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In microbial electrolysis cells (MECs), microbial communities catalyze conversions between dissolved organic compounds, electrical energy, and energy carriers such as hydrogen and methane. Bacteria and archaea, which catalyze reactions on the anode and cathode of MECs, interact with phages; however, phage communities have previously not been examined in MECs. In this study, we used metagenomic sequencing to study prokaryotes and phages in nine MECs. A total of 852 prokaryotic draft genomes representing 278 species, and 1476 phage contigs representing 873 phage species were assembled. Among high quality prokaryotic genomes (>95% completion), 55% carried a prophage, and the three Desulfobacterota spp. that dominated the anode communities all carried prophages. Geobacter anodireducens , one of the bacteria dominating the anode communities, carried a CRISPR spacer showing evidence of a previous infection by a Peduoviridae phage present in the liquid of some MECs. Methanobacteriaceae spp. and an Acetobacterium sp., which dominated the cathodes, had several associations with Straboviridae spp. The results of this study show that phage communities in MECs are diverse and interact with functional microorganisms on both the anode and cathode., Competing Interests: The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Aerobic granular sludge is a compact and efficient biofilm process used for wastewater treatment which has received much attention and is currently being implemented worldwide. The microbial associations and their ecological implications occurring during granule development, especially those involving inter-kingdom interactions, are poorly understood. In this work, we monitored the prokaryote and eukaryote community composition and structure during the granulation of activated sludge for 343 days in a sequencing batch reactor (SBR) and investigated the influence of abiotic and biotic factors on the granule development. Sludge granulation was accomplished with low-wash-out dynamics at long settling times, allowing for the microbial communities to adapt to the SBR environmental conditions. The sludge granulation and associated changes in microbial community structure could be divided into three stages: floccular, intermediate, and granular. The eukaryotic and prokaryotic communities showed parallel successional dynamics, with three main sub-communities identified for each kingdom, dominating in each stage of sludge granulation. Although inter-kingdom interactions were shown to affect community succession during the whole experiment, during granule development random factors like the availability of settlement sites or drift acquired increasing importance. The prokaryotic community was more affected by deterministic factors, including reactor conditions, while the eukaryotic community was to a larger extent shaped by biotic interactions (including inter-kingdom interactions) and stochasticity., (© 2024. The Author(s).)

Geosmin and 2-methylisoborneol (MIB) are known to cause taste-and-odour problems in recirculating aquaculture systems (RAS). Both geosmin and MIB are microbial metabolites belonging to terpenoids. Precursors for terpenoids are biosynthesized via the methylerythritol phosphate (MEP) and the mevalonate (MVA) pathways. We carried out a metagenomic analysis of 50 samples from five RAS to investigate terpenoid biosynthesis and metabolic potential for geosmin and MIB production in RAS microbiomes. A total of 1008 metagenome-assembled genomes (MAGs) representing 26 bacterial and three archaeal phyla were recovered. Although most archaea are thought to use the MVA pathway for terpenoid precursor biosynthesis, an Iainarchaeota archaeal MAG is shown to harbour a complete set of genes encoding the MEP pathway but lacking genes associated with the MVA pathway. In this study, a total of 16 MAGs affiliated with five bacterial phyla (Acidobacteriota, Actinobacteriota, Bacteroidota, Chloroflexota, and Myxococcota) were identified as possessing potential geosmin or MIB synthases. These putative taste and odour producers were diverse, many were taxonomically unidentified at the genus or species level, and their relative abundance differed between the investigated RAS farms. The metagenomic study of the RAS microbiomes revealed a previously unknown phylogenetic diversity of the potential to produce geosmin and MIB., (© 2024 The Author(s). Environmental Microbiology published by John Wiley & Sons Ltd.)

The deterioration of fibre-reinforced sprayed concrete was studied in the Oslofjord subsea tunnel (Norway). At sites with intrusion of saline groundwater resulting in biofilm growth, the concrete exhibited significant concrete deterioration and steel fibre corrosion. Using amplicon sequencing and shotgun metagenomics, the microbial taxa and surveyed potential microbial mechanisms of concrete degradation at two sites over five years were identified. The concrete beneath the biofilm was investigated with polarised light microscopy, scanning electron microscopy and X-ray diffraction. The oxic environment in the tunnel favoured aerobic oxidation processes in nitrogen, sulfur and metal biogeochemical cycling as evidenced by large abundances of metagenome-assembled genomes (MAGs) with potential for oxidation of nitrogen, sulfur, manganese and iron, observed mild acidification of the concrete, and the presence of manganese- and iron oxides. These results suggest that autotrophic microbial populations involved in the cycling of several elements contributed to the corrosion of steel fibres and acidification causing concrete deterioration., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

