms
According to current European Union regulations (Aa.Vv.
2012), a biocide is defined as “any substance or mixture, in
the form in which it is supplied to the user, consisting of,
containing or generating one or more active substances, with
the intention of destroying, deterring, rendering harmless,
preventing the action of, or otherwise exerting a controlling
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1 3
effect on, any harmful organism by any means other than
mere physical or mechanical action.” Examples of biocides
include disinfectants, preservatives, antiseptics, pesticides,
herbicides, fungicides, and insecticides. Biocides can be
classified in several groups according to their functional
chemical groups or their targets of action (Gnanadhas etal.
2013).
Biocides based on quaternary ammonium compounds
(QACs), used as pesticides as well as biocides and with
benzalkonium chloride (BAC) and didecyldimethylammo-
nium chloride (DDAC) as main referents, have been used
to treat microbial colonization on cultural heritage objects
and are still widely used, mainly in stone conservation (Lo
Schiavo etal. 2020). QACs target the microbial cell mem-
brane, and electrostatic interactions between the positively
charged QAC head and the negatively charged microbial cel-
lular membrane are followed by permeation of the QAC side
chains into the intramembrane region, ultimately leading to
leakage of cytoplasmic material and cellular lysis (Gnana-
dhas etal. 2013).
Microorganisms on the surface of cultural heritage
objects form communities called subaerial biofilms (SABs,
Gorbushina 2007). Several microorganisms have the ability
to secrete extracellular polymeric substances (EPS), which
generally gives biofilms their adhesive properties, as well as
reducing desiccation and providing organisms with a favora-
ble microenvironment (Flemming 2016). Lichens (especially
crustose lichens) and mature biofilms are extremely diffi-
cult to remove because they penetrate the surface even for a
few millimeters adhering firmly to the substrate and also to
eradicate because the presence of EPS and cells of low meta-
bolic activity interfere with the action of biocides reducing
their efficacy (Pinna 2022). Nevertheless, the application of
biocide treatments with QACs as the last stage in the clean-
ing treatment of cultural heritage stone surfaces, to maintain
the results longer, is a standard practice. But, what occurs at
later stages? Does the colonization arise again? Is it the same
as before? How does it affect conservation of the material,
especially in the long term?
It has been shown that re-colonization of the surface
might occur after the application of QACs, particularly on
porous substrates (highly bioreceptive, i.e., substrates with
high capacity to be colonized by living organisms) and areas
that remain humid for long periods (Sanmartín etal. 2021;
Pinna 2022). Re-colonization can be favored by new micro-
organisms colonizing the surface and using the residues
of dead biomass and biocide compounds as nutrients, or
by indigenous microorganisms, previously present on the
surface and exposed to the biocide, which has developed
resistance and now increase their presence (Pinna 2017;
Sanmartín and Carballeira 2021). Some researchers have
described the re-colonization of outdoor objects by lichens
after treatment with different biocides, such as Biotin T®,
Biotin R®, tributyltin oxide, dibutyltin dilaurate, and cop-
per nanoparticles, identifying substrate bioreceptivity and
climatic conditions as the main factors involved in the revi-
talization of viable fungal, green algal, and cyanobacterial
cells (e.g., De los Ríos etal. 2012; Pinna etal. 2018). Other
examples include algae and cyanobacteria, which were able
to re-colonize the surface of stone and majolica-glazed tiles
(Coutinho etal. 2016; Pfendler etal. 2018). Fungi can also
be considered as newly emerging re-colonizers of cultural
and artistic stone surfaces treated with biocides (Isola etal.
