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Abstract Background Pure bacterial strains give better yields when producing H2 than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H2 production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research. Results Four pure Clostridium cultures, including C. butyricum CWBI1009, C. pasteurianum DSM525, C. beijerinckii DSM1820 and C. felsineum DSM749, and three different co-cultures composed of (1) C. pasteurianum and C. felsineum, (2) C. butyricum and C. felsineum, (3) C. butyricum and C. pasteurianum, were grown in 20 L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H2 production (sequence 1); and the H2-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H2 yields were obtained for starch fermentations, and the highest yield of 2.91 mol H2/ mol hexose was reported for C. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5 L biogas/ h was observed for the co-culture (1). In general co-cultures produced H2 at higher rates than the pure Clostridium cultures, without negatively affecting the H2 yields. Interestingly, all the Clostridium strains and co-cultures were shown to utilize lactate (present in a starch-containing medium), and C. beijerinckii was able to re-consume formate producing additional H2. In the second part of the study the co-culture (3) was used to produce H2 during 13 days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence of C. pasteurianum and C. butyricum during this fermentation. Conclusions The four pure Clostridium strains and the artificial co-cultures tested in this study were shown to efficiently produce H2 using glucose and starch as carbon sources. The artificial co-cultures produced H2 at higher rates than the pure strains, while the H2 yields were only slightly affected.

Abstract Background Aquaporins are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. These proteins are vital for maintaining water homeostasis in living organisms. In mammals, thirteen aquaporins (AQP0-12) have been characterized, but in lower vertebrates, such as fish, the diversity, structure and substrate specificity of these membrane channel proteins are largely unknown. Results The screening and isolation of transcripts from the zebrafish (Danio rerio) genome revealed eighteen sequences structurally related to the four subfamilies of tetrapod aquaporins, i.e., aquaporins (AQP0, -1 and -4), water and glycerol transporters or aquaglyceroporins (Glps; AQP3 and AQP7-10), a water and urea transporter (AQP8), and two unorthodox aquaporins (AQP11 and -12). Phylogenetic analyses of nucleotide and deduced amino acid sequences demonstrated dual paralogy between teleost and human aquaporins. Three of the duplicated zebrafish isoforms have unlinked loci, two have linked loci, while DrAqp8 was found in triplicate across two chromosomes. Genomic sequencing, structural analysis, and maximum likelihood reconstruction, further revealed the presence of a putative pseudogene that displays hybrid exons similar to tetrapod AQP5 and -1. Ectopic expression of the cloned transcripts in Xenopus laevis oocytes demonstrated that zebrafish aquaporins and Glps transport water or water, glycerol and urea, respectively, whereas DrAqp11b and -12 were not functional in oocytes. Contrary to humans and some rodents, intrachromosomal duplicates of zebrafish AQP8 were water and urea permeable, while the genomic duplicate only transported water. All aquaporin transcripts were expressed in adult tissues and found to have divergent expression patterns. In some tissues, however, redundant expression of transcripts encoding two duplicated paralogs seems to occur. Conclusion The zebrafish genome encodes the largest repertoire of functional vertebrate aquaporins with dual paralogy to human isoforms. Our data reveal an early and specific diversification of these integral membrane proteins at the root of the crown-clade of Teleostei. Despite the increase in gene copy number, zebrafish aquaporins mostly retain the substrate specificity characteristic of the tetrapod counterparts. Based upon the integration of phylogenetic, genomic and functional data we propose a new classification for the piscine aquaporin superfamily.

Polymer production is rapidly increasing, but there are no large-scale technologies available to effectively mitigate the massive accumulation of these recalcitrant materials. One potential solution is the development of a carbon-neutral polymer life cycle, where microorganisms convert plant biomass to chemicals, which are used to synthesize biodegradable materials that ultimately contribute to the growth of new plants. Realizing a circular carbon life cycle requires the integration of knowledge across microbiology, bioengineering, materials science, and organic chemistry, which itself has hindered large-scale industrial advances. This review addresses the biodegradation status of common synthetic polymers, identifying novel microbes and enzymes capable of metabolizing these recalcitrant materials and engineering approaches to enhance their biodegradation pathways. Design considerations for the next generation of biodegradable polymers are also reviewed, and finally, opportunities to apply findings from lignocellulosic biodegradation to the design and biodegradation of similarly recalcitrant synthetic polymers are discussed.

Background: Members of the Planctomycetota phylum harbour an outstanding potential for carbohydrate degradation given the abundance and diversity of carbohydrate-active enzymes (CAZymes) encoded in their genomes. However, mainly members of the Planctomycetia class have been characterised up to now, and little is known about the degrading capacities of the other Planctomycetota. Here, we present a comprehensive comparative analysis of all available planctomycetotal genome representatives and detail encoded carbohydrolytic potential across phylogenetic groups and different habitats., Results: Our in-depth characterisation of the available planctomycetotal genomic resources increases our knowledge of the carbohydrolytic capacities of Planctomycetota. We show that this single phylum encompasses a wide variety of the currently known CAZyme diversity assigned to glycoside hydrolase families and that many members encode a versatile enzymatic machinery towards complex carbohydrate degradation, including lignocellulose. We highlight members of the Isosphaerales, Pirellulales, Sedimentisphaerales and Tepidisphaerales orders as having the highest encoded hydrolytic potential of the Planctomycetota. Furthermore, members of a yet uncultivated group affiliated to the Phycisphaerales order could represent an interesting source of novel lytic polysaccharide monooxygenases to boost lignocellulose degradation. Surprisingly, many Planctomycetota from anaerobic digestion reactors encode CAZymes targeting algal polysaccharides - this opens new perspectives for algal biomass valorisation in biogas processes., Conclusions: Our study provides a new perspective on planctomycetotal carbohydrolytic potential, highlighting distinct phylogenetic groups which could provide a wealth of diverse, potentially novel CAZymes of industrial interest., (© 2024. The Author(s).)

