Comparison of the bacterial community composition in the granular and the suspended phase of sequencing batch reactors

Wastewater treatment by aerobic granular sludge is a low-footprint technology that allows effective pollutant removal even at high loading rates (Beun et al. 1999; de Bruin et al. 2004). During granulation, bacterial cells are self-immobilized in an EPS (extracellular polymeric substances) matrix, resulting in a dense, compact structure with an anaerobic/anoxic core and an aerobic outer layer (Beun et al. 2001; de Kreuk et al. 2005).

Granulation is usually achieved by high hydraulic selection pressure, i.e. a sequencing batch reactor (SBR) is operated either with short settling time (variable volume operation mode) or with high upflow velocity (constant volume operation/fill-draw mode), both resulting in the wash-out of slow settling particles. Low effluent quality due to high suspended solids (SS) concentration has frequently been reported both in laboratory-scale and pilot-scale applications, fed with synthetic and/or real wastewater (Inizan et al. 2005; Yilmaz et al. 2008; Morales et al. 2013; Rocktäschel et al. 2015; Derlon et al. 2016). Strategies to decrease the effluent SS concentration include lower upflow velocity combined with selective sludge removal (Pronk et al. 2015; Derlon et al. 2016) or longer settling time and lower degree of granulation (Rocktäschel et al. 2015).

The ecological implications of the strong wash-out conditions on the microbial community are not yet fully understood, despite being one of the strongest selective forces applied in SBRs for aerobic granulation. It has been shown that the wash-out rate of different bacterial groups can be different, depending on their spatial distribution within the granule (Winkler et al. 2012). Bacterial populations growing on the surface of the granules may be eroded and washed out in higher numbers than bacteria growing deeper in the granule. Thus, bacterial groups situated in the exterior layer of the granules are likely to contribute more to the suspended solids content in the effluent than bacteria situated in the interior layer. However, if a granule breaks up, exterior and interior bacterial populations will be washed out equally, presuming that the particles are not too dense. The density of the broken granule particle depends on the type(s) of bacteria it is comprised of (Gonzalez-Gil and Holliger 2014), and on the predominant granulation mechanisms—self-aggregation of floccular biomass, self-aggregation of small granules (microcolony aggregation), microcolony outgrowth, or attachment of floccular biomass to granular biomass (Barr et al. 2010; Verawaty et al. 2012; Zhou et al. 2014). Depending on process conditions and the bacteria dominating the microbial community, different granulation mechanisms were reported to prevail (Weissbrodt et al. 2013).

In this study, we followed the community composition in granular sludge reactors at different operational conditions for 12 weeks, both in the granular phase and the suspended phase (effluent). This work is, to our knowledge, the first report about the microbial community composition in the effluent of granular sludge reactors. We assessed how the spatial distribution of certain taxa affected its wash-out, and compared the bacterial community in the granular and suspended phases to gain better understanding of the successional patterns in granular sludge reactors.

Based on the dissimilarity matrices and the correlation analyses, it appears that the community composition of the effluent is similar but not identical to the community composition of the granular sludge. By calculating the retention ratios, the similarities and differences could be examined more thoroughly. Some genera became more abundant in the effluent as the experiment progressed. Other genera, on the contrary, were increasingly retained as the flocculated biomass turned into granules.

During start-up (the first 6 weeks of operation), many genera seemed to be washed out proportionally to their relative abundance in the biomass. We assume that during start-up a large fraction of the biomass is still flocculated, and every floc has similar settleability. Thus the probability of cells being discharged with the effluent is similar for every taxa, irrespective of which floc they are attached to or where in the floc they are situated. As soon as granules start to develop, differences in settleability emerge, and depending on the spatial location and the granulation mechanisms some cells are more likely to be washed out than others.

During steady-state operation (weeks 7–12), most of the biomass is granulated and dense enough to be retained in the reactor. Nonetheless, certain genera had a retention ratio significantly lower than one (Fig. 3), i.e. they were more abundant in the effluent than in the granules. A possible explanation is that these genera were situated on the surface of the granule, and were therefore exposed to granule erosion, as suggested by Winkler et al. (2012). They found that bacterial groups situated on the surface (e.g. ammonium oxidizing bacteria) were more prone to be washed out than bacterial groups situated in the core of the granules. We found that two abundant groups of bacteria, Meganema sp. and Zoogloea sp., which had a low retention ratio in all three reactors during steady-state operation (Fig. 3), were situated mostly in the outer layer of the granules, where the biomass was loosely packed (Fig. 4) and therefore more likely to be washed out due to granule erosion. Meganema sp. is usually found in aerobic environments, therefore it is not expected to grow in the inner parts of the granules (Kragelund et al. 2005). Zoogloea sp. has been reported to produce EPS containing high amounts of water and to grow as slimy colonies, thus the high abundance of these bacteria in the outer layer might explain the lower density of this part of the granule (Thomsen et al. 2007; Nielsen et al. 2010). Moreover, the typical morphological characteristics of these genera (filamentous growth for Meganema sp. (Kragelund et al. 2005), and finger-like structures in case of Zoogloea sp. (Rosselló-Mora et al. 1995) make them even more likely to detach when exposed to high shear force. Another possible explanation for the high abundance in the effluent is that these bacteria grew not only attached to the granule, but also in the bulk liquid as free floating cells. Both Meganema and Zoogloea have high substrate uptake rate and growth rate (Roinestad and Yall 1970; Kragelund et al. 2005), therefore they may also grow in suspended phase even in SBRs operated at 2 min settling time and 9.3 h HRT. It has also been shown that suspended biomass can be retained under wash-out conditions if attached to the rough surfaces of broken granules (Verawaty et al. 2012) or sheltered in indentations on the granule surface (Gonzalez-Gil and Holliger 2014).