2022). The most famous and representative example of re-
colonization by fungi is probably that of the Lascaux Caves
(SW France) after years of successive treatments with bioc-
ides and antibiotics, following the first occurrence (between
1955 and 1960) of “the white disease” and “the green dis-
ease” or “Maladie Verte” (in French). The latter, caused by
the unicellular alga Bracteacoccus minor (Chlorophyta,
Chlorococcales), was first treated (summer 1963) with a
mixture of antibiotics, i.e., penicillin, streptomycin, and
ABC
Fig. 1 A Negative staining of a purified tobavirus product (in the
form of rods) isolated in the laboratory. Bar
=
100 µ. Photo: Vicente
Medina. B Violet stains caused by Streptomyces sp. bacterium in the
Circular Mausoleum, Roman Necropolis of Carmona, Spain. Photo:
Cesáreo Saiz-Jiménez. C Bacteria of the order Rhizobiales on the
paintings in Tomba del Colle (Etruscan tomb, near Chiusi, Italy).
Photo: Cesáreo Saiz-Jiménez
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1 3
kanamycin, which were dissolved in sterile double distilled
water and delivered to the environment in the form of an
aerosol, followed by a biocide treatment with an aqueous
formaldehyde solution (Lefèvre 1974). Between 2001 and
2003, benzalkonium chloride (50%) was sprayed on the cave
surfaces to eradicate (albeit not successfully) the white fun-
gus Fusarium solani, which had begun to appear. During the
same period, Pseudomonas fluorescens also appeared and
was treated with antibiotics (streptomycin sulfates and poly-
myxin). Between 2007 and 2011, more than 30 strains of
fungi were isolated from the caves. Of these, two belonged
to new species of melanized fungi, described as Scolecoba-
sidium (= Ochroconis) lascauxensis and Scolecobasidium
(= Ochroconis) anomala, and also human opportunistic
fungi such as Exophiala castellanii and Exophiala moniliae
(Saiz-Jimenez etal. 2012).
The long-term effects of QACs on the substrate are still
poorly understood, neither in relation to its own stability
(i.e., no physical–chemical degradation) over time, nor in
relation to how this affects re-colonization. Nevertheless,
it has been shown that the repeated application of QACs is
not advisable because this can lead to the development of
some species biocide-tolerant microorganisms, versus whom
QACs become ineffective. In this sense, bacteria are capable
of re-colonizing surfaces previously treated with QAC bioc-
ides (Urzì etal. 2016; Kakakhel etal. 2019; Lo Schiavo etal.
2020; Sanmartín and Carballeira 2021). Moreover, there are
some evidences that bacteria have developed mechanisms to
resist QACs. Bacterial mechanisms of resistance to QACs
have begun to be studied as the resistant bacteria may repre-
sent a threat to public health (Meade etal. 2021). In addition,
a dangerous relationship between resistance to QACs and to
antibiotics was noted in bacteria and found to be due to the
genetic linkage of genes for QAC tolerance and antibiotic
resistance (Mulder etal. 2018). Genes conferring reduced
susceptibility to QACs are called qac. These bacterial genes
encode efflux pumps, which are capable of expelling many
QAC structures from bacterial cells. This serves to decrease
the susceptibility of bacteria to QAC disinfectants. The qac
genes are commonly found in class 1 integrons, which can
occur as part of mobile genetic elements such as plasmids
and transposons. Consequently, qac genes can be transferred
horizontally via mobile genetic elements to other bacteria.
This process can occur at the same time as the transfer of
other antibiotic-resistant genes (Mulder etal. 2018; Vijaya-
kumar and Sandle 2019). Research focused on detecting
these types of genes in cultural heritage objects has only
been attempted recently (He etal. 2022), and members of the
genera Pseudonocardia, Sphingomonas, and Streptomyces
have been recognized as major hosts of genes conferring
resistance to antibiotics and biocides.
The literature search conducted for this review showed
that very few studies have focused on unveiling the
mechanisms of QAC biocide resistance by fungi, cyano-
bacteria, algae, and other types of microbes colonizing
the surfaces of cultural heritage objects. Recent studies
attempted to assess resistance to QACs in dark-pigmented
fungi isolated from stone surfaces (Isola etal. 2021; 2022).
Several of the strains isolated (Acremonium-like, Clad-
osporium spp., Exophiala bonariae, Aureobasidium pul-
lulans, Exophiala oligosperma, and Verrucocladosporium
dirinae) displayed high tolerance to QAC-based biocides.
