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.

Resource recovery assessment at a pulp mill wastewater treatment plant in Uruguay.

Using a mathematical model, a resource recovery assessment was carried out at a pulp mill activated sludge wastewater treatment plant (WWTP) located in Uruguay. Through the evaluation of different scenarios, the potential production of methane from secondary sludge, with its inherent energy savings, and the recovery of phosphorus (P) as struvite were estimated. Considering the current WWTP configuration with a sludge retention time (SRT) of 32 days, and according to the model, which is a simplification of reality, the assessment indicates that the implementation of an anaerobic digester (AD) to treat the excess sludge can lead to a methane production of approximately 1736 m3 CH4 d-1, being a promising alternative to increase the WWTP treatment performance. Furthermore, the model predictions suggest that by shortening the SRT from 32 to 5 days, the methane production could increase by up to 5568 m3 CH4 d-1. If the methane produced is used to generate electrical energy to operate the WWTP, energy savings of about 88% can be achieved. Regarding the potential recovery of P as struvite, the addition of a struvite reactor could be an efficient option to recover approximately 1611 mg L-1 of struvite (corresponding to a load of about 433 kg d-1). By optimizing the process performance, these findings highlight the potential recovery of resources in pulp mill WWTP, while complying with stringent effluent discharge standards. In addition, further research activities such as pilot-test or detailed laboratory studies may be needed to validate the previous recommendations for industrial scale application.

"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.

Effect of Artificial Solar Radiation on the Die-Off of Pathogen Indicator Organisms in Urban Floods.

In the last decade, flooding has caused the death of over 60,000 people and affected over 900 million people globally. This is expected to increase as a result of climate change, increased populations and urbanisation. Floods can cause infections due to the release of water-borne pathogenic microorganisms from surcharged combined sewers and other sources of fecal contamination. This research contributes to a better understanding of how the occurrence of water-borne pathogens in contaminated shallow water bodies is affected by different environmental conditions. The inactivation of fecal indicator bacteria Escherichia coli was studied in an open stirred reactor, under controlled exposure to simulated sunlight, mimicking the effect of different latitudes and seasons, and different concentrations of total suspended solids (TSS) corresponding to different levels of dilution and runoff. While attachment of bacteria on the solid particles did not take place, the decay rate coefficient, k (d-1), was found to depend on light intensity, I (W m-2), and duration of exposure to sunlight, T (h d-1), in a linear way (k = k D+ 0.03·I and k = k D+ 0.65·T, respectively) and on the concentration of TSS (mg L-1), in an inversely proportional exponential way (k = k D+ 14.57·e-0.02·[TSS] ). The first-order inactivation rate coefficient in dark conditions, k D= 0.37 d-1, represents the effect of stresses other than light. This study suggests that given the sunlight conditions during an urban flood, and the concentration of indicator organisms and TSS, the above equations can give an estimate of the fate of selected pathogens, allowing rapid implementation of appropriate measures to mitigate public health risks.

Sludge reduction by ozone: Insights and modeling of the dose-response effects.

Applying ozone to the return flow in an activated sludge (AS) process is a way for reducing the residual solids production. To be able to extend the activated sludge models to the ozone-AS process, adequate prediction of the tri-atoms effects on the particulate COD fractions is needed. In this study, the biomass inactivation, COD mineralization, and solids dissolution were quantified in batch tests and dose-response models were developed as a function of the reacted ozone doses (ROD). Three kinds of model-sludge were used. S1 was a lab-cultivated synthetic sludge with two components (heterotrophs XH and XP). S2 was a digestate of S1 almost made by the endogenous residues, XP. S3 was from a municipal activated sludge plant. The specific ozone uptake rate (SO3UR, mgO3/gCOD.h) was determined as a tool for characterizing the reactivity of the sludges. SO3UR increased with the XH fraction and decreased with more XP. Biomass inactivation was exponential (e-ß.ROD) as a function of the ROD doses. The percentage of solids reduction was predictable through a linear model (CMiner + Ysol ROD), with a fixed part due to mineralization (CMiner) and a variable part from the solubilization process. The parameters of the models, i.e. the inactivation and the dissolution yields (ß, 0.008-0.029 (mgO3/mgCODini)-1 vs Ysol, 0.5-2.8 mg CODsol/mgO3) varied in magnitude, depending on the intensity of the scavenging reactions and potentially the compactness of the flocs for each sludge.

Nitrification by microalgal-bacterial consortia for ammonium removal in flat panel sequencing batch photo-bioreactors.

