Modeling Versatile and Dynamic Anaerobic Metabolism for PAOs/GAOs Competition Using Agent-based Model and Verification via Single Cell Raman Micro-spectroscopy

Abstract

Side-stream enhanced biological phosphorus removal process (S2EBPR) refers to modified EBPR configurations that have been demonstrated to improve the performance stability and offer a suite of advantages compared to conventional EBPR design. Design and optimization of S2EBPR requires modification of the current EBPR models that were not able to fully reflect the metabolic functions of and competition between the PAOs and GAOs under extended anaerobic conditions as in the S2EBPR conditions. In this study, we proposed and validated an improved iEBPR model for simulating PAO and GAO competition that incorporated heterogeneity and versatility in PAO sequential polymer usage, staged maintenance-decay and glycolysis-TCA pathway shifts. The iEBPR model was first calibrated against a bulk batch test experimental data. The improved iEBPR model performed better than the previous EBPR model for predicting the soluble orthoP, ammonia, biomass glycogen and PHA temporal profiles in a batch starvation testing under prolong anaerobic conditions. We further validated the model with another independent set of batch anaerobic batch testing data that included high-resolution cellular and population-level intracellular polymers measurements enabled by single-cell Raman microspectroscopy technique. The model accurately predicted the temporal changes in the intracellular polymers at cellular and population levels within PAOs and GAOs, further confirmed the proposed mechanism of sequential polymer utilization, and polymer availability-dependent and staged maintenance and decay in PAOs. These results indicate that under extended anaerobic phases as in S2EBPR, the PAOs may gain competitive advantage over GAOs due to the possession of multiple intracellular polymers and the adaptive switching of the anaerobic metabolic pathways that consequently lead to the later and slower decay in PAOs than GAOs. The iEBPR model can be applied to facilitate and optimize the design and operations of S2EBPR for more reliable nutrient removal and recovery from wastewater.

Unrevealed roles of polyphosphate‐accumulating microorganisms

“…This metabolic versatility allows Accumulibacter PAOs to adapt to and gain a competitive advantage under a wide range of substrate composition and intracellular polymer availability conditions while maintaining their PHA accumulation activity. Using single‐cell Raman microspectroscopy and agent‐based modelling, we recently confirmed others’ observations (Zhou et al ., 2009; da Silva et al ., 2018) and provided cellular‐level evidences for the adaptive metabolic pathways in PAOs, who can generate the energy needed for cell maintenance and decay depending on the availability of different intracellular polymers (PolyP and glycogen) via glycolysis pathway and/or alternative TCA cycle operations (Li et al ., 2018b, 2020; Majed and Gu, 2020). A completely different metabolic pattern for the novel PAO Tetrasphaera was recently demonstrated (Kristiansen et al ., 2013).…”

Side-Stream Enhanced Biological Phosphorus Removal (S2EBPR) Enables Effective Phosphorus Removal in a Pilot-scale Shortcut Nitrogen Removal System

“…Compared to conventional EBPR, S2EBPR offers unique conditions that favor PAOs over GAOs, which holds the key to EBPR performance and stability, by introducing a more complex composition of VFAs (i.e., a mixture of acetate and propionate) that leads to higher PAO diversity (Onnis-Hayden et al 2020, Srinivasan et al 2021, and consequently, different P release/uptake kinetics. In addition, extended anaerobic conditions in S2EBPR further give PAOs competitive advantages due to their distinct maintenance and delayed decay associated with the versatility of metabolic pathways and the possession of multiple intracellular polymers (i.e., PHA, glycogen, and polyP) (Li et al 2020a). Furthermore, S2EBPR allows for more flexible carbon supplement and improved carbon utilization efficiency, and thus EBPR can be achieved for systems under previously considered unfavorable C/P ratios (Sabba et al 2023.…”

Sidestream bio-P and mainstream anammox in a BNR process with upstream carbon capture

“…Sidestream bio-P (also referred to as sidestream enhanced biological phosphorus removal, S2EBPR) processes are configurations in which the influent is bypassed around the anaerobic zone (Barnard et al, 2017). Sidestream bio-P configurations can include RAS fermentation, in which a portion of the RAS (5-10%) is held in an anaerobic reactor with a retention time of one to two days (Barnard et al, 2017;Gu et al, 2019;Onnis-Hayden et al, 2020;Tooker et al, 2016;Vollertsen et al, 2006). Alternatively, the retention time of the sidestream reactor can be considerably shorter (1-4 hours) with the addition of supplemental carbon (since fermentation is not occurring in the reactor), such as primary sludge fermentate (Cavanaugh et al, 2012;Tooker et al, 2016).…”

“…RAS fermentation was initially investigated as a means to decrease sludge production and nutrient loading to the nutrient removal step, compared to primary sludge fermentation (Andreasen et al, 1997;Novak et al, 2007). It has also been shown that sidestream processes result in more stable bio-P performance (Lanham et al, 2013;Onnis-Hayden et al, 2020). Sidestream processes protect the anaerobic zone from wet weather flows, provide VFA from hydrolysis and fermentation, and offer possible suppression of GAO (Vollertsen et al, 2006;Barnard et al, 2017;Li et al, 2020).…”

“…It has also been shown that sidestream processes result in more stable bio-P performance (Lanham et al, 2013;Onnis-Hayden et al, 2020). Sidestream processes protect the anaerobic zone from wet weather flows, provide VFA from hydrolysis and fermentation, and offer possible suppression of GAO (Vollertsen et al, 2006;Barnard et al, 2017;Li et al, 2020).…”