Other studies have shed light on the toxic effect of QACs
on cyanobacteria and algae (Wu etal. 2021; Qian etal.
2022). Wu etal. (2021) tested alkyl trimethylammonium
compounds (ATMAs), a class of QAC cationic surfactants,
on two cyanobacteria: Aphanizomenon ovalisporum
(recently identified as Chrysosporum ovalisporum) and
Microcystis aeruginosa, as well as, two chlorophytes:
Chlorella sp. and Scenedesmus sp. Results showed that
cyanobacteria have higher sensitivity to ATMA surfactants
than that of green algae. On the former, the growth was
inhibited by depression of photosynthesis (via damage to
the thylakoid membranes), induction of oxidative stress,
and breakage of the cell membrane. Previously, the same
authors demonstrated that the octadecyltrimethyl ammo-
nium (ODTMA), a quaternary ammonium cation, imposed
inactivation of photosynthesis in cyanobacteria, followed
by cell lysis and cellular degradation (Sukenik etal. 2017).
On algae, moderate damage to the thylakoid membranes
and consequently to the photosynthetic process was
observed after treatment with ATMA cations. The greater
resistance displayed by the green algae is explained by
the authors because they contain an acetolysis-resistant
biopolymer, algaenan (Allard and Templier 2000). Qian
etal. (2022) revealed the toxic effects and mechanism
of a type of benzalkonium chloride on the cyanobacte-
rium Microcystis aeruginosa. The quaternary ammonium
compound depressed the photosynthetic activities via
denaturing-related organelle, caused oxidative damage by
producing superoxide radicals, and destroyed the integrity
of cell membrane.
Impairments to the photosynthetic system, promoted by
the application of two different QAC-based biocides (Biotin
T® and Preventol RI80®, at the highest concentration sug-
gested by the producer, i.e., 3% and 2%, respectively), were
also studied in the photobiont of the lichen Xanthoria pari-
etina (Vannini etal. 2018). These researchers demonstrated
the low content of ergosterol in the mycobiont and that the
lack of recovery of the lichen 90days after treatment with
the biocide may be related to blockage of the biosynthetic
pathway leading to synthesis of this important membrane
component. They concluded that the biocides used are effec-
tive for eradicating lichen and also produced satisfactory
results in preventing re-colonization. However, the persis-
tence of tolerant and resistant forms in stone fissures may
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1 3
promote re-colonization of the rock by lichens and/or fungi,
making surface cleaning effective during only a short period
(De los Ríos etal. 2012).
Microbial antagonists
Microbial antagonism occurs as a result of competition
between microorganisms for nutrients and space, leading
to one microorganism inhibiting growth of the other. It is
based on the principle of biological competition (Gause’s
law 1934) whereby two different species of bacteria and/or
fungi, existing in the same ecological space, cannot coexist
in stable equilibrium if they require the same nutrient sub-
strates; one of them, usually the less demanding in terms of
nutrition, will become dominant over (and possibly cause
extinction of) the other. These procedures can be referred to
as “biostabilization techniques,” which are exerted by one
species over another, and therefore they do not imply gen-
eralized biocidal action, as the final effect is against specific
microbial species (Marin etal. 2016). In the field of plant
pathology, the use of microbial antagonists to control post-
harvest fungal diseases of different fruits and vegetables has
been applied since the end of the 1970s, and there has since
been a boom in the search for safer and more eco-friendly
alternative approaches to biological control (e.g., Dukare
etal. 2019; Reyes-Estebanez etal. 2020). In the cultural
heritage field, the use of microbial antagonists or biocontrol
agents as safe, green biocides as alternatives to the tradi-
tional and commercial biocides usually applied to stone-built
heritage, although green alternatives may have a toxicologi-
cal profile similar to their traditional counterparts (see e.g.,
Silva etal. 2016a), remains very limited. In a recent study,
Marin etal. (2016) evaluated two biological biocides on the
early twentieth century handmade bricks, using the Euro-
pean standard protocol for evaluation of cleaning methods
for cultural heritage (UNI 11551–1:2014): Natria (Bayer) a
herbicide based on pelargonic acid and that can be used to
control weeds on exterior surfaces such as garden paths, and
NewFloorCleaner (Chrisal Cleaning Products, Belgium),
a product based on probiotics (spores of Bacillus subtilis,
Bacillus megaterium, and Bacillus pumilus) and used for dis-
infecting surfaces in hospitals. The biocides tested are cur-
rently not used in cultural heritage conservation, but seem
reasonably adequate for this type of use. The study findings
suggest that both products can potentially be used as novel
biocides on stone surfaces, although Natria has a strong,
unpleasant odor and can leave a white patina on the treated
surface. Both disappear over time, when the material absorbs
water. In addition, at the microscopic level product, Natria
left residues that affected the microporosity of the stone, and
NewFloorCleaner caused an increase in conductivity and
in the concentrations of sulfates and calcium, sodium, and
magnesium cations on the treated surface. The authors do
not indicate whether these residues disappear over time. In
this connection, note the importance of long-term monitor-
ing to know what happens over time, if the residues or aes-
thetic problems, such as a visible color change, disappear or
increase. In general, there is a need for more well-designed
long-term experiments to fully explore the dynamic of the
materials and developed microbial communities over time.
