Percutaneous Coronary Intervention Versus Coronary Artery Bypass Grafting in Patients With Left Main Disease With and Without Diabetes: Findings From a Pooled Analysis of 4 Randomized Clinical Trials.

BACKGROUND: Diabetes may be associated with differential outcomes in patients undergoing left main coronary revascularization with percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG). The aim of this study was to investigate outcomes in patients with left main disease with and without diabetes randomized to PCI versus CABG. METHODS: Individual patient data were pooled from 4 trials (SYNTAX [Synergy Between PCI With Taxus and Cardiac Surgery], PRECOMBAT [Premier of Randomized Comparison of Bypass Surgery Versus Angioplasty Using Sirolimus-Eluting Stent in Patients With Left Main Coronary Artery Disease], NOBLE [Nordic-Baltic-British Left Main Revascularisation Study], and EXCEL [Evaluation of XIENCE Versus Coronary Artery Bypass Surgery for Effectiveness of Left Main Revascularization]) that randomized patients with left main disease to PCI or CABG. Patients were considered suitable for either approach. Patients were categorized by diabetes status. Kaplan-Meier event rates, Cox model hazard ratios, and interactions were assessed. RESULTS: Among 4393 patients, 1104 (25.1%) had diabetes. Patients with diabetes experienced higher rates of 5-year death (158/1104 [Kaplan-Meier rate, 14.7%] versus 297/3289 [9.3%]; P<0.001), spontaneous myocardial infarction (MI; 67/1104 [6.7%] versus 114/3289 [3.7%]; P<0.001), and repeat revascularization (189/1104 [18.5%] versus 410/3289 [13.2%]; P<0.001). Rates of all-cause mortality did not differ after PCI versus CABG in those with (84/563 [15.3%] versus 74/541 [14.1%]; hazard ratio, 1.11 [95% CI, 0.82-1.52]) or without (155/1634 [9.7%] versus 142/1655 [8.9%]; hazard ratio, 1.08 [95% CI, 0.86-1.36; PintHR=0.87) diabetes. Rates of stroke within 1 year were lower with PCI versus CABG in the entire population, with no heterogeneity based on diabetes status (PintHR=0.51). The 5-year rates of spontaneous MI and repeat coronary revascularization were higher after PCI regardless of diabetes status (spontaneous MI: 45/563 [8.9%] versus 22/541 [4.4%] in diabetes and 82/1634 [5.3%] versus 32/1655 [2.1%] in no diabetes, PintHR=0.47; repeat revascularization: 127/563 [24.5%] versus 62/541 [12.4%] in diabetes and 254/1634 [16.3%] versus 156/1655 [10.1%] in no diabetes, PintHR=0.18). For spontaneous MI and repeat revascularization, there were greater absolute risk differences beyond 1 year in patients with diabetes (4.9% and 9.9%) compared with those without (2.1% and 4.3%; PintARD=0.047 and 0.016). CONCLUSIONS: In patients with left main disease considered equally suitable for PCI or CABG and with largely low to intermediate SYNTAX scores, diabetes was associated with higher rates of death and cardiovascular events through 5 years. Compared with CABG, PCI resulted in no difference in the risk of death and a lower risk of early stroke regardless of diabetes status, and a higher risk of spontaneous MI and repeat coronary revascularization, with larger late absolute excess risks in patients with diabetes. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifiers: NCT01205776, NCT0146651, NCT00422968, and NCT00114972.

Amplicon-guided isolation and cultivation of previously uncultured microbial species from activated sludge.

IMPORTANCE: Biological wastewater treatment relies on complex microbial communities that assimilate nutrients and break down pollutants in the wastewater. Knowledge about the physiology and metabolism of bacteria in wastewater treatment plants (WWTPs) may therefore be used to improve the efficacy and economy of wastewater treatment. Our current knowledge is largely based on 16S rRNA gene amplicon profiling, fluorescence in situ hybridization studies, and predictions based on metagenome-assembled genomes. Bacterial isolates are often required to validate genome-based predictions as they allow researchers to analyze a specific species without interference from other bacteria and with simple bulk measurements. Unfortunately, there are currently very few pure cultures representing the microbes commonly found in WWTPs. To address this, we introduce an isolation strategy that takes advantage of state-of-the-art microbial profiling techniques to uncover suitable growth conditions for key WWTP microbes. We furthermore demonstrate that this information can be used to isolate key organisms representing global WWTPs.

