Extracellular proteins are supposed to play crucial roles in the formation and structure of biofilms and aggregates. However, often little is known about these proteins, in particular for microbial communities. Here, we use two advanced metaproteomic approaches to study the extracellular proteome in a granular Candidatus Accumulibacter enrichment as a proxy for microbial communities that form solid microbial granules, such as those used in biological wastewater treatment. Limited proteolysis of whole granules and metaproteome isolation from the culture's supernatant successfully classified over 50% of the identified protein biomass to be secreted. Moreover, structural and sequence-based classification identified 387 proteins, corresponding to over 50% of the secreted protein biomass, with characteristics that could aid the formation of aggregates, including filamentous, beta-barrel containing, and cell surface proteins. While various of these aggregate-forming proteins originated from Ca. Accumulibacter, some proteins associated with other taxa. This suggests that not only a range of different proteins but also multiple organisms contribute to granular biofilm formation. Therefore, the obtained extracellular metaproteome data from the granular Ca. Accumulibacter enrichment provides a resource for exploring proteins that potentially support the formation and stability of granular biofilms, whereas the demonstrated approaches can be applied to explore biofilms of microbial communities in general.

Enhanced biological phosphate removal and aerobic sludge granulation are commonly studied with fatty acids as substrate. Fermentative substrates such as glucose have received limited attention. In this work, glucose conversion by aerobic granular sludge and its impact on phosphate removal was studied. Long-term stable phosphate removal and successful granulation were achieved. Glucose was rapidly taken up (273 mg/gVSS/h) at the start of the anaerobic phase, while phosphate was released during the full anaerobic phase. Some lactate was produced during glucose consumption, which was anaerobically consumed once glucose was depleted. The phosphate release appeared to be directly proportional to the uptake of lactate. The ratio of phosphorus released to glucose carbon taken up over the full anaerobic phase was 0.25 Pmol/Cmol. Along with glucose and lactate uptake in the anaerobic phase, poly‑hydroxy-alkanoates and glycogen storage were observed. There was a linear correlation between glucose consumption and lactate formation. While lactate accounted for approximately 89 % of the observed products in the bulk liquid, minor quantities of formate (5 %), propionate (4 %), and acetate (3 %) were also detected (mass fraction). Formate was not consumed anaerobically. Quantitative fluorescence in-situ hybridization (qFISH) revealed that polyphosphate accumulating organisms (PAO) accounted for 61 ± 15 % of the total biovolume. Metagenome evaluation of the biomass indicated a high abundance of Micropruina and Ca. Accumulibacter in the system, which was in accordance with the microscopic observations and the protein mass fraction from metaproteome analysis. Anaerobic conversions were evaluated based on theoretical ATP balances to provide the substrate distribution amongst the dominant genera. This research shows that aerobic granular sludge technology can be applied to glucose-containing effluents and that glucose is a suitable substrate for achieving phosphate removal. The results also show that for fermentable substrates a microbial community consisting of fermentative organisms and PAO develop.

The tremendous progress in sequencing technologies has made DNA sequencing routine for microbiome studies. Additionally, advances in mass spectrometric techniques have extended conventional proteomics into the field of microbial ecology. However, systematic studies that provide a better understanding of the complementary nature of these 'omics' approaches, particularly for complex environments such as wastewater treatment sludge, are urgently needed. Here, we describe a comparative metaomics study on aerobic granular sludge from three different wastewater treatment plants. For this, we employed metaproteomics, whole metagenome, and 16S rRNA amplicon sequencing to study the same granule material with uniform size. We furthermore compare the taxonomic profiles using the Genome Taxonomy Database (GTDB) to enhance the comparability between the different approaches. Though the major taxonomies were consistently identified in the different aerobic granular sludge samples, the taxonomic composition obtained by the different omics techniques varied significantly at the lower taxonomic levels, which impacts the interpretation of the nutrient removal processes. Nevertheless, as demonstrated by metaproteomics, the genera that were consistently identified in all techniques cover the majority of the protein biomass. The established metaomics data and the contig classification pipeline are publicly available, which provides a valuable resource for further studies on metabolic processes in aerobic granular sludge.

