Core-genome multilocus sequence typing and core-SNP analysis of Clostridium neonatale strains isolated in different spatio-temporal settings.

IMPORTANCE: Clostridium neonatale has been isolated from the fecal samples of asymptomatic neonates and cases of necrotizing enterocolitis (NEC). Taking advantage of a large collection of independent strains isolated from different spatio-temporal settings, we developed and established a cgMLST scheme for the molecular typing of C. neonatale. Both the cgMLST and cgSNP methods demonstrate comparable discrimination power. Results indicate geographic- and temporal- independent clustering of C. neonatale NEC-associated strains. No specific cgMLST clade of C. neonatale was genetically associated with NEC.

Revealing the beneficial effects of a dairy infant formula on the gut microbiota of early childhood children with autistic spectrum disorder using static and SHIME® fermentation models.

This study evaluated the impact of the Milnutri Profutura® (MNP) dairy infant formula on the gut microbiota of early childhood children (three to five years) with Autistic Spectrum Disorder (ASD) using static fermentation (time zero, 24, and 48 h) and the Simulator of the Human Intestinal Microbiol Ecosystem (SHIME®) (time zero, 72 h, and 7 days). The relative abundance of selected intestinal bacterial groups, pH values, organic acids, and sugars were verified at time zero, 24, and 48 h using flow cytometry and measurements. In addition, the diversity and changes in the gut microbiota, and the amounts of acetic, butyric, and propionic acids and ammonium ions (NH4+) in fermentation using the SHIME® were measured at time zero, 72 h, and 7 days. MNP increased Lactobacillus/Enterococcus and Bifidobacterium populations and decreased Bacteroides/Prevotella, Clostridium histolyticum and Eubacterium rectale/Clostridium coccoides populations (p < 0.05) at 24 and 48 h of static fermentation, showing a positive prebiotic activity score (65.18 ± 0.07). The pH, fructose and glucose decreased, while lactic, butyric, and propionic acids increased (p < 0.05) at 48 h of static fermentation. MNP increased (p < 0.05) the Firmicutes phylum during the fermentation in SHIME®. MNP decreased the diversity at 72 h of fermentation, mostly by the increase (p < 0.05) in the Lactobacillus genus. Microbial groups considered harmful such as Lachnospiraceae, Negativicoccus, and Lachnoclostridium were inhibited after administration with MNP. Propionic and butyric acids increased at 72 h and NH4+ decreased (p < 0.05) at the end of fermentation with MNP. The results indicate MNP as an infant formula which may benefit the gut microbiota of children with ASD.

Maternal Diet May Modulate Breast Milk Microbiota-A Case Study in a Group of Colombian Women.

There is increasing evidence that the diet and nutritional status of women during pregnancy and lactation can modulate the microbiota of their milk and, therefore, the microbiota of the infant. An observational, descriptive, and cross-sectional study was carried out in a group of lactating women. Dietary intake during gestation and the first trimester of lactation was evaluated, and the microbiota was analyzed by 16S ribosomal RNA (rRNA) sequencing using the Illumina platform. Globally, Streptococcus spp. (32%), Staphylococcus spp. (17.3%), Corynebacterium spp. (5.1%) and Veillonella spp. (3.1%) were the predominant bacterial genera. The consumption of simple carbohydrates in gestation (rho = 0.55, p ≤ 0.01) and lactation (rho = 0.50, p ≤ 0.01) were positively correlated with Enterobacter spp. In lactation, a negative correlation was observed between the intake of simple carbohydrates and the genus Bifidobacterium spp. (rho = -0.51 p ≤ 0.01); furthermore, a positive correlation was identified between the intake of folic acid and Akkermansia spp. (rho = 0.47, p ≤ 0.01). Amplicon sequence variants (ASVs) associated with the delivery mode, employment relationship, the baby's gender, birth weight, the Body Mass Index (BMI) of the breastfeeding woman, and gestational weight gain were recovered as covariates in a linear mixed model. The results of this research showed that the maternal nutritional status and diet of women during gestation and lactation could modulate the microbiota of breast milk.

Exploring the Association between Citrus Nutraceutical Eriocitrin and Metformin for Improving Pre-Diabetes in a Dynamic Microbiome Model.