The oceans harbour a myriad of unknown micro-organisms that remain unstudied because of a failure to establish the right growth conditions under laboratory conditions. To overcome this limitation, an isolation effort inspired by the iChip was performed using marine sediments from Memória beach, Portugal. A novel strain, PMIC_1C1B T , was obtained and subjected to a polyphasic study. Cells of strain PMIC_1C1B T were Gram-positive, rod-shaped, divided by binary fission and formed colonies that were shiny light-yellow. Based on its full 16S rRNA gene sequence, strain PMIC_1C1B T was phylogenetically associated to the genus Microbacterium and its closest relatives were Microbacterium aurum KACC 15219 T (98.55 %), Microbacterium diaminobutyricum RZ63 T (98.48 %) and Microbacterium hatanonis JCM 14558 T (98.13 %). Strain PMIC_1C1B T had a genome size of 2 761 607 bp with 67.71 mol% of G+C content and 2582 coding sequences, which is lower than the genus average. Strain PMIC_1C1B T grew from 15 to 30 °C, optimally at 25 °C, at pH 6.0 to 11.0, optimally between pH 6.0 and 8.0, and from 0 to 5 % (w/v) NaCl, optimally between 2.0 and 3.0 %. It grew with casamino acids, glutamine, methionine, N -acetylglucosamine, sodium nitrate, tryptophan, urea and valine as sole nitrogen sources, and arabinose and cellobiose as sole carbon sources. The major cellular fatty acids were anteiso-C 15 : 0 , iso-C 16 : 0 and iso-C 17 : 0 . Genome mining revealed the presence of four biosynthetic gene clusters (BGCs) with low similarities to other known BCGs. Based on the polyphasic data, strain PMIC_1C1B T is proposed to represent a novel species, for which the name Microbacterium memoriense sp. nov. (=CECT 30366 T =LMG 32350 T ) is proposed.

Background: Biogas and biomethane production from the on-farm anaerobic digestion (AD) of animal manure and agri-food wastes could play a key role in transforming Europe's energy system by mitigating its dependence on fossil fuels and tackling the climate crisis. Although ammonia is essential for microbial growth, it inhibits the AD process if present in high concentrations, especially under its free form, thus leading to economic losses. In this study, which includes both metabolic and microbial monitoring, we tested a strategy to restore substrate conversion to methane in AD reactors facing critical free ammonia intoxication., Results: The AD process of three mesophilic semi-continuous 100L reactors critically intoxicated by free ammonia (> 3.5 g_N L -1 ; inhibited hydrolysis and heterotrophic acetogenesis; interrupted methanogenesis) was restored by applying a strategy that included reducing pH using acetic acid, washing out total ammonia with water, re-inoculation with active microbial flora and progressively re-introducing sugar beet pulp as a feed substrate. After 5 weeks, two reactors restarted to hydrolyse the pulp and produced CH 4 from the methylotrophic methanogenesis pathway. The acetoclastic pathway remained inhibited due to the transient dominance of a strictly methylotrophic methanogen (Candidatus Methanoplasma genus) to the detriment of Methanosarcina. Concomitantly, the third reactor, in which Methanosarcina remained dominant, produced CH 4 from the acetoclastic pathway but faced hydrolysis inhibition. After 11 weeks, the hydrolysis, the acetoclastic pathway and possibly the hydrogenotrophic pathway were functional in all reactors. The methylotrophic pathway was no longer favoured. Although syntrophic propionate oxidation remained suboptimal, the final pulp to CH 4 conversion ratio (0.41 ± 0.10 L N _CH 4 g_VS -1 ) was analogous to the pulp biochemical methane potential (0.38 ± 0.03 L N _CH 4 g_VS -1 )., Conclusions: Despite an extreme free ammonia intoxication, the proposed process recovery strategy allowed CH 4 production to be restored in three intoxicated reactors within 8 weeks, a period during which re-inoculation appeared to be crucial to sustain the process. Introducing acetic acid allowed substantial CH 4 production during the recovery period. Furthermore, the initial pH reduction promoted ammonium capture in the slurry, which could allow the field application of the effluents produced by full-scale digesters recovering from ammonia intoxication., (© 2023. The Author(s).)

A bacterial strain was isolated from a brackish water sample of Tagus river, Alcochete, Portugal and was designated TO1_6 T . It forms light pink colonies on M13 medium supplemented with N-acetylglucosamine. Cells are pear-shaped to spherical, form rosettes and divide by budding. Strain TO1_6 T presents a mesophilic and neutrophilic profile, with optimum growth at 20 to 25 °C and pH 7.0 to 7.5, and vitamin supplementation is not required to promote its growth. The genome of the novel isolate is 7.77 Mbp in size and has a DNA G + C content of 56.3%. Based on its 16S rRNA gene sequence, this strain is affiliated with the phylum Planctomycetota. Further taxonomic characterization using additional phylogenetic markers, namely rpoB gene sequence (encoding the β-subunit of the DNA-dependent RNA polymerase), as well as Percentage of conserved proteins, average nucleotide identity and average amino acid identity, suggest the affiliation of strain TO1_6 T to the genus Stieleria, a recently described taxon in the family Pirellulaceae, order Pirellulales and class Planctomycetia. Based on the genotypic, phylogenetic and physiological characterization, we here describe a new species represented by the type strain TO1_6 T (= CECT 30432 T , = LMG 32465 T ), for which the name Stieleria tagensis sp. nov. is proposed., (© 2023. The Author(s).)

