Development of a novel biochar-made porous monolith for enhanced C1 and H2 fermentation.

Biochar is a carbonaceous porous material that is produced through the thermal processing of biomass under oxygen-limited environment. Nevertheless, biochar is known to be an inexpensive and sustainable raw material with a wide range of possible applications. Recently, biochar has been discovered as an efficient biological catalyst for anaerobic conversion, mainly due to its highly porous structure with micro and macro channels, which procures a viable living area for attached-grown microorganisms. Whereas it is never applied to improve the biological conversion of gas substances such as C1 (e.g., CO, CO2) and H2, which is a promising research area with increasing commercial interest. However, considering that biological reaction is limited by the target water solubility of gas substrates, special attention is required when combining biochar for gas fermentation. The goal was to create a novel gas sparger where the biofilm grows on biochar, thus improving the interaction with the gaseous substrate. For this purpose, polystyrene foam and powdered biochar were compounded to form a mouldable composite, which was then cast as a porous monolith.•Biochar-made sparger (BS) was investigated for the homoacetogenic conversion of H2 gas via microbial mixed cultures as opposed to a control test equipped with a stone sparger.•BS showed a significantly better performance in terms of biological gas fixation rate (36% more than control) and productivity (8.5 gCOD L-1 d-1).

Spineless and overlooked: DNA metabarcoding of autonomous reef monitoring structures reveals intra- and interspecific genetic diversity in Mediterranean invertebrates.

The ability to gather genetic information using DNA metabarcoding of bulk samples obtained directly from the environment is crucial to determine biodiversity baselines and understand population dynamics in the marine realm. While DNA metabarcoding is effective in evaluating biodiversity at community level, genetic patterns within species are often concealed in metabarcoding studies and overlooked for marine invertebrates. In the present study, we implement recently developed bioinformatics tools to investigate intraspecific genetic variability for invertebrate taxa in the Mediterranean Sea. Using metabarcoding samples from Autonomous Reef Monitoring Structures (ARMS) deployed in three locations, we present haplotypes and diversity estimates for 145 unique species. While overall genetic diversity was low, we identified several species with high diversity records and potential cryptic lineages. Further, we emphasize the spatial scale of genetic variability, which was observed from locations to individual sampling units (ARMS). We carried out a population genetic analysis of several important yet understudied species, which highlights the current knowledge gap concerning intraspecific genetic patterns for the target taxa in the Mediterranean basin. Our approach considerably enhances biodiversity monitoring of charismatic and understudied Mediterranean species, which can be incorporated into ARMS surveys.

Be positive: customized reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studies.

Background: In metabarcoding analyses, the taxonomic assignment is crucial to place sequencing data in biological and ecological contexts. This fundamental step depends on a reference database, which should have a good taxonomic coverage to avoid unassigned sequences. However, this goal is rarely achieved in many geographic regions and for several taxonomic groups. On the other hand, more is not necessarily better, as sequences in reference databases belonging to taxonomic groups out of the studied region/environment context might lead to false assignments. Methods: We investigated the effect of using several subsets of a cytochrome c oxidase subunit I (COI) reference database on taxonomic assignment. Published metabarcoding sequences from the Mediterranean Sea were assigned to taxa using COInr, which is a comprehensive, non-redundant and recent database of COI sequences obtained both from BOLD and NCBI, and two of its subsets: (i) all sequences except insects (COInr-WO-Insecta), which represent the overwhelming majority of COInr database, but are irrelevant for marine samples, and (ii) all sequences from taxonomic families present in the Mediterranean Sea (COInr-Med). Four different algorithms for taxonomic assignment were employed in parallel to evaluate differences in their output and data consistency. Results: The reduction of the database to more specific custom subsets increased the number of unassigned sequences. Nevertheless, since most of them were incorrectly assigned by the less specific databases, this is a positive outcome. Moreover, the taxonomic resolution (the lowest taxonomic level to which a sequence is attributed) of several sequences tended to increase when using customized databases. These findings clearly indicated the need for customized databases adapted to each study. However, the very high proportion of unassigned sequences points to the need to enrich the local database with new barcodes specifically obtained from the studied region and/or taxonomic group. Including novel local barcodes to the COI database proved to be very profitable: by adding only 116 new barcodes sequenced in our laboratory, thus increasing the reference database by only 0.04%, we were able to improve the resolution for ca. 0.6-1% of the Amplicon Sequence Variants (ASVs).

Insight on bacteria communities in outdoor bronze and marble artefacts in a changing environment.

