The biogeography of the mucosa-associated microbiome in health and disease.

Introduction: Little is known about the biogeography of the mucosa associated microbiome (MAM) in patients with inflammatory bowel disease (IBD) versus controls in different segments of the gastrointestinal tract, as well as the links between the MAM, gastrointestinal symptoms, and use of proton pump inhibitors (PPI). Methods: We recruited 59 controls (without structural abnormalities and gastrointestinal symptoms), 44 patients with ulcerative colitis (UC) and 31 with Crohn's disease (CD). Biopsies from various segments of the upper and lower gastrointestinal tract were collected. Microbial composition was assessed via 16S rRNA gene amplicon analysis and the bacterial load of the mucosal biopsies were assessed via qPCR. The MAM was examined in the context of disease status, PPI usage, the severity of gastrointestinal symptoms, and the symptom response to a standardised nutrient challenge (SNC). Results: Microbial communities of the MAM in the upper and lower gastrointestinal tract differed. IBD patients were characterised by relative and absolute depletion of numerous genera known to produce butyrate and/or propionate, with the largest differentiation being the depletion of Faecalibacterium in the lower gastrointestinal tract of CD patients. Notably, PPI users exhibited an enrichment of Faecalibacterium in the lower gastrointestinal tract. The severity of gastrointestinal symptoms, as well as the symptom response to the SNC, were significantly associated with MAM composition in the gastrointestinal tract. Conclusion: The absolute and relative composition of the MAM is variable across different segments of the gastrointestinal tract. These quantitative changes indicates that MAM can be targeted in specific segments of the GI tract to improve patient outcomes.

Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress.

Climate change-driven sea level rise threatens freshwater ecosystems and elicits salinity stress in microbiomes. Methane emissions in these systems are largely mitigated by methane-oxidizing microorganisms. Here, we characterized the physiological and metabolic response of freshwater methanotrophic archaea to salt stress. In our microcosm experiments, inhibition of methanotrophic archaea started at 1%. However, during gradual increase of salt up to 3% in a reactor over 12 weeks, the culture continued to oxidize methane. Using gene expression profiles and metabolomics, we identified a pathway for salt-stress response that produces the osmolyte of anaerobic methanotrophic archaea: N(ε)-acetyl-ß-L-lysine. An extensive phylogenomic analysis on N(ε)-acetyl-ß-L-lysine-producing enzymes revealed that they are widespread across both bacteria and archaea, indicating a potential horizontal gene transfer and a link to BORG extrachromosomal elements. Physicochemical analysis of bioreactor biomass further indicated the presence of sialic acids and the consumption of intracellular polyhydroxyalkanoates in anaerobic methanotrophs during salt stress.

A genome-centric view of the role of the Acropora kenti microbiome in coral health and resilience.

Microbial diversity has been extensively explored in reef-building corals. However, the functional roles of coral-associated microorganisms remain poorly elucidated. Here, we recover 191 bacterial and 10 archaeal metagenome-assembled genomes (MAGs) from the coral Acropora kenti (formerly A. tenuis) and adjacent seawater, to identify microbial functions and metabolic interactions within the holobiont. We show that 82 MAGs were specific to the A. kenti holobiont, including members of the Pseudomonadota, Bacteroidota, and Desulfobacterota. A. kenti-specific MAGs displayed significant differences in their genomic features and functional potential relative to seawater-specific MAGs, with a higher prevalence of genes involved in host immune system evasion, nitrogen and carbon fixation, and synthesis of five essential B-vitamins. We find a diversity of A. kenti-specific MAGs encode the biosynthesis of essential amino acids, such as tryptophan, histidine, and lysine, which cannot be de novo synthesised by the host or Symbiodiniaceae. Across a water quality gradient spanning 2° of latitude, A. kenti microbial community composition is correlated to increased temperature and dissolved inorganic nitrogen, with corresponding enrichment in molecular chaperones, nitrate reductases, and a heat-shock protein. We reveal mechanisms of A. kenti-microbiome-symbiosis on the Great Barrier Reef, highlighting the interactions underpinning the health of this keystone holobiont.

