Bacterial interactions on nutrient-rich surfaces in the gut lumen.

The intestinal lumen is a turbulent, semi-fluid landscape where microbial cells and nutrient-rich particles are distributed with high heterogeneity. Major questions regarding the basic physical structure of this dynamic microbial ecosystem remain unanswered. Most gut microbes are non-motile, and it is unclear how they achieve optimum localization relative to concentrated aggregations of dietary glycans that serve as their primary source of energy. In addition, a random spatial arrangement of cells in this environment is predicted to limit sustained interactions that drive co-evolution of microbial genomes. The ecological consequences of random versus organized microbial localization have the potential to control both the metabolic outputs of the microbiota and the propensity for enteric pathogens to participate in proximity-dependent microbial interactions. Here, we review evidence suggesting that several bacterial species adopt organized spatial arrangements in the gut via adhesion. We highlight examples where localization could contribute to antagonism or metabolic interdependency in nutrient degradation, and we discuss imaging- and sequencing-based technologies that have been used to assess the spatial positions of cells within complex microbial communities.

Promethearchaeum syntrophicum gen. nov., sp. nov., an anaerobic, obligately syntrophic archaeon, the first isolate of the lineage 'Asgard' archaea, and proposal of the new archaeal phylum Promethearchaeota phyl. nov. and kingdom Promethearchaeati regn. nov.

An anaerobic, mesophilic, syntrophic, archaeon strain MK-D1T, was isolated as a pure co-culture with Methanogenium sp. strain MK-MG from deep-sea methane seep sediment. This organism is, to our knowledge, the first cultured representative of 'Asgard' archaea, an archaeal group closely related to eukaryotes. Here, we describe the detailed physiology and phylogeny of MK-D1T and propose Promethearchaeum syntrophicum gen. nov., sp. nov. to accommodate this strain. Cells were non-motile, small cocci, approximately 300-750 nm in diameter and produced membrane vesicles, chains of blebs and membrane-based protrusions. MK-D1T grew at 4-30 °C with optimum growth at 20 °C. The strain grew chemoorganotrophically with amino acids, peptides and yeast extract with obligate dependence on syntrophy with H2-/formate-utilizing organisms. MK-D1T showed the fastest growth and highest maximum cell yield when grown with yeast extract as the substrate: approximately 3 months to full growth, reaching up to 6.7×106 16S rRNA gene copies ml-1. MK-D1T had a circular 4.32 Mb chromosome with a DNA G+C content of 31.1 mol%. The results of phylogenetic analyses of the 16S rRNA gene and conserved marker proteins indicated that the strain is affiliated with 'Asgard' archaea and more specifically DHVC1/DSAG/MBG-B and 'Lokiarchaeota'/'Lokiarchaeia'. On the basis of the results of 16S rRNA gene sequence analysis, the most closely related isolated relatives were Infirmifilum lucidum 3507LTT (76.09 %) and Methanothermobacter tenebrarum RMAST (77.45 %) and the closest relative in enrichment culture was Candidatus 'Lokiarchaeum ossiferum' (95.39 %). The type strain of the type species is MK-D1T (JCM 39240T and JAMSTEC no. 115508). We propose the associated family, order, class, phylum, and kingdom as Promethearchaeaceae fam. nov., Promethearchaeales ord. nov., Promethearchaeia class. nov., Promethearchaeota phyl. nov., and Promethearchaeati regn. nov., respectively. These are in accordance with ICNP Rules 8 and 22 for nomenclature, Rule 30(3)(b) for validation and maintenance of the type strain, and Rule 31a for description as a member of an unambiguous syntrophic association.

Impact of additives on syntrophic propionate and acetate enrichments under high-ammonia conditions.