Important advances have been made in studies carried
out in Portugal in the use of bacteria of the genera Bacil-
lus, which are capable of producing secondary metabolites
with antagonistic activities against fungal isolates that cause
biodeterioration on heritage monuments (Caldeira 2021 and
references therein). Some strains of Bacillus subtilis and
Bacillus amyloliquefaciens have been reported to produce
antifungal peptides. Bacillus subtilis is one of the most ver-
satile producers of cyclic lipopeptides, such as surfactin,
iturin, and fengycin, amphiphilic membrane-active biosur-
factants with potent antifungal activity. These lipopeptides
are produced on sporulation of Bacillus at the end of the
resting stage, which makes bacterial culture a key process
in this respect. In addition, Silva etal. (2016b) proposed a
simple, rapid method for detecting and characterizing bio-
active compounds produced by Bacillus strains with high
antagonistic activity, particularly against Penicillium sp.
and Cladosporium sp., and to a lesser extent Alternaria sp.,
Mucor sp., and Fusarium oxysporum. The same authors per-
formed a further study collecting plants such as Pouteria
ramiflora, known for its antimicrobial, anti-inflammatory,
and antifungal activity, and other plants in the families Apo-
cynaceae and Fabaceae in the Brazilian Cerrado, a tropi-
cal highland savanna in the midwestern region of Brazil, in
order to extract potentially bioactive compounds (Silva etal.
2019). Furthermore, the same research group has success-
fully combined the bioactive compounds produced by Bacil-
lus sp. CCLBH 1053 and Pouteria ramiflora (Sapotaceae)
extracts as an antifungal agent simultaneously against bio-
deteriogenic yeast and filamentous fungi (Silva etal. 2019).
The genus Bacillus comprises around 380 species with a
remarkable potential to produce a large variety of second-
ary metabolites, including ribosomally and non-ribosomally
synthesized antimicrobial peptides. Two terpenes (isoprene
and monoterpene α-terpineol) produced by B. subtilis exhibit
antagonistic activity against cyanobacteria and nematodes
(Caulier etal. 2019).
In aqueous ecosystems, bacteria are capable of controlling
massive growth of algae and cyanobacteria through physi-
cal association or the production of algicidal compounds
(Coyne etal. 2022). The aquatic bacterium Streptomyces
neyagawaensis has been shown to be capable of suppressing
the growth of cyanobacteria (such as Microcystis aerugi-
nosa) and of a wide range of algae, including Chlorella spp.
(May etal. 2009). The bacterium Phaeobacter inhibens can
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1 3
promote growth of the marine alga Emiliania huxleyi at an
early stage (mutualistic interaction) but then kill it at a later
stage (antagonistic interaction). Bacteria isolated from dia-
toms of the genus Pseudo-nitzschia display algicidal activity,
and a few bacteria exhibit lytic activity against both cyano-
bacteria and microcystins (Coyne etal. 2022).