Anaerobic oxidation of ammonium and short-chain gaseous alkanes coupled to nitrate reduction by a bacterial consortium.

The bacterial species "Candidatus Alkanivorans nitratireducens" was recently demonstrated to mediate nitrate-dependent anaerobic oxidation of short-chain gaseous alkanes (SCGAs). In previous bioreactor enrichment studies, the species appeared to reduce nitrate in two phases, switching from denitrification to dissimilatory nitrate reduction to ammonium (DNRA) in response to nitrite accumulation. The regulation of this switch or the nature of potential syntrophic partnerships with other microorganisms remains unclear. Here, we describe anaerobic multispecies cultures of bacteria that couple the oxidation of propane and butane to nitrate reduction and the oxidation of ammonium (anammox). Batch tests with 15N-isotope labelling and multi-omic analyses collectively supported a syntrophic partnership between "Ca. A. nitratireducens" and anammox bacteria, with the former species mediating nitrate-driven oxidation of SCGAs, supplying the latter with nitrite for the oxidation of ammonium. The elimination of nitrite accumulation by the anammox substantially increased SCGA and nitrate consumption rates, whereas it suppressed DNRA. Removing ammonium supply led to its eventual production, the accumulation of nitrite, and the upregulation of DNRA gene expression for the abundant "Ca. A. nitratireducens". Increasing the supply of SCGA had a similar effect in promoting DNRA. Our results suggest that "Ca. A. nitratireducens" switches to DNRA to alleviate oxidative stress caused by nitrite accumulation, giving further insight into adaptability and ecology of this microorganism. Our findings also have important implications for the understanding of the fate of nitrogen and SCGAs in anaerobic environments.

Nitrate-driven anaerobic oxidation of ethane and butane by bacteria.

The short-chain gaseous alkanes (ethane, propane, and butane; SCGAs) are important components of natural gas, yet their fate in environmental systems is poorly understood. Microbially mediated anaerobic oxidation of SCGAs coupled to nitrate reduction has been demonstrated for propane, but is yet to be shown for ethane or butane-despite being energetically feasible. Here we report two independent bacterial enrichments performing anaerobic ethane and butane oxidation, respectively, coupled to nitrate reduction to dinitrogen gas and ammonium. Isotopic 13C- and 15N-labelling experiments, mass and electron balance tests, and metabolite and meta-omics analyses collectively reveal that the recently described propane-oxidizing "Candidatus Alkanivorans nitratireducens" was also responsible for nitrate-dependent anaerobic oxidation of the SCGAs in both these enrichments. The complete genome of this species encodes alkylsuccinate synthase genes for the activation of ethane/butane via fumarate addition. Further substrate range tests confirm that "Ca. A. nitratireducens" is metabolically versatile, being able to degrade ethane, propane, and butane under anoxic conditions. Moreover, our study proves nitrate as an additional electron sink for ethane and butane in anaerobic environments, and for the first time demonstrates the use of the fumarate addition pathway in anaerobic ethane oxidation. These findings contribute to our understanding of microbial metabolism of SCGAs in anaerobic environments.

Pyrogenic Carbon Promotes Anaerobic Oxidation of Methane Coupled with Iron Reduction via the Redox-Cycling Mechanism.

Pyrogenic carbon (PC) can mediate electron transfer and thus catalyze biogeochemical processes to impact greenhouse gas (GHG) emissions. Here, we demonstrate that PC can contribute to mitigating GHG emissions by promoting the Fe(III)-dependent anaerobic oxidation of methane (AOM). It was found that the amendment PCs in microcosms dominated by Methanoperedenaceae performing Fe(III)-dependent AOM simultaneously promoted the rate of AOM and Fe(III) reduction with a consistent ratio close to the theoretical stoichiometry of 1:8. Further correlation analysis showed that the AOM rate was linearly correlated with the electron exchange capacity, but not the conductivity, of added PC materials, indicating the redox-cycling electron transfer mechanism to promote the Fe(III)-dependent AOM. The mass content of the C═O moiety from differentially treated PCs was well correlated with the AOM rate, suggesting that surface redox-active quinone groups on PCs contribute to facilitating Fe(III)-dependent AOM. Further microbial analyses indicate that PC likely shuttles direct electron transfer from Methanoperedenaceae to Fe(III) reduction. This study provides new insight into the climate-cooling impact of PCs, and our evaluation indicates that the PC-facilitated Fe(III)-dependent AOM could have a significant contribution to suppressing methane emissions from the world's reservoirs.