High ammonia concentrations in anaerobic degradation systems cause volatile fatty acid accumulation and reduced methane yield, which often derive from restricted activity of syntrophic acid-oxidising bacteria and hydrogenotrophic methanogens. Inclusion of additives that facilitate the electron transfer or increase cell proximity of syntrophic species by flocculation can be a suitable strategy to counteract these problems, but its actual impact on syntrophic interactions has yet to be determined. In this study, microbial cultivation and molecular and microscopic analysis were performed to evaluate the impact of conductive (graphene, iron oxide) and non-conductive (zeolite) additives on the degradation rate of acetate and propionate to methane by highly enriched ammonia-tolerant syntrophic cultures derived from a biogas process. All additives had a low impact on the lag phase but resulted in a higher rate of acetate (except graphene) and propionate degradation. The syntrophic bacteria 'Candidatus Syntrophopropionicum ammoniitolerans', Syntrophaceticus schinkii and a novel hydrogenotrophic methanogen were found in higher relative abundance and higher gene copy numbers in flocculating communities than in planktonic communities in the cultures, indicating benefits to syntrophs of living in close proximity to their cooperating partner. Microscopy and element analysis showed precipitation of phosphates and biofilm formation in all batches except on the graphene batches, possibly enhancing the rate of acetate and propionate degradation. Overall, the concordance of responses observed in both acetate- and propionate-fed cultures highlight the suitability of the addition of iron oxide or zeolites to enhance acid conversion to methane in high-ammonia biogas processes. KEY POINTS: • All additives promoted acetate (except graphene) and propionate degradation. • A preference for floc formation by ammonia-tolerant syntrophs was revealed. • Microbes colonised the surfaces of iron oxide and zeolite, but not graphene.

Effect of micro-aeration on syntrophic and methanogenic activity in anaerobic sludge.

Micro-aeration was shown to improve anaerobic digestion (AD) processes, although oxygen is known to inhibit obligate anaerobes, such as syntrophic communities of bacteria and methanogens. The effect of micro-aeration on the activity and microbial interaction in syntrophic communities, as well as on the potential establishment of synergetic relationships with facultative anaerobic bacteria (FAB) or aerobic bacteria (AB), was investigated. Anaerobic sludge was incubated with ethanol and increasing oxygen concentrations (0-5% in the headspace). Assays with acetate or H2/CO2 (direct substrates for methanogens) were also performed. When compared with the controls (0% O2), oxygen significantly decreased substrate consumption and initial methane production rate (MPR) from acetate or H2/CO2. At 0.5% O2, MPR from these substrates was inhibited 30-40%, and close to 100% at 5% O2. With ethanol, significant inhibition (>36%) was only observed for oxygen concentrations higher than 2.5%. Oxygen was consumed in the assays, pointing to the stimulation of AB/FAB by ethanol, which helped to protect the syntrophic consortia under micro-aerobic conditions. This highlights the importance of AB/FAB in maintaining functional and resilient syntrophic communities, which is relevant for real AD systems (in which vestigial O2 amounts are frequently present), as well as for AD systems using micro-aeration as a process strategy. KEY POINTS: •Micro-aeration impacts syntrophic communities of bacteria and methanogens. •Oxygen stimulates AB/FAB, maintaining functional and resilient consortia. •Micro-aeration studies are critical for systems using micro-aeration as a process strategy.

Mechanisms of microbial co-aggregation in mixed anaerobic cultures.

Co-aggregation of anaerobic microorganisms into suspended microbial biofilms (aggregates) serves ecological and biotechnological functions. Tightly packed aggregates of metabolically interdependent bacteria and archaea play key roles in cycling of carbon and nitrogen. Additionally, in biotechnological applications, such as wastewater treatment, microbial aggregates provide a complete metabolic network to convert complex organic material. Currently, experimental data explaining the mechanisms behind microbial co-aggregation in anoxic environments is scarce and scattered across the literature. To what extent does this process resemble co-aggregation in aerobic environments? Does the limited availability of terminal electron acceptors drive mutualistic microbial relationships, contrary to the commensal relationships observed in oxygen-rich environments? And do co-aggregating bacteria and archaea, which depend on each other to harvest the bare minimum Gibbs energy from energy-poor substrates, use similar cellular mechanisms as those used by pathogenic bacteria that form biofilms? Here, we provide an overview of the current understanding of why and how mixed anaerobic microbial communities co-aggregate and discuss potential future scientific advancements that could improve the study of anaerobic suspended aggregates. KEY POINTS: • Metabolic dependency promotes aggregation of anaerobic bacteria and archaea • Flagella, pili, and adhesins play a role in the formation of anaerobic aggregates • Cyclic di-GMP/AMP signaling may trigger the polysaccharides production in anaerobes.

