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Innovative approaches to accurately assess the effectiveness of biocide-based treatments to fight biodeterioration of Cultural Heritage monuments
Abstract
The development of diagnostic methods to accurately assess the effects of treatments on lithobiont colonization remains a challenge for the conservation of Cultural Heritage monuments. In this study, we tested the efficacy of biocide-based treatments on microbial colonization of a dolostone quarry, in the short and long-term, using a dual analytical strategy. We applied a metabarcoding approach to characterize fungal and bacterial communities over time, integrated with microscopy techniques to analyze the interactions of microorganisms with the substrate and evaluate the effectiveness. These communities were dominated by the bacterial phyla Actinobacteriota, Proteobacteria and Cyanobacteria, and the fungal order Verrucariales, which include taxa previously reported as biodeteriogenic agents and observed here associated with biodeterioration processes. Following the treatments, changes over time in the abundance profiles depend on taxa. While Cyanobacteriales, Cytophagales and Verrucariales decreased in abundance, other groups, such as Solirubrobacteriales, Thermomicrobiales and Pleosporales increased. These patterns could be related not only to the specific effects of the biocide on the different taxa, but also to different recolonization abilities of those organisms. The different susceptibility to treatments could be associated with the inherent cellular properties of different taxa, but differences in biocide penetration to endolithic microhabitats could be involved. Our results demonstrate the importance of both removing epilithic colonization and applying biocides to act against endolithic forms. Recolonization processes could also explain some of the taxon-dependent responses, especially in the long-term. Taxa showing resistance, and those benefiting from nutrient accumulation in the form of cellular debris following treatments, may have an advantage in colonizing treated areas, pointing to the need for long-term monitoring of a wide range of taxa. This study highlights the potential utility of combining metabarcoding and microscopy to analyze the effects of treatments and design appropriate strategies to combat biodeterioration and establish preventive conservation protocols.
... However, biocide application cannot have a long-lasting efficiency, and repetitive use may result in microbial resistance, leading to a resurgence of microbes on treated surfaces [23,29,30]. Moreover, their potential risks to human health and the environment, as well as substrate interference [31,32], have prompted a quest for alternative ecofriendly solutions [33][34][35]. ...
Microbial colonization can lead to various biodeterioration phenomena in outdoor stone monuments. To prevent these issues and mitigate the negative effects of chemical control, recent research has focused on encapsulating biocides in nanostructures. We tested the antifouling efficiency of the new multifunctional coating through in situ experiments performed on the Aurelian Walls in Rome. We selected two different biocides (2-mercaptobenzothiazole and zosteric sodium salt) and loaded them into two different silica nanocontainers (nanocapsules and mesoporous particles), which were dispersed in TEOS coatings. These coatings were applied to four common lithotypes (marble, travertine, mortar, and brick). Subsequently, we accelerated the colonization by inoculating microorganisms onto half of the samples, subjecting all samples to aging cycles. We conducted colorimetric, infrared spectroscopy, and other microscopic analyses to assess their durability and antimicrobial action. Our 3-year survey indicates that the coatings demonstrated antifoulant efficiency across all samples, with higher effectiveness observed on brick and mortar compared to travertine and marble. Among the nanostructures, nanocapsules exhibited greater efficiency than mesoporous particles, and regarding the biocides, the commercial one outperformed the natural one. These findings highlighted the potential of such systems in preserving cultural heritage; however, further research and product development is imperative for practical implementation.
... However, biocide application cannot have a long-lasting efficiency, and repetitive use may result in microbial resistance, leading to a resurgence of microbes on treated surfaces [23,29,30]. Moreover, their potential risks to human health and the environment, as well as substrate interference [31,32], have prompted a quest for alternative ecofriendly solutions [33][34][35]. ...
Caves represent unique habitats for the development of microbial communities due to particular environmental conditions. Mural paintings occurring in caves suffer from degradation caused by natural processes in which microorganisms are significantly involved. The characterization of the microbial biofilm with non-destructive analysis is important to better preserve and safeguard these artifacts. This study aimed at investigating the microbial communities colonising the frescoes in the Fornelle cave (Calvi Risorta, Caserta, Italy) using a metabarcoding approach. We found a high representation of eubacteria, followed by cyanobacteria, fungi, and microalgae. This study confirmed the potential of non-invasive techniques as DNA metabarcoding as powerful tool for conservation and restoration of artworks.
Black fungi are considered as one of the main group of microorganisms responsible for the biodeterioration of stone cultural heritage artifacts. In this paper, we provide a critical analysis and review of more than 30 years of studies on black fungi isolated from stone-built heritage from 1990 to date. More than 109 papers concerning the fungal biodeterioration activity of stone were analysed. The main findings were a check list of the black fungal taxa involved in the biodeterioration of stone-built heritage, with a particular reference to meristematic black fungi, the main biodeterioration pattern attributed to them, and the methods of study including the new molecular advances. A particular focus was to discuss the current approaches to control black fungi from stone-built heritage and future perspectives. Black fungi are notoriously hard to remove or mitigate, so new methods of study and of control are needed, but it is also important to combine classical methods with new approaches to improve current knowledge to implement future conservation strategies.
The continuous introduction of cleaning products containing benzalkonium chloride (BAC) from household discharges can mold the microbial communities in wastewater treatment plants (WWTPs) in a way still poorly understood. In this study, we performed an in vitro exposure of activated sludge from a WWTP in Costa Rica to BAC, quantified the changes in intI1, sul2, and qacE/qacEΔ1 gene profiles, and determined alterations in the bacterial community composition. The analysis of the qPCR data revealed elevated charges of antibiotic resistance genes in the microbial community; after BAC's exposure, a significant increase in the qacE/qacEΔ1 gene, which is related to ammonium quaternary resistance, was observed. The 16S rRNA gene sequences’ analysis showed pronounced variations in the structure of the bacterial communities, including reduction of the alpha diversity values and an increase of the relative abundance of Alphaproteobacteria, particularly of Rhodospseudomonas and Rhodobacter. We confirmed that the microbial communities presented high resilience to BAC at the mg/mL concentration, probably due to constant exposure to this pollutant. They also presented antibiotic resistance-related genes with similar mechanisms to tolerate this substance. These mechanisms should be explored more thoroughly, especially in the context of high use of disinfectant. HIGHLIGHTS
In vitro BAC's exposure enhances qacE/qacEΔ1 gene presence in bacterial communities from AS.;
AR related genes in AS's microbial communities from tropical countries are reported.;
The BAC exposure can alter the AS microbial community composition.;
Putative nitrifiers are enhanced by BAC's exposure.;
The presence of intI-1 gene in AS could indicate the anthropogenic spread of microbial resistance into the environment.;
Throughout the Negev Desert highlands, thousands of ancient petroglyphs sites are susceptible to deterioration processes that may result in the loss of this unique rock art. Therefore, the overarching goal of the current study was to characterize the composition, diversity and effects of microbial colonization of the rocks to find ways of protecting these unique treasures. The spatial organization of the microbial colonizers and their relationships with the lithic substrate were analysed using scanning electron microscopy. This approach revealed extensive epilithic and endolithic colonization and close microbial–mineral interactions. Shotgun sequencing analysis revealed various taxa from the archaea, bacteria and some eukaryotes. Metagenomic coding sequences (CDS) of these microbial lithobionts exhibited specific metabolic pathways involved in the rock elements' cycles and uptake processes. Thus, our results provide evidence for the potential participation of the microorganisms colonizing these rocks during different solubilization and mineralization processes. These damaging actions may contribute to the deterioration of this extraordinary rock art and thus threaten this valuable heritage. Shotgun metagenomic sequencing, in conjunction with the in situ scanning electron microscopy study, can thus be considered an effective strategy to understand the complexity of the weathering processes occurring at petroglyph sites and other cultural heritage assets.