In a microbial electrolysis cell (MEC), the oxidization of organic compounds is facilitated by an electrogenic biofilm on the anode surface. The biofilm community composition determines the function of the system. Both deterministic and stochastic factors affect the community, but the relative importance of different factors is poorly understood. Anode material is a deterministic factor as materials with different properties may select for different microorganisms. Ecological drift is a stochastic factor, which is amplified by dispersal limitation between communities. Here, we compared the effects of three anode materials (graphene, carbon cloth, and nickel) with the effect of dispersal limitation on the function and biofilm community assembly. Twelve MECs were operated for 56 days in four hydraulically connected loops and shotgun metagenomic sequencing was used to analyse the microbial community composition on the anode surfaces at the end of the experiment. The anode material was the most important factor affecting the performance of the MECs, explaining 54-80 % of the variance observed in peak current density, total electric charge generation, and start-up lag time, while dispersal limitation explained 10-16 % of the variance. Carbon cloth anodes had the highest current generation and shortest lag time. However, dispersal limitation was the most important factor affecting microbial community structure, explaining 61-98 % of the variance in community diversity, evenness, and the relative abundance of the most abundant taxa, while anode material explained 0-20 % of the variance. The biofilms contained nine Desulfobacterota metagenome-assembled genomes (MAGs), which made up 64-89 % of the communities and were likely responsible for electricity generation in the MECs. Different MAGs dominated in different MECs. Particularly two different genotypes related to Geobacter benzoatilyticus competed for dominance on the anodes and reached relative abundances up to 83 %. The winning genotype was the same in all MECs that were hydraulically connected irrespective of anode material used., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Sustainability assessment using multicriteria analysis (MCA) is a structured way of including criteria from the three sustainability dimensions (environmental, economic, and social) when comparing different alternatives. A problem with the conventional MCA methods is that the consequences of the weights given to different criteria are not transparent. Here, we amend the simple additive weighting MCA method with weighted score ratios (WSRs), which are used during the sustainability assessment to show how the weights affect the valuation of the criteria (e.g., cost per kg CO 2 e). This enables comparisons to other sustainability assessments and reference values from society, which increases the transparency and can make weighting more objective. We applied the method to a comparison of technologies for removal of pharmaceutical residues from wastewater. Due to growing concern about the effects that pharmaceutical residues can have on our environment, implementations of advanced technologies are increasing. However, they entail high requirements of energy and resources. Therefore, many aspects must be considered to make a sustainable choice of technology. In this study, a sustainability assessment was performed of ozonation, powdered activated carbon and granular activated carbon for removal of pharmaceutical residues at a large wastewater treatment plant (WWTP) in Sweden. The outcome showed that powdered activated carbon is the least sustainable choice for the studied WWTP. Whether ozonation or granular activated carbon is most sustainable depends on how climate impact and energy use are valued. The total sustainability of ozonation is affected by how the electricity is assumed to be produced, whereas for granular activated carbon it depends on whether the carbon source is of renewable or fossil origin. Using WSRs allowed the participants in the assessment to make conscious choices on how they weighted different criteria in relation to how these criteria are valued in society at large., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Granular sludge is a biofilm process used for wastewater treatment which is currently being implemented worldwide. It is important to understand how disturbances affect the microbial community and performance of reactors. Here, two acetate-fed replicate reactors were inoculated with acclimatized sludge and the reactor performance, and the granular sludge microbial community succession were studied for 149 days. During this time, the microbial community was challenged by periodically removing half of the reactor biomass, subsequently increasing the food-to-microorganism (F/M) ratio. Diversity analysis together with null models show that overall, the microbial communities were resistant to the disturbances, observing some minor effects on polyphosphate-accumulating and denitrifying microbial communities and their associated reactor functions. Community turnover was driven by drift and random granule loss, and stochasticity was the governing ecological process for community assembly. These results evidence the aerobic granular sludge process as a robust system for wastewater treatment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

The occurrence of pharmaceuticals in the aquatic environment is a global water quality challenge for several reasons, such as deleterious effects on ecological and human health, antibiotic resistance development, and endocrine-disrupting effects on aquatic organisms. To optimize their removal from the water cycle, understanding the processes during biological wastewater treatment is crucial. Time-of-flight secondary ion mass spectrometry imaging was successfully applied to investigate and analyze the distribution of pharmaceuticals as well as endogenous molecules in the complex biological matrix of biofilms for wastewater treatment. Several compounds and their localization were identified in the biofilm section, including citalopram, ketoconazole, ketoconazole transformation products, and sertraline. The images revealed the pharmaceuticals gathered in distinct sites of the biofilm matrix. While citalopram penetrated the biofilm deeply, sertraline remained confined in its outer layer. Both pharmaceuticals seemed to mainly colocalize with phosphocholine lipids. Ketoconazole concentrated in small areas with high signal intensity. The approach outlined here presents a powerful strategy for visualizing the chemical composition of biofilms for wastewater treatment and demonstrates its promising utility for elucidating the mechanisms behind pharmaceutical and antimicrobial removal in biological wastewater treatment.

In single-chamber microbial electrolysis cells (MECs), organic compounds are oxidized at the anode, liberating electrons that are used for hydrogen evolution at the cathode. Microbial communities on the anode and cathode surfaces and in the bulk liquid determine the function of the MEC. The communities are complex, and their assembly processes are poorly understood. We investigated MEC performance and community composition in nine MECs with a carbon cloth anode and a cathode of carbon nanoparticles, titanium, or stainless steel. Differences in lag time during the startup of replicate MECs suggested that the initial colonization by electrogenic bacteria was stochastic. A network analysis revealed negative correlations between different putatively electrogenic Deltaproteobacteria on the anode. Proximity to the conductive anode surface is important for electrogens, so the competition for space could explain the observed negative correlations. The cathode communities were dominated by hydrogen-utilizing taxa such as Methanobacterium and had a much lower proportion of negative correlations than the anodes. This could be explained by the diffusion of hydrogen throughout the cathode biofilms, reducing the need to compete for space., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Abadikhah, Rodriguez, Persson, Wilén, Farewell and Modin.)

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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Wastewater infrastructure has a long lifetime and is subject to changing conditions and demands. When plans are made to upgrade or build new infrastructure, transdisciplinary planning processes and a robust analysis of future conditions are needed to make sustainable choices. Here, we provide a stepwise collaborative planning process in which future scenarios are developed together with local stakeholders and expert groups. The process was implemented at one of the largest wastewater treatment plants (WWTPs) in Scandinavia. With a combination of workshops and the use of a web-based digital tool, future scenarios including flows, pollutant loads, and treatment requirements could be created. Furthermore, sustainability prioritizations affecting the WWTP, were identified. The future scenarios developed for the WWTP in the case study, predict stricter and new regulations, constant or lower future loads and ambiguous future flows. The highest ranked sustainability priority was low resource and energy consumption together with low CO 2 footprint. The quantified future scenarios developed in the planning process were used as input to a process model to show the consequences they would have on the WWTP in the case study. Applying this collaborative process revealed future scenarios with many, sometimes conflicting, expectations on future WWTPs. It also highlighted needs for improvements of both the collection system and the WWTP., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s). Published by Elsevier Ltd.)

