Effect of Ethanol and Glycerol on the diversity of polyphosphate and glycogen accumulating organisms in enhanced biological phosphorus removal systems

Effect of Ethanol and Glycerol on the diversity of polyphosphate and glycogen accumulating organisms in enhanced biological phosphorus removal systems Valerie Liese*, Jonas Lapp**, Johannes Gescher**, Tobias Morck* *Chair of Urban Water Engineering, University of Kassel, Kurt-Wolters-Street 3, 34125 Kassel, Germany (E-mail: [email protected], [email protected]) **Institute for Technical Microbiology (TMI), Hamburg University of Technology (TUHH), Kasernenstraße 12, 21073 Hamburg, Germany (E-mail: [email protected], [email protected]) Abstract To investigate the impact of glycerol and ethanol dosing on the performance of the Enhanced Biological Phosphorus Removal (EBPR) process and the diversity of polyphosphate (PAOs) and glycogen accumulating organisms (GAOs), a series of studies with multiple adaptation phases was conducted using an automated sequencing batch reactor system. Overall, phosphorus release driven by glycerol and ethanol is significantly limited. Whole native DNA sequencing emphazises the underestimated role of Dechloromonas and Tessaracoccus in EBPR. Keywords EBPR; Ethanol; Glycerol; Glycogen accumulating Organism; Polyphosphate accumulating Organism INTRODUCTION Enhanced Biological Phosphorus Removal (EBPR) has regained prominence in research as a key factor enabling phosphorus recovery from wet sludge and maintaining high stability of granules in aerobic granular systems (AGS) (de Kreuk and van Loosdrecht, 2004; Egle et al., 2016). In wastewater treatment plants (WWTP) chemical precipitation remains the prioritized method due to the variable performance of EBPR, despite its cost effectiveness and environmental sustainability. To fully comprehend and optimize the process, it is not only important to investigate the influence of different boundary conditions on the sensitivity of EBPR performance and on specific genera of polyphosphate accumulating organisms (PAOs), but crucially to broaden the perspective on the linkage of PAOs and glycogen accumulating organisms (GAOs) biodiversity, as the interactions in the biocoenosis exert a decisive influence on the system functionality. However, despite advances in gene sequencing technologies, the costs associated with comprehensive biodiversity analysis remain significant (Li et al., 2024). In regard to boundary conditions, the external addition of carbon sources is one approach to support PAOs and stabilize EBPR performance. Affordable and readily available carbon sources, such as glycerol by-products from biodiesel production and brewery wastewater containing ethanol, may offer attractive options for WWTP operators (Shen and Zhou, 2016). This study aims to investigate the effects of glycerol and ethanol as carbon sources on EBPR performance and to relate these effects to the diversity and abundance of PAOs and GAOs. MATERIALS AND METHODS To adapt PAOs, a lab-scale Sequencing Batch Reactor (SBR) system comprising three automated reactors, each with an 8 L working volume, was operated on an 8-hour cycle. The cycle consisted of a filling phase (10 Min) including N2 sparging, an anaerobic phase (180 Min), an aerobic phase (240 Min), settling (40 Min) and decanting (10 Min). The experimental trial (Figure 1) started by inoculating the reactors with two distinct activated sludges: one from the WWTP Ulm-Steinhäule (US) for SBR1 and another from the WWTP Langenhagen for SBR2 and SBR3. After inoculation, multi-week adaptation phases were conducted with the following substrates: (I) acetate as a reference carbon source, (II) pure ethanol for SBR1 and SBR2, and pure glycerol for SBR3, and (III) denatured ethanol sourced from brewery wastewater for SBR1 and SBR2, alongside a commercial glycerol product used as an external carbon source at the WWTP Langenhagen for SBR3. PO4-P and DOC were measured weekly as the primary dissolved components. Volatile Suspended Solids (VSS) were also determined. Additionally, intracellular polyhydroxyalkanoates (PHA) were analysed as the sum of PHB and PHV at the beginning and end of each adaptation phase. Sludge samples were assessed for biodiversity using nanopore gene sequencing at the end of each adaptation phase. Figure 1. Design of SBR trials (SBR1-3), including one inoculation (US: Inoculum Ulm-Steinhäule, LG: Langenhagen) and three adaption phases (I-III) with different carbon sources RESULTS AND DISCUSSION The use of acetate as the reference carbon source resulted in high specific P release in all three SBRs within three weeks (Prel,VSS,SBR1 = 13.2 mg P/g VSS, Prel,VSS,SBR2 = 15.4 mg P/g, Prel,VSS,SBR3 = VSS 22.2 mg P/g VSS). Simultaneously, a marked increase in PHA production was observed (Figure 2). With regard to gene sequencing, significant abundances were noted for Cand. Accumulibacter and Dechloromonas as PAO representative, and Competibacter and Contendobacter as GAO representative. Despite the observed increase in P release across all SBRs, gene sequencing reveals distinct enrichment patterns depending on the inoculum. In SBR 1, Cand. Accumulibacter and Dechloromonas exhibit significant enrichment, whereas GAOs remain at relatively low abundance (Figure 3). Conversely, in SBRs 2 and 3 Competibacter and Contendobacter experience a marked