Percutaneous coronary intervention with drug-eluting stents versus coronary artery bypass grafting in left main coronary artery disease: an individual patient data meta-analysis.

BACKGROUND: The optimal revascularisation strategy for patients with left main coronary artery disease is uncertain. We therefore aimed to evaluate long-term outcomes for patients treated with percutaneous coronary intervention (PCI) with drug-eluting stents versus coronary artery bypass grafting (CABG). METHODS: In this individual patient data meta-analysis, we searched MEDLINE, Embase, and the Cochrane database using the search terms "left main", "percutaneous coronary intervention" or "stent", and "coronary artery bypass graft*" to identify randomised controlled trials (RCTs) published in English between database inception and Aug 31, 2021, comparing PCI with drug-eluting stents with CABG in patients with left main coronary artery disease that had at least 5 years of patient follow-up for all-cause mortality. Two authors (MSS and BAB) identified studies meeting the criteria. The primary endpoint was 5-year all-cause mortality. Secondary endpoints were cardiovascular death, spontaneous myocardial infarction, procedural myocardial infarction, stroke, and repeat revascularisation. We used a one-stage approach; event rates were calculated by use of the Kaplan-Meier method and treatment group comparisons were made by use of a Cox frailty model, with trial as a random effect. In Bayesian analyses, the probabilities of absolute risk differences in the primary endpoint between PCI and CABG being more than 0·0%, and at least 1·0%, 2·5%, or 5·0%, were calculated. FINDINGS: Our literature search yielded 1599 results, of which four RCTs-SYNTAX, PRECOMBAT, NOBLE, and EXCEL-meeting our inclusion criteria were included in our meta-analysis. 4394 patients, with a median SYNTAX score of 25·0 (IQR 18·0-31·0), were randomly assigned to PCI (n=2197) or CABG (n=2197). The Kaplan-Meier estimate of 5-year all-cause death was 11·2% (95% CI 9·9-12·6) with PCI and 10·2% (9·0-11·6) with CABG (hazard ratio 1·10, 95% CI 0·91-1·32; p=0·33), resulting in a non-statistically significant absolute risk difference of 0·9% (95% CI -0·9 to 2·8). In Bayesian analyses, there was an 85·7% probability that death at 5 years was greater with PCI than with CABG; this difference was more likely than not less than 1·0% (<0·2% per year). The numerical difference in mortality was comprised more of non-cardiovascular than cardiovascular death. Spontaneous myocardial infarction (6·2%, 95% CI 5·2-7·3 vs 2·6%, 2·0-3·4; hazard ratio [HR] 2·35, 95% CI 1·71-3·23; p<0·0001) and repeat revascularisation (18·3%, 16·7-20·0 vs 10·7%, 9·4-12·1; HR 1·78, 1·51-2·10; p<0·0001) were more common with PCI than with CABG. Differences in procedural myocardial infarction between strategies depended on the definition used. Overall, there was no difference in the risk of stroke between PCI (2·7%, 2·0-3·5) and CABG (3·1%, 2·4-3·9; HR 0·84, 0·59-1·21; p=0·36), but the risk was lower with PCI in the first year after randomisation (HR 0·37, 0·19-0·69). INTERPRETATION: Among patients with left main coronary artery disease and, largely, low or intermediate coronary anatomical complexity, there was no statistically significant difference in 5-year all-cause death between PCI and CABG, although a Bayesian approach suggested a difference probably exists (more likely than not <0·2% per year) favouring CABG. There were trade-offs in terms of the risk of myocardial infarction, stroke, and revascularisation. A heart team approach to communicate expected outcome differences might be useful to assist patients in reaching a treatment decision. FUNDING: No external funding.

On the evolution and physiology of cable bacteria.

