isolation-identification-bacterial-strains-from-skin-terrestrial

Isolation and Identification of Bacterial Strains from Skin of Terrestrial Amphibians

The microbiota is a vital element of higher organisms, with the cutaneous microbiota significantly contributing to the organism&#39;s defense against ...

UNIVERSITY OF L'AQUILA

Bacterially induced carbonate mineralization has been proposed as a new method for the restoration of limestones in historic buildings and monuments. We describe here the formation of calcite crystals by extracellular polymeric substances isolated from Bacillus firmus and Bacillus sphaericus. We isolated bacterial outer structures (glycocalix and parietal polymers), such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) and checked for their influence on calcite precipitation. CPS and EPS extracted from both B. firmus and B. sphaericus were able to mediate CaCO3 precipitation in vitro. X-ray microanalysis showed that in all cases the formed crystals were calcite. Scanning electron microscopy showed that the shape of the crystals depended on the fractions utilized. These results suggest the possibility that biochemical composition of CPS or EPS influences the resulting morphology of CaCO3. There were no precipitates in the blank samples. CPS and EPS comprised of proteins and glycoproteins. Positive alcian blue staining also reveals acidic polysaccharides in CPS and EPS fractions. Proteins with molecular masses of 25-40 kDa and 70 kDa in the CPS fraction were highly expressed in the presence of calcium oxalate. This high level of synthesis could be related to the binding of calcium ions and carbonate deposition.

Bacterially induced mineralization of calcium carbonate: the role of exopolysaccharides and capsular polysaccharides.

This study highlights the role of specific outer bacterial structures, such as the glycocalix, in calcium carbonate crystallization in vitro. We describe the formation of calcite crystals by extracellular polymeric materials, such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) isolated from Bacillus firmus and Nocardia calcarea. Organic matrices were isolated from calcifying bacteria grown on synthetic medium--in the presence or absence of calcium ions--and their effect on calcite precipitation was assessed. Scanning electron microscopy observations and energy dispersive X-ray spectrometry analysis showed that CPS and EPS fractions were involved in calcium carbonate precipitation, not only serving as nucleation sites but also through a direct role in crystal formation. The utilization of different synthetic media, with and without addition of calcium ions, influenced the biofilm production and protein profile of extracellular polymeric materials. Proteins of CPS fractions with a molecular mass between 25 and 70 kDa were overexpressed when calcium ions were present in the medium. This higher level of protein synthesis could be related to the active process of bioprecipitation.

Calcium carbonate mineralization: involvement of extracellular polymeric materials isolated from calcifying bacteria.

Four bacteria selected on the basis of their capability of fixing atmospheric nitrogen, stimulating plant-growth, and protecting the host plant from pathogens - Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, Burkholderia ambifaria - were inoculated on tomato seeds either singularly, in couple and in a four bacteria mixer. Aim of this research was to evaluate: (1) effect of single and mixed cultures on the inoculated plant - plant growth, dry weight, root length and surface, number of leaves, among others; (2) colonization and interactions of the bacteria inside the host plant; (3) localization inside the host of single bacterial strains marked with the gusA reporter gene. The results obtained indicate that all selected microbial strains have colonized Lycopersicon esculentum but in a different way, depending on the single species. A. brasilense, G. diazotrophicus inoculated in vitro singularly and together were the best plant colonizers. In vivo essays, instead, B. ambifaria and the four-bacteria mixer gave the best results. It was possible to localize both A. brasilense and H. seropedicae inside the plant by the gusA reporter gene. The bacterial strains occur along the root axis from the apical zone until to the basal stem, on the shoot from the base up to the leaves. The four bacteria actively colonize tomato seeds and establish an endophytic community inside the plant. This review gives new information about colonization processes, in particular how bacteria interact with plants and whether they are likely to establish themselves in the plant environment after field application as biofertilizers or biocontrol agents.

In vitro and in vivo inoculation of four endophytic bacteria on Lycopersicon esculentum.

