Soils as natural filters for GHG: an imbalance between the expected CH4 fluxes and the direct measurements

The final composition of the atmosphere results from several processes and exchanges between all the Earth’s spheres. Some of these are widely known and others, such as the methane degassing from hydrothermal areas, are still understudied. Methane plays a key role in climate change being an efficient greenhouse gas. Although it would be crucial, the total CH4 output from geogenic sources is still not well defined; limitations in CH4 output estimations are due to many factors concerning a scarce dataset availability, difficulty in direct measurements, and interaction with methane-consuming microbiota in the soil. Often, the CH4 flux estimation was obtained indirectly, e.g. cross-correlating …

Heterogeneity within and between primary colorectal carcinomas and matched metastases as revealed by analysis of Ki-ras and p53 mutations

Analysis of the genetic status of Ki-ras and p53 in primary colorectal carcinomas and matched colorectal liver metastasis from 30 patients reveals an overall heterogeneity both within and between the two tumoral tissues. Both genes were found mutated with a similar frequency in both tissues; however, identical mutations in primary tumor and matched metastasis were found less frequently in the case of the Ki-ras than the p53 gene. Only in three cases the same p53 and Ki-ras mutations found in the primary tumor were found also in the metastasis. In several metastatic specimens the DNA bearing a mutation detected also in the primary tumor appears significantly less abundant than the wild-type …

Methanotrophic activity and bacterial diversity in volcanic-geothermal soils at Pantelleria island (Italy)

Abstract. Volcanic and geothermal systems emit endogenous gases by widespread degassing from soils, including CH4, a greenhouse gas twenty-five times as potent as CO2. Recently, it has been demonstrated that volcanic/geothermal soils are source of methane, but also sites of methanotrophic activity. Methanotrophs are able to consume 10–40 Tg of CH4 a−1 and to trap more than 50% of the methane degassing through the soils. We report on methane microbial oxidation in the geothermally most active site of Pantelleria island (Italy), Favara Grande, whose total methane emission was previously estimated in about 2.5 t a−1. Laboratory incubation experiments with three top-soil samples from Favara Gra…

A modified culture medium for improved isolation of marine vibrios

Abstract Marine Vibrio members are of great interest for both ecological and biotechnological research, which often relies on their isolation. Whereas many efforts have been made for the detection of food‐borne pathogenic species, much less is known about the performances of standard culture media toward environmental vibrios. We show that the isolation/enumeration of marine vibrios using thiosulfate‐citrate‐bile salts‐sucrose agar (TCBS) as selective medium may be hampered by the variable adaptability of different taxa to the medium, which may result even in isolation failure and/or in substantial total count underestimation. We propose a modified TCBS as isolation medium, adjusted for mar…

Temporal dynamic of biofilms enhances the settlement of the central-1 Mediterranean reef2 builder Dendropoma cristatum (Biondi, 1859)

Research on marine invertebrate settlement provides baseline knowledge for restoration technique implementation, especially for biogenic engineers with limited dispersion ability. Previously, we determined that the maturity of a biofilm strongly enhances the settlement of the vermetid reef-builder Dendropoma cristatum . In order to elucidate which biofilm features support a higher settlement of this species, we analyse the structure and composition of a marine biofilm over time, through microscopic observations, eukaryotic and prokaryotic fingerprinting analyses and 16S rDNA Illumina sequencing. The vermetid settlement temporal increase matched with the higher biofilm extent on the substrat…

Profiling microbial communities in hyperalkaline waters of the Kizildag ophiolite complex (Turkey).