Methanonatronarchaeia represents a deep-branching phylogenetic lineage of extremely halo(alkali)philic and moderately thermophilic methyl-reducing methanogens belonging to the phylum Halobacteriota. It includes two genera, the alkaliphilic Methanonatronarchaeum and the neutrophilic Ca. Methanohalarchaeum. The former is represented by multiple closely related pure culture isolates from hypersaline soda lakes, while the knowledge about the latter is limited to a few mixed cultures with anaerobic haloarchaea. To get more insight into the distribution and ecophysiology of this enigmatic group of extremophilic methanogens, potential activity tests and enrichment cultivation with different substrates and at different conditions were performed with anaerobic sediment slurries from various hypersaline lakes in Russia. Methanonatronarchaeum proliferated exclusively in hypersaline soda lake samples mostly at elevated temperature, while at mesophilic conditions it coexisted with the extremely salt-tolerant methylotroph Methanosalsum natronophilum. Methanonatronarchaeum was also able to serve as a methylotrophic or hydrogenotrophic partner in several thermophilic enrichment cultures with fermentative bacteria. Ca. Methanohalarchaeum did not proliferate at mesophilic conditions and at thermophilic conditions it competed with extremely halophilic and moderately thermophilic methylotroph Methanohalobium, which it outcompeted at a combination of elevated temperature and methyl-reducing conditions. Overall, the results demonstrated that Methanonatronarchaeia are specialized extremophiles specifically proliferating in conditions of elevated temperature coupled with extreme salinity and simultaneous availability of a wide range of C₁-methylated compounds and H₂/formate.

Metaproteomics has emerged as one of the most promising approaches for determining the composition and metabolic functions of complete microbial communities. Conventional metaproteomics approaches rely on the construction of protein sequence databases and efficient peptide-spectrum-matching algorithms, an approach that is intrinsically biased towards the content of the constructed sequence database. Here, we introduce a highly efficient, database-independent de novo metaproteomics approach and systematically evaluate its quantitative performance using synthetic and natural microbial communities comprising dozens of taxonomic families. Our work demonstrates that the de novo sequencing approach can vastly expand many metaproteomics applications by enabling rapid quantitative profiling and by capturing unsequenced community members that otherwise remain inaccessible for further interpretation. Kleikamp et al., describe a novel de novo metaproteomics pipeline (NovoBridge) that enables rapid community profiling without the need for constructing protein sequence databases.

Polyhydroxyalkanoate (PHA) accumulating microbial enrichment was established on volatile fatty acids (VFAs) containing leachate derived from the organic fraction of municipal solid waste (OFMSW). The enrichment was based on a 12-h feast-famine batch cycle and an exchange ratio of 50% in which VFAs were completely consumed in less than 50 min during stable periods of operation. No pH control was applied in the system, and the pH went as high as 9 due to the presence of amongst others, ammonia [500 mg·L-1 total ammonia nitrogen (TAN) on average]. The degree of enrichment was evaluated with fluorescence in situ hybridization (FISH), and a yet unknown genus of large (3-5 μm diameter) beta-proteobacteria appeared dominant in the culture. A method for estimating the fraction of PHA accumulating active biomass in the total volatile suspended solids was established, and the results indicated an increase of this fraction from 25% to 56% after implementing two modifications in the operational protocol: (1) a pretreatment of the substrate removing virtually all settleable solids; and (2) a settling phase in the enrichment reactor after the feast phase, selectively removing nonsettleable solids and slowly degradable substrates. The PHA accumulation potential of the culture was 77±18 wt% PHA (n=3) after 3 h in batch accumulation experiments. The results suggest the potential feasibility of PHA production under conditions that were previously considered economically favorable but technically difficult.

