Microbiome structure and function in parallel full-scale aerobic granular sludge and activated sludge processes.

Aerobic granular sludge (AGS) and conventional activated sludge (CAS) are two different biological wastewater treatment processes. AGS consists of self-immobilised microorganisms that are transformed into spherical biofilms, whereas CAS has floccular sludge of lower density. In this study, we investigated the treatment performance and microbiome dynamics of two full-scale AGS reactors and a parallel CAS system at a municipal WWTP in Sweden. Both systems produced low effluent concentrations, with some fluctuations in phosphate and nitrate mainly due to variations in organic substrate availability. The microbial diversity was slightly higher in the AGS, with different dynamics in the microbiome over time. Seasonal periodicity was observed in both sludge types, with a larger shift in the CAS microbiome compared to the AGS. Groups important for reactor function, such as ammonia-oxidising bacteria (AOB), nitrite-oxidising bacteria (NOB), polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs), followed similar trends in both systems, with higher relative abundances of PAOs and GAOs in the AGS. However, microbial composition and dynamics differed between the two systems at the genus level. For instance, among PAOs, Tetrasphaera was more prevalent in the AGS, while Dechloromonas was more common in the CAS. Among NOB, Ca. Nitrotoga had a higher relative abundance in the AGS, while Nitrospira was the main nitrifier in the CAS. Furthermore, network analysis revealed the clustering of the various genera within the guilds to modules with different temporal patterns, suggesting functional redundancy in both AGS and CAS. KEY POINTS: • Microbial community succession in parallel full-scale aerobic granular sludge (AGS) and conventional activated sludge (CAS) processes. • Higher periodicity in microbial community structure in CAS compared to in AGS. • Similar functional groups between AGS and CAS but different composition and dynamics at genus level.

Primary filtration of municipal wastewater with sludge fermentation - Impacts on biological nutrient removal.

Primary filtration is a compact pre-treatment process for municipal wastewater, which can lead to high removal of total suspended solids (TSS) if polymer is added prior to filtration. Extensive carbon removal with rotating belt filter (RBF) can be combined with filter primary sludge fermentation at ambient temperature, in order to produce volatile fatty acids (VFAs) as carbon source for biological nutrient removal (BNR). This process was implemented at large pilot-scale and operated for more than a year. The results showed that the RBF efficiently removed particles >10 µm, and that the TSS removal had a strong linear correlation to the influent TSS concentration. Fermentation of the sludge at ambient temperature and five days retention time and addition of the fermentate to the wastewater could nearly double the VFA concentration in the wastewater by adding 31 ± 9 mg VFA-COD/L. Meanwhile, an increase of 2 mg/L of ammonium nitrogen, and 0.7 mg /L of phosphate phosphorus would be added to the wastewater with the fermentate. Adding the fermented sludge to the wastewater stream and removing the particles with RBF makes it possible to utilize nearly all the produced VFAs for BNR, and the feasibility of this configuration was shown at pilot-scale. According to simulations of subsequent BNR, the pre-treatment would lead to lower effluent total nitrogen concentrations. Alternatively, the required BNR volume could be reduced by 11-18 %. The estimated total biogas production was similar for pre-treatment with primary settler and RBF with fermentation. RBF without fermentation gave the most favourable energy balance, but did not reach the same low effluent value for total nitrogen as RBF with fermentation.

Case study of aerobic granular sludge and activated sludge-Energy usage, footprint, and nutrient removal.

