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.

Primary settling changes the microbial community of influent wastewater to wastewater treatment plants.

The continuous immigration of bacteria in influent wastewater strongly impacts the microbial community of activated sludge (AS) in wastewater treatment plants (WWTP), both in terms of species composition and their abundance. Therefore, it is of interest to elucidate the route of immigrating bacteria into the biological tanks, including the effect of primary settlers. These are commonly used pretreatment units that can possibly selectively increase or reduce the relative abundance of certain bacteria. Species-level identification of the microbial composition of influent wastewater before and after primary settling was carried out in four full-scale municipal WWTPs biweekly over one year by 16S rRNA gene amplicon sequencing. Overall, 37-49% of incoming COD was removed in the primary settlers. Most genera and species were present in the wastewater to all four plants and the trend of these were investigated across the primary settlers. Approximately 50% of the genera had the same trend across at least three WWTPs. Few genera significantly increased in relative read abundance (3.7%) after settling, while 22.3% showed a significant reduction in relative abundance. We investigated process-critical species in AS, such as known nitrifiers, polyphosphate-accumulating organisms, and filamentous bacteria. Most taxa were affected similarly in all WWTPs including multiple genera involved in bulking in AS. However, some genera, e.g., important polyphosphate-accumulating bacteria, had inconsistent trends across WWTPs, suggesting that the characteristics of the wastewater are important for the trend of some bacteria through primary settling. In all cases, primary settling changed the microbial community of the influent wastewater, posing an obvious candidate for upstream control to optimize the assembly of the microbial communities in activated sludge.

Full-scale activated sludge transplantation reveals a highly resilient community structure.

Well-functioning and stable microbial communities are critical for the operation of activated sludge (AS) wastewater treatment plants (WWTPs). Bioaugmentation represents a potentially useful approach to recover deteriorated systems or to support specific AS processes, but its application in full-scale WWTPs is generally problematic. We conducted a massive transplantation (in one day) exchanging AS from a donor to a recipient full-scale WWTP with similar process type (biological removal of nitrogen and phosphorus) and performance, but with differences in microbial community structure. The treatment performance in the recipient plant was not compromised and the effluent quality remained stable. The AS community structure of the recipient plant was initially very similar to the donor AS, but it almost completely restored the pre-transplantation structure approximately 40 days after transplantation, corresponding to 3 times the solid retention time. Most of the unique species of donor AS added to recipient AS disappeared quickly, although some disappeared more slowly the following months, indicating some survival and potentially a time limited function in the recipient plant. Moreover, the addition in higher abundance of most species already present in the recipient AS (e.g., the polyphosphate accumulating organisms) or the reduction of the abundance of unwanted bacteria (e.g., filamentous bacteria) in the recipient plant was not successful. Moreover, we observed similar abundance patterns after transplantation for species belonging to different functional guilds, so we did not observe an increase of the functional redundancy. Investigations of the microbial community structure in influent wastewater revealed that for some species the abundance trends in the recipient plant were closely correlated to their abundance in the influent. We showed that a very resilient microbial community was responsible for the outcome of the transplantation of AS at full-scale WWTP, potentially as a consequence of mass-immigration from influent wastewater. The overall results imply that massive transplantation of AS across different WWTPs is not a promising strategy to permanently solve operational problems. However, by choosing a compatible AS donor, short term mitigation of serious operational problems may be possible.

Diversity and Ecophysiology of the Genus OLB8 and Other Abundant Uncultured Saprospiraceae Genera in Global Wastewater Treatment Systems.

The Saprospiraceae family within the phylum Bacteroidota is commonly present and highly abundant in wastewater treatment plants (WWTPs) worldwide, but little is known about its role. In this study, we used MiDAS 4 global survey with samples from 30 countries to analyze the abundance and distribution of members of Saprospiraceae. Phylogenomics were used to delineate five new genera from a set of 31 high-quality metagenome-assembled genomes from Danish WWTPs. Newly designed probes for fluorescence in situ hybridization (FISH) revealed rod-shaped morphologies for all genera analyzed, including OLB8, present mostly inside the activated sludge flocs. The genomes revealed potential metabolic capabilities for the degradation of polysaccharides, proteins, and other complex molecules; partial denitrification; and storage of intracellular polymers (glycogen, polyphosphate, and polyhydroxyalkanoates). FISH in combination with Raman microspectroscopy confirmed the presence of intracellular glycogen in Candidatus Brachybacter, Candidatus Parvibacillus calidus (both from the former genus OLB8), and Candidatus Opimibacter, and the presence of polyhydroxyalkanoates in Candidatus Defluviibacterium haderslevense and Candidatus Vicinibacter. These results provide the first overview of the most abundant novel Saprospiraceae genera present in WWTPs across the world and their potential involvement in nutrient removal and the degradation of macromolecules.

