Evolution of microbial communities and nutrient removal performances in aerobic granular sludge sequencing batch reactor during change of substrate

• Aerobic granular sludge (AGS) is a promising alternative wastewater treatment to the conventional activated sludge system. As AGS has enhanced settling abilities and provides different redox conditions across the granules at the same time, the processes based on AGS allow substantial space, energy and chemical products savings. A few wastewater treatment plants (mainly hybrid or pilot plants) are already using AGS. Nevertheless the performances of lab-scale reactors fed with simple synthetic wastewater are usually different from those of plants treating real wastewater where nutrient removal performances are more versatile and granules have a fluffy structure. Moreover, if the microbial community structures from AGS reactor fed with simple synthetic substrate have been extensively studied, it is less the case of the biomass from AGS reactors fed with more complex substrates or real wastewater. Material and Methods • In order to make a step toward the comprehension of AGS used to treat municipal wastewater, this experiment was designed to study the impact of polymeric organic compounds on nutrient removal performances and microbial communities. These properties were monitored on a lab scale AGS sequencing batch reactor during a progressive substrate transition from volatile fatty acids (VFAs), to a more complex substrate mixture containing VFAs, glucose and amino acids, and finally to a synthetic wastewater containing VFAs, starch and oligo-peptides. Results and Conclusions • With the lowered VFA concentrations, phosphorus (P)-removal performances were impaired. At the same time, amino acids fermentation significantly increased the ammonium concentration, thus extending the time required for total nitrification. The composition of the artificial wastewater was further adapted in order to recover efficient P- and nitrogen (N)-removal. Molecular analyses will show how the changes in substrate composition have influenced the AGS bacterial community structure that has kept its high settling ability and the granule size distribution throughout the first substrate transition

Evolution of microbial communities in aerobic granular sludge during changes in wastewater composition

Aerobic granular sludge (AGS) is an emerging technology offering an alternative wastewater treatment with a reduced footprint compared to conventional activated sludge systems. Basic understanding of AGS has mainly been obtained in lab-scale reactors fed with simple synthetic wastewaters. Yet, the properties and performances of AGS cultivated in these model systems are different from those obtained in reactors treating real wastewater. The composition of the synthetic wastewater of an AGS reactor was progressively changed from a simple wastewater containing mainly volatile fatty acids to a complex monomeric wastewater containing amino acids and glucose (1), from complex monomeric wastewater to a complex polymeric wastewater containing starch and peptone (2), and from complex monomeric back to simple wastewater (3). The microbial community of the sludge, its performances and properties were monitored. During transition (1), a clear shift from the dominant class of β-Proteobacteria to Actinobacteria was first observed, followed by a second phase where the two classes were present in high abundance. During transition (2), a slight increase of members of the Saccharibacteria phylum and the class of Sphingobacteria was noticed. After transition (3) the microbial community was similar to the initial one. The nutrient removal performances and granulation remained stable with the monomeric wastewaters. However, denitrification and granulation were impaired with the addition of polymeric compounds (2). As conclusion, changes in the influent wastewater composition led to reversible changes in the microbial communities of AGS while denitrification performances and granulation were affected by the presence of polymeric compounds in the influent wastewate

Phage-dependent variability of Candidatus ‘Accumulibacter phosphatis’ populations in aerobic granular sludge

During the past 20 years, aerobic granular sludge (AGS) has been extensively studied with the aim to develop an attractive alternative to conventional activated sludge for wastewater treatment. The phosphate-accumulating organism (PAO) Candidatus ‘Accumulibacter phosphatis’ is often found with significant abundance in AGS as well as in other enhanced biological phosporus removal (EPBR) systems. Although they have never been isolated in pure culture, members of this bacterial genus appear to be genetically and physiologically more diversified than initially expected. Impaired EBPR performances observed in lab- and full-scale reactors have often been correlated to a decrease in Accumulibacter populations. This phenomenon has mainly been linked to the presence of bacterial competitors such as glycogen-accumulating organisms (GAO), and bacteriophages have only rarely been suspected to be responsible for this depletion (1). In the present study, the metagenome of 46 individual granules from a lab-scale AGS sequencing batch reactor was sequenced. The results showed a surprisingly variable relative abundance of Accumulibacter populations amongst the different granules that could only be partially explained by the “phenotype” of these granules. A co-occurrence analysis revealed a strong negative correlation between the number of Accumulibacter sequencing reads with the relative abundance of two bacteriophages, namely EBPR podovirus 1 (EPV1) and EBPR podovirus 3 (EPV3), that have been previously detected in a lab-scale EBPR reactor (2). These results suggest that these phages are the major reason for the variability of Accumulibacter relative abundance in the sampled granules which raises the question whether the Accumulibacter populations in the different granules have different sensitivities towards these phages. (1) Barr et al., 2010, Fems Microbiology Ecology, 631-642 (2) Skennerton et al., 2011, Plos On

Influence of Wastewater Composition on Microbial Communities of Aerobic Granules and their Nutrient Removal Performances

ABSTRACT Basic understanding of aerobic granular sludge (AGS) processes has mainly been obtained in laboratory-scale studies with simple synthetic wastewaters. Two approaches were applied here to make a step toward the comprehension of AGS systems treating real municipal wastewater. One approach consisted in increasing the complexity of the influent composition of an AGS sequencing batch reactor (SBR) fed with volatile fatty acids, the other in starting up four AGS SBRs with four different wastewaters. Nutrient removal could be maintained in the first approach and indications for a change in the population responsible for biological phosphorous removal were obtained (P-removal). The four reactors started up with different wastewaters showed different granulation behaviour and P-removal was impaired in the reactors fed with municipal wastewater. More detailed investigations of the microbial communities will allow to elucidate the reasons behind the observations made in this preliminary study