Bacteria in the order 'Candidatus Brocadiales' within the phylum Planctomycetes (Planctomycetota) have the remarkable ability to perform anaerobic ammonium oxidation (anammox). Two families of anammox bacteria with different biogeographical distributions have been reported, marine Ca. Scalinduaceae and freshwater Ca. Brocadiaceae. Here we report evidence of three new species within a novel genus and family of anammox bacteria, which were discovered in biofilms of a subsea road tunnel under a fjord in Norway. In this particular ecosystem, the nitrogen cycle is likely fuelled by ammonia from organic matter degradation in the fjord sediments and the rock mass above the tunnel, resulting in the growth of biofilms where anammox bacteria can thrive under oxygen limitation. We resolved several metagenome-assembled genomes (MAGs) of anammox bacteria, including three Ca. Brocadiales MAGs that could not be classified at the family level. MAGs of this novel family had all the diagnostic genes for a full anaerobic ammonium oxidation pathway in which nitrite was probably reduced by a NirK-like reductase. A survey of published molecular data indicated that this new family of anammox bacteria occurs in many marine sediments, where its members presumably would contribute to nitrogen loss., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: High-throughput amplicon sequencing of marker genes, such as the 16S rRNA gene in Bacteria and Archaea, provides a wealth of information about the composition of microbial communities. To quantify differences between samples and draw conclusions about factors affecting community assembly, dissimilarity indices are typically used. However, results are subject to several biases, and data interpretation can be challenging. The Jaccard and Bray-Curtis indices, which are often used to quantify taxonomic dissimilarity, are not necessarily the most logical choices. Instead, we argue that Hill-based indices, which make it possible to systematically investigate the impact of relative abundance on dissimilarity, should be used for robust analysis of data. In combination with a null model, mechanisms of microbial community assembly can be analyzed. Here, we also introduce a new software, qdiv, which enables rapid calculations of Hill-based dissimilarity indices in combination with null models., Results: Using amplicon sequencing data from two experimental systems, aerobic granular sludge (AGS) reactors and microbial fuel cells (MFC), we show that the choice of dissimilarity index can have considerable impact on results and conclusions. High dissimilarity between replicates because of random sampling effects make incidence-based indices less suited for identifying differences between groups of samples. Determining a consensus table based on count tables generated with different bioinformatic pipelines reduced the number of low-abundant, potentially spurious amplicon sequence variants (ASVs) in the data sets, which led to lower dissimilarity between replicates. Analysis with a combination of Hill-based indices and a null model allowed us to show that different ecological mechanisms acted on different fractions of the microbial communities in the experimental systems., Conclusions: Hill-based indices provide a rational framework for analysis of dissimilarity between microbial community samples. In combination with a null model, the effects of deterministic and stochastic community assembly factors on taxa of different relative abundances can be systematically investigated. Calculations of Hill-based dissimilarity indices in combination with a null model can be done in qdiv, which is freely available as a Python package ( https://github.com/omvatten/qdiv ). In qdiv, a consensus table can also be determined from several count tables generated with different bioinformatic pipelines. Video Abstract.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier B.V.)

The aerobic granular sludge (AGS) technology is growing towards becoming a mature option for new municipal wastewater treatment plants and capacity extensions. A process based on AGS was compared to conventional activated sludge processes (with and without enhanced biological phosphorus removal), an integrated fixed-film activated sludge (IFAS) process and a membrane bioreactor (MBR) by estimating the land area demand (footprint), electricity demand and chemicals' consumption. The process alternatives compared included pre-settling, sludge digestion and necessary post-treatment to achieve effluent concentrations of 8 mg/L nitrogen and 0.2 mg/L phosphorus at 7°C. The alternative based on AGS was estimated to have a 40-50% smaller footprint and 23% less electricity requirement than conventional activated sludge. In relation to the other compact treatment options IFAS and MBR, the AGS process had an estimated electricity usage that was 35-70% lower. This suggests a favourable potential for processes based on AGS although more available experience of AGS operation and performance at full scale is desired.

In microbial fuel cells (MFCs), microorganisms generate electrical current by oxidizing organic compounds. MFCs operated with different electron donors harbour different microbial communities, and it is unknown how that affects their response to starvation. We analysed the microbial communities in acetate- and glucose-fed MFCs and compared their responses to 10 days starvation periods. Each starvation period resulted in a 4.2 ± 1.4% reduction in electrical current in the acetate-fed MFCs and a 10.8 ± 3.9% reduction in the glucose-fed MFCs. When feed was resumed, the acetate-fed MFCs recovered immediately, whereas the glucose-fed MFCs required 1 day to recover. The acetate-fed bioanodes were dominated by Desulfuromonas spp. converting acetate into electrical current. The glucose-fed bioanodes were dominated by Trichococcus sp., functioning as a fermenter, and a member of Desulfuromonadales, using the fermentation products to generate electrical current. Suspended biomass and biofilm growing on non-conductive regions within the MFCs had different community composition than the bioanodes. However, null models showed that homogenizing dispersal of microorganisms within the MFCs affected the community composition, and in the glucose-fed MFCs, the Trichococcus sp. was abundant in all locations. The different responses to starvation can be explained by the more complex pathway requiring microbial interactions to convert glucose into electrical current., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Two nitrifying MBBR reactors were operated in parallel, one with PAC dosing and one without, to determine the effects of PAC dosing on nitrification and micropollutant adsorption in municipal wastewater. The removal of micropollutants was evaluated for several doses of PAC and batch experiments were performed to measure adsorption kinetics and nitrification rates. The influence of PAC on the nitrifying microbial community was examined by high-throughput amplicon sequencing. Long-term operation of the pilot reactors showed that nitrification could be maintained while supplying PAC at increasing doses, as confirmed by high nitrification rates and significant abundance of nitrifying bacteria. The adsorption of organic micropollutants could be controlled by the PAC dose, and increased dosing resulted in corresponding improvements in removal efficiency. Biomass, suspended or attached to carriers, did not interfere with the adsorption of organic micropollutants. Freundlich isotherms obtained from the batch experiments were used to predict removal of organic micropollutants in the pilot reactors, suggesting that batch adsorption experiments can be used to predict micropollutant removal on a full scale. Collectively, the results show that nitrification and adsorption of organic micropollutants can be performed simultaneously in an MBBR., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Granular sludge is an efficient and compact biofilm process for wastewater treatment. However, the ecological factors involved in microbial community assembly during the granular biofilm formation are poorly understood, and little is known about the reproducibility of the process. Here, three replicate bioreactors were used to investigate microbial succession during the formation of granular biofilms. We identified three successional phases. During the initial phase, the successional turnover was high and α-diversity decreased as a result of the selection of taxa adapted to grow on acetate and form aggregates. Despite these dynamic changes, the microbial communities in the replicate reactors were similar. The second successional phase occurred when the settling time was rapidly decreased to selectively retain granules in the reactors. The influence of stochasticity on succession increased and new niches were created as granules emerged, resulting in temporarily increased α-diversity. The third successional phase occurred when the settling time was kept stable and granules dominated the biomass. Turnover was low, and selection resulted in the same abundant taxa in the reactors, but drift, which mostly affected low-abundant community members, caused the community in one reactor to diverge from the other two. Even so, performance was stable and similar between reactors.