It has recently been hypothesized that life on Earth could have been originated in hyperalkaline waters related to serpentinization of ophiolitic rocks, despite their extreme conditions (high pH and very low levels of nutrients). Five hyperalkaline springs of the Kizildag ophiolite complex (Turkey) were characterized. The dominant gases are either H2, CH4 or N2 . Bacterial diversity, analysed by RISA (Ribosomal Intergenic Spacer Analysis) revealed different profles for each spring. Sequencing of excised DNA bands allowed to identify the presence of Bacillus, Ralstonia, Pseudoalteromonas., Ureibacillus, Alicycliphilus, Anaerococcus. 16S ribosomal DNA sequencing by Illumina is in progress. Th…

Characterization of Translationally Controlled Tumour Protein from the Sea Anemone Anemonia viridis and Transcriptome Wide Identification of Cnidarian Homologues

Gene family encoding translationally controlled tumour protein (TCTP) is defined as highly conserved among organisms; however, there is limited knowledge of non-bilateria. In this study, the first TCTP homologue from anthozoan was characterised in the Mediterranean Sea anemone, Anemonia viridis. The release of the genome sequence of Acropora digitifera, Exaiptasia pallida, Nematostella vectensis and Hydra vulgaris enabled a comprehensive study of the molecular evolution of TCTP family among cnidarians. A comparison among TCTP members from Cnidaria and Bilateria showed conserved intron exon organization, evolutionary conserved TCTP signatures and 3D protein structure. The pattern of mRNA exp…

Neural Crest-Derived Chondrocytes Isolation for Tissue Engineering in Regenerative Medicine

Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone …

Intrachromosomal recombination of the c-myc locus leading to gene amplification

Possible relation between genetic recombination and amplification within the c-myc locus in a case of primary colorectal carcinoma

The gut microbiota of larvae of Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae)

Background: The red palm weevil (RPW) Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) is one of the major pests of palms. The larvae bore into the palm trunk and feed on the palm tender tissues and sap, leading the host tree to death. The gut microbiota of insects plays a remarkable role in the host life and understanding the relationship dynamics between insects and their microbiota may improve the biological control of insect pests. The purpose of this study was to analyse the diversity of the gut microbiota of field-caught RPW larvae sampled in Sicily (Italy). Results: The 16S rRNA gene-based Temporal Thermal Gradient Gel Electrophoresis (TTGE) of the gut microbiota of RPW …

Effects of human disturbance on cave-nesting seabirds: The case of the storm petrel

We tested the effects of human disturbance in two sub-colonies of Mediterranean storm petrel. We conducted three experiments to measure the capacity of the storm petrels to respond to stress. The part of the colony exposed to human disturbance resulted to be habituated and did not show chronic stress related to anthropogenic disturbance.

Biological diversity of the microbial film associated to the central-Mediterranean Dendropoma cristatum (Biondi, 1859) reefs.

Microbial films may provide physical and bio-chemical cues which positively affect the settlement dynamic of a variety of benthic marine organisms, driving the development of ecosystems. Also for the Mediterranean intertidal reef-builder Dendropoma cristatum (Biondi, 1859), biofilm maturity has been found to enhance the settlement pattern. However, the microbial diversity associated with these bioconstructions has never been described. This study investigates the D. cristatum reef bacterial assemblage composition and temporal evolution in two localities in the northwest of Sicily. Biological diversity of the reef-associated biofilm and of 3 progressively older biofims obtained on artificial…

The gut bacterial community of the red palm weevil Rhynchophorus ferrugineus (Olivier, Coleoptera, Curculionidae) larvae

The red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier, Coleoptera, Curculionidae) is the major pest of palm trees in the Mediterranean area. Damages are caused mainly by the endophytophagous larvae that consume the palm tender soft tissues making galleries and producing a wet “frass” with a characteristic fermented odour. The culturable bacterial community associated to the frass produced by RPW larvae inside Phoenix canariensis trees is mainly composed of facultative anaerobe gamma-proteobacteria that have their closest phylogenetic relatives in the gut microbiome of other endophytophagous and xilophagous coleopteran as bark beetles and other plant biomass-degrading insects (leaf-c…

Vibrio Proteases for Biomedical Applications: Modulating the Proteolytic Secretome of V. alginolyticus and V. parahaemolyticus for Improved Enzymes Production

Proteolytic enzymes are of great interest for biotechnological purposes, and their large-scale production, as well as the discovery of strains producing new molecules, is a relevant issue. Collagenases are employed for biomedical and pharmaceutical purposes. The high specificity of collagenase-based preparations toward the substrate strongly relies on the enzyme purity. However, the overall activity may depend on the cooperation with other proteases, the presence of which may be essential for the overall enzymatic activity, but potentially harmful for cells and tissues. Vibrios produce some of the most promising bacterial proteases (including collagenases), and their exo-proteome includes s…