This study demonstrates a comparison of energy usage, land footprint, and volumetric requirements of municipal wastewater treatment with aerobic granular sludge (AGS) and conventional activated sludge (CAS) at a full-scale wastewater treatment plant characterized by large fluctuations in nutrient loadings and temperature. The concentration of organic matter in the influent to the AGS was increased by means of hydrolysis and bypassing the pre-settler. Both treatment lines produced effluent concentrations below 5 mg BOD7 L-1 , 10 mg TN L-1 , and 1 mg TP L-1 , by enhanced biological nitrogen- and phosphorus removal. In this case study, the averages of volumetric energy usage over 1 year were 0.22 ± 0.08 and 0.26 ± 0.07 kWh m-3 for the AGS and CAS, respectively. A larger difference was observed for the energy usage per reduced population equivalents (P.E.), which was on average 0.19 ± 0.08 kWh P.E.-1 for the AGS and 0.30 ± 0.08 kWh P.E.-1 for the CAS. However, both processes had the potential for decreased energy usage. Over 1 year, both processes showed similar fluctuations in energy usage, related to variations in loading, temperature, and DO. The AGS had a lower specific area, 0.3 m2  m-3 d-1 , compared to 0.6 m2  m-3 d-1 of the CAS, and also a lower specific volume, 1.3 m3  m-3 d-1 compared to 2.0 m3  m-3 d-1 . This study confirms that AGS at full-scale can be compact and still have comparable energy usage as CAS. PRACTITIONER POINTS: Full-scale case study comparison of aerobic granular sludge (AGS) and conventional activated sludge (CAS), operated in parallel. AGS had 50 % lower footprint compared to CAS. Energy usage was lower in the AGS, but both processes had potential to improve the energy usage efficiency. Both processes showed low average effluent concentrations.

Seasonal variations in acidogenic fermentation of filter primary sludge.

Primary treatment of municipal wastewater by rotating belt filtration followed by hydrolysis and acidogenic fermentation of the filter primary sludge (FPS) at ambient temperature was studied at pilot-scale during one year. The seasonal variations of volatile fatty acids (VFAs), nutrient release and soluble COD production as well as microbial community assembly were assessed, leading to novel findings for fermentation at ambient temperature. The reproducibility of VFA production performance was first established by operating the two fermentation reactors under the same conditions, showing similar results regarding VFA production and microbial community structure. One year of operation at 5 d retention time (RT) and 16-29 °C resulted in an average VFA yield of 180±35 mg COD/g VSin and soluble COD yield of 242±40 mg COD/g VSin. The VFA formation was temperature-dependent, with Ï´=1.033±0.005 ( [Formula: see text] . The seasonal variations of the acetic and propionic acid productions were pronounced, whereas the productions of VFAs with longer chains were more stable regardless of temperature. The community structure of the reactor microbiomes was also clearly affected by season and temperature and linked with the production spectrum of VFAs. The ammonium and phosphate releases were stable during the year, leading to a decrease in ratios of soluble COD to NH4+-N and PO43--P during winter. The soluble COD yield was 11% and 27% higher at 5 d RT compared to 3 and 2 d RT respectively, but the corresponding volumetric productivities were lower. The dissimilarities between microbiomes in influent FPS and fermenters were significant even at a short RT of 2 d, and increased with longer RT of 3 and 5 d, primarily caused by selection of bacteria within Bacteroidota in the fermentation reactors.

Removal of organic micropollutants from municipal wastewater by aerobic granular sludge and conventional activated sludge.

Removal performances of organic micropollutants by conventional activated sludge (CAS) and aerobic granular sludge (AGS) were investigated at a full-scale wastewater treatment plant. Lab-scale kinetic experiments were performed to assess the micropollutant transformation rates under oxic and anoxic conditions. Transformation rates were used to model the micropollutant removal in the full-scale processes. Metagenomic sequencing was used to compare the microbial communities and antimicrobial resistance genes of the CAS and AGS systems. Higher transformation ability was observed for CAS compared to AGS for most compounds, both at the full-scale plant and in the complementary batch experiments. Oxic conditions supported the transformation of several micropollutants with faster and/or comparable rates compared to anoxic conditions. The estimated transformation rates from batch experiments adequately predicted the removal for most micropollutants in the full-scale processes. While the compositions in microbial communities differed between AGS and CAS, the full-scale biological reactors shared similar resistome profiles. Even though granular biomass showed lower potential for micropollutant transformation, AGS systems had somewhat higher gene cluster diversity compared to CAS, which could be related to a higher functional diversity. Micropollutant exposure to biomass or mass transfer limitations, therefore played more important roles in the observed differences in OMP removal.