Fuente de Cibeles of Madrid (Spain) represents Cybele goddess sitting on a carriage drawn by two lions. This sculptural set was carved in Montesclaros (Toledo-Spain) marble between 1777 and 1782. The sculptural set has experienced some modifications throughout its history. Two zoomorphic water-spouts carved in Colmenar de Oreja limestone, one in the shape of a dragon and another one in the shape of a bear, were installed in 1798. In 1895, the fountain changed of location, and two putti of Carrara marble were installed on the back of the fountain, expanding the rocky surface promontory on which the sculptural set stands. Two tiered basins made of granite were added in 1968, when the fountain acquired its current form.A geotouristic route was created based on Fuente de Cibeles and its main building stone, Montesclaros marble. An exhaustive description of the fountain was made, and its construction history was reviewed to elaborate the route. In this way, alternatives that diversify the conventional tourist options of the city of Madrid are presented. In addition, scientific data on historical quarries and the marble that forms part of the emblematic fountain is provided.
The cryptic lifestyle of most fungi necessitates molecular identiication of the guild in environmental studies. Over the past
decades, rapid development and afordability of molecular tools have tremendously improved insights of the fungal diversity
in all ecosystems and habitats. Yet, in spite of the progress of molecular methods, knowledge about functional properties of
the fungal taxa is vague and interpretation of environmental studies in an ecologically meaningful manner remains challenging.
In order to facilitate functional assignments and ecological interpretation of environmental studies we introduce a user
friendly traits and character database FungalTraits operating at genus and species hypothesis levels. Combining the information
from previous eforts such as FUNGuild and FunFun together with involvement of expert knowledge, we reannotated
10,210 and 151 fungal and Stramenopila genera, respectively. This resulted in a stand-alone spreadsheet dataset covering 17
lifestyle related traits of fungal and Stramenopila genera, designed for rapid functional assignments of environmental studies. In order to assign the trait states to fungal species hypotheses, the scientiic community of experts manually categorised
and assigned available trait information to 697,413 fungal ITS sequences. On the basis of those sequences we were able to
summarise trait and host information into 92,623 fungal species hypotheses at 1% dissimilarity threshold.
An experimental study was conducted to assess the nature and extent of the biodeterioration of the limestone in the Batalha Monastery in Portugal. Stone fragments covered with microbial biofilms and lichenous crusts were investigated using Optical Microscopy (OM), Low Vacuum Scanning Electron Microscopy with Energy Dispersive Spectroscopy (LV-SEM + EDS), and X-ray micro-Diffractometry (μ-XRD). Microbial samples were collected from the stone surface, cultured, and analyzed with NGS metagenomic DNA test to classify the bacterial communities associated with the formation of the biofilms. Particulate air pollutants collected on Pall GN-6 paper filters using a cascade impactor were characterized by SEM-EDS + NGS. The results showed that lichens play a major role in biodeterioration by promoting both physical and chemical attack on the limestone substrate via hyphae mechanical penetration along calcite inter-crystalline spaces, the dissolution/leaching of calcite minerals, and the precipitation of secondary minerals such as Ca-oxalates within the stone porosity framework. DNA analyses identified the bacterial communities within the biofilms and their relative abundances. Air quality monitoring results suggest that the microbial population colonizing the monastery limestone could at least partially be derived from the dry and wet deposition of airborne biological particles on the stone surfaces and that S, N, and P-rich air pollutants may have provided nutrients and energy for the bacteria communities, thus indirectly facilitating biofilm formation, the growth of a lichenous crusts, and limestone biodeterioration effects.
Cultural heritage objects constitute a very diverse environment, inhabited by various bacteria and fungi. The impact of these microorganisms on the degradation of artworks is undeniable, but at the same time, some of them may be applied for the efficient biotreatment of cultural heritage assets. Interventions with microorganisms have been proven to be useful in restoration of artworks, when classical chemical and mechanical methods fail or produce poor or short-term effects. The path to understanding the impact of microbes on historical objects relies mostly on multidisciplinary approaches, combining novel meta-omic technologies with classical cultivation experiments, and physico-chemical characterization of artworks. In particular, the development of metabolomic- and metatranscriptomic-based analyses associated with metagenomic studies may significantly increase our understanding of the microbial processes occurring on different materials and under various environmental conditions. Moreover, the progress in environmental microbiology and biotechnology may enable more effective application of microorganisms in the biotreatment of historical objects, creating an alternative to highly invasive chemical and mechanical methods.
High soil salinity is the main factor that limits soil microbial activity in the Yellow River Delta (YRD); however, its effects on fungal
community and ecological function are unknown. Here, we comparatively investigated the diversity and structures of soil fungal communities targeting the internally transcribed fungal spacer gene using Illumina MiSeq sequencing methods under a salt gradient with five levels, namely, Low: low-salinity soil, Medium: medium-salinity soil, High: high-salinity soil, Extreme: extreme-salinity soil, and a non-salted site as the control (Non-saline). The results show that bulk density (BD) values significantly increased (p < 0.05), while significantly lower values of soil total carbon (TC), nitrogen (TN), and fungal Shannon and Chao indexes were observed as the salinization gradient increased (p < 0.05). The relatively high levels of the families Nectriaceae and Cladosporiaceae distinguished two of the clusters, indicating two enterotypes of low (Non-saline and Low) and high (Medium, High, and Extreme) salinity soils, respectively. The family Nectriaceae was most abundant in the networks, and the positive correlations were more pronounced than negative correlations; however, Cladosporiaceae was the family most negatively correlated with others based on the network analysis. At the ecological function level, plant saprotroph and litter saprotroph were significantly less abundant in extremely saline soil than non-saline soil. The change in soil properties (TC, TN, and BD) caused by soil salinization (salt and EC) regulated the diversity of soil fungal communities, and ecological function, as indicated by Pearson correlation analyses. We suggest further investigation into the ecological functions of soil microorganisms in the extremely saline-alkaline soils of the YRD.
The microbial deterioration of cultural heritage includes physical and chemical damage as well as aesthetic alteration. With the technological advancement, a plethora of techniques for removing unwanted microorganisms have opened up new opportunities for microbiologists and conservators. This article reviews the most applied, up-to-date, and sustainable techniques developed for the control of cultural heritage microbial deterioration presenting noteworthy case studies. These techniques include chemical methods, i.e., traditional biocides and nanoparticles; physical methods, such as mechanical removal, UV irradiation, gamma radiation, laser cleaning, heat shocking, microwaves, and dry ice treatment; and biological methods, such as natural molecules with biocidal activity, enzymes, and microorganisms. The application of control systems requires the comprehension of their behavior toward the unwanted microorganisms and possible interactions with the heritage materials. This overview shows also the control methods drawbacks for the purpose of creating awareness in selecting the most suitable technique or combination of techniques.