The operational efficiency of activated sludge wastewater treatment plants depends to a large extent on the microbial community structure of the activated sludge. The aims of this paper are to describe the composition of the bacterial community in a Swedish full-scale activated sludge wastewater treatment plant, to describe the dynamics of the community and to elucidate possible causes for bacterial community composition changes. The bacterial community composition in the activated sludge was described using 16S rRNA gene libraries and monitored for 15 months by a terminal restriction fragment (T-RF) length polymorphism (T-RFLP) analysis of the 16S rRNA gene. Despite variable environmental conditions, a large fraction of the observed T-RFs were present at all times, making up at least 50% in all samples, possibly representing a relatively stable core fraction of the bacterial community. However, the proportions of the different T-RFs in this fraction as well as the T-RFs in the more variable fraction showed a significant variation over time and temperature. The difference in community composition between summer and winter coincided with observed differences in floc structure. These observations suggest a relationship between floc properties and bacterial community composition, although additional experiments are required to determine causality.

Biocathodes where living microorganisms catalyse reduction of CO 2 can potentially be used to produce valuable chemicals. Microorganisms harbouring hydrogenases may play a key role for biocathode performance since H 2 generated on the electrode surface can act as an electron donor for CO 2 reduction. In this study, the possibility of catalysing cathodic reactions by hydrogenotrophic methanogens, acetogens, sulfate-reducers, denitrifiers, and acetotrophic methanogens was investigated. The cultures were enriched from an activated sludge inoculum and performed the expected metabolic functions. All enrichments formed distinct microbial communities depending on their electron donor and electron acceptor. When the cultures were added to an electrochemical cell, linear sweep voltammograms showed a shift in current generation close to the hydrogen evolution potential (-1 V versus SHE) with higher cathodic current produced at a more positive potential. All enrichment cultures except the denitrifiers were also used to inoculate biocathodes of microbial electrolysis cells operated with H + and bicarbonate as electron acceptors and this resulted in current densities between 0.1-1 A/m 2 . The microbial community composition of biocathodes inoculated with different enrichment cultures were as different from each other as they were different from their suspended culture inoculum. It was noteworthy that Methanobacterium sp. appeared on all the biocathodes suggesting that it is a key microorganism catalysing biocathode reactions.

Deterioration of concrete is a large societal cost. In the Oslofjord subsea tunnel (Norway), deterioration of sprayed concrete and corrosion of reinforcing steel fibres occur under biofilm formed at sites with intrusion of saline groundwater. In this study, the microbial community structure, in situ environmental gradients and chemical composition of the biofilms were examined at three tunnel sites. Ammonia- and nitrite-oxidising microorganisms, in particular Nitrosopumilus sp., and iron-oxidising bacteria within Mariprofundus sp., were omnipresent, together with a diversity of presumably heterotrophic bacteria. Alpha- and beta diversity measures showed significant differences in richness and community structure between the sites as well as over time and null-models suggested that deterministic factors were important for the community assembly. The superficial flow of water over the biofilm had a strong effect on oxygen penetration in the biofilm and was identified as one major environmental gradient that varied between the sites, likely being important for shaping the microbial communities.

Environmental deterioration together with the need for water reuse and the increasingly restrictive legislation of water quality standards have led to a demand for compact, efficient and less energy consuming technologies for wastewater treatment. Aerobic granular sludge and membrane bioreactors (MBRs) are two technologies with several advantages, such as small footprint, high-microbial density and activity, ability to operate at high organic- and nitrogen-loading rates, and tolerance to toxicity. However, they also have some disadvantages. The aerobic granular sludge process generally requires post-treatment in order to fulfill effluent standards and MBRs suffer from fouling of the membranes. Integrating the two technologies could be a way of combining the advantages and addressing the main problems associated with both processes. The use of membranes to separate the aerobic granules from the treated water would ensure high-quality effluents suitable for reuse. Moreover, the use of granular sludge in MBRs has been shown to reduce fouling. Several recent studies have shown that the aerobic granular membrane bioreactor (AGMBR) is a promising hybrid process with many attractive features. However, major challenges that have to be addressed include how to achieve granulation and maintain granular stability during continuous operation of reactors. This paper aims to review the current state of research on AGMBR technology while drawing attention to relevant findings and highlight current limitations.

Wastewater management in developing countries is a challenge, especially in small towns with rapid population growth. This study aims at assessing the performance and management of five treatment plants (TPs) in rural areas of Cochabamba, Bolivia. Pollutants' concentrations, wastewater flows, hydraulic and organic loads and hydraulic retention times were determined in three small treatment plants (2000-10,000 population equivalent [p.e.]; flow > 432 m 3 /d) and two very small treatment plants (<2000 p.e.; flow < 432 m 3 /d). The performance assessment was based on operational parameters, treatment efficiency and effluent quality. Management data were collected through semi-structured interviews with managers of local water associations. The results support that the poor performance of the TPs is due to lack of operational expertise and financial resources for adequate operation and maintenance (O&M). Additionally, effective treatment was affected by the type of technology used and whether the plant design included plans for O&M with available resources. This study contributes to a better understanding of actual operating conditions of wastewater TPs in small towns, thus providing needed information regarding technology selection, design, implementation and operation.

Granular activated sludge has gained increasing interest due to its potential in treating wastewater in a compact and efficient way. It is well-established that activated sludge can form granules under certain environmental conditions such as batch-wise operation with feast-famine feeding, high hydrodynamic shear forces, and short settling time which select for dense microbial aggregates. Aerobic granules with stable structure and functionality have been obtained with a range of different wastewaters seeded with different sources of sludge at different operational conditions, but the microbial communities developed differed substantially. In spite of this, granule instability occurs. In this review, the available literature on the mechanisms involved in granulation and how it affects the effluent quality is assessed with special attention given to the microbial interactions involved. To be able to optimize the process further, more knowledge is needed regarding the influence of microbial communities and their metabolism on granule stability and functionality. Studies performed at conditions similar to full-scale such as fluctuation in organic loading rate, hydrodynamic conditions, temperature, incoming particles, and feed water microorganisms need further investigations.

