Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium, and several new genera described in this study. Potentially interesting functions discovered in these organisms include a fucosyltransferase encoded in the genome of the novel species Clostridium porci, and prevalent gene clusters for biosynthesis of sactipeptide-like peptides. Many strains deconjugate primary bile acids in in vitro assays, and a Clostridium scindens strain produces secondary bile acids via dehydroxylation. In addition, cells of the novel species Bullifex porci are coccoidal or spherical under the culture conditions tested, in contrast with the usual helical shape of other members of the family Spirochaetaceae. The strain collection, called ‘Pig intestinal bacterial collection’ (PiBAC), is publicly available at www.dsmz.de/pibac and opens new avenues for functional studies of the pig gut microbiota.Our knowledge about the gut microbiota of pigs is still scarce, despite the importance of these animals for biomedical research and agriculture. Here, we present a collection of cultured bacteria from the pig gut, including 110 species across 40 families and nine phyla. We provide taxonomic descriptions for 22 novel species and 16 genera. Meta-analysis of 16S rRNA amplicon sequence data and metagenome-assembled genomes reveal prevalent and pig-specific species within  The authors present a public collection of 117 bacterial isolates from the pig gut, including the description of 38 novel taxa. Interesting functions discovered in these organisms include a new fucosyltransferease and sactipeptide-like molecules encoded by biosynthetic gene clusters. For instance, transgenic pig models of colorectal cancer4 and diabetes5 help generating useful preclinical data on molecular mechanisms underlying disease. Pigs are also very important agricultural domestic animals, with an estimated worldwide population nearing one billion individuals. Both biomedical research and agriculture would benefit from enhanced understanding of the pig gut microbiome. Intestinal microorganisms are known to regulate the onset of chronic diseases such as cancer8 and metabolic disorders11. They also influence animal growth13 and play an important role in resistance against enteric infections15. However, underlying mechanisms remain poorly described and microbiome-based applications are hampered due to our still limited knowledge about gut microbiota members17.Pigs are invaluable animals in biomedical research. The physiology of their digestive tract and omnivorous feeding behaviour make them ideal models for studying gastrointestinal and metabolic pathologies where mice hold limited value18. In agreement with other reports20, this study highlights the necessity to assess gut microbiomes in a host-specific manner considering the narrow overlap in terms of gene diversity and the occurrence of bacterial taxa between host species, despite the high similarity of functional potential between ecosystems. The study by Xiao et al.18. also underlines the urgent need to describe novel bacteria, considering that <10% of the genes detected could be taxonomically assigned at the genus level. The renaissance of cultivation has already generated valuable data pertaining to the human and mouse gut microbiota22. In contrast, there has so far been no comprehensive study of cultured bacteria from the pig intestine, despite obvious added value: providing access to well-described bacterial strains will facilitate (i) functional studies to dissect microbe–host interactions underlying diseases in preclinical contexts, and (ii) intervention trials with defined cocktails of commensals as an alternative to antibiotics use. Such trials could particularly influence enteric infection-associated post-weaning diarrhoea that affect millions of pigs worldwide and is linked to the use of colistin, a last-resort antibiotic in human medicine24.Sequencing studies have provided detailed insights into the pig gut microbiome, including a reference catalogue of ~8 million microbial genes using stool from almost 300 pigsIn this work, we aimed to establish the pig intestinal bacterial collection (PiBAC), a publicly available repository of cultured commensal strains from the pig intestine. This includes the formal description of 38 novel bacterial taxa complemented by metagenomic investigations. A detailed survey of the ecological distribution of all taxa as well as new functional insights are presented.n = 91) were detected in 16S rRNA gene amplicon data from 1346 samples at a prevalence >10%, indicating their presence in dominant communities within pig gut microbiota. Seventeen species had a prevalence >50% and a relative abundance >0.5%, suggesting that they are key members of the pig gut microbiome . To help researchers looking for pig gut bacteria besides our own isolates, this online list also contains 31 type strains isolated previously by others from the intestine of pigs and available at the DSMZ  were also determined . Both 16S rRNA gene-based phylogeny 29, we searched the genomes of B. porci and Spirochaetaceae for genes involved in cell morphology and division, peptidoglycan synthesis, and cell wall formation  involved in bacterial binary fission30, ftsQ and ftsX were absent in B. porci .Among the 38 novel taxa, lls Fig. . Anotherogy Fig. . Multiplogy Fig. . As fourion Fig. 34. Interrci Fig. . Both gerci Fig. . Analysirci Fig. . AltogetGenome-based mapping of the collection on KEGG pathways is shown in Supplementary Fig. 36, we bioinformatically analysed the genomes of all 38 novel taxa for the presence of biosynthetic gene clusters (BGCs). Sixty-four putative BGCs were identified in 32 of the 38 strains, encoding a broad variety of biosynthetic systems for natural products such as terpenes, arylpolyones, β-lactones, non-ribosomal peptides (NRPs), and ribosomally synthesized and post-translationally modified peptides (RiPPs)  sequence motif initially defined in clostridia37. However, the sequences were significantly different from precursor peptides of previously characterized sactipeptide-encoding BGCs for subtilosin A38, thurincin H39, sporulation killing factor A (Skf-A)40, thuricin CD 41, and ruminococcin (RumC)42  Fig. . Whereas)42 Fig. . Despite)42 Fig. , which p)42 Fig. . The ant44. Annotation of genomes against the CAZy database (www.cazy.org) revealed that all isolates encoded CAZymes, with a diversity of up to 421 enzymes in the novel species Victivalis lenta compared with an average number of 106 ± 67 for all other species . This and two other species encoded a GT of family 10, which includes alpha--fucosyltransferases (FucT) that are of particular biotechnological interest45 . Its putative FucT contained the FxxxFEN motif reported as conserved within alpha--FucT46. Structural modelling identified the highest structural similarity to 2NZW , a known alpha--FucT. Gene ontology analysis via I-TASSER predicted the highest scoring molecular function as ‘fucosyltransferase activity’ . Heterologous expression and enrichment of this protein from C. porci allowed in vitro testing. The substrate LacNAc type 2 . Whilst detailed biochemistry of this enzyme requires additional work, its identification is a further proof of the functional bacterial diversity present within our strain collection.We then assessed carbohydrate-active enzymes (CAZymes) considering their importance for both ecological interactions in the gut and biotechnological applicationsies Fig. . When lot45 Fig. . One of 47 with >5000 sequences . Sequence mapping to the PiBAC species at 97% identity revealed a median cultured fraction of 35.8% in the pig gut vs. 24.4% and 10.5% in human and mouse, respectively    Fig. . Further03) Fig. . The con03) Fig. .Fig. 4HoFloccifex porci, Clostridium porci, Mogibacterium kristiansenii, and Peptostreptococcus porci  identified in the thousands of amplicon datasets. OTU mapping (97% identity) revealed only a small overlap (6.8%) between the three host species  were reconstructed from previously published pig gut metagenomes18 using a newly described workflow50 (see ‘Methods’ section) , Floccifex porci , Hallerella porci , Hallerella succinigenes , Mogibacterium kristiansenii , Oliverpabstia intestinalis , Selenomonas montiformis , Sharpea porci , Sodaliphilus pleomorphus , Stecheria intestinalis .The amplicon-based findings above were complemented by metagenomic analysis. A total of 589 high-quality on) Fig. . These hing Fig. : Holdema51. Genomes were assigned to species-level genome bins (SGBs) at average nucleotide identity (ANI) values >95% as described in the original work cited above and implemented in an updated version of PhyloPhlAn52. Seventeen of the 38 genomes matched human SGBs, with ten having no cultured representative, confirming them to be unexplored even in the human microbiome (Supplementary Data n = 11), three of which comprised >100 MAGs  and can thus be considered as prevalent gut bacterial species in human. More than half of the strains representing new taxa in the collection (n = 21) had however no match to the human SGBs catalogue and may thus represent pig-specific species  represented unknown taxa . This highlights the novel richness within this metagenome-based species catalogue and the utility to further explore pig microbiomes by cultivation. Most of these yet-uncultured bacteria belong to the phylum Firmicutes and a list of ten ‘most-wanted’ taxa is provided ded Fig. . Searchi.2% Fig. . MoreoveTaken together, these findings highlight the value of combining cultivation with metagenomics to study microbial diversity in the pig intestine and the need to both widen the pool of pig gut microbiomes studied and continue efforts in cultivating pig gut bacteria to obtain important taxonomic groups not currently represented by isolates.55 highlight the usefulness of developing simplified communities of cultured microbes that can be used to study molecular mechanisms in controlled experimental settings57.The high diversity and still substantial unknown fraction of microbes within gut microbiomes18 were used as a comprehensive pool of data to infer the composition of minimal communities of isolates that best represent faecal pig microbiomes functionally. Binary (presence/absence) profiling of 284 metagenomes at the level of protein families (PFAMs)58 indicated a significant clustering according to the country of origin  showed bile salt hydrolase (BSH) activity (see next section). Additional functions covered by this consortium include: complex carbohydrate metabolism ; secondary bile acid production (Clostridium scindens); trans-aconitate oxidation and anaerobic glutamate fermentation (Acidaminococcus fermentans); butyrate production from amino acids (Intestinimonas butyriciproducens). The sole species picked in 100% of the pigs across all three countries was S. pleomorphus, followed closely by Prevotella mizrahii (>98%), which may reflect the importance of these novel taxa in pig microbiomes. In Chinese pigs, Streptococcus alactolyticus was selected in all samples vs. 50% in samples from France and 71% from Denmark. This may be explained by common usage of antibiotics in the Chinese pigs studied59 and the detection of multiple antibiotic resistance genes such as lsaE, lnuB, ant(9)-Ia, and ant(6)-Ia in the isolate’s genome  based on an iterative scoring system that optimizes the number of matches vs. mismatches between the reference genomes and the given metagenomes (see details in the ‘Methods’ section). The proportion of minimal communities in which each species was selected, both across the entire cohort and per country, was then plotted: a total of 23 species were characterized by a prevalence >50% in the pigs of at least one country Fig. . They sp60. The composition of minimal consortia will have to be amended according to future progress in isolating and describing additional strains.In summary, the proposed species have a high degree of phylogenetic and functional diversity and can now be used as minimal bacterial consortia in experimental and applied studies, e.g. prevent antibiotic use linked to enteric infections in pig farming61. In contrast to numerous sequencing studies65, knowledge of cultured bile acid-metabolizing bacteria is scarce, which hampers applications towards targeted modulation of secondary bile acid metabolism. Nearly half of the collection (n = 50 strains across six phyla) showed cleavage of taurine residues from bile acids in vitro  of the species Clostridium scindens was able to produce the secondary bile acid deoxycholic acid (DCA) from cholic acid (CA) via 7-alpha-dehydroxylation   of unknown function or mobile genetic elements  operon, responsible for 7-alpha-dehydroxylation of primary bile acids, was nearly identical in both strains  .Among the 3655 protein-coding genes detected in its closed genome, comparative genomics with the human isolate ps) Fig. 72. A sub49) Fig. . In DSM T (ref. 72), indicating that additional nutrients required for growth remain to be determined. We then performed RNA-Seq analysis of strain DSM 100975 in the absence (PYF control medium) or presence of bile acids (0.1 mM CA or 0.1 mM DCA). A total of 1393 genes  were significantly upregulated by CA , whilst 1336  were downregulated  .The pig-derived strain did not grow in the defined medium recently reported for ATCC 35704ted Fig. . Signifi−5) Fig. . Express77, although a higher number of strains will be required for comprehensive analysis. PiBAC can now be used as a toolbox for in vitro and in vivo studies of the impact of bile acids on microbe–microbe and microbe–host interactions.In conclusion, these in vitro assays provide multiple strains able to metabolize bile acids and highlight differences between a pig and a human isolate. This fits with the concept of functional adaptation of intestinal bacteria to a specific host and associated environmental conditions55. Contemporary loss of bacterial diversity is a real concern79, stressing the need to archive strains and describe novel taxa, as presented in this study. We focused on pig gut microbiota considering the importance of pigs in biomedical research and agriculture and the host specificity of intestinal microbiomes81. The collection of isolates presented here reveals a substantial taxonomic and functional diversity of bacteria that opens avenues for mechanistic studies and biotechnological applications. Important members of the pig gut microbiome such as Archaea, fungi, and strains of yet unknown bacterial phyla are not included and the current resource does not address strain-level diversity. Hence, this work also shows that efforts to isolate and characterize known and yet uncultured microbial taxa from the intestine must be continued.The pool of yet undescribed bacteria is still very largeDetailed information about the origin of all strains, including isolation site, donors, and gut region are provided in Supplementary Data 1311/+ pigs82 from the Technical University of Munich , and one Aachen minipig83 hosted at the RWTH University Hospital . Fresh faecal samples were collected directly from the rectum of individual pigs using sterile gloves and prepared immediately as described below in the section ‘Bacterial isolation, cultivation, and storage’. Besides faeces, intestinal content was collected using sterile equipment from pigs euthanized for the purpose of other experiments. Animal use was approved by the Federal Government of Bavaria .For the 98 strains isolated in Germany, gut samples from 19 pigs were used, including German Landrace, German Landrace crossed to minipigs, p53 and APCn = 6 isolates). After euthanasia, samples were collected from the lumen and mucosa of the ileum, caecum, and mid-colon for bacterial cultivation. Piglets were acquired and managed in accordance with the National Animal Disease Centre Animal Care and Use Committee guidelines; (ii) at the University of Guelph (Canada) from farmed ‘wild’ boars, i.e., living in a free-range enclosure and left to forage, breed, and raise their young with minimal human intervention (n = 10 isolates). Permission of the farmer was asked prior to sampling. Defecation was watched from a safe distance, and droppings were collected from three separate boars once they had moved away, and within 10 min of voiding. Faecal samples were immediately placed in sterile sample collection bags and onto ice packs, transferred to the laboratory and stored at −80 °C. At the time of culture, samples were carefully thawed, and a ‘core’ region from inside of the dropping was removed for further work, to minimize contamination from the ground; (iii) at the Department of Bacteriology, University of Wisconsin-Madison, USA, as described in detail previously (n = 3 isolates)84.Additional strains were isolated: (i) at