Aggregate Size and Architecture Determine Microbial Activity Balance for One-Stage Partial Nitritation and Anammox

Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing.In the last few years, autotrophic nitrogen removal via partial nitritation and anoxic ammonium oxidation (anammox) has evolved from lab- to full-scale treatment of nitrogenous wastewaters with a low biodegradable organic compound content, and this evolution has been driven mainly by a significant decrease in the operational costs compared to the costs of conventional nitrification and heterotrophic denitrification (11, 23). Oxygen-limited autotrophic nitrification and denitrification (OLAND) is one of the autotrophic processes used and is a one-stage procedure; i.e., partial nitritation and anammox occur in the same reactor (30). The “functional” autotrophic microorganisms in OLAND include aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB). With oxygen, AerAOB oxidize ammonium to nitrite (nitritation), and with the nitrite AnAOB oxidize the residual ammonium to form dinitrogen gas and some nitrate (anammox). Additional aerobic nitrite oxidation to nitrate (nitratation) by nitrite-oxidizing bacteria (NOB) lowers the nitrogen removal efficiency, but it can, for instance, be prevented at low dissolved oxygen (DO) levels because the oxygen affinity of AerAOB is higher than that of NOB (16). Reactor configurations for the OLAND process can be based on suspended biomass growing in aggregates, like that in a sequencing batch reactor (SBR) (37) or a gas lift or upflow reactor (32). For suspended-growth systems there are two important challenges: biomass retention and equilibrated microbial activities.High biomass retention efficiency is a prerequisite in anammox technologies because of the slow growth of AnAOB (33). In suspended biomass systems, settling properties determine the retention of biomass and are related to the microbial aggregate morphology (floc or granule) and size. Granules can be defined as compact and dense aggregates with an approximately spherical external appearance that do not coagulate under decreased hydrodynamic shear conditions and settle significantly faster than flocs (18). Toh and coworkers calculated a lower sludge volume index for aerobic granules than for aerobic flocs and also showed that there was an increase in the settling velocity with increasing granule size (35). Hence, in terms of physical properties, large granules are preferable for suspended-growth applications.OLAND aggregate size not only influences settling properties but also affects the proportion of microbial nitrite production and consumption; lower AerAOB activity and higher AnAOB activity were observed with larger aggregates (25, 37). Theoretically, a microbial aggregate with equal nitrite production and nitrite consumption can remove ammonium autonomously, because of its independence from other aggregates for acquisition and conversion of nitrite. Hence, with an increasing aggregate size and thus with a decreasing ratio of nitrite production to nitrite consumption, three functional categories of aggregates can be distinguished: nitrite sources, autonomous nitrogen removers, and nitrite sinks. Because minimal nitrite accumulation is one of the prerequisites for high nitrogen removal efficiency in OLAND reactors, the presence of excess small aggregates is undesirable (9, 37).Although large granular aggregates are desirable for biomass retention and activity balance, so far no formation mechanisms have been proposed for OLAND granules, in contrast to the well-studied anaerobic (13) and aerobic (1) granules. In order to determine general and environment-specific determinants for aggregate size and architecture, three suspended-growth OLAND reactors with different inoculation and operation (mixing and aeration) parameters were selected, and these reactors were designated reactors A, B, and C (Table ​(Table1).1). The first objective of this study was to gain more insight into the relationship between OLAND aggregate size, AerAOB and AnAOB abundance, and the activity balance. The second objective was to propose pathways for aggregation and granulation by relating (dis)similarities in aggregate size distribution, morphology, and architecture to differences in reactor inoculation and operation.

TABLE 1.

Overview of the three OLAND reactor systems from which suspended biomass samples were obtained

The time response of anaerobic digestion microbiome during an organic loading rate shock

Knowledge of connections between operational conditions, process stability, and microbial community dynamics is essential to enhance anaerobic digestion (AD) process efficiency and management. In this study, the detailed temporal effects of a sudden glycerol-based organic overloading on the AD microbial community and process imbalance were investigated in two replicate anaerobic digesters by a time-intensive sampling scheme. The microbial community time response to the overloading event was shorter than the shifts of reactor performance parameters. An increase in bacterial community dynamics and in the abundances of several microbial taxa, mainly within the Firmicutes, Tenericutes, and Chloroflexi phyla and Methanoculleus genera, could be detected prior to any shift on the reactor operational parameters. Reactor acidification already started within the first 24 h of the shock and headed the AD process to total inhibition in 72 h alongside with the largest shifts on microbiome, mostly the increase of Anaerosinus sp. and hydrogenotrophic methanogenic Archaea. In sum, this work proved that AD microbial community reacts very quickly to an organic overloading and some shifts occur prior to alterations on the performance parameters. The latter is very interesting as it can be used to improve AD process management protocols.