With the expansion of the green products market and the worldwide policies and strategies directed toward a green revolution and ecological transition, the demand for innovative approaches is always on the rise. Among the sustainable agricultural approaches, microbial-based products are emerging over time as effective and feasible alternatives to agrochemicals. However, the production, formulation, and commercialization of some products can be challenging. Among the main challenges are the industrial production processes that ensure the quality of the product and its cost on the market. In the context of a circular economy, solid-state fermentation (SSF) might represent a smart approach to obtaining valuable products from waste and by-products. SSF enables the growth of various microorganisms on solid surfaces in the absence or near absence of free-flowing water. It is a valuable and practical method and is used in the food, pharmaceutical, energy, and chemical industries. Nevertheless, the application of this technology in the production of formulations useful in agriculture is still limited. This review summarizes the literature dealing with SSF agricultural applications and the future perspective of its use in sustainable agriculture. The survey showed good potential for SSF to produce biostimulants and biopesticides useful in agriculture.

Solid-State Fermentation: Applications and Future Perspectives for Biostimulant and Biopesticides Production.

Numerous microbial species participate in precipitation of carbonates in various natural environments, including soils, geological formations, freshwater biofilms and oceans. Despite the geochemical interest of such a biomineralization process, its molecular mechanisms and adaptive aspects remain poorly known. Many Gram-negative bacteria use cell-to-cell communication systems relying on N-acylhomoserine lactone (AHLs) signal molecules to express certain phenotypic traits in a density-dependent manner, a phenomenon referred as to quorum-sensing (QS). In this work, bacterial isolates collected from cave and rhizosphere soil were analyzed to study the occurrence of the AHL-mediated QS in bacterial calcium carbonate (CaCO) precipitation. To test the production of AHLs signal molecules, we cross-streaked Gram-negative calcifying strains, selected among the environmental strains studied, with the AHL-negative mutant  strain CV026. Only  LMG 11351 was able to restore violacein production in CV026 among the tested strains. The constructed AHL-negative mutant of  LMG 11351 could not precipitate CaCO on B-4 agar. Scanning Electron Microscopy (SEM) analysis on CaCO crystals obtained  shows crystals of different morphologies, calcified biofilms and bacteria in close contact with the precipitated crystals. In the inner layers of the bioliths deposited by  LMG 11351, a stream-like organization of the  imprints was not detected by SEM. Our data provide preliminary evidence that the activation of AHL-regulated genes may be a prerequisite for  bacterial carbonatogenesis, in some cases, confirming the specific role of bacteria as CaCO precipitating agents. We enhance the understanding of bacterial CaCO biomineralization and its potential biotechnology implications for QS-based strategies to enhance or decrease CaCO precipitation through specific bacterial processes. The AHL-negative mutant of  LMG 11351 (a well-known plant growth-promoting bacterium) could also be used to study plant-bacteria interactions. The adaptive role of bacterial CaCO biomineralization was also discussed.

Preliminary indication of the role of AHL-dependent quorum sensing systems in calcium carbonate precipitation in Gram-negative bacteria.

Sustainable alternatives are essential to improving agriculture production to meet the growing world's critical demands. Cyanobacteria and microalgae are considered renewable resources with a wide range of potential uses in the agricultural sector. We aimed to isolate cyanobacteria and microalgae from the mud of a carbon dioxide-rich sulfur pond and to investigate their plant growth-promoting (PGP) and soil bio-consolidating ability. Mud samples were subjected to DNA extraction and 16S rRNA gene sequencing to characterize the prokaryotic community. Phototrophic culturable microbiota was isolated and evaluated for different PGP properties. The most relevant isolates were combined in a consortium and used for  bioconsolidation activity. In a greenhouse experiment, the isolates were evaluated for their ability to promote salinity stress tolerance in sunflower plants. Metabarcoding results showed that most Amplicon Sequence Variants (ASV) were associated with Actinobacteriota (35%), Proteobacteria (19%), and Acidobacteriota (11%) at the phylum level and  (32%) and uncultured (31%) lineages at the genus level. The culture-dependent method yielded eight isolates associated with cyanobacteria and microalgae genera. The isolates obtained showed interesting PGP activities. Isolates C1, C2, and M1 were selected based on phosphate solubilization (85.6 µg PO  mL on average), indoles (C1 and M1 0.54 µg mL IAA equivalents on average), and ACC deaminase activity (C2 and M1 6.00 µmol α-KB mg proteins h. The consortium efficiently consolidated sand particles in the presence of calcium carbonate by forming biomineralized aggregates.  results showed positive effects of the consortium on ., plant growth under normal conditions and salt stress. The positive effects on soil and plants indicated their effectiveness as bioconsolidants and biostimulant agents. Our findings highlight the interesting potential of cyanobacteria and microalgae applications in sustainable agriculture.