Free-living and mycorrhizal fungi are able to enhance the weathering of rock and other solid substrates. Deciphering the exact mechanisms of these natural processes requires their experimental simulation. Moreover, by performing these simulations with genetically amenable rock-weathering fungi, one can knock-out certain fungal traits and consequently identify their weathering-relevant function. Here, the effect of the rock-inhabiting fungus, Knufia petricola A95, on the dissolution kinetics of an Fe-bearing olivine (Mg1.86Fe0.19SiO4) is investigated at 25°C and pH 6 using reproducible batch and mixed flow experiments. The availability of a melanin-deficient mutant (ΔKppks) of K. petricola A95, which produces more extracellular polymeric substances (EPS) than the wild type (WT), enables the comparative study of the role of melanin and EPS in olivine dissolution. In abiotic dissolution experiments, the olivine dissolution rate decreased considerably over time at pH 6 but not at pH 3.5. This inhibition of abiotic olivine dissolution at pH 6 was most likely caused by the in-situ oxidation of ferrous Fe and/or the precipitation of ferric hydroxides at the olivine surface. In corresponding biotic experiments at pH 6, both the wild type K. petricola and its melanin-deficient mutant ΔKppks solubilised and bound significant amounts of Fe released by olivine dissolution. Fe oxidation and precipitation were thus prevented, and olivine dissolution proceeded faster than in the abiotic experiments. By sequestering Fe directly at the olivine surface, the attached wild type K. petricola cells were particularly efficient at preventing the oxidation of Fe at the mineral surface: the slowdown of olivine dissolution almost completely disappeared. The attachment capacity of these wild type cells is most likely mediated by wild type-specific EPS. Our presented experimental systems allow the oxidation of mineral-released Fe and include a rock-inhabiting fungus, thus simulating chemical, physical and biological conditions that set dissolution rates in a way that is relevant to natural ecosystems.
Microbiological methodologies allow understanding the causes that lead to the development of a certain microbial community colonizing an artistic surface, to characterize its composition and describe its role in the deterioration of the constituent materials. Metagenomics allows identifying microbial communities directly in their natural environments, bypassing the need for isolation and cultivation of individual species, thus providing a more comprehensive picture of the biodiversity present on a surface compared with standard cultivation methods. Furthermore, molecular analyses require small amounts of material, favoring the preservation of the artistic surface during sampling. Here, we verified the suitability of a protocol consisting in DNA extraction with micro-invasive sampling, using adhesive tape, PCR amplification with universal primers [bacteria (16S), fungi (ITS), and Viridiplantae (18S)], and amplicon sequencing by Oxford Nanopore Technologies (ONT) in the hypogeum of Basilica di San Nicola in Carcere Church (Rome, Italy). Sequence data were analyzed with a bioinformatic pipeline customized for pinpointing cultural heritage spoiling organisms, named “AmpLIcon SequencIng Analysis” (ALISIA). These data were integrated with traditional microbiology techniques that allowed the isolation of cultivable bacteria; three species were also characterized through their capability of biofilm formation and antibiotic resistance. Further, Fourier-transform infrared spectroscopy (FTIR) spectroscopy was performed to characterize the main products present on the masonry surface providing indications on the type of decay present. This novel biological workflow represents a powerful opportunity to investigate the microbial colonization of artistic surfaces aimed at implementing preservation strategies of cultural heritage from bio-spoilage.
Glacier forefields provide a unique chronosequence to assess microbial or plant colonization and ecological succession on previously uncolonized substrates. Patterns of microbial succession in soils of alpine and subpolar glacier forefields are well documented but those affecting high polar systems, including moraine rocks, remain largely unexplored. In this study, we examine succession patterns in pioneering bacterial, fungal and algal communities developing on moraine rocks and soil at the Hurd Glacier forefield (Livingston Island, Antarctica). Over time, changes were produced in the microbial community structure of rocks and soils (ice-free for different lengths of time), which differed between both substrates across the entire chronosequence, especially for bacteria and fungi. In addition, fungal and bacterial communities showed more compositional consistency in soils than rocks, suggesting community assembly in each niche could be controlled by processes operating at different temporal and spatial scales. Microscopy revealed a patchy distribution of epilithic and endolithic lithobionts, and increasing endolithic colonization and microbial community complexity along the chronosequence. We conclude that, within relatively short time intervals, primary succession processes at polar latitudes involve significant and distinct changes in edaphic and lithic microbial communities associated with soil development and cryptogamic colonization.
Despite the massive presence of biofilms causing aesthetic alteration to the façade of the Monza Cathedral, our team in a previous work proved that the biocolonization was not a primary damaging factor if compared to chemical-physical deterioration due to the impact of air pollution. Nonetheless, the conservators tried to remove the sessile dwelling microorganisms to reduce discolouration. In this research, two nearby sculpted leaves made of Candoglia marble were selected to study the effects of a chemical treatment combining the biocides benzalkonium chloride, hydrogen peroxide and Algophase® and mechanical cleaning procedures. One leaf was cleaned with the biocides and mechanically, and the other was left untreated as control. The impact of the treatment was investigated after 1 month from the cleaning by digital microscopy, environmental scanning electron microscopy, confocal microscopy and molecular methods to determine the composition and the functional profiles of the bacterial communities. Despite the acceptable aesthetic results obtained, the overall cleaning treatment was only partially effective in removing the biofilm from the colonized surfaces and, therefore, not adequately suitable for the specific substrate. Furthermore, the cleaning process selected microorganisms potentially more resistant to biocides so that the efficacy of future re-treatment by antimicrobial agents could be negatively affected.
Black patinas are very common biological deterioration phenomena on lapideous artworks in outdoor environments. These substrates, exposed to sunlight, and atmospheric and environmental agents (i.e. wind and temperature changes), represent extreme environments that can only be colonized by highly versatile and adaptable microorganisms. Black patinas comprise a wide variety of microorganisms, but the morphological plasticity of most of these microorganisms hinders their identification by optical microscopy. This study used Next-Generation Sequencing (NGS) (including shotgun and amplicon sequencing) to characterize the black patina of the travertine embankments (muraglioni) of the Tiber River in Rome (Italy). Overall, the sequencing highlighted the rich diversity of bacterial and fungal communities and allowed the identification of more than one hundred taxa. NGS confirmed the relevance of coccoid and filamentous cyanobacteria observed by optical microscopy and revealed an informative landscape of the fungal community underlining the presence of microcolonial fungi and phylloplane yeasts. For the first time high-throughput sequencing allowed the exploration of the expansive diversity of bacteria in black patina, which has so far been overlooked in routine analyses. Furthermore, the identification of euendolithic microorganisms and weathering agents underlines the biodegradative role of black patina, which has often been underestimated. Therefore, the use of NGS to characterize black patinas could be useful in choosing appropriate conservation treatments and in the monitoring of stone colonization after the restoration interventions.
Biocides are added to or applied on building materials to prevent microorganisms from growing on their surface or to treat them. They are leached into building runoff and contribute to diffuse contamination of receiving waters. This review aimed at summarizing the current state of knowledge concerning the impact of biocides from buildings on the aquatic environment. The objectives were (i) to assess the key parameters influencing the leaching of biocides and to quantify their emission from buildings, (ii) to determine the different pathways from urban sources into receiving waters and (iii) to assess the associated environmental risk. Based on consumption data and leaching studies, a list of substances to monitor in receiving water was established. Literature review of their concentrations in the urban water cycle showed evidences of contamination and risk for aquatic life, which should put them into consideration for inclusion to European or international monitoring programs. However, some biocide concentration data in urban and receiving waters is still missing to fully assess their environmental risk, especially for isothiazolinones, iodopropynyl carbamate, zinc pyrithione and quaternary ammonium compounds, and little is known about their transformation products. Although some models supported by actual data were developed to extrapolate emissions on larger scales (watershed or city scales), they are not sufficient to prioritize the pathways of biocides from urban sources into receiving waters during both dry and wet weathers. Our review highlights the need to reduce emissions and limit their transfer into rivers and reports several solutions to address these issues.
