WHO Laboratory Biosafety Manual: A New Approach to Security.

The paper aims to highlight the new indications introduced in the 4th edition of the "Laboratory Biosafety Manual" of World Health Organization. The authors propose a new vision to improve biosafety and biosecurity in the laboratory aligned with the technical standards ISO 35001:2019 "Biorisk management for laboratories and other related organizations" and ISO 45001:2018 "Occupational health and safety management systems-Requirements with guidelines for use" framework. The current edition has a more innovative approach compared to the previous ones, more attention is given to training awareness and providing skills, to promote the culture of safety by adopting an approach based on risk analysis, rather than the prescriptive approach that has been used previously.

Profiling microalgal cultures growing on municipal wastewater and fertilizer media in raceway photobioreactors.

Microalgae cultivation is proposed as an effective system for pathogens reduction and wastewater depuration, however, a full characterisation of the risks is still needed. Two raceways were inoculated with Scenedesmus, one using wastewater and the other using a fertilizer medium. Microbial community and pathogen presence were explored by next generation sequencing (NGS), commercial qPCR array and plate counts. These methods proved to be complementary for a full characterization of community structure and potential risks. Media and sampling locations contributed to shape communities and pathogenic loads. The main pathogenic genera detected were Arcobacter and Elizabethkingia (mainly in wastewater) with an important presence of Aeromonas (all samples). A lower presence of pathogens was detected in fertilizer samples, while wastewater showed a reduction from inlet to outlet. Raceways showed potential as an effective biotreatment, with most of the retained pathogens released in the outlet and only a minor part settled in the biomass.

Containment of a genetically modified microorganism by an activated sludge system.

The effectiveness of physical, chemical and biological barriers to the diffusion of genetically modified microorganisms (GMMs) to prevent their release into the environment is currently under scrutiny worldwide because of the associated potential ecological impacts. An industrial discharge of a non-sterilized fermentation broth containing GMM biomass into a conventional municipal wastewater treatment plant would deliver the GMMs into the activated sludge system process (ASSP). The present work aimed to model and evaluate the containment capability of a small ASSP (part of a 20,000 people equivalent municipal plant) in the event of receiving GMM biomass from a medium-small biotechnological plant dedicated to the production of polyhydroxyalkanoates (3000 t/year of biopolymer). An actual GMM (Pseudomonas putida KTOY06) was injected into a bench-scale ASSP (ASSPLab) in a quantity proportional to the relative dimensions of the plants mentioned. The experimental and model results indicated that the ASSP of the target municipal treatment plant would not be capable of holding back such a sudden input of GMM; 6 h after the discharge, 11-15 % of injected GMM cells were released through the clarified stream of the ASSPLab, with the rest being gradually released over time. Since the GMM employed did not exhibit any growth in the ASSPLab, its concentration in the clarified water stream would not represent a substantial risk of release into the environment if appropriate tertiary treatments were integrated. This study confirmed the necessity of a thorough risk assessment of biotechnological processes prior to their implementation.

Victoriomyces antarcticus gen. nov., sp. nov., a distinct evolutionary lineage of the Cephalothecaceae (Ascomycota) based on sequence-based phylogeny and morphology.

In this study, we propose a new genus, Victoriomyces, with a new species, Victoriomyces antarcticus, isolated from soil samples collected in Victoria Land, Antarctica. To determine its taxonomic status and evolutionary relationships, phylogenetic analysis was performed on DNA sequences from the nuclear 18S rRNA, 28S rRNA and the second largest subunit of RNA polymerase II (RPB2) genes. Victoriomyces antarcticus constitutes one well-supported distinct lineage within the Cephalothecaceae (family incertae sedis in Sordariomycetes), in which the only recognised asexual morphs belong to the genus Phialemonium and to Acremonium thermophilum. Victoriomyces antarcticus can be clearly distinguished from these taxa by means of DNA sequence analysis and its morphological traits that consist in having a Metarhizium-like asexual morph, dark red-coloured disk-like structures, immature bodies and the production of an intense red pigment in the growth media. Finally, we inferred the divergence time of V. antarcticus and the Cephalothecaceae using Bayesian analysis and secondary calibration. The holotype of V. antarcticus is FBL 165. The ex-type strain has been deposited as MUT 3686T and CCF 6158T. An additional strain of the species is FBL 577. The MycoBank number is MB 823713 for the genus and MB 823714 for the species.

An integrated study on Gammarus elvirae (Crustacea, Amphipoda): perspectives for toxicology of arsenic-contaminated freshwater.

