Critical evaluation of heat extraction temperature on soluble microbial products (SMP) and extracellular polymeric substances (EPS) quantification in wastewater processes

Soluble microbial products (SMP) and extracellular polymeric substances (EPS) have been widely studied within the context of various biological wastewater treatment configurations as they can exert major influence on the chemical and physical characteristics of the suspended solids or mixed liquor, including flocculation, settleability, and surface charge, in addition to impacting effluent quality (Morgan et al. 1990; Sheng et al. 2010; Kunacheva & Stuckey 2014; Hu et al. 2019). Understanding the behavior of SMP and EPS continues to gain importance, especially with the widespread adoption of membrane bioreactor (MBR) technology for wastewater treatment due to their impacts on treatment performance as well as their relationship with the critical issue of membrane fouling (Ishiguro et al. 1994; Huang et al. 2010; Xu et al. 2020). However, there is major difficulty in comparing data between studies due to the lack of a standard protocol and inconsistent definitions of SMP and EPS (Laspidou & Rittmann 2002; Sheng et al. 2010; Kunacheva & Stuckey 2014).

EPS has commonly been defined as the largely insoluble proteins, carbohydrates, lipids, nucleic acids, and other macromolecules secreted by bacteria, and are found surrounding the surface of bacteria within a floc or biofilm community as a hydrated gel matrix (Laspidou & Rittmann 2002; Gao et al. 2010; Judd 2010). In contrast with the relatively insoluble EPS, SMP has been defined as soluble cellular components that may be released during cell lysis or otherwise excreted for cellular functions as well as the products resulting from substrate breakdown (Namkung & Rittmann 1986; Laspidou & Rittmann 2002). SMPs are associated with soluble proteins, polysaccharides, and humic-like substances, and it is generally accepted that soluble EPS and SMP refer to the same components (Laspidou & Rittmann 2002; Gao et al. 2010). In addition to loose definitions for EPS and SMP, there are a variety of extraction techniques, including cation exchange, chemical extractions, and thermal extractions, each with their own operational definitions.

Due to its broad applicability and ease of execution, heat extraction, either standalone or as part of a procedure, is a common EPS and SMP extraction method that has been used with anaerobic, anoxic, and aerobic wastewater sample matrices (Morgan et al. 1990; Dereli et al. 2015; Hu et al. 2019). While heating is a common extraction step among many procedures including NaOH, sonication, and thermal extraction methods, exposure to excessively high temperatures leads to the lysing of the microbial biomass, demonstrating a need to determine an acceptable extraction temperature (Redmile-Gordon et al. 2014; Dai et al. 2016; Lv et al. 2019). Additionally, the heat extraction temperature varied by study, typically between 80 °C and 100 °C for 10 min to 1 h, which makes comparisons between studies difficult due to differences both in extraction efficiency and extent of lysis; in one pure culture study by Lv et al. 2019, heat extractions were performed at 45 °C, which extracted less than 5 mg/g of carbohydrates, and 60 °C, which extracted 55 mg/g, which highlights the difficulty even between comparisons of the same sample (Gao et al. 2010; Lv et al. 2019). This study aims to broadly investigate the effect of temperature on SMP and EPS extraction and cell lysis when applied to sludge samples encountered in wastewater biological treatment processes.

After extraction, the dissolved organic matter (DOM) can be analyzed for a range of constituents, but the carbohydrate and protein profiles and their overall properties are often the focus (Kunacheva & Stuckey 2014). Colorimetric analysis remains the predominant method of quantifying carbohydrates and proteins (Felz et al. 2019). However, despite the improvements made through modifications or adaptations for use with a microplate to improve their accuracy and repeatability, colorimetric measurements have been shown to be inadequate. This is primarily due to the dependence of these quantification methods on the standard compound selected and interfering substances, particularly humic-like and phenolic compounds that are characteristically present in SMP and EPS samples, which leads to the need for advanced tools for analysis (Felz et al. 2019).