An isolation effort focused on sporogenous Actinomycetota from the Tagus estuary in Alcochete, Portugal, yielded a novel actinomycetal strain, designated MTZ3.1 T , which was subjected to a polyphasic taxonomic study. MTZ3.1 T is characterised by morphology typical of members of the genus Streptomyces , with light beige coloured substrate mycelium, which does not release pigments to the culture medium and with helicoidal aerial hyphae that differentiate into spores with a light-grey colour. The phylogeny of MTZ3.1 T , based on the full 16S rRNA gene sequence, indicated that its closest relatives were Streptomyces alkaliterrae OF1 T (98.48 %), Streptomyces chumphonensis KK1-2 T (98.41 %), Streptomyces albofaciens JCM 4342 T (98.34 %), Streoptomyces paromomycinus NBRC 15454 T (98.34 %) and Streptomyces chrestomyceticus NRBC 13444 T (98.34 %). Moreover, average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridisation (dDDH) are below the species cutoff values (ANI 67.70 and 68.35 %, AAI 77.06 and 76.71 % and dDDH 22.10 and 21.50 % for S. alkaliterrae OF1 T and S. chumphonensis KK1-2 T , respectively). Whole genome sequencing revealed that MTZ3.1 T has a genome of 5 644 485 bp with a DNA G+C content of 71.29 mol% and 5044 coding sequences. Physiologically, MTZ3.1 T is strictly aerobic, able to grow at 15-37 °C, optimally at 25 °C and between pH5 and 8 and showed high salinity tolerance, growing with 0-10 %(w/v) NaCl. Major cellular fatty acids are C 15 : 0 , iso-C 15 : 0 , anteiso-C 15 : 0 and iso-C 16 : 0 . Furthermore, it was able to utilise a variety of nitrogen and carbon sources. Antimicrobial screening indicated that MTZ3.1 T has potent anti- Staphylococcus aureus activity. On the basis of the polyphasic data, MTZ3.1 T is proposed to represent a novel species, Streptomyces meridianus sp. nov. (= CECT 30416 T = DSM 114037 T =LMG 32463 T ).

Background: Termites are among the most successful insects on Earth and can feed on a broad range of organic matter at various stages of decomposition. The termite gut system is often referred to as a micro-reactor and is a complex structure consisting of several components. It includes the host, its gut microbiome and fungal gardens, in the case of fungi-growing higher termites. The digestive tract of soil-feeding higher termites is characterised by radial and axial gradients of physicochemical parameters (e.g. pH, O 2 and H 2 partial pressure), and also differs in the density and structure of residing microbial communities. Although soil-feeding termites account for 60% of the known termite species, their biomass degradation strategies are far less known compared to their wood-feeding counterparts., Results: In this work, we applied an integrative multi-omics approach for the first time at the holobiont level to study the highly compartmentalised gut system of the soil-feeding higher termite Labiotermes labralis. We relied on 16S rRNA gene community profiling, metagenomics and (meta)transcriptomics to uncover the distribution of functional roles, in particular those related to carbohydrate hydrolysis, across different gut compartments and among the members of the bacterial community and the host itself. We showed that the Labiotermes gut was dominated by members of the Firmicutes phylum, whose abundance gradually decreased towards the posterior segments of the hindgut, in favour of Bacteroidetes, Proteobacteria and Verrucomicrobia. Contrary to expectations, we observed that L. labralis gut microbes expressed a high diversity of carbohydrate active enzymes involved in cellulose and hemicelluloses degradation, making the soil-feeding termite gut a unique reservoir of lignocellulolytic enzymes with considerable biotechnological potential. We also evidenced that the host cellulases have different phylogenetic origins and structures, which is possibly translated into their different specificities towards cellulose. From an ecological perspective, we could speculate that the capacity to feed on distinct polymorphs of cellulose retained in soil might have enabled this termite species to widely colonise the different habitats of the Amazon basin., Conclusions: Our study provides interesting insights into the distribution of the hydrolytic potential of the highly compartmentalised higher termite gut. The large number of expressed enzymes targeting the different lignocellulose components make the Labiotermes worker gut a relevant lignocellulose-valorising model to mimic by biomass conversion industries., (© 2023. The Author(s).)

Bacteria within the phylum Planctomycetota are biologically relevant due to unique characteristics among prokaryotes. Members of the genus Rhodopirellula can be abundant in marine habitats, however, only six species are currently validly described. In this study, we expand the explored genus diversity by formally describing a novel species. The pink-coloured strain ICT_H3.1 T was isolated from brackish sediments collected in the Tagus estuary (Portugal) and a 16S rRNA gene sequence-based analysis placed this strain into the genus Rhodopirellula (family Pirellulaceae). The closest type strain is Rhodopirellula rubra LF2 T , suggested by a similarity of 98.4% of the 16S rRNA gene sequence. Strain ICT_H3.1 T is heterotrophic, aerobic and able to grow under microaerobic conditions. The strain grows between 15 and 37 °C, over a range of pH 6.5 to 11.0 and from 1 to 8% (w/v) NaCl. Several nitrogen and carbon sources were utilized by the novel isolate. Cells have an elongated pear-shape with 2.0 ± 0.3 × 0.9 ± 0.2 µm in size. Cells of strain ICT_H3.1 T cluster in rosettes through a holdfast structure and divide by budding. Younger cells are motile. Ultrathin cell sections show cytoplasmic membrane invaginations and polar fimbriae. The genome size is 9,072,081 base pairs with a DNA G + C content of 56.1 mol%. Genomic, physiological and morphological comparison of strain ICT_H3.1 T with its relatives suggest that it belongs to a novel species within the genus Rhodopirellula. Hence, we propose the name Rhodopirellula aestuarii sp. nov., represented by ICT_H3.1 T (=CECT30431 T  = LMG32464 T ) as the type strain of this novel species. 16S rRNA gene accession number: GenBank = OK001858. Genome accession number: The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAMQBK000000000. The version described in this paper is version JAMQBK010000000., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