The Italian region Latium is characterized by extensive quaternary volcanic systems that contribute greatly to arsenic (As) contamination of freshwater, including drinking water supplies. However, knowledge of the possible toxic effects in these aquatic environments is, despite being highly relevant to public health, still limited. In this paper, we approach this issue using Gammarus elvirae, an amphipod species that inhabits rivers and streams in central Italy, including Latium. We explored the possibility of using G. elvirae in the toxicology of freshwater by addressing the most relevant issues. First, we tested the usefulness of hemocytes from G. elvirae in determining non-specific DNA damage by means of the Comet assay after exposure (24 h and 7 days) to different river water samples in Latium; second, we provided an interpretative overview of the usefulness of hepatopancreatic epithelial cells of G. elvirae as a means of assessing toxicity after long-term exposure to As and other pollutants; third, the LC (50-240 h) value for G. elvirae was estimated for arsenate, which is usually the dominant arsenic species in surface waters. Our study sheds light on G. elvirae at different levels, providing a background for future toxicological research of freshwater.

Green job bio-aerosol exposure during anaerobic digestion for biomass energetic valorisation.

The continued expansion of the green economy increases the risk profile for green occupational jobs. One of the broadest green sectors in terms of growth is the anaerobic digestion of biomasses. In recent years, this development has also interested Italian regions. The management of biomass includes biological risk and the risk of particulate and endotoxin exposure. In the present study, we evaluated airborne exposure for anaerobic digestion workers at two real-scale plants. Digested biomass has different origins, ranging from cattle sludge and manure to poultry manure to agricultural harvesting or processing residues, particularly from maize and fruits. Two sampling points were chosen: at the first, the input biomasses were stored, and the hopper was loaded; at the second, the digested sludge exited the digester. The microbiological parameters, assessed using an active sampler and cultural method, were the total bacteria counts (at 22, 37, and 55°C), yeasts, fungi, Pseudomonaceae, Clostridia spp., Enterobacteriaceae and Actinomycetes. Moreover, at the same sampling points, we evaluated six PM10 fraction levels (10.0-7.2, 7.2-3.0, 3.0-1.5, 1.5-0.95, 0.95-0.49, and <0.49µm) and the endotoxin content of each fraction. In this investigation, the microbe contamination of the air varied from low to high levels, while the PM10 and endotoxin levels were limited, reaching rural environmental levels (61.40µg/m(3) and 18.88EU/m(3), respectively). However, contamination and occupational risk must be evaluated individually for each plant because numerous variables influence the risk magnitude, particularly digested sludge treatments, such as input biomass nature, storage, movement conditions, building configuration and technological processes.

Environmental effects of using chelating agents in polluted sediment remediation.

The results of laboratory scale experimental tests of contaminant extraction from marine sediment slurries are presented and discussed. The objective of this study was to compare the effectiveness of EDTA and rhamnolipid in copper removal from an artificially contaminated sediment. The comparison was made in terms of metal extraction yield, and in the evaluation of its mobilization towards the more exchangeable fractions in the sediment. Results show that, under acidic conditions established during washing, EDTA ensured higher extractions efficiencies of Cu (up to 95 %) than rhamnolipid, although there was less mobilization into bioavailable forms with the use of rhamnolipid. In addition, in the view of a biological treatment of the spent solution, the use of rhamnolipid resulted in a lower decrease of the specific oxygen uptake rate with respect to EDTA. In fact, the low surfactants concentration required, partially compensated the toxic effect of Cu towards biomass.

A molecular and bioinformatic study on the ochratoxin A (OTA)-producing Aspergillus affinis (section Circumdati).

Aspergillus affinis (section Circumdati) is a novel ochratoxin A (OTA)-producing species found in submerged riparian decomposing leaves. However, very little is known about its role on the breakdown of plant debris and its ability to degrade carbohydrate polymers. Moreover, its OTA biosynthetic pathway has not yet been explored. In the present paper, we investigated the gene encoding the extracellular alpha-amylase (amyAa) of A. affinis within the evolution of the Aspergillus lineages in relation to the possible use of this enzyme in starch processing. The novel amyAa, despite being related to branches of the Aspergillus species of the sections Terrei and Flavi, formed a distinct phylogenetic branch, which may be of outstanding importance from a biotechnological point of view. Moreover, we identified the polyketide synthase gene (pks) putatively required for the first step of OTA biosynthesis in A. affinis. This otapks was examined in relation to a limited number of orthologous genes available from Aspergillus species of the sections Circumdati and Nigri. Our study highlights the importance of otapks as target genes in the treatment of ochratoxigenic Aspergillus species on a more comprehensive evolutionary basis.

