Maddalena Del Gallo

Microbial Formation of Oxalate Films on Monument Surfaces: Bioprotection or Biodeterioration?

Oxalate films observed on stone monument surfaces deserve greater interest because of their possible role in protecting against deterioration. Their origin remains controversial. We present here the results of research conducted on production of oxalic acid and other organic acids by bacterial communities isolated from two monuments. Both communities were developed in vitro, and oxalate production was evaluated in a context of global metabolic activities that could eventually lead to protection or to degradation of the surface itself. HPLC analyses of organic acids production revealed that all mixed cultures produced oxalic acid but in different amounts. Besides oxalic acid, other organic acids are released that can solubilize stone calcium carbonate and have a deteriorating activity. Calcium carbonate solubilization, evaluated both by mixed cultures and isolated strains, was stronger with mixed cultures than with single strains. Our data show that oxalate production is promoted by the bacterial communiti...

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

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.

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.

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 moonmilk. 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, Burk- holderia ,a ndPasteurella 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 d 13 C values of about - 5.00%, 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.

BIOGENICITY AND CHARACTERIZATION OF MOONMILK IN THE GROTTA NERA (MAJELLA NATIONAL PARK, ABRUZZI, CENTRAL ITALY)

Observations and hypotheses on the possible influence of unidentified calcifying bacteria on moonmilk speleothem formation in the Grotta Nera are reported for the first time. The Majella Massif hosts a complex karst system of several caves; the accessible Grotta Nera is the most interesting one. Despite its name, the cave is characterized by particularly abundant ivory-white deposits of moonmilk. Two samples of moonmilk were analyzed to determine the geochemistry, fabric, depositional setting, and extent of biogenicity. For this, we combined geochemical, scanning electron microscopic, microbiological, and in vitro precipitation studies. X-ray diffraction of the moonmilk deposits gave clear evidence for the presence of calcite. Scanning electron microscopy showed that moonmilk in the Grotta Nera consists of a network of calcite fibers oriented in all directions, resembling a felted mat. The cultivation on specific medium of moonmilk and drip-water samples showed the presence of fungi, actinomycetes, and other bacteria, but the dominant cultivable microorganisms were bacteria, which produced significant crystallization. Examination of Gram-stained smears taken from the fifteen different colony types showed that the majority (66.7%) of the bacterial isolates were Gram-negative. Single small rods and rod chains were the most common bacteria isolated from the Grotta Nera. None of the molds isolated from the Grotta Nera samples were able to precipitate CaCO3 crystals, suggesting a major bacterial contribution to moonmilk deposition in the cave. Bacteria were capable of precipitating CaCO3 on B-4 solid medium at 15 (cave temperature), 22, and 32 uC. The calcifying bacteria isolated from the Grotta Nera showed a greater capability to solubilize CaCO3 than those associated with consolidated stalactites sampled from previously studied caves. The electron microscopy and microbiological evidences, together with the geochemistry and environmental data, allowed us to postulate the biogenic nature of the moonmilk in the Grotta Nera Cave.

A study of chlorinated solvent contamination of the aquifers of an industrial area in central Italy: a possibility of bioremediation

Perchloroethene, trichloroethene, and other chlorinated solvents are widespread groundwater pollutants. They form dense non-aqueous phase liquids that sink through permeable groundwater aquifers until non-permeable zone is reached. In Italy, there are many situations of serious contamination of groundwater that might compromise their use in industry, agriculture, private, as the critical case of a Central Italy valley located in the province of Teramo (“Val Vibrata”), characterized by a significant chlorinated solvents contamination. Data from the various monitoring campaigns that have taken place over time were collected, and new samplings were carried out, resulting in a complete database. The data matrix was processed with a multivariate statistic analysis (in particular principal component analysis, PCA) and was then imported into geographic information system (GIS), to obtain a model of the contamination. A microcosm anaerobic study was utilized to assess the potential for in situ natural or enhanced bioremediation. Most of the microcosms were positive for dechlorination, particularly those inoculated with a mineral medium. This indicate the presence of an active native dechlorinating population in the subsurface, probably inhibited by co-contaminants in the groundwater, or more likely by the absence or lack of nutritional factors. Among the tested electron donors (i.e., yeast extract, lactate, and butyrate) lactate and butyrate enhanced dechlorination of chlorinated compounds. PCA and GIS studies allowed delimiting the contamination; the microcosm study helped to identify the conditions to promote the bioremediation of the area.