Cable bacteria of the family Desulfobulbaceae form centimeter-long filaments comprising thousands of cells. They occur worldwide in the surface of aquatic sediments, where they connect sulfide oxidation with oxygen or nitrate reduction via long-distance electron transport. In the absence of pure cultures, we used single-filament genomics and metagenomics to retrieve draft genomes of 3 marine Candidatus Electrothrix and 1 freshwater Ca. Electronema species. These genomes contain >50% unknown genes but still share their core genomic makeup with sulfate-reducing and sulfur-disproportionating Desulfobulbaceae, with few core genes lost and 212 unique genes (from 197 gene families) conserved among cable bacteria. Last common ancestor analysis indicates gene divergence and lateral gene transfer as equally important origins of these unique genes. With support from metaproteomics of a Ca. Electronema enrichment, the genomes suggest that cable bacteria oxidize sulfide by reversing the canonical sulfate reduction pathway and fix CO2 using the Wood-Ljungdahl pathway. Cable bacteria show limited organotrophic potential, may assimilate smaller organic acids and alcohols, fix N2, and synthesize polyphosphates and polyglucose as storage compounds; several of these traits were confirmed by cell-level experimental analyses. We propose a model for electron flow from sulfide to oxygen that involves periplasmic cytochromes, yet-unidentified conductive periplasmic fibers, and periplasmic oxygen reduction. This model proposes that an active cable bacterium gains energy in the anodic, sulfide-oxidizing cells, whereas cells in the oxic zone flare off electrons through intense cathodic oxygen respiration without energy conservation; this peculiar form of multicellularity seems unparalleled in the microbial world.

Percutaneous coronary angioplasty versus coronary artery bypass grafting in the treatment of unprotected left main stenosis: updated 5-year outcomes from the randomised, non-inferiority NOBLE trial.

BACKGROUND: Percutaneous coronary intervention (PCI) is increasingly used in revascularisation of patients with left main coronary artery disease in place of the standard treatment, coronary artery bypass grafting (CABG). The NOBLE trial aimed to evaluate whether PCI was non-inferior to CABG in the treatment of left main coronary artery disease and reported outcomes after a median follow-up of 3·1 years. We now report updated 5-year outcomes of the trial. METHODS: The prospective, randomised, open-label, non-inferiority NOBLE trial was done at 36 hospitals in nine northern European countries. Patients with left main coronary artery disease requiring revascularisation were enrolled and randomly assigned (1:1) to receive PCI or CABG. The primary endpoint was major adverse cardiac or cerebrovascular events (MACCE), a composite of all-cause mortality, non-procedural myocardial infarction, repeat revascularisation, and stroke. Non-inferiority of PCI to CABG was defined as the upper limit of the 95% CI of the hazard ratio (HR) not exceeding 1·35 after 275 MACCE had occurred. Secondary endpoints included all-cause mortality, non-procedural myocardial infarction, and repeat revascularisation. Outcomes were analysed in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT01496651. FINDINGS: Between Dec 9, 2008, and Jan 21, 2015, 1201 patients were enrolled and allocated to PCI (n=598) or CABG (n=603), with 17 subsequently lost to early follow-up. 592 patients in each group were included in this analysis. At a median of 4·9 years of follow-up, the predefined number of events was reached for adequate power to assess the primary endpoint. Kaplan-Meier 5-year estimates of MACCE were 28% (165 events) for PCI and 19% (110 events) for CABG (HR 1·58 [95% CI 1·24-2·01]); the HR exceeded the limit for non-inferiority of PCI compared to CABG. CABG was found to be superior to PCI for the primary composite endpoint (p=0·0002). All-cause mortality was estimated in 9% after PCI versus 9% after CABG (HR 1·08 [95% CI 0·74-1·59]; p=0·68); non-procedural myocardial infarction was estimated in 8% after PCI versus 3% after CABG (HR 2·99 [95% CI 1·66-5·39]; p=0·0002); and repeat revascularisation was estimated in 17% after PCI versus 10% after CABG (HR 1·73 [95% CI 1·25-2·40]; p=0·0009). INTERPRETATION: In revascularisation of left main coronary artery disease, PCI was associated with an inferior clinical outcome at 5 years compared with CABG. Mortality was similar after the two procedures but patients treated with PCI had higher rates of non-procedural myocardial infarction and repeat revascularisation. FUNDING: Biosensors.

Complete nitrification by a single microorganism.

Nitrification is a two-step process where ammonia is first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite-oxidizing bacteria. Already described by Winogradsky in 1890, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle. Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible, and it was postulated that this process could occur under conditions selecting for species with lower growth rates but higher growth yields than canonical ammonia-oxidizing microorganisms. Still, organisms catalysing this process have not yet been discovered. Here we report the enrichment and initial characterization of two Nitrospira species that encode all the enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding of the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.

