Importance of subaerial biofilms and airborne microflora in the deterioration of stonework: a molecular study

The study characterized the sessile microbial communities on mortar and stone in Milan University's Richini's Courtyard and investigated the relationship between airborne and surface-associated microbial communities. Active colonization was found in three locations: green and black patinas were present on mortar and black spots on stone. Confocal laser scanning microscopy, scanning electron microscopy and culture-independent molecular methods revealed that the biofilm causing deterioration was dominated by green algae and black fungi. The mortar used for restoration contained acrylic and siloxane resins that could be used by microorganisms as carbon and energy sources thereby causing proliferation of the biofilm. Epifluorescence microscopy and culture-based methods highlighted a variety of airborne microflora. Bacterial and fungal counts were quantitatively similar to those reported in other investigations of urban areas, the exception being fungi during summer (1–2 orders of magnitude higher). For the first time in the cultural heritage field, culture-independent molecular methods were used to resolve the structure of airborne communities near discoloured surfaces, and to investigate the relationship between such communities and surface-associated biofilms.     

Characterizing the Microbial Colonization of a Dolostone Quarry: Implications for Stone Biodeterioration and Response to Biocide Treatments

This study examines the microbial colonization of three fronts of an abandoned dolostone quarry (Redueña, Madrid, Spain) exposed to atmospheric conditions for different time periods since Roman times to the present. Through scanning electron microscopy in backscattered electron mode (SEM-BSE), endolithic colonization was predominantly detected in the most recently exposed front, while in the longer exposed quarry fronts, epilithic forms of growth were most often observed. These observations were confirmed by denaturing gradient gel electrophoresis (DGGE) analysis. Based on the distribution pattern of microbial colonization in the different quarry fronts, we then established a sequence of colonization events that took place over this long time frame. Bioalteration processes related to this sequential colonization were also identified. Characterizing these sequential processes can be useful for interpreting biodeterioration processes in historic dolostone monuments, especially those affecting constructions in the area of the Redueña stone quarry. In a second experimental stage, different biocide treatments were tested on this quarry rock to find the best way to avoid the microbial colonization effects identified. Through combined SEM-BSE/DGGE analysis, the efficacy of several biocides against the microorganisms inhabiting the dolostones was assessed after 4 and 16 months treatment. In general, all treatments were effective at reducing around 80% of the lichen cover, although effects on endolithic lithobiontic communities were dependant on how well the rock surface had been mechanically cleaned prior to treatment and gradually disappeared over time.     

Non-random processes determine the colonization of groundwater sediments by microbial communities in a pristine porous aquifer

Sediments accommodate the dominating share of groundwater microbiomes, however the processes that govern the assembly and succession of sediment-attached microbial communities in groundwater aquifers are not well understood. To elucidate these processes, we followed the microbial colonization of sterile sediments in in situ microcosms that were exposed to groundwater for almost 1 year at two distant but hydrologically connected sites of a pristine, shallow, porous aquifer. Our results revealed intriguing similarities between the community succession on the newly-colonized sediments and succession patterns previously observed for biofilms in other more dynamic aquatic environments, indicating that the assembly of microbial communities on surfaces may be governed by similar underlying mechanisms across a wide range of different habitats. Null model simulations on spatiotemporally resolved 16S rRNA amplicon sequencing data further indicated selection of specific OTUs rather than random colonization as the main driver of community assembly. A small fraction of persistent OTUs that had established on the sediments during the first 115 days dominated the final communities (68%–85%), suggesting a key role of these early-colonizing organisms, in particular specific genera within the Comamonadaceae and Oxalobacteraceae, for community assembly and succession during the colonization of the sediments. Overall, our study suggests that differences between planktonic and sediment-attached communities often reported for groundwater environments are not the result of purely stochastic events, but that sediment surfaces select for specific groups of microorganisms that assemble over time in a reproducible, non-random way.     