Disturbance-based management of ecosystem services and disservices in partial nitritation-anammox biofilms.

The resistance and resilience provided by functional redundancy, a common feature of microbial communities, is not always advantageous. An example is nitrite oxidation in partial nitritation-anammox (PNA) reactors designed for nitrogen removal in wastewater treatment, where suppression of nitrite oxidizers like Nitrospira is sought. In these ecosystems, biofilms provide microhabitats with oxygen gradients, allowing the coexistence of aerobic and anaerobic bacteria. We designed a disturbance experiment where PNA biofilms, treating water from a high-rate activated sludge process, were constantly or intermittently exposed to anaerobic sidestream wastewater, which has been proposed to inhibit nitrite oxidizers. With increasing sidestream exposure we observed decreased abundance, alpha-diversity, functional versatility, and hence functional redundancy, among Nitrospira in the PNA biofilms, while the opposite patterns were observed for anammox bacteria within Brocadia. At the same time, species turnover was observed for aerobic ammonia-oxidizing Nitrosomonas populations. The different exposure regimens were associated with metagenomic assembled genomes of Nitrosomonas, Nitrospira, and Brocadia, encoding genes related to N-cycling, substrate usage, and osmotic stress response, possibly explaining the three different patterns by niche differentiation. These findings imply that disturbances can be used to manage the functional redundancy of biofilm microbiomes in a desirable direction, which should be considered when designing operational strategies for wastewater treatment.

A relationship between phages and organic carbon in wastewater treatment plant effluents.

With stringent effluent requirements and the implementation of new processes for micropollutant removal, it is increasingly important for wastewater treatment plants (WWTPs) to understand the factors affecting effluent quality. Phages (viruses infecting prokaryotes) are abundant in the biological treatment processes. They can contribute to organic carbon in the treated effluent both because they are organic in nature and occur in the effluent and because they cause lysis of microorganisms. Today very little is known about the effects of phages on effluent quality. The goal of this study was, therefore, to determine the relationship between phages and organic carbon in WWTP effluents. We also examined the diversity, taxonomy, and host-association of DNA phages using metagenomics. Effluent samples were collected from four WWTPs treating municipal wastewater. Significant differences in both organic carbon and virus-like particle concentrations were observed between the plants and there was a linear relationship between the two parameters. The phage communities were diverse with many members being taxonomically unclassified. Putative hosts were dominated by bacteria known to be abundant in activated sludge systems such as Comamonadaceae. The composition of phages differed between the WWTPs, suggesting that local conditions shape the communities. Overall, our findings suggest that the abundance and composition of phages are related to effluent quality. Thus, there is a need for further research clarifying the association between phage dynamics and WWTP function.

Ex-situ manometric activity test for evaluation of mainstream anammox in Moving Bed Biofilm Reactors.

Implementing anammox based processes in the mainstream at municipal wastewater treatment plants is challenging and requires carefully constructed control strategies in order to be successful. A method to measure the specific anammox activity was further developed and implemented in the mainstream anammox pilot plant at Sjölunda Wastewater Treatment Plant. The method was found to be reliable and reproducible. The influence of initial nitrite and ammonium concentrations on the activity as well as temperature dependency were analysed. The continuously monitored manometric batch tests were performed on Moving Bed Biofilm Reactor carriers type K1®. The activity showed a dependency of initial nitrite concentrations below 75 mg N L-1 whereas the activity was independent of initial nitrite concentrations in the interval of 75-125 mg N L-1. Temperature dependency of the specific anammox activity as expressed in activation energy Ea increased at lower temperatures (10-20°C) compared to higher temperatures (20-30°C). Decreasing temperature from 30°C to 10°C resulted in 95-98% loss of the anammox activity.

The role of the external mass transfer resistance in nitrite oxidizing bacteria repression in biofilm-based partial nitritation/anammox reactors.