DUE DIFFERENTI MECCANISMI MOLECOLARI COINVOLTI NEL FENOMENO DELL'AMPLIFICAZIONE GENICA IN UN CASO DI CARCINOMA COLORETTALE

Signals from the deep-sea: Genetic structure, morphometric analysis, and ecological implications of Cyclothone braueri (Pisces, Gonostomatidae) early life stages in the Central Mediterranean Sea

Abstract Cyclothone braueri (Stomiiformes, Gonostomatidae) is a widely distributed fish inhabiting the mesopelagic zone of marine tropical and temperate waters. Constituting one of the largest biomasses of the ocean, C. braueri is a key element in most of the ecological processes occurring in the twilight layer. We focused on the ecological processes linked to early life stages in relation to marine pelagic environmental drivers (temperature, salinity, food availability and geostrophic currents) considering different regions of the Central Mediterranean Sea. A multivariate morphometric analysis was carried out using six parameters with the aim of discerning different larval morphotypes, whi…

Intrachromosomal recombination of c-myc locus leading to gene amplification

Aberrant gene expression profiles in Mediterranean sea urchin reproductive tissues after metal exposures

Abstract Marine organisms are simultaneously exposed to numerous pollutants, among which metals probably represent the most abundant in marine environments. In order to evaluate the effects of metal exposure at molecular level in reproductive tissues, we profiled the sea urchin transcriptional response after non-lethal exposures using pathway-focused mRNA expression analyses. Herein, we show that exposures to relatively high concentrations of both essential and toxic metals hugely affected the gonadic expression of several genes involved in stress-response, detoxification, transcriptional and post-transcriptional regulation, without significant changes in gonadosomatic indices. Even though …

Optimized RNA extraction and northern hybridization in streptomycetes.

Abstract Northern blot hybridization is a useful tool for analyzing transcript patterns. To get a picture of what really occurs in vivo, it is necessary to use a protocol allowing full protection of the RNA integrity and recovery and unbiased transfer of the entire transcripts population. Many protocols suffer from severe limitations including only partial protection of the RNA integrity and/or loss of small sized molecules. Moreover, some of them do not allow an efficient and even transfer in the entire sizes range. These difficulties become more prominent in streptomycetes, where an initial quick lysis step is difficult to obtain. We present here an optimized northern hybridization protoc…

Advanced Strategies for Food-Grade Protein Production: A New E. coli/Lactic Acid Bacteria Shuttle Vector for Improved Cloning and Food-Grade Expression

Food-grade production of recombinant proteins in Gram-positive bacteria, especially in LAB (i.e., Lactococcus, Lactobacillus, and Streptococcus), is of great interest in the areas of recombinant enzyme production, industrial food fermentation, gene and metabolic engineering, as well as antigen delivery for oral vaccination. Food-grade expression relies on hosts generally considered as safe organisms and on clone selection not dependent on antibiotic markers, which limit the overall DNA manipulation workflow, as it can be carried out only in the expression host and not in E. coli. Moreover, many commercial expression vectors lack useful elements for protein purification. We constructed a &ld…

So close, so different: geothermal flux shapes divergent soil microbial communities at neighbouring sites

This study is focused on the (micro)biogeochemical features of two close geothermal sites (FAV1 and FAV2), both selected at the main exhalative area of Pantelleria Island, Italy. A previous biogeochemical survey revealed high CH4 consumption and the presence of a diverse community of methanotrophs at FAV2 site, whereas the close site FAV1 was apparently devoid of methanotrophs and recorded no CH4 consumption. Next-Generation Sequencing (NGS) techniques were applied to describe the bacterial and archaeal communities which have been linked to the physicochemical conditions and the geothermal sources of energy available at the two sites. Both sites are dominated by Bacteria and host a negligib…

Exploring methanotrophic activity in geothermal soils from Pantelleria Island (Italy)