Ammonium removal from artificial wastewater by microalgal-bacterial consortia in a flat-panel reactor (FPR1) was compared with a microalgae-only flat-panel reactor (FPR2). The microalgal-bacterial consortia removed ammonium at higher rates (100±18mgNH4+-NL-1d-1) than the microalgae (44±16mgNH4+-NL-1d-1), after the system achieved a stable performance at a 2days hydraulic retention time. Nitrifiers present in the microalgae-bacteria consortia increased the ammonium removal: the ammonium removal rate by nitrifiers and by algae in FPR1 was, respectively, 50(±18) and 49(±22)mgNH4+-NL-1d-1. Apparently ammonium removal by algae was not significantly different between FPR1 and FPR2. The activity of the nitrifiers did not negatively affect the nitrogen uptake by algae, but improved the total ammonium removal rate of FPR1.

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.

Prevalence of 'Candidatus Accumulibacter phosphatis' type II under phosphate limiting conditions.

P-limitation in enhanced biological phosphorus removal (EBPR) systems fed with acetate, has generally been considered as a condition leading to enrichment of organisms of the genotype' Candidatus Competibacter phosphatis' expressing the glycogen-accumulating organisms (GAO) phenotype. Recent studies have demonstrated in short-term experiments that organisms of the genotype 'Candidatus Accumulibacter phosphatis' clade I and II, known to express the polyphosphate-accumulating organisms (PAO) phenotype can switch to the GAO phenotype when poly-P is absent, but are performing the HAc-uptake at lower kinetic rates, where clade I showed the lowest rates. The objective of this study was to verify whether organisms of the genotype 'Candidatus Accumulibacter phosphatis' can also be enriched under P-limiting conditions while expressing a GAO phenotype and more specifically to see which specific clade prevails. A sequencing batch reactor was inoculated with activated sludge to enrich an EBPR culture for a cultivation period of 128 days (16 times the solids retention time) under P-limiting conditions. A mixed culture was obtained comprising of 49 % 'Candidatus Accumulibacter phosphatis' clade II and 46 % 'Candidatus Competibacter phosphatis'. The culture performed a full GAO metabolism for anaerobic HAc-uptake, but was still able to switch to a PAO metabolism, taking up excessive amounts of phosphate during the aerobic phase when it became available in the influent. These findings show that P-limitation, often used as strategy for enrichment of 'Candidatus Competibacter phosphatis', does not always lead to enrichment of only 'Candidatus Competibacter phosphatis'. Furthermore, it demonstrates that 'Candidatus Accumulibacter phosphatis' are able to proliferate in activated sludge systems for periods of up to 128 days or longer when the influent phosphate concentrations are just enough for assimilation purposes and no poly-P is formed. The 'Candidatus Accumulibacter phosphatis' retain the ability to switch to the PAO phenotype, taking up phosphate from the influent as soon as it becomes available.

Nitrite reduction and methanogenesis in a single-stage UASB reactor.

In this study, nitrite reduction and methanogenesis in a single-stage upflow anaerobic sludge blanket (UASB) reactor was investigated, using high-strength synthetic domestic wastewater as substrate. To assess long-term effects and evaluate the mechanisms that allow successful nitrite reduction and methanogenesis in a single-stage UASB, sludge was exposed to relatively high nitrite loading rates (315 ± 13 mgNO(2)(-)-N/(l.d)), using a chemical oxygen demand (COD) to nitrogen ratio of 18 gCOD/gNO(2)(-)-N, and an organic loading rate of 5.4 ± 0.2 gCOD/(l.d). In parallel, the effects of sludge morphology on methanogenesis inhibition were studied by performing short-term batch activity tests at different COD/NO(2)(-)-N ratios with anaerobic sludge samples. In long-term tests, denitrification was practically complete and COD removal efficiency did not change significantly after nitrite addition. Furthermore, methane production only decreased by 13%, agreeing with the reducing equivalents requirement for complete NO(2)(-) reduction to N2. Apparently, the spatial separation of denitrification and methanogenesis zones inside the UASB reactor allowed nitrite reduction and methanogenesis to occur at the same moment. Batch tests showed that granules seem to protect methanogens from nitrite inhibition, probably due to transport limitations. Combined COD and N removal via nitrite in a single-stage UASB reactor could be a feasible technology to treat high-strength domestic wastewater.

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.

Autotrophic nitrogen removal over nitrite in a sponge-bed trickling filter.