Pre-diabetes is recognized as an altered metabolic state, which precedes type 2 diabetes, and it is associated with great dysfunction of the intestinal microbiota, known as dysbiosis. Natural compounds, capable of reducing blood glucose without side effects and with a beneficial effect on the microbiota, have been studied as substitutes or adjuvants to conventional hypoglycemic agents, such as metformin. In this work, the effect of the nutraceutical Eriomin®, a mixture of citrus flavonoids (eriocitrin, hesperidin, naringin, and didymin), which reduces glycemia and increases glucagon-like peptide-1 (GLP-1) in pre-diabetic patients, was tested in the Simulator of Human Intestinal Microbial Ecosystem (SHIME®), inoculated with pre-diabetic microbiota. After treatment with Eriomin® plus metformin, a significant increase in acetate and butyrate production was observed. Furthermore, sequencing of the 16S rRNA gene of the microorganisms showed that Eriomin® plus metformin stimulated the growth of Bacteroides and Subdoligranulum genera. Bacteroides are the largest fraction of the intestinal microbiota and are potential colonizers of the colon, with some species producing acetic and propionic fatty acids. In addition, Subdoligranulum species are associated with better host glycemic metabolism. In conclusion, Eriomin® associated with metformin improved the composition and metabolism of the intestinal microbiota, suggesting a potential use in pre-diabetes therapy.

Exploring the Potential of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 as Promising Psychobiotics Using SHIME.

Psychobiotics are probiotics that have the characteristics of modulating central nervous system (CNS) functions or reconciled actions by the gut-brain axis (GBA) through neural, humoral and metabolic pathways to improve gastrointestinal activity as well as anxiolytic and even antidepressant abilities. The aim of this work was to evaluate the effect of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 on the gut microbiota of mildly anxious adults using SHIME®. The protocol included a one-week control period and two weeks of treatment with L. helveticus R0052 and B. longum R0175. Ammonia (NH4+), short chain fatty acids (SCFAs), gamma-aminobutyric acid (GABA), cytokines and microbiota composition were determined. Probiotic strains decreased significantly throughout the gastric phase. The highest survival rates were exhibited by L. helveticus R0052 (81.58%; 77.22%) after the gastric and intestinal phase when compared to B. longum (68.80%; 64.64%). At the genus level, a taxonomic assignment performed in the ascending colon in the SHIME® model showed that probiotics (7 and 14 days) significantly (p < 0.005) increased the abundance of Lactobacillus and Olsenella and significantly decreased Lachnospira and Escheria-Shigella. The probiotic treatment (7 and 14 days) decreased (p < 0.001) NH4+ production when compared to the control period. For SCFAs, we observed after probiotic treatment (14 days) an increase (p < 0.001) in acetic acid production and total SCFAs when compared to the control period. Probiotic treatment increased (p < 0.001) the secretion of anti-inflammatory (IL-6 and IL-10) and decreased (p < 0.001) pro-inflammatory cytokines (TNF-alpha) when compared to the control period. The gut-brain axis plays an important role in the gut microbiota, producing SCFAs and GABA, stimulating the production of anti-anxiety homeostasis. The signature of the microbiota in anxiety disorders provides a promising direction for the prevention of mental illness and opens a new perspective for using the psychobiotic as a main actor of therapeutic targets.

Gut Microbiota Diversity of Preterm Neonates Is Associated With Clostridioides Difficile Colonization.

In adults, Clostridioides difficile infections are associated with alterations of the intestinal bacterial populations. Although preterm neonates (PN) are frequently colonized by C. difficile, limited data are available regarding the relationship between C. difficile and the intestinal microbiota of this specific population. Therefore, we studied the intestinal microbiota of PN from two multicenter cohorts using high-throughput sequencing of the bacterial 16S rRNA gene. Our results showed that alpha diversity was significantly higher in children colonized by C. difficile than those without colonization. Beta diversity significantly differed between the groups. In multivariate analysis, C. difficile colonization was significantly associated with the absence of postnatal antibiotherapy and higher gestational age. Taxa belonging to the Lachnospiraceae, Enterobacteriaceae, Oscillospiraceae families and Veillonella sp. were positively associated with C. difficile colonization, whereas Bacteroidales and Bifidobacterium breve were negatively associated with C. difficile colonization. After adjustment for covariables, Clostridioides, Rothia, Bifidobacterium, Veillonella, Eisenbergiella genera and Enterobacterales were more abundant in the gut microbiota of colonized children. There was no significant association between C. difficile colonization and necrotizing enterocolitis in PN. Our results suggest that C. difficile colonization in PN is related to the establishment of physiological microbiota.