Biodeterioration is a serious threat to cultural heritage objects and buildings. The deterioration of a given material often incurs irreparable losses in terms of uniqueness and historical value. Hence preventive actions should be taken. One important challenge is to identify microbes involved in the biodeterioration process. In this study, we analyzed the microbial diversity of an ancient architectonical structure of the Rotunda of Sts. Felix and Adauctus, which is a part of the Wawel Royal Castle located in Krakow, Poland. The Rotunda is unavailable to tourists and could be treated as an extreme habitat due to the low content of nutrients coming either from sandstone plates bound with lime mortar or air movement. Microbial diversity was analyzed with the use of the high-throughput sequencing of marker genes corresponding to fragments of 16S rDNA (for Bacteria) and ITS2 (internal transcribed spacer 2) (for Fungi). The results showed that the microbial community adhered to wall surfaces is, to a large extent, endemic. Furthermore, alongside many microorganisms that could be destructive to masonry and mortar (e. g., Pseudomonas, Aspergillus), there were also bacteria, such as species of genera Bacillus, Paenisporosarcina, and Amycolatopsis, that can positively affect wall surface properties by reducing the damage caused by the presence of other microorganisms. We also showed that airborne microorganisms probably have little impact on the biodeterioration process as their abundance in the microbial community adhered to the ancient walls was very low.
UNITE (https://unite.ut.ee/) is a web-based database and sequence management environment for the molecular identification of fungi. It targets the formal fungal barcode-the nuclear ribosomal internal transcribed spacer (ITS) region-and offers all ∼1 000 000 public fungal ITS sequences for reference. These are clustered into ∼459 000 species hypotheses and assigned digital object identifiers (DOIs) to promote unambiguous reference across studies. In-house and web-based third-party sequence curation and annotation have resulted in more than 275 000 improvements to the data over the past 15 years. UNITE serves as a data provider for a range of metabarcoding software pipelines and regularly exchanges data with all major fungal sequence databases and other community resources. Recent improvements include redesigned handling of unclassifiable species hypotheses, integration with the taxonomic backbone of the Global Biodiversity Information Facility, and support for an unlimited number of parallel taxonomic classification systems.
The internally transcribed spacer (ITS) region between the small subunit ribosomal RNA gene and large subunit ribosomal RNA gene is a widely used phylogenetic marker for fungi and other taxa. The eukaryotic ITS contains the conserved 5.8S rRNA and is divided into the ITS1 and ITS2 hypervariable regions. These regions are variable in length and are amplified using primers complementary to the conserved regions of their flanking genes. Previous work has shown that removing the conserved regions results in more accurate taxonomic classification. An existing software program, ITSx, is capable of trimming FASTA sequences by matching hidden Markov model profiles to the ends of the conserved genes using the software suite HMMER. ITSxpress was developed to extend this technique from marker gene studies using Operational Taxonomic Units (OTU’s) to studies using exact sequence variants; a method used by the software packages Dada2, Deblur, QIIME 2, and Unoise. The sequence variant approach uses the quality scores of each read to identify sequences that are statistically likely to represent real sequences. ITSxpress enables this by processing FASTQ rather than FASTA files. The software also speeds up the trimming of reads by a factor of 14-23 times on a 4-core computer by temporarily clustering highly similar sequences that are common in amplicon data and utilizing optimized parameters for Hmmsearch. ITSxpress is available as a QIIME 2 plugin and a stand-alone application installable from the Python package index, Bioconda, and Github.
Background:
Taxonomic classification of marker-gene sequences is an important step in microbiome analysis.
Results:
We present q2-feature-classifier ( https://github.com/qiime2/q2-feature-classifier ), a QIIME 2 plugin containing several novel machine-learning and alignment-based methods for taxonomy classification. We evaluated and optimized several commonly used classification methods implemented in QIIME 1 (RDP, BLAST, UCLUST, and SortMeRNA) and several new methods implemented in QIIME 2 (a scikit-learn naive Bayes machine-learning classifier, and alignment-based taxonomy consensus methods based on VSEARCH, and BLAST+) for classification of bacterial 16S rRNA and fungal ITS marker-gene amplicon sequence data. The naive-Bayes, BLAST+-based, and VSEARCH-based classifiers implemented in QIIME 2 meet or exceed the species-level accuracy of other commonly used methods designed for classification of marker gene sequences that were evaluated in this work. These evaluations, based on 19 mock communities and error-free sequence simulations, including classification of simulated "novel" marker-gene sequences, are available in our extensible benchmarking framework, tax-credit ( https://github.com/caporaso-lab/tax-credit-data ).
Conclusions:
Our results illustrate the importance of parameter tuning for optimizing classifier performance, and we make recommendations regarding parameter choices for these classifiers under a range of standard operating conditions. q2-feature-classifier and tax-credit are both free, open-source, BSD-licensed packages available on GitHub.
Bacterial community and diversity in a long-term petroleum-contaminated soil of an oilfield were characterized using 16S rRNA gene-based Illumina MiSeq high-throughput sequencing. Results indicated that Proteobacteria (49.11%) and Actinobacteria (24.24%) were the most dominant phyla, and the most abundant genera were Pseudoxanthomonas (8.47%), Luteimonas (3.64%), Alkanindiges (9.76%), Acinetobacter (5.26%) and Agromyces (8.56%) in the soil. Meanwhile a series of cultivations were carried out for isolation of alkane degraders from petroleum-contaminated soil with gellan gum and agar as gelling agents. And the isolates were classified by their 16S rRNA genes. Nine of the isolates including Enterobacter, Pseudomonas,Acinetobacter, Rhizobium, Bacillus, Sphingomonas, Paenibacillus, Variovorax and Rhodococcus showed strong biodegradability of alkane mixture (C9–C30) in a wide range of chain-length, which could be potentially applied in enhancement of bioremediation.
Melanised cell walls and extracellular polymeric matrices protect rock-inhabiting microcolonial fungi from hostile environmental conditions. How extracellular polymeric substances (EPS) perform this protective role was investigated by following development of the model microcolonial black fungus Knufia petricola A95 grown as a sub-aerial biofilm. Extracellular substances were extracted with NaOH/formaldehyde and the structures of two excreted polymers studied by methylation as well as NMR analyses. The main polysaccharide (~ 80%) was pullulan, also known as α-1,4-; α-1,6-glucan, with different degrees of polymerisation. Αlpha-(1,4)-linked-Glcp and α-(1,6)-linked-Glcp were present in the molar ratios of 2:1. A branched galactofuromannan with an α-(1,2)-linked Manp main chain and a β-(1,6)-linked Galf side chain formed a minor fraction (~ 20%). To further understand the roles of EPS in the weathering of minerals and rocks, viscosity along with corrosive properties were studied using atomic force microscopy (AFM). The kinetic viscosity of extracellular K. petricola A95 polysaccharides (≈ 0.97 × 10⁻⁶ m² s⁻¹) ranged from the equivalent of 2% (w/v) to 5% glycerine, and could thus profoundly affect diffusion-dominated processes. The corrosive nature of rock-inhabiting fungal EPS was also demonstrated by its effects on the aluminium coating of the AFM cantilever and the silicon layer below.
