Microbiomes of Biofilms on Decorative Siliceous Stone: Drawbacks and Advantages of Next Generation Sequencing.

Next Generation Sequencing (NGS), using the Illumina® metabarcoding system, showed differences between biofilm communities on three degraded siliceous stone church façades in central Rio de Janeiro. Two church biofilms (on granite and augen gneiss) were dominated by Actinobacteria; the third (granite), surrounded by trees and further from intense vehicular traffic, by Gammaproteobacteria. Yeast-like forms of Basidiomycetes and Ascomycetes were major fungi on all facades, but 22.8% of Operational Taxonomic Units could not be assigned to any fungal taxon after DNA amplification with ITS primers and analysis with the UNITE database, indicating the need for more fungal NGS studies. The pipeline used in analysis of the V4 region of rRNA bacterial gene sequences influenced the taxa detected, with two major classes and many genera identified only by the pipeline using the Greengenes, and not the Silva, database. Principal Components Analysis separated façade biofilms into the appropriate three groups and indicated greater dissimilarity of the tree-surrounded church biofilm from the others, confirmed by Jaccard Similarity coefficients, suggesting that local environment influences community composition more than stone type. NGS allows rapid and detailed analysis of microbiomes, but results must be carefully assessed and must not be used as the sole indication of community composition.

Epilithic and endolithic microorganisms and deterioration on stone church facades subject to urban pollution in a sub-tropical climate.

Weathering of two church facades in Rio de Janeiro was caused substantially by salts, mainly halite and gypsum, detected by SEM and chemical analyses, which cause physical stresses by deposition within the rock. Biofilm populations, determined by SEM and as operational taxonomic units (OTUs), degraded stone by penetration, solubilization and redeposition of minerals on their surfaces. Endolithic cyanobacteria were associated with gypsum deposits. Microbiomes were typical for high-stress environments, high salt, intense insolation, low water and low nutrients (eg halophilic Rubrobacter, Salinicola, Sterigmatomyces). The main colonizers on the church most affected by traffic (Nossa Senhora da Candelária - CA) were Actinobacteria; Gammaproteobacteria (chiefly Pseudomonas) were predominant on the site situated in a leafy square (São Francisco de Paula - SF). Major Gammaproteobacteria on CA were halophilic Halomonas and Rhodobacteriaceae. Fungal OTUs on both churches were principally dimorphic, yeast-like basidiomycetes. Many OTUs of thermophilic microorganisms (eg the Thermomicrobia class, Chloroflexi) were present. This is the first use of next generation sequencing (NGS) to study microbial biofilm interactions with metamorphic and granite buildings in an intensely urban, sub-tropical climate.

16S rRNA gene profiling of planktonic and biofilm microbial populations in the Gulf of Guinea using Illumina NGS.

16S rRNA gene profiling using a pipeline involving the Greengenes database revealed that bacterial populations in innermost (proximal to the steel surface) and outer regions of biofilms on carbon steel exposed 3 m below the surface at an offshore site in the Gulf of Guinea differed from one another and from seawater. There was a preponderance of gammaproteobacterial sequences, representing organisms known for hydrocarbon degradation. Total DNA from the innermost layer was 1500 times that recovered from the outermost. Stramenopiles (diatom) sequences were prevalent in the former. Rhodobacteriaceae, key biofilm formers, comprised 14.9% and 4.22% OTUs of inner and outer layers, respectively. Photosynthetic anaerobic sulfur oxidizer sequences were also prominent in the biofilms. Analysis of data using a different pipeline with Silva111 allowed detection of 0.3-0.4% SRB in the biofilms. The high abundance of aerobic micro-algal sequences in inner biofilm suggests they are initial colonizers of carbon steel surfaces in a marine environment. This is the first time that the microbial population of the strongly attached inner layer of the biofilm on steel has been differentiated from the outer, readily removed layer. The accepted scraping removal method is obviously inadequate and the resulting microbial analysis does not offer complete information on the biofilm community structure.

Metabolomic and high-throughput sequencing analysis-modern approach for the assessment of biodeterioration of materials from historic buildings.

Preservation of cultural heritage is of paramount importance worldwide. Microbial colonization of construction materials, such as wood, brick, mortar, and stone in historic buildings can lead to severe deterioration. The aim of the present study was to give modern insight into the phylogenetic diversity and activated metabolic pathways of microbial communities colonized historic objects located in the former Auschwitz II-Birkenau concentration and extermination camp in Oswiecim, Poland. For this purpose we combined molecular, microscopic and chemical methods. Selected specimens were examined using Field Emission Scanning Electron Microscopy (FESEM), metabolomic analysis and high-throughput Illumina sequencing. FESEM imaging revealed the presence of complex microbial communities comprising diatoms, fungi and bacteria, mainly cyanobacteria and actinobacteria, on sample surfaces. Microbial diversity of brick specimens appeared higher than that of the wood and was dominated by algae and cyanobacteria, while wood was mainly colonized by fungi. DNA sequences documented the presence of 15 bacterial phyla representing 99 genera including Halomonas, Halorhodospira, Salinisphaera, Salinibacterium, Rubrobacter, Streptomyces, Arthrobacter and nine fungal classes represented by 113 genera including Cladosporium, Acremonium, Alternaria, Engyodontium, Penicillium, Rhizopus, and Aureobasidium. Most of the identified sequences were characteristic of organisms implicated in deterioration of wood and brick. Metabolomic data indicated the activation of numerous metabolic pathways, including those regulating the production of primary and secondary metabolites, for example, metabolites associated with the production of antibiotics, organic acids and deterioration of organic compounds. The study demonstrated that a combination of electron microscopy imaging with metabolomic and genomic techniques allows to link the phylogenetic information and metabolic profiles of microbial communities and to shed new light on biodeterioration processes.

Sharp transition in the immunoimmobilization of E. coli O157:H7.

This work focuses on immobilization of living enterohemorrhagic Escherichia coli O157:H7 on a gold surface as a function of the concentration of antibody tethered to the surface in the physiological environment of the organisms. Experiments are conducted using antibodies raised against bacterial surface lipopolysaccharides (LPS) tethered to gold-coated silicon wafers at surface concentrations spanning a range from submonolayers of antibodies to full coverage, an estimated 1 antibody per ∼100 nm(2). A careful optimization of surface chemistry is conducted to obtain the most efficient tethering of the antibodies to the surface. The mechanism of immobilizing the bacteria is antibody-antigen interactions between the tethered antibodies on the surface and the bacterial surface LPS firmly attached to the bacteria. This type of attachment is known as immunoimmobilization. The experiments suggest no noticeable bacterial attachment until the surface antibody concentration reaches ∼70% of a full monolayer of coverage. Above this critical antibody density, a sharp increase in immunoimmobilized bacteria is observed as they populate nearly 80% to 100% of the available surface area, reaching ∼1.2 cells/10 µm(2). This sharp increase in population is tentatively explained in terms of the minimum number of antibody-antigen interactions required per bacterium to immobilize the cell. This critical number is estimated to be ∼6000-8000 antibodies per bacterium (having a 1 µm(2) footprint on the surface) under the assumption that a full monolayer of antibodies is about 1 antibody per ∼100 nm(2). However, the large majority of the 6000-8000 antibodies are not expected to participate in antibody-antigen interactions, in that the loose LPS in solution will saturate many of these antibodies before bacteria have a chance to interact with them. Furthermore, the geometric considerations will further restrict the majority of the active antibodies from interacting with the surface antigens of the cell, reducing its effective contact area with the antibodies considerably.