Multi-heme cytochrome-mediated extracellular electron transfer by the anaerobic methanotroph 'Candidatus Methanoperedens nitroreducens'.

Anaerobic methanotrophic archaea (ANME) carry out anaerobic oxidation of methane, thus playing a crucial role in the methane cycle. Previous genomic evidence indicates that multi-heme c-type cytochromes (MHCs) may facilitate the extracellular electron transfer (EET) from ANME to different electron sinks. Here, we provide experimental evidence supporting cytochrome-mediated EET for the reduction of metals and electrodes by 'Candidatus Methanoperedens nitroreducens', an ANME acclimated to nitrate reduction. Ferrous iron-targeted fluorescent assays, metatranscriptomics, and single-cell imaging suggest that 'Ca. M. nitroreducens' uses surface-localized redox-active cytochromes for metal reduction. Electrochemical and Raman spectroscopic analyses also support the involvement of c-type cytochrome-mediated EET for electrode reduction. Furthermore, several genes encoding menaquinone cytochrome type-c oxidoreductases and extracellular MHCs are differentially expressed when different electron acceptors are used.

Phylogenetic and metabolic diversity of microbial communities performing anaerobic ammonium and methane oxidations under different nitrogen loadings.

The microbial guild coupling anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is an innovative process to achieve energy-efficient nitrogen removal with the beneficial use of methane in biogas or in anaerobically treated wastewater. Here, metagenomics and metatranscriptomics were used to reveal the microbial ecology of two biofilm systems, which incorporate anammox and n-DAMO for high-level nitrogen removal in low-strength domestic sewage and high-strength sidestream wastewater, respectively. We find that different nitrogen loadings (i.e., 0.1 vs. 1.0 kg N/m3/d) lead to different combinations of anammox bacteria and anaerobic methanotrophs ("Candidatus Methanoperedens" and "Candidatus Methylomirabilis"), which play primary roles for carbon and nitrogen transformations therein. Despite methane being the only exogenous organic carbon supplied, heterotrophic populations (e.g., Verrucomicrobiota and Bacteroidota) co-exist and actively perform partial denitrification or dissimilatory nitrate reduction to ammonium (DNRA), likely using organic intermediates from the breakdown of methane and biomass as carbon sources. More importantly, two novel genomes belonging to "Ca. Methylomirabilis" are recovered, while one surprisingly expresses nitrate reductases, which we designate as "Ca. Methylomirabilis nitratireducens" representing its inferred capability in performing nitrate-dependent anaerobic methane oxidation. This finding not only suggests a previously neglected possibility of "Ca. Methylomirabilis" bacteria in performing methane-dependent nitrate reduction, and also challenges the previous understanding that the methane-dependent complete denitrification from nitrate to dinitrogen gas is carried out by the consortium of bacteria and archaea.

Anaerobic methanotroph 'Candidatus Methanoperedens nitroreducens' has a pleomorphic life cycle.

'Candidatus Methanoperedens' are anaerobic methanotrophic (ANME) archaea with global importance to methane cycling. Here meta-omics and fluorescence in situ hybridization (FISH) were applied to characterize a bioreactor dominated by 'Candidatus Methanoperedens nitroreducens' performing anaerobic methane oxidation coupled to nitrate reduction. Unexpectedly, FISH revealed the stable co-existence of two 'Ca. M. nitroreducens' morphotypes: the archetypal coccobacilli microcolonies and previously unreported planktonic rods. Metagenomic analysis showed that the 'Ca. M. nitroreducens' morphotypes were genomically identical but had distinct gene expression profiles for proteins associated with carbon metabolism, motility and cell division. In addition, a third distinct phenotype was observed, with some coccobacilli 'Ca. M. nitroreducens' storing carbon as polyhydroxyalkanoates. The phenotypic variation of 'Ca. M. nitroreducens' probably aids their survival and dispersal in the face of sub-optimal environmental conditions. These findings further demonstrate the remarkable ability of members of the 'Ca. Methanoperedens' to adapt to their environment.

