Fabrizio Cappa

Soil bacterial diversity screening using single 16S rRNA gene V regions coupled with multi-million read generating sequencing technologies.

The novel multi-million read generating sequencing technologies are very promising for resolving the immense soil 16S rRNA gene bacterial diversity. Yet they have a limited maximum sequence length screening ability, restricting studies in screening DNA stretches of single 16S rRNA gene hypervariable (V) regions. The aim of the present study was to assess the effects of properties of four consecutive V regions (V3-6) on commonly applied analytical methodologies in bacterial ecology studies. Using an in silico approach, the performance of each V region was compared with the complete 16S rRNA gene stretch. We assessed related properties of the soil derived bacterial sequence collection of the Ribosomal Database Project (RDP) database and concomitantly performed simulations based on published datasets. Results indicate that overall the most prominent V region for soil bacterial diversity studies was V3, even though it was outperformed in some of the tests. Despite its high performance during most tests, V4 was less conserved along flanking sites, thus reducing its ability for bacterial diversity coverage. V5 performed well in the non-redundant RDP database based analysis. However V5 did not resemble the full-length 16S rRNA gene sequence results as well as V3 and V4 did when the natural sequence frequency and occurrence approximation was considered in the virtual experiment. Although, the highly conserved flanking sequence regions of V6 provide the ability to amplify partial 16S rRNA gene sequences from very diverse owners, it was demonstrated that V6 was the least informative compared to the rest examined V regions. Our results indicate that environment specific database exploration and theoretical assessment of the experimental approach are strongly suggested in 16S rRNA gene based bacterial diversity studies.

Identification of fungi from dairy products by means of 18S rRNA analysis.

The role of fungi as cause of spoilage of dairy products, such as cheese and yoghurt, has been clearly demonstrated. Despite of this, there is still a lack in rapid methods for the identification of food-associated fungi. In the course of the present work, molecular taxonomical techniques were developed and used to identify yeasts involved in the spoilage of yoghurt and moulds responsible for spoilage of vacuum-packaged hard cheese. Three methods for DNA extraction and purification were evaluated and the fungus-specific primers TR1 and TR2 were used to amplify a 581-bp fragment within the gene, coding for the small ribosomal subunit (18S rRNA) of fungi. The 18S rRNA sequence analysis of fungi isolated from yoghurt and packaged cheese allowed to identify yeast belonging to Zygosaccharomyces microellipsoides and moulds belonging to Penicillium chrysogenum and Cladosporium cladosporoides.

Molecular characterization of the Lactobacillus community in traditional processing of Mozzarella cheese

The natural Lactobacillus community involved in traditional Mozzarella cheese production has been investigated. The bacterial associations of whey, curd before stretching and Mozzarella were analyzed using randomly amplified polymorphic DNA (RAPD) to follow growth kinetics, and 16S rDNA sequencing to identify the taxonomical position of isolated strains. Analysis of RAPD fingerprints revealed that the Lactobacillus community was composed of 13 different biotypes and the sequence analysis of 16S rDNA demonstrated that the isolated strains belong to L. plantarum, L. fermentum, L. helveticus and L. casei subsp. casei. In addition, two strains of Weissella hellenica were isolated on selective media for lactobacilli. The four L. casei subsp. casei strains and W. hellenica contained sequences related to the prtP gene coding for proteinase, and the highest proteolytic activity in milk was found in one strain of L. casei subsp.casei.

Surface microbiota analysis of Taleggio, Gorgonzola, Casera, Scimudin and Formaggio di Fossa Italian cheeses.