The enrichment of CO 2 -reducing microbial biocathodes is challenging. Previous research has shown that a promising approach could be to first enrich bioanodes and then lower the potential so the electrodes are converted into biocathodes. However, the effect of such a transition on the microbial community on the electrode has not been studied. The goal of this study was thus to compare the start-up of biocathodes from preenriched anodes with direct start-up from bare electrodes and to investigate changes in microbial community composition. The effect of three electrode materials on the long-term performance of the biocathodes was also investigated. In this study, preenrichment of acetate-oxidizing bioanodes did not facilitate the start-up of biocathodes. It took about 170 days for the preenriched electrodes to generate substantial cathodic current, compared to 83 days for the bare electrodes. Graphite foil and carbon felt cathodes produced higher current at the beginning of the experiment than did graphite rods. However, all electrodes produced similar current densities at the end of the over 1-year-long study (2.5 A/m 2 ). Methane was the only product detected during operation of the biocathodes. Acetate was the only product detected after inhibition of the methanogens. Microbial community analysis showed that Geobacter sp. dominated the bioanodes. On the biocathodes, the Geobacter sp. was succeeded by Methanobacterium spp., which made up more than 80% of the population. After inhibition of the methanogens, Acetobacterium sp. became dominant on the electrodes (40% relative abundance). The results suggested that bioelectrochemically generated H 2 acted as an electron donor for CO 2 reduction. IMPORTANCE In microbial electrochemical systems, living microorganisms function as catalysts for reactions on the anode and/or the cathode. There is a variety of potential applications, ranging from wastewater treatment and biogas generation to production of chemicals. Systems with biocathodes could be used to reduce CO 2 to methane, acetate, or other high-value chemicals. The technique can be used to convert solar energy to chemicals. However, enriching biocathodes that are capable of CO 2 reduction is more difficult and less studied than enriching bioanodes. The effect of different start-up strategies and electrode materials on the microbial communities that are enriched on biocathodes has not been studied. The purpose of this study was to investigate two different start-up strategies and three different electrode materials for start-up and long-term operation of biocathodes capable of reducing CO 2 to valuable biochemicals., (Copyright © 2018 Saheb-Alam et al.)

Granulation of activated sludge is an increasingly important area within the field of wastewater treatment. Granulation is usually achieved by high hydraulic selection pressure, which results in the wash-out of slow settling particles. The effect of the harsh wash-out conditions on the granular sludge ecosystem is not yet fully understood, but different bacterial groups may be affected to varying degrees. In this study, we used high-throughput amplicon sequencing to follow the community composition in granular sludge reactors for 12 weeks, both in the granular phase and the suspended phase (effluent). The microbiome of the washed out biomass was similar but not identical to the microbiome of the granular biomass. Certain taxa (e.g. Flavobacterium spp. and Bdellovibrio spp.) had significantly (p < 0.05) higher relative abundance in the granules compared to the effluent. Fluorescence in situ hybridization images indicated that these taxa were mainly located in the interior of granules and therefore protected from erosion. Other taxa (e.g. Meganema sp. and Zooglea sp.) had significantly lower relative abundance in the granules compared to the effluent, and appeared to be mainly located on the surface of granules and therefore subject to erosion. Despite being washed out, these taxa were among the most abundant members of the granular sludge communities and were likely growing fast in the reactors. The ratio between relative abundance in the granular biomass and in the effluent did not predict temporal variation of the taxa in the reactors, but it did appear to predict the spatial location of the taxa in the granules.

Vacuum degassing of activated sludge was tested at eight different Swedish wastewater treatment plants with laboratory-scale equipment in batch mode in order to evaluate its efficiency on improvement of sludge compaction and settling properties. The results show that the efficiency of the degassing technique is mainly dependent on the initial sludge volume index (SVI) of the target sludge which was found to be related to its process configuration. Facilities with full activated sludge-based nitrogen removal processes, including both nitrification and denitrification, had high SVIs (>300 mL g -1 ) and were strongly affected by vacuum degassing with reduction of SVI up to 30%. Nitrogen removal facilities also including biological phosphorus removal showed better compaction and settling properties with relatively lower SVIs and were affected to a lesser extent by degassing with SVI reduction of 10-20%. Wastewater treatment plants without full biological nitrogen removal, lacking either nitrification or denitrification (or both) processes in the activated sludge had the lowest SVIs observed with almost no effect of vacuum degassing.

Partial nitritation-anammox (PNA) permits energy effective nitrogen removal. Today PNA is used for treatment of concentrated and warm side streams at wastewater treatment plants, but not the more diluted and colder main stream. To implement PNA in the main stream, better knowledge about microbial communities at the typical environmental conditions is necessary. In order to investigate the response of PNA microbial communities to decreasing substrate availability, we have operated a moving bed biofilm reactor (MBBR) at decreasing reactor concentrations (311-27 mg-N l -1 of ammonium) and low temperature (13°C) for 302 days and investigated the biofilm community using high throughput amplicon sequencing; quantitative PCR; and fluorescence in situ hybridization. The anammox bacteria (Ca. Brocadia) constituted a large fraction of the biomass with fewer aerobic ammonia oxidizing bacteria (AOB) and even less nitrite oxidizing bacteria (NOB; Nitrotoga, Nitrospira and Nitrobacter). Still, NOB had considerable impact on the process performance. The anammox bacteria, AOB and NOB all harboured more than one population, indicating some diversity, and the heterotrophic bacterial community was diverse (seven phyla). Despite the downshifts in substrate availability, changes in the relative abundance and composition of anammox bacteria, AOB and NOB were small and also the heterotrophic community showed little changes in composition. This indicates stability of PNA MBBR communities towards decreasing substrate availability and suggests that even heterotrophic bacteria are integral components of these communities., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