Rhizosphere and Endosphere Bacterial Communities Survey by Metagenomics Approach.

The diversity of microbes associated with plant roots is in the order of tens of thousands of species. It is estimated that only 0.1-1.0% of the living bacteria present in soils can be cultured under standard conditions. The microbial marker-gene sequence data and the next-generation sequencing technologies have enabled systemic studies of root-associated microbiomes. Molecular techniques can be used to generate comprehensive taxonomic profiles of the microorganisms present in roots. The aim of this chapter is to provide a standard method for the obtention of rhizosphere and endosphere fractions, and a generic workflow of the Quantitative Insights Into Microbial Ecology version 2 (QIIME2) software to analysis of 16S rRNA marker-gene.

Core-, pan- and accessory genome analyses of Clostridium neonatale: insights into genetic diversity.

Clostridium neonatale is a potential opportunistic pathogen recovered from faecal samples in cases of necrotizing enterocolitis (NEC), a gastrointestinal disease affecting preterm neonates. Although the C. neonatale species description and name validation were published in 2018, comparative genomics are lacking. In the present study, we provide the closed genome assembly of the C. neonatale ATCC BAA-265T (=250.09) reference strain with a manually curated functional annotation of the coding sequences. Pan-, core- and accessory genome analyses were performed using the complete 250.09 genome (4.7 Mb), three new assemblies (4.6-5.6 Mb), and five publicly available draft genome assemblies (4.6-4.7 Mb). The C. neonatale pan-genome contains 6840 genes, while the core-genome has 3387 genes. Pan-genome analysis revealed an 'open' state and genomic diversity. The strain-specific gene families ranged from five to 742 genes. Multiple mobile genetic elements were predicted, including a total of 201 genomic islands, 13 insertion sequence families, one CRISPR-Cas type I-B system and 15 predicted intact prophage signatures. Primary virulence classes including offensive, defensive, regulation of virulence-associated genes and non-specific virulence factors were identified. The presence of a tet(W/N/W) gene encoding a tetracycline resistance ribosomal protection protein and a 23S rRNA methyltransferase ermQ gene were identified in two different strains. Together, our results revealed a genetic diversity and plasticity of C. neonatale genomes and provide a comprehensive view of this species genomic features, paving the way for the characterization of its biological capabilities.

Spirulina platensis biomass enhances the proliferation rate of Lactobacillus acidophilus 5 (La-5) and combined with La-5 impact the gut microbiota of medium-age healthy individuals through an in vitro gut microbiome model.

This study first evaluated the stimulatory effect of S. platensis biomass on the growth of L. acidophilus and the metabolic activity during fermentation (37 °C, 72 h) in a culture medium. The results demonstrated a higher impact of S. platensis biomass than fructooligosaccharide (FOS), an established prebiotic. Higher L. acidophilus proliferation rates and metabolic activity were observed (lower pH values and higher concentrations of acetic, lactic, and propionic acids) in the presence of S. platensis. Then, we evaluated the effects of the S. platensis biomass (1.5 g, twice a day, 5 days) in association with L. acidophilus (106 CFU/g) on the gut microbiota composition of medium-age healthy individuals through the Simulator of Human Intestinal Microbial Ecosystem (SHIME®) and measurement of metabolites. L. acidophilus (La5) and L. acidophilus + S. platensis (Spi-La5) could positively modulate the intestinal microbiota. The administration of La5 resulted in increases in Bacteroides, Megasphaera, Lactobacillus, and Parabacteroides genus abundance, with a consequent decrease in ammonium ions. The administration of Spi-La5 increased the abundance of the genus Erysipelatoclostridium, Roseburia, Enterococcus, Bifidobacterium, Coriobacteriaceae UCG-003, Enterobacter, and Paraclostridium. The results demonstrate that the intestinal microbiota was differently modified by administrating La5 and Spi-La5 and indicate the latter as an alternative for microbiota positive modulation in healthy individuals.

Bacterial inoculant-assisted phytoremediation affects trace element uptake and metabolite content in Salix atrocinerea.