The composition of the bacterial consortia of the smear Italian cheeses and their role on quality and safety is still poorly understood. The objective of this study was to identify and characterize the bacterial communities present on the surface of five traditional Italian cheeses, Casera Valtellina, Scimudin, Formaggio di Fossa, Gorgonzola and Taleggio. DGGE analysis performed using total DNA obtained from cheese surfaces enabled us to identify the dominant bacterial populations. Bands showing different intensity and identified as Staphylococcus, Micrococcus, Psychrobacter, Enterococcus and Brevibacterium species were detected on the surface of cheeses. The cluster analysis showed that Gorgonzola, Taleggio and Formaggio di Fossa cheeses present high similarity in their surface bacterial composition while major differences in the DGGE profiles were observed in Scimudin and Casera. The molecular taxonomical identification among the Gram positive isolates, reveals the presence of the following bacterial genera: Staphylococcus, Micrococcus, Macrococcus, Enterococcus, Lactobacillus, Carnobacterium, Leuconostoc, Brevibacterium, Corynebacterium, Brochothrix, Bacillus. The combination of culture dependent and independent techniques allowed us to obtain information about the bacterial species covering the surface of five different traditional Italian cheeses.

Two 2[5H]-Furanones as Possible Signaling Molecules in Lactobacillus helveticus

ABSTRACT Two 2[5H]-furanones, in association with medium-chain fatty acids, were released in whey by Lactobacillus helveticus exposed to oxidative and heat stresses. This species plays an important role in cheese technology, particularly for Swiss-type cheeses and Grana cheese. Moreover, it significantly contributes to cheese ripening by means of an early autolysis and the release of enzymes during processing. Experimental evidence of the involvement of the two 2[5H]-furanones, detected by a gas chromatography-mass spectrometry/solid-phase microextraction technique, in the autolysis phenomenon has been obtained. Zymograms performed by using renaturing sodium dodecyl sulfate-polyacrylamide gels were used to detect the bioactivity of the supernatants containing the two furanones on fresh cells of the same strain. In addition to bands corresponding to known autolysins, new autolysins were detected concomitant with the exposure of Lactobacillus helveticus to the supernatants, which can be regarded as conditioned media (CM), and to a commercial furanone, 5-ethyl-3-hydroxy-4-methyl-2[5H]-furanone (HEMFi), having spectral data similar to those of the newly described 2[5H]-furanones. Morphological changes were observed when fresh cells were exposed to CM containing the two 2[5H]-furanones and HEMFi. The two furanones produced by Lactobacillus helveticus, which met a number of criteria to be included in cell-cell signaling molecules, have a presumptive molecular mass lower than those of already known 3[2H]-furanones having an autolytic activity and being produced by gram-negative bacteria. Moreover, they present a different chemical structure with respect to the furanones already identified as products of Lactococcus lactis subsp. cremoris or to those identified in some cheeses with Lactobacillus helveticus as a starter culture.

Effects of a humic acid and its size-fractions on the bacterial community of soil rhizosphere under maize (Zea mays L.).

The effects of a humic acid (HA) and its size-fractions on plants carbon deposition and the structure of microbial communities in the rhizosphere soil of maize (Zea mays L.) plants were studied. Experiments were conducted in rhizobox systems that separate an upper soil-plant compartment from a lower compartment, where roots are excluded from the rhizosphere soil by a nylon membrane. The upper rhizobox compartment received the humic additions, whereas, after roots development, the rhizosphere soil in the lower compartment was sampled and sliced into thin layers. The lux-marked biosensor Pseudomonas fluorescens 10586 pUCD607 biosensor showed a significant increase in the deposition of bioavailable sources of carbon in the rhizosphere of soils when treated with bulk HA, but no response was found for treatments with the separated size-fractions. PCR-DGGE molecular fingerprintings revealed that the structure of rhizosphere microbial communities was changed by all humic treatments and that the smaller and more bioavailable size-fractions were more easily degraded by microbial activity than the bulk HA. On the other hand, highly hydrophobic and strongly associated humic molecules in the bulk HA required additional plant rhizodeposition before their bio-transformation could occur. This work highlights the importance of applying advanced biological and biotechnological methods to notice changes occurring in plant rhizodeposition and rhizosphere microbial activity. Moreover, it suggests correlations between the molecular properties of humic matter and their effects on microbial communities in the rhizosphere as mediated by root exudation.

