Aerobic granular sludge phosphate removal using glucose.

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, poly­hydroxy-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.

Enhancing phosphorus removal of photogranules by incorporating polyphosphate accumulating organisms.

Photogranules are a novel wastewater treatment technology that can utilize the sun's energy to treat water with lower energy input and have great potential for nutrient recovery applications. They have been proven to efficiently remove nitrogen and carbon but show lower conversion rates for phosphorus compared to established treatment systems, such as aerobic granular sludge. In this study, we successfully introduced polyphosphate accumulating organisms (PAOs) to an established photogranular culture. We operated photobioreactors in sequencing batch mode with six cycles per day and alternating anaerobic (dark) and aerobic (light) phases. We were able to increase phosphorus removal/recovery by 6 times from 5.4 to 30 mg/L/d while maintaining similar nitrogen and carbon removal compared to photogranules without PAOs. To maintain PAOs activity, alternating anaerobic feast and aerobic famine conditions were required. In future applications, where aerobic conditions are dependent on in-situ oxygenation via photosynthesis, the process will rely on sunlight availability. Therefore, we investigated the feasibility of the process under diurnal cycles with a 12-h anaerobic phase during nighttime and six short cycles during the 12 h daytime. The 12-h anaerobic phase had no adverse effect on the PAOs and phototrophs. Due to the extension of one anaerobic phase to 12 h the six aerobic phases were shortened by 47% and consequently decreased the light hours per day. This resulted in a decrease of phototrophs, which reduced nitrogen removal and biomass productivity up to 30%. Finally, we discuss and suggest strategies to apply PAO-enriched photogranules at large-scale.

Glycerol conversion by aerobic granular sludge.

Glycerol is abundantly present in wastewater from industries such as biodiesel production facilities. Glycerol is also a potential carbon source for microbes that are involved in wastewater nutrient removal processes. The conversion of glycerol in biological phosphorus removal of aerobic granular sludge processes has not been explored to date. The current study describes glycerol utilization by aerobic granular sludge and enhanced biological phosphorus removal (EBPR). Robust granules with good phosphorus removal capabilities were formed in an aerobic granular sludge sequencing batch reactor fed with glycerol. The interaction between the fermentative conversion of glycerol and product uptake by polyphosphate accumulating organisms (PAO) was studied using stoichiometric and microbial community analysis. Metagenomic, metaproteomic and microscopic analysis identified a community dominated by Actinobacteria (Tessaracoccus and Micropruina) and a typical PAO known as Ca. Accumulibacter. Glycerol uptake facilitator (glpF) and glycerol kinase (glpK), two proteins involved in the transport of glycerol into the cellular metabolism, were only observed in the genome of the Actinobacteria. The anaerobic conversion appeared to be a combination of a substrate fermentation and product uptake-type reaction. Initially, glycerol fermentation led mainly to the production of 1,3-propanediol (1,3-PDO) which was not taken up under anaerobic conditions. Despite the aerobic conversion of 1,3-PDO stable granulation was observed. Over time, 1,3-PDO production decreased and complete anaerobic COD uptake was observed. The results demonstrate that glycerol-containing wastewater can effectively be treated by the aerobic granular sludge process and that fermentative and polyphosphate accumulating organisms can form a food chain in glycerol-based EBPR processes.

Trehalose as an osmolyte in Candidatus Accumulibacter phosphatis.