Seasonal microbial community dynamics complicates the evaluation of filamentous bulking mitigation strategies in full-scale WRRFs.

The activated sludge wastewater treatment process has been thoroughly researched in more than 100 years, yet there are still operational challenges that have not been fully resolved. Such a challenge is the control of filamentous bulking caused by the overgrowth of certain filamentous bacteria. In this study, we tested different mitigation strategies to reduce filamentous bulking, caused by two common filamentous genera found in full-scale water resource recovery facilities (WRRF), Candidatus Microthrix and Candidatus Amarolinea. PAX dosing, ozone addition, hydrocyclone implementation, and the addition of nano-coagulants were tested as mitigation strategies in four parallel treatment lines in a full-scale WRRF over three consecutive years. Unexpectedly, the activated sludge settleability was not affected by any of the mitigation strategies. Some of the strategies appeared to have a strong mitigating effect on the two filamentous species. However, detailed analyses of the microbial communities revealed strong recurrent seasonal variations in all four lines, including the control line which masked the real effect. After removing the effect of the seasonal variation by using a time-series decomposition approach, it was clear that the filamentous bacteria were mostly unaffected by the mitigation strategies. Only PAX dosing had some effect on Ca. Microthrix, but only on one species, Ca. Microthrix subdominans, and not on the most common Ca. Microthrix parvicella. Overall, our study shows the importance of long-term monitoring of microbial communities at species level to understand the normal seasonal pattern to effectively plan and execute full-scale experiments. Moreover, the results highlight the importance of using parallel reference treatment lines when evaluating the effect of mitigation strategies in full-scale treatment plants.

MiDAS 4: A global catalogue of full-length 16S rRNA gene sequences and taxonomy for studies of bacterial communities in wastewater treatment plants.

Microbial communities are responsible for biological wastewater treatment, but our knowledge of their diversity and function is still poor. Here, we sequence more than 5 million high-quality, full-length 16S rRNA gene sequences from 740 wastewater treatment plants (WWTPs) across the world and use the sequences to construct the 'MiDAS 4' database. MiDAS 4 is an amplicon sequence variant resolved, full-length 16S rRNA gene reference database with a comprehensive taxonomy from domain to species level for all sequences. We use an independent dataset (269 WWTPs) to show that MiDAS 4, compared to commonly used universal reference databases, provides a better coverage for WWTP bacteria and an improved rate of genus and species level classification. Taking advantage of MiDAS 4, we carry out an amplicon-based, global-scale microbial community profiling of activated sludge plants using two common sets of primers targeting regions of the 16S rRNA gene, revealing how environmental conditions and biogeography shape the activated sludge microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 966 genera and 1530 species that represent approximately 80% and 50% of the accumulated read abundance, respectively. Finally, we show that for well-studied functional guilds, such as nitrifiers or polyphosphate-accumulating organisms, the same genera are prevalent worldwide, with only a few abundant species in each genus.

Quantification of Biologically and Chemically Bound Phosphorus in Activated Sludge from Full-Scale Plants with Biological P-Removal.

Phosphorus (P) is present in activated sludge from wastewater treatment plants in the form of metal salt precipitates, extracellular polymeric substances, or bound into the biomass, for example, as intracellular polyphosphate (poly-P). Several methods for a reliable quantification of the different P-fractions have recently been developed, and this study combines them to obtain a comprehensive P mass-balance of activated sludge from four enhanced biological phosphate removal (EBPR) plants. Chemical characterization by ICP-OES and sequential P fractionation showed that chemically bound P constituted 38-69% of total P, most likely in the form of Fe, Mg, or Al minerals. Raman microspectroscopy, solution state 31P NMR, and 31P MAS NMR spectroscopy applied before and after anaerobic P-release experiments, were used to quantify poly-P, which constituted 22-54% of total P and was found in approximately 25% of all bacterial cells. Raman microspectroscopy in combination with fluorescence in situ hybridization was used to quantify poly-P in known polyphosphate-accumulating organisms (PAO) (Tetrasphaera, Candidatus Accumulibacter, and Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. Interestingly, only 1-13% of total P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are known.

Microbial communities across activated sludge plants show recurring species-level seasonal patterns.