Polyhydroxyalkanoate (PHB) as a slow-release electron donor for advanced in situ bioremediation of chlorinated solvent-contaminated aquifers.

During the last two decades permeable reactive barriers (PRBs) established as robust alternatives to traditional pump & treat approaches for groundwater remediation. Zero-valent iron (ZVI) is currently the most frequently employed reactive media, especially for treating plumes polluted by chlorinated hydrocarbons. However PRB-ZVI technology is affected by some problems such as the long-term performance decrease, loss of porosity and no applicability to some important compounds, such as 1,2-dichloroetane (1,2-DCA). In this study we wanted to investigate whether the coupling of ZVI with a long-lasting slow-release substrate (i.e. poly-hydroxybutyrate, PHB) could be a strategy to enhance the degradation performance of ZVI barriers towards chlorinated ethanes especially stimulating biological reductive dechlorination downgradient the PRB. Results here presented clearly demonstrate the feasibility of the proposed approach and the possibility that a biodegradable polymer, usually produced for different commercial sectors, could be advantageously used in the groundwater remediation market.

A molecular study on bacterial resistance to arsenic-toxicity in surface and underground waters of Latium (Italy).

Latium, a region in central Italy, is known for its extensive volcanic areas that make a significant contribution to the arsenic (As) contamination of freshwater environments, even though some degree of As water pollution may be caused by human activities. The information available on indigenous As-resistant prokaryotes in aquatic environments of Latium is, however, still limited. In this study, we describe new bacteria that are resistant to arsenic toxicity and were isolated from the surface waters of Lake Vico and the Sacco River, two groundwater systems in Latium, as well as from bottled natural mineral water from the same region. The 16S rRNA gene sequence analysis for the As-resistant strains in lake and river waters points to a prevalence of ß- and γ-Proteobacteria, while α-Proteobacteria, Firmicutes and Bacteroidetes are represented to a lesser extent. By contrast, solely γ-Proteobacteria were isolated from groundwater samples. The presence of Actinobacteria was documented exclusively in bottled mineral water. In addition, we conducted a DNA sequence-based study on the gene codifying arsB, an As(III) efflux membrane protein pump related to arsenic resistance, for all the As-resistant bacterial isolates. A phylogenetic analysis was carried out on the newly sequenced 16S rRNA genes and arsB in the present study as well as on an additional 16S rRNA/arsB dataset we obtained previously from Lake Albano, from the Tiber and from a well in Bassano Romano located in Latium (Davolos and Pietrangeli, 2011). Overall, the phylogenetic diversity of As-resistant bacteria in underground water was very limited if compared with lentic and lotic waters. Lastly, our molecular data support the hypothesis that the horizontal gene transfer of ars in As-containing freshwater environments is not limited to closely-related genomes, but also occurs between bacteria that are distant from an evolutionary viewpoint, thereby indicating that such genetic events may be considered a source of microbial resistance to arsenic-toxicity.

CARD-FISH analysis of a TCE-dechlorinating biocathode operated at different set potentials.

Bioelectrochemical systems (BES) are increasingly being considered for bioremediation applications, such as the reductive transformation of chlorinated hydrocarbons in subsurface environments. These systems typically rely on a polarized solid-state electrode (i.e. a cathode) serving as electron donor for the microbially catalyzed reductive dechlorination of chlorinated contaminants. The microorganisms involved in dechlorinating biocathodes are not still identified. Particularly, it is not clear whether the same microorganisms responsible for the reductive dechlorination in 'conventional' bioremediation systems (i.e. those based on the supply of soluble substrates as electron donors) also play a role in BES. Here, we analyzed by CARD-FISH, the microbial composition of a dechlorinating biocathode operated at different set potential, in the range from -250 mV to -750 mV (vs. the standard hydrogen electrode, SHE). The rate and extent of TCE dechlorination, as well as of competing metabolisms (i.e. methanogenesis), were found to increase as the cathode potential decreased. The higher metabolic activities observed at the more reducing cathode potentials were mirrored by a higher total biomass concentration (as DAPI-stained cells) in the cathode effluent. CARD-FISH analysis revealed that Dehalococcoides was the dominant dechlorinating bacterial genus (from 65% to 100% of Bacteria) in the range from -550 mV to -750 mV, whereas it was abruptly outcompeted by other (yet unidentified) members of the Chloroflexi phylum, when the cathode was controlled in the range from -250 mV to -450 mV. Most probably, the observed changes in the microbial composition of the biocathode were driven by changes in the dominant mechanisms of electron transfer to TCE: mediated by the electrolytic production of H(2) gas (in the range from -550 mV to -750 mV), or direct (in the range of cathode potentials from -250 mV to -450 mV).

