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Anaerobic microorganisms are thought to play a critical role in regulating the flux of short-chain gaseous alkanes (SCGAs; including ethane, propane and butane) from terrestrial and aquatic ecosystems to the atmosphere. Sulfate has been confirmed to act as electron acceptor supporting microbial anaerobic oxidation of SCGAs, yet several other energetically more favourable acceptors co-exist with these gases in anaerobic environments. Here, we show that a bioreactor seeded with biomass from a wastewater treatment facility can perform anaerobic propane oxidation coupled to nitrate reduction to dinitrogen gas and ammonium. The bioreactor was operated for more than 1000 days, and we used 13C- and 15N-labelling experiments, metagenomic, metatranscriptomic, metaproteomic and metabolite analyses to characterize the microbial community and the metabolic processes. The data collectively suggest that a species representing a novel order within the bacterial class Symbiobacteriia is responsible for the observed nitrate-dependent propane oxidation. The closed genome of this organism, which we designate as 'Candidatus Alkanivorans nitratireducens', encodes pathways for oxidation of propane to CO2 via fumarate addition, and for nitrate reduction, with all the key genes expressed during nitrate-dependent propane oxidation. Our results suggest that nitrate is a relevant electron sink for SCGA oxidation in anaerobic environments, constituting a new microbially-mediated link between the carbon and nitrogen cycles. Anaerobic microorganisms can oxidize short-chain gaseous alkanes such as ethane, propane and butane using sulfate as electron acceptor. Here, the authors show that a bioreactor enrichment of a wastewater microbial community can perform anaerobic propane oxidation coupled to nitrate reduction. [ABSTRACT FROM AUTHOR]

A sequencing batch reactor (SBR) was used for the treatment of abattoir wastewater to produce effluent with desirable nitrogen and phosphorus levels for irrigation. The SBR cycle consisted of an anaerobic phase with wastewater feeding, a relatively short aerobic period (allowing full ammonium oxidation), a second anoxic period with feeding, followed by settling and decanting. This design of operation allowed biological nitrification and denitrification via nitrite, and therefore with reduced demand for aeration and COD for nitrogen removal. The design also allowed ammonium, rather than oxidized nitrogen, being the primary nitrogen species in the effluent. Biological phosphorus removal was also achieved, with an effluent level desirable for irrigation. A high-level of nitrite accumulation (40 mg N/L) in the reactor caused inhibition to the biological P uptake. This problem was solved through process optimization. The cycle time of the SBR was reduced, with the wastewater load per cycle also reduced, while the daily hydraulic loading maintained. This modification proved to be an effective method to ensure reliable N and P removal. N2O accumulation was measured in two experiments simulating the anoxic phase of the SBR and using nitrite and nitrate respectively as electron donors. The estimated N2O emissions for both experiments were very low. [ABSTRACT FROM AUTHOR]

Wastewater treatment is a huge industry worldwide. Despite the massive capital and operating costs, only a relatively small amount of R&D investment is made. This might have been related to the limited demands in terms of effluent quality in the past, but today's environmental awareness requires much stricter effluent standards to be achieved. This in turn should give sufficient incentives, together with the possible large cost savings, to increase the R&D activities in this field. There are certainly significant knowledge gaps to be filled and substantial benefits could be gained from this. A range of knowledge gaps are identified in this paper, extending from the role of intermediates in nutrient removal over parameter estimation in modelling and simulation to understanding the microbial metabolic pathways at a genetic and enzymatic level. These gaps are opportunities and challenges for all researchers and professionals in this field. Addressing them will help substantially in the continuing development of wastewater treatment technologies. The complexity of biological wastewater treatment processes requires a broad range of tools and expertise to address the knowledge gaps. Novel process analysis tools are critically important to investigate biological treatment processes in future. They will come from different expertise areas and will need to be used in close integration to gain maximal benefits from the efforts. These tools will likely include respirometry, novel chemical analyses, microsensors, gene-based identification, microbial physiology techniques and integrated modelling and simulation. Examples of the application of such techniques are provided to demonstrate the way these techniques may be used in future. In the next few years, there is likely an exciting and highly interactive period of research and development for the wastewater industry. [ABSTRACT FROM AUTHOR]

