Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase

A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1

Metaproteomics and metabolomics analyses of chronically petroleum-polluted sites reveal the importance of general anaerobic processes uncoupled with degradation

Crude oil is one of the most important natural assets for humankind, yet it is a major environmental pollutant, notably in marine environments. One of the largest crude oil polluted areas in the word is the semi-enclosed Mediterranean Sea, in which the metabolic potential of indigenous microbial populations towards the large-scale chronic pollution is yet to be defined, particularly in anaerobic and micro-aerophilic sites. Here, we provide an insight into the microbial metabolism in sediments from three chronically polluted marine sites along the coastline of Italy: the Priolo oil terminal/refinery site (near Siracuse, Sicily), harbour of Messina (Sicily) and shipwreck of MT Haven (near Genoa). Using shotgun metaproteomics and community metabolomics approaches, the presence of 651 microbial proteins and 4776 metabolite mass features have been detected in these three environments, revealing a high metabolic heterogeneity between the investigated sites. The proteomes displayed the prevalence of anaerobic metabolisms that were not directly related with petroleum biodegradation, indicating that in the absence of oxygen, biodegradation is significantly suppressed. This suppression was also suggested by examining the metabolome patterns. The proteome analysis further highlighted the metabolic coupling between methylotrophs and sulphate reducers in oxygen-depleted petroleum-polluted sediments

ULIXES, unravelling and exploiting Mediterranean Sea microbial diversity and ecology for xenobiotics' and pollutants' clean up

The civilizations in the Mediterranean Sea have deeply changed the local environment, especially with the extraction of subsurface oil and gas, their refinery and transportation. Major environmental impacts are affecting all the sides of the basin with actual and potential natural and socio-economic problems. Events like the recent BP\u2019s oil disaster in the Gulf of Mexico would have a tremendous impact on a close basin like the Mediterranean Sea. The recently EU-funded project ULIXES (http://www.ulixes.unimi.it/) aims to unravel, categorize, catalogue, exploit and manage the microbial diversity available in the Mediterranean Sea for addressing bioremediation of polluted marine sites. The rationale of the project is based on the multiple diverse environmental niches of the Mediterranean Sea and the huge range of microorganisms inhabiting therein. Microbial consortia and their ecology, their components or products are used for designing novel pollutant- and site-tailored bioremediation approaches. ULIXES exploits microbial resource mining by the isolation of novel microorganisms as well as by novel advanced \u2018meta-omics\u2019 technologies for solving pollution of three major high priority pollutant classes, petroleum hydrocarbons, chlorinated compounds and heavy metals. A network of twelve European and Southern Mediterranean partners is exploring the microbial diversity and ecology associated to a large set of polluted environmental matrices including seashore sands, lagoons, harbors and deep-sea sediments, oil tanker shipwreck sites, as well as coastal and deep sea natural sites where hydrocarbon seepages occur. The mined collections are exploited for developing novel bioremediation processes to be tested in ex situ and in situ field bioremediation trials

Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins

Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70°C of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90°C, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80°C). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical a/b-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of a N-terminal b-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. Copyright © 2023 Distaso et al.This study was conducted under the auspices of the FuturEnzyme Project funded by the European Union’s Horizon 2020 Research and Innovation Program under grant agreement 101000327. M.F. and F.J.P. also acknowledge grants PID2020-112758RB-I00 (M.F.), PDC2021- 121534-I00 (M.F.), TED2021-130544B-I00 (M.F.), and PID2019-105838RB-C31 (F.J.P.) from MCIN/AEI/10.13039/501100011033 and the European Union (“NextGenerationEU/PRTR”). M.A.D., T.N.C., R.B., A.N.K., O.V.G., A.F.Y., and P.N.G. are thankful for support fromthe European Regional Development Fund (ERDF) through the Welsh Government to the Centre for Environmental Biotechnology, project number 81280. P.N.G. and A.F.Y. acknowledge the Natural Environment Research Council UK-funded Plastic Vectors project NE/S004548/1 and the Sêr Cymru program partly funded by the ERDF through the Welsh Government for support of the project BioPOL4Life.We are indebted to Connie Tulloch and GwionWilliams for their technical support.Supporting InformationPeer reviewe