A new molecular approach based on qPCR for the quantification of fecal bacteria in contaminated marine sediments.

Harbour sediments are periodically subjected to dredging operations and their management is mainly based on the assessment of the chemical contamination levels, but the potential risks posed by the presence of pathogenic microorganisms have been largely neglected. Here we first developed new molecular protocols based on the use of Real Time Quantitative PCR (qPCR), targeting both bacterial DNA and the transcription product (rRNA), for the identification and quantification of bacteria of fecal origin (Escherichia coli, Enterococcus spp. and Salmonella spp.) in contaminated harbour sediments. Then, we assessed the effects of bioremediation treatments, conventionally utilized for abating the hydrocarbon contamination in the sediment, on the abundance of fecal bacteria (FB). The qPCR technique was highly specific, sensitive and reproducible, and detected a number of fecal bacteria significantly higher than the classical cultivation techniques. Sediments subjected to bioremediation experiments by biostimulation with inorganic nutrients at different temperatures displayed a significant increase of the abundance of E. coli and Enterococcus spp. These findings suggest the risk of a potential increase of the contamination by pathogenic microorganisms of fecal origin during bioremediation and, as such, highlight the importance of careful monitoring this biological component in harbour sediments when subjected to bio-treatments.

Penicillium simile sp. nov. revealed by morphological and phylogenetic analysis.

The morphology of three phenetically identical Penicillium isolates, collected from the bioaerosol in a restoration laboratory in Italy, displayed macro- and microscopic characteristics that were similar though not completely ascribable to Penicillium raistrickii. For this reason, a phylogenetic approach based on DNA sequencing analysis was performed to establish both the taxonomic status and the evolutionary relationships of these three peculiar isolates in relation to previously described species of the genus Penicillium. We used four nuclear loci (both rRNA and protein coding genes) that have previously proved useful for the molecular investigation of taxa belonging to the genus Penicillium at various evolutionary levels. The internal transcribed spacer region (ITS1-5.8S-ITS2), domains D1 and D2 of the 28S rDNA, a region of the tubulin beta chain gene (benA) and part of the calmodulin gene (cmd) were amplified by PCR and sequenced. Analysis of the rRNA genes and of the benA and cmd sequence data indicates the presence of three isogenic isolates belonging to a genetically distinct species of the genus Penicillium, here described and named Penicillium simile sp. nov. (ATCC MYA-4591(T)  = CBS 129191(T)). This novel species is phylogenetically different from P. raistrickii and other related species of the genus Penicillium (e.g. Penicillium scabrosum), from which it can be distinguished on the basis of morphological trait analysis.

Aspergillus affinis sp. nov., a novel ochratoxin A-producing Aspergillus species (section Circumdati) isolated from decomposing leaves.

Two ochratoxin A (OTA)-producing Aspergillus isolates, recently collected from submerged riparian decomposing leaves in Italy, were found to have a similar morphology to Aspergillus cretensis (subgenus Circumdati, section Circumdati). However, marked differences emerged between these two novel isolates and A. cretensis as the former displayed different colony features and had larger vesicles, metulae, phialides and conidia, as well as a distinct sclerotial form and size. In order to determine the taxonomic status and to infer the evolutionary relationships of these two morphologically identical isolates, a molecular phylogenetic analysis was performed on all the officially recognized lineages in the section Circumdati. The DNA sequences and the deduced amino acid residues from the nuclear loci were analysed. Both rRNA and protein coding genes were assessed, which are widely used to differentiate taxa belonging to genus Aspergillus at various evolutionary levels. The 5.8S rDNA gene and internal transcribed spacers (ITS), the D1/D2 domains of the 28S rDNA gene, a region of the tubulin beta chain gene (benA) and part of the calmodulin gene (cmd) were amplified by PCR and then sequenced. The analysis of the rRNA regions and of the benA and cmd sequence data indicated that the two isogenic isolates belonged to a genetically distinct OTA-producing species of the genus Aspergillus. The isolates are proposed as representing a novel species, Aspergillus affinis sp. nov., with the type strain ATCC MYA-4773T=CBS 129190=417). Phylogenetically, A. affinis sp. nov. appeared to be very closely related to A. cretensis, from which it could be distinguished by means of a morphological trait analysis.