Competition for electrons among different steps of denitrification has previously been shown to occur, and to play an important role in the accumulation and emission of N2O in wastewater treatment. However, this electron competition is not recognized in the current denitrification models, limiting their ability to predict N2O accumulation during denitrification. In this work, a new denitrification model is developed for wastewater treatment processes. It describes electron competition among the four steps of denitrification, through modeling the carbon oxidation and nitrogen reduction processes separately, in contrast to the existing models that directly couple these two types of processes. Electron carriers are introduced to link carbon oxidation, which donates electrons to carriers, and nitrogen oxides reduction, which receives electrons from these carriers. The relative ability of each denitrification step to compete for electrons is modeled through the use of different affinity constants with reduced carriers. Model calibration and validation results demonstrate that the developed model is able to reasonably describe the nitrate, nitrite, and N2O reduction rates of a methanol-utilizing denitrifying culture under various carbon and nitrogen oxides supplying conditions. The model proposed, while subject to further validation, is expected to enhance our ability to predict N2O accumulation in denitrification. [ABSTRACT FROM AUTHOR]

Sulfide is produced in sewer networks, and previous studies suggest that sulfide in sewage could alter the activity of heterotrophic denitrification and lead to N2O accumulation during biological wastewater treatment. However, the details of this phenomenon are poorly understood. In this study, the potential inhibitory effects of sulfide on nitrate, nitrite, and N2O reduction were assessed with a methanol-utilizing denitrifying culture both prior to and after its exposure and adaptation to sulfide. Hydrogen sulfide was found to be strongly inhibitory to N2O reduction, with 50% inhibition observed at H2S concentrations of 0.04 mg H2S-S/L and 0.1 mg H2S-S/L for the unadapted and adapted cultures, respectively. In comparison, both nitrate and nitrite reduction was more tolerant to H2S. A 50% inhibition of nitrite reduction was observed at approximately 2.0 mg H2S-S/L for both unadapted and adapted cultures, while no inhibition of nitrate reduction occurred at the highest H2S concentrations applied (2.0 mg H2S-S/L) to either culture. N2O accumulation was observed during nitrate and nitrite reduction by the adapted culture when H2S concentrations were above 0.5 and 0.2 mg H2S-S/L, respectively. Additionally, we reveal that hydrogen sulfide (H2S), rather than sulfide, was likely the true inhibitor of N2O reduction, and the inhibitory effect was reversible. These findings suggest that sulfide management in sewers could potentially have a significant impact on N2O emission from wastewater treatment plants. [ABSTRACT FROM AUTHOR]

Abstract: In full-scale wastewater treatment systems, phosphorus removal typically occurs together with nitrogen removal. Nitrite, an intermediate of both the nitrification and denitrification processes, can accumulate in the reactor. The inhibitory effect of nitrite/free nitrous acid (FNA) on the aerobic metabolism of poly-phosphate accumulating organisms (PAOs) is investigated. A culture highly enriched (90 ± 5%) in Candidatus “Accummulibacter phosphatis”, a well-known PAO, was used to perform a series of batch experiments at various nitrite and pH levels. FNA was found to inhibit all key aerobic metabolic processes performed by PAOs, namely PHA oxidation, phosphate uptake, glycogen replenishment and growth. The inhibitory effect on the anabolic processes (growth, phosphate uptake and glycogen production) was much stronger than that on the catabolic processes (PHA oxidation). 50% inhibition on all anabolic processes occurred at FNA concentrations of approximately 0.5 × 10−3 mg HNO2–N/L (equivalent to 2.0 mg NO2 −–N/L at pH 7.0), while full inhibition occurred at FNA concentrations of approximately 6.0 × 10−3 mg HNO2–N. These concentrations could be found in full-scale wastewater treatment systems that achieve nitrogen removal via the nitrite pathway. In comparison, PHA oxidation remained at 40–50% of the highest rate at FNA concentrations in the range 2.0 × 10−3–10.0 × 10−3 mg HNO2–N/L. Interestingly, PAOs were able to reduce nitrite under aerobic conditions (DO ≈ 3 mg/L), with the rate increasing substantially with the FNA concentration. The inhibition on phosphate uptake was found to be reversible. [ABSTRACT FROM AUTHOR]