Epilithic bacteria play a fundamental role in the conservation of cultural heritage (CH) materials. On stones, bacterial communities cause both degradation and bioprotection actions. Bronze biocorrosion in non-burial conditions is rarely studied. Only few studies have examined the relationship between bacteria communities and the chemical composition of patinas (surface degradation layers). A better comprehension of bacterial communities growing on our CH is fundamental not only to understand the related decay mechanisms but also to foresee possible shifts in their composition due to climate change. The present study aims at (1) characterizing bacterial communities on bronze and marble statues; (2) evaluating the differences in bacterial communities' composition and abundance occurring between different patina types on different statues; and (3) providing indications about a representative bacterial community which can be used in laboratory tests to better understand their influence on artefact decay. Chemical and biological characterization of different patinas were carried out by sampling bronze and marble statues in Bologna and Ravenna (Italy), using EDS/Raman spectroscopy and MinION-based 16SrRNA sequencing. Significant statistical differences were found in bacterial composition between marble and bronze statues, and among marble patinas in different statues and in the same statue. Marble surfaces showed high microbial diversity and were characterized mainly by Cyanobacteria, Proteobacteria and Deinococcus-Thermus. Bronze patinas showed low taxa diversity and were dominated by copper-resistant Proteobacteria. The copper biocidal effect is evident in greenish marble areas affected by the leaching of copper salts, where the bacterial community is absent. Here, Ca and Cu oxalates are present because of the biological reaction of living organisms to Cu ions, leading to metabolic product secretions, such as oxalic acid. Therefore, a better knowledge on the interaction between bacteria communities and patinas has been achieved.

Coral-ID: A forensically validated genetic test to identify precious coral material and its application to objects seized from illegal traffic.

The production and trade of objects manufactured from the skeletal axis of coralid precious corals is a historically, culturally and economically important global industry. Coralids are members of the diverse Coralliidae family, which contains several species complexes and morphospecies. For most precious coral found in the jewelry trade, the color remains the sole clue and link to the taxonomic identity of the individual. Different coralid species have however similar or overlapping colors resulting in difficulty to taxonomically identify jewelry objects, including four species listed by the Convention on the International Trade of Endangered Species (CITES) whose international transport and trade requires species-specific and country of origin documentation. We aimed at developing a reliable method to taxonomically identify coralid material with the objective of distinguishing CITES protected species from their non-protected counterparts. We present Coral-ID, a genetic assay to taxonomically classify coralid objects using quasi non-destructive sampling. The assay classifies the analyzed sample in one of six taxonomic categories and performs at least presumptive separation of CITES-listed and non-listed species in all cases. Developmental validation experiments prove that Coral-ID is a specific, accurate and very sensitive method. As the first attempt to randomly sample corals in the trade to identify them, we applied Coral-ID on 20 precious coral objects seized by custom authorities upon import to in Switzerland. Thirteen (65%) of these samples could be analyzed; three of these were found to be presumptively CITES-listed, and 10 of them have proven to originate from non-CITES-listed species.

Evidence of genetic isolation between two Mediterranean morphotypes of Parazoanthus axinellae.

Coralligenous assemblages are among the most species-rich and vulnerable habitats of the Mediterranean Sea. Nevertheless, data on connectivity patterns on species inhabiting these habitats, crucial to define management and protection priorities, are largely lacking. Moreover, unreliable species-level taxonomy can confound ecological studies and mislead management strategies. In the northwestern Mediterranean two Parazoanthus axinellae morphotypes differing in size, color and preferred substrate are found in sympatry. In this study, we used COI and ITS sequence polymorphism to assess (1) the genetic divergence between the two morphotypes, (2) their connectivity patterns and (3) their phylogenetic position within the Parazoanthidae. Specimens of P. axinellae were sampled in 11 locations along the northwestern Mediterranean; in 6 locations, samples of the two morphotypes were collected in sympatry. Small genetic diversity and structure were found within morphotypes, while marked and consistent differentiation was detected between them. Moreover, the less widespread morphotype appeared to be closer to Pacific species as P. juanfernandezii and P. elongatus. Our findings confirmed the limited knowledge on Parazoanthus species complex, and how this gap can have important implication for the conservation strategies of this widespread and valuable genus in the Mediterranean Sea.

Why do mesophotic coral ecosystems have to be protected?