Unnatural Direct Interspecies Electron Transfer Enabled by Living Cell-Cell Click Chemistry.

Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR-1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell-cell click chemistry strategy. By introducing alkyne- or azide-modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C-type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell-cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications.

A global metagenomics-based analysis of BPA degradation and its coupling with nitrogen, sulfur, and methane metabolism in landfill leachates.

Microbial metabolism in landfill leachate systems is critically important in driving the degradation reactions of organic pollutants, including the emerging pollutant bisphenol A (BPA). However, little research has addressed the microbial degradation of BPA in landfill leachate and its interactions with nitrogen (N), sulfur (S), and methane (CH4) metabolism on a global scale. To this end, in this study on a global scale, an extremely high concentration of BPA was detected throughout the global landfill leachates. Subsequent reconstructive analyses of metagenomic datasets from 113 sites worldwide revealed that the predominant BPA-degrading microflora included Proteobacteria, Firmicutes, and Bacteroidota. Further metabolic analyses revealed that all four biochemical pathways involved in the degradation of BPA were achieved through biochemical cooperation between different bacterial members of the community. In addition, BPA degraders have also been found to actively collaborate synergistically with non-BPA degraders in the N and S removal as well as CH4 catabolism in landfill leachates. Collectively, this study not only provides insights into the dominant microbial communities and specific types of BPA-degrading microbial members in the community of landfill leachates worldwide, but also reveals the synergistic interactions between BPA mineralization and N, S, and CH4 metabolism. These findings offer valuable and important insights for future comprehensive and in-depth investigations into BPA metabolism in different environments.

CO-driven electron and carbon flux fuels synergistic microbial reductive dechlorination.

BACKGROUND: Carbon monoxide (CO), hypothetically linked to prebiotic biosynthesis and possibly the origin of the life, emerges as a substantive growth substrate for numerous microorganisms. In anoxic environments, the coupling of CO oxidation with hydrogen (H2) production is an essential source of electrons, which can subsequently be utilized by hydrogenotrophic bacteria (e.g., organohalide-respring bacteria). While Dehalococcoides strains assume pivotal roles in the natural turnover of halogenated organics and the bioremediation of chlorinated ethenes, relying on external H2 as their electron donor and acetate as their carbon source, the synergistic dynamics within the anaerobic microbiome have received comparatively less scrutiny. This study delves into the intriguing prospect of CO serving as both the exclusive carbon source and electron donor, thereby supporting the reductive dechlorination of trichloroethene (TCE). RESULTS: The metabolic pathway involved anaerobic CO oxidation, specifically the Wood-Ljungdahl pathway, which produced H2 and acetate as primary metabolic products. In an intricate microbial interplay, these H2 and acetate were subsequently utilized by Dehalococcoides, facilitating the dechlorination of TCE. Notably, Acetobacterium emerged as one of the pivotal collaborators for Dehalococcoides, furnishing not only a crucial carbon source essential for its growth and proliferation but also providing a defense against CO inhibition. CONCLUSIONS: This research expands our understanding of CO's versatility as a microbial energy and carbon source and unveils the intricate syntrophic dynamics underlying reductive dechlorination.

Methanogenic symbionts of anaerobic ciliates are host- and habitat-specific.

The association between anaerobic ciliates and methanogenic archaea has been recognized for over a century. Nevertheless, knowledge of these associations is limited to a few ciliate species, and so the identification of patterns of host-symbiont specificity has been largely speculative. In this study, we integrated microscopy and genetic identification to survey the methanogenic symbionts of 32 free-living anaerobic ciliate species, mainly from the order Metopida. Based on Sanger and Illumina sequencing of the 16S rRNA gene, our results show that a single methanogenic symbiont population, belonging to Methanobacterium, Methanoregula, or Methanocorpusculum, is dominant in each host strain. Moreover, the host's taxonomy (genus and above) and environment (i.e., endobiotic, marine/brackish, or freshwater) are linked with the methanogen identity at the genus level, demonstrating a strong specificity and fidelity in the association. We also established cultures containing artificially co-occurring anaerobic ciliate species harboring different methanogenic symbionts. This revealed that the host-methanogen relationship is stable over short timescales in cultures without evidence of methanogenic symbiont exchanges, though, our intraspecific survey indicated that metopids also tend to replace their methanogens over longer evolutionary timescales. Therefore, anaerobic ciliates have adapted a mixed transmission mode to maintain and replace their methanogenic symbionts, allowing them to thrive in oxygen-depleted environments.