the National Animal Disease Centre of the United State Department of Agriculture  from mixed bred pigs obtained from a commercial supplier .BBE: l-cysteine, 0.5 g; dithiothreitol (DTT), 0.2 g.BHI: Brain Heart Infusion , 37.0 g; Bifidobacterium Selective Agar .Bifido: Blood agar: Columbia Agar with Sheep Blood PLUS .BSM: Bifidus Selective Medium , 42.5 g; BSM Supplement  116 mg.2PO4, 1 g; MgSO4, 0.1 g; NaCl, 0.25 g; CaCl2, 0.05 g, cellulose, 0.3 g.CA: Cellulose Agar: KH2, 0.8 g (dissolved first in 1 M HCl). After autoclaving : bile salts (Fluka ref. 48305) 4 g; mucin type III , 250 mg; arginine, glycine, histidine, and lysine, each 6 g; ampicillin, 50 mg; neomycin 50 mg/l; l-cysteine, 0.5 g; DTT, 0.2 g; sheep blood , 50 ml.Corio2: M9 minimal salts , 11.3 g; Tween-80, 1 ml; PdClI) nalidixic acid and colistin, 10 mg each; (II) defibrinated sheep blood, 5% (v/v); (III) filter-sterilized spent culture medium (from a bioreactor inoculated with human faeces)85, 3%.FAA: Fastidious Anaerobe Agar , 46 g; as such or supplemented with either ; Tween-20, 1 ml; tryptone, 2 g. After autoclaving: filter-sterilized pig faecal water, 6 ml; rumen fluid , 50 ml; vitamin solution (as in DSMZ medium 141), 1 ml; Fusobacterium Selective Agar .FSA: l-cysteine, 0.5 g; DTT, 0.2 g.GAM-mod: Gifu Anaerobic Medium, modified , 41.7 g; 86; hog gastric mucin, 10 g; CaCl2, 0.45 g; MgSO4, 0.45 g; KH2PO4, 2.25 g; K2HPO4, 2.25 g; NaCl, 4.5 g; (NH4)2SO4, 4.5 g; cysteine, 0.05 %; haemin, 0.05 g; resazurin, 0.0001 %; Noble agar 1.6 %.HM: Hog gastric mucin mediumLKV: Laked Brucella Blood Agar with kanamycin and vancomycin .McConkey: Roth, ref. X922.1.MDM: Modified Dehority Medium (DSMZ medium 1668) supplemented with cellobiose, 10 g.Medium 78: Chopped Meat Medium (DSMZ medium 78).MRS: Lactobacillus Broth according to De Man, Rogosa, and Sharpe (DSMZ medium 11).2PO4, 0.4 g; Na2HPO4, 0.53 g; NH4Cl, 0.3 g; NaCl, 0.3 g; MgCl2·6H2O, 0.1 g; CaCl2, 0.11 g; NaHCO3, 4 g; Na2S·9H2O, 0.25 g; alkaline trace element solution, 1 ml; trace element solution (according to DSMZ medium 141), 1 ml; mucin type III , 2.5 g. After autoclaving : vitamin solution (according to DSMZ medium 141), 1 ml; filter-sterilized human faecal water, 7 ml.MUC: KHNM: M9 minimal salts , 11.3 g; yeast extract, 0.2 g. After autoclaving : penicillin and streptomycin, each 25 mg; trace element and vitamin solutions (as DSMZ medium 141); ethanol and methanol (each 3 ml).Nutrient agar: DSMZ medium 1.Oil-RF-PBS: Phosphate-buffered saline, 500 ml; rumen fluid, 500 ml; Tween-20, 0.5 ml; olive oil, 5 ml. Used for pre-cultivation (5 days) prior to plating on other media.87: KH2PO4, 0.5 g; NH4Cl, 1 g; Na2SO4, 2 g; CaCl2·6H2O, 0.1 g; MgSO4·7H2O, 1 g; sodium lactate, 3.5 g; yeast extract, 1 g; thioglycolic acid, 0.1 g; FeSO4·7H2O, 0.5 g; NaHCO3, 2 g; PdCl2, 2 mg. After autoclaving : penicillin, 25 mg; trace element and vitamin solutions (as in DSMZ medium 141).PG: Postgate MediumRF-PBS: Phosphate-buffered saline, 500 ml; rumen fluid, 500 ml. RF-PBAS was used for pre-cultivation (5 days) prior to plating onto other media.2·6H2O, 1.5 mg; FeSO4·H2O, 0.5 mg; MgSO4·7H2O, 0.8 g; KH2PO4, 0.5 g; K2HPO4, 0.5 g; KCl, 0.05 g; CaCl2·7H2O, 0.05 g; NaCl, 1.5 g; NH4Cl, 1 g; MnSO4·7H2O, 0.6 mg; ZnSO4·7H2O, 0.1 mg; CuSO4·5H2O, 0.02 mg; KAl(SO4)2·12H2O, 0.2 mg; H3BO3, 7 mg; CoSO4·7H2O, 4 mg; Na2MoO4·2H2O, 0.5 mg; Na2SeO3·5H2O, 3 mg; Na2WO4·2H2O, 4 mg; nitrilotriacetic acid, 0.15 mg; resazurin, 1 mg. The following compounds were aseptically added to the medium after autoclaving: streptomycin, 100 mg; tetracycline, 100 mg; penicillin G, 100 mg; NaHCO3, 2 g; Na2S, 0.4 g; methanol, 3.3 ml; sodium formate, 11 g; and vitamin solution (DSMZ medium 141), 1 ml. SAB medium was used for pre-cultivation (5 days) prior to plating onto other media.SAB: sodium acetate, 1 g; trypticase, 2 g; yeast extract, 2 g; L-cysteine hydrochloride monohydrate, 0.5 g; valeric acid, 5 mM; isovaleric acid, 5 mM; 2-methylbutyric acid, 5 mM; isobutyric acid, 6 mM; 2-methyl valeric acid, 5 mM; NiCl2HPO4, 2 g; Tween-80, 1 ml; salt solution (as in DSMZ medium 104), 40 ml. After autoclaving : hemin (dissolved in 1 M NaOH), 5 mg; vitamin K1 (dissolved in 95% ethanol), 1 µl; glucose, 5 g; l-cysteine, 0.5 g.SM: Tryptic peptone, 5 g; peptone, 5 g; yeast extract, 10 g; beef extract, 5 g; Kl-cysteine, 0.5 g; DTT, 0.2 g. Either used as such or supplemented with mucin, 18.5 g (WCA-MUC) or with bromoethanesulfonic acid, 0.221 g, and sodium molybdate, 0.205 g (WCA1).WCA: Wilkins–Chalgren Anaerobe broth , 33.0 g; 92, but rather that the diversity of microbes to be catalogued is tremendous and that the required cultivation efforts are colossal if not tailored. As in recently published studies95, in order to capture as much diversity as possible, we employed an array of rich and selective culture media (see listing and composition in the previous section) in combination with samples from different gut locations  and pigs of various origins . After manual homogenization by vigorous shaking, glass flasks were left to stand for max. 1 min to sediment debris. The slurry was diluted again 1:10 in the same anoxic buffered solution contained in another glass flask using a sterile syringe fitted with a needle to inoculate through the sterile rubber stopper and thereby guarantee anoxic conditions. Samples were transported to the lab on ice prior to further processing, which occurred max. 1.5 h after sample collection.For all strains isolated in Germany, the procedure followed to collect intestinal samples and keep them under strictly anaerobic conditions as soon as possible is shown in Supplementary Fig. −2 to 10−6) were prepared in an anaerobic workstation  containing an atmosphere of N2 (89.3%), CO2 (6%), and H2 (4.7%). Dilutions (10 µl) were pipetted onto one of the culture media listed above and spread on the agar by tilting plates. Cells were incubated at 37 °C for 1–40 days prior to picking. Pure cultures were obtained by re-streaking single colonies at least three times before transfer into broth medium in Hungate tubes98 and observation of colony morphology and cells by light microscopy. Strictly anaerobic strains were subjected to growth under aerobic conditions to check for potential latent contaminations with facultative anaerobes. For aerobic isolation, the same procedure as above was followed using aerobic media and incubation conditions. For enrichment procedures prior to plating, faecal slurries were prepared as above and transferred (100 µl) directly into Hungate tubes containing the desired culture medium and incubated for 7–21 days at 37 °C on a shaker. The obtained mixed cultures were then plated as above.For direct isolation, tenfold dilution series  in broth to enhance the growth of mucolytic bacteria before inoculation on solid media. Bacteria were isolated on solid HM medium after incubation at 39 °C for 5 days.For strains isolated in Ames, Iowa, USA, a previously published protocol−2 to 10−8) in saline (0.9% NaCl) were plated (100 µl each) onto different solid media types  in duplicate and incubated either aerobically or anaerobically at 37 °C. After 72 h and every 24 h up to 7 days, isolated colonies were re-streaked onto FAA plates. Bacterial stocks were frozen and stored at −80 °C in freezing media .For strains isolated in Canada, 0.5 g of thawed faecal sample was homogenized in degassed Tryptic Soy Broth in an anaerobic chamber. Tenfold dilution series (10l-cysteine and DTT (0.05% and 0.02% (w/v), respectively). Rumen fluid or 6% CO2 in the atmosphere were added if necessary (Supplementary Data www.dsmz.de/pibac). For storage, cryo-stocks were prepared by mixing freshly grown cultures 1:1 in a sterile, 40% (v/v) glycerol-containing medium. Cryo-aliquots (400 µl) were dispensed into sterile Eppendorf tubes kept under sterile conditions in a laminar flow cabinet under constant flow of the anaerobic gas mixture before freezing on dry-ice. For long-term storage, all strains were deposited at the Leibniz-Institute DSMZ .For maintaining all strains in culture, the standard medium used was anoxic WCA supplemented with 99, sequencing using primer 27f after PCR-product cleaning, and searching for most closely related species with a valid name using EzBioCloud100. For all strains included in the collection, nearly full-length 16S rRNA gene sequences were generated by re-sequencing the aforementioned PCR products using primers 338r, 785f, and 1492r. Contigs were built in MEGA (v10)101 and ambiguous positions were double-checked using electropherograms. All sequences, their length, and accession numbers are listed in Supplementary Data 100 was first considered as an indication for novel taxa, and draft genomes were generated for corresponding strains (see method below). For digital DNA:DNA hybridization (dDDH), the Genome-to-Genome Distance Calculator 2.0 (GGDC), a web service freely available at http://ggdc.dsmz.de, provided a genome-based delineation of species by reporting dDDH estimates as well as their confidence intervals. Additionally, species-level delineation was confirmed using ANI values as calculated by FastANI102. Genus-level delineation was inferred via percentage of conserved proteins (POCP) using BLASTP103 where only matches with an E-value <1e−5, identity >40%, and query coverage of >50% were counted104. Genome based-phylogenomic trees were created using PhyloPhlAn (v0.99)52 based on 400 marker genes.Cultures were first identified using a MALDI-Biotyper following the manufacturer’s instructions (Bruker). The identity of interesting strains was confirmed by amplifying the 16S rRNA gene using primers 27f and 1492rwww.dsmz.de/pibac). Approximately 100 mg (wet weight) of cell biomass was extracted according to the standard protocol of the Microbial Identification System . The composition of cellular fatty acids was identified by comparison with the TSBA40 naming table.Cellular fatty acids were determined at the Leibniz Institute DSMZ for all novel taxa under their respective optimal growth conditions .The identity of strain DSM 106279 was confirmed to be Fusobacterium mortiferum DSM 108838 (= FSA-380-WT-2B) was used as positive control.BSH activity was determined using WCA or BHI medium (depending on strains) supplemented with 0.5% (w/v) taurodeoxycholic acid (TDCA)  from filter-sterilized aqueous stock solutions. For plate-based assays (1.5% g/l agar), a single colony was picked and transferred onto solid medium with and without TDCA and incubated for at least 5 days at 37 °C (under anaerobic conditions whenever appropriate) until growth was observed. Isolates were considered positive in case of halos surrounding and/or whitening of colonies due to precipitation after deconjugation Fig. . For liqC. scindens DSM 100975 and two strains of Collinsella aerofaciens to metabolize bile acids, duplicate fresh cultures were transferred (1:10) in anoxic WCA broth containing 50 µM CA and incubated at 37 °C for 48 h under anaerobic conditions. Samples were taken at 0 h and 48 h, centrifuged  and supernatants were collected and stored at −80 °C until analysed by chromatography coupled with mass spectrometry according to our previously described protocols66. Extibacter muris DSM 28560T was used as positive control105.To test the ability of g, 10 min, RT) and supernatants were collected and stored at −20 °C for a maximum of 7 days prior to measurement. Before HPLC analysis, samples were filtered into 2-ml short thread vials with screw caps  using non-sterile 0.2-µm cellulose membrane filters . Vials were then placed in the refrigerated autosampler of the HPLC system, a Hitachi Chromaster 5450  fitted with a Refractive Index detector and a Shodex SUGAR SH1011 column (300 × 8.0 mm) . A Shodex SUGAR SH-G (6.0 × 50 mm) was used as guard column. The injection volume was 40 µl. The running temperature was 40 °C. The eluent was 10 mM H2SO4 with a constant flow of 0.6 ml/min. Concentrations were determined using external standards via comparison of the retention time . Peaks were integrated using the Chromaster System Manager software . For each of the tested strains, only SCFA concentrations >0.8 mM (limit of detection (LOD) for succinate, lactate, and acetate) or >0.5 mM  in at least one of the triplicates were considered for calculation. Results are summarized in Supplementary Fig. For novel taxa, the concentration of SCFAs , branched SCFAs , and intermediate metabolites  were determined using high-performance liquid chromatography (HPLC). Bacteria were grown in modified YCFA broth (DSMZ medium 1611) supplemented with 0.02% DTT in Hungate tubes for 48 h or 96 h at 37 °C under anaerobic conditions and with shaking (200 r.p.m.). Triplicate cultures were measured for each strain. Negative controls consisted of medium without bacteria. After incubation, samples were centrifuged  at 37 °C with constant shaking (220 r.p.m.) under anaerobic conditions in Hungate tubes containing 10 ml of culture. For dilution assays, a gradient of medium dilutions  was obtained by mixing the basal medium (BHI broth supplemented with 10% rumen fluid) with autoclaved and gassed distilled water in appropriate volume ratios. For increasing osmolarity, the medium was supplemented with the appropriate amount of NaCl  prior to gassing and autoclaving.600 nm directly within the Hungate tubes using a CO8000 Cell Density meter. After 1 week of growth, in order to increase cell density for microscopy, cells were centrifuged  and re-suspended in a lower volume of the corresponding growth medium. Cell morphology was observed by phase contrast microscopy (see below).Triplicate cultures were tested for each condition. The osmolarity of all media was measured using an OSMO Station OM-6050 . Growth was followed overtime by measuring the ODThe microscopic images in Fig. 2b1 and in Supplementary Figs. B. porci DSM 105750T was grown at 37 °C for 4 days in BHI broth supplemented with 10% rumen fluid , cysteine (0.05% w/v), and DTT (0.02%) under anaerobic conditions . Cultures (1 ml) were centrifuged  and washed once with PBS. Harvested cells were incubated for 10 min in the dark at room temperature with the membrane stain FM4-64 (1 µg/ml) and the DNA stain 4′,6-diamidino-2-phenylindole (DAPI) (2 µg/ml). After final PBS wash, cells were placed onto microscope slides and visualized by phase contrast and fluorescence microscopy using a Zeiss Axio Imager.Z2 microscope  equipped with a Plan-Apochromat x 63 phase contrast objective lens, appropriate filter sets, and an ORCA-Flash 4.0 LT digital CMOS camera  using the Zeiss Zen 2 (Blue Edition) software. Brightness and contrast level of the images were adjusted using ImageJ (version 1.50g).Bullifex porci DSM 105750T, Pseudoramibacter porci DSM 106894T, Stecheria intestinalis DSM 109718T, and Tissierella pigra DSM 105185T were analysed via scanning electron microscopy at the University Hospital of RWTH Aachen and via transmission electron microscopy at both the University Hospital of RWTH Aachen and at Wageningen University in order to validate results in independent laboratories using different sample preparations based on classical fixation or high-pressure freezing followed by freeze substitution. The strains were cultured at 37 °C under anaerobic conditions in Hungate tubes with constant shaking (200 r.p.m.) as follows: B. porci, BHI broth supplemented with 10% rumen fluid, 148 h; P. porci, GAM broth, 36 h; S. intestinalis, GAM modified broth supplemented with 5% rumen fluid, 96 h; T. piger, BHI broth, 24 h. Cell were pelleted  and immediately processed.Bacteria in their growth medium were allowed to adsorb on formvar-carbon-coated nickel grids  for 10 min prior to washing with distilled water. Samples were then stained by placing a drop of 1% phosphotungstic acid  in distilled water (pH 7.2) onto the grid for a few seconds. The grid edge was carefully laid onto a filter paper to remove the staining solution from the grid. After air drying, samples were examined at the electron microscopy facility of the University Hospital of RWTH Aachen using a Hitachi HT7800 transmission electron microscope operating at an acceleration voltage of 100 kV.2  and sputter coated  with a 10-nm gold/palladium layer. Samples were analysed using an environmental scanning electron microscope  with a 10-kV acceleration voltage in a high vacuum environment.At the Electron Microscopy Facility of the RWTH University Hospital, bacteria were fixed with 3% (v/v) glutaraldehyde  in 0.1 M Soerensen’s phosphate buffer, washed in phosphate buffer for 15 min, and dehydrated by incubating consecutively in an ascending ethanol series  for 10 min each and the last step thrice. The samples were critical point dried in liquid COAt the Electron Microscopy Facility of the RWTH University Hospital, bacterial cells were fixed with either 