Microbial communities in activated sludge (AS) are the core of sanitation in wastewater treatment plants (WWTPs). Microbial communities in AS have shown seasonal changes, however, long-term experiments (>2 years) are rarely conducted, limiting our understanding of the true seasonal dynamics in WWTPs. In this study, we resolved the microbial seasonal dynamics at the species level in four municipal full-scale WWTPs, sampled every 7-10 days, during 3-5 consecutive years. By applying a new time-series analysis approach, we revealed that the seasonal pattern was species-specific, where species belonging to the same functional guild or genus may show different seasonal dynamics. Species could be grouped into cohorts according to their seasonal patterns, where seasonal cohorts showed repeatable annual dynamics across years and plants. Species were also grouped according to their net growth rate in the AS (i.e., growing species and disappearing species). Growing species were more prevailing in spring and autumn cohorts, while disappearing species, which were only present due to the continuous immigration from influent wastewater, were mostly associated with winter and spring cohorts. Most known process-critical species, such as nitrifiers, polyphosphate accumulating organisms and filamentous organisms, showed distinct species-specific patterns. Overall, our study showed that overarching seasonal patterns affected microbial species in full-scale AS plants, with similar seasonal patterns across plants for many dominant species. These recurrent seasonal variations should be taken into account in the operation, understanding and management of the WWTPs.

Low Global Diversity of Candidatus Microthrix, a Troublesome Filamentous Organism in Full-Scale WWTPs.

Candidatus Microthrix is one of the most common bulking filamentous microorganisms found in activated sludge wastewater treatment plants (WWTPs) across the globe. One species, Ca. M. parvicella, is frequently observed, but global genus diversity, as well as important aspects of its ecology and physiology, are still unknown. Here, we use the MiDAS ecosystem-specific 16S rRNA gene database in combination with amplicon sequencing of Danish and global WWTPs to investigate Ca. Microthrix spp. diversity, distribution, and factors affecting their global presence. Only two species were abundant across the world confirming low diversity of the genus: the dominant Ca. M. parvicella and an unknown species typically present along with Ca. M. parvicella, although usually in lower abundances. Both species were mostly found in Europe at low-to-moderate temperatures and their growth was favored in municipal WWTPs with advanced process designs. As no isolate is available for the novel species, we propose the name "Candidatus Microthrix subdominans." Ten high-quality metagenome-assembled genomes recovered from Danish WWTPs, including 6 representing the novel Ca. M. subdominans, demonstrated high genetic similarity between the two species with a likely preference for lipids, a putative capability to reduce nitrate and nitrite, and the potential to store lipids and poly-P. Ca. M. subdominans had a potentially more versatile metabolism including additional sugar transporters, higher oxygen tolerance, and the potential to use carbon monoxide as energy source. Newly designed fluorescence in situ hybridization probes revealed similar filamentous morphology for both species. Raman microspectroscopy was used to quantify the in situ levels of intracellular poly-P. Despite the observed similarities in their physiology (both by genomes and in situ), the two species showed different seasonal dynamics in Danish WWTPs through a 13-years survey, possibly indicating occupation of slightly different niches. The genomic information provides the basis for future research into in situ gene expression and regulation, while the new FISH probes provide a useful tool for further characterization in situ. This study is an important step toward understanding the ecology of Ca. Microthrix in WWTPs, which may eventually lead to optimization of control strategies for its growth in this ecosystem.

Mass-immigration determines the assembly of activated sludge microbial communities.

The assembly of bacterial communities in wastewater treatment plants (WWTPs) is affected by immigration via wastewater streams, but the impact and extent of bacterial immigrants are still unknown. Here, we quantify the effect of immigration at the species level in 11 Danish full-scale activated sludge (AS) plants. All plants have different source communities but have very similar process design, defining the same overall environmental growth conditions. The AS community composition in each plant was strongly reflected by the corresponding influent wastewater (IWW) microbial composition. Most species in AS across the plants were detected and quantified in the corresponding IWW, allowing us to identify their fate in the AS: growing, disappearing, or surviving. Most of the abundant species in IWW disappeared in AS, so their presence in the AS biomass was only due to continuous mass-immigration. In AS, most of the abundant growing species were present in the IWW at very low abundances. We predicted the AS species abundances from their abundance in IWW by using a partial least square regression model. Some species in AS were predicted by their own abundance in IWW, while others by multiple species abundances. Detailed analyses of functional guilds revealed different prediction patterns for different species. We show, in contrast to the present understanding, that the AS microbial communities were strongly controlled by the IWW source community and could be quantitatively predicted by taking into account immigration. This highlights a need to revise the way we understand, design, and manage the microbial communities in WWTPs.