A combination of a SEM technique and X-ray microanalysis for studying the spore germination process of Clostridium tyrobutyricum

Clostridium tyrobutyricum is an anaerobic bacterium responsible for late blowing defects during cheese ripening and it is of scientific interest for biological hydrogen production. A scanning electron microscopy (SEM) coating technique and X-ray microanalysis were developed to analyze the architecture and chemical composition of spores upon germination in response to environmental changes. In addition, we investigated the effects of different compounds on this process. Agents and environmental conditions inducing germination were characterized monitoring changes in optical density (OD). Among all tested conditions, the greatest drop in OD(625) (57.4%) was obtained when spores were incubated in l-alanine/l-lactate buffer, pH 4.6. In addition, a carbon-coating SEM technique and X-ray microanalysis were used to observe the architecture of spores and to examine calcium dipicolinate release. Conditions inducing C. tyrobutyricum spore germination were identified and SEM X-ray microanalysis clearly distinguished germinating from dormant spores. We confirmed that calcium dipicolinate release is one of the first events occurring. These microscopy methods could be considered sensitive tools for evaluating morphological and chemical changes in spores of C. tyrobutyricum during the initial phase of germination. Information gathered from this work may provide new data for further research on germination.

Soil microbial diversity patterns of a lowland spring environment

The Po river plain lowland springs represent unique paradigms of managed environments. Their current locations used to be swamps that were drained 6-7 centuries ago, and they have been in constant use ever since. Our aims were to identify the effects of land use on the microbial communities of these soils, look for associated diversity drivers, and assess the applicability of ecology theories with respect to identified patterns. We screened the microbial diversity across a land use transect via high-throughput sequencing of partial 16S rrRNA gene amplicons. Land use had a major effect on soil properties and microbial community structures. Total organic carbon and pH were major diversity drivers for Bacteria, and pH was important for Archaea. We identified the potential contribution of soil amendments to the indigenous microbial communities, and also gained insights into potential roles of taxa in the organic carbon turnover. Verrucomicrobia coincided with the higher values of the recalcitrant organic carbon. Actinobacteria and Acidobacteria correlated with the more labile organic carbon. Finally, the higher diversity found in the soils less enzymatically active and relatively poorer in nutrients, may be explained to an extent by niche-based theories such as the resource heterogeneity hypothesis and Connells intermediate disturbance hypothesis.

Draft Genome Sequence of Clostridium tyrobutyricum Strain UC7086, Isolated from Grana Padano Cheese with Late-Blowing Defect

ABSTRACT Clostridium tyrobutyricum is considered the main agent of late-blowing defect in the production of hard cheese. Here, we described the draft genome sequences and annotation of C. tyrobutyricum strain UC7086, which was isolated from Grana Padano cheese with blowing defect, and C. tyrobutyricum DSM 2637 type strain in a comparative study.

Effects of geographic area, feedstock, temperature, and operating time on microbial communities of six full-scale biogas plants

The objective of this study was to investigate the effect of different animal feedings operated in two distinct PDO (protected designation of origin) cheese production areas (Parmigiano Reggiano and Grana Padano) on the microbiome of six full-scale biogas plants, by means of Illumina sequencing and qPCR techniques. The effects of feedstock (cattle slurry manure, energy crops, agro-industrial by-products), temperature (mesophilic/thermophilic), and operating time were also examined, as were the relationships between the predominant bacterial and archaeal taxa and process parameters. The different feedstocks and temperatures strongly affected the microbiomes. A more biodiverse archaeal population was highlighted in Parmigiano Reggiano area plants, suggesting an influence of the different animal feedings. Methanosarcina and Methanosaeta showed an opposite distribution among anaerobic plants, with the former found to be related to ammonium concentration. The Methanoculleus genus was more abundant in the thermophilic digester whereas representation of the Thermotogales order correlated with hydraulic retention time.