Natural plant-associated microorganisms are of critical importance to plant growth and survival in field conditions under toxic concentrations of trace elements (TE) and these plant-microbial processes can be harnessed to enhance phytoremediation. The total bacterial diversity from grey willow (Salix atrocinerea) on a brownfield heavily-polluted with lead (Pb) and arsenic (As) was studied through pyrosequencing. Culturable bacteria were isolated and in vitro tested for plant growth-promotion (PGP) traits, arsenic (As) tolerance and impact on As speciation. Two of the most promising bacterial strains - the root endophyte Pantoea sp. AV62 and the rhizospheric strain Rhodococcus erythropolis AV96 - were inoculated in field to S. atrocinerea. This bioaugmentation resulted in higher As and Pb concentrations in both, roots and leaves of bacterial-inoculated plants as compared to non-inoculated plants. In consequence, bacterial bioaugmentation also affected parameters related to plant growth, oxidative stress, the levels of phytochelatins and phenylpropanoids, together with the differential expression of genes related to these tolerance mechanisms to TE in leaves. This study extends our understanding about plant-bacterial interactions and provides a solid basis for further bioaugmentation studies aiming to improve TE phytoremediation efficiency and predictability in the field.

Effect of probiotic, prebiotic, and synbiotic on the gut microbiota of autistic children using an in vitro gut microbiome model.

Imbalances in gut microbiota composition occur in individuals with autism spectrum disorder (ASD). The administration of probiotics, prebiotics, and synbiotics is emerging as a potential and promising strategy for regulating the gut microbiota and improving ASD-related symptoms. We first investigated the survival of the probiotics Limosilactobacillus (L.) reuteri and Bifidobacterium (B.) longum alone, mixed and combined with a galacto-oligosaccharide (GOS) under simulated gastrointestinal conditions. Next, we evaluated the impact of probiotics (L. reuteri + B. longum), prebiotic (GOS), and synbiotic (L. reuteri + B. longum + GOS) on gut microbiota composition and metabolism of children with ASD using an in vitro fermentation model (SHIME®). The combination of L. reuteri, B. longum, and GOS showed elevated gastrointestinal resistance. The probiotic, prebiotic, and synbiotic treatments resulted in a positive modulation of the gut microbiota and metabolic activity of children with ASD. More specifically, the probiotic treatment increased the relative abundance of Lactobacillus, while the prebiotic treatment increased the relative abundance of Bifidobacterium and decreased the relative abundance of Lachnoclostridium. Changes in microbial metabolism were associated with increased short-chain fatty acid concentrations and reduced ammonium levels, particularly in the prebiotic and synbiotic treatments.

Enzymatic synthesis of capric acid-rich structured lipids and their effects on mice with high-fat diet-induced obesity.

The objective of this study was to produce structured lipids (SLs) by enzymatic acidolysis using Rhizopus oryzae lipase covalently immobilized in a low-cost material. Grape seed oil was used to synthesize SLs containing the medium-chain fatty acid (C10:0) capric acid. SL synthesis led to 38.8% medium-chain fatty acid incorporation with 5 reuses of the enzymatic derivative. The reaction conditions for the synthesis of MLM-TAGs (triacylglycerols with one long- and two medium-chain acyl residues) were at a molar ratio of fatty acid:oil of 3:1, performed at 40 °C and lipase immobilized load of 5% (w/w). The in vivo effects of SLs were studied in Swiss mice fed premade diets: control (C) diet, high-fat diet (HFD) with 100% lipid content as lard, HFD with 50% lipid content as grape seed oil (HG) or HFD with 50% lipid content as capric acid-containing SLs produced from grape seed oil (HG-MCT). Mice from HG and HG-MCT groups had decreases in body weight gain and reductions in the weights of white adipose tissues. In addition, HG and HG-MCT mice had low plasma levels of glucose and total cholesterol, and improvements in the glucose tolerance. HG and HG-MCT diets have remarkable antioxidant properties, since low plasma levels of TBARS (thiobarbituric acid reactive substances, biomarkers of lipid peroxidation) were found in mice fed these diets. Interestingly, TBARS levels in HG-MCT mice were further decreased than values of HG mice. Mice fed HG and HG-MCT diets also showed preservation in the activity of the antioxidant enzyme paraoxonase 1. Both HG and HG-MCT diets promoted reduction of IL-6 and IL-10 production by splenocytes. The capric acid-containing SLs produced from grape seed oil emerges as a functional oil capable to mitigate obesity complications resulting from oxidative stress and inflammation.