"Candidatus Dechloromonas phosphoritropha" and "Ca. D. phosphorivorans", novel polyphosphate accumulating organisms abundant in wastewater treatment systems.

Members of the genus Dechloromonas are often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) to investigate the abundance and distribution of Dechloromonas spp. in Danish and global wastewater treatment plants. The two most abundant species worldwide revealed in situ dynamics of important intracellular storage polymers, measured by FISH-Raman in activated sludge from four full-scale EBPR plants and from a lab-scale reactor fed with different substrates. Moreover, seven distinct Dechloromonas species were determined from a set of ten high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for polyphosphate (poly-P), glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two species exhibited an in situ phenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic-aerobic cycling, legitimately placing these microorganisms among the important PAOs. They are potentially involved in denitrification showing niche partitioning within the genus and with other important PAOs. As no isolates are available for the two species, we propose the names Candidatus Dechloromonas phosphoritropha and Candidatus Dechloromonas phosphorivorans.

Characterizing the growing microorganisms at species level in 46 anaerobic digesters at Danish wastewater treatment plants: A six-year survey on microbial community structure and key drivers.

Anaerobic digestion (AD) is a key technology at many wastewater treatment plants (WWTPs) for converting primary and surplus activated sludge to methane-rich biogas. However, the limited number of surveys and the lack of comprehensive datasets have hindered a deeper understanding of the characteristics and associations between key variables and the microbial community composition. Here, we present a six-year survey of 46 anaerobic digesters, located at 22 WWTPs in Denmark, which is the first and largest known study of the microbial ecology of AD at WWTPs at a regional scale. For three types of AD (mesophilic, mesophilic with thermal hydrolysis pretreatment, and thermophilic), we present the typical value range of 12 key parameters including operational variables and performance parameters. High-resolution bacterial and archaeal community analyses were carried out at species level using amplicon sequencing of >1,000 samples and the new ecosystem-specific MiDAS 3 reference database. We detected 42 phyla, 1,600 genera, and 3,584 species in the bacterial community, where 70% of the genera and 93% of the species represented environmental taxa that were only classified based on MiDAS 3 de novo placeholder taxonomy. More than 40% of the bacterial species were found not to grow in the mesophilic and thermophilic digesters and were only present due to immigration with the feed sludge. Ammonium concentration was the main driver shaping the bacterial community while temperature and pH were main drivers for the archaea in the three types of ADs. Sub-setting for the growing microbes improved significantly the correlation analyses and revealed the main drivers for the presence of specific species. Within mesophilic digesters, feed sludge composition and other key parameters (organic loading rate, biogas yield, and ammonium concentration) correlated with specific growing species. This survey provides a comprehensive insight into community structure at species level, providing a foundation for future studies of the ecological significance/characteristics and function of the many novel or poorly described taxa.

Fouling of membranes in membrane bioreactors for wastewater treatment: Planktonic bacteria can have a significant contribution.

Membrane bioreactors (MBRs) for wastewater treatment show great potentials in the sustainable development of urban environments. However, fouling of membranes remains the largest challenge of MBR technology. Dissolved extracellular polymeric substances (EPS) are often assumed be the main foulant in MBRs. However, single bacterial cells are often erroneously measured as EPS in traditional spectrophotometric analysis of EPS in activated sludge, so we hypothesized that single cells in many cases could be the true foulants in MBRs for wastewater treatment. To study this, raw MBR sludge and sludge supernatant with varying concentrations of planktonic cells were filtered on microfiltration (MF) membranes, and we found a direct correlation between the cell count and rate of flux decline. Addition of planktonic cells to fresh MBR sludge dramatically increased the flux decline. The identity of the most abundant planktonic cells in a full-scale MBR water resource recovery facility was determined by DNA fingerprinting. Many of these genera are known to be abundant in influent wastewater suggesting that the influent bacterial cells may have a direct effect on the fouling propensity in MBR systems. This new knowledge may lead to new anti-fouling strategies targeting incoming planktonic bacteria from the wastewater feed. PRACTITIONER POINTS: Planktonic cells constituted up to 60% of the total protein content of "soluble extracellular polymeric substances" in membrane bioreactor sludge. Planktonic cells are hidden under a surrogate concentration of extracellular polymeric substances which is often associated with fouling. Membrane fouling rate is directly proportional to amount of free planktonic cells suspended in sludge. Several influent bacterial genera are enriched in the water phase of membrane bioreactor sludge. Removing these may mitigate fouling.

MiDAS 3: An ecosystem-specific reference database, taxonomy and knowledge platform for activated sludge and anaerobic digesters reveals species-level microbiome composition of activated sludge.

