Effects of substrate stress and light intensity on enhanced biological phosphorus removal in a photo-activated sludge system.

Photo-activated sludge (PAS) systems are an emerging wastewater treatment technology where microalgae provide oxygen to bacteria without the need for external aeration. There is limited knowledge on the optimal conditions for enhanced biological phosphorus removal (EBPR) in systems containing a mixture of polyphosphate accumulating organisms (PAOs) and microalgae. This research aimed to study the effects of substrate composition and light intensity on the performance of a laboratory-scale EBPR-PAS system. Initially, a model-based design was developed to study the effect of organic carbon (COD), inorganic carbon (HCO3) and ammonium-nitrogen (NH4-N) in nitrification deprived conditions on phosphorus (P) removal. Based on the mathematical model, two different synthetic wastewater compositions (COD:HCO3:NH4-N: 10:20:1 and 10:10:4) were examined at a light intensity of 350 µmol m-2 sec-1. Add to this, the performance of the system was also investigated at light intensities: 87.5, 175, and 262.5 µmol m-2 sec-1 for short terms. Results showed that wastewater having a high level of HCO3 and low level of NH4-N (ratio of 10:20:1) favored only microalgal growth, and had poor P removal due to a shortage of NH4-N for PAOs growth. However, lowering the HCO3 level and increasing the NH4-N level (ratio of 10:10:4) balanced PAOs and microalgae symbiosis, and had a positive influence on P removal. Under this mode of operation, the system was able to operate without external aeration and achieved a net P removal of 10.33 ±1.45 mg L-1 at an influent COD of 100 mg L-1. No significant variation was observed in the reactor performance for different light intensities, indicating the EBPR-PAS system can be operated at low light intensities with a positive influence on P removal.

"Candidatus Accumulibacter delftensis": A clade IC novel polyphosphate-accumulating organism without denitrifying activity on nitrate.

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.

Cooperation between Candidatus Competibacter and Candidatus Accumulibacter clade I, in denitrification and phosphate removal processes.

Although simultaneous P-removal and nitrate reduction has been observed in laboratory studies as well as full-scale plants, there are contradictory reports on the ability of PAO I to efficiently use nitrate as electron acceptor. Such discrepancy could be due to other microbial groups performing partial denitrification from nitrate to nitrite. The denitrification capacities of two different cultures, a highly enriched PAO I and a PAO I-GAO cultures were assessed through batch activity tests conducted before and after acclimatization to nitrate. Negligible anoxic phosphate uptake coupled with a reduction of nitrate was observed in the highly enriched PAO I culture. On the opposite, the PAO I-GAO culture showed a higher anoxic phosphate uptake activity. Both cultures exhibited good anoxic phosphate uptake activity with nitrite (8.7 ± 0.3 and 9.6 ± 1.8 mgPO4-P/gVSS.h in the PAO I and PAO I-GAO cultures, respectively). These findings suggest that other microbial populations, such as GAOs, were responsible to reduce nitrate to nitrite in this EBPR system, and that PAO I used the nitrite generated for anoxic phosphate uptake. Moreover, the simultaneous denitrification and phosphate removal process using nitrite as electron acceptor may be a more sustainable process as can: i) reduce the carbon consumption, ii) reduce oxygen demand of WWTP, and iii) due to a lower growth yield contribute to a lower sludge production.

Long-term effects of sulphide on the enhanced biological removal of phosphorus: The symbiotic role of Thiothrix caldifontis.

Thiothrix caldifontis was the dominant microorganism (with an estimated bio-volume of 65 ± 3%) in a lab-scale enhanced biological phosphorus removal (EBPR) system containing 100 mg of sulphide per litre in the influent. After a gradual exposure to the presence of sulphide, the EBPR system initially dominated by Candidatus Accumulibacter phosphatis Clade I (98 ± 3% bio-volume) (a known polyphosphate accumulating organism, PAO) became enriched with T. caldifontis. Throughout the different operating conditions studied, practically 100% phosphate removal was always achieved. The gradual increase of the sulphide content in the medium (added to the anaerobic stage of the alternating anaerobic-aerobic sequencing batch reactor) and the adjustment of the aerobic hydraulic retention time played a major role in the enrichment of T. caldifontis. T. caldifontis exhibited a mixotrophic metabolism by storing carbon anaerobically as poly-ß-hydroxy-alkanoates (PHA) and generating the required energy through the hydrolysis of polyphosphate. PHA was used in the aerobic period as carbon and energy source for growth, polyphosphate, and glycogen formation. Apparently, extra energy was obtained by the initial accumulation of sulphide as an intracellular sulphur, followed by its gradual oxidation to sulphate. The culture enriched with T. caldifontis was able to store approximately 100 mg P/g VSS. This research suggests that T. caldifontis could behave like PAO with a mixotrophic metabolism for phosphorus removal using an intracellular sulphur pool as energy source. These findings can be of major interest for the biological removal of phosphorus from wastewaters with low organic carbon concentrations containing reduced S-compounds like those (pre-)treated in anaerobic systems or from anaerobic sewers.