Anaerobic oxidation of propane coupled to nitrate reduction by a lineage within the class Symbiobacteriia.

Anaerobic microorganisms are thought to play a critical role in regulating the flux of short-chain gaseous alkanes (SCGAs; including ethane, propane and butane) from terrestrial and aquatic ecosystems to the atmosphere. Sulfate has been confirmed to act as electron acceptor supporting microbial anaerobic oxidation of SCGAs, yet several other energetically more favourable acceptors co-exist with these gases in anaerobic environments. Here, we show that a bioreactor seeded with biomass from a wastewater treatment facility can perform anaerobic propane oxidation coupled to nitrate reduction to dinitrogen gas and ammonium. The bioreactor was operated for more than 1000 days, and we used 13C- and 15N-labelling experiments, metagenomic, metatranscriptomic, metaproteomic and metabolite analyses to characterize the microbial community and the metabolic processes. The data collectively suggest that a species representing a novel order within the bacterial class Symbiobacteriia is responsible for the observed nitrate-dependent propane oxidation. The closed genome of this organism, which we designate as 'Candidatus Alkanivorans nitratireducens', encodes pathways for oxidation of propane to CO2 via fumarate addition, and for nitrate reduction, with all the key genes expressed during nitrate-dependent propane oxidation. Our results suggest that nitrate is a relevant electron sink for SCGA oxidation in anaerobic environments, constituting a new microbially-mediated link between the carbon and nitrogen cycles.

Expanding the phylogenetic distribution of cytochrome b-containing methanogenic archaea sheds light on the evolution of methanogenesis.

Methane produced by methanogenic archaea has an important influence on Earth's changing climate. Methanogenic archaea are phylogenetically diverse and widespread in anoxic environments. These microorganisms can be divided into two subgroups based on whether or not they use b-type cytochromes for energy conservation. Methanogens with b-type cytochromes have a wider substrate range and higher growth yields than those without them. To date, methanogens with b-type cytochromes were found exclusively in the phylum "Ca. Halobacteriota" (formerly part of the phylum Euryarchaeota). Here, we present the discovery of metagenome-assembled genomes harboring methyl-coenzyme M reductase genes reconstructed from mesophilic anoxic sediments, together with the previously reported thermophilic "Ca. Methylarchaeum tengchongensis", representing a novel archaeal order, designated the "Ca. Methylarchaeales", of the phylum Thermoproteota (formerly the TACK superphylum). These microorganisms contain genes required for methyl-reducing methanogenesis and the Wood-Ljundahl pathway. Importantly, the genus "Ca. Methanotowutia" of the "Ca. Methylarchaeales" encode a cytochrome b-containing heterodisulfide reductase (HdrDE) and methanophenazine-reducing hydrogenase complex that have similar gene arrangements to those found in methanogenic Methanosarcinales. Our results indicate that members of the "Ca. Methylarchaeales" are methanogens with cytochromes and can conserve energy via membrane-bound electron transport chains. Phylogenetic and amalgamated likelihood estimation analyses indicate that methanogens with cytochrome b-containing electron transfer complexes likely evolved before diversification of Thermoproteota or "Ca. Halobacteriota" in the early Archean Eon. Surveys of public sequence databases suggest that members of the lineage are globally distributed in anoxic sediments and may be important players in the methane cycle.

Polyhydroxyalkanoate-driven current generation via acetate by an anaerobic methanotrophic consortium.