0.2% or 1.5% glutaraldehyde in 0.1 M Soerensen’s phosphate buffer for 30 min and then embedded in 1% low melting agarose . They were frozen in a high-pressure freezer  and subsequently embedded into Epon  via freeze substitution. Samples were then incubated in ethanol at −80 °C for 50 h, brought slowly to room temperature, incubated in fresh ethanol three times for 10 min followed by 30 min in propylene oxide (PO), 2 h in a 1:1 mixture of PO and Epon, 2 h in pure Epon, and finally cured in pure Epon for 2 h at 90 °C. Ultrahin sections (70–100 nm) were cut with an ultramicrotome , picked up on Cu/Rh grids  and stained with 0.5 uranyl acetate and 1% lead citrate  to enhance contrast. Samples were examined using a LEO 906 transmission electron microscope  operating at an acceleration voltage of 60 kV.At the Electron Microscopy Centre of Wageningen University, two different protocols were used:B. porci in anoxic WCA medium were pelleted by centrifugation  and fixed  using 2% (v/v) formaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer [Na(CH3)2 AsO2·3H2O] (CB). Pellets were washed in CB, fixed (1 h on ice) using 1% osmium tetroxide in CB, and then washed twice in CB. The buffer was removed and replaced with 70% (v/v) ethanol overnight and then embedded in a small volume (100–200 µl) of 1% agarose. Ethanol was increased to 90% and then 100% (v/v) with 30-min intervals at room temperature. An equal volume of a 1:1 mixture of 100% PO and ethanol was added and replaced after 30 min by 100% PO for a further 30 min. Finally, PO was replaced by Epon in PO (1:4 mixture) overnight at 4 °C. The specimens were then placed in Epon in PO (1:1 mixture) for 2 h at room temperature and finally in pure Epon at 4 °C prior to transfer to a casting mould for polymerization at 60 °C. Thin sections were cut from the Epon blocks with a diamond knife using a Reichart Ultracut S apparatus (Leica). The sections were transferred to a 50 mesh copper grid, which was dried and stained by immersion in 1% uranyl acetate in distilled water for 10 min and then Reynolds lead citrate stain for 2 min. Whenever ruthenium red was used for staining, the fixation buffer CB included 0.075% (w/v) ruthenium red and the copper grids were stained only in 1% uranyl acetate in distilled water for 10 min. Cells were imaged using a transmission electron microscope (JEOL 1400 plus).For protocol 1, cultures of B. porci was cultured as mentioned above in the section: ‘Bacterial cultures for further microscopy analyses’. Cells were pelleted by centrifugation  and small aliquots of the pellet were frozen in a Leica EM HPM100 high-pressure freezing system. Freeze substitution was carried out with 0.2% osmium tetroxide and 0.1% uranyl acetate in acetone for 24 h at −90 °C, 24 h at −80 °,C and 24 h at −60 °C using a Leica EM AFS2 equipped with a freeze substitution processor. The temperature was then raised to −20 °C in 12 h and the samples were washed six times with acetone. The temperature was raised further to 4 °C and the samples were infiltrated with acetone:Spurr resin mixtures 2:1, 1:1, and 1:2 each for 6 h. Finally, the samples were infiltrated with pure Spurr for 3 × 6 h at 20 °C and the resin was polymerized for 8 h at 70 °C. Thin sections were cut from the Spurr blocks with a diamond knife using a Leica Ultramicrotome UC7. The sections were transferred to 50 mesh copper grids and stained by UranyLess (10 min) and lead citrate (10 min) both ready-to-use staining solutions from Electron Microscopy Sciences . Cells were imaged using a JEOL 1400 plus transmission electron microscope operating at an acceleration voltage of 120 kV.For protocol 2, 107. Two treatments were tested before muramidase digestion: (i) DNase, RNase, α-amylase, and α-chymotrypsin alone or (ii) with addition of LiCl, EDTA, and HF treatments after protease inactivation. The soluble fraction obtained from muramidase digestion was reduced using 0.5 M sodium borate (pH 9.5) and sodium borohydride (10 mg/ml) and the pH was adjusted to 3.5 with phosphoric acid for liquid chromatography (LC). Solubilized muropeptides were detected and analysed by LC-MS using UPLC coupled with a Xevo G2/XS Q-TOF mass spectrometer (Waters Corp.) operated in positive ionization mode108. Data were acquired and processed using the UNIFI software package (Waters Corp.). The molecular structure of muropeptides included in the UNIFI compound library was obtained using ChemSketch (www.adclabs.com).Sacculi samples obtained by boiling bacterial biomass in 5% SDS were used for peptidoglycan isolation47. All 16S rRNA amplicon samples that contained >5000 sequences and were labelled as ‘pig gut’ (n = 1346), ‘mouse gut’ , or ‘human gut’  were used. Random subsampling of both the mouse and human samples to a number of 1346 samples was conducted to ensure comparative results pertaining to host specificity. The 16S rRNA genes of all isolates were compared to IMNGS-derived OTU sequences using blastn . Annotation of OTUs to the Living Tree Project database48 was conducted using the same parameters. Collection members were considered as being enriched within the pig vs. mouse and human gut if detected in at least 20% of the pig samples and at a higher median relative abundance as determined by Wilcoxon Rank Sum test after Benjamini–Hochberg correction . To determine common and unique IMNGS-derived OTUs across the host species, the SILVA database (v132)109 was reduced to single representatives per species via clustering at 97.0% using Usearch (v8.1)110. The subsampled host-specific datasets (n = 1346 samples each) were annotated against the reduced SILVA database using Usearch at 97% identity to provide a common reference point for species occurrence.To evaluate the occurrence of isolates, datasets were downloaded from the IMNGS database111 investigating the impact of dietary protein content on the faecal microbiota of castrated male Duroc pigs at the age of 165 days; (ii) stool samples from ten German landrace pigs from the animal facility of Thalhausen  at the age of 8, 24, and 52 weeks; (iii) samples from six wildtype pigs from the MIDY biobank112, including five locations within the gastro-intestinal tract. For studies 2 and 3, stool samples were processed and sequenced as previously reported in detail113 and briefly described in the next section. All data were analysed using IMNGS47 and Rhea114. Cultured fractions were determined by searching for matches between the amplicon sequences of all study-specific OTUs (those occurring at a relative abundance of ≥0.25% in at least one sample within the respective dataset) and 16S rRNA gene sequences of the isolates using blastn  at two different sequence identity thresholds: 97% (as proxy for species level) and 95% (genus level).Besides the large-scale analysis described in the previous paragraph, in order to assess the coverage of sequence-based diversity by all isolates in relation to parameters such as diet, age, and gut locations, we processed three additional datasets: (i) published data115. Aliquots of stool mixed in DNA stabilization solution (Invitek) (600 µl) were supplemented with 250 µl of Guanidinethiocyanat (4 M) and 500 µl of N-laurolylsarcosine (5%), mixed and incubated for 1 h at 70 °C under moderate shaking (700 r.p.m.). Cell were mechanically lysed by bead-beating  with 0.1-mm glass beads using a Fast-Prep-24 fitted with a cooling adapter. The cell lysate was then vortexed with 15 mg of Poly(vinylpolypyrrolidone) and centrifuged . The clear intermediary phase was transferred into a new tube and centrifuged again. After addition of 5 µl of RNase (10 mg/ml), samples were incubated  followed by DNA purification using the NucleoSpin gDNA kit  according to the manufacturer’s instructions. The V3–V4 regions were amplified (25 cycles) from 24 ng of template DNA in a two-step process116 using primer 341F and 785R117 and a combinatorial dual indexing strategy. PCR products were purified using magnetic beads (Beckman Coulter) and pooled in an equimolar amount of 2 nM. The multiplexed samples were sequenced on an Illumina MiSeq in paired-end mode (2 × 275 bp) using the Rapid v2 chemistry.Metagenomic DNA was isolated using a modified version of the protocol by GodonSodaliphilus pleomorphus DSM 108610T to refine taxonomic assignment and for C. scindens DSM 100975 to optimize RNAseq and comparative genomic analyses.Genomes were generated for all strains within the collection, including 115 draft genomes and two closed genomes for 118 or using the NEBNextra Ultra II DNA Library Prep Kit (New England Biolabs) otherwise. Libraries were sequenced using the Illumina MiSeq system according to the manufacturer´s instructions.For draft genomes, DNA libraries were prepared using either the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina) following a protocol optimized (DNA shearing and fragment size selection) to improve assembly quality when sufficient amounts of DNA were availableS. pleomorphus was carried out on a PacBio RSII  using P6 chemistry. The genome was assembled using the ‘RS_HGAP_Assembly.3’ protocol included in SMRT Portal version 2.3.0, utilizing 68,258 postfiltered reads with an average length of 11,462 bp. The obtained chromosomal contig was trimmed, circularized, and adjusted to dnaA . For short-read correction, sequencing was also carried out on a NextSeq  in a 150-bp paired-end run. Quality improvement was performed with the Burrows-Wheeler Aligner (BWA) mapping the Illumina reads onto the obtained chromosome119. The final genome sequence was annotated using Prokka120.Whole-genome sequencing of C. scindens DSM 100975, we used a combination of Oxford nanopore  and Illumina  sequencing. DNA was converted into Nanopore libraries using the NBD104 barcoding kit and 1D library kit SQK-LSK109. Libraries were sequenced on a SpotON R9.4.1 FLO-MIN106 flowcell for 48 h using a GridION x 5 sequencer. Basecalling was performed with Guppy 3.0.3 . Demultiplexing and adaptor trimming from the fastq files was performed with Porechops 0.2.3 (https://github.com/rrwick/Porechop). Shotgun genomic libraries were prepared with the Hyper Library construction kit from Kapa Biosystems . The libraries were quantitated by qPCR and sequenced on one Illumina MiSeq flowcell for 251 cycles from each end of the fragments (250 nt length) using a MiSeq 500-cycle sequencing kit Nano. Fastq files were generated and demultiplexed with the bcl2fastq v2.20 conversion software .To generate a closed genome for Genome sequences were submitted to the NCBI and are available within BioProject PRJNA561470. Individual genome accession numbers are given in Supplementary Data 121 with activated BayesHammer tool for error correction and MismatchCorrector module for post-assembly mismatch and indel corrections. Assemblies were evaluated using Quast (v3.1)122 and CheckM (v1.0.12)123. Whenever appropriate, genomes were circularized using Circlator-1.4.1 (https://github.com/sangerpathogens/circlator). Genes were predicted and annotated using Prokka, accepting only closed end CDS (default setting)120. Pathway analysis, including the identification of cell wall and divisome proteins, was conducted by converting Prokka output into KEGG orthologue IDs using Prokka2KEGG (https://github.com/SilentGene/Bio-py/tree/master/prokka2kegg) and the KEGG Mapper tool124. In addition, genomes lacking a KO for FtsK were instead searched for the SpoIIIE gene and, if found, assigned to the FtsK/SpoIIIE function, as SpoIIIE is a highly related protein commonly grouped with FtsK due to their shared functionality. The presence or absence of all functions included in Fig. 125). EggNOG annotations were obtained using eggNOG-Mapper (v2)126. Proteins within the pig metagenomic gene catalogue were assigned to isolate- or MAG-derived proteomes using BLASTP (bitscore >100). Comparative genomics of the C. scindens strains was performed using OrthoFinder127 to infer orthologous groups and in-house scripts for data processing. Functional category analysis was done with the eggNOG-mapper function using bacteria as taxonomic scope and DIAMOND as the search program128. Graphing and statistical analysis (Fisher’s exact test with Bonferroni correction) were performed in R.Illumina reads were assembled using Spades (v3.6.1)129) with a query and subject coverage of 40% along with 40% sequence identity against the GT family 10 subset of the CAZy database (www.cazy.org). Variants of known FucT motifs  were then searched and matched proteins were entered into the online I-TASSER server130 for structural modelling and homology analysis.All proteins from the 38 novel taxa identified and extracted via Prokka (see genome processing above) were annotated in a targeted manner using DIAMOND v0.9.13.114 (ref. Escherichia coli BL21 (DE3) was transformed with a pET21a vector containing the target gene and cultured in TB medium at 37 °C. At OD600 of 0.5–0.8, expression was induced with 1 mM IPTG, after which the temperature was reduced to 25 °C and cells were incubated for another 24 h. Cells were harvested by centrifugation and stored at −20 °C until further processing. For purification, 4 g of cells were suspended in Tris-HCl lysis buffer , sonicated  and centrifuged. Crude extracts were filtered and loaded onto a HisTrap column . The column was washed with lysis buffer, and the enzyme was eluted with elution buffer (lysis buffer with 500 mM imidazole). The enzyme was dialyzed overnight in Tris-HCl buffer  and stored at 4 °C. Purification was assessed by SDS-PAGE (12%) and Western Blot.N-Acetyllactosamine (LacNAc) carrying a tBoc linker was produced as described recently131. The enzyme (50 µg/ml) was incubated at 37 °C for 24 h in 50 mM MOPS (pH 6), 25 mM KCl, 5 mM MgCl2, 6.5 mM GDP-Fucose, 5 mM tetrasaccharide, and 0.02 U/mL FastAP. Negative controls included reactions without GDP-Fucose and with an enzyme from a rumen Lachnospiraceae isolate (protein accession SFH53651.1) cloned and expressed as described above for the target protein44. Reactions were stopped by heat treatment  and products were analysed using a Dionex RP-HPLC system  at a wavelength of 254 nm, using an analytic MultoKrom 100-5 C18 column  and a flow rate of 1 ml/min (isocratic eluent of 15% acetonitrile in water). Product peaks were collected and further analysed by ESI-MS , using a Multospher 120 RP 18 HP-3µ columns  and a flow rate of 0.2 ml/min with 50% acetonitrile . The product from the GT assay was treated  with a α3-fucosidase  according to the manufacturer’s instructions to determine the position and glycosidic bond type of fucose. The reaction was analysed via HPLC as described above.For GT assays, 132). The function of individual genes within BGCs was further validated by manual curation using BLAST103 against the NCBI nr database to identify core genes for biosynthesis. The sequence alignment of the sactipeptide precursor peptide was performed using Clustal Omega133 and visualized by Weblogo134. Clustal Omega was also use for phylogenetic analyses based on 16S rRNA gene, SCIFF, and rSAM sequences.Putative specialized metabolite BGCs were discovered by mining the draft genomes of novel taxa within the collection using antiSMASH 5.0 (ref. 18. All codes are publicly available at https://github.com/strowig-lab/PIBAC (ref. 50), referenced under 10.5281/zenodo.4075065. Briefly, the bioinformatic pipeline removed host-derived reads with BBMap using the corresponding Ensembl masked genome from pig and phiX. Megahit135 with specific parameters  was then applied using a SGI-UV2000 cluster with 256 cores and 2 TB shared memory for an all-in-one assembly of all metagenomic libraries. Contigs >1000 bp were used as reference to map all libraries using BWA119 with default settings. Binning was done with MetaBAT (version 0.32) using -verysensitive -pB 20 -B 100 -minclustersize 200000. Clusters were evaluated with CheckM123. Minimum quality thresholds were >50% completeness and <10% contamination, whilst >90% completeness and <5% contamination were indicators for high-quality MAGs. Their identity was tested against GTDB (release 04-RS89)53 and the 38 new taxa from the present study using ANI values <95% as species-level delineation. dRep136 was used for genome dereplication (95% Mash ANI). For depicting evolutionary relationships between taxa, analyses were conducted in MEGA7137 using the Neighbour-Joining method138 and bacteria marker gene alignment from GTDB-Tk. The evolutionary distances were computed using the Poisson correction method139 and expressed in the units of the number of amino acid substitutions per site. The analysis involved 609 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 5040 positions in the final dataset. The occurrence  of the 38 new taxa in PiBAC was estimated using bbmap140 by transforming mapped reads from