Fundamental drivers for endolithic microbial community assemblies in the hyperarid Atacama Desert

In hyperarid deserts, endolithic microbial communities colonize the rocks’ interior as a survival strategy. Yet, the composition of these communities and the drivers promoting their assembly are still poorly understood. We analysed the diversity and community composition of endoliths from four different lithic substrates – calcite, gypsum, ignimbrite and granite – collected in the hyperarid zone of the Atacama Desert, Chile. By combining microscopy, mineralogy, spectroscopy and high throughput sequencing, we found these communities to be highly specific to their lithic substrate, although they were all dominated by the same four main phyla, Cyanobacteria, Actinobacteria, Chloroflexi and Proteobacteria. Our finding indicates a fine scale diversification of the microbial reservoir driven by substrate properties. The data suggest that the overall rock chemistry and the light transmission properties of the substrates are not essential drivers of community structure and composition. Instead, we propose that the architecture of the rock, i.e., the space available for colonization and its physical structure, linked to water retention capabilities, is ultimately the driver of community diversity and composition at the dry limit of life.     

Mapping the lithic colonization at the boundaries of life in Northern Victoria Land,Antarctica

The endolithic microbial communities of the Antarctic represent a borderline lifestyle in the most hostile ice-free areas of the continent. The extreme adaptation of microbes in these communities renders them very sensitive to environmental changes. To date, the actual distribution of these communities has never been investigated; yet, this information would define the geographic limits for life at present and supply a useful tool for monitoring any possible future variation related to climate change. In this study, most of the outcrops of Northern and of one site in Southern Victoria Land were recorded by altitudinal and sea distance gradients. The presence of endolithic life was determined by in situ observation, by microscopic observation of rock fragments in the laboratory, and, for doubtful samples, by culture experiment. Colonizers were present in more than 87 % of the visited sites. The presence of lithic life in Victoria Land appears to be wider than that reported 14 years earlier. The colonization trend follows climatic variation, with epiliths prevailing in coastal sites and decreasing towards the interior, while chasmoendoliths and cryptoendoliths increase and become predominant from the coast towards the inland sites. Typical cryptoendolithic colonization was exclusive on porous rocks as sandstone, chasmoendolithic colonization occurred even in less porous but translucent rocks as granite and quartz. Multivariate analysis of the combined results clearly indicates the pivotal role of the rock type in the colonization of endolithic micro-organisms; sandstone allows lithobionts to push themselves towards areas characterized by harsher conditions.     

Biofilm formation on the surface of monazite and xenotime during bioleaching

Microbial attachment and biofilm formation is a ubiquitous behaviour of microorganisms and is the most crucial prerequisite of contact bioleaching. Monazite and xenotime are two commercially exploitable minerals containing rare earth elements (REEs). Bioleaching using phosphate solubilizing microorganisms is a green biotechnological approach for the extraction of REEs. In this study, microbial attachment and biofilm formation of Klebsiella aerogenes ATCC 13048 on the surface of these minerals were investigated using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). In a batch culture system, K. aerogenes was able to attach and form biofilms on the surface of three phosphate minerals. The microscopy records showed three distinctive stages of biofilm development for K. aerogenes commencing with initial attachment to the surface occurring in the first minutes of microbial inoculation. This was followed by colonization of the surface and formation of a mature biofilm as the second distinguishable stage, with progression to dispersion as the final stage. The biofilm had a thin-layer structure. The colonization and biofilm formation were localized toward physical surface imperfections such as cracks, pits, grooves and dents. In comparison to monazite and xenotime crystals, a higher proportion of the surface of the high-grade monazite ore was covered by biofilm which could be due to its higher surface roughness. No selective attachment or colonization toward specific mineralogy or chemical composition of the minerals was detected. Finally, in contrast to abiotic leaching of control samples, microbial activity resulted in extensive microbial erosion on the high-grade monazite ore.     

Rock geochemistry induces stress and starvation responses in the bacterial proteome

Interactions between microorganisms and rocks play an important role in Earth system processes. However, little is known about the molecular capabilities microorganisms require to live in rocky environments. Using a quantitative label‐free proteomics approach, we show that a model bacterium (Cupriavidus metallidurans CH34) can use volcanic rock to satisfy some elemental requirements, resulting in increased rates of cell division in both magnesium‐ and iron‐limited media. However, the rocks also introduced multiple new stresses via chemical changes associated with pH, elemental leaching and surface adsorption of nutrients that were reflected in the proteome. For example, the loss of bioavailable phosphorus was observed and resulted in the upregulation of diverse phosphate limitation proteins, which facilitate increase phosphate uptake and scavenging within the cell. Our results revealed that despite the provision of essential elements, rock chemistry drives complex metabolic reorganization within rock‐dwelling organisms, requiring tight regulation of cellular processes at the protein level. This study advances our ability to identify key microbial responses that enable life to persist in rock environments.     