Partial nitritation in sponge-bed trickling filters (STF) under natural air circulation was studied in two reactors: STF-1 and STF-2 operated at 30°C with sponge thickness of 0.75 and 1.50cm, respectively. The coexistence of nitrifiers and Anammox bacteria was obtained and attributed to the favorable environment created by the reactors' design and operational regimes. After 114days of operation, the STF-1 had an average NH4(+)-N removal of 69.3% (1.17kgN/m(3)sponged) and a total nitrogen removal of 52.2% (0.88kgN/m(3)sponged) at a Nitrogen Loading Rate (NLR) of 1.68kgN/m(3)sponged and Hydraulic Retention Time (HRT) of 1.71h. The STF-2 showed an average NH4(+)-N removal of 81.6 % (0.77kgN/m(3)sponged) and a total nitrogen removal of 54% (0.51kgN/m(3)sponged), at an NLR of 0.95kgN/m(3)sponged and HRT of 2.96h. The findings suggest that autotrophic nitrogen removal over nitrite in STF systems is a feasible alternative.

Anammox cultivation in a closed sponge-bed trickling filter.

A feasibility study was carried out to assess the cultivation of Anammox bacteria in lab-scale closed sponge-bed trickling filter (CSTF) reactors, namely: CSTF-1 at 20°C and CSTF-2 at 30°C. Stable conditions were reached from day 66 in CSTF-2 and from day 104 in CSTF-1. The early stability of CSTF-2 is attributable to the influence of temperature; nevertheless, by day 405, the nitrogen removal performed by CSTF-1 increased up to similar values of CSTF-2. The maximum total nitrogen removal efficiency was 82% in CSTF-1 and 84% in CSTF-2. After more than 400 days of operation, CSTF-1 and CSTF-2 were capable to attain a total nitrogen removal efficiency of 74±5% and 78±4% with a total nitrogen conversion rate of 1.52 and 1.60kg-N/m(sponge)(3)d, respectively. The proposed technology could be a suitable alternative for mainstream nitrogen removal in post-treatment units via the Anammox conversion pathway.

Impact of salinity on the aerobic metabolism of phosphate-accumulating organisms.

The use of saline water in urban areas for non-potable purposes to cope with fresh water scarcity, intrusion of saline water, and disposal of industrial saline wastewater into the sewerage lead to elevated salinity levels in wastewaters. Consequently, saline wastewater is generated, which needs to be treated before its discharge into surface water bodies. The objective of this research was to study the effects of salinity on the aerobic metabolism of phosphate-accumulating organisms (PAO), which belong to the microbial populations responsible for enhanced biological phosphorus removal (EBPR) in activated sludge systems. In this study, the short-term impact (hours) of salinity (as NaCl) was assessed on the aerobic metabolism of a PAO culture, enriched in a sequencing batch reactor (SBR). All aerobic PAO metabolic processes were drastically affected by elevated salinity concentrations. The aerobic maintenance energy requirement increased, when the salinity concentration rose up to a threshold concentration of 2 % salinity (on a W/V basis as NaCl), while above this concentration, the maintenance energy requirements seemed to decrease. All initial rates were affected by salinity, with the NH4- and PO4-uptake rates being the most sensitive. A salinity increase from 0 to 0.18 % caused a 25, 46, and 63 % inhibition of the O2, PO4, and NH4-uptake rates. The stoichiometric ratios of the aerobic conversions confirmed that growth was the process with the highest inhibition, followed by poly-P and glycogen formation. The study indicates that shock loads of 0.18 % salt, which corresponds to the use or intrusion of about 5 % seawater may severely affect the EBPR process already in wastewater treatment plants not exposed regularly to high salinity concentrations.

Effects of organic carbon source, chemical oxygen demand/N ratio and temperature on autotrophic nitrogen removal.

To assess the feasibility of the Anammox process as a cost-effective post-treatment step for anaerobic sewage treatment, the simultaneous effects of organic carbon source, chemical oxygen demand (COD)/N ratio, and temperature on autotrophic nitrogen removal was studied. In batch experiments, three operating conditions were evaluated at 14, 22 and 30 °C, and at COD/N ratios of 2 and 6. For each operating condition, containing 32 ± 2 mg NH4(+)-N/L and 25 ± 2 mg NO2(-)-N/L, three different substrate combinations were tested to simulate the presence of readily biodegradable and slowly biodegradable organic matter (RBCOD and SBCOD, respectively): (i) acetate (RBCOD); (ii) starch (SBCOD); and (iii) acetate + starch. The observed stoichiometric NO2(-)-N/NH4(+)-N conversion ratios were in the range of 1.19-1.43, and the single or simultaneous presence of acetate and starch did not affect the Anammox metabolism. High Anammox nitrogen removal was observed at 22 °C (77-84%) and 30 °C (73-79%), whereas there was no nitrogen removal at 14 °C; the Anammox activity was strongly influenced by temperature, in spite of the COD source and COD/N ratios applied. These results suggest that the Anammox process could be applied as a nitrogen removal post-treatment for anaerobic sewage systems in warm climates.