The phylum Planctomycetota is known for having uncommon biological features. Recently, biotechnological applications of its members have started to be explored, namely in the genus Stieleria . Here, we formally describe a novel Stieleria isolate designated as strain ICT_E10.1 T , obtained from sediments collected in the Tagus estuary (Portugal). Strain ICT_E10.1 T is pink-pigmented, spherical to ovoid in shape, and 1.7 µm ± 0.3 × 1.4 µm ± 0.3 in size. Cells cluster strongly in aggregates or small chains, divide by budding, and have prominent fimbriae. Strain ICT_E10.1 T is heterotrophic and aerobic. Growth occurs from 20 to 30 °C, from 0.5 to 3% ( w / v ) NaCl, and from pH 6.5 to 11.0. The analysis of the 16S rRNA gene sequence placed strain ICT_E10.1 T into the genus Stieleria with Stieleria neptunia Enr13 T as the closest validly described relative. The genome size is 9,813,311 bp and the DNA G+C content is 58.8 mol%. Morphological, physiological, and genomic analyses support the separation of this strain into a novel species, for which we propose the name Stieleria sedimenti represented by strain ICT_E10.1 T as the type of strain (=CECT 30514 T = DSM 113784 T ). Furthermore, this isolate showed biotechnological potential by displaying relevant biosynthetic gene clusters and potent activity against Staphylococcus aureus ., Competing Interests: The authors declare no conflict of interest.

The mutual nutritional cooperation underpinning syntrophic propionate degradation provides a scant amount of energy for the microorganisms involved, so propionate degradation often acts as a bottleneck in methanogenic systems. Understanding the ecology, physiology and metabolic capacities of syntrophic propionate-oxidizing bacteria (SPOB) is of interest in both engineered and natural ecosystems, as it offers prospects to guide further development of technologies for biogas production and biomass-derived chemicals, and is important in forecasting contributions by biogenic methane emissions to climate change. SPOB are distributed across different phyla. They can exhibit broad metabolic capabilities in addition to syntrophy (e.g. fermentative, sulfidogenic and acetogenic metabolism) and demonstrate variations in interplay with cooperating partners, indicating nuances in their syntrophic lifestyle. In this review, we discuss distinctions in gene repertoire and organization for the methylmalonyl-CoA pathway, hydrogenases and formate dehydrogenases, and emerging facets of (formate/hydrogen/direct) electron transfer mechanisms. We also use information from cultivations, thermodynamic calculations and omic analyses as the basis for identifying environmental conditions governing propionate oxidation in various ecosystems. Overall, this review improves basic and applied understanding of SPOB and highlights knowledge gaps, hopefully encouraging future research and engineering on propionate metabolism in biotechnological processes., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Real-world evaluations of metagenomic reconstructions are challenged by distinguishing reconstruction artifacts from genes and proteins present in situ. Here, we evaluate short-read-only, long-read-only and hybrid assembly approaches on four different metagenomic samples of varying complexity. We demonstrate how different assembly approaches affect gene and protein inference, which is particularly relevant for downstream functional analyses. For a human gut microbiome sample, we use complementary metatranscriptomic and metaproteomic data to assess the metagenomic data-based protein predictions. Our findings pave the way for critical assessments of metagenomic reconstructions. We propose a reference-independent solution, which exploits the synergistic effects of multi-omic data integration for the in situ study of microbiomes using long-read sequencing data., (© The Author(s) 2021. Published by Oxford University Press.)

Background: Termites are among the most successful insect lineages on the globe and are responsible for providing numerous ecosystem services. They mainly feed on wood and other plant material at different stages of humification. Lignocellulose is often a principal component of such plant diet, and termites largely rely on their symbiotic microbiota and associated enzymes to decompose their food efficiently. While lower termites and their gut flagellates were given larger scientific attention in the past, the gut lignocellulolytic bacteria of higher termites remain less explored. Therefore, in this study, we investigated the structure and function of gut prokaryotic microbiomes from 11 higher termite genera representative of Syntermitinae, Apicotermitinae, Termitidae and Nasutitermitinae subfamilies, broadly grouped into plant fibre- and soil-feeding termite categories., Results: Despite the different compositional structures of the studied termite gut microbiomes, reflecting well the diet and host lineage, we observed a surprisingly high functional congruency between gut metatranscriptomes from both feeding groups. The abundance of transcripts encoding for carbohydrate active enzymes as well as expression and diversity profiles of assigned glycoside hydrolase families were also similar between plant fibre- and soil-feeding termites. Yet, dietary imprints highlighted subtle metabolic differences specific to each feeding category. Roughly, 0.18% of de novo re-constructed gene transcripts were shared between the different termite gut microbiomes, making each termite gut a unique reservoir of genes encoding for potentially industrially applicable enzymes, e.g. relevant to biomass degradation. Taken together, we demonstrated the functional equivalence in microbial populations across different termite hosts., Conclusions: Our results provide valuable insight into the bacterial component of the termite gut system and significantly expand the inventory of termite prokaryotic genes participating in the deconstruction of plant biomass. Video Abstract.

Miscanthus sp. biomass could satisfy future biorefinery value chains. However, its use is largely untapped due to high recalcitrance. The termite and its gut microbiome are considered the most efficient lignocellulose degrading system in nature. Here, we investigate at holobiont level the dynamic adaptation of Cortaritermes sp. to imposed Miscanthus diet, with a long-term objective of overcoming lignocellulose recalcitrance. We use an integrative omics approach combined with enzymatic characterisation of carbohydrate active enzymes from termite gut Fibrobacteres and Spirochaetae. Modified gene expression profiles of gut bacteria suggest a shift towards utilisation of cellulose and arabinoxylan, two main components of Miscanthus lignocellulose. Low identity of reconstructed microbial genomes to closely related species supports the hypothesis of a strong phylogenetic relationship between host and its gut microbiome. This study provides a framework for better understanding the complex lignocellulose degradation by the higher termite gut system and paves a road towards its future bioprospecting.