Some genera had a retention ratio significantly larger than one during steady-state operation (Fig. 3). These genera had a higher relative abundance in the granules than in the effluent, and were not washed out of the reactor. However, this does not necessarily mean that their relative abundance in the granules increased with time. For example, Brevundimonas in R1, Acidovorax in R2, or Comamonas in R3 had an average retention ratio above one during steady-state operation (Fig. 3; Table 3), but their relative abundance decreased with time (Additional file 1: Figure S7). In general, no statistically significant correlation was found between the retention ratio and the temporal variation of relative abundance. Slow growing species located in the interior of the granule may be protected from erosion and thus have a high retention ratio (>1), but their relative abundance is likely to decrease because they become outnumbered by other species with higher growth rate. Taxa with consistently high or increasing relative abundance (Additional file 1: Figure S7) and high retention ratio (e.g. Flavobacterium and Bdellovibrio in R1, or OTU_6 in R2 and R3) are presumably growing well under the respective operational conditions, and they are probably growing in the core of the granules (protected from erosion). Indeed, Flavobacterium and Bdellovibrio were located in the inner parts of the granule (Fig. 5), thus suggesting that genera with high retention ratios are actually growing in deeper parts of the granules. Bdellovibrio is a bacterial predator (Rosenberg et al. 2014), while Flavobacterium spp. have been reported to hydrolyze various substrates (Bernardet et al. 1996), thus they are likely to grow on living or dead biomass, soluble microbial products, and EPS. These substrates can be found in the core of the granule. The presence of predatory bacteria like Bdellovibrio has been reported earlier in granular sludge (Wan et al. 2014; Weissbrodt et al. 2014; Li et al. 2014), but the effect of predation on the ecosystem is not yet fully understood. Despite being obligate aerobes (Rosenberg et al. 2014), Bdellovibrio spp. were located in the inner parts of the granule. It has been reported earlier that certain Bdellovibrio species can predate under anoxic conditions (Monnappa et al. 2013). Moreover, we have found that oxygen can penetrate the deeper regions of the granules through channels (Szabó et al. 2017), where most of the Bdellovibrio cells were found (Fig. 5).

The core of the granule is not only protected from erosion, but also provides microaerobic and anaerobic niches [above a certain diameter, i.e. after a certain number of weeks of operation, and depending on the bulk COD concentration (Szabó et al. 2017)]. Thus, denitrifying organisms (e.g. Acidovorax spp., Pseudorhodobacter spp., Rhodobacter spp., Fig. 3; Table 3) can be expected to have a high retention ratio (low relative read abundance in the effluent). Bacteria situated in the core are washed out only in case the granule breaks up, and due to the young age (<100 days) of our granules it is likely that very few granules broke up during the experiment (Gonzalez-Gil and Holliger 2014).

EPS producing taxa (e.g. Meganema, Thauera, and Zoogloea) were observed in high abundance in every reactor, which suggests that the predominant aggregation mechanism was microcolony outgrowth (Weissbrodt et al. 2013). These genera are usually present in conventional activated sludge ecosystems, but in lower abundances (in the seed sludge the cumulative relative read abundance of EPS producers was only 13%). Many of the EPS producing genera typical for sewage treatment plants are mixotrophic bacteria capable of denitrification and/or PHA production (Etchebehere et al. 2003; Lu et al. 2014; McIlroy et al. 2015; Inoue et al. 2016; McIlroy et al. 2016). EPS was earlier reported to play an important role in the formation and (mechanical) stability of granular sludge (Weber et al. 2007; Lemaire et al. 2008; Tan et al. 2014), while denitrification was reported to accelerate granule formation (Wan and Sperandio 2009; Suja et al. 2015). The EPS producing functional group was dominated by different genera in the three reactors (Thauera in R1, Meganema in R2 and Zoogloea in R3) due to the different organic loading rates applied.

It can be concluded that the community composition of the suspended phase is similar but not identical to the community composition of the granular phase. Complex patterns were observed for the dynamics of the reactor and effluent microbial communities. During the first part of the experiment, most genera showed similar relative abundance in the reactor and effluent samples. Once granules were developed, bacterial groups located in the interior of the granules tended to be present in lower numbers in the effluent, while taxa growing on the surface and/or in the bulk phase were more abundant in the effluent. However there was no correlation between the degree of wash-out and the temporal dynamics of individual taxa in the granules. Bacteria that were well retained in the granules may be numerically outcompeted over time, as seen by their decreasing relative abundance. On the other hand, bacteria that may appear to be washed out of the system, as seen by their high relative abundance in the suspended phase, may in fact grow in suspension and might even reattach to the granules.