Sulphide effects on the physiology of Candidatus Accumulibacter phosphatis type I.

Sulphate-rich wastewaters can be generated due to (i) use of saline water as secondary-quality water for sanitation in urban environments (e.g. toilet flushing), (ii) discharge of industrial effluents, (iii) sea and brackish water infiltration into the sewage and (iv) use of chemicals, which contain sulphate, in drinking water production. In the presence of an electron donor and absence of oxygen or nitrate, sulphate can be reduced to sulphide. Sulphide can inhibit microbial processes in biological wastewater treatment systems. The objective of the present study was to assess the effects of sulphide concentration on the anaerobic and aerobic physiology of polyphosphate-accumulating organisms (PAOs). For this purpose, a PAO culture, dominated by Candidatus Accumulibacter phosphatis clade I (PAO I), was enriched in a sequencing batch reactor (SBR) fed with acetate and propionate. To assess the direct inhibition effects and their reversibility, a series of batch activity tests were conducted during and after the exposure of a PAO I culture to different sulphide concentrations. Sulphide affected each physiological process of PAO I in a different manner. At 189 mg TS-S/L, volatile fatty acid uptake was 55% slower and the phosphate release due to anaerobic maintenance increased from 8 to 18 mg PO4-P/g VSS/h. Up to 8 mg H2S-S/L, the decrease in aerobic phosphorus uptake rate was reversible (Ic60). At higher concentrations of sulphide, potassium (>16 mg H2S-S/L) and phosphate (>36 mg H2S-S/L) were released under aerobic conditions. Ammonia uptake, an indicator of microbial growth, was not observed at any sulphide concentration. This study provides new insights into the potential failure of enhanced biological phosphorus removal sewage plants receiving sulphate- or sulphide-rich wastewaters when sulphide concentrations exceed 8 mg H2S-S/L, as PAO I could be potentially inhibited.

Accumulibacter clades Type I and II performing kinetically different glycogen-accumulating organisms metabolisms for anaerobic substrate uptake.

The anaerobic acetate (HAc) uptake stoichiometry of phosphorus-accumulating organisms (PAO) in enhanced biological phosphorus removal (EBPR) systems has been an extensive subject of study due to the highly variable reported stoichiometric values (e.g. anaerobic P-release/HAc-uptake ratios ranging from 0.01 up to 0.93 P-mol/C-mol). Often, such differences have been explained by the different applied operating conditions (e.g. pH) or occurrence of glycogen-accumulating organisms (GAO). The present study investigated the ability of biomass highly enriched with specific PAO clades ('Candidatus Accumulibacter phosphatis' Clade I and II, hereafter PAO I and PAO II) to adopt a GAO metabolism. Based on long-term experiments, when Poly-P is not stoichiometrically limiting for the anaerobic VFA uptake, PAO I performed the typical PAO metabolism (with a P/HAc ratio of 0.64 P-mol/C-mol); whereas PAO II performed a mixed PAO-GAO metabolism (showing a P/HAc ratio of 0.22 P-mol/C-mol). In short-term batch tests, both PAO I and II gradually shifted their metabolism to a GAO metabolism when the Poly-P content decreased, but the HAc-uptake rate of PAO I was 4 times lower than that of PAO II, indicating that PAO II has a strong competitive advantage over PAO I when Poly-P is stoichiometrically limiting the VFA uptake. Thus, metabolic flexibility of PAO clades as well as their intrinsic differences are additional factors leading to the controversial anaerobic stoichiometry and kinetic rates observed in previous studies. From a practical perspective, the dominant type of PAO prevailing in full-scale EBPR systems may affect the P-release processes for biological or combined biological and chemical P-removal and recovery and consequently the process performance.