Environmental fluctuations in the availability of nutrients lead to intricate metabolic strategies. "Candidatus Accumulibacter phosphatis," a polyphosphate-accumulating organism (PAO) responsible for enhanced biological phosphorus removal (EBPR) from wastewater treatment systems, is prevalent in aerobic/anaerobic environments. While the overall metabolic traits of these bacteria are well described, the nonavailability of isolates has led to controversial conclusions on the metabolic pathways used. In this study, we experimentally determined the redox cofactor preferences of different oxidoreductases in the central carbon metabolism of a highly enriched "Ca Accumulibacter phosphatis" culture. Remarkably, we observed that the acetoacetyl coenzyme A reductase engaged in polyhydroxyalkanoate (PHA) synthesis is NADH preferring instead of showing the generally assumed NADPH dependency. This allows rethinking of the ecological role of PHA accumulation as a fermentation product under anaerobic conditions and not just a stress response. Based on previously published metaomics data and the results of enzymatic assays, a reduced central carbon metabolic network was constructed and used for simulating different metabolic operating modes. In particular, scenarios with different acetate-to-glycogen consumption ratios were simulated, which demonstrated optima using different combinations of glycolysis, glyoxylate shunt, or branches of the tricarboxylic acid (TCA) cycle. Thus, optimal metabolic flux strategies will depend on the environment (acetate uptake) and on intracellular storage compound availability (polyphosphate/glycogen). This NADH-related metabolic flexibility is enabled by the NADH-driven PHA synthesis. It allows for maintaining metabolic activity under various environmental substrate conditions, with high carbon conservation and lower energetic costs than for NADPH-dependent PHA synthesis. Such (flexible) metabolic redox coupling can explain the competitiveness of PAOs under oxygen-fluctuating environments.IMPORTANCE Here, we demonstrate how microbial storage metabolism can adjust to a wide range of environmental conditions. Such flexibility generates a selective advantage under fluctuating environmental conditions. It can also explain the different observations reported in PAO literature, including the capacity of "Ca Accumulibacter phosphatis" to act like glycogen-accumulating organisms (GAOs). These observations stem from slightly different experimental conditions, and controversy arises only when one assumes that metabolism can operate only in a single mode. Furthermore, we also show how the study of metabolic strategies is possible when combining omics data with functional cofactor assays and modeling. Genomic information can only provide the potential of a microorganism. The environmental context and other complementary approaches are still needed to study and predict the functional expression of such metabolic potential.

Arsenic contamination of groundwater aquifers is an issue of global concern. Among the affected sites, in several Italian groundwater aquifers arsenic levels above the WHO limits for drinking water are present, with consequent issues of public concern. In this study, for the first time, the role of microbial communities in metalloid cycling in groundwater samples from Northern Italy lying on Pleistocene sediments deriving from Alps mountains has been investigated combining environmental genomics and cultivation approaches. 16S rRNA gene libraries revealed a high number of yet uncultured species, which in some of the study sites accounted for more of the 50% of the total community. Sequences related to arsenic-resistant bacteria (arsenate-reducing and arsenite-oxidizing) were abundant in most of the sites, while arsenate-respiring bacteria were negligible. In some of the sites, sulfur-oxidizing bacteria of the genus Sulfuricurvum accounted for more than 50% of the microbial community, whereas iron-cycling bacteria were less represented. In some aquifers, arsenotrophy, growth coupled to autotrophic arsenite oxidation, was suggested by detection of arsenite monooxygenase (aioA) and 1,5-ribulose bisphosphate carboxylase (RuBisCO) cbbL genes of microorganisms belonging to Rhizobiales and Burkholderiales. Enrichment cultures established from sampled groundwaters in laboratory conditions with 1.5 mmol L-1 of arsenite as sole electron donor were able to oxidize up to 100% of arsenite, suggesting that this metabolism is active in groundwaters. The presence of heterotrophic arsenic resistant bacteria was confirmed by enrichment cultures in most of the sites. The overall results provided a first overview of the microorganisms inhabiting arsenic-contaminated aquifers in Northern Italy and suggested the importance of sulfur-cycling bacteria in the biogeochemistry of arsenic in these ecosystems. The presence of active arsenite-oxidizing bacteria indicates that biological oxidation of arsenite, in combination with arsenate-adsorbing materials, could be employed for metalloid removal.

