Aldo Lepidi

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.

A biosensor for Escherichia coli based on a potentiometric alternating biosensing (PAB) transducer

Routinary methods for the detection of bacterial contamination in table water are costly and time consuming. In the present paper we describe a biosensor based on an immunoassay test. Escherichia coli cells present in water samples were detected by a complete Potentiometric alternating biosensor (PAB). The PAB system consists of a measuring chamber, acquisition and driving electronics and the appropriate software: the apparatus is computer controlled in order to obtain on-line acquisitions and recording of data. The transducer principle is based on a LAPS (Light Addressable Potentiometrie Sensor) technology which, in our case, reveals the production of NH3 by a urease-E. coli antibody conjugate. The proposed system appears to be very sensitive: concentrations of 10 cells per mL were clearly detected over blank of E. coli free water. No signals were detected from other bacteria, eventually polluting drinking water such as Pseudomonas marina, and also from bacteria such as Klebsiella oxytoca phyletically related to E. coli.

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...

Biological treatment of alkaline industrial waste waters

Abstract The biotechnological treatment of alkaline waste waters (AWW) resulting from the production of caprolactam by the SNIA-viscosa process has been studied. The pollutant in the AWW is 80–120 g litre −1 cyclohexanecarboxysulphonate (CECS) sodium salt with a COD up to 325 000 mg litre −1 . Bacterial strains have been isolated which are able to grow on AWW and to degrade the largest possible range of organic compounds. These strains have been screened for their performance in lowering the COD and degrade the sulphonic bonds. Combinations of strains have also been verified. The strains have been compared in cultures both in shake flasks and in a laboratory scale fermenter. The results showed that: (a) a 1/10 dilution of AWW with water permitted microbial growth coupled with decrease in COD and carboxylic concentration (representative of several organic compounds such as cyclohexanecarboxysulphonate, 2-aminocapronic and e-aminocapronic acids); (b) the polymerised caprolactam molecules are exhaustively degraded; (c) very similar results are found both in shake flask tests and in the lab scale fermenter but with different kinetics; and (d) pretreatment of alkaline waters with CaCl 2 and lowering the pH with H 3 PO 4 , implement the kinetics and yields of the process in terms of degradation of COD and carboxylic compounds. The experiments gave very preliminary results and led to some suggestions for the development of a chemical and biological process to treat this kind of AWW.

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.

Fractal analysis to discriminate between biotic and abiotic attacks on chalcopyrite and pyrolusite

In the present paper, a model describing release and mobility of copper and manganese in chalcopyrite and pyrolusite powders due to bacterial bioleaching, i.e. Thiobacillus ferrooxidans and Arthrobacter sp., is proposed. Sites where copper and manganese were released were examined by scanning electron microscopy (SEM). The resulting micrographs were scanned and submitted to a point by point fractal analysis to verify if a discrimination between biological and chemical attack could be established on the basis of the distribution of the fractional part of the fractal dimension over the whole surface. We demonstrate that such a method is able to discriminate among the different attacks.

Influence of Manganese Dioxide and Manganic Ions on the Production of Two Proteins in Arthrobacter sp.

The production by Arthrobacter sp. of a 30-kDa surface protein and a 25-kDa cytoplasmic protein was increased by the presence of MnO2 in the medium. This high production was also observed in the presence of MnO4- (Mn VII). N-terminal and partial internal sequences of the 30-kDa surface protein have shown no homology with other known proteins. The role of this protein is still unknown, but its highly induced synthesis is possibly related to the binding or the processing of manganic ion by the cells. The 25-kDa cytoplasmic protein has been identified by its N-terminal matching sequence as a superoxide dismutase isoenzyme (Mn-SOD). SOD activity measurements performed on cytoplasmic fractions are related to the protein amounts observed by gel electrophoresis. Arthrobacter sp. synthesized and exhibited SOD activity in both aerobic and anaerobic conditions, thus suggesting other or additional physiological functions for this enzyme.