Impact of salinity on the anaerobic metabolism of phosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO).

The use of saline water as secondary quality water in urban environments for sanitation is a promising alternative towards mitigating fresh water scarcity. However, this alternative will increase the salinity in the wastewater generated that may affect the biological wastewater treatment processes, such as biological phosphorus removal. In addition to the production of saline wastewater by the direct use of saline water in urban environments, saline wastewater is also generated by some industries. Intrusion of saline water into the sewers is another source of salinity entering the wastewater treatment plant. In this study, the short-term effects of salinity on the anaerobic metabolism of phosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) were investigated to assess the impact of salinity on enhanced biological phosphorus removal. Hereto, PAO and GAO cultures enriched at a relatively low salinity level (0.02 % W/V) were exposed to salinity concentrations of up to 6 % (as NaCl) in anaerobic batch tests. It was demonstrated that both PAO and GAO are affected by higher salinity levels, with PAO being the more sensitive organisms to the increasing salinity. The maximum acetate uptake rate of PAO decreased by 71 % when the salinity increased from 0 to 1 %, while that of GAO decreased by 41 % for the same salinity increase. Regarding the stoichiometry of PAO, a decrease in the P-release/HAc uptake ratio accompanied with an increase in the glycogen consumption/HAc uptake ratio was observed for PAO when the salinity increased from 0 to 2 % salinity, indicating a metabolic shift from a poly-P-dependent to a glycogen-dependent metabolism. The anaerobic maintenance requirements of PAO and GAO increased as the salinity concentrations risen up to 4 % salinity.

Thermophilic biological nitrogen removal in industrial wastewater treatment.

Nitrification is an integral part of biological nitrogen removal processes and usually the limiting step in wastewater treatment systems. Since nitrification is often considered not feasible at temperatures higher than 40 °C, warm industrial effluents (with operating temperatures higher than 40 °C) need to be cooled down prior to biological treatment, which increases the energy and operating costs of the plants for cooling purposes. This study describes the occurrence of thermophilic biological nitrogen removal activity (nitritation, nitratation, and denitrification) at a temperature as high as 50 °C in an activated sludge wastewater treatment plant treating wastewater from an oil refinery. Using a modified two-step nitrification-two-step denitrification mathematical model extended with the incorporation of double Arrhenius equations, the nitrification (nitrititation and nitratation) and denitrification activities were described including the cease in biomass activity at 55 °C. Fluorescence in situ hybridization (FISH) analyses revealed that Nitrosomonas halotolerant and obligatehalophilic and Nitrosomonas oligotropha (known ammonia-oxidizing organisms) and Nitrospira sublineage II (nitrite-oxidizing organism (NOB)) were observed using the FISH probes applied in this study. In particular, this is the first time that Nitrospira sublineage II, a moderatedly thermophilic NOB, is observed in an engineered full-scale (industrial) wastewater treatment system at temperatures as high as 50 °C. These observations suggest that thermophilic biological nitrogen removal can be attained in wastewater treatment systems, which may further contribute to the optimization of the biological nitrogen removal processes in wastewater treatment systems that treat warm wastewater streams.

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.

Incorporating microbial ecology into the metabolic modelling of polyphosphate accumulating organisms and glycogen accumulating organisms.

In the enhanced biological phosphorus removal (EBPR) process, the competition between polyphosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO) has been studied intensively in recent years by both microbiologists and engineers, due to its important effects on phosphorus removal performance and efficiency. This study addresses the impact of microbial ecology on assessing the PAO-GAO competition through metabolic modelling, focussing on reviewing recent developments, discussion of how the results from molecular studies can impact the way we model the process, and offering perspectives for future research opportunities based on unanswered questions concerning PAO and GAO metabolism. Indeed, numerous findings that are seemingly contradictory could in fact be explained by the metabolic behaviour of different sub-groups of PAOs and/or GAOs exposed to different environmental and operational conditions. Some examples include the glycolysis pathway (i.e. Embden-Meyerhof-Parnas (EMP) vs. Entner-Doudoroff (ED)), denitrification capacity, anaerobic tricarboxylic acid (TCA) cycle activity and PAOs' ability to adjust their metabolism to e.g. a GAO-like metabolism. Metabolic modelling may further yield far-reaching influences on practical applications as well, and serves as a bridge between molecular/biochemical research studies and the optimisation of wastewater treatment plant operation.