Mesophotic coral ecosystems (MCEs; ~30-150 m depth) are among the most biologically diverse and least protected ecosystems in the world's oceans. However, discussions regarding the conservation of these unique ecosystems are scarce. To address this issue, we identified the features of MCEs that demonstrate they should be considered as a global conservation priority. Some MCEs are characterized by their well-preserved and unique seascapes; their narrow environmental tolerance and high vulnerability to anthropogenic effects; and their slow recovery and reduced reproductive performance. The unique biodiversity of MCEs includes depth-adapted specialist species and new species, most of which are threatened or important fishery resources. MCEs also provide refuge against human stressors, valuable ecosystem services, and ecological connectivity. MCEs generally meet the criteria to be classified as Ecologically and Biologically Significant Marine Areas under the Convention on Biological Diversity. However, we highlight that many MCEs worldwide are threatened and not yet adequately protected by fishery regulations, marine protected areas, or considered in marine spatial planning. Establishing MCEs as a global conservation priority requires the designation of national, international, transnational, public, and private policies.

Charcot Neuroarthropathy: From the Laboratory to the Bedside.

BACKGROUND: The diabetic Charcot foot syndrome is a serious and potentially limbthreatening lower-extremity complication of diabetes. INTRODUCTION: The present review provides a concise account of the advances made over the last twentyfive years in understanding the pathogenesis and management of Charcot neuroarthropathy (CN). METHODS: In this study, the widely known pathogenetic mechanisms underpinning CN are brought into focus, particularly the role of RANKL/RANK/OPG system and advanced glycation end production in the pathogenesis of CN. Furthermore, other potential triggering factors, namely nitric oxide, endothelial dysfunction, macro calcifications and body weight that influence CN have also been discussed. RESULTS: The wide range of diagnostic tools available to clinicians for accurate staging of this pathology has been examined, particularly radiological and nuclear medicine imaging. Additionally, the difficult differential diagnosis between osteomyelitis and CN is also elucidated. CONCLUSION: The review concludes with the comprehensive summary of the major promising therapeutic strategies, including conservative treatment involving orthopedic devices, pharmacological approach, and the most common surgical techniques currently employed in the diagnosis and treatment of this acute disease.

Mediterranean Bioconstructions Along the Italian Coast.

Marine bioconstructions are biodiversity-rich, three-dimensional biogenic structures, regulating key ecological functions of benthic ecosystems worldwide. Tropical coral reefs are outstanding for their beauty, diversity and complexity, but analogous types of bioconstructions are also present in temperate seas. The main bioconstructions in the Mediterranean Sea are represented by coralligenous formations, vermetid reefs, deep-sea cold-water corals, Lithophyllum byssoides trottoirs, coral banks formed by the shallow-water corals Cladocora caespitosa or Astroides calycularis, and sabellariid or serpulid worm reefs. Bioconstructions change the morphological and chemicophysical features of primary substrates and create new habitats for a large variety of organisms, playing pivotal roles in ecosystem functioning. In spite of their importance, Mediterranean bioconstructions have not received the same attention that tropical coral reefs have, and the knowledge of their biology, ecology and distribution is still fragmentary. All existing data about the spatial distribution of Italian bioconstructions have been collected, together with information about their growth patterns, dynamics and connectivity. The degradation of these habitats as a consequence of anthropogenic pressures (pollution, organic enrichment, fishery, coastal development, direct physical disturbance), climate change and the spread of invasive species was also investigated. The study of bioconstructions requires a holistic approach leading to a better understanding of their ecology and the application of more insightful management and conservation measures at basin scale, within ecologically coherent units based on connectivity: the cells of ecosystem functioning.

Living upside down: patterns of red coral settlement in a cave.

BACKGROUND: Larval settlement and intra-specific interactions during the recruitment phase are crucial in determining the distribution and density of sessile marine populations. Marine caves are confined and stable habitats. As such, they provide a natural laboratory to study the settlement and recruitment processes in sessile invertebrates, including the valuable Mediterranean red coral Corallium rubrum. In the present study, the spatial and temporal variability of red coral settlers in an underwater cave was investigated by demographic and genetic approaches. METHODS: Sixteen PVC tiles were positioned on the walls and ceiling of the Colombara Cave, Ligurian Sea, and recovered after twenty months. A total of 372 individuals of red coral belonging to two different reproductive events were recorded. Basal diameter, height, and number of polyps were measured, and seven microsatellites loci were used to evaluate the genetic relationships among individuals and the genetic structure. RESULTS: Significant differences in the colonization rate were observed both between the two temporal cohorts and between ceiling and walls. No genetic structuring was observed between cohorts. Overall, high levels of relatedness among individuals were found. CONCLUSION: The results show that C. rubrumindividuals on tiles are highly related at very small spatial scales, suggesting that nearby recruits are likely to be sibs. Self-recruitment and the synchronous settlement of clouds of larvae could be possible explanations for the observed pattern.