One potential tool to address this deficiency is fluorescence spectroscopy, which has increasingly been used to characterize DOM in the wastewater field (Ramesh et al. 2006; Sheng et al. 2010; Lim et al. 2020). Fluorometry provides a field-deployable, sensitive, non-destructive and rapid analytical method that can show a profile of the various DOM constituents, including protein-like materials, humics, and fulvics, presented using excitation-emission matrices (EEMs) and provides insight into the physicochemical properties of the SMP and EPS that colorimetric methods may fail to capture (Henderson et al. 2009; Sheng et al. 2010). However, fluorometry falls short for use as a standalone tool to quantify SMP and EPS constituents, mainly due to the need for visual interpretation of peaks and principal component analysis only providing qualitative or semi-quantitative data, unless more extensive analyses such as parallel factor (PARAFAC) analysis are employed (Stedmon & Bro 2008). Pairing EEMs with quantitative analyses, such as colorimetry, total carbon (TC) or total nitrogen (TN) measurements can be a simple check that each analysis has not been excessively compromised by the interfering effects of the constituents present in the sample matrix and provides a deeper investigation into the SMP and EPS profiles.

Because of the difficulties in comparing SMP and EPS data between studies due to the inadequacies of current analytical methodologies as well as the inconsistent operational definitions based on extraction procedures, this study seeks to address these drawbacks through the following objectives: (1) determine the effects of extraction temperature on SMP and EPS profiles, (2) determine the effects of extraction temperature on cell lysis, (3) compare the commonly obtained extraction data obtained through colorimetry, TC, and TN analyses, versus fluorometry, and (4) compare and contrast the response profiles across common wastewater sludges. This study will particularly examine the effects of temperature on SMP and EPS extraction from sludges from various zones of a biological nutrient removal (BNR) process, i.e., activated sludge, anoxic sludge, and anaerobic sludge, to achieve an acceptable extraction efficiency while minimizing cell lysis and preserving the structure of the constituents for protein and carbohydrate quantification. The findings from the BNR sludge experiments will then be used to estimate a range of possible acceptable extraction temperatures for SMP and EPS extractions from MBR sludge.

The experiment was designed to evaluate the effect of extraction temperature on activated sludge (AS), anoxic (ANX) sludge, anaerobic (ANA) sludge, collectively referred to as the BNR sludges, and aerobic MBR (AeMBR) sludge. Key information about the sampling can be found in Table 1. The Manhattan water resource recovery facility's BNR system is typical for a treatment system designed for a medium-sized midwestern city's municipal wastewater, but notably is operated to accommodate for a transient student and military population. BioMicrobics’ AeMBR in De Soto, Kansas also treats municipal wastewater, but the residential flow primarily originates from a retirement community. There are two notable differences between AeMBR0 compared with AeMBR1 and AeMBR2. Firstly, AeMBR0's comparatively higher solids concentrations are due to being collected before a major sludge wasting event. Secondly, AeMBR1 and AeMBR2 were experiencing foaming during the sampling dates; the reactors smelled of detergent, and the bubble morphology likely indicated that it was caused by surfactants. The unplanned conditions in the two latter samples provided a valuable coincidence to test the robustness of the procedures used in this study. Total suspended solids (TSS) and volatile suspended solids (VSS) analyses were performed on the sludge samples according to standard methods for wastewater 2540D and 2540E, respectively (Eaton et al. 2005).

The procedural outline of this study can be found in Figure 1. All centrifugation steps were performed using an Eppendorf 5920 R (Eppendorf, Hamburg, Germany) at 4,000 g and 4 °C for 15 min; the centrifuge speed was determined by a series of experiments to find the minimal speed that could settle all the tested sludges into stable pellets. Notably, AS and AeMBR samples settled more compactly than ANX and ANA samples. Heat extraction was performed at room temperature, 40 °C, 60 °C, and 80 °C to determine the effect of temperature on SMP and EPS extraction.

The SMP and EPS extraction data consists of total carbon and total nitrogen (TC-TN) analysis, and 96-well microplate colorimetric analysis methods for carbohydrates and proteins. Sample processing and analyses began within 2 h of sample collection and immediately continued to completion without interruption. TC-TN analyses were performed using a Shimadzu TOC-L and TNM-L (Shimadzu, Kyoto, Japan) analyzer and all data were reported as mgC/L and mgN/L for the TC and TN values, respectively. Carbohydrate and protein analyses, using the phenol sulfuric acid and BCA methods, respectively, were performed in triplicate on a BioTek microplate reader (BioTek Epoch 2, VT, USA) with Greiner Bio-One μClear Bottom 96-well plates (Greiner Bio-One, Kremsmünster, Austria). All concentrations reported in this study have been normalized to the 45 mL sludge samples collected, as SMP has no solid weight to normalize to, and normalization to the sludge volume allows for a better demonstration of the relative concentrations of SMP versus EPS. In addition to the extraction data, fluorometry was performed on a Horiba Aqualog fluorometer (Horiba, Kyoto, Japan) with the samples aliquoted in quartz cuvettes (Starna 3-Q-10, Ilford, UK). Statistical analysis was completed using the PROC GLM procedure on SAS Studio 3.8 (Enterprise Edition).