the queried genomes to TPM (transcripts per million) values with adjustment to the fraction of unmapped reads for each library. Details and codes are provided here: https://github.com/strowig-lab/PIBAC.Genomes were reconstructed using shotgun metagenomic reads from the literature previously used for establishing the first reference gene catalogue of the pig microbiome18 were expressed into binary vectors (presence/absence) of PFAMs58. Iterative search against a local database of the isolates’ genomes returned those isolates that best matched each of the original profiles, considering matches and mismatches to the given metagenome. The number of species picked within minimal communities was determined in a sample-specific manner based on knee-points of cumulative metagenomic PFAM coverage curves using the ‘inflection’ package in R. The prevalence of picked species across the entire pig cohort and their median picking rank were then reported.The same pig gut metagenomes as in the previous sectionC. scindens DSM 100975 was cultivated in PYG medium, induced with 0.1 mM CA or DCA and mRNAs were isolated, enriched, sequenced, and analysed as in our previously published studies141. Raw data were submitted to the NCBI and are available under the BioSample accession numbers SAMN13016626-31.142. dDDH values <70% and ANI values <95% were considered as an indication for separate species. As within-species differences in the genome-based G+C content of DNA are almost exclusively <1%, larger differences also supported the status of distinct species143. POCP values <50% were considered as an indication for separate genera104. The classification of isolates based on genomes was also assessed using GTDB-Tk53 and by constructing phylogenomic trees to either confirm or deny novelty and genus delineation.Thresholds of 98.7%, 94.5%, and 86.5% 16S rRNA gene sequence identities were considered as indications for novel species, genera, and families, respectivelyAnaerococcus porci sp. nov.: Anaerococcus porci (por’ci. L. gen. n. porci of a pig). The closest phylogenetic neighbour within family Peptoniphilaceae (phylum Firmicutes) is Anaerococcus vaginalis, which shares a 16S rRNA gene sequence identity of 96.7%. The dDDH and ANI values of 22.2% and 81%, respectively, between the two genomes confirm the separate species status. Values between the isolate and Anaerococcus prevotii, the type species of the genus, are 22.5% and <80.0%. Cells grow as diplococci, are strictly anaerobic and stain Gram-positive. The genome encodes transporter systems for phosphate, alkanesulfonate, proline, monosaccharides, biotin, zinc, and d-methionine. Starch, trehalose, N-acetylmuramate, cellobiose, maltose, arbutin, salicin, and sucrose were identified as potential substrates. However, only starch, maltose, and trehalose enter glycolysis whilst the others undergo initial conversion and no subsequent reactions. Myo-inositiol can be converted into acetyl-CoA and enter the TCA cycle via a multistep pathway including conversion to scyllo-inosose (EC: 1.1.1.18) and malonic semialdehyde , which is finally converted into acetyl-CoA (EC: 1.2.1.18). After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (10.2 ± 0.1 mM) and acetate (9.6 ± 0.9 mM) and produced 16.8 ± 0.8 mM butyrate, 9.3 ± 1.5 mM formate, 5.5 ± 0.7 mM lactate, and 2.1 ± 0.1 mM propionate. The predominant cellular fatty acids are C16:0 (20%), iso-C17:1/anteiso-C17:1 (20%), C18:1 ω9c (14%), and C14:0 (12%). Other fatty acids are C16:1 ω7c/iso-C15:0 2-OH (4.4%), C12:0 (3.3%), and C18:1 ω7c (3.3%). The type strain is WCA-380-WT-2BT (=DSM 101005T). It was isolated from faeces of a German Landrace pig in Freising, Germany. Its G+C content of genomic DNA is 29.9 mol%.Description of Anaerovibrio slackiae sp. nov.: Anaerovibrio slackiae . According to 16S rRNA gene-based phylogeny, the species is placed into the family Selenomonadaceae (phylum Firmicutes). The closest phylogenetic neighbour is Anaerovibrio lipolyticus, which shares 94.7% sequence identity (1504 bp) and a POCP value of 54.6%, suggesting that the two species belong to the same genus, which was confirmed using GTDB-Tk. The ANI and dDDH values of <80% and 22.8%, respectively, together with a difference in the G+C content of genomic DNA of 5.3 mol% between the two genomes clearly confirm the status of a new taxon and the need to create a novel species to accommodate the isolate. It grows as short, motile rods that are slightly pear-shaped and curved. Occurs as single cells or in pairs. Passage intervals longer than 2–3 days result in strong reduction or cessation of growth in WCA and BHI broth. Within the genome, five transporters were identified along with ABC transporters for phosphate, alkanesulfonate, teichoic acid, d-methionine, cobalt, and biotin. Arbutin, salicin, d-glucose, sucrose, starch, cellobiose, cellulose, trehalose, and maltose were all indicated to be carbohydrate sources. The detection of genes encoding for FliA/C/D/F/G/M/N/P/S, FlhA/B, FlgB/C/D/E/G/K/L, and MotB indicate the presence of flagella. The RpfC/G two-component system along with the biofilm dispersal gene manA suggest that this species is able to form biofilms. This is further supported by the presence of the glycogen biosynthesis genes, glgA/C/P, along with its regulator, csrA. After 48 h at 37 °C in YCFA medium under anaerobic conditions, the species consumed glucose (11.3 ± 1.0 mM) and produced 19.3 ± 1.4 mM propionate, 11.0 ± 1.4 mM acetate, and smaller amounts of lactate (1.1 ± 0.1 mM). Major cellular fatty acids are C15:0 (20%), C13:0 3OH/C15:1 i I/H (15%), and C17:1 ω8c (13%). Lower amounts of the following fatty acids were detected: C17:0 (7.5%), C15:1 ω8c (7.4%), C16:1 ω9c (6.7%), C11:0 (5.4%), C13:0 (4.9%), C14:0 3OH/iso-C16:1 (4.7%), C16:0 (4.2%), and iso-C18:1 (3.8%). The type strain is WCA-693-APC-5D-AT (=DSM 108025T). It was isolated from faeces of an APC1311/+ pig in Freising, Germany. Its G+C content of genomic DNA is 48.7 mol%.Description of Baileyella gen. nov.: Baileyella . Based on 16S rRNA gene sequence analysis, the genus is phylogenetically placed into the yet undescribed family ‘Mogibacteriaceae’ (phylum Firmicutes). Phylogenomic analysis via GTDB-Tk classifies the isolate within the genus Mogibacterium and the POCP value to Mogibacterium pumilum, the type species of the genus, is 51.2%, which is nearing the genus-level threshold of 50%. However, we consider it not sound to propose a novel species within this genus to accommodate the isolate because its closest 16S rRNA gene-based phylogenetic neighbour M. pumilum shares only 92.3% 16S rRNA gene sequence identity (1430 bp), which is markedly below established genera delineation thresholds. The difference in the G+C content of genomic DNA between the two species is 5.4 mol%. The corresponding ANI and dDDH values are <80% and 20.1%, respectively. These findings clearly demonstrate the status of a novel taxon. Taken together, we propose the creation of a novel genus, for which the name Baileyella is proposed. Members of the genus are strictly anaerobic. The type species is Baileyella intestinalis.Description of Baileyella intestinalis sp. nov.: Baileyella intestinalis . The species has all the features of the genus. It forms short, straight rods (ca. 1 µm) that grow as single cells. Within the genome, nine transporters were identified along with ABC transporter systems for phosphate, osmoprotectants, and zinc. Starch was the only predicted carbohydrate source potentially utilized. Folate biosynthesis was identified from serine via conversion into 5,10-methylene-THMPT . Serine production from hydroxy-pyruvate was identified via 2-phospho-d-glycerate and phosphoserine . Serine can also be directly converted into l-tryptophane (EC: 4.2.1.20). The species is positive for lipase production using the Rhodamine B agar assay. The predominant cellular fatty acids are iso-C19:1 (19%), C18:0 (18%), C18:1 ω9c (14%), C18:1 ω6c (11%), and C16:0 (11%). Other fatty acids measured at lower amounts included iso/anteiso-C17:1 (7.7%), C17:1 ω9c (7.3%), C12:0 (3.3%), and C14:1 ω5c (2.3%). The type strain is RF-744-FAT-WT-3T (=DSM 106896T). It was isolated from faeces of an Aachen minipig83 in Germany. Its G+C content of genomic DNA is 29.9 mol%.Description of Berryella gen. nov.: Berryella . The 16S rRNA gene sequence of this isolate (1389 bp) shows highest identity with species of the genus Adlercreutzia, with values ranging from 92.6% for Adlercreutzia caecimuris to 94.1% for Adlercreutzia equolifaciens, the type species of the genus. The isolate is thus placed into the family Eggerthellaceae within the phylum Actinobacteria. POCP values between its genome and the genome of any of the Adlercreutzia spp. range from 48.5% to 51.9% and thus oscillate around the proposed genus-delineation threshold of 50%. GTDB-Tk analysis confirmed the status of novel genus within the family Eggerthellaceae. Of note, one strain designated Marseille-P7992 shares 99.8% 16S rRNA gene sequence identity as well as ANI, dDDH, and POCP values of 97.0%, 77.0%, and 84.6% with our isolate, respectively. This indicates that the two isolates belong to the same species. However, strain Marseille-P7992 has no official standing in the nomenclature. The type species of our proposed genus is Berryella intestinalis.Description of Berryella intestinalis sp. nov.: Berryella intestinalis . As many members of the family Eggerthellaceae, the species forms small rods that occur as single cells or in pairs, stain Gram-positive, and show low turbidity after growth in broth. Within the genome, 24 transporters along with ABC transporter systems for phosphate, d-methionine, zinc, biotin, aspartate, glutamate, and glutamine were detected. No carbohydrate sources were identified to be utilized by this species. Formate production from CO2 (EC: 1.17.1.10) and subsequent conversion into pyruvate via serine may be one method by which this species produces energy . Ammonia was able to be converted into l-glutamine (EC: 6.3.1.2), which in-turn can be utilized to form l-glutamate (EC: 1.4.1.13). Glutamate can be used to produce ornithine , which may be converted into citruline (EC: 2.1.3.3) and then to arginine (EC: 3.5.3.6). The co-factor siroheme was identified to be produced from glutamate . The main cellular fatty acids are C18:1 ω9c (30%), C16:0 (19%), and C18:0 (13%). Fatty acids with lower proportions include C14:0 (9.1%), C13:0 3OH/C15:1 i I/H (6.0%), and iso/anteiso-C17:1 (3.8%). The type strain is 68-1-3T (=DSM 104960T). It was isolated from the ileal content of a pig in Ames, Iowa, USA. Its G+C content of genomic DNA is 63.6 mol%.Description of Bilifractor gen. nov.: Bilifractor . Based on 16S rRNA gene-based phylogeny, the genus falls into the family Lachnospiraceae (phylum Firmicutes). The closest phylogenetic neighbours are Syntrophococcus sucromutans, Eubacterium cellulosolvens, and Blautia coccoides, which share 94.3%, 92.4%, and 91.7% sequence identity, respectively (1501 bp). The POCP values between the genome of our isolate and that of E. cellulosolvens and B. coccoides are 21.6% and 12.8%, respectively, suggesting a separate genus status. The corresponding dDDH values of 27.4% and 23.3% confirm that the isolate represents a different taxon. As no genome is yet available for S. sucromutans, corresponding analysis could not be performed. The type species is Bilifractor porci.Description of Bilifractor porci sp. nov.: Bilifractor porci (por’ci. L. gen. n. porci of a pig). The species grows under anaerobic conditions as diplococci that can form long, irregular chains. They stain Gram-negative. A single transporter was identified within the genome along with ABC transporter systems for phosphate, cystine, teichoic acid, biotin, and the RbsA/B/C/D transport system for ribose, autoinducer 2 (AI-2) and d-xylose. AI-2-based quorum signalling is likely to be utilized by this species due to the presence of LuxS and LsrA/B/D. Starch and trehalose were identified to be potential carbohydrate sources. Indole biosynthesis from shikimate may occur via conversion of shikimate to chorismate  and choristmate to indole . Biofilm formation may be favoured via the production of glycogen, as suggested by the presence of the GlgA/C/P genes. The species is considered to deconjugate primary bile acids as determined in vitro by cleavage of taurine residues on WCA agar medium containing TDCA. It is also positive for lipase production using the Rhodamine B agar assay. After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species did not use glucose and produced only small amounts of lactate (0.4 ± 0.1 mM). The most dominant cellular fatty acid is iso/anteiso-C17:1 (44%). Other fatty acids include C16:0 (19.1%), C16:0 N alcohol (5.8%), C18:0 (4.2%), and C13:0 3OH/C15:1 I I/H (3.3%). The type strain is Oil+RF-744-WCA-WT-13T (=DSM 106898T). It was isolated from faeces of an Aachen minipig83 in Germany. Its G+C content of genomic DNA is 50.4 mol%.Description of Bullifex gen. nov.: Bullifex ; N.L. masc. n. Bullifex a producer of bubbles, pertaining to the very peculiar cell morphology of the type strain of the type species). This new taxon belongs to the family Spirochaetaceae (phylum Spirochaetes). Whilst members of the phylum Spirochaetes are usually helically shaped, this isolate is coccoidal to spherical under the growth conditions tested. It shares 88.4% 16S rRNA gene sequence identity (1499 bp) with Sphaerochaeta pleomorpha and 87.7% with Sphaerochaeta globosa, the type species of the genus Sphaerochaeta. The corresponding ANI/dDDH values of <80%/17.8% and <80%/19.4% between the genome of our isolate and that of the two species aforementioned confirm the discovery of a new taxon. The POCP values of 38.5% and 38.7% justify the creation of a new genus to accommodate this isolate. This is supported by the GTDB-Tk classification within family ‘Sphaerochaetaceae’ without further information at the genus level. The type species is Bullifex porci.Description of Bullifex porci sp. nov.: Bullifex porci (por’ci. L. gen. n. porci of a pig). Cells are coccoidal to spherical with a size that varies between 1 and 10 µm. After several days of growth, they tend to form inflated round cells with an opaque, half-moon-shaped compartment on one side of the cell that stains positive with DAPI. The isolate is characterized by slow growth that can be improved by the addition of rumen fluid to the culture medium. Growth and cell morphology are not affected by β-lactam antibiotics . Within the genome, three transporters were identified along with ABC transporter systems for phosphate, cystine, d-methionine, arabino-oligosaccharide, methyl-galactoside, and biotin. Sucrose and starch were the only carbohydrates sources predicted to be utilized. Glutamate biosynthesis may occur from ammonia , which itself is produced from nitrite (EC: 1.7.1.15). Glutamate can then be used for the biosynthesis of ornithine . l-serine can be converted into O-acetyl-l-serine (EC: 2.3.1.30) and combined with sulfide to form l-cysteine (EC: 2.5.1.47). The absence of the flagella genes flgK and flgC within the genome corroborates the non-motile behaviour of the species as observed in vitro. The major cellular fatty acids (11–14%) are C18:0, C12:0, C16:0, and C18:1 ω9c. Other fatty acids include iso/anteiso-C17:1 (8.8%), iso-C19:1 (7.3%), C18:1 ω6c (4.8%), C20:1 ω9c (4.7%), C18:2 ω6,9c/anteiso-C18:0 (4.2%), C18:1 ω7c (3.9%), C14:0 (3.7%), and anteiso-C15:0 (3.0%). The type strain is NM-380-WT-3C1T (=DSM 105750T). It was isolated from faeces of a German Landrace pig in Freising, Germany. Its G+C content of genomic DNA is 35.9 mol%.Description of Clostridium porci sp. nov.: Clostridium porci (por’ci. L. gen. n. porci of a pig). According to 16S rRNA gene sequence analysis, the closest neighbours of this isolate are Clostridium clostridioforme, Clostridium aldenense, and Clostridium citroniae with 95.9%, 95.6%, and 95.5% sequence identity, respectively (1468 bp). All three species are placed into family Lachnospiraceae (phylum Firmicutes), apart from the type species of the genus Clostridium, Clostridium butyricum. ANI values between the genome of the three species aforementioned and that of the isolate were all <80%. The corresponding dDDH values were 22.8%, 22.3%, and 21.0%, respectively. These data clearly indicate the status of a separate species. Cells are rod-shaped and often occur in pairs. They grow only under strictly anoxic conditions. The genome encodes transporter systems for alkanesulfonate, arabinooligosaccharides, monosaccharides, methyl-galactoside, phosphate, aspartate, d-methionine, oligopeptide, zinc, cobalt, biotin, and teichoic acid. N-acetylmuramate and N-acetyl-d-glucosamine can be converted into N-acetyl-d-glucosamine-6-phosphate , which can in-turn be converted into fructose-6-phosphate and enter glycolysis . Cellobiose can also be utilized via conversion into cellobiose-6-phosphate (EC: 2.7.1.205) and subsequent conversion into d-glucose (EC: 3.2.1.86) or direct conversion into d-glucose (EC: 2.4.1.20) for use in glycolysis. Starch can also be utilized as a carbon source (EC: 2.4.1.1). The motility of this species as observed by light microscopy may be explained by the presence of many proteins involved in flagella assembly . After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (10.2 ± 0.6 mM) and produced 12.5 ± 0.7 mM acetate, 7.0 ± 1.1 mM formate, and trace amounts of isobutyrate (0.5 ± 0.2 mM). The major cellular fatty acids are C16:0 (31%) and C18:1 ω9c (21%). Other fatty acids detected at lower proportions include C14:0 (8.7%), C18:0 (8.3%), C18:1 ω7c (8.2%), C16:1 ω7c/iso-C15:1 2OH (5.6%), iso-C17:1/anteiso B-C17:1 (5.0%), and anteiso-C18:0/C18:2 ω6,9c (3.3%). The type strain is WCA-389-WT-23D1T (=DSM 100959T). It was isolated from faeces of a German Landrace pig in Freising, Germany. Its G+C content of genomic DNA is 46.2 mol%.Descriptin of Cutibacterium porci sp. nov.: Cutibacterium porci (por’ci. L. gen. n. porci of a pig). According to 16S rRNA gene-based phylogeny, the isolate is placed within family Propionibacteriaceae (phylum Actinobacteria). Its closest relative is Cutibacterium namnetense144, which shares a 16S rRNA gene sequence identity of 99.0%. The status of separate species is suggested by ANI and dDDH values between the two genomes of 82.6% and 25.6%, respectively. Moreover, the difference in G+C content of the genomic DNA is 1.2%. The isolate shares 98.4% sequence identity with Cutibacterium acnes, the type species of the genus Cutibacterium. Six transporters were identified within its genome along with ABC transporter systems for phosphate, maltose/maltodextrin, d-methionine, glutathione, ribose/AI-2/D-xylose, cobalt, and biotin. Arbutin, salicin, d-glucose, sucrose, trehalose, maltose, and starch were identified as potential carbohydrate sources. Metabolism of l-aspartate into fumarate via adenylo-succinate facilitates its entry into the citrate cycle . Conversion of sulfide into l-cysteine was identified, utilizing O-acetyl-l-serine produced from l-serine . Production of riboflavin from GTP and ribulose-5-phosphate was identified . After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed 11.4 ± 0.8 mM glucose and produced 24.4 ± 0.3 mM propionate, 10.0 ± 0.4 mM acetate, and trace amounts of formate (0.6 ± 0.1 mM). The predominant cellular fatty acid is iso-C15:0 (42%). Other fatty acids included C15:0 (15%), iso-C14:0 3-OH (10%), anteiso-C15:0 (7.4%), iso-C16:0/C14:0 3-OH (7.0%), and iso-C17:0 (4.5%). The type strain is WCA-380-WT-3AT (=DSM 101006T). It was isolated from faeces of a German Landrace pig in Freising, Germany. Its G+C content of genomic DNA is 58.8 mol%.Description of Desulfovibrio porci sp. nov.: Desulfovibrio porci (por’ci. L. gen. n. porci of a pig). The closest phylogenetic neighbour within family Desulfovibrionaceae  is Desulfovibrio desulfuricans, which shares a 16S rRNA gene sequence identity of 96.7%. The dDDH value of 21.4%, ANI value <80%, and difference in the G+C content of DNA of 4.2% between the two genomes confirmed the separate species status. Cells are rod-shaped and motile. A total of 52 ABC transporters were detected within the genome, including those for teichoic acid, lipoprotein, alkanesulfonate, phosphate, and d-methionine. Genes coding for the HydH/G zinc/lead two-component system were detected, suggesting a resistance to high environmental zinc and lead levels. Starch utilization may occur via conversion into alpha-d-glucose-1P (EC: 2.4.1.1), which can directly enter glycolysis or be converted into UDP-glucose (EC: 2.7.7.9) and then amylose (EC: 2.4.1.11). The complete dissimilatory sulfate reduction and oxidation reaction was detected . l-cysteine formation was identified using either sulfide (EC: 2.5.1.47) or l-serine via O-acetyl-l-serine . Siroheme biosynthesis from glutamate  suggests that siroheme may act as a cofactor during both sulfur and nitrogen reduction. The predominant cellular fatty acids are C15:0 ISO (44%) and iso-17:1 ω9c (21%). Other fatty acids include iso-C17:0 (7.4%), C16:0 (7.0%), iso-C14:0 3OH (3.5%), cyclo-C17:0 (3.0%), anteiso-C15:0 (2.6%), and C18:1 ω9c (2.1%). The type strain is PG-178-WT-4T (=DSM 105247T). It was isolated from faeces of a German Landrace pig in Freising, Germany. Its G+C content of genomic DNA is 61.4 mol%.Description of Eisenbergiella porci sp. nov.: Eisenbergiella porci (por’ci. L. gen. n. porci of a pig). The 16S rRNA gene sequence of this species (1432 bp) shares 97.8% identity with the sequence of Eisenbergiella tayi, the type species of the genus Eisenbergiella within family Lachnospiraceae (phylum Firmicutes). Difference in the G+C content of genomic DNA between the two species is 1.6%. The ANI value <95% and dDDH value of 22.9% clearly favour the creation of a novel species to accommodate the isolate. Of note, a strain previously isolated by others and tentatively named ‘Eisenbergiella massiliensis’ shares 99.4% 16S rRNA gene sequence identity, an ANI value of 96.7% and dDDH value of 76.5% with the isolate, indicating that they belong to the same species. However, to the best of our knowledge until the time of submission of the present manuscript, ‘E. massiliensis’ is not a valid name. Cells grow under anaerobic conditions and form 2–5-µm-long, straight, thick rods that often grow as long chains. A single transporter was identified within the genome along with ABC transporter systems for phosphate, teichoic acid, bacitracin, osmoprotectants, maltose, d-methionine, oligo-peptides, glutathione, arabino-oligosaccharide, zinc, methyl-galactoside, cobalt, and biotin. Two-component systems for phosphate and potassium were identified along with both the BceA/B and VraD/E bacitracin efflux systems. Trehalose, starch, fructan, cellobiose, and d-glucose were identified as potential carbohydrate sources. Folate biosynthesis was identified from serine via conversion into 5,10-methylene-THMPT . Indole biosynthesis from shikimate was identified via conversion of shikimate to chorismate  and choristmate to indole . Ammonia may be converted into l-glutamine (EC: 6.3.1.2) which in-turn can be utilized to form l-glutamate (EC: 1.4.1.13). The co-factor siroheme was identified to be produced from glutamate . After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed 11.6 ± 0.2 mM glucose and trace amounts of propionate (0.8 ± 0.5 mM) and produced 26.3 ± 1.7 mM formate and 10.4 ± 0.1 mM butyrate. Smaller amounts of acetate (6.0 ± 4.2 mM) and traces of isobutyrate (0.5 ± 0.1 mM) were also detected. The predominant cellular fatty acid is C18:1 ω9c (46%). Additional fatty acids are C16:0 (17.0%), C14:0 (11.8%), C18:1 ω7c (8.9%), and C17:1 ω9c (6.7%). The type strain is WCA-389-WT-23BT (=DSM 101007T). It was isolated from faeces of a German Landrace pig in Freising, Germany. Its G+C content of genomic DNA is 48.4 mol%.Description of Floccifex gen. nov.: Floccifex ; N.L. masc. n. Floccifex a producer of flakes, pertaining to the growth behaviour of the type strain of the type species in liquid medium). The isolate is phylogenetically placed into the family Erysipelotrichaceae (phylum Firmicutes). Based on 16S rRNA gene sequence analysis, it clusters apart from Catenisphaera adipataccumulans, with which it shares 90.5% sequence identity. The species with the closest gene sequence identity is Faecalitalea pleomorphus (93.0%), which is characterized by a POCP value of 53.1% to the genome of the isolate. In contrast, GTDB-Tk analysis classifies the isolate within the genus Holdemanella. There is no genome of C. adipataccumulans available to date, preventing further analyses. Considering these discrepancies based on genomes and the aforementioned 16S rRNA gene sequence identities clearly below genus delineation thresholds, we propose the creation of a novel genus, Floccifex, to accommodate this isolate. The type species is Floccifex porci.Description of Floccifex porci sp. nov.: Floccifex porci (por’ci. L. gen. n. porci of a pig). Cells are irregular rods of ca. 1 µm in length that form short to long chains. After 7–14 days of growth in BHI broth at 37 °C under anaerobic conditions, the species forms characteristic amalgams of cells that appear as flakes floating in the liquid medium. Within the genome, 18 transporters were detected along with ABC transporter systems for phosphate, teichoic acid, d-methionine, oligopeptides, and biotin. The CssS/R, HtrA two-component system for detection and removal of misfolded proteins was present. d-glucose, arbutin, salicin, fructan, starch, cellobiose, maltose, and trehalose were all identified as potential carbohydrate substrates for this species. Production of ornithine, valine, and leucine was identified from glutamate, pyruvate, and 2-oxoisocaproate, respectively. Metabolism of l-aspartate into fumarate allowed for entry into the citrate cycle. Glutamate biosynthesis was identified from ammonia , which itself was produced from nitrile (EC: 3.5.5.1). Main cellular fatty acids are C18:1 ω9c (23%), iso-anteiso-C17:1 (18%), and C16:0 (16.3%). Other fatty acids were detected in lower proportions: C18:2 ω6,9c/anteiso-C18:0 (9.4%), C18:0 (8.5%), and iso-C17:0 3-OH (8.1%). The type strain is LKV-178-WT-2GT (=DSM 104670T). It was isolated from faeces of a German Landrace pig in Freising, Germany. Its G+C content of genomic DNA is 33.5 mol%.Description of Hallerella gen. nov.: Hallerella . The nearest phylogenetic neighbour of these strictly anaerobic bacteria within the family Fibrobacteriaceae (phylum Fibrobacteres) is Fibrobacter intestinalis. The POCP values >70% between the two isolated strains and F. intestinalis clearly indicate the status of these three bacteria as members of the same genus. The POCP value between the genome of F. intestinalis and that of Fibrobacter succinogenes, the type species of the genus Fibrobacter, is 52.1%, which is nearing the genus-specific delineation threshold. Although the isolates showed POCP values of 54.7 and 55.8% to F. succinogenes, GTDB-Tk proposed a genus distinct from Fibrobacter. Moreover, 16S rRNA gene sequence identities between the two isolates and F. succinogenes are <92%, well below established genus delineation thresholds. According to these findings, we suggest that a novel genus distinct from Fibrobacter must be created to accommodate the two isolated strains and that F. intestinalis may have to be re-classified within this genus, for which the name Hallerella is proposed. The type species is Hallerella porci.Description of Hallerella porci sp. nov.: Hallerella porci (por’ci. L. gen. n. porci of a pig). The species shares 94.2% 16S rRNA gene sequence identity with F. intestinalis and <91% identity with F. succinogenes, the type species of the genus Fibrobacter. The species grows as straight rods of 1–2 µm in length that occur as single cells. It grows best on solid media. Within the genome, 11 transporters were identified along with ABC transporter systems for phosphate, alkanesulfonate, teichoic acid, and lipoproteins. Starch and cellulose were identified as potential carbohydrate substrates. Both the complete pathway from pyruvate to valine and to leucine via 2-oxoisovalerate were identified. Conversion of folate to and from both 7,8-dihydrofolate and 5,6,7,8-tetrahydrofolate was also identified (EC: 1.5.1.3). After 96 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (13.1 ± 0.4 mM) and produced succinate (10.4 ± 0.5 mM), acetate (5.3 ± 0.6 mM), formate (2.5 ± 0.3 mM), and traces of isobutyrate (0.7 ± 0.1 mM). The main cellular fatty acid is C16:0 (40%). Other cellular fatty acids include C14:0 2-OH (17.9%), C15:0 (12.4%), iso-C13:0 3-OH (10.6%), and iso-C16:0 (4.8%). The type strain is UWS4T (=DSM 104699T). It was isolated from the caecum of a pig in Madison, Wisconsin, USA. Its G+C content of genomic DNA is 44.8 mol%.Description of Hallerella succinigenes sp. nov.: Hallerella succinigenes  producing; N.L. part. adj. succinigenes succinic acid-producing). The species shares 97.0% 16S rRNA gene sequence identity with F. intestinalis, 95.0% with H. porci, the type species of the proposed genus Hallerella, and 91.5% with F. succinogenes, the type species of the genus Fibrobacter. The dDDH and ANI values of 22.8% and 80.2% to H. porci as well as 21.8% and <80% to F. intestinalis confirm the status of the isolate as a separate species. Cells are tiny rods that grow only under strictly anoxic conditions. Within the genome, 14 transporters along with transporter systems for alkanesulfonate, zinc, phosphonate, phosphate, teichoic acid, Na+, and lipoprotein were found. Cellulose was predicted to be converted into d-glucose and utilized in glycolysis via conversion into cellobiose (EC: 3.2.1.4) and then d-glucose (EC: 2.4.1.20). Starch may also be utilized as a carbon source (EC: 2.4.1.1). Pyruvate may be utilized to produce either valine  or leucine . DAP-type peptidoglycan production was predicted to occur from UDP-N-acetyl-alpha-d-glucosamine . Propanoate production was identified to be formed from propanoyl-CoA via propanoyl-phosphate  or propionyl-adenylate (EC: 6.2.1.1). After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (11.5 ± 0.5 mM) and produced succinate (8.2 ± 1.9 mM), acetate (3.2 ± 1.9 mM), and small amounts of formate (0.9 ± 0.1 mM). The cellular fatty acids profile consists of iso-C13:0 3-OH (24%), C14:0 2-OH (24%) and C15:0 (18%), C16:0 (13%), iso-C16:0 (6.2%), and anteiso-C15:1 (5.9%). The type strain is UWS3T (=DSM 104698T). It was isolated from the caecum of a pig in Madison, Wisconsin, USA. Its G+C content of genomic DNA is 49.2 mol%.Description of Holdemanella porci sp. nov.: Holdemanella porci (por’ci. L. gen. n. porci of a pig). The closest phylogenetic neighbour within family Erysipelotrichaceae (phylum Firmicutes) is Holdemanella biformis, the type species of the genus, which shares a 16S rRNA gene sequence identity of 98.4%. The dDDH and ANI value of 39.8% and 90.1%, respectively, confirm the separate species status. The species is strictly anaerobic and grows as bacilli that stain Gram-positive and form chains of up to 10 µm. A total of 18 transporters along with transporter systems for phosphate, d-methionine, and teichoic acid were detected in the genome. The two-component system CssRS for removal of misfolded proteins along with the HtrA serine protease were also detected. Cellobiose was suggested to be an energy source via conversion into cellobiose-6-phosphate (EC: 2.7.1.205) and subsequent conversion into d-glucose (EC: 3.2.1.86). Extracellular trehalose can also be utilized via conversion to trehalose-6-phosphate (EC: 2.7.1.201) and then to d-glucose-6-phosphate (EC: 3.2.1.122). Arbutin and salicin can be converted into beta-d-glucose-6-phosphate  which then enters glycolysis via conversion into beta-d-fructose-6-phosphate (EC: 5.3.1.9). Starch can also be utilized as a carbon source (EC: 2.4.1.1). Ammonia was identified to be utilized for production of l-glutamate  via l-glutamine (EC: 6.3.1.2). Based on the analysis of a large catalogue of metagenome-reconstructed genomes, the species is proposed to be a prevalent member of the intestinal microbiome also in human. The cellular fatty acid profile comprises C18:1 ω9c (35%), C16:0 (20%), C18:0 (15%) as major components. Minor fatty acids included C18:2 ω6,9c/anteiso-C18:0 (6.9%), iso-C17:1/anteiso-C17:1 (4.0%), C16:1 ω6,9c/iso-C15:0 2OH (3.5%), and C18:1 ω7c (3.2%). The type strain is LKV-472-APC-3T (=DSM 105256T). It was isolated from faeces of an APC1311/+ pig in Freising, Germany. Its G+C content of genomic DNA is 34.1 mol%.Description of Hornefia gen. nov.: Hornefia . Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbour for both isolates is Eubacterium nodatum (<93.5% sequence identity). They are placed within the yet undescribed family ‘Mogibacteriaceae’, distant from the type species of the type genus within family Eubacteriaceae, Eubacterium limosum. With POCP values of ca. 24 and 53% against the genome of E. limosum and E. nodatum together with a POCP value of 36–38% against Mogibacterium pumilum, the type species of the genus Mogibacterium, the isolates can be considered to represent a novel genus, which was confirmed by GTDB-Tk classification. Both isolates grow only under strictly anaerobic conditions. Genome features that are consistent across species of this genus include: (i) the production of O-acetyl-l-homoserine and O-succinyl-l-homoserine from l-aspartate; (ii) the conversion of CO2 into formate; (iii) starch was identified as the sole consistent carbohydrate source; (iv) ABC transport systems for phosphate, teichoic acid, osmoprotectants, cobalt, and biotin; (v) the AtoA/B/C/D/E/S two-component system which detects acetoacetate and leads to SCFA metabolism; (vi) the SCFAs butyric, acetic, and propionic acid, were all identified as products; (vii) propionic acid production occurs from acryloyl-CoA , acetic acid from acetyl-CoA  and butyric acid from butanal . The genus name Hornefia is proposed to accommodate both isolates. The type species is Hornefia butyriciproducens.Description of Hornefia butyriciproducens sp. nov.: Hornefia butyriciproducens . This species shares all the functional features associated with the genus. Cells are irregular rods with a length of 1–3 µm. Whilst both species of the genus are capable of producing O-acetyl-l-homoserine and O-succinyl-l-homoserine from l-aspartate, only H. butyriciproducens is able to convert l-aspartate into 2-ocobutanoate using l-homoserine as an intermediate , which can then be utilized for production of propionic acid . H. butyriciproducens is also able to utilize sucrose. The species is considered to deconjugate primary bile acids as determined in vitro by cleavage of taurine residues on WCA agar medium containing TDCA. After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed traces of acetate (1.4 ± 1.1 mM) and produced 6.0 ± 1.8 mM butyrate and small amounts of formate (1.3 ± 0.4 mM) and succinate (0.4 ± 0.2 mM). Cellular fatty acids mostly consist of C16:0 (26%), iso-C17:1/anteiso-C17:0 (17%), iso-C19:1 (16%), and C16:1 ω7c/iso-C15:0 2OH (10%). Minor fatty acids include C14:0 (8.6%), C18:1 ω7c (4.8%), C18:0 (4.1%), and C18:1 ω7c (3.7%). The type strain is WCA-MUC-591-APC-3HT (=DSM 104962T). It was isolated from faeces of an APC1311/+ pig in Freising, Germany. Its G+C content of genomic DNA is 51.6 mol%.Description of Hornefia porci sp. nov.: Hornefia porci (por’ci. L. gen. n. porci of a pig). This species shares 99.3% sequence identity with the type species of the genus, H. butyriciproducens, according to their the 16S rRNA genes. However, their dDDH and ANI values of 49.4% and 92.8% indicate a separate species status, whilst the POCP value of 81.5% confirms their classification within the same genus. This species possesses all functional features of the genus. It is able to utilize CO2 for the production of both CO (EC: 1.2.7.4) and formate (EC: 1.17.1.10). The species is considered to deconjugate primary bile acids as determined in vitro by cleavage of taurine residues on WCA agar medium containing TDCA. After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed traces of acetate (1.9 ± 0.7 mM) and produced 5.6 ± 0.4 mM butyrate and 2.2 ± 0.3 mM formate. Compared with H. butyriciproducens, cells of H. porci contain higher amounts of the cellular fatty acid iso-C17:1/anteiso-C17:0 (31%) at the expense of iso-C19:1 (8.7%), C14:0 (5.4%), and C16:1 ω7c/iso-C15:0 2OH (4.34%). C17:1 ω8c (3.7%) was detected only in H. porci. The type strain is 68-3-10T (=DSM 104948T). It was isolated from the colon content of a pig in Ames, Iowa, USA. Its G+C content of genomic DNA is 52.3 mol%.Description of  Inconstantimicrobium gen. nov.: Inconstantimicrobium . The genus is phylogenetically placed into the family Clostridiaceae (phylum Firmicutes) based on 16S rRNA gene analysis. Clostridium sardiniense is the closest relative, with a sequence identity of 94.4%. The isolate clusters away from Clostridium butyricum, the type species of the genus Clostridium, with which it shares 93.4% sequence identity. There is no genome available for C. sardiniense, which prevents further analysis. The POCP values of 45.6% between the genome of the isolate and that of C. butyricum confirm a genus status distinct from Clostridium, for which the name Inconstantimicrobium is proposed. The type species is Inconstantimicrobium porci.Description ofInconstantimicrobium porci sp. nov.: Inconstantimicrobium porci (por’ci. L. gen. n. porci of a pig). The species has all the features of the genus. Cells are motile, thick rods with a length of 2–10 µm. They have a very characteristic swarming growth behaviour on solid media. They grow only under strictly anoxic conditions and stain Gram-positive. Within the genome, a single transporter was identified along with ABC transporter systems for phosphate, Na+, bacitracin, d-methionine, zinc, and biotin. Sucrose, cellobiose, maltose, trehalose, starch, arbutin, salicin, and d-glucose were identified as potential carbohydrate sources. Motility may be conferred by flagella as indicated by the presence of the following genome-encoded proteins: FliA/C/D/E/F/G/M/N/S/P, FlhA/B, FlgB/C/E/G/K, and MotB. Motility direction is predicted to be governed via the CheA/B/C/D/R/V/W/Y/X and MCP genes. Glycogen production via GlgA/C/P along with quorum sensing genes including LuxS may indicate that this species is able to form biofilm. The major cellular fatty acid is C18:1 ω9c (86%). Smaller proportions of C18:1 ω7c (6.0%) and C18:1 (3.5%) were detected. The type strain is WCA-383-APC-5BT (=DSM 108839T). It was isolated from faeces of an APC1311/+ pig in Freising, Germany. Its G+C content of genomic DNA is 31.4 mol%.Description of Mobiluncus porci sp. nov.: Mobiluncus porci (por’ci. L. gen. n. porci of a pig). The closest phylogenetic neighbour within family Actinomycetaceae (phylum Actinobacteria) is Mobiluncus curtisii, the type species of the genus Mobiluncus, which shares a 16S rRNA gene sequence identity of 98.6%. The dDDH value of 20.6% and ANI value <80% between the two genomes indicate that M. porci is a novel species. Cells are motile, rod-shaped, curved, and 2–4 µm long. Twenty transporters along with transporter systems for d-methionine, phosphate, and teichoic acid were detected in the genome. The phosphate detection and assimilation two-component system SenX3/RegX3 along with the phoA and pstS genes were also detected. Starch seems to be the only extracellular carbon source (EC: 2.4.1.1) utilized by this species, apart from d-glucose, both of which enter glycolysis. Sulfide and l-serine can be converted into l-cysteine  or converted into sulfur, releasing hydrogen (EC: 1.8.2.3). Propanoate production was identified to occur via the utilization of propanoyl-CoA via propanoyl-phosphate . The motility of this species may be due to the presence of flagella as multiple flagella assembly genes were identified within the genome . The major cellular fatty acids are C16:0 (22%), C18:0 (15%), and C18:1 ω9c (14%). Other fatty acids included C12:0 (8.5%), iso I-C19:1 (4.3%), C18:1 ω6c (3.9%), C14:0 (3.7%), C18:2 ω6,9c/anteiso-C18:0 (3.2%), C17:1 ω9c (2.9%), C16:1 ω7c/iso-C15:0 2OH (2.8%), iso I/anteiso B-C17:1 (2.4%), C13:0 3OH/C15:1 i I/H (2.4%), and C10:0 (2.2%). The type strain is RF-GAM-744-WT-7T (=DSM 108840T). It was isolated from faeces of an Aachen minipig83 in Germany. Its G+C content of genomic DNA is 56.3 mol%.Description of Mogibacterium kristiansenii sp. nov.: Mogibacterium kristiansenii . Based on 16S rRNA gene sequence analysis, the nearest phylogenetic neighbour is Mogibacterium diversum, a member of the yet undescribed family ‘Mogibacteriaceae’ within the phylum Firmicutes , which shares 94.4% sequence identity. Analysis of the two genomes revealed a POCP value of 52.0% and GTDB-Tk classified the isolate as member of the genus Mogibacterium. The difference in G+C content of genomic DNA of 9.1 mol% together with ANI and dDDH values of <80% and 20.4%, respectively, clearly indicate the status of a separate species. Cells are 1–1.5 µm long, thin rods that grow only under strictly anoxic conditions and stain Gram-positive. Within the genome, four transporters were identified along with ABC transporter systems for phosphate, teichoic acid, and biotin. No carbohydrate sources were identified to be used by this species. However, glucose-6-phosphate utilization within glycolysis was present, suggesting an alternative method for the production of energy. Formate production from CO2 (EC: 1.17.1.10) and subsequent conversion into pyruvate via serine may be one way by which this species produces energy . The most dominant cellular fatty acid is C18:1 ω9c (64%). Lower proportions of other fatty acids were measured: C16:0 (10.2%), C18:1 ω6c (5.7%), C18:0 (5.2%), and C17:1 ω9c (4.1%) could be detected as well. The type strain is WCA-MUC-591-APC-4BT (=DSM 106282T). It was isolated from faeces of an APC1311/+ pig in Freising, Germany. Its G+C content of genomic DNA is 50.0 mol%.Description of Oliverpabstia gen. nov.: Oliverpabstia . The genus is phylogenetically placed into the family Lachnospiraceae (phylum Firmicutes). Based on 16S rRNA sequence identity, Blautia stercoris is the closest phylogenetic neighbour (94.3%). The corresponding ANI and dDDH values of <80% and 24.5% confirm that the isolate represents a different taxon. The POCP value between the two genomes is 54.9%. However, GTDB-Tk places the isolates into a new genus within the Lachnospiraceae and the 16S rRNA gene sequence identity to Blautia coccoides, the type species of this genus, is 92.7%. Hence, we propose the name Oliverpabstia for this novel genus. The type species is Oliverpabstia intestinalis.Description of Oliverpabstia intestinalis sp. nov.: Oliverpabstia intestinalis . Cells are strictly anaerobic coccobacilli with conic ends that stain Gram-positive and often grow in pairs. The genome encodes two transporters along with ABC transporter systems for phosphate, teichoic acid, arabino-oligosaccharide, and biotin. The RpfC/G, Clp two-component system for biofilm production was detected within the genome, along with LuxS for quorum signalling. Indole biosynthesis from shikimate was identified via conversion of shikimate to chorismate  and choristmate to indole . The species is considered to deconjugate primary bile acids as determined in vitro by cleavage of taurine residues on WCA agar medium containing TDCA. The major cellular fatty acid is C16:0 (34%). Other fatty acids are C14:0 (16.6%), C18:1 ω7c (15.6%), iso/anteiso-C17:1 (8.8%), C13:0 3OH/C15:1 I I/H (4.0%), and C16:1 ω7C/iso-C15:1 2OH (3.3%). The type strain is BSM-380-WT-5A T (=DSM 106162T). It was isolated from faeces of a German Landrace pig in Freising, Germany. Its G+C content of genomic DNA is 44.1 mol%.Description of Olsenella porci sp. nov.: Olsenella porci (por’ci. L. gen. n. porci of a pig). The closest phylogenetic neighbour within family Atopobiaceae (phylum Actinobacteria) is Olsenella uli, the type species of the genus Olsenella, which shares a 16S rRNA gene sequence identity of 96.7% (1463 bp). The dDDH value of 20.8% and ANI value <80% between the two genomes confirm that O. porci is a novel species. The bacterium grows as irregular rods with a length of 0.5–1 µm that may form chains. Eight transporters along with transporter systems for phosphate, aspartate/glutamate/glutamine, and cystine were present in the genomes. Starch, cellobiose, maltose, arbutin, salicin, and d-glucose were identified as potential substrates. l-cysteine may be converted into pyruvate (EC: 4.4.1.13), potentially acting as a source of energy. l-cysteine can also be produced internally using sulfide and acetyl-l-serine (EC: 2.5.1.47). Additionally, l-aspartate may also enter the TCA cycle via conversion into oxaloacetate (EC: 2.6.1.1) or to fumarate using either adenylo-succinate  or l-arginino-succinate  as intermediates. The TCA cycle itself is incomplete with the steps from 2-oxo-glutarate to fumarate and the conversion of malate to oxaloacetate missing. The species is considered to deconjugate primary bile acids as determined in vitro by cleavage of taurine residues on WCA agar medium containing TDCA. After 96 h at 37 °C in modified YCFA medium under anaerobic conditions, no glucose was utilized and only small amounts of acetate (1.9 ± 0.2 mM) were detected. The predominant cellular fatty acid is C18:1 ω9c (33%). Other fatty acids include C12:0 (11.2%), iso-C19:1 (10.6%), C16:0 (9.6%), C18:2 ω6/9c/anteiso-C18:0 (6.6%), C18:0 (6.4%), C18:1 ω7c (5.8%), C17:1 ω9c (3.7%), iso/anteiso-C17:1 (3.3%), and C16:1 ω7c/iso-C15:0 2-OH (2.3%). The type strain is CA-Schmier-601-WT-1T (=DSM 105246T). It was isolated from faeces of a German Landrace pig (minipig breed) in Freising, Germany. Its G+C content of genomic DNA is 64.9 mol%.Description of Peptoniphilus porci sp. nov.: Peptoniphilus porci (por’ci. L. gen. n. porci of a pig). Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbour with a valid name is Peptoniphilus gorbachii within family Peptoniphilaceae (phylum Firmicutes), with a sequence identity of 97.1% (1412 bp). There is no genome available for P. gorbachii, preventing further analysis. Sequence identity to the next relative, Peptoniphilus harei, is 96.0% and to Peptoniphilus asaccharolyticus, the type species of the genus, 88.5%. The latter species is characterized by a POCP value of 55.2 to the isolate, suggesting that they indeed belong to the same genus despite the low 16S rRNA gene sequence identity outlined above. The ANI/dDDH values of 81.1%/20.6% (isolate to P. harei) and <80%/33.1% (isolate to P. asaccharolyticus) together with corresponding differences in the G+C content of genomic DNA of 0.9 mol% and 3.2 mol% confirm the status of our isolate as a new species. Of note, due to an ANI value >95%, the isolate is proposed to belong to the same species as strain ph5 (= DSM 25475), previously isolated by others from human faeces145. However, by the time of submission of this manuscript, the proposed name of the latter isolate, ‘Peptinophilus grossensis’, is still not valid. Moreover, the two isolates share only 96.9% 16S rRNA gene sequence identity. Hence, we think it is appropriate to propose a novel species to accommodate this pig isolate. Altogether, Peptoniphilus appears to be a phylogenetically and phylogenomically heterogeneous genus that may require taxonomic rearrangement in the future. Cells of this new species are cocci that stain Gram-positive and grow single or in pairs. Within the genome, a single transporter was identified; however, transporter systems for osmoprotectants, zinc, biotin, d-methionine, and phosphate were present. A two-component system for phosphate limitation was detected, which consists of PhoR, PhoB, PhoA, and PhoP. A distinct lack of carbohydrate metabolism genes was noticed with no extracellular carbohydrate sources being utilized. Alternatively, it appeared that both d-serine and l-serine may act as an energy source via conversion into pyruvate and ammonia . Pyruvate can then be converted into acetyl-CoA (EC: 1.2.7.11) that can be used to produce acetate . Propanoate production was also identified from acryloyl-CoA via conversion to propanoyl-CoA (EC: 1.3.8.1), propanoyl phosphate  and then propanoate (EC: 2.7.2.1). After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species did not consume glucose and produced 9.5 ± 0.5 mM acetate, 3.5 ± 0.2 mM butyrate, 3.0 ± 0.5 mM propionate, and smaller amounts of formate (0.6 ± 0.2 mM). The dominant cellular fatty acids are: C16:0 (28%), C18:1 ω7c (15%), C14:0 (13%), iso/anteiso-C17:1, and C16:1 ω7c/iso-C15:1 2OH (12% each). Lower proportions of the following fatty acids were detected: C16:1 ω9c (5.7%), C18:0 (2.2%), and C16:1 ω5c (2.1%). The type strain is 35-6-1T (=DSM 104947T). It was isolated from the colonic mucosa of a pig in Ames, Iowa, USA. Its G+C content of genomic DNA is 31.2 mol%.Description of Peptostreptococcus porci sp. nov.: Peptostreptococcus porci (por’ci. L. gen. n. porci of a pig). The closest phylogenetic neighbours within family Peptostreptococcaceae (phylum Firmicutes) are Peptostreptococcus anaerobius, the type species of the genus Peptostreptococcus, which shares a 16S rRNA gene sequence identity of 98.4% and Peptostreptococcus canis (97.9% sequence identity). Whilst there is no genome available for P. canis, the dDDH value of 22.6%, ANI value <80%, and difference in the G+C content of genomic DNA (2.4%) between the genomes of our isolate and P. anaerobius support a separate species. Cells are irregular bacilli with a length of 0.5–1 µm. They stain Gram-positive and are strictly anaerobic. Within the genome, two transporters were identified along with ABC transporter systems for phosphate, osmoprotectants, zinc, biotin, cobalt, and d-methionine. Both the arlS and arlP genes were detected, suggesting a potential two-component system for adhesion. Starch was identified as the sole carbohydrate source. Alternatively, l-serine and sulfide may act as an energy source via conversion into acetate and l-cysteine (EC: 2.5.1.47). l-serine may first be converted into O-acetyl-l-serine (EC: 2.5.1.47). Acetate can be converted into acetyl-CoA , which can be used for fatty acid synthesis. Propanoate production was also identified from acryloyl-CoA