Different plant parts (roots, stems, flowers, fruit) have
traditionally been used to treat some human diseases because
they contain several phytochemicals such as flavonoids,
alkaloids, tannin, and terpenoids, which possess antimicro-
bial and antioxidant properties. In the cultural heritage field,
invitro bioactivity testing has been successfully carried out
with extracts of spontaneous plants, i.e., Solanum nigrum
(Solanaceae family), Moricandia arvensis (Brassicaceae
family), Volutaria lippii (L.), and Pulicaria inuloides (both
belonging to Asteraceae family), which showed strong bioc-
idal activity against bryophytes, green algae, and lichens iso-
lated from two rupestrian churches belonging to a UNESCO
World Heritage site in Matera (southern Italy) (Scrano etal.
2020).
Not all viruses are bad: phages
Bacteriophages (phages), i.e., viruses that infect bacteria,
are probably the most abundant life form in the biosphere
(Summers 2012; Sadekuzzaman etal. 2015). Viruses have
a bad reputation, exacerbated in recent times by the recent
global SARS-CoV-2 pandemic. Nevertheless, as stated in
an ancient proverb, “The enemy of my enemy is my friend,”
and when the bacteria that viruses target are also within our
firing range, they become our allies in “viral remediation”
or “phage therapy” treatment (Abedon etal. 2017), as long
as they are safe for human and environmental health (Sof-
fritti etal. 2019). The potential of phages as antibacterial
agents has been known since 1917, i.e., before the discovery
of antibiotics by Fleming, in 1928. There is now renewed
interest in the use of phages due to the increasing emer-
gence of tolerant and resistant bacteria and to the advantages
of phages over antibiotics and chemical agents, especially
for inhibiting or disrupting biofilms (Sadekuzzaman etal.
2015), including those formed by Escherichia coli (Meng
etal. 2011).
Phage therapy has been poorly exploited, despite the fact
that it has been demonstrated to be safe and effective, with
rapid, simple, and inexpensive phage isolation and produc-
tion, and highly specific self-replication in the target host
bacterium because they work in a key/lock way (Sadekuz-
zaman etal. 2015). It is sometimes used in the agricultural,
food, clinical, and veterinary sectors (e.g., Ni etal. 2020),
although infrequently, due to the dominance of antibiotics,
which are much more familiar, stable, easy to mass pro-
duce and administer, and display broad spectrum efficacy. In
addition, the lack of regulatory approval and political aspects
has also prevented wide use of phage therapy.
In the cultural heritage field, the few studies involving
phages published to date report two different lines of action:
(1) phages as protective agents preventing bacterial decay
of wood in archaeological sites (Klaassen 2005; Cappitelli
etal. 2020) and, (2) eukaryotic viruses (not phages) that
infect algae, including the chloroviruses that infect Chlo-
rella-like green algae growing on stone surfaces, such as
Portland limestone gravestones (Kang etal. 2005; May etal.
2009; Cappitelli etal. 2020). In the first case, the greatest
advances have arisen within the framework of the EU pro-
ject BACPOLES (2002–2005), which is based on the idea
that bacteriophages are frequently found in nature, wherever
bacteria are present, and that wood-degrading bacteria may
already be infected by strain-specific (target) phages. Unfor-
tunately, screening assays aimed at obtaining pure cultures
of wood-degrading bacteria and then monoclonal antibodies
against bacteria were unsuccessful, and the development of
a phage-based wood preservative did not proceed further
(Klaassen 2005). In the second case, much of the work on
algal viruses has been done in aquatic systems (May etal.
2009). Viruses with antialgal activity have been isolated
from sediments, and mature biofilms have been also found
on sediment surfaces. However, the aforementioned authors
were not able to isolate the viruses from Portland limestone
in the laboratory for use in removing biofilms. As in the first
case, the laboratory studies were very problematic; the algae
grew very slowly and did not reach the required densities
in liquid cultures, which, in addition, were frequently con-
taminated by bacteria and fungi. However, positive results
were obtained in laboratory assays using paired algal hosts
(Chlorella strains, NC64A, Pbi, and Sag 3.83) and viruses
(PBCV-1, MT325, and ATCV-1) derived from aquatic sys-
tems, confirming the possibility of virus-induced biore-
mediation to inhibit growth of the alga Chlorella on stone.