enrichment, especially in SBR 2, these GAOs surpass the abundance of PAOs. Here, the widely accepted narrative of competition between PAOs and GAOs does not seem to apply universally. Instead, this confirms Nielsen et al. (2019) and that a stable EBPR performance appears to be achievable under conditions of coexistence. Following the switch of carbon sources in adaptation phase II, a significant decline in specific P release was recorded compared to acetate. Although some adaptation was to be assumed within the first two weeks, the specific P release remained significantly lower throughout the remainder of the adaptation phase for both pure ethanol (Prel,VSS,SBR1 = 5.6 mg P/g VSS, Prel,VSS,SBR2 = 2.0 mg P/g VSS) and pure glycerol (Prel,VSS,SBR3 = 5.0 mg P/g VSS). Notably, performance in SBR1 was higher than in SBR2, suggesting a potential influence of the inoculum: GAOs proliferate extensively in SBR 2, whereas their abundance remains low in SBR 1. When denatured alcohol and the glycerol product were used, the P release remained at a lower level throughout the entirety of adaptation phase III (Prel,VSS,SBR1 = 4.3 mg P/g VSS, Prel,VSS,SBR2 = 1.1 mg P/g, Prel,VSS,SBR3 = VSS 4.5 mg P/g VSS). In SBR 1, the diversity shifts as Dechloromonas shows significant enrichment, while Contendobacter and Competibacter exhibit pronounced growth. It is plausible that the substantial proliferation of GAOs also contributes to the pronounced increase in PHA production observed during adaptation phase III. In SBR 3, the abundance of GAOs remains stable when pure glycerol or glycerol-derived products are administered, while Dechloromonas shows a notable decrease in prevalence. Figure 2. Specific P release (Prel/VSS) and specific PHA formation (PHAform/VSS) during anaerobic phase as a weekly measurement over the period of the adaptation phases I-III Figure 3. Composition of the microbial community across different inocula and adaptation phases in SBR 1–3. Relevant PAOs (Tetrasphaera, Tessaracoccus, Cand. Accumulibacter, Dechloromonas) and GAOs (Micropruina, Competibacter, Contendobacter, Propionivibrio, Zoogloea) are displayed as individual genera The interactions between PAOs and GAOs do not follow a consistent pattern. While a lower PAO/GAO ratio generally correlates with reduced P release, it remains unclear whether this is due to direct competition or the ability of GAOs to occupy distinct ecological niches. For instance, during adaptation phase I, high PAO activity is observed despite a substantial abundance of GAOs in both SBR 1 and SBR 2. In contrast, during adaptation phase II in SBR 1, PAO activity is low even when GAOs are present in low abundance. Regarding glycerol and ethanol as carbon sources, it appears that GAOs have a higher acceptance of these substrates compared to PAOs, which is reflected in the limited P release (Liese et al., 2025). CONCLUSIONS The analysis indicates that ethanol and glycerol are effective in promoting P release, although their positive effect is limited compared to acetate. Whole native DNA sequencing reveals that Dechloromonas plays a crucial role in the EBPR process, while Tessaracoccus appears to be involved in glycerol fermentation. These findings underscore the previously underestimated contribution of these genera to EBPR. Furthermore, the coexistence of PAOs and GAOs does not appear to adversely affect EBPR performance, as substantial P release is achieved in the presence of GAOs. ACKNOWLEDGEMENTS This study as part of the project “RoKKa: Die Kläranlage als Rohstoffquelle und Bioraffinerie” received funding by the Baden-Württemberg Ministry for the Environment, Climate and Energy Sector together with the EU Commission as part of the ERDF funding program “Bioeconomy - Biorefineries for the recovery of raw materials from waste and wastewater - Bio-Ab-Cycling”. REFERENCES Egle, L. et al. (2016) ‘Phosphorus recovery from municipal wastewater: An integrated comparative technological, environmental and economic assessment of P recovery technologies’, Science of The Total Environment, 571, pp. 522–542. Available at: https://doi.org/10.1016/j.scitotenv.2016.07.019. de Kreuk, M. and van Loosdrecht, M. (2004) ‘Selection of slow growing organisms as a means for improving aerobic granular sludge stability’, Water Science and Technology, 49(11–12), pp. 9–17. Available at: https://doi.org/10.2166/wst.2004.0792. Li, G. et al. (2024) ‘Distinct microdiversity of phosphate accumulating organisms (PAOs) between side-stream and conventional enhanced biological phosphorus removal (EBPR) systems with performance implications’, Water Research, 266, p. 122280. Available at: https://doi.org/10.1016/j.watres.2024.122280. Liese, V. et al. (2025). ‘EBPR process dynamics under variable conditions: interaction between PAOs and the microbial community’. Bioresource Technology 133468. Available at: https://doi.org/10.1016/j.biortech.2025.133468 Nielsen, P.H. et al. (2019) ‘Re-evaluating the microbiology of the enhanced biological phosphorus removal process’, Current Opinion in Biotechnology, 57, pp. 111–118. Available at: https://doi.org/10.1016/j.copbio.2019.03.008. Shen, N. and Zhou, Y. (2016) ‘Enhanced biological phosphorus removal with different carbon sources’, Applied Microbiology and Biotechnology, 100(11), pp. 4735–4745. Available at: https://doi.org/10.1007/s00253-016-7518-4.