Quaternary ammonium compounds (QACs) are surface-active, antimicrobial, high production volume (HPV) chemicals with a broad application in agriculture. This review provides a comprehensive overview of (1) predicted and measured concentrations of QACs in soils including their analysis, (2) sequestration mechanisms in soils based on their physicochemical properties and chemical structure, and (3) implications of concentrations and fate of QACs in soils for the proliferation of antibiotic resistance in the environment. Predicted environmental concentrations (PEC) for QACs that are applied to soils with manure are in the order of 3.5 mg kg⁻¹. Based on literature data, the median PEC of QAC in sewage sludge amended soils is 25 µg kg⁻¹. The positively charged QACs are mainly sorbed to clay minerals. We propose that QACs might be sequestered in the interlayer regions of layered silicates in clay-rich soils, reducing their acute toxicity, while increasing their persistence. The release of sequestered QACs from soil can still potentially maintain concentration levels that are sufficient to develop antibiotic resistance in the environment.
Biodeterioration represents a revealing problem for the conservation of cultural heritage. It can be identified as a complex interaction within the ecosystem of a microbial community and its substrate and involves physical and chemical alterations resulting from biological and metabolic activity. Designing a diagnostic approach for evaluating the extent of the damage, identifying the biological community, and opting for an efficient methodology aimed at eliminating deteriogens is equally complicated. The correct approach would require understanding the nature of the biodeterioration and implementing methodologies respectful of human health which, however, avoid the indiscriminate killing of organisms. Different preventive or remedial methods are used for this purpose. They include well-known physical and mechanical methods with their operating limitations and the most frequently used chemical methods, supported by biocide products for the elimination or growth inhibition of target organisms. Unfortunately, most—if not all—biocides applied on artworks are toxic or otherwise polluting substances, and their degradation is frequently difficult, being persistent in the natural environment. Moreover, due to the fact that there are no specific formulations destined for conservation practice, commercial biocide products come from the medical or agricultural field, carrying with them their well-known negative effects. Research in this sector focuses on ways to replace toxic products with natural molecules that do not cause adverse effects, in addition to the application of alternative methods and the support of formulations for safe nontoxic novel compounds.KeywordsMicrobial colonizationMinimum biocidal concentrationEssential oilsHydroalcoholic extractin vitro analysis
The surfaces of historical stone monuments are visibly covered with a layer of colonizing microorganisms and their degradation products. In this study, a metadata analysis was conducted using the microbial sequencing data available from NCBI database to determine the diversity, biodeterioration potential and functionality of the stone microbiome on important world cultural heritage sites under four different climatic conditions. The retrieved stone microbial community composition in these metagenomes shows a clear association between climate types of the historical monuments and the diversity and taxonomic composition of the stone microbiomes. Shannon diversity values showed that microbial communities on stone monuments exposed to dry climate were more diverse than those under humid ones. In particular, functions associated with photosynthesis and UV resistance were identified from geographical locations under different climate types. The distribution of key microbial determinants responsible for stone deterioration was linked to survival under extreme environmental conditions and biochemical capabilities and reactions. Among them, biochemical reactions of the microbial nitrogen and sulfur cycles were most predominant. These stone-dwelling microbiomes on historical stone monuments were highly diverse and self-sustaining driven by energy metabolism and biomass accumulation. And metabolic products of the internal geomicrobiological nitrogen cycling on these ancient monuments play a unique role in the biodeterioration of stone monuments. These results highlight the significance of identifying the essential microbial biochemical reactions to advance the understanding of stone biodeterioration for protection management.
The devitalization of lithobionts prior to their removal from engraved rocks is a common conservation practice periodically undertaken in rock art sites. In this study, we assessed in situ the efficacy of three traditional biocides and of an innovative microwave heating system, and compared different application protocols to devitalize foliose and crustose lichens and a cyanobacteria-dominated biofilm on the rock engravings of Valle Camonica (UNESCO site n.94, Italy). The analysis of their vitality and stress responses by monitoring chlorophyll a fluorescence parameters (Fv/Fm, F0, OJIP transient) showed that the common application of biocides by brush is rather ineffective, particularly in the case of the resistant crustose lichens. The heating of rock surfaces to 70 °C for a few minutes by the microwave system caused devitalization of lithobionts to a similar extent as the biocide application with cellulose poultice, which, however, introduced high amounts of chemicals in the environment. The microwave irradiation overcame any lithobiontic stress resistance and avoided useless or excessive spread of biocides, appearing a promising sustainable approach for the parallel conservation of rock art and its surrounding natural environment.
All types of building materials are rapidly colonized by microorganisms, initially through an invisible and then later a visible biofilm that leads to their biodeterioration. Over centuries, this natural phenomenon has been managed using mechanical procedures, oils, or even wax. In modern history, many treatments such as high-pressure cleaners, biocides (mainly isothiazolinones and quaternary ammonium compounds) are commercially available, as well as preventive ones, such as the use of water-repellent coatings in the fabrication process. While all these cleaning techniques offer excellent cost-benefit ratios, their limitations are numerous. Indeed, building materials are often quickly recolonized after application, and microorganisms are increasingly reported as resistant to chemical treatments. Furthermore, many antifouling compounds are ecotoxic, harmful to human health and the environment, and new regulations tend to limit their use and constrain their commercialization. The current state-of-the-art highlights an urgent need to develop innovative antifouling strategies and the widespread use of safe and eco-friendly solutions to biodeterioration. Interestingly, innovative approaches and compounds have recently been identified, including the use of photocatalysts or natural compounds such as essential oils or quorum sensing inhibitors. Most of these solutions developed in laboratory settings appear very promising, although their efficiency and ecotoxicological features remain to be further tested before being widely marketed. This review highlights the complexity of choosing the adequate antifouling compounds when fighting biodeterioration and proposes developing case-to-case innovative strategies to raise this challenge, relying on integrative and multidisciplinary approaches.
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Permutational multivariate analysis of variance (PERMANOVA) is a geometric partitioning of variation across a multivariate data cloud, defined explicitly in the space of a chosen dissimilarity measure, in response to one or more factors in an analysis of variance design. Statistical inferences are made in a distribution‐free setting using permutational algorithms. The PERMANOVA framework is readily extended to accommodate random effects, hierarchical models, mixed models, quantitative covariates, repeated measures, unbalanced and/or asymmetrical designs, and, most recently, heterogeneous dispersions among groups. Plots to accompany PERMANOVA models include ordinations of either fitted or residualized distance matrices, including multivariate analogues to main effects and interaction plots, to visualize results.
This work analyzed the influence of different application protocols on the efficacy of two biocides against the
foliose lichen Xanthoparmelia tinctina on the sandstones of the Roman Archaeological site of Luni (Italy). The
hypotheses that (a) biocide application tools (brush vs. poultice), (b) pre-treatment hydration, and (c) posttreatment
washing may affect devitalization success were verified by monitoring chlorophyll a fluorescence of
thalli, both in situ and in laboratory conditions. The hypothesis that (d) stone substrate may act as reservoir for
later biocide release under repeated cycles of wetting and drying was also assayed. Analyses confirmed the
importance of the application tool, with cellulose poultice being more effective than brush. Hydration influenced
the biocide absorption by thalli. Moreover it modulated the metabolic activity and susceptibility to the available
toxic compound, hindering lichens from entering a dormant state to tolerate stress. Depending on the preparation
solvent (water vs. white spirit), the biocide application benefited from pre-treatment hydration and/or a posttreatment
washing. Lastly, we showed that different sandstones variously adsorb the biocides and potentially
contribute as a reservoir for their long-term release at low concentrations during successive hydration events.
Geomicrobially induced deterioration of stone monuments and buildings contributes to a considerable loss of world cultural heritage, especially when exposed to a changing climate or environment. The active biodeterioration processes typically involve biochemical activities and cooperation among functional microorganisms in epilithic biofilms, which assimilate mineral nutrients and metabolize anthropogenic pollutants through biogeochemical cycles. Development of any effective mitigation strategies requires the comprehensive understanding of such processes. We focus on how microbes contribute to the biodeterioration processes through their activities and biogeochemical cycles of elements, discuss biochemical mechanisms involved and provide innovative strategies for sustainable conservation of stone monuments and buildings. Biodeterioration of stone monuments and buildings can lead to a loss of world cultural heritage. This Review discusses the role of microbes in the deterioration processes, the biochemical mechanisms involved and possible strategies for sustainable conservation of stone monuments and buildings.
A holistic perspective on the universe of living microorganisms, their metabolism and environment, is called systems biology. This framework is a compelling way to diagnose the biodeterioration of cultural heritage (CH). It is important to understand both the types of microorganisms present on objects, their metabolic functions, and the impact of the environment on microorganisms. Such an understanding generates answers to key questions concerning the mechanisms of biodeterioration by microbial communities and will help to select appropriate strategies for restoration and maintenance. In recent years, systemic methods called “-omics” have completely changed the approach to environmental research. These methods include metagenomics, transcriptomics, metabolomics and proteomics. Article reviews newly-available -omic techniques that can be used to assess CH biodeterioration and discusses the application of molecular and chemical techniques, including next generation sequencing (Pyrosequencing 454, Ion Torrent Sequencing, Illumina MiSeq), mass spectrometry techniques such as mass spectrometry coupled with liquid chromatography (LCMS) and surface-assisted desorption/ionization (SALDI) MS, in biodeterioration studies. Finally, article presents future perspectives for the evaluation of CH biodeterioration.
Feilaifeng is listed as a cultural heritage site because of its abundant Buddhist statues possibly dating back to the Five Dynasties period (907 AD–960 AD). However, microorganism growth on the surface of stone results in esthetic and structural damage.
Biocide treatment is commonly used to eradicate the microorganisms, and we assessed the effectiveness of four chemicals (benzalkonium chloride, octhilinone, tebuconazole, and thiabendazole) and two commercial biocides (ACTICIDE® 50 X (A) and AW-600) in inhibiting microbial colonization.
The minimal inhibitory concentrations of various biocides were determined, and their efficacies were compared using the inhibition zone method. The most effective biocides were applied in situ to Feilaifeng Limestone, and the overall effects were monitored by photographic recordings and microbial cultures for a month. The factors affecting the efficacy of each biocide, such as thermal stability and loss-resistance, were also evaluated.
The biocides varied markedly in their ability to inhibit the proliferation of different microorganisms. The three species of fungi tested herein demonstrated the highest susceptibility to octhilinone, but benzalkonium chloride showed the best antibacterial activity to the bacteria, Pseudomonas sp. Based on the results of in vitro assays, octhilinone, as well as its respective commercial product AW-600, were the best biocides for treating microorganisms, and 0.5% was the optimal concentration. Although the biocides on disc papers could be easily diluted by water, they were resistant to degradation by high temperatures. These findings suggest that frequent rainfall might have been the reason for the recolonization of microorganisms in a short time after octhilinone and AW-600 treatment in situ. Our results provide new insights on how to protect Feilaifeng Limestone, which is prone to damage caused by various microorganisms.
This study evaluated the medium-term performance of some of the most widely used methods of cleaning stone: dry brushing or washing with water or with an ethanol-water (1:1) mixture, benzalkonium chloride or commercial products based on quaternary ammonium salts with biocidal properties (Biotin R®, Biotin T®, Preventol RI80®, and New Des 50®). The ability of the different methods to remove an algal biofilm formed on a granite-built historical monument was compared. The biofilm has been shown to affect the appearance, but not the integrity, of the building material. In the cleaning trials, the presence and vitality of the organisms colonizing the substrate were determined, immediately after treatment and after one, three, six and twelve months, by colour spectrophotometry and chlorophyll-a fluorescence (ChlaF) analysis. The quantity and physiological state of the biofilm-forming phototrophs in the test areas were also determined one year after the treatments. Although recolonization of the test areas was barely noticeable after all of the treatments, Preventol RI80® proved to be the most effective, followed by benzalkonium chloride and the ethanol-water (1:1) mixture, which yielded similar results. New Des 50® caused clearly visible colour changes on the granite wall, and the biocidal efficacy was poor. Biotin R® performed better than Biotin T®, which yielded similar results to New Des 50® regarding recolonization. Considering the overall results and the potential toxicity of the products tested, cleaning with water is recommended if and when required.
Biodeterioration is when living organisms chemically or physically change or alter the appearance of materials objects. Organisms can colonize and destroy valuable cultural heritage. New advances in biotechnology and applied microbiology provide important information on conserving cultural heritage. Various physical and mechanical methods have previously been used, but they are incapable of preventing the growth of organisms entirely. Organic biocides, particularly commercial formulations, do not last long because they can be utilized as a nutrient source by indigenous microflora after these microflora are exposed to biocides and develop resistance. Therefore, inorganic nanoparticles have a better chance to protect cultural heritage. Silver (Ag2O) and titanium (TiO2) oxides are effective against biofilm, and nanoparticles of zinc oxide (ZnO) are effective antimicrobial agents. This new generation of biocides is much smaller in size and extremely active to damage DNA or RNA. In addition, green biocides from natural sources offer an alternative to chemical ones, having low toxicity compared to chemically synthesized biocides. Future research on biofilm control technologies may contribute to a broader understanding of and new perspectives on a future generation of biocontrol agents and methods with the potential for sustainable development.
Endolithic growth within rocks is a critical adaptation of microbes living in harsh environments where exposure to extreme temperature, radiation, and desiccation limits the predominant life‐forms, such as in the ice‐free regions of Continental Antarctica. The microbial diversity of the endolithic communities in these areas has been sparsely examined. In this work, diversity and composition of bacterial assemblages in the cryptoendolithic lichen‐dominated communities of Victoria Land (Continental Antarctica) were explored using a high‐throughput metabarcoding approach, targeting the V4 region of 16S rDNA. Rocks were collected in 12 different localities (from 14 different sites), along a gradient ranging from 1,000 to 3,300 m a.s.l. and at a sea distance ranging from 29 to 96 km. The results indicate Actinobacteria and Proteobacteria are the dominant taxa in all samples and defined a ‘core’ group of bacterial taxa across all sites. The structure of bacteria communities is correlated with the fungal counterpart and among the environmental parameters considered, altitude was found to influence bacterial biodiversity, while distance from sea had no evident influence. This article is protected by copyright. All rights reserved.
Fungi are powerful biodeteriogens and when colonizing stone monuments are often responsible for severe physical, chemical and aesthetical modifications. Recently the old cathedral of Coimbra (Portugal) was awarded the UNESCO World Heritage Site distinction as an integrant part of the “University of Coimbra- Alta and Sofia” area. The limestone walls in this cathedral show clear visual signs of biological colonization and biodeterioration. The aim of this work was to obtain an extensive fingerprint of the biodeteriorating fungal agents in this affected ancient limestone through the application of traditional cultivation and modern Next Generation Sequencing techniques. Due to high diversity of distinctive biodeterioration phenomena affecting this site, a rapport between the identified fungal populations and the different biodeterioration types found was also established and their importance for the monument preservation discussed. The results obtained through the application of this approach, not only allowed a robust characterization of the fungal communities colonizing this monument, but also elucidated that distinct fungal communities are dissimilar according to the type of biodeterioration analyzed. In addition, we verified that both cultivation and metagenomics methodologies should be employed synergistically in limestone biodeterioration studies in order to tackle inherent limitations regarding both techniques.
Fungi with dark-coloured cells and mycelia—also known as black fungi—form a ubiquitous fraction of microbial communities on rock surfaces and often occur on rock-inhabiting lichens as well. The diversity and lifestyles of these fungi are still insufficiently known. In this chapter we review the current state of knowledge about the diversity and biology of these fungi focusing on rock and lichen habitats. The production of melanin pigments in cell walls shields black fungi effectively against radiation stress. High osmotic tolerance to withstand a wide range of environmental challenges, such as drought, temperature and pH extremes, is mediated by osmoprotective small compounds (osmolytes). Most of the black fungi demonstrate a high degree of phenotypic plasticity and heterogeneity, which we regard as important to adapt and thrive in a wide range of ecological conditions, including extremes of temperatures and radiation. First genomic data from black fungi provide insights in the variation of gene families that are potentially involved in stress tolerance, but reconciliation with transcriptomic data is required to elucidate the gene functions. Both culture-dependent and culture-independent approaches demonstrate that black fungi are frequently associated with lichens. To better understand their association with the algal partners of lichen symbioses, we established co-culture experiments. These showed that some of the black fungi have a transient capacity to optionally develop lichen-like associations with algae (or cyanobacteria), which can also be observed in natural and urban surfaces of rocks and concrete, respectively. This chapter indicates that extremotolerant fungi creatively use a wide range of survival strategies to adapt to stressful environments.
Stone surfaces are extreme environments that support microbial life. This microbial growth occurs despite unfavorable conditions associated with stone including limited sources of nutrients and water, high pH, and exposure to extreme variations in temperature, humidity and irradiation. These stone‐dwelling microbes are often resistant to extreme environments including exposure to desiccation,heavy metals, UV, and Gamma irradiation. Here, we report on the effects of climate and stone geochemistry on microbiomes of Roman stone ruins in North Africa. Stone microbiomes were dominated by Actinobacteria, Cyanobacteria, and Proteobacteria but were heavily impacted by climate variables that influenced water availability. Stone geochemistry also influenced community diversity, particularly through biologically available P, Mn, and Zn. Functions associated with photosynthesis and UV protection were enriched in the metagenomes, indicating the significance of these functions for community survival on stones. Core members of the stone microbial communities were also identified and included Geodermatophilaceae, Rubrobacter, Sphingomonas, and others. Our research has helped to expand the understanding of stone microbial community structure and functional capacity within the context of varying climates, geochemical properties, and stone conditions.
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In 2013, UNESCO classified the ‘University of Coimbra – Alta and Sofia’ (Portugal) as World Heritage site. Some of the limestone monuments that compose the site, such as the Old Cathedral of Coimbra, show clear visual effects of biological colonization by cyanobacteria and microalgae. The aim of this study was to thoroughly characterize the structural diversity of the photoautotrophic community at this site, combining isolation and molecular identification with next-generation sequencing techniques. Culture-dependent approaches resulted on the identification of green microalgae belonging to Prasiolales, Chlorellales, Watanabea, Chlamydomonadales and Sphaeropleales clades, and cyanobacteria belonging to Nostocales and Synechococcales clades. Culture-independent methods using Illumina Platform resulted in the identification of 610 Operational Taxonomic Units (OTUs), revealing the presence of populations within phyla Chlorophyta, Cyanobacteria, Rhodophyta and Stramenopiles/Bacillariophyta. In addition, principal component analysis (PCA) and a network analysis were generated and successfully related the different populations to specific biodeterioration scenarios observed at this monument. The use of culture-dependent and -independent techniques together with PCA and network analysis allowed us to understand the distribution patterns of the photoautotrophic community among the different biofilms observed at this monument, as well as their ecological specifications. This study highlights that both cultivation and metagenomics methodologies should be employed synergistically in similar biodeterioration studies.
The present study analyzed the dark patina present on the surface of a marble statue exposed in the Gardens of the Quirinal Palace in Rome, with a special focus on euendolithic black meristematic fungi. The study of the spatial distribution of microorganisms and their identification were performed by using a multistep approach based on microscopy, cultural analyses, molecular techniques, and embedding resin-casting technique. Black meristematic fungi were observed in the patina. Since morphological features are not sufficient to identify fungi belonging to this group, cultural and molecular analyses were performed. The results highlighted the presence of the species Coniosporium apollinis and of strains related to the genus Knufia (order of Chaetothyriales). The resin-casting embedding technique demonstrated the active penetration of these fungi into the marble grains, thus documenting their true endolithic behavior. The involvement of phototrophic microorganisms present on the surface in the active penetration of the marble was excluded.
On the stone cultural heritage, the influence of architecture-related microenvironmental features on lichen diversity, abundance and consequent threats for conservation has been still poorly characterized to support management plans. Such relationships were here investigated on the vertical surfaces of the House of the Ancient
Hunt in Pompeii, archaeological site in S-Italy where the variability of lichen saxicolous communities has been still completely neglected despite their widespread occurrence. Lichen colonization in semiconfined rooms was sporadic and limited to Dirina massiliensis, while a remarkable turnover of six communities, encompassing 22
species, characterized mortar, painted and plastered surfaces in outdoor environments, with local covers up to 80%. Microscopic and spectroscopic analyses displayed the deteriogenic potential of three dominant species, due to hyphal penetration within paint and plaster layers (Verrucaria macrostoma) and the release of oxalic acid and/
or secondary metabolites with acidic and chelating functions (D. massiliensis, Lepraria lobificans). A higher vertical distance of surfaces from the ground and a larger room dimension were the main conditional factors related to a higher lichen abundance and the distribution of the different communities. Such knowledge on architecture-related microenvironmental features driving lichen distribution and biodeterioration threats may contribute to address restoration priorities and conservation strategies.
Benzalkonium chloride (BAC) is mostly bactericidal at 1% and yeasticidal at 0.2% (5 min). A mycobactericidal activity cannot be expected. Epidemiological cut-off values to determine acquired resistance have been proposed for Salmonella spp. (128 mg/l), E. coli (64 mg/l), K. pneumoniae and Enterobacter spp. (32 mg/l), S. aureus and C. albicans (16 mg/l), and E. faecalis and E. faecium (8 mg/l). Elevated MIC values suggestive of BAC resistance have been reported among numerous species including A. hydrophila (≤31,300 mg/l), B. cereus and E. meningoseptica (≤7,800 mg/l), P. aeruginosa (≤5,000 mg/l) and L. monocytogenes (≤625 mg/l). Specific resistance mechanisms are often known, e.g. resistance genes, efflux pumps, membrane changes or plasmids. Cross-tolerance to chlorhexidine, triclosan, hexachlorophene and selected antibiotics can occur in numerous species. Low-level exposure leads to no MIC change in 19 species, a weak MIC change in 25 species and a strong MIC change in 31 species (14 of them being stable) resulting in MIC values as high as 3,000 mg/l (S. Typhimurium) or 2,500 mg/l (P. aeruginosa). Bacterial biofilm formation is rather inhibited than enhanced by BAC. Biofilm removal by BAC is poor.
Stone, one of the earliest testimonies of human artistic expression, is susceptible to biodeterioration by microorganisms. The most frequent stone colonizing agents are algae, cyanobacteria, bacteria, fungi and lichens, each with their own set of adaptive traits, which allow them to prosper and consequently damage the stone substrate. Limestone is particularly susceptible to biological agents; therefore, in order to act towards the protection and prevention of colonization by microorganisms, it is crucial to understand the microbial communities thriving in limestone heritage buildings. Data regarding the biodiversity and biological activity in Portuguese limestone monuments is, however, still scarce and the scattered knowledge on the subject impairs a full comprehension of the complex and relevant phenomena associated with this particular setting. This review presents and discusses the available studies performed in Portuguese limestone. In addition, the state of the art methodologies to be used, as well as the future studies to be considered, in order to effectively protect such invaluable witnesses of our history, are discussed.
This study evaluated whether exposing samples of granite colonized by a natural biofilm to artificial daylight or UV-A/B/C irradiation for 48 h enhanced removal of the biofilm with a chemical product previously approved for conservation of monuments by the European Biocide Directive. Rodas granite, which is commonly found in stone-built heritage monuments in Galicia (NW Spain), was naturally colonized by a sub-aerial biofilm. The efficacy of the cleaning method was evaluated relative to uncolonized surfaces and colonized control samples without previous irradiation, treated by dry-brushing or with benzalkonium chloride. The effect of UV irradiation in the combined treatment was evident, as comparable cleaning levels were not reached in the controls. Although the biofilm was not totally removed by any of the treatments, UV-B irradiation followed by benzalkonium chloride was potentially useful for cleaning stone, with results comparable to those achieved by UV-C irradiation, which is known to have germicidal effects.
There has been an increasing demand for fresh fruit and vegetables in recent years. Along the processing line in fresh-cut vegetable production, disinfection is one of the most important processing steps affecting the quality and safety, and the shelf-life of the end produce. Although a range of antimicrobial compounds commonly termed biocides or disinfectants are available, chlorine has long been used to disinfect washing waters of fresh-cut vegetables. However, since chlorine reactions with organic matter lead to the production of by-products, alternative disinfectants to chlorine must be evaluated. A synthetic washing water formula has been developed to determine the antimicrobial efficiency of different families of potential disinfectants: quaternary ammonium compounds (QACs) as benzalkonium chloride (BZK), and didecyldimethylammonium chloride (DDAC); isothiazolinones (mixture of methylchloroisothiazolinone and methylisothiazolinone, CMIT:MIT 3:1 and 1:1); and essential oils (carvacrol, CAR). The twin configuration and higher length of the chains of alkyl groups of DDAC compared to BZK have led to a higher antimicrobial efficiency. In both cases, Gram-positive bacteria seemed to be much more sensitive to the QAC attack than Gram-negative. The opposite happened for CMIT:MIT. The chloro-substituted isothiazolinone (CMIT) has been proven to be much more effective than its unsubstituted form (MIT). In addition, in contrast to chlorine, its antimicrobial activity together with that of DDAC was not decreased when increasing the organic matter content of the water. Synergetic antimicrobial effects have been confirmed when combining BZK and CAR. MBC values were determined in SWW, during 90 s of contact time and Salmonella concentration of 103 CFU/mL, corresponding to: 100 (BZK), 30 (DDAC), 50 (CMIT:MIT 3:1), 100 (CMIT:MIT 1:1), 300 (CAR), 75 (BZK)-200 (CAR), and 9 (free chlorine) mg/L. MBC values for inactivating similar concentration of E. faecalis corresponded to: 50 (BZK), and 10 (DDAC) mg/L. Increasing contact times up to 5 min did not lead to higher antimicrobial efficiencies. CMIT:MIT 3:1 together with DDAC, and combinations of BZK-CAR seem to be a plausible alternative to chlorine.
The archaeological site of Herculaneum (Campania, Italy), which was buried as a result of the eruption of Mt. Vesuvius in the first Century C.E., was first excavated in the XVIII century. It has been included on the UNESCO World Heritage List since 1997, and in 2001 the Herculaneum Conservation Project (HCP) was started, which has carried out, among other activities, studies and conservation interventions across the site. Up until this time, little data has been available on the growth of biological agents that could cause biodeterioration of wall paintings and archaeological structures. Particularly, the presence of rosy discoloration is frequent on ancient monuments of the Vesuvian area, even if such phenomenon has so far been largely neglected. In this study, we describe, for the first time, the pink patina distribution and the microbial species isolated from the House of the Bicentenary in Herculaneum. By combining culture-based approaches with molecular and phylogenetic analyses we reliably isolated the pink-producing bacterial species and attributed them primarily to Arthrobacter agilis, and secondarily to Rhodococcus corynebacterioides, and Methylobacterium extorquens. Strains closely related to Dietzia maris and Gordonia rubripertincta were also isolated. With the exception of M. extorquens, a proteobacterium, all of the other isolates belong to the phylum Actinobacteria. All isolates produced carotenoid pigments, suggesting that they can participate in the development of such peculiar coloration. Our data indicate the presence of a large number of pink-pigmented bacterial species in the community, even if the presence of bacteria in viable but not-culturable state, such as Rubrobacter radiotolerans, is not excludible. The culture-based approach had the advantage of (i) obtaining bacterial isolates, (ii) showing their differential ability to produce pink discolorations, and (iii) testing conditions for in vitro growth. Moreover, field observations showed an association of pink patinas with dry conditions, saline efflorescence and moderate solar radiation. Some seasonal variations were also detected, with an increase in late spring and summer.
Ecologists have long studied primary succession, the changes that occur in biological communities after initial colonization of an environment. Most of this work has focused on succession in plant communities, laying the conceptual foundation for much of what we currently know about community assembly patterns over time. Because of their prevalence and importance in ecosystems, an increasing number of studies have focused on microbial community dynamics during succession. Here, we conducted a meta-analysis of bacterial primary succession patterns across a range of distinct habitats, including the infant gut, plant surfaces, soil chronosequences, and aquatic environments, to determine whether consistent changes in bacterial diversity, community composition, and functional traits are evident over the course of succession. Although these distinct
habitats harbor unique bacterial communities, we were able to identify patterns in community assembly that were shared across habitat types. We found an increase in taxonomic and functional diversity with time while the taxonomic composition and functional profiles of communities became less variable (lower beta diversity) in late successional stages. In addition, we found consistent decreases in the rRNA operon copy number and in the high-efficient phosphate assimilation process (Pst system) suggesting that reductions in resource availability during succession select for taxa adapted to low-resource conditions. Together, these results highlight that, like many plant communities, microbial communities also exhibit predictable patterns during primary succession.