This study utilized the phenol sulfuric microplate method previously developed for carbohydrate analysis (Masuko et al. 2005). It was performed by adding 50 μL of sample followed by forcefully and quickly adding 150 μL of 18.4M H₂SO₄ into each well and shaking until the solution develops a homogeneous yellow-brown color that indicates the presence of sugars. 30 μL of 0.53 M phenol was immediately added into each well following the sulfuric acid, and the solution was then floated in a 90 °C in a water bath, incubating for 5 min before reading the absorbances at 490 nm using a microplate reader. Glucose was chosen as the carbohydrate calibration standard, as it was the most commonly used standard encountered in wastewater literature (Kunacheva & Stuckey 2014; Berkessa et al. 2018). While the anthrone method is a common alternative to the phenol sulfuric method, the latter has a more rapid and simpler procedure, which contributes to its popularity (Masuko et al. 2005; Gao et al. 2010; Dai et al. 2016).

Protein analysis was performed using the BCA assay with methods from Sigma Aldrich (BCA1, MO, USA), which included their BCA solution (B9643, MO, USA), containing bicinchoninic acid (BCA), sodium carbonate, sodium tartrate, and sodium bicarbonate in 0.1 M NaOH. 25 μL of each sample was added to the well plate, followed by forcefully and quickly adding 200 μL of BCA reagent and then sealed and incubated at 37 °C for 30 min. The plates were then cooled to 20 °C before having absorbances read at 562 nm. BSA was used as the protein calibration standard, as it was the most commonly used standard encountered in wastewater literature (Felz et al. 2019; Hu et al. 2019). While the Lowry method is a common alternative, also relying on the conversion of Cu²⁺ to Cu⁺ for protein quantification, its reliance on the Folin-Ciocalteu reagent causes interference from reducing agents, which the BCA method and its modified procedures replace with bicinchoninic acid, to help minimize analytical artifacts (Le et al. 2016).

Lysis was investigated using the BacLight Bacterial Viability Kit (L7012, Thermo Fisher, MA, USA) live/dead staining assay. After heat extracting each sample, each pellet was resuspended, then diluted with 0.154 M NaCl to achieve an OD₆₇₀ of ∼0.6. A 1:1 staining solution of 3.34 mM SYTO 9 and 20 mM propidium iodide (PI) was used for this experiment. 100 μL of each sample was then mixed with 100 μL of the working solution and incubated in the dark for 15 min prior to its analysis using a fluorescent microplate reader (BioTek Synergy H1, VT, USA).

Temperature did not have a statistically significant impact on the SMP values in any of the BNR sludges tested. For all samples tested, there was no statistically significant impact of temperature on SMP measurements for carbohydrates or proteins, and the TC and TN results were similarly unresponsive (Table 2). These results could be expected based on a prior knowledge that SMP constituents are, theoretically, already solubilized and do not require further extraction. The EPS extractions, in contrast to the SMP observations, responded greatly to the increasing temperatures applied (Figure 2).

AS, ANX, and ANA samples all showed increases in concentrations for extracted carbohydrates and proteins as well as TC and TN with temperature, with the steepest increase occurring between 40 °C and 60 °C, which indicates that the bulk of the extraction occurs somewhere between these temperatures. While direct comparisons between the absolute concentration values obtained between different days can vary (Figure 2, Figure S1), the influence of temperature produced consistent trends in the measured parameters for each sample matrix. Additionally, in all samples, the TC and TN data generally corroborated the trends in the carbohydrate and protein data, respectively. The extraction data for activated sludge are shown in Figure 2(a) and 2(b). The four parameters generally followed the same trend: a slight increase from 20 °C to 40 °C, followed by the steep increase from 40 °C to 60 °C, and a slightly less steep increase from 60 °C to 80 °C. The overall shape resembles the exponential-linear range of a sigmoidal curve.