Complete nitrification by Nitrospira bacteria.

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.

Metabolic Traits of Candidatus Accumulibacter clade IIF Strain SCELSE-1 Using Amino Acids As Carbon Sources for Enhanced Biological Phosphorus Removal.

Despite recent evidence from full-scale plants suggesting that Candidatus Accumulibacter may be capable of using amino acids, this metabolic trait has never been confirmed in a bioreactor experiment. Here we show that an enriched culture of Ca. Accumulibacter clade IIF strain SCELSE-1 could metabolize 11 of 20 α-amino acids, with aspartate, glutamate, asparagine, and glutamine resulting in the highest phosphorus removal. The anaerobic uptake of aspartate and glutamate was achieved through a glutamate/aspartate-proton symporter fully powered by the proton motive force (PMF). Under anaerobic conditions aspartate was deaminized and routed into core carbon metabolic pathways to form polyhydroxyalkanoates (PHA). The lack of genes encoding NADH dependent isocitrate dehydrogenase in the Ca. Accumulibacter genome resulted in a kinetic barrier for glutamate to be channelled to the TCA cycle. Glutamate was stored as glutamate polymer. When amino acids (aspartate or glutamate) and acetate were supplied together, Ca. Accumulibacter took up both carbon sources simultaneously, with the uptake rate of each carbon source largely preserved. Overall energy savings (up to 17%) were achieved under mixed carbon scenarios, due to the ability of Ca. Accumulibacter to rearrange its anaerobic carbon metabolism based on the reducing power, PMF and ATP balance.

Denitrification activity of polyphosphate accumulating organisms (PAOs) in full-scale wastewater treatment plants.

A comprehensive assessment of full-scale enhanced biological phosphorus removal (EBPR) plants (five plants, 19 independent tests) was undertaken to determine their effectiveness in terms of aerobic and anoxic P removal. By comparing parallel P uptake tests under only aerobic or under anoxic-aerobic conditions, results revealed that introducing an anoxic stage led to an overall P removal of on average 90% of the P removed under only aerobic conditions. This was achieved with negligible higher PHA and glycogen requirements, 30% lower overall oxygen consumption and with the simultaneous removal of nitrate, reducing up to an estimate of 70% of carbon requirements for simultaneous N and P removal. Varying fractions of denitrifying polyphosphate accumulating organisms (DPAOs), from an average of 25% to 84%, were found in different plants. No correlation was found between the DPAO fractions and EBPR configuration, season, or the concentration of any of the microbial groups measured via quantitative fluorescence in situ hybridisation. These included Type I and Type II Ca. Accumulibacter and glycogen accumulating organisms, suggesting that chemical batch tests are the best methodology for quantifying the potential of anoxic P removal in full-scale wastewater treatment plants.

Percutaneous coronary angioplasty versus coronary artery bypass grafting in treatment of unprotected left main stenosis (NOBLE): a prospective, randomised, open-label, non-inferiority trial.

BACKGROUND: Coronary artery bypass grafting (CABG) is the standard treatment for revascularisation in patients with left main coronary artery disease, but use of percutaneous coronary intervention (PCI) for this indication is increasing. We aimed to compare PCI and CABG for treatment of left main coronary artery disease. METHODS: In this prospective, randomised, open-label, non-inferiority trial, patients with left main coronary artery disease were enrolled in 36 centres in northern Europe and randomised 1:1 to treatment with PCI or CABG. Eligible patients had stable angina pectoris, unstable angina pectoris, or non-ST-elevation myocardial infarction. Exclusion criteria were ST-elevation myocardial infarction within 24 h, being considered too high risk for CABG or PCI, or expected survival of less than 1 year. The primary endpoint was major adverse cardiac or cerebrovascular events (MACCE), a composite of all-cause mortality, non-procedural myocardial infarction, any repeat coronary revascularisation, and stroke. Non-inferiority of PCI to CABG required the lower end of the 95% CI not to exceed a hazard ratio (HR) of 1·35 after up to 5 years of follow-up. The intention-to-treat principle was used in the analysis if not specified otherwise. This trial is registered with ClinicalTrials.gov identifier, number NCT01496651. FINDINGS: Between Dec 9, 2008, and Jan 21, 2015, 1201 patients were randomly assigned, 598 to PCI and 603 to CABG, and 592 in each group entered analysis by intention to treat. Kaplan-Meier 5 year estimates of MACCE were 29% for PCI (121 events) and 19% for CABG (81 events), HR 1·48 (95% CI 1·11-1·96), exceeding the limit for non-inferiority, and CABG was significantly better than PCI (p=0·0066). As-treated estimates were 28% versus 19% (1·55, 1·18-2·04, p=0·0015). Comparing PCI with CABG, 5 year estimates were 12% versus 9% (1·07, 0·67-1·72, p=0·77) for all-cause mortality, 7% versus 2% (2·88, 1·40-5·90, p=0·0040) for non-procedural myocardial infarction, 16% versus 10% (1·50, 1·04-2·17, p=0·032) for any revascularisation, and 5% versus 2% (2·25, 0·93-5·48, p=0·073) for stroke. INTERPRETATION: The findings of this study suggest that CABG might be better than PCI for treatment of left main stem coronary artery disease. FUNDING: Biosensors, Aarhus University Hospital, and participating sites.