Abstract Inline sludge treatment using free nitrous acid (FNA) was recently shown to be effective in establishing the nitrite pathway in a biological nitrogen removal system. However, the effects of FNA treatment conditions on the nitrite pathway performance remained to be investigated. In this study, three different FNA treatment frequencies (daily sludge treatment ratios of 0.22, 0.31 and 0.38, respectively), two FNA concentrations (1.35 mgN/L and 4.23 mgN/L, respectively) and two influent feeding regimes (one- and two-step feeding) were investigated in four laboratory-scale sequencing batch reactors. The nitrite accumulation ratio was positively correlated to the FNA treatment frequency. However, when a high treatment frequency was used e.g., daily sludge treatment ratio of 0.38, a significant reduction in ammonia oxidizing bacteria (AOB) activity occurred, leading to poor ammonium oxidation. AOB were able to acclimatise to FNA concentrations up to of 4.23 mgN/L, whereas nitrite oxidizing bacteria (NOB) were limited by an FNA concentration of 1.35 mgN/L over the duration of the study (up to 120 days). This difference in sensitivity to FNA could be used to further enhance nitrite accumulation, with 90% accumulation achieved at an FNA concentration of 4.23 mgN/L and a daily sludge treatment ratio of 0.31 in this study. However, this high level of nitrite accumulation led to increased N 2 O emission, with emission factors of up to 3.9% observed. The N 2 O emission was mitigated (reduced to 1.3%) by applying two-step feeding resulting in a nitrite accumulation ratio of 45.1%. Economic analysis showed that choosing the optimal FNA treatment conditions depends on a combination of the wastewater characteristics, the nitrogen discharge standards, and the operational costs. This study provides important information for the optimisation and practical application of FNA-based sludge treatment technology for achieving the mainstream stable nitrite pathway. Graphical abstract Unlabelled Image Highlights • Study result and analysis guide free nitrous acid treatment condition determination • Stable mainstream nitrite pathway reached with nitrite accumulation ratio over 90%. • Distinct nitrifying bacteria acclimation behaviours to high treatment concentration • Treatment frequency negatively related to in-reactor nitrifying bacteria activities. • N 2 O emission mainly from nitrifier denitrification, can be reduced by multi-feeding. [ABSTRACT FROM AUTHOR]

The presence of a high level of dissolved methane (e.g., 20–26 g m −3 ) in the anaerobic sludge digestion liquor represents a major challenge to the treatment of this stream, as its emission to the atmosphere contributes significantly to the carbon footprint of wastewater treatment. Here we propose a new approach to simultaneous ammonium and dissolved methane removal from the anaerobic digestion liquor through integrating partial nitritation-Anammox and denitrifying anaerobic methane oxidation (DAMO) processes in a single-stage membrane biofilm reactor (MBfR). In such an MBfR, the anaerobic digestion liquor is provided in the bulk liquid, while oxygen is supplied through gas-permeable membranes to avoid dissolved methane stripping. A previously developed model with appropriate extensions was applied to assess the system performance under different operational conditions and the corresponding microbial interactions. Both influent surface loading (or hydraulic retention time) and oxygen surface loading are found to significantly influence the total nitrogen (TN) and dissolved methane removal, which jointly determine the overall system performance. The counter diffusion and concentration gradients of substrates cause microbial stratification in the biofilm, where ammonia-oxidizing bacteria (AOB) attach close to the membrane surface (biofilm base) where oxygen and ammonium are available, while Anammox and DAMO microorganisms jointly grow in the biofilm layer close to the bulk liquid where methane, ammonium, and nitrite are available with the latter produced by AOB. These results provide first insights and useful information for the design and operation of this new technology for simultaneous ammonium and dissolved methane removal in its potential future applications. [ABSTRACT FROM AUTHOR]

Autotrophic ammonia oxidizing bacteria (AOB) have been recognized as a major contributor to N2O production in wastewater treatment systems. However, so far N2O models have been proposed based on a single N2O production pathway by AOB, and there is still a lack of effective approach for the integration of these models. In this work, an integrated mathematical model that considers multiple production pathways is developed to describe N2O production by AOB. The pathways considered include the nitrifier denitrification pathway (N2O as the final product of AOB denitrification with NO2- as the terminal electron acceptor) and the hydroxylamine (NH2OH) pathway (N2O as a byproduct of incomplete oxidation of NH2OH to NO2-). In this model, the oxidation and reduction processes are modeled separately, with intracellular electron carriers introduced to link the two types of processes. The model is calibrated and validated using experimental data obtained with two independent nitrifying cultures. The model satisfactorily describes the N2O data from both systems. The model also predicts shifts of the dominating pathway at various dissolved oxygen (DO) and nitrite levels, consistent with previous hypotheses. This unified model is expected to enhance our ability to predict N2O production by AOB in wastewater treatment systems under varying operational conditions. [ABSTRACT FROM AUTHOR]