Populations of “Candidatus Accumulibacter”, a known polyphosphate-accumulating organism, within clade IC have been proposed to perform anoxic P-uptake activity in enhanced biological phosphorus removal (EBPR) systems using nitrate as electron acceptor. However, no consensus has been reached on the ability of “Ca. Accumulibacter” members of clade IC to reduce nitrate to nitrite. Discrepancies might relate to the diverse operational conditions which could trigger the expression of the Nap and/or Nar enzyme and/or to the accuracy in clade classification. This study aimed to assess whether and how certain operational conditions could lead to the enrichment and enhance the denitrification capacity of “Ca. Accumulibacter” within clade IC. To study the potential induction of the denitrifying enzyme, an EBPR culture was enriched under anaerobic–anoxic–oxic (A2O) conditions that, based on fluorescence in situ hybridization and ppk gene sequencing, was composed of around 97% (on a biovolume basis) of affiliates of “Ca. Accumulibacter” clade IC. The influence of the medium composition, sludge retention time (SRT), polyphosphate content of the biomass (poly-P), nitrate dosing approach, and minimal aerobic SRT on potential nitrate reduction were studied. Despite the different studied conditions applied, only a negligible anoxic P-uptake rate was observed, equivalent to maximum 13% of the aerobic P-uptake rate. An increase in the anoxic SRT at the expenses of the aerobic SRT resulted in deterioration of P-removal with limited aerobic P-uptake and insufficient acetate uptake in the anaerobic phase. A near-complete genome (completeness = 100%, contamination = 0.187%) was extracted from the metagenome of the EBPR biomass for the here-proposed “Ca. Accumulibacter delftensis” clade IC. According to full-genome-based phylogenetic analysis, this lineage was distant from the canonical “Ca. Accumulibacter phosphatis”, with closest neighbor “Ca. Accumulibacter sp. UW-LDO-IC” within clade IC. This was cross-validated with taxonomic classification of the ppk1 gene sequences. The genome-centric metagenomic analysis highlighted the presence of genes for assimilatory nitrate reductase (nas) and periplasmic nitrate reductase (nap) but no gene for respiratory nitrate reductases (nar). This suggests that “Ca. Accumulibacter delftensis” clade IC was not capable to generate the required energy (ATP) from nitrate under strict anaerobic-anoxic conditions to support an anoxic EBPR metabolism. Definitely, this study stresses the incongruence in denitrification abilities of “Ca. Accumulibacter” clades and reflects the true intra-clade diversity, which requires a thorough investigation within this lineage.

The existence of sulfate-reducing bacteria (SRB) is a major concern in oil industry due to the detrimental effects of SRB in oil technology. SRB are co-habited with diverse microbial populations in oil fields. The presence of other bacterial groups in oil fields may alter SRB activity in different ways. Therefore, understanding this coexistence may provide insights into problems induced by SRB activity and possible solutions to these problems. To investigate this aspect, not only the presence and abundance of SRB but also bacterial population that coexists with SRB in sulfate-reducing enrichment cultures obtained from the Diyarbakır oil fields in southeast of Turkey was determined by using cultivation- and molecular-based approaches. The most probable number technique (MPN) was used to determine the number of sulfidogenic bacteria in the enrichments. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction-amplified 16S rRNA gene fragments was performed to examine the bacterial diversity of the enrichments. The results demonstrated that the number of sulfidogenic bacteria in the enrichments was low (<10³ cells/mL). The DGGE analysis indicated that community members belonging to the Firmicutes were more abundant than those of other phyla. Members belonging to SRB mainly consisted of the genera Desulfosporosinus, Desulfovibrio, Thermodesulfovibrio, and Desulfotomaculum. Fermentative bacteria, acetogens, nitrate reducers, and sulfur reducers were also detected in the enrichments. The results of this study not only provide information regarding the diversity of the cultivable portion of the bacterial community that coexists with cultivable SRB, but they also offer insights into the interactions of bacteria in complex microbial communities that inhabit natural environments.