The extraction data of ANX (Figure 2(c) and 2(d)) had some similarities, but also showed several differences compared to AS. The EPS protein concentrations at different temperatures followed the same exponential-linear range of a sigmoidal curve similar to what was observed in the AS samples. However, the carbohydrates, TC, and TN concentrations in the ANX EPS measurements appeared to follow a diauxic pattern – a full sigmoidal curve with an apparent asymptote at the higher temperatures. However, the steepest increase still occurred between 40 °C and 60 °C. ANA's extraction data (Figure 2(e) and 2(f)) shows more similarities to ANX than AS. This is to be expected since the anaerobic and anoxic zones are located sequentially next to each other and the microbes experience mostly similar redox conditions in these zones due to the absence of dissolved oxygen. The extracted EPS concentrations for the parameters all followed a full sigmoidal curve. The shapes of the curves from each sample, and even the measured parameters within the same sample, can differ, suggesting that there may not be a single temperature that can be broadly applied for all samples for heat extraction procedures. The difference in curve shape between the aerobic basin's sludge from the anaerobic and anoxic sludges, which are more similar to each other, may be due to metabolic differences and sludge age, both of which can affect floc strength.

Despite higher temperature extractions incurring an increased risk of potentially lysing cells and releasing intracellular materials into the bulk solution, it is not uncommon to encounter studies that heat extract samples between 80 °C and 100 °C, which could lead to an overestimation of SMP and EPS concentrations (Morgan et al. 1990; Comte et al. 2007). The threshold for complete cell inactivation and thermal lysis in activated sludge has been noted in literature to occur around 60 °C, which suggests that heat extraction above 60 °C should be avoided if possible (Rocher et al. 1999; Kim et al. 2013).

The live/dead assay corroborates the findings in literature, as 60 °C appears to be an inflection point before dramatic rises in cell lysis for both anaerobic sludge and activated sludge, as shown in Figure 3. The anoxic sludge exhibited similar behavior but saw significantly more lysis than the other samples. While more research is required to more accurately determine the temperature after which aggressive onset of lysis begins, all of the samples experiencing excessive lysis at 80 °C suggests that, despite its popularity as a heat extraction temperature, the results may be skewed by intracellular components (Morgan et al. 1990; Comte et al. 2007).

These thermal lysis problems compound with issues stemming from the overestimation of protein and carbohydrate concentrations in the SMP and EPS using colorimetric microplate assays. While the reasons for the various colorimetric analyses overestimating the SMP and EPS concentrations are not fully understood, notable interfering substances, including detergents, lipids, iron, and creatinine, which are known to be present in the wastewater matrix, could obfuscate results (Stuckey & McCarty 1984; Sigma-Aldrich 2011). Further complicating matters is the fact that, proteins have been noted to interfere with the carbohydrate measurement by reacting with sulfuric acid (Chow & Landhäusser 2004). Because colorimetric methods rely on absorbance, and thus cannot distinguish between interfering substances and the intended analyte, more advanced analytical tools such as fluorometry, are required (Stedmon & Bro 2008; Felz et al. 2019).

Because fluorometry is sensitive to different compounds, it can provide semi-quantitative and qualitative information about the substances in the wastewater matrix, especially with respect to proteins, which is the primary biologically associated macromolecule with strong intrinsic fluorescence characteristics (Lakowicz 2006). The EEMs of the SMP extractions for AS, ANX, and ANA can be found in Figures S2, S3, and S4, respectively. Across all the BNR sludges, there appeared to be marginal increases in peak intensities, corroborating the extraction data's finding that SMP samples derive little benefit from additional heat extraction. These increases were slight, but likely more noticeable due to the specificity and sensitivity of fluorometric analysis compared to colorimetry. However, as the peak intensity increase was relatively negligible, the heat treatment step for general SMP analyses can usually be foregone.

In contrast to the SMP samples, the EEMs for AS and ANA EPS extractions showed significant responses with different temperatures (Figure 4); the EEMs for ANX EPS follow the same trend as ANA and can be found in Figure S5. In all three BNR samples, the most apparent fluorophores present were T1, indicative of tryptophan-like compounds, and B2, which is characteristic of tyrosine-like compounds (Hudson & Reynolds 2007; Henderson et al. 2009). While the fluorometer did not show any distinct peaks associated with humic-like (fluorophore A) or fulvic-like (fluorophore D) substances on the BNR extracts, their presence can be seen in the more diffuse profiles at 60 °C and 80 °C (Hudson & Reynolds 2007; Henderson et al. 2009). One important note on fluorescent spectroscopy is that the peak intensities of each fluorophore does not necessarily indicate its abundance relative to other fluorophores. For example, it may not be appropriate to conclude that tyrosine-like compounds occur in lower concentrations than tryptophan-like compounds because of its lower peak signal because each has different spectral properties.