Abstract: Sludge treatment and disposal is one of the major challenges for biological wastewater treatment plants and can represent up to 60% of their total operating costs. This study presents a novel strategy based on free nitrous acid (FNA or HNO2) treatment to achieve sludge reduction. Two sequencing batch reactors treating synthetic domestic wastewater were used, with one serving as an experimental reactor and the other as a control. In the experimental system, 50% of the excess sludge was exposed to FNA at 2.0 mg N/L for 24–42 h in an FNA treatment unit and then returned to the parent reactor. The sludge wastage flow from the experimental reactor was adjusted to obtain a similar mixed liquor suspended solids concentration to that in the control reactor, which was operated at a sludge retention time of 15 days. It was found that sludge production in the experimental system was 28% lower in comparison to the control. The addition of the FNA-treated sludge did not negatively affect the treatment performance and sludge properties of the experimental reactor, namely the organic carbon and nitrogen removal, nitrous oxide (N2O) emission and sludge settleability. Endogenous denitrification batch tests indicate that FNA-treated sludge is available as a carbon source for denitrification. [Copyright &y& Elsevier]

Abstract: Limited availability of carbon sources has been regarded as an important factor leading to N2O accumulation during denitrification in wastewater treatment. By varying the carbon (methanol) loading rate to a methanol utilizing denitrifying culture in the presence of various electron acceptors (nitrate, nitrite, N2O and their combinations), this study quantitatively investigated the electron distribution among different nitrogen oxide reductases during denitrification. The results showed that electron competition occurs under not only carbon limiting but also carbon abundant conditions. The electron distribution among the nitrogen oxide reductases is affected by the carbon loading rate, with a lower fraction of electrons distributed to the N2O reductase with reduced carbon loading rate. N2O accumulation occurs when the electron flux going to nitrite reduction is higher than that going to N2O reduction. The study also showed that, for the culture investigated, the carbon to nitrogen ratio is not a key factor leading to N2O accumulation. [Copyright &y& Elsevier]

Nitrogen removal via nitrite (the nitrite pathway) is beneficial for carbon-limited biological wastewater treatment plants. However, partial nitrification to nitrite has proven difficult in continuous processes treating domestic wastewater. The nitrite pathway is achieved in this study in a pilot-scale continuous pre-denitrification plant (V =300L) treating domestic wastewater by controlling the dissolved oxygen (DO) concentration at 0.4–0.7mg/L. It is demonstrated that the nitrite pathway could be repeatedly and reliably achieved, with over 95% of the oxidized nitrogen compounds at the end of the aerobic zone being nitrite. The nitrite pathway improved the total nitrogen (TN) removal by about 20% in comparison to the nitrate pathway, and also reduced aeration costs by 24%. FISH analysis showed that the nitrite oxidizing bacteria (NOB) population gradually reduced at low DO levels, and reached negligible levels when stable nitrite pathway was established. It is hypothesized that NOB was washed out due to its relatively lower affinity with oxygen. A lag phase was observed in the establishment of the nitrite pathway. Several sludge ages were required for the onset of the nitrite pathway after the application of low DO levels. However, nitrite accumulation increased rapidly after that. A similar lag phase was observed for the upset of the nitrite pathway when a DO concentration of 2–3mg/L was applied. The nitrite pathway negatively impacted on the sludge settleability. A strong correlation between the sludge volume index and the degree of nitrite accumulation was observed. [Copyright &y& Elsevier]

The enhanced biological phosphorus removal (EBPR) process has been implemented in many wastewater treatment plants worldwide. While the EBPR process is indeed capable of efficient phosphorus (P) removal performance, disturbances and prolonged periods of insufficient P removal have been observed at full-scale plants on numerous occasions under conditions that are seemingly favourable for EBPR. Recent studies in this field have utilised a wide range of approaches to address this problem, from studying the microorganisms that are primarily responsible for or detrimental to this process, to determining their biochemical pathways and developing mathematical models that facilitate better prediction of process performance. The overall goal of each of these studies is to obtain a more detailed insight into how the EBPR process works, where the best way of achieving this objective is through linking together the information obtained using these different approaches. This review paper critically assesses the recent advances that have been achieved in this field, particularly relating to the areas of EBPR microbiology, biochemistry, process operation and process modelling. Potential areas for future research are also proposed. Although previous research in this field has undoubtedly improved our level of understanding, it is clear that much remains to be learned about the process, as many unanswered questions still remain. One of the challenges appears to be the integration of the existing and growing scientific knowledge base with the observations and applications in practice, which this paper hopes to partially achieve. [Copyright &y& Elsevier]