Microbiological and biochemical aspects of inland Pecorino Abruzzese cheese

Little is known on physicochemical and biochemical characteristics of “Pecorino” Abruzzese cheese in L’Aquila province, an artisanal cheese produced from ewe raw full-cream milk. Three batches of inland “Pecorino” Abruzzese cheese were examined for microbiological, compositional, biochemical and sensory characteristics at the aim of isolating and storing in a bacterial collection, indigenous strain to preserve the microbial biodiversity present in this cheese, to a possible definition of a PDO. Cheese samples from three dairies, at different stages of production were collected and 148 colonies were characterized. Physicochemical assays, species-specific PCR and 16S rRNA gene sequencing revealed that the majority of the lactic acid bacteria (LAB) isolates were Enterococcus faecium and En. faecalis. They were highly prevalent, accounting for 48% of the isolates. The lactic microflora consisted of lactobacilli and lactococci from the species Lactobacillus plantarum (12.2%), Lactobacillus brevis (10.1%), Lactococcus lactis subsp. cremoris (11.5%), respectively. Urea-PAGE electrophoresis showed extensive degradation of αS1-casein (CN) and moderate hydrolysis of β-CN. Formation of γ-CNs from β-CN were highlighted. RP-HPLC profiles of the ethanol-soluble and ethanol-insoluble fractions of the pH 4.6-soluble nitrogen showed only minor differences between the three farms: lower proteolysis in the soluble fraction than the insoluble. Leucine, glutamic acid, lysine, valine were the free amino acids present at the highest levels in all the cheeses. Flavour and texture profile were characterized through a sensory analysis.

Microbial community distribution and genetic analysis in a sludge active treatment for a complex industrial wastewater: a study using microbiological and molecular analysis and principal component analysis

Liquid industrial chemical wastes are often treated with complex and integrated chemical and biological processes to attain the required standard for water discharge into the environment. Such wastewaters are potentially dangerous and of unknown composition. Biological treatment represents the cheapest means of pollution abatement. In the present work, a microbiological and genetic analysis was performed for the biological pool of an industrial wastewater treatment plant located in Civita Castellana (Viterbo, Italy). This study focused on active sludge treatment and on the subsequent secondary sedimentation system. The parameters of the biological section (the sludge active plant) are normally constantly monitored, but the biological efficiency of the microbial community should be better evaluated to understand the dynamics of the microbial community and its relationship to the overall standard quality parameters that are usually monitored. For this study, Biolog community level physiological profiling (CLPP) on EcoPlates and PCR-amplified 16S rRNA denaturing gradient gel electrophoresis (DGGE) were used in comparison and combined as ecological techniques to characterize an anthropic closed ecosystem. Biolog CLPP provides the potential metabolic pattern and DGGE analyses helps to explain the structure and complexity of the microbial community. The results suggest that these techniques could be predictive and more useful when used together than alone. In addition, a principal component analysis (PCA) performed on the Biolog assay over time can relate principal components predictive wells to the biological and standard chemical analysis used to control and monitor the activity of this type of industrial wastewater treatment plant, providing a selection method for further DGGE community analysis.

Selection of a Very Active Microbial Community for the Coupled Treatment of Tetramethylammonium Hydroxide and Photoresist in Aqueous Solutions

Aerobic treatment of wastewater containing Tetramethylammonium hydroxide (TMAH) and photoresist was investigated using a lab scale reactor inoculated with activated sludge coming from urban wastewater treatment that never received TMAH before. The consumption of TMAH was monitored by liquid ion chromatography. Biodiversity indices were calculated from Denaturing Gradient Gel Electrophoresis (DGGE) bands distribution and used to estimate changes in community composition related to adaptation to the new feeding compound. The first week of adaptation was crucial, and it was analyzed in detail: many organisms died, and the microbial community suffered a great shock. TMAH levels remained constant through the first four days, and then suddenly dropped to undetectable, and at the same time NH4+ increased. When the community showed complete adaptation, predominant groups of bacteria were obtained by the Illumina sequencing of 16s rDNA amplicons, to provide insights on ecology of the adapted community, focusing on the main actors of TMAH abatement. Richness of species (Rr) peaks suggest that the development of TMAH-consuming bacteria leads to persistent consortia that maintain toxicity resistance over time. This showed adaptation and changes of the population to the different feeding conditions, and it opens new perspectives in the in situ treatment of these important residues of industrial processes without relying on external processing plants.

Immunocytochemical characterisation of endophytic bacteriaAzospirillum brasilense, Herbaspirillum seropedicae, Burkholderia ambifaria andGluconacetobacter diazotrophicus

In the present work an immunocytochemical characterisation of four endophytic bacterial species has been made by using polyclonal antiserum produced against each of the four bacterial strains previously heated at 60 °C. The aim of this researchsito identify common elements among bacteria associated with their endophytic behaviour. Analysis of extracts of each strain by immunoblotting and ELISA confirmed the presence of proteins from different bacterial strains made up of common epitopes. However, antisaproduced againstHerbaspirillum seropedicae andBurkholderia ambifaria show a high number of bands recognised on each extracts, while antisera againstAzospirillum brasilense andGluconacetobacter diazotrophicus show a low number of bands recognised on each extract. Immunogold labelling showed that epitopes are located both on the cell wall and in the cytoplasm; most likely they could be preursor cell wall proteins synthesized inside the cytoplasm and subsequently transported onto cell wall. Finally, the common bands amog bacterial strains revealed by immunoblotting could play a role as active hydrolases involved in host tissue penetration.