Miniband-related 1.4–1.8 μm luminescence of Ge/Si quantum dot superlattices

The luminescence properties of highly strained, Sb-doped Ge/Si multi-layer heterostructures with incorporated Ge quantum dots (QDs) are studied. Calculations of the electronic band structure and luminescence measurements prove the existence of an electron miniband within the columns of the QDs. Miniband formation results in a conversion of the indirect to a quasi-direct excitons takes place. The optical transitions between electron states within the miniband and hole states within QDs are responsible for an intense luminescence in the 1.4–1.8 µm range, which is maintained up to room temperature. At 300 K, a light emitting diode based on such Ge/Si QD superlattices demonstrates an external quantum efficiency of 0.04% at a wavelength of 1.55 µm

Phase Behavior of Aqueous Na-K-Mg-Ca-CI-NO3 Mixtures: Isopiestic Measurements and Thermodynamic Modeling

A comprehensive model has been established for calculating thermodynamic properties of multicomponent aqueous systems containing the Na{sup +}, K{sup +}, Mg{sup 2+}, Ca{sup 2+}, Cl{sup -}, and NO{sub 3}{sup -} ions. The thermodynamic framework is based on a previously developed model for mixed-solvent electrolyte solutions. The framework has been designed to reproduce the properties of salt solutions at temperatures ranging from the freezing point to 300 C and concentrations ranging from infinite dilution to the fused salt limit. The model has been parameterized using a combination of an extensive literature database and new isopiestic measurements for thirteen salt mixtures at 140 C. The measurements have been performed using Oak Ridge National Laboratory's (ORNL) previously designed gravimetric isopiestic apparatus, which makes it possible to detect solid phase precipitation. Water activities are reported for mixtures with a fixed ratio of salts as a function of the total apparent salt mole fraction. The isopiestic measurements reported here simultaneously reflect two fundamental properties of the system, i.e., the activity of water as a function of solution concentration and the occurrence of solid-liquid transitions. The thermodynamic model accurately reproduces the new isopiestic data as well as literature data for binary, ternary and higher-order subsystems. Because of its high accuracy in calculating vapor-liquid and solid-liquid equilibria, the model is suitable for studying deliquescence behavior of multicomponent salt systems

<i>Halanaeroarchaeum sulfurireducens</i> gen. nov., sp. nov., the first obligately anaerobic sulfur-respiring haloarchaeon, isolated from a hypersaline lake

Anaerobic enrichments with acetate as electron donor and carbon source, and elemental sulfur as electron acceptor at 4?M NaCl using anaerobic sediments and brines from several hypersaline lakes in Kulunda Steppe (Altai, Russia) resulted in isolation in pure culture of four strains of obligately anaerobic haloarchae growing exclusively by sulfur respiration. Such metabolism has not yet been demonstrated in any known species of Halobacteria, and in the whole archaeal kingdom, acetate oxidation with sulfur as acceptor was not previously demonstrated. The four isolates had nearly identical 16S rRNA gene sequences and formed a novel genus-level branch within the family Halobacteriaceae . The strains had a restricted substrate range limited to acetate and pyruvate as electron donors and elemental sulfur as electron acceptor. In contrast to aerobic haloarchaea, the biomass of anaerobic isolates completely lacked the typical red pigments. Growth with acetate+sulfur was observed between 3–5?M NaCl and at a pH range from 6.7 to 8.0. The membrane core lipids were dominated by archaeols. On the basis of distinct physiological and phylogenetic data, the sulfur-respiring isolates represent a novel species of a new genus in the family Halobacteriaceae, for which the name Halanaeroarchaeaum sulfurireducens gen. nov., sp. nov. is proposed. The type strain of the type species is HSR2T (=JCM 30661T=UNIQEM U935T)

Corrigendum: High representation of archaea across all depths in oxic and low-pH sediment layers underlying an acidic stream

In the original article, there was a mistake. The incorrect Figure 1 was published..