Viruses in Extreme Environments, Current Overview, and Biotechnological Potential.

Virus research has advanced significantly since the discovery of the tobacco mosaic virus (TMV), the characterization of its infection mechanisms and the factors that determine their pathogenicity. However, most viral research has focused on pathogenic viruses to humans, animals and plants, which represent only a small fraction in the virosphere. As a result, the role of most viral genes, and the mechanisms of coevolution between mutualistic viruses, their host and their environment, beyond pathogenicity, remain poorly understood. This review focuses on general aspects of viruses that interact with extremophile organisms, characteristics and examples of mechanisms of adaptation. Finally, this review provides an overview on how knowledge of extremophile viruses sheds light on the application of new tools of relevant use in modern molecular biology, discussing their value in a biotechnological context.

Determinants and Prediction of Esterase Substrate Promiscuity Patterns.

Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases' substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets.

Bacterial, Archaeal, and Eukaryotic Diversity across Distinct Microhabitats in an Acid Mine Drainage.

Acid mine drainages are characterized by their low pH and the presence of dissolved toxic metallic species. Microorganisms survive in different microhabitats within the ecosystem, namely water, sediments, and biofilms. In this report, we surveyed the microbial diversity within all domains of life in the different microhabitats at Los Rueldos abandoned mercury underground mine (NW Spain), and predicted bacterial function based on community composition. Sediment samples contained higher proportions of soil bacteria (AD3, Acidobacteria), as well as Crenarchaeota and Methanomassiliicoccaceae archaea. Oxic and hypoxic biofilm samples were enriched in bacterial iron oxidizers from the genus Leptospirillum, order Acidithiobacillales, class Betaproteobacteria, and archaea from the class Thermoplasmata. Water samples were enriched in Cyanobacteria and Thermoplasmata archaea at a 3-98% of the sunlight influence, whilst Betaproteobacteria, Thermoplasmata archaea, and Micrarchaea dominated in acid water collected in total darkness. Stalactites hanging from the Fe-rich mine ceiling were dominated by the neutrophilic iron oxidizer Gallionella and other lineages that were absent in the rest of the microhabitats (e.g., Chlorobi, Chloroflexi). Eukaryotes were detected in biofilms and open-air water samples, and belonged mainly to clades SAR (Alveolata and Stramenopiles), and Opisthokonta (Fungi). Oxic and hypoxic biofilms displayed higher proportions of ciliates (Gonostomum, Oxytricha), whereas water samples were enriched in fungi (Paramicrosporidium and unknown microbial Helotiales). Predicted function through bacterial community composition suggested adaptive evolutive convergence of function in heterogeneous communities. Our study showcases a broad description of the microbial diversity across different microhabitats in the same environment and expands the knowledge on the diversity of microbial eukaryotes in AMD habitats.

Use of Endophytic and Rhizosphere Bacteria To Improve Phytoremediation of Arsenic-Contaminated Industrial Soils by Autochthonous Betula celtiberica.