Chlorella commonly occurs on historic fountains and other
wet stone surfaces (Bolivar-Galiano etal. 2021) and also in
lampenflora communities (Nikolić etal. 2021).
Despite the interest in the field of study, to the best of
our knowledge, there has been no further progress in this
type of research on cultural heritage field for more than
10years. This may be because despite the advantages of
phage therapy (application before, during, or after growth
of the host microorganism, high specificity, self-replication
of insitu while the host microorganism persists, no reported
adverse effects), various limitations and potential side effects
have also been described. For example, the EPS matrix and
the multi-species or multi-organism nature of the biofilm
reduce the effectiveness of treatment, although the first could
be overcome by including a mixture of viruses and other
substances (like enzymes) that facilitate penetration of the
virus through the biofilm matrix. Another drawback is the
2032 Applied Microbiology and Biotechnology (2023) 107:2027–2037
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1 3
natural emergence of phage-resistant mutants, which could
be minimized by using combined therapies of essential oils
(e.g., carvacrol, Ni etal. 2020. Although it should be noted
here that the long-term effects of essential oils should be also
assessed in relation to their impact on the material, beyond
their impact against re-colonization) and other phages.
Likewise, the potential release of toxins and the potential
incorporation of the virus encoding virulence genes into the
host genome could be addressed by using hydrogels (that
immobilize the release of toxins, Donlan 2009) and the use
of engineered phages lacking virulence genes. Furthermore,
the knowledge on environmental bacteria and the role played
on cultural heritage is very limited.
Overlooked strains: pathogens andnovel
antibiotics
Dark and wet heritage sites, such as caves and chapels, can
act as reservoirs of pathogenic organisms. In a recent study,
Pavlović etal. (2022) detected, for the first time, the danger-
ous amphibian fungal parasite Batrachochytrium on paving
stones in a historical European chapel. A high proportion
of the massive decline in amphibians worldwide has been
associated with this pathogen, and human-mediated trans-
location of the fungus is the main cause of the widespread
distribution of the disease (Rollins-Smith 2020). In Europe,
there has been a dramatic decline in some species that are
highly susceptible to Batrachochytrium, such as the mid-
wife toad (Alytes obstetricans, Fig.2) (Bosch etal. 2020).
Microbiological studies of heritage monuments, particularly
those using metagenomic tools, should focus on detecting
and highlighting the occurrence of emergent pathogens in
cultural sites, to prevent making these frequently visited
site sources of the spread of hazards either to humans or to
other organisms. Along this line, Jurado etal. (2010) focused
their review paper on pathogenic microorganisms in caves,
such as the human pathogenic black yeast fungi Exophiala
castellanii and Exophiala moniliae found in the Lascaux
Caves, as mentioned above. Isolated species of the genera
Amycolatopsis, Aureobacterium, Brevibacterium, Nocardia,
Nocardioides, Rhodococcus, and Streptomyces and members
of the family Micrococcaceae are responsible for different
skin, lung, and brain infections in humans. These microor-
ganisms can also form abscesses that induce sensory, motor,
and behavioral disturbances, as well as nausea, headaches,
and vomiting. The main opportunistic pathogens of Nocar-
dia species include, e.g., N. farcinica, N. nova, N. absces-
sus, and N. cyriacigeorgica, while members of the genus
Gordonia include G. bronchialis, G. otitidis, G. aichiensis,
and G. terrae. Regarding fungal strains, there are references
to pulmonary histoplasmosis caused by the fungus Histo-
plasma capsulatum found in caves inhabited by bats where
it is also common to find the emerging pathogenic fungus
Penicillium marneffei.