Impact of sludge retention time on the fine composition of the microbial community and extracellular polymeric substances in a membrane bioreactor.

Membrane bioreactors (MBRs) are an advanced technology for wastewater treatment whose wide application has been hindered by rapid fouling of the membranes. MBRs can be operated with long sludge retention time (SRT), a crucial parameter impacting microbial selection in the reactor. This also affects filtration performance, since a major fouling agent are the extracellular polymeric substances (EPS). In this study, the impact of the SRT on the ecophysiology of the MBRs and, consequently, on membrane fouling was evaluated. A MBR was operated under a SRT of 60 days followed by a SRT of 20 days. A comprehensive analysis of the microbial community structure and EPS proteins and polysaccharide profiles of the mixed liquor and cake layer was carried out throughout both operation periods. The results of this study showed that the imposition of a shorter SRT led to a shift in the dominant bacterial populations. The mixed liquor and cake layer communities were very different, with Actinomycetales order standing out in the cake layer at SRT of 20 days. Overall, higher EPS concentrations (particularly proteins) were found at this SRT. Furthermore, EPS profiles were clearly affected by the SRT: it was possible to correlate a group of soluble EPS proteins with the SRT of 60 days, and a lower sludge age led to a lower diversity of polysaccharide sugar monomers, with an increase of glucose and galactose in the cake layer. This study improves our knowledge regarding the molecular reasons for fouling, which may contribute to improve MBR design and operation.

Major proteomic changes associated with amyloid-induced biofilm formation in Pseudomonas aeruginosa PAO1.

The newly identified functional amyloids in Pseudomonas (Fap) are associated with increased aggregation and biofilm formation in the opportunistic pathogen P. aeruginosa; however, whether this phenomenon can be simply ascribed to the mechanical properties of the amyloid fibrils remains undetermined. To gain a deeper understanding of the Fap-mediated biofilm formation, the physiological consequences of Fap expression were investigated using label-free protein quantification. The functional amyloids were found to not solely act as inert structural biofilm components. Their presence induced major changes in the global proteome of the bacterium. These included the lowered abundance of classical virulence factors such as elastase B and the secretion system of alkaline protease A. Amyloid-mediated biofilm formation furthermore increased abundance of the alginate and pyoverdine synthesis machinery, which turned P. aeruginosa PAO1 into an unexpected mucoid phenotype. The results imply a significant impact of functional amyloids on the physiology of P. aeruginosa with subsequent implications for biofilm formation and chronic infections.

Low temperature partial nitritation/anammox in a moving bed biofilm reactor treating low strength wastewater.