The granular sludge process is an effective, low-footprint alternative to conventional activated sludge wastewater treatment. The architecture of the microbial granules allows the co-existence of different functional groups, e.g., nitrifying and denitrifying communities, which permits compact reactor design. However, little is known about the factors influencing community assembly in granular sludge, such as the effects of reactor operation strategies and influent wastewater composition. Here, we analyze the development of the microbiomes in parallel laboratory-scale anoxic/aerobic granular sludge reactors operated at low (0.9 kg m -3 d -1 ), moderate (1.9 kg m -3 d -1 ) and high (3.7 kg m -3 d -1 ) organic loading rates (OLRs) and the same ammonium loading rate (0.2 kg NH 4 -N m -3 d -1 ) for 84 days. Complete removal of organic carbon and ammonium was achieved in all three reactors after start-up, while the nitrogen removal (denitrification) efficiency increased with the OLR: 0% at low, 38% at moderate, and 66% at high loading rate. The bacterial communities at different loading rates diverged rapidly after start-up and showed less than 50% similarity after 6 days, and below 40% similarity after 84 days. The three reactor microbiomes were dominated by different genera (mainly Meganema, Thauera, Paracoccus , and Zoogloea ), but these genera have similar ecosystem functions of EPS production, denitrification and polyhydroxyalkanoate (PHA) storage. Many less abundant but persistent taxa were also detected within these functional groups. The bacterial communities were functionally redundant irrespective of the loading rate applied. At steady-state reactor operation, the identity of the core community members was rather stable, but their relative abundances changed considerably over time. Furthermore, nitrifying bacteria were low in relative abundance and diversity in all reactors, despite their large contribution to nitrogen turnover. The results suggest that the OLR has considerable impact on the composition of the granular sludge communities, but also that the granule communities can be dynamic even at steady-state reactor operation due to high functional redundancy of several key guilds. Knowledge about microbial diversity with specific functional guilds under different operating conditions can be important for engineers to predict the stability of reactor functions during the start-up and continued reactor operation.

The activated sludge process is commonly used to treat wastewater by aerobic oxidation of organic pollutants into carbon dioxide and water. However, several nonoxidative mechanisms can also contribute to removal of organics. Sorption onto activated sludge can remove a large fraction of the colloidal and particulate wastewater organics. Intracellular storage of, e.g., polyhydroxyalkanoates (PHA), triacylglycerides (TAG), or wax esters can convert wastewater organics into precursors for high-value products. Recently, several environmental, economic, and technological drivers have stimulated research on nonoxidative removal of organics for wastewater treatment. In this paper, we review these nonoxidative removal mechanisms as well as the existing and emerging process configurations that make use of them for wastewater treatment. Better utilization of nonoxidative processes in activated sludge could reduce the wasteful aerobic oxidation of organic compounds and lead to more resource-efficient wastewater treatment plants.

Storage methods are important to preserve the viability and biochemical characteristics of microbial cultures between experiments or during periods when bioreactors are inactive. Most of the research on storage has focused on isolates; however, there is an increasing interest in methods for mixed cultures, which are of relevance in environmental biotechnology. The purpose of this study was to investigate the effect of different storage methods on electrochemically active enrichment cultures. Acetate-oxidizing bioanodes generating a current density of about 5 A m(-2) were enriched in a microbial electrolysis cell. The effect of five weeks of storage was evaluated using electrochemical techniques and microbial community analysis. Storage by refrigeration resulted in quicker re-activation than freezing in 10% glycerol, while the bioelectrochemical activity was entirely lost after storage using dehydration. The results showed that the bioelectrochemical activity of bioanodes stored at low temperature could be retained. However, during the re-activation period the bioanodes only recovered 75% of the current density generated before storage and the bacterial communities were different in composition and more diverse after storage than before.

New activated sludge processes that utilize sorption as a major mechanism for organics removal are being developed to maximize energy recovery from wastewater organics, or as enhanced primary treatment technologies. To model and optimize sorption-based activated sludge processes, further knowledge about sorption of organics onto sludge is needed. This study compared primary-, anaerobic-, and aerobic activated sludge as sorbents, determined sorption capacity and kinetics, and investigated some characteristics of the organics being sorbed. Batch sorption assays were carried out without aeration at a mixing velocity of 200 rpm. Only aerobic activated sludge showed net sorption of organics. Sorption of dissolved organics occurred by a near-instantaneous sorption event followed by a slower process that obeyed 1st order kinetics. Sorption of particulates also followed 1st order kinetics but there was no instantaneous sorption event; instead there was a release of particles upon mixing. The 5-min sorption capacity of activated sludge was 6.5±10.8 mg total organic carbon (TOC) per g volatile suspend solids (VSS) for particulate organics and 5.0±4.7 mgTOC/gVSS for dissolved organics. The observed instantaneous sorption appeared to be mainly due to organics larger than 20 kDa in size being sorbed, although molecules with a size of about 200 Da with strong UV absorbance at 215-230 nm were also rapidly removed.

Background: Terminal restriction fragment length polymorphism (T-RFLP) analysis is a DNA-fingerprinting method that can be used for comparisons of the microbial community composition in a large number of samples. There is no consensus on how T-RFLP data should be treated and analyzed before comparisons between samples are made, and several different approaches have been proposed in the literature. The analysis of T-RFLP data can be cumbersome and time-consuming, and for large datasets manual data analysis is not feasible. The currently available tools for automated T-RFLP analysis, although valuable, offer little flexibility, and few, if any, options regarding what methods to use. To enable comparisons and combinations of different data treatment methods an analysis template and an extensive collection of macros for T-RFLP data analysis using Microsoft Excel were developed., Results: The Tools for T-RFLP data analysis template provides procedures for the analysis of large T-RFLP datasets including application of a noise baseline threshold and setting of the analysis range, normalization and alignment of replicate profiles, generation of consensus profiles, normalization and alignment of consensus profiles and final analysis of the samples including calculation of association coefficients and diversity index. The procedures are designed so that in all analysis steps, from the initial preparation of the data to the final comparison of the samples, there are various different options available. The parameters regarding analysis range, noise baseline, T-RF alignment and generation of consensus profiles are all given by the user and several different methods are available for normalization of the T-RF profiles. In each step, the user can also choose to base the calculations on either peak height data or peak area data., Conclusions: The Tools for T-RFLP data analysis template enables an objective and flexible analysis of large T-RFLP datasets in a widely used spreadsheet application.