The aim of this study was to investigate the potential of indigenous arsenic-tolerant bacteria to enhance arsenic phytoremediation by the autochthonous pseudometallophyte Betula celtiberica The first goal was to perform an initial analysis of the entire rhizosphere and endophytic bacterial communities of the above-named accumulator plant, including the cultivable bacterial species. B. celtiberica's microbiome was dominated by taxa related to Flavobacteriales, Burkholderiales, and Pseudomonadales, especially the Pseudomonas and Flavobacterium genera. A total of 54 cultivable rhizobacteria and 41 root endophytes, mainly affiliated with the phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria, were isolated and characterized with respect to several potentially useful features for metal plant accumulation, such as the ability to promote plant growth, metal chelation, and/or mitigation of heavy-metal stress. Seven bacterial isolates were further selected and tested for in vitro accumulation of arsenic in plants; four of them were finally assayed in field-scale bioaugmentation experiments. The exposure to arsenic in vitro caused an increase in the total nonprotein thiol compound content in roots, suggesting a detoxification mechanism through phytochelatin complexation. In the contaminated field, the siderophore and indole-3-acetic acid producers of the endophytic bacterial consortium enhanced arsenic accumulation in the leaves and roots of Betula celtiberica, whereas the rhizosphere isolate Ensifer adhaerens strain 91R mainly promoted plant growth. Field experimentation showed that additional factors, such as soil arsenic content and pH, influenced arsenic uptake in the plant, attesting to the relevance of field conditions in the success of phytoextraction strategies.IMPORTANCE Microorganisms and plants have developed several ways of dealing with arsenic, allowing them to resist and metabolize this metalloid. These properties form the basis of phytoremediation treatments and the understanding that the interactions of plants with soil bacteria are crucial for the optimization of arsenic uptake. To address this in our work, we initially performed a microbiome analysis of the autochthonous Betula celtiberica plants growing in arsenic-contaminated soils, including endosphere and rhizosphere bacterial communities. We then proceeded to isolate and characterize the cultivable bacteria that were potentially better suited to enhance phytoextraction efficiency. Eventually, we went to the field application stage. Our results corroborated the idea that recovery of pseudometallophyte-associated bacteria adapted to a large historically contaminated site and their use in bioaugmentation technologies are affordable experimental approaches and potentially very useful for implementing effective phytoremediation strategies with plants and their indigenous bacteria.

Microbial diversity and metabolic networks in acid mine drainage habitats.

Acid mine drainage (AMD) emplacements are low-complexity natural systems. Low-pH conditions appear to be the main factor underlying the limited diversity of the microbial populations thriving in these environments, although temperature, ionic composition, total organic carbon, and dissolved oxygen are also considered to significantly influence their microbial life. This natural reduction in diversity driven by extreme conditions was reflected in several studies on the microbial populations inhabiting the various micro-environments present in such ecosystems. Early studies based on the physiology of the autochthonous microbiota and the growing success of omics-based methodologies have enabled a better understanding of microbial ecology and function in low-pH mine outflows; however, complementary omics-derived data should be included to completely describe their microbial ecology. Furthermore, recent updates on the distribution of eukaryotes and archaea recovered through sterile filtering (herein referred to as filterable fraction) in these environments demand their inclusion in the microbial characterization of AMD systems. In this review, we present a complete overview of the bacterial, archaeal (including filterable fraction), and eukaryotic diversity in these ecosystems, and include a thorough depiction of the metabolism and element cycling in AMD habitats. We also review different metabolic network structures at the organismal level, which is necessary to disentangle the role of each member of the AMD communities described thus far.

Microbial stratification in low pH oxic and suboxic macroscopic growths along an acid mine drainage.

Macroscopic growths at geographically separated acid mine drainages (AMDs) exhibit distinct populations. Yet, local heterogeneities are poorly understood. To gain novel mechanistic insights into this, we used OMICs tools to profile microbial populations coexisting in a single pyrite gallery AMD (pH ∼2) in three distinct compartments: two from a stratified streamer (uppermost oxic and lowermost anoxic sediment-attached strata) and one from a submerged anoxic non-stratified mat biofilm. The communities colonising pyrite and those in the mature formations appear to be populated by the greatest diversity of bacteria and archaea (including 'ARMAN' (archaeal Richmond Mine acidophilic nano-organisms)-related), as compared with the known AMD, with ∼44.9% unclassified sequences. We propose that the thick polymeric matrix may provide a safety shield against the prevailing extreme condition and also a massive carbon source, enabling non-typical acidophiles to develop more easily. Only 1 of 39 species were shared, suggesting a high metabolic heterogeneity in local microenvironments, defined by the O2 concentration, spatial location and biofilm architecture. The suboxic mats, compositionally most similar to each other, are more diverse and active for S, CO2, CH4, fatty acid and lipopolysaccharide metabolism. The oxic stratum of the streamer, displaying a higher diversity of the so-called 'ARMAN'-related Euryarchaeota, shows a higher expression level of proteins involved in signal transduction, cell growth and N, H2, Fe, aromatic amino acids, sphingolipid and peptidoglycan metabolism. Our study is the first to highlight profound taxonomic and functional shifts in single AMD formations, as well as new microbial species and the importance of H2 in acidic suboxic macroscopic growths.