Microcolonial fungi from antique marbles in Perge/Side/Termessos (Antalya/Turkey)

In this study rock surfaces of archaeological sites in Antalya were investigated with a focus on black fungi for the first time. Black, meristematic fungi were isolated from surfaces of antique marble monuments in Antalya (Side, Perge, Termessos). Their morphology was characterized, their diversity was documented and the taxonomy and phylogeny of new isolates was clarified based on molecular methods, that is, by sequencing parts of the small ribosomal subunit (18S) and internal transcribed spacer (ITS) regions. From a total of around 250 samples 99 different fungal strains were isolated. In most of 99 strains the rDNA sequencing data and the lack of homologies in “Genbank” gave strong evidence that these strains have to be described as new species/subspecies and/or genera. All of them, however, clustered within the ascomycete orders of Dothideales, Chaetothyriales, and Pleosporales. Field studies show that these organisms cause color changes, black spots, crater shaped lesions as well as chipping and exfoliation of the rock surfaces in Antalya and thus they have to be regarded as a serious threat to the antique cultural heritage in Turkey.     

Deep Crustal Communities of the Juan de Fuca Ridge Are Governed by Mineralogy

Volcanic ocean crust contains a global chemosynthetic microbial ecosystem that impacts ocean productivity, seawater chemistry and geochemical cycling. We examined the mineralogical effect on community structure in the aquifer ecosystem by using a four-year in situ colonization experiment with igneous minerals and glasses in Integrated Ocean Drilling Program Hole 1301A on the Juan de Fuca Ridge. Microbial community analysis and scanning electron microscopy revealed that olivine phases and iron-bearing minerals bore communities that were distinct from iron-poor phases. Communities were dominated by Archaeoglobaceae, Clostridia, Thermosipho, Desulforudis and OP1 lineages. Our results suggest that mineralogy determines microbial composition in the subseafloor aquifer ecosystem.     

Subaerial biofilms on granitic historic buildings: microbial diversity and development of phototrophic multi-species cultures

Microbial communities of natural subaerial biofilms developed on granitic historic buildings of a World Heritage Site (Santiago de Compostela, NW Spain) were characterized and cultured in liquid BG11 medium. Environmental barcoding through next-generation sequencing (Pacific Biosciences) revealed that the biofilms were mainly composed of species of Chlorophyta (green algae) and Ascomycota (fungi) commonly associated with rock substrata. Richness and diversity were higher for the fungal than for the algal assemblages and fungi showed higher heterogeneity among samples. Cultures derived from natural biofilms showed the establishment of stable microbial communities mainly composed of Chlorophyta and Cyanobacteria. Although most taxa found in these cultures were not common in the original biofilms, they are likely common pioneer colonizers of building stone surfaces, including granite. Stable phototrophic multi-species cultures of known microbial diversity were thus obtained and their reliability to emulate natural colonization on granite should be confirmed in further experiments.     

文化遗产微生物研究方法进展

文化遗产的生物退化与生物降解问题普遍存在。从方法学的角度回顾了针对不同类型文化遗产微生物病害与防治研究中常用的方法和技术,总结了国内外当前所取得的主要研究进展,提出了该领域研究中未来面临的问题和挑战,并展望了应对策略和今后发展趋势。文化遗产微生物研究方法经历了传统培养、显微镜分析、分子生物学技术、酶活性和代谢产物分析以及快速发展的多组学及生物信息学分析等多个阶段,在微生物多样性、群落特征、生理生态学及其退化机理方面的认识已逐渐深入,以文化遗产保护为目标的病害微生物防治技术不断发展。近年来通过新一代测序和宏基因组学技术获得了文化遗产微生物的诸多重要信息,但其作用机理和影响因素的研究还不够。本综述突出了结合传统与现代方法技术开展研究的重要性,旨在为文化遗产微生物学领域研究提供参考和借鉴。     

Bacterial Communities Involved in Soil Formation and Plant Establishment Triggered by Pyrite Bioweathering on Arctic Moraines

In arctic glacier moraines, bioweathering primed by microbial iron oxidizers creates fertility gradients that accelerate soil development and plant establishment. With the aim of investigating the change of bacterial diversity in a pyrite-weathered gradient, we analyzed the composition of the bacterial communities involved in the process by sequencing 16S rRNA gene libraries from different biological soil crusts (BSC). Bacterial communities in three BSC of different morphology, located within 1 m distance downstream a pyritic conglomerate rock, were significantly diverse. The glacier moraine surrounding the weathered site showed wide phylogenetic diversity and high evenness with 15 represented bacterial classes, dominated by Alphaproteobacteria and pioneer Cyanobacteria colonizers. The bioweathered area showed the lowest diversity indexes and only nine bacterial families, largely dominated by Acidobacteriaceae and Acetobacteraceae typical of acidic environments, in accordance with the low pH of the BSC. In the weathered BSC, iron-oxidizing bacteria were cultivated, with counts decreasing along with the increase of distance from the rock, and nutrient release from the rock was revealed by environmental scanning electron microscopy-energy dispersive X-ray analyses. The vegetated area showed the presence of Actinomycetales, Verrucomicrobiales, Gemmatimonadales, Burkholderiales, and Rhizobiales, denoting a bacterial community typical of developed soils and indicating that the lithoid substrate of the bare moraine was here subjected to an accelerated colonization, driven by iron-oxidizing activity.     

Rock-degrading endophytic bacteria in cacti

A plant–bacterium association of the cardon cactus (Pachycereus pringlei) and endophytic bacteria promotes establishment of seedlings and growth on igneous rocks without soil. These bacteria weather several rock types and minerals, unbind significant amounts of useful minerals for plants from the rocks, fix in vitro N₂, produce volatile and non-volatile organic acids, and reduce rock particle size to form mineral soil. This study revealed the presence of large populations of culturable endophytic bacteria inside the seeds extracted from wild plants, from seeds extracted from the guano of bats feeding on cactus fruit, in seedlings growing from these seeds, in the pulp of fruit, and in small, mature wild plants, and are comparable in size to populations of endophytic populations in some agricultural crops. The dominant culturable endophytes were isolates of the genera Bacillus spp., Klebsiella spp., Staphylococcus spp., and Pseudomonas spp. Based on partial sequencing of the 16s rRNA gene, the isolated strains had low similarity to known strains in these genera. However, these strains have higher molecular similarity among endophytes obtained from seeds, endophytes from roots, and some bacterial strains from the rhizoplane. Seedlings developed from seeds with endophytes contain the similar species of endophytes in their shoots, possibly derived from the seeds. This study shows the involvement of endophytic bacteria in rock weathering by cacti in a hot, subtropical desert and their possible contribution to primary colonization of barren rock. This study proposes that cacti capable of acquiring diverse populations of endophytes may give them an evolutionary advantage to gain a foothold on highly uncompromising terrain.     

Microbial populations and activities in the rhizoplane of rock-weathering desert plants. I. Root colonization and weathering of igneous rocks

Dense layers of bacteria and fungi in the rhizoplane of three species of cactus (Pachycereus pringlei, Stenocereus thurberi, Opuntia cholla) and a wild fig tree (Ficus palmeri) growing in rocks devoid of soil were revealed by bright-field and fluorescence microscopy and field emission scanning electron microscopy. These desert plants are responsible for rock weathering in an ancient lava flow at La Purisima-San Isidro and in sedimentary rock in the Sierra de La Paz, both in Baja California Sur, Mexico. The dominant bacterial groups colonizing the rhizoplane were fluorescent pseudomonads and bacilli. Seven of these bacterial species were identified by the 16S rRNA molecular method. Unidentified fungal and actimomycete species were also present. Some of the root-colonizing microorganisms fixed in vitro N(2), produced volatile and non-volatile organic acids that subsequently reduced the pH of the rock medium in which the bacteria grew, and significantly dissolved insoluble phosphates, extrusive igneous rock, marble, and limestone. The bacteria were able to release significant amounts of useful minerals, such as P, K, Mg, Mn, Fe, Cu, and Zn from the rocks and were thermo-tolerant, halo-tolerant, and drought-tolerant. The microbial community survived in the rhizoplane of cacti during the annual 10-month dry season. This study indicates that rhizoplane bacteria on cacti roots in rock may be involved in chemical weathering in hot, subtropical deserts.     

