Paola Cacchio

Bacterially Induced Mineralization of Calcium Carbonate: The Role of Exopolysaccharides and Capsular Polysaccharides

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.

Calcium Carbonate Precipitation by Bacterial Strains Isolated from a Limestone Cave and from a Loamy Soil

To study the role of calcifying bacteria in monument protection, 31 calcifying bacterial strains were isolated from natural habitats: 64% were of the genus Bacillus , 16% of the genus Arthrobacter , and 2 of the remaining isolates were identified as Kingella and Xanthomonas . The ability to form CaCO 3 crystals, the extent of the precipitation, and the type of crystals formed were determined at incubation temperatures of 4, 22, and 32°C. The highest of these temperatures favored CaCO 3 formation. Most of the bacteria precipitated CaCO 3 in the form of calcite. This activity was strictly controlled by the growth of microbial colonies on a solid substrate. The role of the calcifying bacteria in natural precipitation of carbonates is discussed. Further experiments are in progress in order to select the most suitable bacterial strains for a controlled production of calcareous crusts.

Involvement of Microorganisms in the Formation of Carbonate Speleothems in the Cervo Cave (L'Aquila-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.

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.