Chasing genetic structure in coralligenous reef invertebrates: patterns, criticalities and conservation issues.

Conservation of coastal habitats is a global issue, yet biogenic reefs in temperate regions have received very little attention. They have a broad geographic distribution and are a key habitat in marine ecosystems impacted by human activities. In the Mediterranean Sea coralligenous reefs are biodiversity hot spots and are classified as sensitive habitats deserving conservation. Genetic diversity and structure influence demographic, ecological and evolutionary processes in populations and play a crucial role in conservation strategies. Nevertheless, a comprehensive view of population genetic structure of coralligenous species is lacking. Here, we reviewed the literature on the genetic structure of sessile and sedentary invertebrates of the Mediterranean coralligenous reefs. Linear regression models and meta-analytic approaches are used to assess the contributions of genetic markers, phylum, pelagic larval duration (PLD) and geographical distance to the population genetic structure. Our quantitative approach highlight that 1) most species show a significant genetic structure, 2) structuring differs between phyla, and 3) PLD does not appear to be a major driver of the structuring. We discuss the implication of these finding for the management and conservation, suggesting research areas that deserve attention, and providing recommendations for broad assessment and monitoring of genetic diversity in biogenic reefs species.

Inter- and Intraspecific Variability of Nitrogenated Compounds in Gorgonian Corals via Application of a Fast One-Step Analytical Protocol.

Gorgonian corals play a structural role in temperate and tropical biogenic reefs, forming animal forests and creating biodiversity hot spots. In the Mediterranean Sea, slow-growing and long-lived gorgonian species are threatened by human disturbances and global environmental changes and concern about their conservation is rising. Alkaloid metabolites have proven to be essential in protecting these species from environmental stressors. Traditional profiling methodologies to detect these metabolites require a large quantity of living tissue. Here, the chemodiversity of gorgonian alkaloids was investigated by applying a fast and effective protocol combining extraction and derivatization using small-scale tissue samples and GC/MS analysis. The method was effective in identifying and quantifying alkaloids and guanine-based compounds. Eight N-heterocyclic compounds were found in six Mediterranean gorgonians differing for types and quantity. The metabolomic profile was conservative in species of the Eunicella genus, with three species sharing the same pattern. Conversely, Paramuricea clavata displayed a noticeable spatial pattern of variation among colonies collected in different locations. The analytical approach presented here proved to be effective, allowing rare, endangered, and small-sized species to be screened rapidly for detection of new compounds in order to explore their biological and ecological functions.

Autoantibodies to post-translationally modified type I and II collagen in Charcot neuroarthropathy in subjects with type 2 diabetes mellitus.

AIMS: Charcot neuroarthropathy (CN) is a disabling complication, culminating in bone destruction and involving joints and articular cartilage with high inflammatory environment. Its real pathogenesis is as yet unknown. In autoinflammatory diseases, such as rheumatoid arthritis, characterized by inflammation and joint involvement, autoantibodies against oxidative post-translationally modified (oxPTM) collagen type I (CI) and type II (CII) were detected. Therefore, the aim of our study was to assess the potential involvement of autoimmunity in charcot neuroarthropathy, investigating the presence of autoantibodies oxPTM-CI and oxPTM-CII, in participants with charcot neuroarthropathy. METHODS: In this case-control study, we enrolled 124 participants with type 2 diabetes mellitus (47 with charcot neuroarthropathy, 37 with diabetic peripheral neuropathy without charcot neuroarthropathy, and 40 with uncomplicated diabetes), and 32 healthy controls. The CI and CII were modified with ribose and other oxidant species, and the modifications were evaluated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding of sera from the participants was analyzed with enzyme-linked immunosorbent assay. RESULTS: Age, body mass index, waist and hip circumferences, and lipid profile were similar across the 4 groups, as well as glycated hemoglobin and duration of diabetes among people with diabetes. An increased binding to both native and all oxidation-modified forms of CII was found in participants with CN and diabetic neuropathy. Conversely, for CI, an aspecific increased reactivity was noted. CONCLUSIONS: Our results detected the presence of autoantibodies against oxidative post-translational modified collagen, particularly type 2 collagen, in participants with charcot neuroarthropathy and diabetic neuropathy, suggesting the possible involvement of autoimmunity. Further studies are required to understand the role of autoimmunity in the pathogenesis of charcot neuroarthropathy.