Methane is released to the atmosphere by a wide number of natural (geological and biological) and anthropogenic sources, and is the second most important greenhouse gas after CO2. Microbial oxidation in soils by methanotrophic bacteria contributes to the removal of CH4 from the atmosphere and methanotrophic activity was also detected in volcanic/geothermal areas where degassing of endogenous gases occurs. Our aim is to describe the methanotrophs at the main exhalative area of Le Favare site at Pantelleria Island, where high CH4 consumption (up to 950 ng/g/ per h) was measured. Total soil bacterial diversity was analysed by TTGE of amplified 16S rRNA genes and the diversity of proteobacteria…

High-Performance PCR for Alleles Discrimination of Chromo-Helicase-DNA Binding Protein (CHD1) Gene in Bird Sexing

Genetic analyses aiming at assessing the presence of specific sequences or alleles are often carried out by PCR. Sexing of most birds is nowadays based on PCR with “universal” primers and relies on the assessment of the presence of the sex-linked CHD1-Z and -W alleles. The entire workflow is relatively time-consuming, especially for batch analyses, whereas methods that allow carrying out the entire procedure in a short time are highly desirable. The only method for outdoor analyses reported so far relies on LAMP; however; it fails to work properly in Procellariiformes. Besides improving the LAMP test; we have developed a PCR-based DNA amplification procedure (named high-performance PCR); wh…

On the identity of Tettigonia krugeri Massa from Libya with some remarks on variability of Tettigonia species (Orthoptera: Tettigoniidae; Tettigoniinae).

Morphological and genetic data allowed the authors to resurrect the name Tettigonia krugeri Massa, 1998 as a valid species. It is currently known only from two specimens (one male and one female) collected in Cirenaica (Libya).

Complete decontamination and regeneration of DNA purification silica colum

Silica columns are among the most used DNA purification systems, allowing a good yield of high-quality nucleic acids without organic extractions. Silica column regeneration protocols reported up to now to remove DNA traces are time-consuming, and their effectiveness on genomic DNA has not been demonstrated. Here we report a very rapid regeneration procedure that ensures no DNA carryover, independent of its size, without impairing column efficiency. The method takes advantage of the improved DNA removal by low concentrations of Triton X-100.

DNA Extraction From Orthoptera Museum Specimens

We describe a procedure for rapid purification of high quality DNA from either fresh or dry Orthoptera, suitable for the PCR amplification of DNA regions more than 800 bp long (even from oldest specimens), which allows genetic analyses on animals from collections without the complete specimen disruption.

Methanotrophy in geothermal soils, an overlooked process: The example of Nisyros island (Greece)

Abstract A multidisciplinary field campaign was carried out at Nisyros Island (Greece). Hydrothermal gases were sampled and analysed, and CH4 and CO2 fluxes from the soils were measured with the accumulation chamber method. The sampling area (Lakki plain) covers an area of about 0.08 km2, and includes the main fumarolic areas of Kaminakia, Stefanos, Ramos, Lofos and Phlegeton. Flux values measured at 130 sites range from −3.4 to 1420 mg m−2 d−1 for CH4 and from 0.1 to 383 g m−2 d−1 for CO2. The fumarolic areas show very different CH4 degassing patterns, Kaminakia showing the highest CH4 output values (about 0.8 t a−1 from an area of about 30,000 m2) and Phlegeton the lowest (about 0.01 t a−…

Methanotrophic activity and diversity of methanotrophs in volcanic geothermal soils at Pantelleria (Italy)

Volcanic and geothermal systems emit endogenous gases by widespread degassing from soils, including CH4, a greenhouse gas twenty-five times as potent as CO2. Recently, it has been demonstrated that volcanic or geothermal soils are not only a source of methane, but are also sites of methanotrophic activity. Methanotrophs are able to consume 10–40 Tg of CH4 a−1 and to trap more than 50% of the methane degassing through the soils. We report on methane microbial oxidation in the geothermally most active site of Pantelleria (Italy), Favara Grande, whose total methane emission was previously estimated at about 2.5 Mg a−1 (t a−1). Laboratory incubation experiments with three top-soil samples from …

Gene expression changes after parental exposure to metals in the sea urchin affect timing of genetic programme of embryo development

Simple Summary Intergenerational and transgenerational effects, in which exposure to stressors in a parental generation affects the phenotype of the offspring have been connected to anthropic impacts on biological systems. Therefore, environmental stress experienced inside a generation, particularly during gametogenesis, may lead to erroneous patterns in their offspring just emerging at early developmental stages. In this scenario, the sea urchin embryo represents a suitable model for integrating analyses of gene expression through embryogenesis with developmental alteration induced by environmental stressors. Herein we provide pieces of evidence for the alteration of the gene regulatory ne…

Two distinct amplification events of the c-myc locus in a colorectal tumour.