Methanogenic enrichments from hypersaline lakes at moderate thermophilic conditions have resulted in the cultivation of an unknown deep lineage of euryarchaeota related to the class Halobacteria. Eleven soda lake isolates and three salt lake enrichment cultures were methyl-reducing methanogens that utilize C₁ methylated compounds as electron acceptors and H₂ or formate as electron donors, but they were unable to grow on either substrates alone or to form methane from acetate. They are extreme halophiles, growing optimally at 4 M total Na⁺ and the first representatives of methanogens employing the ‘salt-in’ osmoprotective mechanism. The salt lake subgroup is neutrophilic, whereas the soda lake isolates are obligate alkaliphiles, with an optimum around pH 9.5. Both grow optimally at 50°C. The genetic diversity inside the two subgroups is very low, indicating that the soda and salt lake clusters consist of a single genetic species each. The phylogenetic distance between the two subgroups is in the range of distant genera, whereas the distance to other euryarchaea is below 83 % identity of the 16S rRNA gene. These isolates and enriched methanogens, together with closely related environmental clones from hypersaline habitats (the SA1 group), form a novel class-level clade in the phylum Euryarchaeota. On the basis of distinct phenotypic and genetic properties, the soda lake isolates are classified into a new genus and species, Methanonatronarchaeum thermophilum, with the type strain AMET1^T (DSM 28684^T=NBRC 110805^T=UNIQEM U982^T), and the salt lake methanogens into a candidate genus and species ‘Candidatus Methanohalarchaeum thermophilum’. These organisms are proposed to form novel family, order and class Methanonatronarchaeaceae fam. nov., Methanonatronarchaeales ord. nov. and Methanonatronarchaeia classis nov., within the phylum Euryarchaeota.

The concentration of sulphate present in wastewater can vary from 10 to 500 mg SO₄ ²⁻/L. During anaerobic conditions, sulphate is reduced to sulphide by sulphate-reducing bacteria (SRB). Sulphide generation is undesired in wastewater treatment plants (WWTPs). Previous research indicated that SRB are inhibited by the presence of electron acceptors (such as O₂, NO₃ and NO₂). However, the contact times and concentrations used in those studies are by far higher than occur in WWTPs. Since sulphide can influence the biological nitrogen and phosphorus removal processes, this research aimed to understand how the different electron acceptors commonly present in biological nutrient removal (BNR) systems can affect the proliferation of SRB. For this purpose, a culture of SRB was enriched in a sequencing batch reactor (approx. 88% of the total bacteria population). Once enriched, the SRB were exposed for 2 h to typical concentrations of electron acceptors like those observed in BNR systems. Their activity was assessed using three different types of electron donors (acetate, propionate and lactate). Oxygen was the most inhibiting electron acceptor regardless the carbon source used. After exposure to oxygen and when feeding acetate, an inactivation time in the sulphate reduction activity was observed for 1.75 h. Once the sulphate reduction activity resumed, only 60% of the original activity was recovered. It is suggested that the proliferation of SRB is most likely to occur in BNR plants with an anaerobic fraction higher than 15% and operating at sludge retention times higher than 20 days (at a temperature of 20 °C). These results can be used to implement strategies to control the growth of sulphate reducers that might compete for organic carbon with phosphate-accumulating organisms.

Methanogenic archaea are major players in the global carbon cycle and in the biotechnology of anaerobic digestion. The phylum Euryarchaeota includes diverse groups of methanogens that are interspersed with non-methanogenic lineages. So far, methanogens inhabiting hypersaline environments have been identified only within the order Methanosarcinales. We report the discovery of a deep phylogenetic lineage of extremophilic methanogens in hypersaline lakes and present analysis of two nearly complete genomes from this group. Within the phylum Euryarchaeota, these isolates form a separate, class-level lineage 'Methanonatronarchaeia' that is most closely related to the class Halobacteria. Similar to the Halobacteria, 'Methanonatronarchaeia' are extremely halophilic and do not accumulate organic osmoprotectants. The high intracellular concentration of potassium implies that 'Methanonatronarchaeia' employ the 'salt-in' osmoprotection strategy. These methanogens are heterotrophic methyl-reducers that use C 1 -methylated compounds as electron acceptors and formate or hydrogen as electron donors. The genomes contain an incomplete and apparently inactivated set of genes encoding the upper branch of methyl group oxidation to CO₂ as well as membrane-bound heterodisulfide reductase and cytochromes. These features differentiate 'Methanonatronarchaeia' from all known methyl-reducing methanogens. The discovery of extremely halophilic, methyl-reducing methanogens related to haloarchaea provides insights into the origin of methanogenesis and shows that the strategies employed by methanogens to thrive in salt-saturating conditions are not limited to the classical methylotrophic pathway.