Historically, soil has always been the main source of
antibiotics. Thousands of bioactive compounds (e.g., chlo-
ramphenicol, tetracycline, and erythromycin) have been
extracted from soil bacteria (Cycoń etal. 2019). However,
cultural heritage monuments and sites can also be consid-
ered as sources of antibiotics. Approximately, two-thirds
of all known antibiotics are produced by Actinobacteria (a
large part of the stone heritage communities), particularly
by species of the genus Streptomyces (Mast and Stegmann
2018), and many authors consider Actinobacteria an inex-
haustible source of naturally occurring antibiotics. Historical
caves and mines are currently considered emergent sources
of novel antibiotics, acting as excellent reservoirs of new
species of Actinobacteria (Cheeptham and Saiz-Jimenez
2015). Methanotrophic and heterotrophic bacteria found
in caves and mines can produce bioactive compounds and
may be potential sources of metabolites with antibacterial,
antifungal, and anticancer properties. Some examples of
drug discovery in caves include the chemical structure of
cervimycin A-D; a polyketide glycoside complex obtained
from Streptomyces tendae, isolated from Grotta dei Cervi
(Italy); cyclodysidin D and chaxalactin B produced by a
Streptomyces sp. isolated from moonmilk (cave milk); and
the huanglongmycin A-C complex, synthesized by a strain
of Streptomyces, found in a cave in China (Martín-Pozas
etal. 2020).
Use ofplants tomitigate theeects
ofclimate change onstone heritage
Temperature change is a major factor in stone weathering;
and therefore, knowing and controlling micrometeorologi-
cal conditions, i.e., the actual climatic conditions around
the stone surfaces, is a determining factor in heritage con-
servation. Plant landscaping has been suggested to be effec-
tive for regulating the cultural heritage microenvironment
Fig. 2 Midwife toad (Alytes obstetricans), one of the amphibian
species most threatened by chytridiomycosis, is a disease caused by
Batrachochytrium spp. Photo: Miguel Serrano
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1 3
(Li etal. 2021). Comparison of unshaded areas and areas
shaded by trees along the historical Nanjing wall (China)
showed that trees could reduce evaporation by 8–18% and
prevent the associated risk of deterioration on wall surfaces
by buffering freeze–thaw cycles and efflorescences. An
ongoing survey, started in the summer of 2022, has shown
that small plants growing between paving stones in the his-
torical city of Santiago de Compostela (Galicia, NW Spain),
a UNESCO World Heritage City since 1985, can help to
counteract the negative effects of global rising temperatures
by controlling the biometeorological conditions within the
plants (Kevan etal. 2019). As plants cannot move to avoid
the effects of hot temperatures, they have evolved mecha-
nisms such as increasing transpiration rates, to cope with
the risk of overheating, which ultimately results in maintain-
ing the surrounding temperatures below those on the bare
pavement or on paving joints filled with cement (Fig.3).
Despite the small size of these plants and the relatively low
cover relative to the whole area (typically less than 15%),
the micrometeorological effects of reducing temperatures
during heatwaves are noticeable, even at heights of 1.80m
above the pavements (with decreases of up to 3°C in areas
with plants growing between paving stone) contributing to
both stone heritage conservation and the habitability of cit-
ies (Serrano, preliminary unpublished results). The small
plants involved have until now been neglected as weeds,
and are routinely removed from cultural sites and histori-
cal city centers. The ecological arrangement of these spe-
cies in the urban fabric (e.g., plants with C4 photosynthetic
metabolism, such as Digitaria spp. and Amaranthus spp.,
and therefore possessing mechanisms to reduce water loss,
prevailing in the more isolated areas) should be studied and
used in urban design to enhance the provision of ecosystem
services, including micrometeorological temperature control
and cohesion of paving stones via lateral root growth.
In addition, climate change is affecting many archaeo-
logical sites scattered through forested areas in Europe as
fire rates increase (Fernández etal. 2021). A recent study
compared the damage, including aesthetic damage, to gran-
ite rocks in cultural sites with megalithic and parietal art in
areas of the NW Iberian Peninsula characterized by different
types of vegetation (Pozo-Antonio etal. 2020); the study
findings showed that oak woods could buffer the damaging
effects of fire in natural areas, and the authors suggested
using oak species as protective belts in conservation plan-
ning for cultural sites in natural areas.