Primary Colonization of Volcanic Rocks by Plants in Arid Baja California, Mexico

Abstract: In an arid region of Baja California Sur, Mexico, field observations, combined with chemical and physical analyses, mineral analysis and scanning electron microscopy of unweathered and weathered volcanic rocks, revealed the presence of rock-colonizing plants (most are tree-shaped cacti, possibly rock weathering), growing in volcanic rocks without benefit of soil. Many are at the seedling stage. At least four cactus species ( Pachycereus pringlei [S. Wats] Britt. and Ross, Stenocereus thurberi [Engelm.] Buxb. subsp. thurberi, Mamillaria fraileana [Britt. and Rose] Boed., Opuntia cholla F. A. C. Weber), and one tree (wild fig, Ficus palmeri [S. Wats]) were capable of cracking, growing in and colonizing cliffs and rocks formed from ancient lava flows and, consequently, forming soil for succession by other plant species. This study shows that plant colonization of volcanic rocks may assist soil formation, which eventually leads to accumulation of soil, water and nutrients in a desert terrestrial ecosystem that otherwise lacks these essential plant-growth variables.     

Using Artificial Rocks to Restore Nonrenewable Shelter Sites in Human‐Degraded Systems: Colonization by Fauna

Many animals spend much of their time within retreat sites underneath loose surface rocks and may be highly selective in terms of the physical characteristics of the sites that they use. If such shelters are eliminated by anthropogenic activities, such as rock removal for landscaping, the only way to restore these nonrenewable habitats may be to replace them with artificial rocks. To construct artificial rock habitats, we need to understand which rock attributes are important for faunal use and develop methods to mimic these important natural retreat site characteristics. Based on our prior understanding of rocky retreat sites used by reptiles in sandstone outcrops of southeastern Australia, we constructed realistic‐looking artificial rocks from fiber‐reinforced cement and evaluated (1) the degree to which they mimicked natural retreat sites in both thermal regime and three‐dimensional crevice structure and (2) their colonization by fauna after deployment in the field. Our results demonstrate that thermal regimes and crevice structures beneath the artificial rocks were similar to those beneath natural rocks. In addition, 100% of the artificial rocks were colonized (by 45 invertebrate, six lizard, and two snake species, including the endangered Broad‐headed snake, Hoplocephalus bungaroides) after only 40 weeks. Together, these results suggest that restoring degraded habitats for rock‐dwelling species is feasible and can provide a rapid means of enhancing shelter‐site availability for such species.     

Towards the human colorectal cancer microbiome

Multiple factors drive the progression from healthy mucosa towards sporadic colorectal carcinomas and accumulating evidence associates intestinal bacteria with disease initiation and progression. Therefore, the aim of this study was to provide a first high-resolution map of colonic dysbiosis that is associated with human colorectal cancer (CRC). To this purpose, the microbiomes colonizing colon tumor tissue and adjacent non-malignant mucosa were compared by deep rRNA sequencing. The results revealed striking differences in microbial colonization patterns between these two sites. Although inter-individual colonization in CRC patients was variable, tumors consistently formed a niche for Coriobacteria and other proposed probiotic bacterial species, while potentially pathogenic Enterobacteria were underrepresented in tumor tissue. As the intestinal microbiota is generally stable during adult life, these findings suggest that CRC-associated physiological and metabolic changes recruit tumor-foraging commensal-like bacteria. These microbes thus have an apparent competitive advantage in the tumor microenvironment and thereby seem to replace pathogenic bacteria that may be implicated in CRC etiology. This first glimpse of the CRC microbiome provides an important step towards full understanding of the dynamic interplay between intestinal microbial ecology and sporadic CRC, which may provide important leads towards novel microbiome-related diagnostic tools and therapeutic interventions.     

Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle.

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.