     

Long-chain acylhomoserine lactones increase the anoxic ammonium oxidation rate in an OLAND biofilm

The oxygen-limited autotrophic nitrification/denitrification (OLAND) process comprises one-stage partial nitritation and anammox, catalyzed by aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB), respectively. The goal of this study was to investigate whether quorum sensing influences anoxic ammonium oxidation in an OLAND biofilm, with AnAOB colonizing 13% of the biofilm, as determined with fluorescent in situ hybridization (FISH). At high biomass concentrations, the specific anoxic ammonium oxidation rate of the OLAND biofilm significantly increased with a factor of 1.5 ± 0.2 compared to low biomass concentrations. Supernatant obtained from the biofilm showed no ammonium-oxidizing activity on itself, but its addition to low OLAND biomass concentrations resulted in a significant activity increase of the biomass. In the biofilm supernatant, the presence of long-chain acylhomoserine lactones (AHLs) was shown using the reporter strain Chromobacterium violaceum CV026, and one specific AHL, N-dodecanoyl homoserine lactone (C₁₂-HSL), was identified via LC-MS/MS. Furthermore, C₁₂-HSL was detected in an AnAOB-enriched community, but not in an AerAOB-enriched community. Addition of C₁₂-HSL to low OLAND biomass concentrations resulted in a significantly higher ammonium oxidation rate (p < 0.05). To our knowledge, this is the first report demonstrating that AHLs enhance the anoxic ammonium oxidation process. Future work should confirm which species are responsible for the in situ production of C₁₂-HSL in AnAOB-based applications.     

Correlations between molecular and operational parameters in continuous lab-scale anaerobic reactors

In this study, the microbial community characteristics in continuous lab-scale anaerobic reactors were correlated to reactor functionality using the microbial resource management (MRM) approach. Two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP), were applied to analyze the bacterial and archaeal communities, and the results obtained have been compared. Clustering analyses showed a similar discrimination of samples with DGGE and T-RFLP data, with a clear separation between the meso- and thermophilic communities. Both techniques indicate that bacterial and mesophilic communities were richer and more even than archaeal and thermophilic communities, respectively. Remarkably, the community composition was highly dynamic for both Bacteria and Archaea, with a rate of change between 30% and 75% per 18 days, also in stable performing periods. A hypothesis to explain the latter in the context of the converging metabolism in anaerobic processes is proposed. Finally, a more even and diverse bacterial community was found to be statistically representative for a well-functioning reactor as evidenced by a low Ripley index and high biogas production.     

Biodegradation kinetic constants and sorption coefficients of micropollutants in membrane bioreactors

In order to elucidate the capability of biomass developed in membrane bioreactors (MBR) to degrade and sorb emerging micropollutants, biodegradation (k_biol) and sorption (k_sor) kinetic constants as well as solid–liquid partition coefficients (K_d) of 13 selected pharmaceutical and personal care products (PPCPs) were determined with MBR heterotrophic biomass adding a pulse (100 ppb of each compound) and following the liquid and solid phase concentrations over time. The results obtained were compared to literature data referring to conventional activated sludge (CAS) systems. Two experiments were performed: one in the MBR itself and the second one in a batch reactor with the same type and concentration of biomass as in the MBR. Overall, both biodegradation and sorption coefficients were in the same range as previously reported by other studies in CAS systems, indicating that MBR biomass does not show better capabilities for the biological degradation and/or sorption of PPCPs compared to the biomass developed in CAS reactors. Therefore, the higher PPCPs removal efficiencies found in MBRs are explained by the high biomass concentrations obtained at the long sludge retention times at which this type of reactors are usually operated.     