Anaerobic oxidation of methane (AOM) is an important microbial process mitigating methane (CH4) emission from natural sediments. Anaerobic methanotrophic archaea (ANME) have been shown to mediate AOM coupled to the reduction of several compounds, either directly (i.e. nitrate, metal oxides) or in consortia with syntrophic bacterial partners (i.e. sulfate). However, the mechanisms underlying extracellular electron transfer (EET) between ANME and their bacterial partners or external electron acceptors are poorly understood. In this study, we investigated electron and carbon flow for an anaerobic methanotrophic consortium dominated by 'Candidatus Methanoperedens nitroreducens' in a CH4-fed microbial electrolysis cell (MEC). Acetate was identified as a likely intermediate for the methanotrophic consortium, which stimulated the growth of the known electroactive genus Geobacter. Electrochemical characterization, stoichiometric calculations of the system, along with stable isotope-based assays, revealed that acetate was not produced from CH4 directly. In the absence of CH4, current was still generated and the microbial community remained largely unchanged. A substantial portion of the generated current in the absence of CH4 was linked to the oxidation of the intracellular polyhydroxybutyrate (PHB) and the breakdown of extracellular polymeric substances (EPSs). The ability of 'Ca. M. nitroreducens' to use stored PHB as a carbon and energy source, and its ability to donate acetate as a diffusible electron carrier expands the known metabolic diversity of this lineage that likely underpins its success in natural systems.

Methane-dependent selenate reduction by a bacterial consortium.

Methanotrophic microorganisms play a critical role in controlling the flux of methane from natural sediments into the atmosphere. Methanotrophs have been shown to couple the oxidation of methane to the reduction of diverse electron acceptors (e.g., oxygen, sulfate, nitrate, and metal oxides), either independently or in consortia with other microbial partners. Although several studies have reported the phenomenon of methane oxidation linked to selenate reduction, neither the microorganisms involved nor the underlying trophic interaction has been clearly identified. Here, we provide the first detailed evidence for interspecies electron transfer between bacterial populations in a bioreactor community where the reduction of selenate is linked to methane oxidation. Metagenomic and metaproteomic analyses of the community revealed a novel species of Methylocystis as the most abundant methanotroph, which actively expressed proteins for oxygen-dependent methane oxidation and fermentation pathways, but lacked the genetic potential for selenate reduction. Pseudoxanthomonas, Piscinibacter, and Rhodocyclaceae populations appeared to be responsible for the observed selenate reduction using proteins initially annotated as periplasmic nitrate reductases, with fermentation by-products released by the methanotrophs as electron donors. The ability for the annotated nitrate reductases to reduce selenate was confirmed by gene knockout studies in an isolate of Pseudoxanthomonas. Overall, this study provides novel insights into the metabolic flexibility of the aerobic methanotrophs that likely allows them to thrive across natural oxygen gradients, and highlights the potential role for similar microbial consortia in linking methane and other biogeochemical cycles in environments where oxygen is limited.

Lateral Gene Transfer Drives Metabolic Flexibility in the Anaerobic Methane-Oxidizing Archaeal Family Methanoperedenaceae.

Anaerobic oxidation of methane (AOM) is an important biological process responsible for controlling the flux of methane into the atmosphere. Members of the archaeal family Methanoperedenaceae (formerly ANME-2d) have been demonstrated to couple AOM to the reduction of nitrate, iron, and manganese. Here, comparative genomic analysis of 16 Methanoperedenaceae metagenome-assembled genomes (MAGs), recovered from diverse environments, revealed novel respiratory strategies acquired through lateral gene transfer (LGT) events from diverse archaea and bacteria. Comprehensive phylogenetic analyses suggests that LGT has allowed members of the Methanoperedenaceae to acquire genes for the oxidation of hydrogen and formate and the reduction of arsenate, selenate, and elemental sulfur. Numerous membrane-bound multiheme c-type cytochrome complexes also appear to have been laterally acquired, which may be involved in the direct transfer of electrons to metal oxides, humic substances, and syntrophic partners.IMPORTANCE AOM by microorganisms limits the atmospheric release of the potent greenhouse gas methane and has consequent importance for the global carbon cycle and climate change modeling. While the oxidation of methane coupled to sulfate by consortia of anaerobic methanotrophic (ANME) archaea and bacteria is well documented, several other potential electron acceptors have also been reported to support AOM. In this study, we identify a number of novel respiratory strategies that appear to have been laterally acquired by members of the Methanoperedenaceae, as they are absent from related archaea and other ANME lineages. Expanding the known metabolic potential for members of the Methanoperedenaceae provides important insight into their ecology and suggests their role in linking methane oxidation to several global biogeochemical cycles.

Candidatus Amarolinea and Candidatus Microthrix Are Mainly Responsible for Filamentous Bulking in Danish Municipal Wastewater Treatment Plants.

Filamentous bulking is a common serious operational problem leading to deteriorated sludge settling that has long been observed in activated sludge biological wastewater treatment systems. A number of bacterial genera found therein possess filamentous morphology, where some have been shown to be implicated in bulking episodes (e.g., Ca. Microthrix), the impact of many others is still not clear. In this study we performed a survey of 17 Danish municipal wastewater treatment plants (WWTPs) with nutrient removal using 16S rRNA amplicon sequencing over a period of 13 years, where all known filamentous bacteria from 30 genera were analyzed. The filamentous community constituted on average 13 ± 6%, and up to 43% of total read abundance with the same genera common to all plants. Ca. Microthrix and several genera belonging to phylum Chloroflexi were among the most abundant filamentous bacteria. The effect of filamentous bacteria on sludge settling properties was analyzed using measurements of the diluted sludge volume index (DSVI). Strong positive correlations with DSVI were observed only for Ca. Microthrix and Ca. Amarolinea, the latter being a novel, recently characterized genus belonging to the phylum Chloroflexi. The bulking potential of other filamentous bacteria was not significant despite their presence in many plants. Low phylogenetic diversity was observed for both Ca. Microthrix and Ca. Amarolinea, making physiological characterization of individual species and potential development of control strategies more feasible. In this study we show that, despite the high diversity of filamentous phylotypes in Danish WWTPs, only few of them were responsible for severe bulking episodes.

Anaerobic methane oxidation coupled to manganese reduction by members of the Methanoperedenaceae.

Anaerobic oxidation of methane (AOM) is a major biological process that reduces global methane emission to the atmosphere. Anaerobic methanotrophic archaea (ANME) mediate this process through the coupling of methane oxidation to different electron acceptors, or in concert with a syntrophic bacterial partner. Recently, ANME belonging to the archaeal family Methanoperedenaceae (formerly known as ANME-2d) were shown to be capable of AOM coupled to nitrate and iron reduction. Here, a freshwater sediment bioreactor fed with methane and Mn(IV) oxides (birnessite) resulted in a microbial community dominated by two novel members of the Methanoperedenaceae, with biochemical profiling of the system demonstrating Mn(IV)-dependent AOM. Genomic and transcriptomic analyses revealed the expression of key genes involved in methane oxidation and several shared multiheme c-type cytochromes (MHCs) that were differentially expressed, indicating the likely use of different extracellular electron transfer pathways. We propose the names "Candidatus Methanoperedens manganicus" and "Candidatus Methanoperedens manganireducens" for the two newly described Methanoperedenaceae species. This study demonstrates the ability of members of the Methanoperedenaceae to couple AOM to the reduction of Mn(IV) oxides, which suggests their potential role in linking methane and manganese cycling in the environment.

Re-evaluating the microbiology of the enhanced biological phosphorus removal process.

We have critically assessed some of the dogmas in the microbiology of enhanced biological phosphorus removal (EBPR) and argue that the genus Tetrasphaera can be as important as Ca. Accumulibacter for phosphorus removal; and that proliferation of their competitors, the glycogen accumulating organisms, does not appear to be a practical problem for EBPR efficiency even under tropical conditions. An increasing number of EBPR-related genomes are changing our understanding of their physiology, for example, their potential to participate in denitrification. Rather than trying to identify organisms that adhere to strict phenotype metabolic models, we advocate for broader analyses of the whole microbial communities in EBPR plants by iterative studies with isolates, lab enrichments, and full-scale systems.

Genomic insights into the metabolism of 'Candidatus Defluviicoccus seviourii', a member of Defluviicoccus cluster III abundant in industrial activated sludge.