Termites are eusocial insects having evolved several feeding, nesting and reproductive strategies. Among them, inquiline termites live in a nest built by other termite species: some of them do not forage outside the nest, but feed on food stored by the host or on the nest material itself. In this study, we characterized some dimensions of the ecological niche of Cavitermes tuberosus (Termitidae: Termitinae), a broad-spectrum inquiline termite with a large neotropical distribution, to explain its ecological success. We used an integrative framework combining ecological measures (physico-chemical parameters, stable isotopic ratios of N and C) and Illumina MiSeq sequencing of 16S rRNA gene to identify bacterial communities and to analyse termites as well as the material from nests constructed by different termite hosts (the builders). Our results show that (1) nests inhabited by C. tuberosus display a different physico-chemical composition when compared to nests inhabited by its builder alone; (2) stable isotopic ratios suggest that C. tuberosus feeds on already processed, more humified, nest organic matter; and (3) the gut microbiomes cluster by termite species, with the one of C. tuberosus being much more diverse and highly similar to the one of its main host, Labiotermes labralis. These results support the hypothesis that C. tuberosus is a generalist nest feeder adapted to colonize nests built by various builders, and explain its ecological success.

Increased hydrolysis of easily digestible biomass may lead to acidosis of anaerobic reactors and decreased methane production. Previously, it was shown that the structure of microbial communities changed during acidosis; however, once the conditions are back to optimal, biogas (initially CO 2 ) production quickly restarts. This suggests the retention of the community functional redundancy during the process failure. In this study, with the use of metagenomics and downstream bioinformatics analyses, we characterize the carbohydrate hydrolytic potential of the microbial community, with a special focus on acidosis. To that purpose, carbohydrate-active enzymes were identified, and to further link the community hydrolytic potential with key microbes, bacterial genomes were reconstructed. In addition, we characterized biochemically the specificity and activity of selected enzymes, thus verifying the accuracy of the in silico predictions. The results confirm the retention of the community hydrolytic potential during acidosis and indicate Bacteroidetes to be largely involved in biomass degradation. Bacteroidetes showed higher diversity and genomic content of carbohydrate hydrolytic enzymes that might favor the dominance of this phylum over other bacteria in some anaerobic reactors. The combination of bioinformatic analyses and activity tests enabled us to propose a model of acetylated glucomannan degradation by Bacteroidetes IMPORTANCE The enzymatic hydrolysis of lignocellulosic biomass is mainly driven by the action of carbohydrate-active enzymes. By characterizing the gene profiles at the different stages of the anaerobic digestion experiment, we showed that the microbiome retains its hydrolytic functional redundancy even during severe acidosis, despite significant changes in taxonomic composition. By analyzing reconstructed bacterial genomes, we demonstrate that Bacteroidetes hydrolytic gene diversity likely favors the abundance of this phylum in some anaerobic digestion systems. Further, we observe genetic redundancy within the Bacteroidetes group, which accounts for the preserved hydrolytic potential during acidosis. This work also uncovers new polysaccharide utilization loci involved in the deconstruction of various biomasses and proposes the model of acetylated glucomannan degradation by Bacteroidetes Acetylated glucomannan-enriched biomass is a common substrate for many industries, including pulp and paper production. Using naturally evolved cocktails of enzymes for biomass pretreatment could be an interesting alternative to the commonly used chemical pretreatments., (Copyright © 2019 Bertucci et al.)

Ultramafic (i.e. serpentine) soils are widespread in the Balkans and particularly in Albania. They account for a large part of plant endemism in that region and host several hyperaccumulator species, which are characterized by leaf nickel concentrations frequently above 1%. This rich nickel hyperaccumulating flora could serve as candidate to be used in phytoextraction and agromining. Despite recent interest in metal hyperaccumulating plants and agromining, very few studies have investigated the bacterial diversity and the influence of environmental factors on microbial gene profiles in the rhizosphere of hyperaccumulator plants growing on ultramafic soils. Because rhizospheric bacteria could be crucial to the success of phytoremediation, we studied a total of 48 nickel-hyperaccumulating plants which were sampled from four species that are widespread in Albania: Noccaea ochroleuca, Odontarrhena smolikana, O. rigida and O. chalcidica. All samples were taken from the ultramafic regions of Librazhd and Pogradec in eastern Albania in October 2015. Our study shows that Proteobacteria, Actinobacteria and Acidobacteria dominated the soil bacterial communities. Of these three phyla, only Proteobacteria was relatively abundant. This study underlines the influence of soil Cation Exchange Capacity on the bacterial community's diversity and structure. Based on the predicted metagenomes, the genes belonging to amino acid, lipid and carbohydrate metabolisms were identified as major gene families. Our study sheds some light on our understanding of how bacterial communities are structured within and affect the rhizosphere of hyperaccumulator plants from ultramafic soils in Albania., (Copyright © 2018. Published by Elsevier B.V.)

Wolbachia has deeply shaped the ecology and evolution of many arthropods, and interactions between the two partners are a continuum ranging from parasitism to mutualism. Non-dispersing queens of the termite Cavitermes tuberosus are parthenogenetically produced through gamete duplication, a mode of ploidy restoration generally induced by Wolbachia. These queens display a bacteriome-like structure in the anterior part of the mesenteron. Our study explores the possibility of a nutritional mutualistic, rather than a parasitic, association between Wolbachia and C. tuberosus. We found a unique strain (wCtub), nested in the supergroup F, in 28 nests collected in French Guiana, the island of Trinidad and the state of Paraíba, Brazil (over 3500 km). wCtub infects individuals regardless of caste, sex or reproductive (sexual versus parthenogenetic) origin. qPCR assays reveal that Wolbachia densities are higher in the bacteriome-like structure and in the surrounding gut compared to other somatic tissues. High-throughput 16S rRNA gene amplicon sequencing reveals that Wolbachia represents over 97% of bacterial reads present in the bacteriome structure. BLAST analyses of 16S rRNA, bioA (a gene of the biosynthetic pathway of B vitamins) and five multilocus sequence typing genes indicated that wCtub shares 99% identity with wCle, an obligate nutritional mutualist of the bedbug Cimex lectularius.