The effect of different organic substrates on the microbial communities of aerobic granular wastewater treatment sludge

Aerobic granular sludge (AGS) is a promising alternative wastewater treatment to the conven- tional activated sludge system, allowing space and energy savings. This process is particularly suited for biological phosphorus removal, avoiding use of coagulant chemicals. Basic under- standing of this process has mainly been obtained in laboratory-scale studies with simple synthetic wastewater containing volatile fatty acids as main carbon source. Yet, the aspect and performance of granular sludge cultivated in such model systems are rather different from those obtained in systems treating real wastewater. In order to make a step toward the comprehension of AGS treating municipal wastewater, two approaches were applied to investigate the impact of the wastewater composition on AGS bacterial communities, settling properties and nutrient removal performance. The first approach was to transform activated sludge performing enhanced biological phosphorus removal into AGS in four parallel lab-scale reactors fed with different wastewater types: simple and complex polymeric synthetic, as well as raw and clarified municipal wastewater. The second approach was to progressively change the composition of the wastewater treated by AGS acclimated to simple synthetic wastewater to complex polymeric wastewater with an intermediary step with complex monomeric wastewater as a control. The whole DNA of biomass corresponding to the three wastewater types was sequenced with PacBio and Illumina technologies. During the two experiments, the bacterial communities, settling properties and nutrient removal performance were monitored. The bacterial communities in AGS treating simple wastewater were drastically different form the ones treating complex wastewater. Several taxa belonging to Actinobacteria and Saccha- ribacteria were largely underrepresented with the simple wastewater. Lower nitrogen removal, lower settling properties and higher proportions of flocs were observed with the polymeric wastewaters compared to the monomeric wastewaters. The lower concentrations of diffusible organic carbon rather than the bacterial community compositions were identified as the cause for these differences. Indeed, genes putatively involved in denitrification and biofilm formation were found in the AGS treating monomeric and polymeric wastewater. Moreover, different denitrification efficiencies and settling properties were observed with AGS having very similar bacterial communities but treating wastewater with a different concentration of organic carbon. The phosphate accumulating organism (PAO) Candidatus (Ca.) Accumulibacter, abundant in most of the AGS samples, was highly diverse and different clade repartitions were found within the AGS treating different wastewater types. The fermentative PAO Tetrasphaera was less diverse and mostly found in the AGS treating complex wastewater. The co-occurrence of two PAO occupying distinct ecological niches likely participated to the quick recovery of the P-removal after the transient but sharp decrease of Ca. Accumulibacter observed during the transition from simple to complex monomeric wastewater and likely due to a bacteriophage attack. The assembly of PacBio sequences associated to a binning based on composition and cov- erage of Illumina sequences produced nearly complete draft genomes attributed to poorly characterized taxa, thus providing information on their potential metabolism and a template for future metatranscriptomic an

Polyphosphate - a key biopolymer in aerobic granular sludge technology

Polyphosphate is the key biopolymer in wastewater treatment (WWT) processes applying enhanced biological phosphorus removal (EBPR) by polyphosphate-accumulating organisms (PAO). Alternating anaerobic (no oxygen and no nitrate) and aerobic phases is used to promote a net phosphorus removal from the wastewater as PAO are capable to take up and store phosphate as intracellular polyphosphate during the aerobic growth phase. In the anaerobic phase, PAO replenish their carbon and energy source in form of polyhydroxyalkanoates (PHA) from volatile fatty acids (VFA) present or formed in the wastewater. The energy and reducing equivalents needed to form PHA come from glycogen and polyphosphate, the polymers that are replenished during the aerobic phase. A few PAO have been already identified, among which Candidatus Accumulibacter phosphatis is a major player in WWT microbial communities enriched with VFA. However, depending on the carbon source the microbial community can strongly fluctuate and other PAO might play a major role in phosphorus removal. We are studying the dynamics of microbial communities in lab-scale aerobic granular sludge sequencing batch reactors subjected to changes of carbon source going from simple VFA to a mixture of VFA, glucose and amino acids. Both metagenomic approaches targeting polyphosphate kinase (ppk) genes and functional analysis of PAO using fluorescence techniques staining the polyphosphate polymer allow us to investigate the key metabolic genes in the synthesis of polyphosphate and expand the knowledge on the diversity of PAO in such engineered systems

Evolution of Microbial Communities and Nutrient Removal Performances of Aerobic Granular Sludge during Change of Substrates

Aerobic granular sludge (AGS) is a promising alternative wastewater treatment to the conventional activated sludge system, allowing substantial space, energy and chemichal products savings. Basic understanding of this processes has mainly been obtained in laboratory-scale studies with soluble synthetic wastewaters. Yet, the aspect and performances of granular sludge grown in such model systems are very different from those obtained in reactor treating real wastewater. It is hypothetized that fermentable compounds and hydrolyzable matter can have a major impact on AGS microbial communities. This experiment aims to study the impact of wastewater composition on the AGS microbial communities and their nutrient removal performances. After the first transition from simple to complex soluble synthetic wastewater, the nutrient removal performances were good, but the microbial community shifted from β-proteobacteria to Actinobacteria. In particular, the phosphate accumulating organism (PAO) Candidatus Accumulibacter decreased drastically letting Tetrasphaera be the only PAO present in significant abundance in the sludge.These results confirm the importance of the synthetic wastewater composition on the labscale models for aerobic granular sludge. In particular, Tetrasphaera seems to play an important role in phosphate removal from complex wastewater