Multistability and Reversibility of Aerobic Granular Sludge Microbial Communities Upon Changes From Simple to Complex Synthetic Wastewater and Back

Aerobic granular sludge (AGS) is a promising alternative wastewater treatment to the conventional activated sludge system allowing space and energy saving. Basic understanding of AGS has mainly been obtained using simple wastewater containing acetate and propionate as carbon source. Yet, the aspect and performances of AGS grown in such model systems are different from those obtained in reactor treating real wastewater. The impact of fermentable and hydrolyzable compounds on already formed AGS was assessed separately by changing the composition of the influent from simple wastewater containing volatile fatty acids to complex monomeric wastewater containing amino acids and glucose, and then to complex polymeric wastewater containing also starch and peptone. The reversibility of the observed changes was assessed by changing the composition of the wastewater from complex monomeric back to simple. The introduction of fermentable compounds in the influent left the settling properties and nutrient removal performance unchanged, but had a significant impact on the bacterial community. The proportion of Gammaproteobacteria diminished to the benefit of Actinobacteria and the Saccharibateria phylum. On the other hand, the introduction of polymeric compounds altered the settling properties and denitrification efficiency, but induced smaller changes in the bacterial community. The changes induced by the wastewater transition were only partly reversed. Seven distinct stables states of the bacterial community were detected during the 921 days of experiment, four of them observed with the complex monomeric wastewater. The transitions between these states were not only caused by wastewater changes but also by operation failures and other incidences. However, the nutrient removal performance and settling properties of the AGS were globally maintained due to the functional redundancy of its bacterial community.

Polyphosphate - a key biopolymer in aerobic granular sludge technology

Aline Sondra Adler, Arnaud Michel Gelb, Christof Holliger

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.

2016

Influence of Wastewater Composition on the Microbial Communities of Aerobic Granular sludge

Aline Sondra Adler, Valérie Berclaz, Christof Holliger, Marie Christelle Horisberger, Julien Maillard, Eva Reynaert

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 processes has mainly been obtained in laboratory-scale studies with simple synthetic wastewaters containing volatile fatty acids (VFA) as main carbon sources. Yet, the bacterial communities of AGS treating municipal wastewater have been much less investigated. Two approaches were applied here to assess the impact of fermentable and polymeric compounds on the microbial communities of the AGS and thus make a step toward the comprehension of AGS systems treating municipal wastewater. The first approach was to run an AGS reactor with a simple synthetic wastewater containing VFA as carbon sources and to make a progressive transition to a complex synthetic wastewater, by adding fermentable and polymeric compounds. The bacterial communities of weekly samples were monitored by 16S rRNA gene amplicon sequencing. The second approach was to start in parallel four AGS reactors fed with distinct wastewaters with activated sludge as inoculum. Two reactors were fed with the two synthetic wastewaters used in the first approach, the simple and complex synthetic wastewaters. The other two reactors were fed with either primary clarifier effluent or raw municipal wastewater. In both experiments, the microbial community from the AGS treating simple synthetic wastewater was clearly different from the one treating complex synthetic wastewater. Similarities within the microbial communities fed with the same carbon source were identified. Interestingly the synthetic complex wastewater led to a bacterial community which was closer to the one obtained in the reactors treating real wastewaters. The results of this study show that the type of carbon source is a major factor shaping the bacterial community structure of AGS and that by working with simple synthetic wastewater, one may bacteria which play an important role in the treatment of municipal wastewater. The use of a complex synthetic wastewater may provide a more appropriate model system for the study of the bacterial communities in AGS.

2019

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

Aline Sondra Adler

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 ana