Polymyxa betae belongs to the Plasmodiophorida (Phytomyxea, Rhizaria). Here, we report the first draft genome sequence of a member of the Polymyxa genus, which includes two obligate root endoparasite species, vectors of important soilborne plant viruses. The genome assembly was represented by 1,001 contigs, with a cumulated length of 27,085,946 bp.

Most of the research dedicated to agromining has focused on cultivating a single hyperaccumulator plant, although plant diversity has been shown to positively modify soil characteristics. Hence, we compared the effect of cropping a nickel-hyperaccumulator Alyssum murale with a legume (Vicia sativa) to A. murale's mono-culture, on the bacterial diversity and physico-chemical characteristics of an ultramafic soil. A pot experiment with 5 replicates was conducted in controlled conditions for 11 months. The treatments studied were: co-cropping and rotation vs. mineral fertilization controls and bare soil. The introduction of legumes induced a clearly positive effect on the soil's microbial biomass carbon and nitrogen. Arylsulfatase and urease activities tended to be enhanced in the co-cropping and rotation treatments and to be lessened in the mineral fertilization treatments. However, β-glucosidase and phosphatase activities were seen to decrease when legumes were used. Our results showed that the rotation treatment induced a higher organic matter content than the fertilized control did. Actinobacteria was the most-represented bacterial phyla and had lower relative abundance in treatments associating legumes. Conversely, the relative abundance of Acidobacteria and Gemmatimonadetes phyla increased but not significantly in treatments with legumes. The relative abundance of Chloroflexi phylum was shown to be significantly higher for the fertilized rotation control. The relative abundance of β-Proteobacteria subphylum increased but not significantly in treatments with legumes. NMDS analysis showed a clear separation between planted treatments and bare soil and between co-cropping and rotation and fertilized controls. Shannon index showed reduction in microbial diversity that was mainly due to chemical inputs in the soil. This study showed that these new cropping systems influenced both the bacterial diversity and the physico-chemical characteristics of an ultramafic soil. In addition, this study provides evidence that mineral fertilization can negatively impact bacterial communities and some of their functions linked to biogeochemical cycles., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Anaerobic digestion (AD) is a microbe-driven process of biomass decomposition to CH 4 and CO 2 . In addition to renewable and cost-effective energy production, AD has emerged in the European Union as an environmentally friendly model of bio-waste valorisation and nutrient recycling. Nevertheless, due to the high diversity of uncharacterised microbes, a typical AD microbiome is still considered as "dark matter"., Results: Using the high-throughput sequencing of small rRNA gene, and a monthly monitoring of the physicochemical parameters for 20 different mesophilic full-scale bioreactors over 1 year, we generated a detailed view of AD microbial ecology towards a better understanding of factors that influence and shape these communities. By studying the broadly distributed OTUs present in over 80% of analysed samples, we identified putatively important core bacteria and archaea to the AD process that accounted for over 70% of the whole microbial community relative abundances. AD reactors localised at the wastewater treatment plants were shown to operate with distinct core microbiomes than the agricultural and bio-waste treating biogas units. We also showed that both the core microbiomes were composed of low (with average community abundance ≤ 1%) and highly abundant microbial populations; the vast majority of which remains yet uncharacterised, e.g. abundant candidate Cloacimonetes . Using non-metric multidimensional scaling, we observed microorganisms grouping into clusters that well reflected the origin of the samples, e.g. wastewater versus agricultural and bio-waste treating biogas units. The calculated diversity patterns differed markedly between the different community clusters, mainly due to the presence of highly diverse and dynamic transient species. Core microbial communities appeared relatively stable over the monitoring period., Conclusions: In this study, we characterised microbial communities in different AD systems that were monitored over a 1-year period. Evidences were shown to support the concept of a core community driving the AD process, whereas the vast majority of dominant microorganisms remain yet to be characterised.

Streptomyces lunaelactis MM109 T is a ferroverdin A (anticholesterol) producer isolated from cave moonmilk deposits. The complete genome sequence of MM109 T was obtained by combining Oxford Nanopore MinION and Illumina HiSeq and MiSeq technologies, revealing an 8.4-Mb linear chromosome and two plasmids, pSLUN1 (127,264 bp, linear) and pSLUN2 (46,827 bp, circular)., (Copyright © 2018 Naômé et al.)

Background: Thanks to specific adaptations developed over millions of years, the efficiency of lignin, cellulose and hemicellulose decomposition of higher termite symbiotic system exceeds that of many other lignocellulose utilizing environments. Especially, the examination of its symbiotic microbes should reveal interesting carbohydrate-active enzymes, which are of primary interest for the industry. Previous metatranscriptomic reports (high-throughput mRNA sequencing) highlight the high representation and overexpression of cellulose and hemicelluloses degrading genes in the termite hindgut digestomes, indicating the potential of this technology in search for new enzymes. Nevertheless, several factors associated with the material sampling and library preparation steps make the metatranscriptomic studies of termite gut prokaryotic symbionts challenging., Methods: In this study, we first examined the influence of the sampling strategy, including the whole termite gut and luminal fluid, on the diversity and the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. Secondly, we evaluated different commercially available kits combined in two library preparative pipelines for the best bacterial mRNA enrichment strategy., Results: We showed that the sampling strategy did not significantly impact the generated results, both in terms of the representation of the microbes and their transcriptomic profiles. Nevertheless collecting luminal fluid reduces the co-amplification of unwanted RNA species of host origin. Furthermore, for the four studied higher termite species, the library preparative pipeline employing Ribo-Zero Gold rRNA Removal Kit "Epidemiology" in combination with Poly(A) Purist MAG kit resulted in a more efficient rRNA and poly-A-mRNAdepletion (up to 98.44% rRNA removed) than the pipeline utilizing MICROBExpress and MICROBEnrich kits. High correlation of both Ribo-Zero and MICROBExpresse depleted gene expression profiles with total non-depleted RNA-seq data has been shown for all studied samples, indicating no systematic skewing of the studied pipelines., Conclusions: We have extensively evaluated the impact of the sampling strategy and library preparation steps on the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. The presented methodological approach has great potential to enhance metatranscriptomic studies of the higher termite intestinal flora and to unravel novel carbohydrate-active enzymes.

Background: Recent advances in high-throughput sequencing allow for much deeper exploitation of natural and engineered microbial communities, and to unravel so-called "microbial dark matter" (microbes that until now have evaded cultivation). Metagenomic analyses result in a large number of genomic fragments (contigs) that need to be grouped (binned) in order to reconstruct draft microbial genomes. While several contig binning algorithms have been developed in the past 2 years, they often lack consensus. Furthermore, these software tools typically lack a provision for the visualization of data and bin characteristics., Results: We present ICoVeR, the Interactive Contig-bin Verification and Refinement tool, which allows the visualization of genome bins. More specifically, ICoVeR allows curation of bin assignments based on multiple binning algorithms. Its visualization window is composed of two connected and interactive main views, including a parallel coordinates view and a dimensionality reduction plot. To demonstrate ICoVeR's utility, we used it to refine disparate genome bins automatically generated using MetaBAT, CONCOCT and MyCC for an anaerobic digestion metagenomic (AD microbiome) dataset. Out of 31 refined genome bins, 23 were characterized with higher completeness and lower contamination in comparison to their respective, automatically generated, genome bins. Additionally, to benchmark ICoVeR against a previously validated dataset, we used Sharon's dataset representing an infant gut metagenome., Conclusions: ICoVeR is an open source software package that allows curation of disparate genome bins generated with automatic binning algorithms. It is freely available under the GPLv3 license at https://git.list.lu/eScience/ICoVeR . The data management and analytical functions of ICoVeR are implemented in R, therefore the software can be easily installed on any system for which R is available. Installation and usage guide together with the example files ready to be visualized are also provided via the project wiki. ICoVeR running instance preloaded with AD microbiome and Sharon's datasets can be accessed via the website.

The goal of this study was to investigate how the microbial community structure establishes during the start-up phase of a full-scale farm anaerobic reactor inoculated with stale and cold cattle slurry. The 16S/18S high-throughput amplicon sequencing results showed an increase of the bacterial, archaeal and eukaryotic diversity, evenness and richness during the settlement of the mesophilic anaerobic conditions. When a steady performing digestion process was reached, the microbial diversity, evenness and richness decreased, indicating the establishment of a few dominant microbial populations, best adapted to biogas production. Interestingly, among the environmental parameters, the temperature, alkalinity, free-NH3, total solids and O2 content were found to be the main drivers of microbial dynamics. Interactions between eukaryotes, characterized by a high number of unknown organisms, and the bacterial and archaeal communities were also evidenced, suggesting that eukaryotes might play important roles in the anaerobic digestion process., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Although viruses are not the key players of the anaerobic digestion process, they may affect the dynamics of bacterial and archaeal populations involved in biogas production. Until now viruses have received very little attention in this specific habitat; therefore, as a first step towards their characterization, we optimized a virus filtration protocol from anaerobic sludge. Afterwards, to assess dsDNA and RNA viral diversity in sludge samples from nine different reactors fed either with waste water, agricultural residues or solid municipal waste plus agro-food residues, we performed metagenomic analyses. As a result we showed that, while the dsDNA viromes (21 assigned families in total) were dominated by dsDNA phages of the order Caudovirales, RNA viruses (14 assigned families in total) were less diverse and were for the main part plant-infecting viruses. Interestingly, less than 2% of annotated contigs were assigned as putative human and animal pathogens. Our study greatly extends the existing view of viral genetic diversity in methanogenic reactors and shows that these viral assemblages are distinct not only among the reactor types but also from nearly 30 other environments already studied, including the human gut, fermented food, deep sea sediments and other aquatic habitats., (© 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: Volatile fatty acid intoxication (acidosis), a common process failure recorded in anaerobic reactors, leads to drastic losses in methane production. Unfortunately, little is known about the microbial mechanisms underlining acidosis and the potential to recover the process. In this study, triplicate mesophilic anaerobic reactors of 100 L were exposed to acidosis resulting from an excessive feeding with sugar beet pulp and were compared to a steady-state reactor., Results: Stable operational conditions at the beginning of the experiment initially led to similar microbial populations in the four reactors, as revealed by 16S rRNA gene T-RFLP and high-throughput amplicon sequencing. Bacteroidetes and Firmicutes were the two dominant phyla, and although they were represented by a high number of operational taxonomic units, only a few were dominant. Once the environment became deterministic (selective pressure from an increased substrate feeding), microbial populations started to diverge between the overfed reactors. Interestingly, most of bacteria and archaea showed redundant functional adaptation to the changing environmental conditions. However, the dominant Bacteroidales were resistant to high volatile fatty acids content and low pH. The severe acidosis did not eradicate archaea and a clear shift in archaeal populations from acetotrophic to hydrogenotrophic methanogenesis occurred in the overfed reactors. After 11 days of severe acidosis (pH 5.2 ± 0.4), the process was quickly recovered (restoration of the biogas production with methane content above 50 %) in the overfed reactors, by adjusting the pH to around 7 using NaOH and NaHCO3., Conclusions: In this study we show that once the replicate reactors are confronted with sub-optimal conditions, their microbial populations start to evolve differentially. Furthermore the alterations of commonly used microbial parameters to monitor the process, such as richness, evenness and diversity indices were unsuccessful to predict the process failure. At the same time, we tentatively propose the replacement of the dominant Methanosaeta sp. in this case by Methanoculleus sp., to be a potential warning indicator of acidosis.