Municipal wastewater collected in areas with moderate climate is subjected to a gradual temperature decrease from around 20 °C in summer to about 10 °C in winter. A lab-scale moving bed biofilm reactor (MBBR) with carrier material (K3 from AnoxKaldnes) was used to test the tolerance of the overall partial nitritation/anammox process to this temperature gradient. A synthetic influent, containing only ammonium and no organic carbon was used to minimize denitrification effects. After stable reactor operation at 20 °C, the temperature was slowly reduced by 2 °C per month and afterward held constant at 10 °C. Along the temperature decrease, the ammonium conversion dropped from an average of 40 gN m(-3) d(-1) (0.2 gN kgTSS h(-1)) at 20 °C to about 15 gN m(-3) d(-1) (0.07 gN kg TSS h(-1)) at 10 °C, while the effluent concentration was kept <8 mg NH4-N l(-1) during the whole operation. This also resulted in doubling of the hydraulic retention time over the temperature ramp. The MBBR with its biofilm on 10 mm thick carriers proved to sufficiently sustain enough biomass to allow anammox activity even at 10 °C. Even though there was a minor nitrite-build up when the temperature dropped below 12.5 °C, reactor performance recovered as the temperature decrease continued. Microbial community analysis by 16S rRNA amplicon analysis revealed a relatively stable community composition over the entire experimental period.

A non-methanogenic archaeon within the order Methanocellales.

Serpentinization, a geochemical process found on modern and ancient Earth, provides an ultra-reducing environment that can support microbial methanogenesis and acetogenesis. Several groups of archaea, such as the order Methanocellales, are characterized by their ability to produce methane. Here, we generate metagenomic sequences from serpentinized springs in The Cedars, California, and construct a circularized metagenome-assembled genome of a Methanocellales archaeon, termed Met12, that lacks essential methanogenesis genes. The genome includes genes for an acetyl-CoA pathway, but lacks genes encoding methanogenesis enzymes such as methyl-coenzyme M reductase, heterodisulfide reductases and hydrogenases. In situ transcriptomic analyses reveal high expression of a multi-heme c-type cytochrome, and heterologous expression of this protein in a model bacterium demonstrates that it is capable of accepting electrons. Our results suggest that Met12, within the order Methanocellales, is not a methanogen but a CO2-reducing, electron-fueled acetogen without electron bifurcation.

Metagenomes obtained by 'deep sequencing' - what do they tell about the enhanced biological phosphorus removal communities?

Metagenomics enables studies of the genomic potential of complex microbial communities by sequencing bulk genomic DNA directly from the environment. Knowledge of the genetic potential of a community can be used to formulate and test ecological hypotheses about stability and performance. In this study deep metagenomics and fluorescence in situ hybridization (FISH) were used to study a full-scale wastewater treatment plant with enhanced biological phosphorus removal (EBPR), and the results were compared to an existing EBPR metagenome. EBPR is a widely used process that relies on a complex community of microorganisms to function properly. Insight into community and species level stability and dynamics is valuable for knowledge-driven optimization of the EBPR process. The metagenomes of the EBPR communities were distinct compared to metagenomes of communities from a wide range of other environments, which could be attributed to selection pressures of the EBPR process. The metabolic potential of one of the key microorganisms in the EPBR process, Accumulibacter, was investigated in more detail in the two plants, revealing a potential importance of phage predation on the dynamics of Accumulibacter populations. The results demonstrate that metagenomics can be used as a powerful tool for system wide characterization of the EBPR community as well as for a deeper understanding of the function of specific community members. Furthermore, we discuss and illustrate some of the general pitfalls in metagenomics and stress the need of additional DNA extraction independent information in metagenome studies.

Modelling the impacts of operational conditions on the performance of a full-scale membrane aerated biofilm reactor.

Membrane Aerated Biofilm Reactors (MABR) are gaining more and more acceptance in the plethora of wastewater process intensification technologies. Mathematical modelling has contributed to show their feasibility in terms of reduced energy consumption and footprint. Nevertheless, most simulation studies published until now are still focused on analyzing MABR as single units and not fully integrated within the flow diagram of the water treatment plant (WWTP). In this paper, the prediction capabilities of an integrated modelling approach is tested using full-scale data from Ejby Mølle WWTP+MABR site (Odense, Denmark). Mass balances, data reconciliation methods, process simulation and the different evaluation criteria were used to adjust influent, effluent and process indicators. Results show 10 % mismatch between flow, COD, N and P predictions and measurements in different plant locations. Using the adopted hydraulic retention time (HRT), nitrogen load (NL), membrane surface area (MA) and oxygen transfer rate (OTR), it was possible to predict nitrification rates (NR) within the interquartile range. This has been done under two different MABR operational conditions: with (#S2) and without (#S1) external aeration (EA) in the bulk liquid. The model provides additional process insights about biofilm structure, substrate gradients, weak acid base chemistry and precipitation potential. More specifically, simulations suggest the potential undesirable effects of sulfate (SRB) and iron reducing bacteria (IRB) on both microbial activity and composition of the biofilm. The latter may have a strong impact on ammonium (NHx), sulfate (SOx) and ferrous ion (Fe+2) conversion processes. The change of operational strategy in the scenario analysis highlights that the denitrifying activity of phosphorus accumulating organisms (PAOs) can enhance nutrient removal in MABR tanks. In addition, it was possible to assess the chance of success (in terms of energetic cost of nitrogen removal) of adding several MABR units in one tank of the WWTP under study before full-scale implementation.