A model-based study was developed to analyse the behaviour of Moving Bed Biofilm Reactor (MBBR) and Integrated Fixed-Film Activated Sludge (IFAS) reactor configurations for the removal of nitrogen in the main water line of municipal wastewater treatment plants via partial nitritation/anammox (PN/AMX). The basic principles and underlying mechanisms linking operating conditions to process performance were investigated, with particular focus on nitrite oxidizing bacteria (NOB) repression and resulting volumetric conversion rates. The external mass transfer resistance is a major factor differentiating granular sludge PN/AMX processes from MBBR or IFAS systems. The external mass transfer resistance was found to promote the metabolic coupling between anammox (AMX) and ammonia oxidizing bacteria (AOB), crucial for NOB repression in the biofilm. Operation at low bulk DO prevents NOB proliferation in the flocs of IFAS systems as AMX activity limits nitrite availability (the so-called AMX nitrite sink). Importantly, the effectiveness of the AMX nitrite sink strongly depends on the AMX sensitivity to oxygen. Also, over a broad range of operational conditions, the seeding of AOB from the biofilm played a crucial role in maintaining their activity in the flocs. From a practical perspective, while low DO promotes NOB repression, lower nitrogen loads have to be applied to maintain the same effluent quality. Thus, a trade-off between NOB repression and volumetric conversion capacity needs to be defined. To this end, IFAS allow for higher volumetric rates, but the window of operating conditions with effective NOB repression is smaller than that for MBBR. Ultimately, this study identified the principles controlling NOB in MBBR and IFAS systems and the key differences with granular reactors, allowing for the interpretation of (seemingly contradictory) published experimental results.

Long-term stability of partial nitritation-anammox for treatment of municipal wastewater in a moving bed biofilm reactor pilot system.

Nitrogen removal from the mainstream of municipal wastewater with partial nitritation-anammox (PNA) would be highly beneficial with regard to the uses of energy and organic carbon. However, the challenges of process instability, low nitrogen removal rates (NRR) and unwanted aerobic nitrite oxidation need to be solved to reach large-scale implementation. Here, we have operated pilot-scale moving bed biofilm reactors (MBBRs) for mainstream treatment, together with sidestream treatment of sludge liquor from anaerobic digestors, for over 900 days to investigate process stability, reactor performance and microbial community structure at realistic conditions. The MBBR biofilm contained stable and high relative abundances of anammox bacteria (10-32%) consisting of two major Brocadia sp. populations, and several populations of aerobic ammonia-oxidising bacteria (AOB) within Nitrosomonas sp. (0.2-3.1%), as assessed by 16S rDNA amplicon sequencing. In addition, nitrite-oxidising bacteria (NOB) consisting of Nitrospira sp. (0.4-0.8%) and Nitrotoga sp. (up to 0.4%) were present. Nitrogen was removed at a peak rate of 0.66 g N m-2 d-1 (0.13 kg N m-3 d-1) with a nitrate production over ammonium consumption of 15% by the NOB, at operation with continuous aeration at 15 °C. However, during most periods with continuous aeration, the NRR was lower (≈ 0.45 g N m-2 d-1), with larger relative nitrate production (≈40%), presumably due to problems to maintain stable residual ammonium concentrations during wet-weather mainstream flows. Changing reactor operation to intermittent aeration decreased the NRR but did not help in suppressing the NOB. The study shows that with MBBRs, stable mainstream PNA can be attained at realistic NRR, but with need for post-treatment of nitrate, since effective NOB suppression was hard to achieve.

Opportunities for microbial electrochemistry in municipal wastewater treatment--an overview.

Microbial bioelectrochemical systems (BESs) utilize living microorganisms to drive oxidation and reduction reactions at solid electrodes. BESs could potentially be used at municipal wastewater treatment plants (WWTPs) to recover the energy content of organic matter, to produce chemicals useful at the site, or to monitor and control biological treatment processes. In this paper, we review bioelectrochemical technologies that could be applied for municipal wastewater treatment. Sjölunda WWTP in Malmö, Sweden, is used as an example to illustrate how the different technologies potentially could be integrated into an existing treatment plant and the impact they could have on the plant's utilization of energy and chemicals.