Divergent marine anaerobic ciliates harbor closely related Methanocorpusculum endosymbionts.

Ciliates are a diverse group of protists known for their ability to establish various partnerships and thrive in a wide variety of oxygen-depleted environments. Most anaerobic ciliates harbor methanogens, one of the few known archaea living intracellularly. These methanogens increase the metabolic efficiency of host fermentation via syntrophic use of host end-product in methanogenesis. Despite the ubiquity of these symbioses in anoxic habitats, patterns of symbiont specificity and fidelity are not well known. We surveyed two unrelated, commonly found groups of anaerobic ciliates, the Plagiopylea and Metopida, isolated from anoxic marine sediments. We sequenced host 18S rRNA and symbiont 16S rRNA marker genes as well as the symbiont internal transcribed spacer region from our cultured ciliates to identify hosts and their associated methanogenic symbionts. We found that marine ciliates from both of these co-occurring, divergent groups harbor closely related yet distinct intracellular archaea within the Methanocorpusculum genus. The symbionts appear to be stable at the host species level, but at higher taxonomic levels, there is evidence that symbiont replacements have occurred. Gaining insight into this unique association will deepen our understanding of the complex transmission modes of marine microbial symbionts, and the mutualistic microbial interactions occurring across domains of life.

High methane potential of oxygenic photogranules decreases after starvation.

Oxygenic photogranules (OPG) are granular biofilms that can treat wastewater without external aeration, making it an advantage over activated sludge. Excess of OPG biomass can serve as energy source through anaerobic digestion. Two sequencing batch photoreactors were operated over 400 days to grow OPG. Biochemical methane potentials (BMP) were obtained from near-infrared spectroscopy. OPGs had an average BMP of 356 mL CH4·gVS-1, much higher than typical BMP from activated sludge. A partial least squares analysis could relate BMP with reactor operating conditions, like light regime, load or biomass concentration. Since organic load was the most influential parameter on BMP, three starvation experiments were set up. An average decrease of BMP by 18.4 % was observed. However, the unexpected growth of biomass during starvation resulted in a higher total methane volume. In conclusion, starvation reduces the BMP of OPGs but anaerobic digestion of OPG biomass remains a promising route for biomass valorization.

Catabolism and interactions of syntrophic propionate- and acetate oxidizing microorganisms under mesophilic, high-ammonia conditions.

Microbial inhibition by high ammonia concentrations is a recurring problem that significantly restricts methane formation from intermediate acids, i.e., propionate and acetate, during anaerobic digestion of protein-rich waste material. Studying the syntrophic communities that perform acid conversion is challenging, due to their relatively low abundance within the microbial communities typically found in biogas processes and disruption of their cooperative behavior in pure cultures. To overcome these limitations, this study examined growth parameters and microbial community dynamics of highly enriched mesophilic and ammonia-tolerant syntrophic propionate and acetate-oxidizing communities and analyzed their metabolic activity and cooperative behavior using metagenomic and metatranscriptomic approaches. Cultivation in batch set-up demonstrated biphasic utilization of propionate, wherein acetate accumulated and underwent oxidation before complete degradation of propionate. Three key species for syntrophic acid degradation were inferred from genomic sequence information and gene expression: a syntrophic propionate-oxidizing bacterium (SPOB) "Candidatus Syntrophopropionicum ammoniitolerans", a syntrophic acetate-oxidizing bacterium (SAOB) Syntrophaceticus schinkii and a novel hydrogenotrophic methanogen, for which we propose the provisional name "Candidatus Methanoculleus ammoniitolerans". The results revealed consistent transcriptional profiles of the SAOB and the methanogen both during propionate and acetate oxidation, regardless of the presence of an active propionate oxidizer. Gene expression indicated versatile capabilities of the two syntrophic bacteria, utilizing both molecular hydrogen and formate as an outlet for reducing equivalents formed during acid oxidation, while conserving energy through build-up of sodium/proton motive force. The methanogen used hydrogen and formate as electron sources. Furthermore, results of the present study provided a framework for future research into ammonia tolerance, mobility, aggregate formation and interspecies cooperation.