via conversion to propanoyl-CoA (EC: 1.3.8.1), propanoyl phosphate  and then propanoate (EC: 2.7.2.1). Whilst production of butanoyl-P and butanoate was not identified, the ability to convert one to the other was present (EC: 2.7.2.7). After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (2.2 ± 4.9 mM) and propionate (1.5 ± 2.1 mM) and produced 15.4 ± 2.0 mM acetate, and smaller amounts of isobutyrate (3.1 ± 0.8 mM), isovalerate (3.0 ± 1.3 mM), formate (1.7 ± 0.6 mM), and lactate (1.7 ± 0.3 mM). The cellular fatty acid composition of P. porci is dominated by iso-C16:0 (20%) and iso-C14:0 (10%). Other fatty acids include iso-C17:1/anteiso-C17:1 (10.3%), iso-C12:0 (6.5%), iso-C18:0 (5.4%), iso-C13:0 3-OH (4.0%), and iso-C17:0 (3.6%). The type strain is WCA-SAB-591-4A-AT (=DSM 106284T). It was isolated from faeces of a German Landrace pig (minipig breed) in Freising, Germany. Its G+C content of genomic DNA is 33.3 mol%.Description of Porcincola gen. nov.: Porcincola . Phylogenetically, the isolate falls into family Lachnospiraceae (phylum Firmicutes). The closest neighbour based on the comparison of partial 16S rRNA gene sequences (1438 bp) is Blautia producta, which shares 90.0% sequence identity. The isolate also shares 89.2% identity with Marvinbryanta formatexigens, the type species of the genus Marvinbryanta within the same family. The POCP values between the genome of the isolate and that of these two related species are 29.3% and 36.3%, respectively, which confirms its separate genus status. Cells grow only under strictly anaerobic conditions. The type species is Porcincola intestinalis.Description of Porcincola intestinalis sp. nov.: Porcincola intestinalis . Cells are coccobacilli that grow in pairs and can form chains up to 10 µm in length. They stain Gram-positive and are non-motile. Two transporters were identified within the genome along with ABC transporter systems for phosphate, cystine, teichoic acid, d-methionine, oligopeptide, arabino-oligosaccharide, ribose, AI-2, d-xylose, methyl-galactoside, biotin, and zinc. Presence of the RbsA/B/C ABC transporter for AI-2 and LuxB/D/S suggests this species may utilize AI-2 as a quorum sensing molecule. Starch and cellulose were identified to be the only carbohydrate sources utilized by this species. An alternative pathway for energy production is conversion of glutamate into ornithine  that can enter the urea cycle, thereby producing fumarate . The species is considered to deconjugate primary bile acids as determined in vitro by cleavage of taurine residues on WCA agar medium containing TDCA. It is also positive for lipase production using the Rhodamine B agar assay. The predominant cellular fatty is C18:1 ω9c (65%). Lower amounts of C16:0 (13.7%), C18:1 ω7c (7.9%), and iso/anteiso-C17:1 (3.5%) were detected. The type strain is Oil+RF-744-WCA-WT-11T (=DSM 106895T). It was isolated from faeces of an Aachen minipig83 in Germany. Its G+C content of genomic DNA is 52.0 mol%.Description of Prevotella mizrahii sp. nov.: Prevotella mizrahii . According to 16S rRNA gene-based phylogeny, the isolate falls into the family Prevotellaceae (phylum Bacteroidetes). The neighbouring species with the highest sequence identity is Prevotella scopos . Identity to Prevotella melaninogenica, the type species of the genus Prevotella, is 90.9%. Additionally, the isolate branches together with Hallella seregens, the type species of this genus, which shares 91.2% identity and was previously proposed to be reclassified within the genus Prevotella146; however, this proposal still has no standing in the nomenclature. The POCP values between the genome of our strain and that of the species aforementioned are 47.8% (P. scopos), 50.8% (P. melaninogenica), and 55.4% (H. seregens), which oscillate around the proposed genus-level threshold of 50%. GTDB-Tk places the isolate within the genus Prevotella. Considering that: (i) these findings on the genus delineation of this isolate are altogether somewhat contradictory ; and (ii) Prevotella appears to be a phylogenetically and phylogenomically heterogeneous genus that will require taxonomic rearrangement in the near future, we propose the creation of a novel species only, named Prevotella mizrahii, to avoid generating even more instability within this genus. Cells are thick rods (0.5–1.5 µm) that can be slightly curved. Growth is good (high turbidity) in anoxic WCA but the strain cannot be maintained in culture unless passaged every second day. Within the genome, one transporter along with ABC transporter systems for phosphate and lipoproteins were identified. Fructan, cellulose, starch, and sucrose are potential carbohydrate substrates. Riboflavin biosynthesis was predicted from GTP and ribulose-5-phosphate . Extracellular nitrite and nitrate were identified to be transported internally via Nrt and then converted into ammonia (EC: 1.7.2.2) and further to l-glutamate . Based on metagenome-based functional predictions, this species is proposed to be a key functional member of pig microbiomes. It is considered to deconjugate primary bile acids as determined in vitro by cleavage of taurine residues on WCA agar medium containing TDCA. After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (10.1 ± 0.3 mM) and produced 16.8 ± 1.4 mM succinate and 10.3 ± 0.6 mM acetate. Small amounts of formate (3.0 ± 0.3 mM) and isobutyrate (0.6 ± 0.1 mM) were also detected. The most abundant cellular fatty acid is anteiso-C15:0 (24.4%). Other fatty acids are C16:0 3-OH (12.5%), C16:0 (11.4%), iso-C17:0 3-OH (11.4%), iso-C15:0 (8.9%), iso/anteiso-C17:1 (8.9%), iso-C17:0 (3.9%), anteiso-C17:0 (3.2%), and iso-C16:0 (3.1%). The type strain is LKV-178-WT-2AT (=DSM 108495T). It was isolated from faeces of a German Landrace pig in Freising, Germany. Its G+C content of genomic DNA is 49.9 mol%.Description of Pseudoramibacter porci sp. nov.: Pseudoramibacter (por’ci. L. gen. n. porci of a pig). Anaerobic bacterium that is phylogenetically placed into the family Eubacteriaceae (phylum Firmicutes) based on 16S rRNA sequence analysis. Pseudoramibacter alactolyticus, the type species of the genus, is the closest phylogenetic neighbour with a sequence identity of 94.3% (1473 bp). The POCP value between the two genomes is 60.8%, which indicates that these species belong to the same genus. The corresponding ANI value <80% and dDDH value of 18.9% confirm that the isolate represents a different species. Cells grow as coccobacilli that can form small chains and stain Gram-positive. Within the genome, six transporters were identified along with ABC transporter systems for phosphate, cobalt, and biotin. Trehalose, starch, and cellulose were identified as potential carbohydrate substrates. Additionally, l-lactate and d-lactate can be converted into pyruvate (EC: 1.1.1.27). Whilst l-aspartate can be converted into fumarate  and enter the citrate cycle, only the initial conversion into (S)-malate was identified within the genome. Ammonia may be converted into l-glutamine (EC:6.3.1.2), which can then be utilized to form l-glutamate . Production of ornithine, valine, and leucine was identified from glutamate, pyruvate, and 2-oxoisocaproate, respectively. The most dominant cellular fatty acids are C14:0 (32%) and iso/anteiso-C17:1 (29%). Other fatty acids include C16:0 (14.0%), iso-C19:1 (5.2%), and C18:0 (4.1%). The type strain is RF-744-FAT-4T (=DSM 106894T). It was isolated from faeces of an Aachen minipig83 in Germany. Its G+C content of genomic DNA is 49.3 mol%.Description of Pyramidobacter porci sp. nov.: Pyramidobacter porci (por’ci. L. gen. n. porci of a pig). The closest phylogenetic neighbour within family Synergistaceae (phylum Synergistetes) is Pyramidobacter piscolens, the type species of the genus Pyramidobacter, which shares a 16S rRNA gene sequence identity of 95.6%. The dDDH value of 46.7% and ANI value of 92.4% indicate that the isolate represents the second cultured bacterium within this genus. Cells are bacilli with a length of ~0.5 µm. They stain Gram-negative and are strictly anaerobic. Within the genome, a single transporter was identified; however, ABC transporter systems for alkanesulfonate, glycine betaine/proline, biotin, zinc, oligopeptides, d-methionine, S-methylcysteine, and phosphate were present. Starch was identified as the sole carbohydrate source. Alternatively, ammonia can be utilized for production of l-glutamine (EC: 6.3.1.2), which can be converted into d-fructose-6-phosphate using d-glucosamine-6-phosphate as an intermediate (EC: 2.6.1.16) and enter glycolysis. Butanoate production was identified to occur via butanoyl-CoA as an intermediate from 3-ocohexanoyl-CoA . After 96 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed small amounts of succinate (1.1 ± 0.1 mM) and produced 10.2 ± 0.4 mM acetate and smaller amounts of isovalerate (2.0 ± 0.1 mM), propionate (1.5 ± 0.0 mM), lactate (1.0 ± 0.0 mM), and isobutyrate (0.5 ± 0.0 mM). The major cellular fatty acid is C18:1 ω9c (19%). Other fatty acids include C14:0 3-OH/iso-C16:1 (9.9%), C13:0 (8.7%), C14:0 (8.6%), iso-C13:0 (6.6%), C18:0 (5.9%), C17:1 ω9c (4.7%), C16:0 (4.0%), iso-C15:0 3-OH (3.9%), 10Me-C18:0 (3.3%), iso-C15:0 (3.0%), and C12:0 (2.9%). The type strain is SM-530-WT-4BT (=DSM 105193T). It was isolated from faeces of a German Landrace pig (minipig breed) in Freising, Germany. Its G+C content of genomic DNA is 59.6 mol%.Description of Roseburia porci sp. nov.: Roseburia porci (por’ci. L. gen. n. porci of a pig). Phylogenetically , this species falls into the family Lachnospiraceae (phylum Firmicutes). Its closest neighbours are Roseburia faecis and Roseburia intestinalis with 95.8% and 95.2% sequence identity, respectively (1432 bp). Any other cultured bacterium with a valid name is characterized by a sequence identity <95%. Of note, the isolate branches close to Agathobacter spp., including the well-known butyrate producing species formerly named Eubacterium rectale. Despite the urgent need to revisit the taxonomy of Eubacterium spp., the placement of E. rectale within the genus Agathobacter and the status of this genus in general have been disputed, despite being still a valid name150. Hence, it is sound to propose that the isolate belongs to the genus Roseburia. The status of a novel species within this genus is supported by ANI values <80% and dDDH values of 20.6% and 24.0% between its genome and that of its closest relatives R. faecis and R. intestinalis. Corresponding differences in the G+C content of genomic DNA are 1.3 mol% and 1.0 mol%, which are equal or above the proposed species-delineation threshold of 1 mol%. The strain grows as motile rods that are 1 µm long on average and slightly curved. Growth only occurs under strictly anoxic conditions. A total of 13 transporters along with transporter systems for phosphate, aspartate/glutamate/glutamine, zinc, Na+, teichoic acid, bacitracin, d-methionine, biotin, and cystine were identified within the genome. The presence of BceA/B and VraD/E suggests the expression of two bacitracin efflux systems. The DegS/U two-component system for salt stress was also identified. A complete two-component system for bacterial chemotaxis and stimulation of flagella action was identified based on MCP and CheW/A/R/Y/V/B. The presence of flagella is further supported by the identification of the following flagellar assembly proteins: FlhA/B, FliA/C/D/F/G/M/N/S/P, and FlgB/C/E/G/L/K. Cellobiose, starch, cellulose, and d-glucose were identified to be potential carbohydrate substrates. Both Spo0F and Spo0A were identified within the genome, suggesting this species may have the ability to produce spores. Whilst no complete quorum sensing pathway was detected, the presence of both the luxQ and luxS genes suggests this species may utilize AI-2-based quorum sensing. After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (11.4 ± 0.3 mM) and acetate (5.1 ± 0.7 mM) and produced 12.3 ± 0.2 mM butyrate, 8.8 ± 0.8 mM lactate, and 5.1 ± 0.6 mM formate. The major cellular fatty acids are C18:0 (38%), C13:0 3-OH/C15:1 i I/H, and iso/anteiso-C17:1 (18% each). Other fatty acids are C13:1 (7.3%) and C14:0 (5.2%). The type strain is MUC/MUC-530-WT-4DT (=DSM 107448T). It was isolated from faeces of a German Landrace pig (minipig breed) in Freising, Germany. Its G+C content of genomic DNA is 41.7 mol%.Description of Scrofimicrobium gen. nov.: Scrofimicrobium . The closest phylogenetic neighbours within family Actinomycetaceae (phylum Actinobacteria) are Actinomyces hyovaginalis and Actinomyces suimastitidis, which share 91.8% and 90.6% 16S rRNA gene sequence identity, respectively. POCP and dDDH values against the genome of S. suimastitidis are 46.5% and 24.1%, respectively, and 38.2% and 22.6% against Actinomyces bovis, the type species of the genus Actinomyces. The respective differences in G+C contents of the genomic DNA are 2.9% and 10.6%. Of note, no genome is available for S. hyovaginalis. All values aforementioned clearly speak in favour of creating a new genus to accommodate strain WB03_NA08, for which the name Scrofimicrobium is proposed. The type species is Scrofimicrobium canadense.Description of Scrofimicrobium canadense sp. nov.: Scrofimicrobium canadense . Cells are rod-shaped that strain Gram-positive and have a length of 1-2 µm. The species is a facultative anaerobe. Transporter systems for arabino-oligosaccharides, monosaccharides, zinc, glutathione, d-methionine, cysteine, phosphate, and glutamate were identified within the genome. The SecYEG system for protein export was also detected. A two-component system for phosphate limitation was present, which consisted of SenX3, RegX3, PhoA, and PstS. A complete TCA cycle was identified with singular input from acetyl-CoA. Acetyl-CoA can be produced from both acetate or glucose using a modified glycolysis pathway. Due to the lack of enzymes for the production or utilization of d-fructose-1,6-bisphosphate, glucose-6-phosphate was identified to be converted into glucono-1,5-lactone-6-phosphate (EC: 1.1.1.49), which is converted into gluconate-6-phosphate (EC: 3.1.1.31). This then becomes ribulose-5-phosphate (EC: 1.1.1.44), which converts to d-xyulose-5-phosphate (EC: 5.1.3.1). d-xyulose-5-phophate is then converted into d-glyceraldehyde-3-phosphate and re-enters glycolysis. After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (8.0 ± 1.6 mM) and produced 9.6 ± 2.0 mM succinate, 3.8 ± 0.5 mM acetate, 3.0 ± 0.3 mM formate, and 1.6 ± 1.0 mM lactate. Major cellular fatty acids are C16:0 (32%), C14:0 (32%), and C18:1 ω9c (10%). Other fatty acids include C12:0 (6.4%), C18:1 ω7c (6.2%), C20:1 ω9c (4.7%), and C18:0 (4.2%). The type strain is WB03_NA08T (=DSM 105338T). It was isolated from faeces of a farmed wild boar in Moserville, Ontario, Canada. Its G+C content of genomic DNA is 53.6 mol%.Description of Selenomonas montiformis sp. nov.: Selenomonas montiformis . Its closest relative based on 16S rRNA gene sequence identity (96.0%) is Selenomonas bovis within family Selenomonadaceae (phylum Firmicutes). The dDDH value of 23.8% and ANI value <80% between the two genomes indicate that the two bacteria represent separate species. Cells are motile, slightly curved rods (1–2 µm) that form colonies resembling mini-mountains on WCA agar under anaerobic conditions. Eight transporters were identified within the genome along with transport systems for phosphate, alkanesulfonate, d-methionine, ribose/AI-2/d-zylose, cobalt, and biotin. The DegS/U two-component system for salt stress was also identified. A complete two-component system for bacterial chemotaxis and stimulation of flagella action was identified based on the detection of genes ecnoding MCP and CheW/A/R/Y/V/B. The presence of flagella is further supported by the identification of the following flagellar assembly proteins: FlhA/B, FliA/C/D/F/G/M/N/S/P, FlgB/C/E/G/L/K, and MotB. This isolate was predicted to utilize a wide range of carbohydrate substrates, including sucrose, cellobiose, cellulose, starch, trehalose, maltose, arbutin, salicin, and d-glucose. Biosynthesis of glycogen via GlgA/C/P may suggest a role for this isolate in biofilm production which is supported by the presence of CsgD, termed the ‘master regulator’ for biofilm production. Indole was identified to be produced from shikimate using chorismate as an intermediate . After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (10.4 ± 0.1 mM) and trace amounts of succinate (0.6 ± 0.3 mM) and valerate (0.4 ± 0.2 mM) and produced 22.9 ± 0.8 mM lactate, 8.9 ± 0.9 mM propionate, 5.7 ± 0.5 mM acetate, and smaller amounts of isobutyrate (0.6 ± 0.1 mM). The major cellular fatty acids are C13:0 3OH/C15:1 i I/H (20%), C15:0 (13%), C15:1 ω8c (13%), and C16:1 ω9c (10%). Lower amounts of C17:1 ω8c (9.8%), C11:0 (9.3%), C13:0 (5.8%), and