Candidatus Accumulibacter phosphatis is an important microorganism for enhanced biological phosphorus removal (EBPR). In a previous study, we found a remarkable flexibility regarding salinity, since this same microorganism could thrive in both freshwater- and seawater-based environments, but the mechanism for the tolerance to saline conditions remained unknown. Here, we identified and described the role of trehalose as an osmolyte in Ca. Accumulibacter phosphatis. A freshwater-adapted culture was exposed to a single batch cycle of hyperosmotic and hypo-osmotic shock, which led to the release of trehalose up to 5.34 mg trehalose/g volatile suspended solids (VSS). Long-term adaptation to 30% seawater-based medium in a sequencing batch reactor (SBR) gave a stable operation with complete anaerobic uptake of acetate and propionate along with phosphate release of 0.73 Pmol/Cmol, and complete aerobic uptake of phosphate. Microbial analysis showed Ca. Accumulibacter phosphatis clade I as the dominant organism in both the freshwater- and seawater-adapted cultures (> 90% presence). Exposure of the seawater-adapted culture to a single batch cycle of hyperosmotic incubation and hypo-osmotic shock led to an increase in trehalose release upon hypo-osmotic shock when higher salinity is used for the hyperosmotic incubation. Maximum trehalose release upon hypo-osmotic shock was achieved after hyperosmotic incubation with 3× salinity increase relative to the salinity in the SBR adaptation reactor, resulting in the release of 11.9 mg trehalose/g VSS. Genome analysis shows the possibility of Ca. Accumulibacter phosphatis to convert glycogen into trehalose by the presence of treX, treY, and treZ genes. Addition of trehalose to the reactor led to its consumption, both during anaerobic and aerobic phases. These results indicate the flexibility of the metabolism of Ca. Accumulibacter phosphatis towards variations in salinity. KEY POINTS: • Trehalose is identified as an osmolyte in Candidatus Accumulibacter phosphatis. • Ca. Accumulibacter phosphatis can convert glycogen into trehalose. • Ca. Accumulibacter phosphatis clade I is present and active in both seawater and freshwater.

Biological phosphorus removal in seawater-adapted aerobic granular sludge.

Seawater can be introduced or intrude in sewer systems and can thereby negatively influence biological wastewater treatment processes. Here we studied the impact of artificial seawater on the enhanced biological phosphate removal (EBPR) process performance by aerobic granular sludge (AGS) with synthetic wastewater. Process performance, granule stability and characteristics as well as microbial community of a seawater-adapted AGS system were observed. In seawater conditions strong and stable granules formed with an SVI5 of 20 mL/g and a lower abrasion coefficient than freshwater-adapted granules. Complete anaerobic uptake of acetate, anaerobic phosphate release of 59.5 ± 4.0 mg/L PO43--P (0.35 mg P/mg HAc), and an aerobic P-uptake rate of 3.1 ± 0.2 mg P/g VSS/h were achieved. The dominant phosphate accumulating organisms (PAO) were the same as for freshwater-based aerobic granular sludge systems with a very high enrichment of Ca. Accumulibacter phosphatis clade I, and complete absence of glycogen accumulating organisms. The effect of osmotic downshocks was tested by replacing influent seawater-based medium by demineralized water-based medium. A temporary decrease of the salinity in the reactor led to a decreased phosphate removal activity, while it also induced a rapid release of COD by the sludge, up to 45.5 ± 1.7 mg COD/g VSS. This is most likely attributed to the release of osmolytes by the cells. Recovery of activity was immediately after restoring the seawater feeding. This work shows that functioning of aerobic granular sludge in seawater conditions is as stable as in freshwater conditions, while past research has shown a negative effect on operation of AGS processes with NaCl-based wastewater at the same salinity as seawater.

Stress-induced assays for polyphosphate quantification by uncoupling acetic acid uptake and anaerobic phosphorus release.

Phosphorus has been successfully eliminated from wastewater by biological techniques of enhanced biological phosphorus removal (EBPR) process, which relies on a specific microbiota of polyphosphate accumulating organisms (PAOs) that accumulate phosphate as polyphosphates (poly-P). Most methods for quantification of poly-P pools suffer from low accuracy and specificity. More powerful and implementable P-analysis tools are required for poly-P quantification, which will help in improved evaluation of processes in laboratory and full-scale EBPR systems. This study developed two methods to quantify poly-P pools by releasing the poly-P from the cell. During experimental optimization, it was observed that two different methods resulted in the highest phosphate release: acetate addition at a pH of 4.8 and exposure to EDTA solution with a concentration of 1% (w/v). Treatment with EDTA resulted in a higher amount of phosphate release from all sludge samples. This was characterized by P-release of 1.5-2.5 times higher than the control tests. In contrast, treatments with acetate addition at a low pH exhibited that P-release depended upon the types of the sludge samples. The highest P-release amount and rate were found in highly-enriched PAO sludge samples, but with fewer influences on the sludge collected from WWTP, which may be attributed to the lower fraction of PAOs in the sludge. Overall, the proposed approaches to quantify the poly-P concentration can be applied in simple, user-friendly, and cost-effective ways.