How are pharmaceutical and personal care products (PPCPs) removed from urban wastewaters?

The presence of bioactive trace pollutants such as pharmaceuticals and ingredients of personal care products (PPCPs) in different environmental compartments (rivers, lakes, groundwaters, sediments, etc.) is an emerging issue due to the lack of existing information about the potential impact associated with their occurrence, fate and ecotoxicological effects. Due to the low PPCP concentrations reported in wastewaters (ppb or ppt) and their complex chemical structure, common technologies used in sewage and drinking water treatment plants may not be efficient enough to accomplish their complete removal. Information about physico-chemical characteristics such as acidity, lipophilicity, volatility and sorption potential is a useful tool to understand the different removal patterns observed. In order to perform an accurate overall mass balance along the different units of sewage treatment plants, it is necessary to gather information not only about the presence of micropollutants in the aqueous phase, but also on the fraction sorbed onto solids. Since only some PPCPs are very well eliminated by conventional sewage treatment configurations, new strategies such as modification of operating conditions (e.g. solids retention time), implementation of new technologies (e.g. biomembrane reactors) or additional advanced post-treatment steps (e.g. oxidation, adsorption, membranes) have been suggested for an increased efficiency.     

Influence of transitional states on the microbial ecology of anaerobic digesters treating solid wastes

A better understanding of the microbial ecology of anaerobic processes during transitional states is important to achieve a long-term efficient reactor operation. Five wastes (pig manure, biodiesel residues, ethanol stillage, molasses residues, and fish canning waste) were treated in five anaerobic reactors under the same operational conditions. The influence of the type of substrate and the effect of modifying feeding composition on the microbial community structure was evaluated. The highest biomethanation efficiency was observed in reactors fed with fish canning waste, which also presented the highest active archaeal population and the most diverse microbial communities. Only two Bacteria populations could be directly related to a particular substrate: Ilyobacter with biodiesel residues and Trichococcus with molasses residues. Results showed that the time to achieve steady-state performance after these transitional states was not dependent on the substrate treated. But reactors needed more time to handle the stress conditions derived from the start-up compared to the adaptation to a new feeding. Cluster analyses showed that the type of substrate had a clear influence on the microbiology of the reactors, and that segregation was related to the reactors performance. Finally, we conclude that the previous inoculum history treating solid waste and higher values of active Archaea population are important factors to face a successful change in substrate not entailing stability failure.     

Improvement of the anaerobic treatment of potato processing wastewater in a UASB reactor by co-digestion with glycerol

The effect of three different types of glycerol on the performance of up-flow anaerobic sludge blanket (UASB) reactors treating potato processing wastewater was investigated. High COD removal efficiencies were obtained in both control and supplemented UASB reactors (around 85%). By adding 2 ml glycerol product per liter of raw wastewater, the biogas production could be increased by 0.74 l biogas ml⁻¹ glycerol product, which leads to energy values in the range of 810–1270 kWh_electric per m³ product. Moreover, a better in-reactor biomass yield was observed for the supplemented UASB reactor (0.012 g VSS g⁻¹ COD_removed) compared to the UASB control (0.002 g VSS g⁻¹ COD_removed), which suggests a positive effect of glycerol on the sludge blanket growth.     

Relationship between microbial activity and microbial community structure in six full-scale anaerobic digesters

High activity levels and balanced anaerobic microbial communities are necessary to attain proper anaerobic digestion performance. Therefore, this work was focused on the kinetic performance and the microbial community structure of six full-scale anaerobic digesters and one lab-scale co-digester. Hydrolytic (0.6–3.5 g COD g^?1 VSS d^?1) and methanogenic (0.01–0.84 g COD g^?1 VSS d^?1) activities depended on the type of biomass, whereas no significant differences were observed among the acidogenic activities (1.5–2.2 g COD g^?1 VSS d^?1). In most cases, the higher the hydrolytic and the methanogenic activity, the higher the Bacteroidetes and Archaea percentages, respectively, in the biomasses. Hydrogenotrophic methanogenic activity was always higher than acetoclastic methanogenic activity, and the highest values were achieved in those biomasses with lower percentages of Methanosaeta. In sum, the combination of molecular tools with activity tests seems to be essential for a better characterization of anaerobic biomasses.