Southern hybridisation of genomic DNA extracted from a human primary colorectal carcinoma revealed amplification of a fragment containing the wild-type c-myc locus. Two additional rearranged DNA fragments, lying upstream of c-myc, fused to distant non-contiguous sequences from the same chromosome, with an opposite configuration (head to head vs. head to tail), were also found to be amplified. Sequences analysis suggested that these rearrangements resulted from illegitimate recombination at two distinct points within the DNA sequence just upstream of the c-myc ORF and further that these events triggered two different amplification mechanisms, only one of which, involving a strand invasion ev…

A taxonomic revision of the Palaearctic genus Roeseliana (Orthoptera: Tettigoniidae: Tettigoniinae: Platycleidini): a case of ongoing Mediterranean speciation

The genus Roeseliana presently includes 10 specific or subspecific taxa, but following different authors some of them are considered synonyms. However, the authors who have treated these taxa often did not agree with the synonymies, in particular, concerning some taxa, such as R. fedtschenkoi (Saussure, 1874) and R. roeselii (Hagenbach, 1822). The present authors examined hundreds of specimens of different taxa, for the first time were able to obtain the translation from the Russian of the description of R. fedtschenkoi, compared the main morphological characters used to discriminate different taxa, biometrics, bioacoustics and genetics of some taxa. This allowed them to conclude that it is…

Greenhouse gas as a nutrient: methanotrophic activity in soils of hydrothermal systems

Methane is the most abundant hydrocarbon in the atmosphere and a significant contributor to the radiative forcing with a global warming potential about 21 times that of CO2. Methane is released to the atmosphere by a wide number of sources, both natural and anthropogenic, with the latter being twice as large as the former. Significant amounts of geological methane, produced within the Earth’s crust (e.g. volcanic/geothermal areas), are currently released into the atmosphere (48 Tg CH4/y). Microbial oxidation in soils by methanotrophic bacteria contributes to the removal of CH4 from the atmosphere for about 3-9%. Methanotrophs belong to the Gamma- and Alpha-proteobacteria and to the recently…

Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years

Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria…

Temporal dynamic of biofilms enhances the settlement of the central-Mediterranean reef-builder Dendropoma cristatum (Biondi, 1859).

Abstract Research on marine invertebrate settlement provides baseline knowledge for restoration technique implementation, especially for biogenic engineers with limited dispersion ability. Previously, we determined that the maturity of a biofilm strongly enhances the settlement of the vermetid reef-builder Dendropoma cristatum. To elucidate settlement-related biofilm features, here we analyse the structure and composition of marine biofilms over time, through microscopic observations, eukaryotic and prokaryotic fingerprinting analyses and 16S rDNA Illumina sequencing. The vermetid settlement temporal increase matched with the higher biofilm coverage on the substratum and the reduction of th…

Development of a biosensor for copper detection in aqueous solutions using an Anemonia sulcata recombinant GFP.

Fluorescent proteins from marine organisms represent potential candidates for biosensor development. In this paper, we described the isolation of a native green fluorescent protein from Anemonia sulcata and the cloning and purification of its equivalent as a recombinant protein in Escherichia coli. Furthermore, the spectroscopic behaviours of the native and recombinant GFPs were investigated as a function of Cu2+, Cd2+, Pb 2+ and Ni2+ concentration. Our results suggest the high selectivity of both proteins at copper than the other metals and, for the recombinant protein, a great sensitivity at a very low concentration (0.1-1 μM). Moreover, starting from these data, using the combination of …