Limited Genetic Connectivity between Gorgonian Morphotypes along a Depth Gradient.

Gorgonian species show a high morphological variability in relation to the environment in which they live. In coastal areas, parameters such as temperature, light, currents, and food availability vary significantly with depth, potentially affecting morphology of the colonies and the structure of the populations, as well as their connectivity patterns. In tropical seas, the existence of connectivity between shallow and deep populations supported the hypothesis that the deep coral reefs could potentially act as (reproductive) refugia fostering re-colonization of shallow areas after mortality events. Moreover, this hypothesis is not so clear accepted in temperate seas. Eunicella singularis is one of the most common gorgonian species in Northwestern Mediterranean Sea, playing an important role as ecosystem engineer by providing biomass and complexity to the coralligenous habitats. It has a wide bathymetric distribution ranging from about 10 m to 100 m. Two depth-related morphotypes have been identified, differing in colony morphology, sclerite size and shape, and occurrence of symbiotic algae, but not in mitochondrial DNA haplotypes. In the present study the genetic structure of E. singularis populations along a horizontal and bathymetric gradient was assessed using microsatellites and ITS1 sequences. Restricted gene flow was found at 30-40 m depth between the two Eunicella morphotypes. Conversely, no genetic structuring has been found among shallow water populations within a spatial scale of ten kilometers. The break in gene flow between shallow and deep populations contributes to explain the morphological variability observed at different depths. Moreover, the limited vertical connectivity hinted that the refugia hypothesis does not apply to E. singularis. Re-colonization of shallow water populations, occasionally affected by mass mortality events, should then be mainly fueled by larvae from other shallow water populations.

Genetic structuring across marine biogeographic boundaries in rocky shore invertebrates.

Biogeography investigates spatial patterns of species distribution. Discontinuities in species distribution are identified as boundaries between biogeographic areas. Do these boundaries affect genetic connectivity? To address this question, a multifactorial hierarchical sampling design, across three of the major marine biogeographic boundaries in the central Mediterranean Sea (Ligurian-Tyrrhenian, Tyrrhenian-Ionian and Ionian-Adriatic) was carried out. Mitochondrial COI sequence polymorphism of seven species of Mediterranean benthic invertebrates was analysed. Two species showed significant genetic structure across the Tyrrhenian-Ionian boundary, as well as two other species across the Ionian Sea, a previously unknown phylogeographic barrier. The hypothesized barrier in the Ligurian-Tyrrhenian cannot be detected in the genetic structure of the investigated species. Connectivity patterns across species at distances up to 800 km apart confirmed that estimates of pelagic larval dispersal were poor predictors of the genetic structure. The detected genetic discontinuities seem more related to the effect of past historical events, though maintained by present day oceanographic processes. Multivariate statistical tools were used to test the consistency of the patterns across species, providing a conceptual framework for across-species barrier locations and strengths. Additional sequences retrieved from public databases supported our findings. Heterogeneity of phylogeographic patterns shown by the 7 investigated species is relevant to the understanding of the genetic diversity, and carry implications for conservation biology.

Quantifying spatial genetic structuring in mesophotic populations of the precious coral Corallium rubrum.

While shallow water red coral populations have been overharvested in the past, nowadays, commercial harvesting shifted its pressure on mesophotic organisms. An understanding of red coral population structure, particularly larval dispersal patterns and connectivity among harvested populations is paramount to the viability of the species. In order to determine patterns of genetic spatial structuring of deep water Corallium rubrum populations, for the first time, colonies found between 58-118 m depth within the Tyrrhenian Sea were collected and analyzed. Ten microsatellite loci and two regions of mitochondrial DNA (mtMSH and mtC) were used to quantify patterns of genetic diversity within populations and to define population structuring at spatial scales from tens of metres to hundreds of kilometres. Microsatellites showed heterozygote deficiencies in all populations. Significant levels of genetic differentiation were observed at all investigated spatial scales, suggesting that populations are likely to be isolated. This differentiation may by the results of biological interactions, occurring within a small spatial scale and/or abiotic factors acting at a larger scale. Mitochondrial markers revealed significant genetic structuring at spatial scales greater then 100 km showing the occurrence of a barrier to gene flow between northern and southern Tyrrhenian populations. These findings provide support for the establishment of marine protected areas in the deep sea and off-shore reefs, in order to effectively maintain genetic diversity of mesophotic red coral populations.