As with the colorimetric protein analysis data, the general trend of the EEMs shows increasing peak intensity with extraction temperature. When plotted on a scale normalized to the concentrations observed for each sample, EPS extraction at 20 °C was below the visualization limit for the fluorometer for AS, and near the limit for ANX and ANA. For AS, the peak intensities of T1 and B2 increased almost linearly from 40 °C to 80 °C, similar to the trends in both the BCA data and TN data. In the ANX and ANA data, however, the peak intensities had a drastic increase between the 40 °C and 60 °C extractions, followed by negligible increases from 60 °C to 80 °C. This more muted response in the EEMs than would be expected based on the BCA data more closely resembles the diauxic peak profile of their respective TN plots for ANX and ANA samples. This likely suggests that by 60 °C, the extracellular proteins have already been fully extracted in these samples (Figures 2 and 4). While further studies involving liquid chromatography and mass spectrometry are required to definitively conclude that the non-fluorescing amino acids have also been extracted, the tryptophan and tyrosine like compounds are likely to have been extracted along with the other amino acids in the protein under the temperature conditions applied, as the onset of thermal decomposition of proteins into component amino acids generally occurs after 130 °C (Kasarda & Black 1968).

An initial sample of aerobic MBR sludge (AeMBR0) was taken to determine its general behavior using the same procedure as with the BNR sludges (Figure S6). The concentrations of protein, carbohydrate, and TC-TN were higher, but the extraction behavior with respect to temperature was similar to what was observed with activated sludge. As with BNR sludges, the EPS concentrations increased with increasing extraction temperatures above 40 °C. The SMP concentrations did not change with temperature, again confirming that the soluble fraction does not benefit from heat extraction; average values and p-values demonstrating that there was no significant difference between temperatures for SMP samples are as follows: 10.6 mg/L-carbohydrate (p=0.3269), 18.7 mg/L-protein (p=0.5552), 29.18 mgC/L (p=0.0627), 2.84 mgN/L (p=0.2290).

After establishing this baseline, two samples were taken to examine the MBR sludge extraction efficiency at temperatures between 45 °C and 60 °C to attempt to determine if a temperature lower than 60 °C would be suitable for this sample (Figures 5 and 6). Based on the AeMBR0 (Figure S6), it appears that extraction at 40 °C is too ineffective to be considered a serious candidate, so 45 °C was the lowest tested temperature in this setup. 60 °C was chosen as the maximum temperature as it is the threshold for thermal degradation of wastewater sludges (Rocher et al. 1999; Kim et al. 2013).

It is important to note that these two samples (AeMBR1 and AeMBR2) were taken during a period where significant amounts of bubbles and foaming was observed in the membrane bioreactor, likely caused by detergents as diagnosed through both visual inspection, odors, and personal communication with the operating staff members. Despite the foaming issue, the extraction procedures used in this study appeared to have reproducible general trends across samples while also finding the presence of surfactants, attesting to the robustness of the procedures and their effectiveness as diagnostic tools to also troubleshoot operational problems (Figures 5 and 6). The overall trends produced by the extraction data for AeMBR1 and AeMBR2 are very similar to each other, despite the difference in absolute concentrations observed. A significant amount of protein extraction occurs by 45 °C, while carbohydrate extraction was not observed until 50 °C. Another significant increase occurred between 55 °C and 60 °C, which shows that the AeMBR sludge extractions show a diauxic response from 20 °C to 60 °C.

Comparing the carbohydrate extraction with the TC extraction at 45 °C reveals an apparent contradiction where carbon is being clearly measured, but carbohydrates are not, with this phenomenon occurring in both AeMBR1 and AeMBR2 samples. A noted issue with colorimetric determination of carbohydrates is the reliance on a single pure substance standard, glucose being the most common, which is the arbitrarily chosen to express the results (Huang et al. 2010; Nielsen 2010). If the carbohydrates extracted at 45 °C have absorptivities significantly different from that of glucose, the calibration may be inappropriate. This potentially highlights a weakness in using colorimetric methods in complex matrices, such as wastewater, which likely contains more than one carbohydrate type and warrants further investigation (Nielsen 2010).