RNA stabillity as determinant of polarity in gene III of phage f1

Regeneration of total RNA purification silica-based columns

Silica-based columns are largely used in RNA purification, allowing fast extractions and good yields of high quality nucleic acid, but their major limitation is the high cost. The reuse of such columns, although desirable, is not recommended because of residual amounts of material from the previous sample trapped in the column matrix, which might be released during further purification. Thus, recycling does need previous complete removal of any detectable RNA trace, but to date no protocol which allows decontamination and reuse is available.We report a very rapid decontamination procedure, based on treatment with warm alkaline solution containing Triton X-100, which ensures no RNA carry-ove…

AMPLIFICAZIONE E RIARRANGIAMENTO NEI TUMORI COLORETTALI

The nucleic acid-binding protein PcCNBP is transcriptionally regulated during the immune response in red swamp crayfish Procambarus clarkii

Gene family encoding cellular nucleic acid binding proteins (CNBP) is well conserved among vertebrates; however, there is limited knowledge in lower organisms. In this study, a CNBP homolog from the red swamp crayfish Procambarus clarkii was characterised. The full-length cDNA of PcCNBP was of 1257 bp with a 5′-untranslated region (UTR) of 63 bp and a 3′-UTR of 331 bp with a poly (A) tail, and an open-reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids with the predicted molecular weight of about 33 kDa. The predicted protein possesses 7 tandem repeats of 14 amino acids containing the CCHC zinc finger consensus sequence, two RGG-rich single-stranded RNA-binding domain an…

Microbial communities and novel taxa in « Continental Smokers » of northern Greece

Analogously to the ocean-bottom black/white smokers, “continental smokers” have recently been defined as sites of mantle degassing through continental crust. The goal of this research, carried out within the Deep Carbon Observatory Community, is to investigate the role of deeplysourced fluids in niche ecosystem differentiation in European continental smokers. A 16S rRNA gene survey of microbial communities was carried out on 11 geothermal manifestations of northern Greece, all showing a distinct contribution of mantle fluids (R/Ra up to 1.2) that allow to classify them as continental smokers. The selected sites cover a wide range of temperatures (15-77 °C), pH (6.11-8.46), Eh (-289 – 40 mV)…

Microbiology meets geochemistry: geothermal flux shapes different microbial communities at the same exhalative area

Volcanic/geothermal areas are sites of complex interactions between geosphere and biosphere. Pantelleria island (Southern Mediterranean Sea) hosts a high enthalpy geothermal system characterized by high CH4 and low H2S fluxes. Two sites, FAV1 and FAV2, located a few meters apart at the main exhalative area of the island (Favara Grande), recorded similar physical conditions (soil temperature 60°C, soil gas composition enriched in CH4, H2 and CO2). However, while high methanotrophic activity (59.2 nmol g-1 h-1) and high diversity of methanotrophs was detected at FAV2, FAV1 was not active and appeared deprived of methanotrophs (1). Our aim was to investigate the main factors influencing methan…

RNA quality control: RppH activity allows selective removal of nonsense messages in E. coli.

Polar effect, the reduced expression level of sequences downstream to mutations reducing translation efficiency, is usually due to transcription termination or inefficient translation reinitiation. Untranslated mRNAs are known to be quickly degraded, probably because of their increased accessibility to degradative enzymes due to the absence of translating ribosomes. In III-VI-I operon of phage f1, a strong polar effect is observed in a gIII 5’ proximal nonsense mutant, resulting in a very fast, RNaseE mediated, degradation of any full-length mRNA. RNaseE is a key component of the E. coli degradosome, the major RNA processing/degrading machinery. Its endonucleolytic activity is strongly enha…

Preliminary results for application of culture-independent molecular methods to olive brines bacterial communities.

Transformation of green table olives requires a brining stage during which olives ferment. In naturally transformed olives, both flavour improvement and debittering are associated to fermentation. Because of brine features, naturally occurring bacteria and yeasts are progressively selected by the high salt concentration, low pH and phenolic compounds with antimicrobial activity. The establishment of an adequate fermenting microflora is essential to obtain a good and safe product; then, during transformation, control of chemical and microbiological parameters is crucial. Currently, microflora is periodically monitored by plate-counting of main groups. These routine analyses, however, don’t s…