Filamentous cluster III Defluviicoccus (DF3) are known to proliferate and cause bulking issues in industrial wastewater treatment plants. Members of the genus Defluviicoccus are also known to exhibit the glycogen accumulating organism (GAO) phenotype, which is suggested to be detrimental to enhanced biological phosphorus removal (EBPR). Despite the reported negative impact members of the DF3 have on activated sludge wastewater treatment systems, limited research has focused on understanding the physiological traits that allow them to compete in these environments. In this study, a near complete genome of an abundant filamentous DF3 named 'Candidatus Defluviicoccus seviourii' was obtained from a full-scale sequencing batch reactor (SBR) treating winery wastewater. Annotation of the 'Ca. D. seviourii' genome revealed interesting metabolic features that help to understand the abundance of this microorganism in industrial wastewater treatment plants. Their potential for the storage of polyhydroxyalkanoates (PHA) is suggested to favour these organisms with the intermittent availability of carbon in these systems. An ability to fix nitrogen and take up urea may provide them with an additional advantage with the characteristically high carbon to nitrogen content of industrial waste. The genome and preliminary findings of this study provide a foundation for further research into these biotechnologically relevant organisms.

Genomic insights into Candidatus Amarolinea aalborgensis gen. nov., sp. nov., associated with settleability problems in wastewater treatment plants.

Settleability of particles in activated sludge systems can be impaired by an overgrowth of filamentous bacteria, a problem known as bulking. These filaments are often members of the phylum Chloroflexi, sometimes reaching abundances in excess of 30% of the biovolume. The uncultured Chloroflexi phylotype, Candidatus Amarolinea, has been observed in high abundances in Danish full-scale activated sludge systems by 16S rRNA gene amplicon surveys, where it has been associated with bulking. In this study, fluorescence in situ hybridization was applied to confirm their high abundance, filamentous morphology, and contribution to the interfloc bridging that characterizes filamentous bulking. Furthermore, genome-centric metagenomics using both Illumina and Oxford Nanopore sequencing was used to obtain a near complete population genome (5.7Mbp) of the Ca. Amarolinea phylotype, which belongs to the proposed novel family Amarolineaceae within the order Caldilineales of Chloroflexi. Annotation of the genome indicated that the phylotype is capable of aerobic respiration, fermentation, and dissimilatory nitrate reduction to ammonia. The genome sequence also gives a better insight into the phylogenetic and evolutionary relationships of the organism. The name Candidatus Amarolinea aalborgensis is proposed for the species.

Genomic and in Situ Analyses Reveal the Micropruina spp. as Abundant Fermentative Glycogen Accumulating Organisms in Enhanced Biological Phosphorus Removal Systems.

Enhanced biological phosphorus removal (EBPR) involves the cycling of biomass through carbon-rich (feast) and carbon-deficient (famine) conditions, promoting the activity of polyphosphate accumulating organisms (PAOs). However, several alternate metabolic strategies, without polyphosphate storage, are possessed by other organisms, which can compete with the PAO for carbon at the potential expense of EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs), which utilize aerobically stored glycogen to energize anaerobic substrate uptake and storage. In full-scale systems the Micropruina spp. are among the most abundant of the proposed GAO, yet little is known about their ecophysiology. In the current study, genomic and metabolomic studies were performed on Micropruina glycogenica str. Lg2T and compared to the in situ physiology of members of the genus in EBPR plants using state-of-the-art single cell techniques. The Micropruina spp. were observed to take up carbon, including sugars and amino acids, under anaerobic conditions, which were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored as glycogen for potential aerobic use. Fermentation was not directly demonstrated for the abundant members of the genus in situ, but was strongly supported by the confirmation of anaerobic uptake of carbon and glycogen storage in the absence of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is markedly different from the classical GAO model. The amount of carbon stored by fermentative organisms has potentially important implications for phosphorus removal - as they compete for substrates with the Tetrasphaera PAO and stored carbon is not made available to the "Candidatus Accumulibacter" PAO under anaerobic conditions. This study shows that the current models of the competition between PAO and GAO are too simplistic and may need to be revised to take into account the impact of potential carbon storage by fermentative organisms.