Background: Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common DNA-fingerprinting technique used for comparisons of complex microbial communities. Although the technique is well established there is no consensus on how to treat T-RFLP data to achieve the highest possible accuracy and reproducibility. This study focused on two critical steps in the T-RFLP data treatment: the alignment of the terminal restriction fragments (T-RFs), which enables comparisons of samples, and the normalization of T-RF profiles, which adjusts for differences in signal strength, total fluorescence, between samples., Results: Variations in the estimation of T-RF sizes were observed and these variations were found to affect the alignment of the T-RFs. A novel method was developed which improved the alignment by adjusting for systematic shifts in the T-RF size estimations between the T-RF profiles. Differences in total fluorescence were shown to be caused by differences in sample concentration and by the gel loading. Five normalization methods were evaluated and the total fluorescence normalization procedure based on peak height data was found to increase the similarity between replicate profiles the most. A high peak detection threshold, alignment correction, normalization and the use of consensus profiles instead of single profiles increased the similarity of replicate T-RF profiles, i.e. lead to an increased reproducibility. The impact of different treatment methods on the outcome of subsequent analyses of T-RFLP data was evaluated using a dataset from a longitudinal study of the bacterial community in an activated sludge wastewater treatment plant. Whether the alignment was corrected or not and if and how the T-RF profiles were normalized had a substantial impact on ordination analyses, assessments of bacterial dynamics and analyses of correlations with environmental parameters., Conclusions: A novel method for the evaluation and correction of the alignment of T-RF profiles was shown to reduce the uncertainty and ambiguity in alignments of T-RF profiles. Large differences in the outcome of assessments of bacterial community structure and dynamics were observed between different alignment and normalization methods. The results of this study can therefore be of value when considering what methods to use in the analysis of T-RFLP data.

It is a challenge to apply anaerobic ammonium oxidation (anammox) for nitrogen removal from wastewater at low temperatures. Maintenance of anammox- and aerobic ammonia oxidizing bacteria (AOB) and suppression of nitrite oxidizing bacteria (NOB) are key issues. In this work, a nitritation-anammox moving bed biofilm pilot reactor was operated at 19-10°C for 300 d. Nitrogen removal was decreasing, but stable, at 19-13°C. At 10°C removal became unstable. Quantitative PCR, fluorescence in situ hybridization and gene sequencing showed that no major microbial community changes were observed with decreased temperature. Anammox bacteria dominated the biofilm (0.9-1.2 × 10(14) 16S rRNA copies m(-2)). Most anammox bacteria were similar to Brocadia sp. 40, but another smaller Brocadia population was present near the biofilm-water interface, where also the AOB community (Nitrosomonas) was concentrated in thin layers (1.8-5.3 × 10(12) amoA copies m(-2)). NOB (Nitrobacter, Nitrospira) were always present at low concentrations (<1.3 × 10(11) 16S rRNA copies m(-2))., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Moving bed biofilm reactors (MBBRs) are increasingly used for nitrogen removal with nitritation-anaerobic ammonium oxidation (anammox) processes in wastewater treatment. Carriers provide protected surfaces where ammonia oxidizing bacteria (AOB) and anammox bacteria form complex biofilms. However, the knowledge about the organization of microbial communities in MBBR biofilms is sparse. We used new cryosectioning and imaging methods for fluorescence in situ hybridization (FISH) to study the structure of biofilms retrieved from carriers in a nitritation-anammox MBBR. The dimensions of the carrier compartments and the biofilm cryosections after FISH showed good correlation, indicating little disturbance of biofilm samples by the treatment. FISH showed that Nitrosomonas europaea/eutropha-related cells dominated the AOB and Candidatus Brocadia fulgida-related cells dominated the anammox guild. New carriers were initially colonized by AOB, followed by anammox bacteria proliferating in the deeper biofilm layers, probably in anaerobic microhabitats created by AOB activity. Mature biofilms showed a pronounced three-dimensional stratification where AOB dominated closer to the biofilm-water interface, whereas anammox were dominant deeper into the carrier space and towards the walls. Our results suggest that current mathematical models may be oversimplifying these three-dimensional systems and unless the multidimensionality of these systems is considered, models may result in suboptimal design of MBBR carriers.

Assessments of bacterial community diversity and dynamics are fundamental for the understanding of microbial ecology as well as biotechnological applications. We show that the choice of PCR primers has great impact on the results of analyses of diversity and dynamics using gene libraries and DNA fingerprinting. Two universal primer pairs targeting the 16S rRNA gene, 27F&1492R and 63F&M1387R, were compared and evaluated by analyzing the bacterial community in the activated sludge of a large-scale wastewater treatment plant. The two primer pairs targeted distinct parts of the bacterial community, none encompassing the other, both with similar richness. Had only one primer pair been used, very different conclusions had been drawn regarding dominant phylogenetic and putative functional groups. With 27F&1492R, Betaproteobacteria would have been determined to be the dominating taxa while 63F&M1387R would have described Alphaproteobacteria as the most common taxa. Microscopy and fluorescence in situ hybridization analysis showed that both Alphaproteobacteria and Betaproteobacteria were abundant in the activated sludge, confirming that the two primer pairs target two different fractions of the bacterial community. Furthermore, terminal restriction fragment polymorphism analyses of a series of four activated sludge samples showed that the two primer pairs would have resulted in different conclusions about community stability and the factors contributing to changes in community composition. In conclusion, different PCR primer pairs, although considered universal, target different ranges of bacteria and will thus show the diversity and dynamics of different fractions of the bacterial community in the analyzed sample. We also show that while a database search can serve as an indicator of how universal a primer pair is, an experimental assessment is necessary to evaluate the suitability for a specific environmental sample.

Biochemical oxygen demand (BOD) is a measure of biodegradable compounds in water and is, for example, a common parameter to design and assess the performance of wastewater treatment plants. The conventional method to measure BOD is time consuming (5 or 7 days) and requires trained personnel. Bioelectrochemical BOD sensors designed as microbial fuel cells (MFCs), which are systems where bacteria convert organic matter into an electrical current, have emerged as an alternative to the conventional technique. In this study, a new type of bioelectrochemical BOD sensor with features that overcome some of the limitations of current MFC-type designs was developed: (1) An external voltage was applied to overcome internal resistances and allow bacteria to generate current at their full capacity, and (2) the ion exchange membrane was omitted to avoid pH shifts that would otherwise limit the applicability of the sensor for wastewaters with low alkalinity. The sensor was calibrated with an aerated nutrient medium containing acetate as the BOD source. Linear correlation (R(2) = 0.97) with charge was obtained for BOD concentrations ranging from 32 to 1280 mg/L in a reaction time of 20 h. Lowering the reaction time to 5 h resulted in lowering the measurable BOD concentration range to 320 mg/L (R(2) = 0.99). Propionate, glucose, and ethanol could also be analyzed by the sensor that was acclimated to acetate. The study demonstrates a way to design more robust and simple bioelectrochemical BOD sensors that do not suffer from the usual limitations of MFCs (high internal resistance and pH shifts)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The activated sludge process is one of the most widely used methods for treatment of wastewater and the microbial community composition in the sludge is important for the process operation. While the bacterial communities have been characterized in various activated sludge systems little is known about archaeal communities in activated sludge. The diversity and dynamics of the Archaea community in a full-scale activated sludge wastewater treatment plant were investigated by fluorescence in situ hybridization, terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16S rRNA genes., Results: The Archaea community was dominated by Methanosaeta-like species. During a 15 month period major changes in the community composition were only observed twice despite seasonal variations in environmental and operating conditions. Water temperature appeared to be the process parameter that affected the community composition the most. Several terminal restriction fragments also showed strong correlations with sludge properties and effluent water properties. The Archaea were estimated to make up 1.6% of total cell numbers in the activated sludge and were present both as single cells and colonies of varying sizes., Conclusions: The results presented here show that Archaea can constitute a constant and integral part of the activated sludge and that it can therefore be useful to include Archaea in future studies of microbial communities in activated sludge.