Isolation and characterization of cyanobacteria and microalgae from a sulfuric pond: Plant growth-promoting and soil bioconsolidation activities.

The present study investigated the contribution of microbial communities in producing "living stones" and the suitability of these clasts as sources of microorganisms with biomineralisation abilities. The calcareous samples were analysed for their microbial community (16S rRNA gene metabarcoding and culturable approach) and in vitro regeneration tests. Scanning electron microscopy and Energy Dispersive Spectroscopy (SEM-EDX) were applied to investigate microbial aggregation structures and footprints in natural and in vitro samples. The metabarcoding unveiled amplicon sequence variants (ASVs) assigned to lineages with biomineralisation abilities (e.g., Proteobacteria and Actinobacteriota). The culturable approach resulted in nineteen calcifying isolates with diverse morphological, metabolic, and mineral precipitation properties. Based on mineralising properties, Stenotrophomonas maltophilia, Lysinbacillus fusiformis, and Microbacterium ginsengiterrae were identified at the molecular level. In vitro regeneration tests and SEM-EDX analyses confirmed the active role of the endogenous microorganisms in forming these "living stones". These findings allow us to hypothesise an essential role of microbial precipitation in forming these "living stones", previously described as of abiotic origin. The current study findings provide a solid scientific foundation for future investigations. The obtained bacterial isolates and their potential applications in bioremediation, construction, and cultural heritage restoration demonstrate the direct applicability of our study in sectors involving biomaterials application. STATEMENT OF SIGNIFICANCE: We studied some "living stones" that can be found worldwide and whose origin is still not completely understood. Geologists have not yet fully explained the origin of these inorganic structures that grow in size over time. The results obtained from our microbiological investigations allowed us to discover that microorganisms play a crucial role in forming these masses. In the investigations of the structures and microbial communities within the stones, we identified specific bacteria that actively contribute to forming minerals and isolated bacteria that can form biominerals. These findings deepen our understanding of natural processes involved in the formation of these structures and show their potential for several applications (e.g., building materials or cultural heritage preservation).

Article	Year
Bacterially induced mineralization of calcium carbonate: the role of exopolysaccharides and capsular polysaccharides. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada\| PubMed ID: 17234036	2007
Calcium carbonate mineralization: involvement of extracellular polymeric materials isolated from calcifying bacteria. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada\| PubMed ID: 22697480	2012
In vitro and in vivo inoculation of four endophytic bacteria on Lycopersicon esculentum. New biotechnology\| PubMed ID: 23352842	2013
Solid-State Fermentation: Applications and Future Perspectives for Biostimulant and Biopesticides Production. Microorganisms\| PubMed ID: 37374910	2023
Preliminary indication of the role of AHL-dependent quorum sensing systems in calcium carbonate precipitation in Gram-negative bacteria. AIMS microbiology\| PubMed ID: 38173968	2023
Isolation and characterization of cyanobacteria and microalgae from a sulfuric pond: Plant growth-promoting and soil bioconsolidation activities. AIMS microbiology\| PubMed ID: 39628714	2024
Erratic calcareous deposits: Biotic formation insights and biomineralising bacterial strain isolation. Acta biomaterialia\| PubMed ID: 39954754	2025

Paola Cacchio

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