The TC measurement, however, does not suffer from this flaw and simply measures the carbon contained in the sample regardless of its chemical form, with results expressed in mg/L of carbon. This downside, while major, does not render the phenol-sulfuric colorimetric method completely unusable for wastewater purposes as there appears to be good correlation with the TC data trends in both the BNR sludges and AeMBR samples (Figures 2 and 5, Figure S6). While the issue of expressing results in terms of a single standard could theoretically be an issue for the BCA method in protein measurement as well, no glaring anomalies have been observed using this dataset between the protein data expressed using BSA as a standard and the TN measurements. One notable difference between the protein and carbohydrate standards is that BSA is a mixed standard, in contrast with glucose being a pure substance standard. Exploring a mixed standard developed for carbohydrates that are applicable to specific matrices may help alleviate these estimation issues but may not address the overestimation due to the lack of specificity of sulfuric acid digestion. Nevertheless, this highlights the need to pair these colorimetric methods with other analytical tools for validation, especially those that can potentially characterize the constituents comprising the sample matrix such as EEMs.

The EPS extraction EEMs of AeMBR1 and AeMBR2 for different temperatures confirm the diauxic behavior observed in the corresponding colorimetric EPS extraction data. Similar to the BNR sludges, T1 (tryptophan-like compounds) was the most dominant fluorophore followed by B (tyrosine-like compounds) and D (fulvics). The intensities of these three constituents increased with temperature, with the peak intensities from 50 °C to 55 °C being rather negligible and a large increase occurring between 55 °C and 60 °C. There is an additional unnamed peak seen at (EX 350, EM 450) which corresponds to surfactants and optical brighteners included in washing powders observed at 55 °C and 60 °C on AeMBR1 and 60 °C for AeMBR2, making it likely that the foaming and bubbling observed was indeed being caused by an unusually high concentration of detergents being present (Henderson et al. 2009; Hartel et al. 2008). Based on the observation that extraction of the surfactants increased with increasing temperature, 60 °C may not have been enough to fully extract and solubilize the detergents. However, increasing the extraction temperatures past 60 °C risks lysing the cells, especially as surfactants by themselves have been known to increase the rate of cell lysis, which could invalidate SMP and EPS measurements by introducing intracellular components (Brown & Audet 2008). This demonstrates a need to incorporate techniques, such as Fourier transform infrared (FTIR) spectroscopy, that can directly characterize solids, and molecules that are either strongly bound to them or unable to be effectively solubilized.

For most wastewater sludge samples, it is unlikely that the SMP would benefit from heat extraction, as the constituents obtained through SMP extraction should already mostly be soluble. The optimal general-use temperature for the heat extraction of EPS in wastewater sludges is likely to occur between 55 °C and 60 °C for wastewater samples, and may vary due to physical factors, such as spatio-temporal variations in the sample, sludge type, and treatment process. Because it is likely that each sample has its own specific optimal extraction temperature, the specific range should be determined through testing similar to the procedure performed in this study, focusing on a temperature range around 60 °C. Additionally, some constituents may not be extractable without risking cell lysis at higher temperatures; analysis of these components may require additional techniques that can directly characterize the solids such as energy dispersive spectroscopy, FTIR, and microscopy.

Known issues with colorimetric analyses’ lack of specificity, particularly with regard to phenol-sulfuric acid method, and their dependence on arbitrarily chosen single compound standards limit their stand-alone reliability, but this can be overcome by supplementing the data with validation from other analytical methods such as TC, TN, and fluorometry. In particular, fluorometry proved to be a useful, rapid method that provided qualitative and semi-quantitative information that could be used to characterize the sample matrix and corroborate or refute the observations from other analytical methods. Taken all together, the combination of colorimetry, TC-TN, and fluorometry can be applied as a rapid, routine monitoring tool for wastewater processes and offer more robust process control in both conventional BNR systems as well as membrane-based systems.

This research was supported by the NSF CBET Environmental Engineering (Program #1440) award #1805631 and the NSF National Research Traineeship (NRT) award #1828571. BNR and AeMBR samples were graciously provided by Keri Brown, an operator at the Manhattan water resource recovery facility, and Dr. Reza Shams of BioMicrobics Inc., respectively. The authors also gratefully acknowledge Emily Randig, Caitlin Swope, and Arvind Damodara Kannan for their assistance with the fluorometric, colorimetric, TC-TN analyses and data visualization. Lastly, this paper acknowledges the late Lemon Lim for their unwavering support.

All relevant data are included in the paper or its Supplementary Information.