Final considerations andfuture prospects
In projections for the future of cultural heritage protection,
efforts should turn towards the green deal, ecosystem ser-
vices, and animal and human health, to provide benefits for
biodiversity and climate change mitigation and adaptation.
This is in accordance with the concept of bioeconomy, as
defined by the European Commission, because it involves
renewable biological resources and conversion of these into
value added products, such as green biocides. It is also con-
sistent with the ONU Sustainable Development Goals (ODS)
as the aim is to promote innovative research and sustainable
Fig. 3 Ecosystem service: urban
plant cool granite pavements in
Santiago de Compostela (NW
Spain), while paving joints
filled with cement accumulate
the highest temperatures. Pho-
tos: Miguel Serrano
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1 3
methods of safeguarding cultural heritage (SDG11, goal
11.4) through the use of natural products that replace tra-
ditional toxic chemical products and therefore promote the
health and well-being of restorers (SDG3, goal 3.9), while
also reducing the use of dangerous chemical substances and
air, water, and soil pollutants (SDG12, goal 12.4) and pro-
moting sustainable and environmentally friendly industri-
alization (SDG9).
Although the mechanisms of resistance to QACs in
microorganisms that affect cultural heritage remain largely
unknown to date, there are studies that explain through them
how green algae are less sensitive than cyanobacteria to cer-
tain QACs compounds. Furthermore, it can be concluded
that the re-colonization promoted by biocides is influenced
by factors such as the cleaning process when an effective
biocide is correctly applied. It is important to conduct pre-
treatment analysis to screen the tolerance to biocides, at least
in the strains isolated, and then to use the most appropriate
biocide. Microbiological monitoring of the cultural heritage
objects after application of biocide treatments would also
help to clarify whether the re-colonization is driven by toler-
ant and resistant microorganisms or by new colonizers, and
also to determine the ratios of these two groups. Combining
microbiological analysis and culture-dependent approaches
will provide a better view of the microbial communities
present and to match these with the detection of potential
biocide-resistant genes. More complex studies could also
focus on the development of multi-strain biofilms in labora-
tory conditions.
Planning historical urban centers should also promote
the growth on pavements of plants that are evolutionarily
adapted to cope with increasing temperatures. Furthermore,
biological control technologies based on viral remediation or
phage therapy is a promising eco-sustainable approach that
deserves further consideration in the cultural heritage field,
but so far, the results have fallen well short of expectations.
Future research lines should aim to confirm the effective-
ness of invivo treatments, the persistence of the effects,
and the lack of side effects on real monuments (lack of
negative interactions with the substrate). Once these crucial
points are elucidated, the methods of application must be
optimized and cost evaluation must be conducted, with the
aim of developing commercially useful, green conservation
products.
Acknowledgements The authors acknowledge the help of Cesáreo
Saiz-Jiménez and Vicente Medina for providing the photographs shown
above.
Author contribution PS, PBR, DP, LK, and MS performed the litera-
ture search, collected the data on various topics, and wrote the review.
PS, PBR, DP, LK, and MS designed the structure of the review and
revised the review. All authors critically revised and approved the final
manuscript.
Funding Open Access funding provided thanks to the CRUE-CSIC
agreement with Springer Nature. The authors would like to express
their gratitude to the Spanish State Research Agency (AEI) of the
Ministry of Science and Innovation (MCIN)for concession of the
BIOXEN project (PID2021-123329NA-I00). This study has been
also supported by the European Regional Development Fund project:
313011V578.P. Sanmartín acknowledges receipt of a Ramón y Cajal
contract (RYC2020-029987-I) financed by theAEI of theMCIN. P.
Sanmartín and M. Serrano are grateful for the financial support from
the Xunta de Galicia (grants ED431C 2022/09 and ED431B 2021/11).
Data availability All data generated or analyzed during this study are
included in this publication (and the supplementary material).
Declarations
Ethics approval This article does not contain any studies involving
human participants or animals.
Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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