A comprehensive study of the dynamics in settling and flocculation properties at a full-scale activated sludge treatment plant with secondary settlers operated at their maximum capacity is presented. An extensive set of process data was analyzed to assess physical parameters affecting the capacity and performance of the secondary settlers. There was a large variation in the settling and compaction properties, and they changed quickly, as a result of the short sludge age. Even though the flocculation properties changed throughout the year, they were generally improved during the summer, with lower values of effluent turbidity and effluent suspended solids concentration. The shear sensitivity, which is a measure of floc stability, was higher during the winter, when the water temperatures were lower. The sludge floc morphology had, in general, a different internal structure, with more round microbial colonies during the warmer summer months and a more net-like open structure during the winter.

The variation in activated sludge floc composition, flocculation and settling properties was studied at a full scale plant over a 2-year period. A comprehensive set of process parameters was analysed and related to the floc properties to increase the understanding of the factors affecting floc formation. The composition of the activated sludge showed a seasonal change with higher concentrations of extractable extracellular polymeric substances (EPS) during the winter months. The protein content of the total sludge and EPS increased significantly during the winter. This coincided with higher effluent suspended solids concentrations and increased shear sensitivity of the sludge flocs. Only poor correlations between EPS contents and stirred sludge volume index (SSVI) could be observed. High iron concentrations in the sludge due to dosage of iron salt to precipitate phosphorus were found to have a negative impact on the settling and compaction properties of the sludge, whereas it had a positive impact on floc stability. Higher organic loading due to by-passed primary settlers leads to improved settling and compaction properties.

The efficiency of activated sludge treatment plants is dependent on the solid-liquid separation properties of the activated sludge. A critical parameter is the stability of the microbial flocs. Weak flocs deflocculate easily leaving increased concentrations of suspended solids in the effluent. The knowledge about how different bacteria are attached to the flocs and their influence on the bioflocculation is limited. In this study, the deflocculation of different phylogenetic groups of bacteria in activated sludge from a full scale plant was investigated. The experiments were carried out by using a shear method where the sludge flocs are deflocculated under controlled shear conditions. The degree of deflocculation was measured as increase in turbidity of the supernatant. Identification and quantification of the microbial community structure of both total activated sludge and deflocculated bacteria were conducted with group-specific gene probes for broad groups of bacteria (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Actinobacteria) and fluorescence in situ hybridization (FISH). The microbial community structure of the deflocculated bacteria was different compared to the total activated sludge with a higher abundance of Gammaproteobacteria in the supernatant indicating that different groups of bacteria are bound with different strength to the floc surface. The results show that the bacteria in the outer parts of the flocs are relatively loosely bound to the floc matrix and can be easily eroded from the surface when exposed to shear.

Floc characteristics were studied at a full scale activated sludge treatment plant with a unique process solution incorporating pre-denitrification with post-nitrification in nitrifying trickling filters. Since greater nitrogen removal is achieved when more secondary settled wastewater is recirculated to the trickling filters, the secondary settlers are always operated close to their maximal capacity. The flocculation and settling properties are therefore crucial and have an effect on the overall plant performance. Since the plant is operated at a short sludge age, these properties change quickly, resulting in variable maximal secondary settler capacity. The dynamics in floc structure and microbial community composition were studied and correlated to the secondary settler performance. Fluorescence in situ hybridisation was used to investigate the microbial community structure and their spatial distribution. The floc structure could to some extent be related to the flocculation and settling properties of the sludge. Even small differences had an influence suggesting that colloidal properties also play a significant role in determining the floc properties. No correlation between microbial community composition and settling properties could be established with the group-specific probes investigated.

The effect of rain events on effluent quality dynamics was studied at a full scale activated sludge wastewater treatment plant which has a process solution incorporating pre-denitrification in activated sludge with post-nitrification in trickling filters. The incoming wastewater flow varies significantly due to a combined sewer system. Changed flow conditions have an impact on the whole treatment process since the recirculation to the trickling filters is set by the hydraulic limitations of the secondary settlers. Apart from causing different hydraulic conditions in the plant, increased flow due to rain or snow-melting, changes the properties of the incoming wastewater which affects process performance and effluent quality, especially the particle removal efficiency. A comprehensive set of on-line and laboratory data were collected and analysed to assess the impact of rain events on the plant performance.

Activated sludge flocs are known to deflocculate under short-term anaerobic conditions, but little is known about possible reflocculation under subsequent aerobic conditions. When activated sludge flocs from two wastewater treatment plants deflocculated under anaerobic conditions with well-defined shear conditions, they could be almost, but not completely, reflocculated by aeration for 1-2 h under the same shear conditions. If the biological activity was reduced by adding azide, chloramphenicol or by decreasing the temperature, no or only very little reflocculation took place. This indicated that the reflocculation was under direct or indirect microbial control. Only a small part of the reflocculation was due to improved flocculation properties obtained by oxidation of Fe(II) to Fe(III), which is a better flocculant. Fe(II) was produced under the anaerobic conditions by microbial iron reduction, and it was oxidized to Fe(III) within less than one hour after the aeration was started. However, by comparing two different sludges with different capabilities for iron reduction, iron oxidation and responses to substrate addition, it was found that the aerobic biological activity most likely was of greatest significance for the observed reflocculation and floc formation under aerobic conditions. This was further supported by adding organic substrates (glucose or ethanol) during the aerobic reflocculation phase, which promoted reflocculation. However, some substrates had the opposite effect (acetate and lactate), where a deterioration of the reflocculation was observed, probably due to different responses from different groups of microorganisms in the sludges.