The role of different methanogen groups evaluated by Real-Time qPCR as high-efficiency bioindicators of wet anaerobic co-digestion of organic waste.

Methanogen populations and their domains are poorly understood; however, in recent years, research on this topic has emerged. The relevance of this field has also been enhanced by the growing economic interest in methanogen skills, particularly the production of methane from organic substrates. Management attention turned to anaerobic wastes digestion because the volume and environmental impact reductions. Methanogenesis is the biochemically limiting step of the process and the industrially interesting phase because it connects to the amount of biogas production. For this reason, several studies have evaluated the structure of methanogen communities during this process. Currently, it is clear that the methanogen load and diversity depend on the feeding characteristics and the process conditions, but not much data is available. In this study, we apply a Real-Time Polymerase Chain Reaction (RT-PCR) method based on mcrA target to evaluate, by specific probes, some subgroups of methanogens during the mesophilic anaerobic digestion process fed wastewater sludge and organic fraction of the municipal solid waste with two different pre-treatments. The obtained data showed the prevalence of Methanomicrobiales and significantly positive correlation between Methanosarcina and Methanosaetae and the biogas production rate (0.744 p < 0.01 and 0.641 p < 0.05). Methanosarcina detected levels are different during the process after the two pre-treatment of the input materials (T-test p < 0.05). Moreover, a role as diagnostic tool could be suggested in digestion optimisation.

The interplay of StyR and IHF regulates substrate-dependent induction and carbon catabolite repression of styrene catabolism genes in Pseudomonas fluorescens ST.

BACKGROUND: In Pseudomonas fluorescens ST, the promoter of the styrene catabolic operon, PstyA, is induced by styrene and is subject to catabolite repression. PstyA regulation relies on the StyS/StyR two-component system and on the IHF global regulator. The phosphorylated response regulator StyR (StyR-P) activates PstyA in inducing conditions when it binds to the high-affinity site STY2, located about -40 bp from the transcription start point. A cis-acting element upstream of STY2, named URE, contains a low-affinity StyR-P binding site (STY1), overlapping the IHF binding site. Deletion of the URE led to a decrease of promoter activity in inducing conditions and to a partial release of catabolite repression. This study was undertaken to assess the relative role played by IHF and StyR-P on the URE, and to clarify if PstyA catabolite repression could rely on the interplay of these regulators. RESULTS: StyR-P and IHF compete for binding to the URE region. PstyA full activity in inducing conditions is achieved when StyR-P and IHF bind to site STY2 and to the URE, respectively. Under catabolite repression conditions, StyR-P binds the STY1 site, replacing IHF at the URE region. StyR-P bound to both STY1 and STY2 sites oligomerizes, likely promoting the formation of a DNA loop that closes the promoter in a repressed conformation. We found that StyR and IHF protein levels did not change in catabolite repression conditions, implying that PstyA repression is achieved through an increase in the StyR-P/StyR ratio. CONCLUSION: We propose a model according to which the activity of the PstyA promoter is determined by conformational changes. An open conformation is operative in inducing conditions when StyR-P is bound to STY2 site and IHF to the URE. Under catabolite repression conditions StyR-P cellular levels would increase, displacing IHF from the URE and closing the promoter in a repressed conformation. The balance between the open and the closed promoter conformation would determine a fine modulation of the promoter activity. Since StyR and IHF protein levels do not vary in the different conditions, the key-factor regulating PstyA catabolite repression is likely the kinase activity of the StyR-cognate sensor protein StyS.

A molecular phylogenetic study of the freshwater talitrid amphipod Orchestia cavimana (Crustacea).

In this paper we performed a molecular phylogenetic study of Orchestia cavimana, the sole talitrid amphipod inhabiting beaches of European freshwater lakes and rivers. For that purpose, we have PCR amplified and sequenced regions of the mitochondrial cytochrome oxidase subunit I (COI) gene, basing our analysis on both nucleotide and amino acid sequences and considering also structural classes of the COI enzyme. Phylogenetic analyses were conducted by neighbour-joining (NJ) and maximum-parsimony (MP) methods comparing homologous sequences of talitrids and other Crustacea. In both NJ and MP trees, O. cavimana shows a basal placement with respect to other talitrid amphipods.