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 Granular Microbiome at Different Organic Loads different operating conditions can be important for engineers to predict the stability of reactor functions during the start-up and continued reactor operation.

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.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0471-5) contains supplementary material, which is available to authorized users.

Abstract:The aerobic granular sludge process is a promising technology for the removal of nutrients and organic contaminants from wastewater. However, a large amount of the sludge is often washed out during the start-up of granular reactors, which results in reduced process performance and a protracted start-up phase. In this study, the possibility of a rapid start-up of the nitrification process through a stepwise decrease of the settling time was investigated, and the bacterial population dynamics in two lab-scale sequencing batch reactors were studied. The results demonstrated that the stepwise decrease of the settling time enabled fast granulation and rapid start-up of the process. Small cores of granules were already observed after 10 days of operation, and the biomass was dominated by granules after 28 days. The removal of organic matter and ammonium was >95% after one day and 14 days, respectively. The bacterial community composition changed rapidly during the first 21 days, resulting in strongly reduced richness and evenness. The diversity increased at a later stage, and the bacterial community continued changing, albeit at a slower pace. The rate of the stepwise decrease in settling time strongly affected the abundance of nitrifying organisms, but not the general composition of the bacterial community. The results of this study support the idea that a stepwise decrease of the settling time is a successful strategy for the rapid start-up of aerobic granular sludge reactors.

Aerobic granular sludge is an energy efficient and compact biofilm process for wastewater treatment which has received much attention during the last decades and is now being implemented in full-scale. However, the factors involved in microbial community assembly during formation of granules are poorly understood and little is known about the reproducibility in treatment performance and community structure. Here we show that both deterministic and stochastic factors exert a dynamic influence during microbial community assembly into granular sludge. During granulation, the microbial communities in three replicate sequencing batch reactors followed similar successional trajectories of the most abundant taxa and showed similar dynamics in diversity. Deterministic factors dominated the assembly of the most abundant community members as the microbial community transitioned from floccular to granular form. Stochastic factors mostly affected rare members of the communities and caused the microbial community structure to diverge in one of the reactors; however, this did not have an impact on the treatment performance. This demonstrates that the reactor function and the dynamics of the most abundant community members are in fact reproducible during the formation of aerobic granules.