iso-C18:1 (4.7%) were detected. The type strain is WCA-380-WT-3B3T (=DSM 106892T). It was isolated from faeces of a German Landrace pig in Freising, Germany. Its G+C content of genomic DNA is 52.6 mol%.Description of Sharpea porci sp. nov.: Sharpea porci (por’ci. L. gen. n. porci of a pig). The closest phylogenetic neighbour is Sharpea azabuensis within the family Erysipelotrichaceae (phylum Firmicutes), which shares 98.4% 16S rRNA gene sequence identity. The dDDH and ANI values of 36.4% and 88.9% between the two genomes confirm that the isolate represents a novel species within the genus Sharpea. The species grows as thick rods with a length of 2–8 µm that often forms chains. A single transporter was identified within the genome along with transporter systems for phosphate, cysteine, teichoic acid, d-methionine, oligopeptides, biotin, and zinc. Cellubiose, starch, maltose, trehalose, sucrose, arbutin, salicin, and d-glucose were predicted to be potential carbohydrate substrates. Indole was identified to be produced from shikimate using chorismate as an intermediate . l-cysteine can also be produced internally using sulfide and acetyl-l-serine (EC:2.5.1.47). The major cellular fatty acids are C16:0 (24%), C18:0 (13%), and C12:0 (12%). Lower amounts of C18:1 ω9c (6.6%), C19:1 ISO I (5.7%), C14:0 (4.8%), C16:1 ω7c/iso-C15:1 2OH (4.7%), iso I/anteiso B-C17:1 (4.7%), C10:0 (3.8%), C17:0 ISO 3OH (3.7%), C17:1 ω9c (3.1%), C18:1 ω7c (2.9%), C18:2 ω6,9c/anteiso-C18:0 (2.8%), and C14:1 ω5c (2.1%) were detected. The type strain is CA-Schmier-601-WT-3T (=DSM 108165T). It was isolated from faeces of a German Landrace pig (minipig breed) in Freising, Germany. Its G+C content of genomic DNA is 30.6 mol%.Description of Sodaliphilus gen. nov.: Sodaliphilus  loving; N.L. masc. n. Sodaliphilus an organism that loves a companion). The isolate is phylogenetically placed into the family Muribaculaceae (phylum Bacteroidetes). Based on 16S rRNA gene sequence identity, Muribaculum intestinale is the closest phylogenetic neighbour (86.4%). Comparison with members of the neighbouring family Porphyromonadaceae (Barnesiella and Coprobacter spp.) revealed 16S rRNA sequence identity values <85.7%. The difference in the G+C content of genomic DNA between the isolate and M. intestinale is 4.31%. The POCP value between the two genomes is 42%, which clearly demonstrates a separate genus status. Compared to members of the three genera aforementioned, Coprobacter secundus shows the highest dDDH value with 42.3%  and Barnesiella viscericola the highest ANI value with 76.6% . These values are clearly below thresholds justifying separate taxa. The type species is Sodaliphilus pleomorphus.Description of Sodaliphilus pleomorphus sp. nov.: Sodaliphilus pleomorphus . Cells grow slowly and only under strictly anaerobic conditions and on solid medium. After 1 week of incubation, cells are irregular rods that can be very long (up to 50 µm). Longer incubation times result in shortening and thickening of cells. Growth of the species was observed to be boosted when co-incubated on plate with other isolates, including Tissierella pigra sp. nov., Hornefia butyriciproducens gen. nov., sp. nov., and Anaerobutyricum soehngenii. Three transporters were identified within the genome along with transport systems for phosphate and lipoproteins. A two-component system (PhoA/B/R) was also identified, which responds to limited phosphate by increasing its assimilation. Starch, amylose, dextrin, and fructan may be utilized by this strain. The glycolysis pathway lacked multiple proteins, suggesting an inability to directly convert phosphoenolpyruvate to pyruvate, instead phosphenolpyruvate can be utilized for production of chorismate , indole, and l-tryptophan . Lipid IV (A) can be produced from lipid X , a hallmark functional feature of Muribaculaceae. Lipid IV (A) can also be converted into lauroyl-KD02-lipid IV (A) . Based on metagenomic prediction, this species is proposed to be a key functional member of pig microbiomes. The biomass collected from three agar plates produced 1.6 ± 0.7 mM formate and traces of isobutyrate (0.5 ± 0.1 mM) after 48 h at 37 °C in modified YCFA medium under anaerobic conditions. The type strain is Oil-RF-744-WCA-WT-10T (=DSM 108610T). It was isolated from faeces of an Aachen minipig83 in Germany. Its G+C content of genomic DNA is 54.4 mol%.Description of Stecheria gen. nov.: Stecheria . Based on 16S rRNA gene phylogeny, the genus is placed into the family Erysipelotrichaceae (phylum Firmicutes). It shares 90.4% sequence identity with both Solobacterium moorei and Bulleidia extructa (1487 bp). The POCP value between the genome of the isolate and the respective genome of these phylogenetic neighbours is 40.0% and 37.1%. This confirms a separate genus status, for which the name Stecheria is proposed. The type species is Stecheria intestinalis.Description of Stecheria intestinalis sp. nov.: Stecheria intestinalis . The species grows as pear-shaped, coccoidal cells that may form pairs or small chains of 2–5 µm in length. They stain Gram-positive and grow only under strictly anaerobic conditions. One single transporter was identified within the genome along with ABC transporter systems for phosphate, teichoic acid, d-methionine, oligopeptides, and methyl-galactoside. Serine protease activity was identified to be modulated by the CssS/R-HtrA two-component system for removal of misfolded proteins. Arbutin, salicin, d-glucose, sucrose, trehalose, maltose, cellobiose, and starch were identified as potential carbohydrate substrates. Propionic acid production was identified to occur from acryloyl-CoA  and 2-ocobutanoate . The species is considered to deconjugate primary bile acids as determined in vitro by cleavage of taurine residues on WCA agar medium containing TDCA. The main cellular fatty acid is C18:1 ω9c (70%). C18:1 ω7c (21.8%) and C18:0 (5.4%) were detected in lower amounts. The type strain is Oil+RF-744-GAM-WT-6T (=DSM 109718T). It was isolated from faeces of an Aachen minipig83 in Germany. Its G+C content of genomic DNA is 49.4 mol%.Description of Suipraoptans gen. nov.: Suipraoptans . Based on 16S rRNA gene sequences, the nearest phylogenetic relatives of this strictly anaerobic bacterium are Ruminococcus lactaris, Faecalicatena fissicatena, and Dorea longicatena (<93% sequence identity). The corresponding POCP values are 41.0%, 44.9%, and 52.3%. Dorea formicigenerans, the type species of this genus, shares 91.4% 16S rRNA gene sequence identity and a POCP value of 48.5% with the isolate, confirming its status as a distinct genus, for which the name Suipraeoptans is proposed. The type species is Suipraeoptans intestinalis.Description of Suipraoptans intestinalis sp. nov.: Suipraoptans intestinalis . The species forms short (0.5 µm) straight rods that grow as single cells or in pairs and stain Gram-positive. Within the genome, 13 transporters were identified along with transporter systems for phosphate, S-methylcysteine, bacitracin, d-methionine, zinc, cobalt, biotin, and d-xylose. Both starch and trehalose were identified as potential substrates. l-tryptophane production from hydroxypyruvate may occur via serine . l-serine could also be converted into O-acetyl-l-serine (EC: 2.3.1.30) and combined with sulfide to form l-cysteine (EC: 2.5.1.47). Anaerobic conversion of Mg-protoporphyrin IX 13-monomethyl ester to divinyl-protochlorophyllide was identified; however, no downstream or upstream production pathways were present. Both cobalamin biosynthesis from cobinamide  and production of folic acid from 7,8-dihydrofolate (EC: 1.5.1.3) were identified. After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (10.2 ± 0.4 mM) and produced 10.9 ± 1.6 mM acetate and traces of formate (0.7 ± 0.0 mM). The major fatty acid is C16:0 (48%). Other fatty acids are C18:0 (11.8%), iso-C17:1/anteiso-C17:1 (10.5%), C14:0 (4.8%), and iso-C19:1 (4.2%). The type strain is 68-1-5T (=DSM 104945T). It was isolated from the ileal content of a pig in Ames, Iowa, USA. Its G+C content of genomic DNA is 47.7 mol%.Description of Tissierella pigra sp. nov.: . The closest phylogenetic relatives within family Tissierellaceae (phylum Firmicutes) are Tissierella praeacuta (97.8% 16S rRNA gene sequence identity), the type species of the genus Tissierella, and Tissierella carlieri (96.3%). The dDDH/ANI values of 22.4%/80.0% and 24.6%/81.1%, respectively, confirm the status of the isolate as a novel species. Cells are rod-shaped with a size of 1.5–2 µm but they can also form long filaments of up to 20 µm. They grow under strictly anaerobic conditions. Only two transporters were identified within the genome with additional transporter systems for phosphate, alkanesulfonate, S-methylcysteine, d-methionine, oligopeptides, glutathione, zinc, cobalt, and biotin. The SecYEG system for protein export was also present. A two-component system for SCFA metabolism based on the detection of acetoacetate was identified (AtoA/B/C/D/S). Another complete two-component system for bacterial chemotaxis and stimulation of flagella action was identified based on MCP and CheW/A/R/Y/V/B. The presence of flagella is further supported by the identification of the assembly proteins FlhA/B, FlgB/C/D/E/G/K, FliA/C/D/E/F/G/M/N/S/P, and MotB, and was confirmed by electron microscopy , taurine (tauACB), and alkanesulfonate (ssuACB) may suggest a sulfur-centric metabolic system. After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species did not consume glucose and produced 16.7 ± 0.6 mM acetate, 10.5 ± 1.0 mM isovalerate, 4.6 ± 0.4 mM butyrate, and 3.0 ± 0.6 mM isobutyrate. The major cellular fatty acid is iso-C15:0 (40%). Other fatty acids include C14:0 (8.5%), C16:0 (7.1%), iso-C17:1 ω9c (6.3%), iso-C13:0 (4.7%), C12:0 (4.1%), iso-C15:1 (4.0%), C18:0 (3.7%), anteiso-C15:0 (3.6%), and C18:1 ω9c (3.3%). The type strain is WCA3-693-APC-4αT (= DSM 105185T). It was isolated from faeces of an APC1311/+ pig in Freising, Germany. Its G+C content of genomic DNA is 36.9 mol%.Description of Velocimicrobium gen. nov.: Velocimicrobium . Strictly anaerobic bacterium that is phylogenetically placed into the family Lachnospiraceae (phylum Firmicutes). Based on 16S rRNA sequence identity, the closest phylogenetic neighbour is Acetivibrio ethanolgignens, which shares 94.2% sequence identity (1484 bp). The isolate is also distantly related to Anaerocolumna cellulosilytica, the types species of this genus (91.5% sequence identity). Whilst no genome is available for A. cellulosilytica, the POCP value between the genome of our isolate and that of A. ethanolgignens is 49.0%, which highlights the need to create a new genus. The corresponding ANI and dDDH values of 78.5% and 21.5% confirm that the isolate represents a different taxon. The type species is Velocimicrobium porci.Description of Velocimicrobium porci sp. nov.: Velocimicrobium porci (por’ci. L. gen. n. porci of a pig). The species grows as straight, thick rods of 2–3 µm in length. They stain Gram-positive. A single transporter was identified within the genome along with ABC transporter systems for phosphate, cystine, teichoic acid, bacitracin, d-methionine, zinc, cobalt, biotin, and methyl-galactoside. Starch, maltose, trehalose, arbutin, salicin, and d-glucose are potential carbohydrate substrates. The motility of this species as observed under light microscopy is confirmed by the presence of the flagella-related genes fliA/C/D/F/G/M/N/P/S, flhA/B, flgB/C/D/E/G/K/L, and motB. Additionally, this species may be a biofilm producer as the glgA/C/P glycogen biosynthesis genes and the CsgD biofilm regulator were detected. The presence of both the ArcB and OxyR biofilm regulator genes suggest oxidative stress may be an inducer for biofilm production. After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (11.1 ± 0.6 mM) and produced 12.6 ± 2.7 mM acetate, 10.2 ± 1.3 mM formate, and small amounts of propionate (1.1 ± 0.9 mM) and isobutyrate (0.9 ± 0.2 mM). The predominant cellular fatty acid is C18:1 ω9c (67%). Other fatty acids are C18:1 ω7c (11.9%), C18:0 (4.8%), C20:0 (4.3%), iso-C19:0 (4.1%), and C16:0 (3.8%). The type strain is WCA-693-APC-MOT-IT (=DSM 107250T). It was isolated from faeces of an APC1311/+ pig in Freising, Germany. Its G+C content of genomic DNA is 34.9 mol%.Description of Victivallis lenta sp. nov.: Victivallis lenta (L. fem. adj. lenta slow). The 16S rRNA gene sequence of this species (1471 bp) shares 95.1% identity with the sequence of Victivallis vadensis, the type species of the genus Victivallis within family Victivallaceae (phylum Lentisphaerae). The ANI and dDDH values are 80.6% and 24.1%, respectively, which support the creation of a novel species to accommodate the isolate. Of note, one strain (CCUG 44730) without a valid name previously isolated by others from the blood of an 80-year-old woman share ANI and dDDH values of 98.8% and 93.3%, respectively, showing that it belongs to the same species as our own isolate. Within the genome, two transporters were identified along with ABC transporter systems for phosphate, glycine betaine, proline, teichoic acid, ribose, AI-2, and d-xylose. Starch, cellulose, and frunctan were identified as potential carbohydrate substrates. Coenzyme-A biosynthesis was identified from pantothenol  and l-aspartate  or l-valine . Ammonia can be converted into l-glutamine (EC: 6.3.1.2), which in turn can be utilized to form l-glutamate (EC: 1.4.1.13). l-aspartate may be converted into fumarate via either l-arginino-succinate  or adenylo-succinate . The HighA-1 antitoxin was identified, which counteracts the effect of the HigB-1 toxin. After 48 h at 37 °C in modified YCFA medium under anaerobic conditions, the species consumed glucose (10.2 ± 0.2 mM) and produced acetate (4.5 ± 0.9 mM), lactate (1.3 ± 0.1 mM), and low amounts of formate (0.5 ± 0.1 mM). The main cellular fatty acids are C18:3 ω6c (30%), iso-C16:0 (22%), and anteiso-C15:0 (18%). Other fatty acids are C18:0 (6.6%), C16:0 (5.6%), iso-C18:0 (4.7%), iso-C14:0 (3.7%), and anteiso-C17:0 (3.5%).The type strain is BBE-744-WT-12T (=DSM 107290T). It was isolated from faeces of an Aachen minipig83 in Germany. Its G+C content of genomic DNA is 59.3 mol%.Description of Waltera gen. nov.: Waltera . This isolate is phylogenetically placed into the family Lachnospiraceae (phylum Firmicutes). It is a strictly anaerobic bacterium that is distantly related to Kineothrix alysoides, the type species of the genus Kineothrix, with which it shares 92.6% 16S rRNA gene sequence identity (1470 bp). The difference in the G+C content of genomic DNA between the two genomes is 3.6%. The ANI value <80% as well as POCP and dDDH values of 43.8% and 18.7%, respectively, clearly demonstrate that the isolate represents a new taxon, for which the creation of a novel genus is required. The type species is Waltera intestinalis.Description of Waltera intestinalis sp. nov.: Waltera intestinalis . Cells are motile, stain Gram-negative, and grow as wavy rods of 2–4 µm in length. Within the genome, four transporters were identified along with ABC transporter systems for phosphate, alkanesulfonate, teichoic acid, Na+, d-methionine, oligopeptides, methyl-galactoside, biotin and the Peb1A/B/C system for aspartate, glutamate, and glutamine. The presence of both the PhoB/R/P and SenX3-RegX3 two-component systems for detecting phosphate limitation leading to phosphate assimilation indicates this species is sensitive to environmental phosphate levels. The MCP, CheA/B/R/Y/V/W two-component system leading to flagella assembly was also detected within the genome along with the MotB, FliA/C/D/F/G//M/N/S/P, FlgB/C/D/E/G/K/L, and FlhA/B flagella proteins. Cellulose, starch, trehalose, maltose, arbutin, salicin, and d-glucose were all identified as potential carbohydrate substrates. Based on the analysis of a large catalogue of metagenome-reconstructed genomes, the species is proposed to be a prevalent member of the intestinal microbiome also in human. The major cellular fatty acid is C10:0 (33%). Lower proportions of the following fatty acids were detected: C16:0 (14.4%), C18:0 (11.0%), alde-C12:0 (8.7%), C12:0 (7.4%), and C18:1 ω9c (5.9%). The type strain is WCA3-601-WT-6HT (=DSM 108985T). It was isolated from faeces of a German Landrace pig (minipig breed) in Freising, Germany. Its G+C content of genomic DNA is 46.3 mol%.Description of p-value <0.05). Transcriptomic analysis as presented in Fig. p-values. Data in Supplementary Fig. https://lagkouvardos.github.io/Rhea). Cell morphological characteristics detected via light and fluorescence microscopy could be repeatedly (>3 replica) observed in independent biological replicates  presented in Fig. es Figs.  and 10. SEM Fig. , b were Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary