Comparative genomic analysis of Planctomycetota potential for polysaccharide degradation identifies biotechnologically relevant microbes.

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.

Microbacterium memoriense sp. nov., a member of the Actinomycetota from marine beach sediment of the north coast of Portugal.

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_1C1BT, was obtained and subjected to a polyphasic study. Cells of strain PMIC_1C1BT 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_1C1BT was phylogenetically associated to the genus Microbacterium and its closest relatives were Microbacterium aurum KACC 15219T (98.55 %), Microbacterium diaminobutyricum RZ63T (98.48 %) and Microbacterium hatanonis JCM 14558T (98.13 %). Strain PMIC_1C1BT 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_1C1BT 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-C15 : 0, iso-C16 : 0 and iso-C17 : 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_1C1BT is proposed to represent a novel species, for which the name Microbacterium memoriense sp. nov. (=CECT 30366T=LMG 32350T) is proposed.

Potential of acetic acid to restore methane production in anaerobic reactors critically intoxicated by ammonia as evidenced by metabolic and microbial monitoring.

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 CH4 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 CH4 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 CH4 conversion ratio (0.41 ± 0.10 LN_CH4 g_VS-1) was analogous to the pulp biochemical methane potential (0.38 ± 0.03 LN_CH4 g_VS-1). CONCLUSIONS: Despite an extreme free ammonia intoxication, the proposed process recovery strategy allowed CH4 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 CH4 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.

Stieleria tagensis sp. nov., a novel member of the phylum Planctomycetota isolated from Tagus River in Portugal.

A bacterial strain was isolated from a brackish water sample of Tagus river, Alcochete, Portugal and was designated TO1_6T. 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_6T 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 ß-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_6T 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_6T (= CECT 30432T, = LMG 32465T), for which the name Stieleria tagensis sp. nov. is proposed.

Streptomyces meridianus sp. nov. isolated from brackish water of the Tagus estuary in Alcochete, Portugal.

An isolation effort focused on sporogenous Actinomycetota from the Tagus estuary in Alcochete, Portugal, yielded a novel actinomycetal strain, designated MTZ3.1T, which was subjected to a polyphasic taxonomic study. MTZ3.1T 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 MTZ3.1T, based on the full 16S rRNA gene sequence, indicated that its closest relatives were Streptomyces alkaliterrae OF1T (98.48 %), Streptomyces chumphonensis KK1-2T (98.41 %), Streptomyces albofaciens JCM 4342T (98.34 %), Streoptomyces paromomycinus NBRC 15454T (98.34 %) and Streptomyces chrestomyceticus NRBC 13444T (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 OF1T and S. chumphonensis KK1-2T, respectively). Whole genome sequencing revealed that MTZ3.1T has a genome of 5 644 485 bp with a DNA G+C content of 71.29 mol% and 5044 coding sequences. Physiologically, MTZ3.1T 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 C15 : 0, iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. Furthermore, it was able to utilise a variety of nitrogen and carbon sources. Antimicrobial screening indicated that MTZ3.1T has potent anti-Staphylococcus aureus activity. On the basis of the polyphasic data, MTZ3.1T is proposed to represent a novel species, Streptomyces meridianus sp. nov. (= CECT 30416T = DSM 114037T=LMG 32463T).

A holobiont approach towards polysaccharide degradation by the highly compartmentalised gut system of the soil-feeding higher termite Labiotermes labralis.

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, O2 and H2 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.

Stieleria sedimenti sp. nov., a Novel Member of the Family Pirellulaceae with Antimicrobial Activity Isolated in Portugal from Brackish Sediments.

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 Stieleriaisolate designated as strain ICT_E10.1T, obtained from sediments collected in the Tagus estuary (Portugal). Strain ICT_E10.1T 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_E10.1T 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_E10.1T into the genus Stieleria with Stieleria neptunia Enr13T 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_E10.1T as the type of strain (=CECT 30514T= DSM 113784T). Furthermore, this isolate showed biotechnological potential by displaying relevant biosynthetic gene clusters and potent activity against Staphylococcus aureus.

Rhodopirellula aestuarii sp. nov., a novel member of the genus Rhodopirellula isolated from brackish sediments collected in the Tagus River estuary, Portugal.

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_H3.1T 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 LF2T, suggested by a similarity of 98.4% of the 16S rRNA gene sequence. Strain ICT_H3.1T 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_H3.1T 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_H3.1T 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_H3.1T (=CECT30431T = LMG32464T) 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.

Integrative omics analysis of the termite gut system adaptation to Miscanthus diet identifies lignocellulose degradation enzymes.

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.