DNA transfer between two different species mediated by heterologous cell fusion in Clostridium coculture.

Prokaryotic evolution is driven by random mutations and horizontal gene transfer (HGT). HGT occurs via transformation, transduction, or conjugation. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, heterologous cell fusion leads to a large-scale exchange of proteins and RNA between the two organisms. Here, we present evidence that heterologous cell fusion facilitates the exchange of DNA between the two organisms. Using selective subculturing, we isolated C. acetobutylicum cells which acquired and integrated into their genome portions of plasmid DNA from a plasmid-carrying C. ljungdahlii strain. Limiting-dilution plating and DNA methylation data based on PacBio Single-Molecule Real Time (SMRT) sequencing support the existence of hybrid C. acetobutylicum/C. ljungdahlii cells. These findings expand our understanding of multi-species microbiomes, their survival strategies, and evolution.IMPORTANCEInvestigations of natural multispecies microbiomes and synthetic microbial cocultures are attracting renewed interest for their potential application in biotechnology, ecology, and medical fields. Previously, we have shown the syntrophic coculture of C. acetobutylicum and C. ljungdahlii undergoes heterologous cell-to-cell fusion, which facilitates the exchange of cytoplasmic protein and RNA between the two organisms. We now show that heterologous cell fusion between the two Clostridium organisms can facilitate the exchange of DNA. By applying selective pressures to this coculture system, we isolated clones of wild-type C. acetobutylicum which acquired the erythromycin resistance (erm) gene from the C. ljungdahlii strain carrying a plasmid with the erm gene. Single-molecule real-time sequencing revealed that the erm gene was integrated into the genome in a mosaic fashion. Our data also support the persistence of hybrid C. acetobutylicum/C. ljungdahlii cells displaying hybrid DNA-methylation patterns.

Development of an internal two-stage upflow anaerobic reactor integrating biostimulation strategies to enhance the degradation of aromatic compounds in wastewater from purified terephthalic acid and dimethyl terephthalate manufacturing processes.

In this study, we aimed to establish high-rate biological treatment of purified terephthalic acid (PTA) and dimethyl terephthalate (DMT) wastewater that minimizes the inhibitory effects of high concentration benzoate and acetate. To achieve this, we developed a novel bioreactor system and biostimulation strategy. An internal two-stage upflow anaerobic (ITUA) reactor was operated with (i) a packed bed containing green tuff medium underlying (ii) a compartment seeded with anaerobic granular sludge. Ethylene glycol was amended to stimulate syntrophic interactions. Continuous operation of the system for 1,026 days achieve an organic removal rate of 11.0 ± 0.6 kg COD/m3/d. The abundance of aromatic degraders significantly increased during operation. Thus, we successfully developed a high-rate treatment system to treat wastewater from the PTA/DMT manufacturing processes by activating syntrophs in an ITUA reactor.

Disentangling a metabolic cross-feeding in a halophilic archaea-bacteria consortium.

Microbial syntrophy, a cooperative metabolic interaction among prokaryotes, serves a critical role in shaping communities, due to the auxotrophic nature of many microorganisms. Syntrophy played a key role in the evolution of life, including the hypothesized origin of eukaryotes. In a recent exploration of the microbial mats within the exceptional and uniquely extreme Cuatro Cienegas Basin (CCB), a halophilic isolate, designated as AD140, emerged as a standout due to its distinct growth pattern. Subsequent genome sequencing revealed AD140 to be a co-culture of a halophilic archaeon from the Halorubrum genus and a marine halophilic bacterium, Marinococcus luteus, both occupying the same ecological niche. This intriguing coexistence hints at an early-stage symbiotic relationship that thrives on adaptability. By delving into their metabolic interdependence through genomic analysis, this study aims to uncover shared characteristics that enhance their symbiotic association, offering insights into the evolution of halophilic microorganisms and their remarkable adaptations to high-salinity environments.