A comprehensive overview of the Chloroflexota community in wastewater treatment plants worldwide.

IMPORTANCE: Chloroflexota are often abundant members of the biomass in wastewater treatment plants (WWTPs) worldwide, typically with a filamentous morphology, forming the backbones of the activated sludge floc. However, their overgrowth can often cause operational issues connected to poor settling or foaming, impairing effluent quality and increasing operational costs. Despite their importance, few Chloroflexota genera have been characterized so far. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide and an in-depth characterization of their morphology, phylogeny, and ecophysiology, obtaining a broad understanding of their ecological role in activated sludge.

The biofilm matrix: multitasking in a shared space.

The biofilm matrix can be considered to be a shared space for the encased microbial cells, comprising a wide variety of extracellular polymeric substances (EPS), such as polysaccharides, proteins, amyloids, lipids and extracellular DNA (eDNA), as well as membrane vesicles and humic-like microbially derived refractory substances. EPS are dynamic in space and time and their components interact in complex ways, fulfilling various functions: to stabilize the matrix, acquire nutrients, retain and protect eDNA or exoenzymes, or offer sorption sites for ions and hydrophobic substances. The retention of exoenzymes effectively renders the biofilm matrix an external digestion system influencing the global turnover of biopolymers, considering the ubiquitous relevance of biofilms. Physico-chemical and biological interactions and environmental conditions enable biofilm systems to morph into films, microcolonies and macrocolonies, films, ridges, ripples, columns, pellicles, bubbles, mushrooms and suspended aggregates - in response to the very diverse conditions confronting a particular biofilm community. Assembly and dynamics of the matrix are mostly coordinated by secondary messengers, signalling molecules or small RNAs, in both medically relevant and environmental biofilms. Fully deciphering how bacteria provide structure to the matrix, and thus facilitate and benefit from extracellular reactions, remains the challenge for future biofilm research.

CRISPR-Cas phage defense systems and prophages in Candidatus Accumulibacter.

Candidatus Accumulibacter plays a major role in enhanced biological phosphorus removal (EBPR) from wastewater. Although bacteriophages have been shown to represent fatal threats to Ca. Accumulibacter organisms and thus interfere with the stability of the EBPR process, little is known about the ability of different Ca. Accumulibacter strains to resist phage infections. We conducted a systematic analysis of the occurrence and characteristics of clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) systems and prophages in Ca. Accumulibacter lineage members (43 in total, including 10 newly recovered genomes). Results indicate that 28 Ca. Accumulibacter genomes encode CRISPR-Cas systems. They were likely acquired via horizontal gene transfer, conveying a distinct adaptivity to phage predation to different Ca. Accumulibacter members. Major differences in the number of spacers show the unique phage resistance of these members. A comparison of the spacers in closely related Ca. Accumulibacter members from distinct geographical locations indicates that habitat isolation may have resulted in the acquisition of resistance to different phages by different Ca. Accumulibacter. Long-term operation of three laboratory-scale EBPR bioreactors revealed high relative abundances of Ca. Accumulibacter with CRISPSR-Cas systems. Their specific resistance to phages in these reactors was indicated by spacer analysis. Metatranscriptomic analyses showed the activation of the CRISPR-Cas system under both anaerobic and aerobic conditions. Additionally, 133 prophage regions were identified in 43 Ca. Accumulibacter genomes. Twenty-seven of them (in 19 genomes) were potentially active. Major differences in the occurrence of CRISPR-Cas systems and prophages in Ca. Accumulibacter will lead to distinct responses to phage predation. This study represents the first systematic analysis of CRISPR-Cas systems and prophages in the Ca. Accumulibacter lineage, providing new perspectives on the potential impacts of phages on Ca. Accumulibacter and EBPR systems.