Organic substrate diffusibility governs microbial community composition, nutrient removal performance and kinetics of granulation of aerobic granular sludge

Abstract

Basic understanding of formation of aerobic granular sludge (AGS) has mainly been derived from lab-scale systems with simple influents containing only highly diffusible volatile fatty acids (VFA) as organic substrate. This study compares start-up of AGS systems fed by different synthetic and municipal wastewaters (WW), characterised by increasing complexity in terms of non-diffusible organic substrate. Four AGS reactors were started with the same inoculum activated sludge and operated for one year. The development of AGS, settling characteristics, nutrient and substrate removal performance as well as microbial community composition were monitored. Our results indicate that the higher the content of diffusible organic substrate in the WW, the faster the formation of AGS. The presence of non-diffusible organic substrate in the influent WW led to the formation of small granules and to the presence of 20–40% (% of total suspended solids) of flocs in the AGS. When AGS was fed with complex influent WW, the classical phosphorus and glycogen accumulating organisms (PAO, GAO) were outcompeted by their fermentative equivalents. Substrate and nutrient removal was observed in all reactors, despite the difference in physical and settling properties of the AGS, but the levels of P and N removal depended on the influent carbon composition. Mechanistically, our results indicate that increased levels of non-diffusible organic substrate in the influent lower the potential for microbial growth deep inside the granules. Additionally, non-diffusible organic substrates give a competitive advantage to the main opponents of AGS formation – ordinary heterotrophic organisms (OHO). Both of these mechanisms are suspected to limit AGS formation. The presented study has relevant implications for both practice and research. Start-up duration of AGS systems treating high complexity WW were one order of magnitude higher than a typical lab-scale system treating VFA-rich synthetic WW, and biomass as flocs persisted as a significant fraction. Finally, the complex synthetic influent WW – composed of VFA, soluble fermentable and particulate substrate - tested here seems to be a more adequate surrogate of real municipal WW for laboratory studies than 100%-VFA WW.

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

“…The statistical analysis and related plots were performed as described (Layer et al, 2019) with R program v. 3.5.0 (R Development Core Team, 2008) using the packages reshape2 and ggplot2 (Wickham, 2007(Wickham, , 2016Warnes et al, 2016). Correlation data were computed with the function rcorr from the package Hmisc v. 4.4-1 (Harrell et al, 2020).…”

“…5 ′ GAGATCTACACT ATGGTAATTCC-AGMGTTYGATYMTGGCTCAG3 ′ and 5 ′ CAAGCAGAAGACGGCATACGAGAT-barcode-AGTCAG TCAGAA-GCTGCCTCCCGTAGGAGT3 ′ , respectively. The DNA was amplified according to the procedure described in Layer et al (2019). The amplified DNA quantified on a labCHip GX (Perkinelmer), with 1-2 µl of each PCR product and the kit DNA 5k (CLS760675).…”

“…The taxonomic affiliation of the 16S rRNA gene sequences was performed similarly to the procedure described (Layer et al, 2019). After demultiplexing, the sequences were trimmed and filtered by using trimmomatic version (v.) 0.36 (Bolger et al, 2014) with a sliding window of 4 base pairs (bp), a quality score threshold of 15 and a minimal length of 100 bp.…”

“…In consequence, the knowledge acquired on AGS treating simple monomeric wastewater cannot be simply transposed to AGS treating more complex wastewater containing polymeric compounds (Wagner et al, 2015). However, to date, only a minority of studies on AGS are performed with wastewater containing substantial proportions of polymeric compounds (Dulekgurgen et al, 2003;Lin et al, 2003;Schwarzenbeck et al, 2004;de Kreuk and van Loosdrecht, 2006;Lemaire et al, 2008;de Kreuk et al, 2010;Layer et al, 2019). Big differences in the physical characteristics (size, density), the settling properties and the nutrient removal efficiency of AGS grown in labscale reactors treating simple monomeric wastewater and fullscale tank treating real wastewater have been reported (Winkler et al, 2013;Wagner et al, 2015;Wang et al, 2018;Layer et al, 2019).…”

“…It can be expected that the bacterial communities of these AGS differ from the ones of AGS treating simple wastewater, which contain mainly classical phosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) from the class Gammaproteobacteria known to favor granulation and good nutrient removal (de Kreuk and van Loosdrecht, 2004). To date, only few studies investigated the microbial communities of AGS treating complex wastewater (Cetin et al, 2018;Kang et al, 2018;Swiatczak and Cydzik-Kwiatkowska, 2018;Wang et al, 2018;Layer et al, 2019;Dasgupta et al, 2020), and the influence of fermentable and polymeric compounds has, to our knowledge, not been studied separately.…”