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

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 (CaCO3) 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 Chromobacterium subtsugae strain CV026. Only Burkholderia ambifaria LMG 11351 was able to restore violacein production in CV026 among the tested strains. The constructed AHL-negative mutant of B. ambifaria LMG 11351 could not precipitate CaCO3 on B-4 agar. Scanning Electron Microscopy (SEM) analysis on CaCO3 crystals obtained in vitro 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 B. ambifaria LMG 11351, a stream-like organization of the Burkholderia imprints was not detected by SEM. Our data provide preliminary evidence that the activation of AHL-regulated genes may be a prerequisite for in vitro bacterial carbonatogenesis, in some cases, confirming the specific role of bacteria as CaCO3 precipitating agents. We enhance the understanding of bacterial CaCO3 biomineralization and its potential biotechnology implications for QS-based strategies to enhance or decrease CaCO3 precipitation through specific bacterial processes. The AHL-negative mutant of B. ambifaria LMG 11351 (a well-known plant growth-promoting bacterium) could also be used to study plant-bacteria interactions. The adaptive role of bacterial CaCO3 biomineralization was also discussed

A Novel Approach to Isolation and Screening of Calcifying Bacteria for Biotechnological Applications

Bacterial calcium-carbonate precipitation (BCP) has been studied for multiple applications such as remediation, consolidation, and cementation. Isolation and screening of strong calcifying bacteria is the main task of BCP-technique. In this paper, we studied CaCO3 precipitation by different bacteria isolated from a rhizospheric soil in both solid and liquid media. It has been found, through culture-depending studies, that bacteria belonging to Actinobacteria, Gammaproteobacteria, and Alphaproteobacteria are the dominant bacteria involved in CaCO3 precipitation in this environment. Pure and mixed cultures of selected strains were applied for sand biocementation experiments. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) analyses of the biotreated samples revealed the biological nature of the cementation and the effectiveness of the biodeposition treatment by mixed cultures. X-ray diffraction (XRD) analysis confirmed that all the calcifying strains selected for sand biocementation precipitated CaCO3, mostly in the form of calcite. In this study, Biolog® EcoPlate is evaluated as a useful method for a more targeted choice of the sampling site with the purpose of obtaining interesting candidates for BCP applications. Furthermore, ImageJ software was investigated, for the first time to our knowledge, as a potential method to screen high CaCO3 producer strains

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

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. © 2012 Copyright Microscopy Society of America

Deposition of calcium carbonate in karst caves: role of bacteria in Stiffe\u27s Cave

Bacteria make a significant contribution to the accumulation of carbonate in severa! natural habitats where large amounts of carbonates are deposited. However, the role played by microbial communities in speleothem formation (stalactites, stalagmites etc.) in caves is still unclear. In bacteria carbonate is formed by autotrophic pathways, which deplete CO2 from the environment, and by heterotrophic pathways, leading to active or passive precipitation. We isolated cultivable heterotrophic microbial strains, able to induce CaCO3 precipitation in vitro, from samples taken from speleothems in the galleries of Stiffe’s cave, L’Aquila, Italy. We found a large number of bacteria in the calcite formations (1 x 104 to 5 x 109 cells g-1). Microscopic examination, in laboratory conditions at different temperatures, showed that most of the isolates were able to form calcium carbonate microcrystals. The most crystalline precipitates were observed at 32°C. No precipitation was detected in un-inoculated controls media or in media that had been inoculated with autoclaved bacterial cells. X-ray diffraction (XRD) analysis showed that most of the carbonate crystals produced were calcite. Bacillus strains were the most common calcifying isolates collected from Stiffe’s Cave. Analysis of carbonate-solubilization capability revealed that the non-calcifying bacteria were carbonate solubilizers

Deposition of calcium carbonate in karst caves: role of bacteria in Stiffe\u27s Cave

Bacteria make a significant contribution to the accumulation of carbonate in severa! natural habitats where large amounts of carbonates are deposited. However, the role played by microbial communities in speleothem formation (stalactites, stalagmites etc.) in caves is still unclear. In bacteria carbonate is formed by autotrophic pathways, which deplete CO2 from the environment, and by heterotrophic pathways, leading to active or passive precipitation. We isolated cultivable heterotrophic microbial strains, able to induce CaCO3 precipitation in vitro, from samples taken from speleothems in the galleries of Stiffe’s cave, L’Aquila, Italy. We found a large number of bacteria in the calcite formations (1 x 104 to 5 x 109 cells g-1). Microscopic examination, in laboratory conditions at different temperatures, showed that most of the isolates were able to form calcium carbonate microcrystals. The most crystalline precipitates were observed at 32°C. No precipitation was detected in un-inoculated controls media or in media that had been inoculated with autoclaved bacterial cells. X-ray diffraction (XRD) analysis showed that most of the carbonate crystals produced were calcite. Bacillus strains were the most common calcifying isolates collected from Stiffe’s Cave. Analysis of carbonate-solubilization capability revealed that the non-calcifying bacteria were carbonate solubilizers

Involvement of Microorganisms in the Formation of Carbonate Speleothems in the Cervo Cave (L\u27Aquila-Italy)

