Enrichment of phosphate-accumulating organisms (PAOs) in a microfluidic model biofilm system by mimicking a typical aerobic granular sludge feast/famine regime

Wastewater treatment using aerobic granular sludge has gained increasing interest due to its advantages compared to conventional activated sludge. The technology allows simultaneous removal of organic carbon, nitrogen, and phosphorus in a single reactor system and is independent of space-intensive settling tanks. However, due to the microscale, an analysis of processes and microbial population along the radius of granules is challenging. Here, we introduce a model system for aerobic granular sludge on a small scale by using a machine-assisted microfluidic cultivation platform. With an implemented logic module that controls solenoid valves, we realized alternating oxic hunger and anoxic feeding phases for the biofilms growing within. Sampling during ongoing anoxic cultivation directly from the cultivation channel was achieved with a robotic sampling device. Analysis of the biofilms was conducted using optical coherence tomography, fluorescence in situ hybridization, and amplicon sequencing. Using this setup, it was possible to significantly enrich the percentage of polyphosphate-accumulating organisms (PAO) belonging to the family Rhodocyclaceae in the community compared to the starting inoculum. With the aid of this miniature model system, it is now possible to investigate the influence of a multitude of process parameters in a highly parallel way to understand and efficiently optimize aerobic granular sludge-based wastewater treatment systems.Key points• Development of a microfluidic model to study EBPR.• Feast-famine regime enriches polyphosphate-accumulating organisms (PAOs).• Microfluidics replace sequencing batch reactors for aerobic granular sludge research.

Recovery of Phosphorus in Wastewater in the Form of Polyphosphates: A Review

As non-renewable resource, the recovery and utilization of phosphorus from wastewater is an enduring topic. Stimulated by the advances in research on polyphosphates (polyP) as well as the development of Enhanced Biological Phosphorus Removal (EBPR) technology to achieve the efficient accumulation of polyP via polyphosphate accumulating organisms (PAOs), a novel phosphorus removal strategy is considered with promising potential for application in real wastewater treatment processes. This review mainly focuses on the mechanism of phosphorus aggregation in the form of polyP during the phosphate removal process. Further discussion about the reuse of polyP with different chain lengths is provided herein so as to suggest possible application pathways for this biosynthetic product.

Simulation of an Oxic-Settling-Anaerobic Pilot Plant Operated under Real Conditions Using the Activated Sludge Model No.2d

Oxic-settling-anaerobic (OSA) process has been introduced into the treatment line of wastewater in order to upgrade activated sludge processes and to reduce the production of excess sludge. The aim of the present study was to simulate the performance of an OSA pilot plant by implementing the Activated Sludge Model No.2d (ASM2d) into a mathematical modelling software (BioWin). The stepwise calibration, performed both by off-line experiments and software dynamic calibration, was carried out in a heuristic way, adjusting the parameters values that showed a major influence to the effluent and internal concentrations. All the reduction factors introduced into ASM2d to simulate the processes occurring in anoxic and anaerobic conditions were lowered in order to reproduce the concentrations of interest. In addition, the values of parameters of the PAOs (polyphosphate accumulating organisms)-related process (namely qPHA and YPO4) were found lower than those usually adopted. In general, theoretical results were in good agreement with the experimental data obtained from plant’s operation, showing an accurate predictive capacity of the model. Good performance was achieved considering the phosphorus removal related process, while some failures were detected in COD and ammonia simulations.

Response of simultaneous nitrification-denitrification to DO increments in continuously aerated biofilm reactors for aquaculture wastewater treatment

Intensive aquaculture usually produces large volumes of nutrient-rich wastewater, which is essential to treat to avoid eutrophication. This study aimed to evaluate the performance of five, continuously aerated, biofilm reactors treating simulated, high-strength, aquaculture wastewater under different dissolved oxygen (DO) levels, and the effects of DO increments on simultaneous nitrification-denitrification (SND). Continuous aeration was beneficial to complete nitrification. Total inorganic nitrogen (TIN), principally ammonium, was mainly removed by SND. The SND rate response to different DO levels was fitted well by the power function of y = 54.81 + 371.58/(1 − 0.16*x)^(−1/0.24) (R2 = 0.897, P = 0.000). When the TIN was removed completely, the optimal SND rate was defined and corresponded to a value of 121.8%. Accordingly, the optimal DO concentration was calculated as 2.10 mg/L, close to the actual level of 1.83 mg/L, at which the highest proportional removals of total nitrogen (58.0%) and TIN (57.3%) were obtained. Phosphorus was also removed by denitrifying polyphosphate-accumulating organisms.

“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.

Influence of Extraction Solvent on Nontargeted Metabolomics Analysis of Enrichment Reactor Cultures Performing Enhanced Biological Phosphorus Removal (EBPR)

Metabolome profiling is becoming more commonly used in the study of complex microbial communities and microbiomes; however, to date, little information is available concerning appropriate extraction procedures. We studied the influence of different extraction solvent mixtures on untargeted metabolomics analysis of two continuous culture enrichment communities performing enhanced biological phosphate removal (EBPR), with each enrichment targeting distinct populations of polyphosphate-accumulating organisms (PAOs). We employed one non-polar solvent and up to four polar solvents for extracting metabolites from biomass. In one of the reactor microbial communities, we surveyed both intracellular and extracellular metabolites using the same set of solvents. All samples were analysed using ultra-performance liquid chromatography mass spectrometry (UPLC-MS). UPLC-MS data obtained from polar and non-polar solvents were analysed separately and evaluated using extent of repeatability, overall extraction capacity and the extent of differential abundance between physiological states. Despite both reactors demonstrating the same bioprocess phenotype, the most appropriate extraction method was biomass specific, with methanol: water (50:50 v/v) and methanol: chloroform: water (40:40:20 v/v) being chosen as the most appropriate for each of the two different bioreactors, respectively. Our approach provides new data on the influence of solvent choice on the untargeted surveys of the metabolome of PAO enriched EBPR communities and suggests that metabolome extraction methods need to be carefully tailored to the specific complex microbial community under study.

High Diversity and Functional Potential of Undescribed “Acidobacteriota” in Danish Wastewater Treatment Plants

Microbial communities in water resource recovery facilities encompass a large diversity of poorly characterized lineages that could have undescribed process-critical functions. Recently, it was shown that taxa belonging to “Acidobacteriota” are abundant in Danish full-scale activated sludge wastewater treatment plants (WWTP), and here we investigated their diversity, distribution, and functional potential. “Acidobacteriota” taxa were identified using a comprehensive full-length 16S rRNA gene reference dataset and amplicon sequencing surveys across 37 WWTPs. Members of this phylum were diverse, belonging to 14 families, eight of which are completely uncharacterized and lack type strains. Several lineages were abundant, with relative abundances of up to 5% of the microbial community. Genome annotation and metabolic reconstruction of 50 high-quality “Acidobacteriota” metagenome-assembled genomes (MAGs) from 19 WWTPs showed high metabolic diversity and potential involvement in nitrogen and phosphorus removal and iron reduction. Fluorescence in situ hybridization (FISH) using newly-designed probes revealed cells with diverse morphologies, predominantly located inside activated sludge flocs. FISH in combination with Raman microspectroscopy revealed ecophysiological traits in probe-defined cells from the families Holophagaceae, Thermoanaerobaculaceae, and Vicinamibacteraceae, and families with the placeholder name of midas_f_502, midas_f_973, and midas_f_1548. Members of these lineages had the potential to be polyphosphate-accumulating organisms (PAOs) as intracellular storage was observed for the key compounds polyphosphate and glycogen.