Background: Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels in future transportation and energy production. However, current industrial hydrogen production processes, such as steam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, in particular the use of fermentative microorganisms, have attracted scientific interest in recent years. However the low overall yield obtained is a major challenge in biological H2 production. Thus, a thorough and detailed understanding of the relationships between genome content, gene expression patterns, pathway utilisation and metabolite synthesis is required to optimise the yield of biohydrogen production pathways., Results: In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridium butyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when the metabolism shifts to H2 production. The growth, H2-production, and glucose-fermentation profiles were monitored in 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differences were observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and they were pH dependent. During unregulated-pH glucose fermentation increased H2 production was associated with concurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation of the [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expression levels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, while the nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmental factors may determine whether H2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/or the nitrogenase., Conclusions: This work, contributing to the field of dark fermentative hydrogen production, provides a multidisciplinary approach for the investigation of the processes involved in the molecular H2 metabolism of clostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways based on genetic engineering techniques.

Pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) act via their cognate glycoprotein hormone receptors (GpHRs), FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR) to regulate gonad physiology. Here, we show that the flatfish Senegalese sole (Solea senegalensis) expresses functional isoforms of fshr and lhcgr, but the genomic origin, ligand activation, and tissue distribution of the receptor transcripts are more complex than expected. By integrating the molecular phylogeny of GpHRs with the syntenic loci of vertebrate orthologs, and by subsequently characterizing the physical maps with the phylogeny of flanking genes, we found that vertebrate GpHRs have undergone a divergent evolution. In Teleostei, fshr genes have a common descent and can be classified as fshra, whereas lhcgrb genes exist as alternatively coded genes even in closely related species. Structural analyses of the receptors revealed that Fshra has an elongated ligand-binding domain, containing an extra leucine-rich repeat that specifically arose in the Acanthomorpha because of exon duplication. Ectopic expression in Xenopus laevis oocytes demonstrated that sole Fshra responded to piscine Fsh and Lh, whereas Lhcgrba was preferentially activated by its cognate hormone. The expression pattern of sole fshra and lhcgrba in gonads during the reproductive cycle was consistent with earlier observations wherein Fshra regulates ovarian growth and spermatogenesis and Lhcgrb triggers gamete maturation, respectively. However, contrary to observations in other teleosts, fshra was localized exclusively in Sertoli cells of the testis, whereas lhcgrba was expressed in Leydig cells as well as in spermatids. These results demonstrate the presence of alternatively coded lhcgr isoforms (lhcgrba and lhcgrbb) in teleosts and suggest a role of the lhcgrba receptor in the differentiation of spermatids into spermatozoa in Senegalese sole.

Among the large variety of micro-organisms capable of fermentative hydrogen production, strict anaerobes such as members of the genus Clostridium are the most widely studied. They can produce hydrogen by a reversible reduction of protons accumulated during fermentation to dihydrogen, a reaction which is catalysed by hydrogenases. Sequenced genomes provide completely new insights into the diversity of clostridial hydrogenases. Building on previous reports, we found that [FeFe] hydrogenases are not a homogeneous group of enzymes, but exist in multiple forms with different modular structures and are especially abundant in members of the genus Clostridium. This unusual diversity seems to support the central role of hydrogenases in cell metabolism. In particular, the presence of multiple putative operons encoding multisubunit [FeFe] hydrogenases highlights the fact that hydrogen metabolism is very complex in this genus. In contrast with [FeFe] hydrogenases, their [NiFe] hydrogenase counterparts, widely represented in other bacteria and archaea, are found in only a few clostridial species. Surprisingly, a heteromultimeric Ech hydrogenase, known to be an energy-converting [NiFe] hydrogenase and previously described only in methanogenic archaea and some sulfur-reducing bacteria, was found to be encoded by the genomes of four cellulolytic strains: Clostridum cellulolyticum, Clostridum papyrosolvens, Clostridum thermocellum and Clostridum phytofermentans.

Background: Aquaporins are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. These proteins are vital for maintaining water homeostasis in living organisms. In mammals, thirteen aquaporins (AQP0-12) have been characterized, but in lower vertebrates, such as fish, the diversity, structure and substrate specificity of these membrane channel proteins are largely unknown., Results: The screening and isolation of transcripts from the zebrafish (Danio rerio) genome revealed eighteen sequences structurally related to the four subfamilies of tetrapod aquaporins, i.e., aquaporins (AQP0, -1 and -4), water and glycerol transporters or aquaglyceroporins (Glps; AQP3 and AQP7-10), a water and urea transporter (AQP8), and two unorthodox aquaporins (AQP11 and -12). Phylogenetic analyses of nucleotide and deduced amino acid sequences demonstrated dual paralogy between teleost and human aquaporins. Three of the duplicated zebrafish isoforms have unlinked loci, two have linked loci, while DrAqp8 was found in triplicate across two chromosomes. Genomic sequencing, structural analysis, and maximum likelihood reconstruction, further revealed the presence of a putative pseudogene that displays hybrid exons similar to tetrapod AQP5 and -1. Ectopic expression of the cloned transcripts in Xenopus laevis oocytes demonstrated that zebrafish aquaporins and Glps transport water or water, glycerol and urea, respectively, whereas DrAqp11b and -12 were not functional in oocytes. Contrary to humans and some rodents, intrachromosomal duplicates of zebrafish AQP8 were water and urea permeable, while the genomic duplicate only transported water. All aquaporin transcripts were expressed in adult tissues and found to have divergent expression patterns. In some tissues, however, redundant expression of transcripts encoding two duplicated paralogs seems to occur., Conclusion: The zebrafish genome encodes the largest repertoire of functional vertebrate aquaporins with dual paralogy to human isoforms. Our data reveal an early and specific diversification of these integral membrane proteins at the root of the crown-clade of Teleostei. Despite the increase in gene copy number, zebrafish aquaporins mostly retain the substrate specificity characteristic of the tetrapod counterparts. Based upon the integration of phylogenetic, genomic and functional data we propose a new classification for the piscine aquaporin superfamily.