Much is known about the bacterial precipitation of carbonate rocks, but comparatively little is known about the involvement of microbes in the formation of secondary mineral structures in caves. We hypothesized that bacteria isolated from calcareous stalactites, which are able to mediate CaCO3 precipitation in vitro, play a role in the formation of carbonate speleothems. We collected numerous cultivable calcifying bacteria from calcareous speleothems from Cervo cave, implying that their presence was not occasional. The relative abundance of calcifying bacteria among total cultivable microflora was found to be related to the calcifying activity in the stalactites. We also determined the δ 13C and δ 18 O values of the Cervo cave speleothems from which bacteria were isolated and of the carbonates obtained in vitro to determine whether bacteria were indeed involved in the formation of secondary mineral structures. We identified three groups of biological carbonates produced in vitro at 11°C on the basis of their carbon isotopic composition: carbonates with δ 13C values (a) slightly more positive, (b) more negative, and (c) much more negative than those of the stalactite carbonates. The carbonates belonging to the first group, characterized by the most similar δ 13C values to stalactites, were produced by the most abundant strains. Most of calcifying isolates belonged to the genus Kocuria. Scanning electron microscopy showed that dominant morphologies of the bioliths were sherulithic with fibrous radiated interiors. We suggest a mechanism of carbonate crystal formation by bacteria

INVOLVEMENT OF BACTERIA IN THE ORIGIN OF A NEWLY DESCRIBED SPELEOTHEM IN THE GYPSUM CAVE OF GRAVE GRUBBO (CROTONE, ITALY)

Microorganisms have been shown to be important active and passive promoters of redox reactions that influence the precipitation of various minerals, including calcite. Many types of secondary minerals thought to be of purely inorganic origin are currently being reevaluated, and microbial involvement has been demonstrated in the formation of pool fingers, stalactites and stalagmites, cave pisoliths, and moontriilk. We studied the possible involvement of bacteria in the formation of a new type of speleothem from Grave Grubbo Cave, the third-largest gypsum cave in Italy. The speleothem we studied consisted of a large aggregate of calcite tubes having a complex morphology, reflecting its possible organic origin. We isolated an abundant heterotrophic microflora associated with this concretion and identified Bacillus, Burkholderia, and Pasteurella spp. among the isolates. All of the isolates precipitated CaCO3 in vitro in the form of calcite. Only one of the isolates solubilized carbonate. The relative abundance of each isolate was found to be directly related to its ability to precipitate CaCO3 at cave temperature. We suggest that hypogean environments select for microbes exhibiting calcifying activity. Isotopic analysis produced speleothem delta C-13 values of about -5.0 parts per thousand, confirming its organic origin. The lightest carbonates purified from B4M agar plates were produced by the most abundant isolates. SEM analysis of the speleothem showed traces of calcified filamentous bacteria interacting with the substrate. Spherical bioliths predominated among the ones produced in vitro. Within the crystals produced in vitro, we observed bacterial imprints, sometimes in a preferred orientation, suggesting the involvement of a quorum-sensing system in the calcium-carbonate precipitation process

Hand movement parameters calculated by the LEAP based Virtual Glove

Hand rehabilitation therapy is fundamental in the recovery process for patients suffering from post-stroke or postsurgery impairments. Beside traditional approaches, that require the presence of therapist during the sessions (with high costs and subjective measurements), alternative approaches can be employed. A novel multi-sensor approach, the Virtual Glove (VG), is based on the simultaneous use of two LEAP motion controllers to track the hand motion and to reconstruct a numerical hand model in real time. In this paper, we illustrate how the VG can be used to calculate and represent graphically static and dynamic hand numerical parameters used to assess the Ranges Of Motion (ROM) of the hand articulations, which are the basis for objective evaluation of a rehabilitation task

Ultrasound-guided procedure for the treatment of trapeziometacarpal osteoarthritis

Initial treatment of trapeziometacarpal osteoarthritis (TMO) often consists of intra-articular injection of corticosteroids or hyaluronic acid. Several studies have shown that ultrasound (US)-guided injections demonstrate greater precision and effectiveness when compared to blind techniques. The aim of our study was to describe a simple and reliable procedure of US-guided intra-articular injection of hyaluronic acid solution in patients with TMO. Thirty-one patients with TMO received one administration per week, for 3 weeks, of US-guided intra-articular injection with 1 ml of hyaluronic acid solution. Patients were evaluated before treatment and at 1, 3, and 6 months after the first injection with visual analog scale (VAS) score and Duruoz Hand Index (DHI). One month after the end of treatment, subjective patient response to therapy was evaluated using a 6-point Likert scale. A statistically significant VAS score reduction was observed at 1 and 3 months after the end of treatment (P < 0.01) but not at 6-month follow-up (P = 0.6). No statistically significant difference was found on the DHI total score at 1- (P = 0.08), 3- (P = 0.1) and 6-month (P = 0.9) follow-ups. One month after the end of treatment, 29 (93.5%) patients reported a Likert scale score of "1" or "2." The described US-guided technique of trapeziometacarpal joint injection is easy to perform and revealed excellent accuracy. Nevertheless, intra-articular injections of hyaluronic acid provided significant pain relief only for a 6-month follow-up period and did not improve hand function. No pain relief was detected at 6-month follow-up, suggesting that periodic injections should be required to gain long-term effectiveness