The function of the microbiomes in wastewater treatment systems and anaerobic digesters is dictated by the physiological activity of their members and complex interactions between them. Since functional traits are often conserved at low taxonomic ranks (genus, species, strain), high resolution taxonomic classification is crucial to understand the role of microbes in any ecosystem. Here we present MiDAS 3, a comprehensive 16S rRNA gene reference database based on full-length 16S rRNA gene amplicon sequence variants (FL-ASVs) derived from activated sludge and anaerobic digester systems in Denmark. The new database proposes unique provisional names for all unclassified microorganisms down to species level, providing a new and much-needed tool for microbiome research. The MiDAS 3 database was used to analyze the microbiome in 20 Danish wastewater treatment plants with nutrient removal, sampled over 13 years. The 50 most abundant species belonged to 42 genera, including 14 genera with provisional 'midas' name. Of those, 20 have no known function in the system, which highlights the need for more efforts towards elucidating the role of important members of wastewater treatment ecosystems. The new MiDAS 3 database also forms the backbone of the MiDAS Field Guide - an online resource linking the identity of microorganisms in wastewater treatment systems to available data related to their functional importance. The new field guide contains a complete list of genera (>1800) and species (>4200) found in activated sludge and anaerobic digesters in Denmark, but is also relevant to wastewater systems across the world. The identity of the microbes is linked to functional information, where available, and the website provides the possibility to BLAST new sequences against the MiDAS 3 database. The MiDAS Field Guide is a collaborative platform acting as an online knowledge repository, facilitating understanding of wastewater treatment ecosystem function.

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.

Bacteria from the Genus Arcobacter Are Abundant in Effluent from Wastewater Treatment Plants.

Pathogenic bacteria in wastewater are generally considered to be efficiently removed in biological wastewater treatment plants. This understanding is almost solely based on culture-based control measures, and here we show, by applying culture-independent methods, that the removal of species in the genus Arcobacter was less effective than for many other abundant genera in the influent wastewater. Arcobacter was one of the most abundant genera in influent wastewater at 14 municipal wastewater treatment plants and was also abundant in the "clean" effluent from all the plants, reaching up to 30% of all bacteria as analyzed by 16S rRNA gene amplicon sequencing. Metagenomic analyses, culturing, genome sequencing of Arcobacter isolates, and visualization by fluorescent in situ hybridization (FISH) confirmed the presence of the human-pathogenic Arcobacter cryaerophilus and A. butzleri in both influent and effluent. The main reason for the high relative abundance in the effluent was probably that Arcobacter cells, compared to those of other abundant genera in the influent, did not flocculate and attach well to the activated sludge flocs, leaving a relatively large fraction dispersed in the water phase. The study shows there is an urgent need for new standardized culture-independent measurements of pathogens in effluent wastewaters, e.g., amplicon sequencing, and an investigation of the problem on a global scale to quantify the risk for humans and livestock.IMPORTANCE The genus Arcobacter was unexpectedly abundant in the effluent from 14 Danish wastewater treatment plants treating municipal wastewater, and the species included the human-pathogenic A. cryaerophilus and A. butzleri Recent studies have shown that Arcobacter is common in wastewater worldwide, so the study indicates that discharge of members of the genus Arcobacter may be a global problem, and further studies are needed to quantify the risk and potentially minimize the discharge. The study also shows that culture-based analyses are insufficient for proper effluent quality control, and new standardized culture-independent measurements of effluent quality encompassing most pathogens should be considered.

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.

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.

Resolving the individual contribution of key microbial populations to enhanced biological phosphorus removal with Raman-FISH.

Enhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms. Candidatus Accumulibacter conform to the classical physiology model for polyphosphate accumulating organisms and are widely believed to be the most important player for the process in full-scale EBPR systems. However, it was impossible till now to quantify the contribution of specific microbial clades to EBPR. In this study, we have developed a new tool to directly link the identity of microbial cells to the absolute quantification of intracellular poly-P and other polymers under in situ conditions, and applied it to eight full-scale EBPR plants. Besides Ca. Accumulibacter, members of the genus Tetrasphaera were found to be important microbes for P accumulation, and in six plants they were the most important. As these Tetrasphaera cells did not exhibit the classical phenotype of poly-P accumulating microbes, our entire understanding of the microbiology of the EBPR process has to be revised. Furthermore, our new single-cell approach can now also be applied to quantify storage polymer dynamics in individual populations in situ in other ecosystems and might become a valuable tool for many environmental microbiologists.