Occurrence of PAOI in a low temperature EBPR system.

The occurrence of Accumulibacter Type I (a known phosphorus-accumulating organism, PAO) has received increased attention due to the potential operating benefits associated with their denitrifying activity in enhanced biological phosphorus removal (EBPR) wastewater treatment plants. In this study, after a shift from an enriched glycogen-accumulating organism (GAO) culture (competitors of PAO) to a PAO-enriched system, Accumulibacter Type I (PAO I) became dominant in an anaerobic-aerobic EBPR system fed with acetate and operated at 10°C with a net aerobic solids retention time (SRT) of 6 d. Since Accumulibacter Type II (PAO II) were not detected, the low temperature in combination with the net aerobic SRT applied appeared to have suppressed their growth as well. The stoichiometry of PAO I was in agreement with previous metabolic models, suggesting that it was the main PAO organisms present in previous studies operated under similar conditions. Moreover, under poly-P limiting conditions, PAO I were unable to switch to a GAO-like metabolism at low temperatures. These results contribute to increase the understanding of the physiology, microbial metabolism and microbial ecology of PAO I.

Modelling the population dynamics and metabolic diversity of organisms relevant in anaerobic/anoxic/aerobic enhanced biological phosphorus removal processes.

In this study, enhanced biological phosphorus removal (EBPR) metabolic models are expanded in order to incorporate the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) under sequential anaerobic/anoxic/aerobic conditions, which are representative of most full-scale EBPR plants. Since PAOs and GAOs display different denitrification tendencies, which is dependent on the phylogenetic identity of the organism, the model was separated into six distinct biomass groups, constituting Accumulibacter Types I and II, as well as denitrifying and non-denitrifying Competibacter and Defluviicoccus GAOs. Denitrification was modelled as a multi-step process, with nitrate (NO(3)), nitrite (NO(2)), nitrous oxide (N(2)O) and di-nitrogen gas (N(2)) being the primary components. The model was calibrated and validated using literature data from enriched cultures of PAOs and GAOs, obtaining a good description of the observed biochemical transformations. A strong correlation was observed between Accumulibacter Types I and II, and nitrate-reducing and non-nitrate-reducing PAOs, respectively, where the abundance of each PAO subgroup was well predicted by the model during an acclimatization period from anaerobic-aerobic to anaerobic-anoxic conditions. Interestingly, a strong interdependency was observed between the anaerobic, anoxic and aerobic kinetic parameters of PAOs and GAOs. This could be exploited when metabolic models are calibrated, since all of these parameters should be changed by an identical factor from their default value. Factors that influence these kinetic parameters include the fraction of active biomass, relative aerobic/anoxic fraction and the ratio of acetyl-CoA to propionyl-CoA. Employing a metabolic approach was found to be advantageous in describing the performance and population dynamics in such complex microbial ecosystems.

Modelling of an oil refinery wastewater treatment plant.

The Activated Sludge Model No. 3 (ASM3) and Dutch calibration guidelines (STOWA) were evaluated in the modelling of an activated sludge system treating effluents from a large oil refinery. The plant was designed to remove suspended solids, organic matter and nitrogen from wastewater at an average water temperature of 34 degrees C. The plant consists of three tanks in series; the first two tanks operate in on-off aeration mode with pure oxygen for N-removal, whilst extra methanol is added for the denitrification, and the third tank is maintained as constantly aerobic. Calibration was performed based on a simplified influent characterisation and extra batch experiments (nitrification and denitrification). With the adjustment of only four parameters the model proved capable of describing the performance of the plant concerning both the liquid phase and the biomass. The model was further used to analyse possible modifications in the plant layout and optimize operational conditions in order to reduce operating costs. Modelling results indicated reduction in methanol dosage by implementing an idle time between aerobic and anoxic phases. In this way, surplus methanol was prevented from entering during the aerobic period. Moreover, simulations showed that the most cost-effective option regarding the denitrification process was a combined pre-post-denitrification scheme, without the need for enlarging existing basins. It can be concluded that although ASM3 and STOWA guidelines were originally developed for domestic wastewater application at a temperature range of 10 to 20 degrees C, they proved well capable of describing the performance of an oil refinery wastewater treatment plant operating at 34 degrees C. Moreover, the plant model proved useful for optimization of the plant performance regarding operational costs.