Markus Andrews

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Effects of imposed disturbance and SRT on QQ-based antifouling efficacyFigure 1 shows the fouling rate profiles of MBRs over time under different operating conditions, summarizing the average fouling rates of each phase. Two representative transmembrane pressure (TMP) profiles, the average fouling time, and the number of MBR runs at each phase are also provided in Supplementary Fig. 1 and Supplementary Table 1. In Phase 1, the average fouling rates for Reactors 1 and 2 were nearly the same, i.e., there was no statistically significant difference between the reactors. This confirms that the MBRs were in identical states, in terms of fouling behavior when operated in the conventional mode. In Phase 2, the average fouling rates of Reactors 1 and 2 were also almost the same, showing that the QQ effect on fouling mitigation appears to be insignificant. Unlike previous reports on the QQ effect on antifouling efficacy35, it was unclear whether QQ played a role in fouling control under Phase 2 conditions. One possible reason for the difference is SRT, which will be further investigated and discussed later. After disturbance (2 d starvation with a high shear rate of 103 s−1) was applied to both MBRs at the beginning of Phase 3, both MBRs experienced severe fouling phenomena, with sharp increases in the average fouling rate at >40 kPa/d. No QQ effect was observed in this phase either. The applied disturbance may have caused a drastic change in mixed liquor characteristics, probably including the microbial community structure, while aggravating fouling propensities, which will be discussed further in later sections.Fig. 1: Membrane-fouling rate.a Its variations with time for Reactor 1 (R1) and Reactor 2 (R2). Each data point indicates the average fouling rate for each MBR run. b Average fouling rates with t-test probability (p) values for each phase. Error bars indicate 1 SD. Details on the experimental conditions are provided in Table 2.Full size imageTo examine how SRT affects membrane fouling in the MBRs, we increased SRT from 50 to 75 d in Phase 4. The average fouling rate of Reactor 1 slightly decreased with vacant beads (which contain no QQ bacteria), whereas that of Reactor 2 decreased more significantly with QQ beads (corresponding to 47% of that of Reactor 1). With the longer SRT, it appears that QQ affected biofouling mitigation. When Reactor 1 was switched to conventional mode in Phase 5, its membrane fouling rate was slightly reduced compared to Phase 4. This implies that the vacant beads had no effect on fouling mitigation and in fact may have caused membrane fouling. A previous study also reported that membrane fouling increased when the media added were trapped inside the membrane fibers36. However, Reactor 2 exhibited a notably slower fouling rate, which corresponds to 55% of that of Reactor 1. Thus, the biofouling control due to QQ was evident at long SRT (75 d). The effect of SRT on QQ will be discussed further in later sections, along with the time-series data of MBR operational performance and microbial community.Effects of QQ, disturbance, and SRT on biopolymer productionFigure 2a–d show EPS and SMP variations during MBR operations with and without QQ at different SRT values. The EPS and SMP data normalized to mixed liquor suspended solids (MLSS) are also provided in Supplementary Fig. 2. During Phase 1, when the MBRs were operated in the conventional mode, the EPS-carbohydrate (EPS-C) and EPS-protein (EPS-P) levels were similar in both MBRs (~20 and 80 mg/L, respectively). Notably, however, there was only ~3% probability that the EPS-P level between Reactors 1 and 2 occurs by chance. One possible explanation is that the microbial communities in both the reactors should change as a result of the provision of synthetic wastewater, leading to alterations in metabolic products. A previous study also reported that fluctuations in EPS-P level at the beginning of MBR operation were observed due to bacterial acclimation to new environments13. In Phase 2, the EPS-C concentration decreased by ~13.5% in Reactor 1 compared with that of Phase 1, but decreased by 33.1% in Reactor 2. However, the EPS-P concentrations in both reactors remained virtually unchanged. It seemed that the lower EPS levels with QQ did not virtually contribute to fouling mitigation, possibly because its levels were still too high to make a perceptible reduction in membrane fouling. In Phase 3, the EPS-C concentration in Reactor 1 increased by ~6%, whereas it increased by ~39.3% in Reactor 2. This substantial EPS-C increase may have resulted in severe membrane fouling, even in the presence of QQ beads. A previous study reported that increased EPS production was strongly correlated with environmental stresses such as shear and starvation37. It was thought that the disturbance at the beginning of Phase 3 may have caused a similar phenomenon. When the SRT was increased to 75 d in Phase 4, the EPS levels in the two MBRs decreased. In Reactor 1, EPS-C and EPS-P concentrations declined by ~15.6% and 11%, respectively, whereas their concentrations decreased by ~74.4% and 21.65%, respectively, in Reactor 2. Similarly, previous studies also reported that EPS production was reduced with long SRT values11,38. In Phase 5, EPS-C and EPS-P contents continued to decrease in Reactor 2; however, no further decrease was observed in Reactor 1. The higher EPS content caused preferential attachment of biomass onto the membrane surface so as to form cake layers39. The reduced EPS production associated with the QQ strategy correlates with previous findings35,40. It is thus believed that membrane fouling could be mitigated by the presence of QQ media.Fig. 2: Biopolymer concentrations and mixed liquor characteristics according to phases in the two MBRs.a EPS-carbohydrates (EPS-C); b EPS-proteins (EPS-P); c SMP-carbohydrates (SMP-C); d SMP-proteins (SMP-P); e mixed liquor suspended solids (MLSS); and f floc size. The box is determined by the 25th and 75th percentiles, whereas the whiskers are determined by the 5th and 95th percentiles. The white square symbol inside each bar represents the average value of each parameter.Full size imageThe SMP-carbohydrate (SMP-C) and SMP-protein (SMP-P) levels were also similar in both reactors in Phase 1 (~5 and 4.5 mg/L, respectively). In Phase 2, there were slight changes in both. When disturbance was applied in Phase 3, the SMP levels in both reactors significantly increased. The SMP-C and SMP-P concentrations in Reactor 1 increased by ~45.3% and ~36.1%, respectively, and their respective increases in Reactor 2 were more significant at ~62.4% and ~110%. These results agree well with previous studies, which reported that the disturbance imposed on microorganisms induced the release of microbial polymeric substances28,37, in addition to substrate limitations41. When the SRT was increased to 75 d in Phases 4 and 5, the SMP-C and SMP-P concentrations started decreasing, and the decline was more significant with QQ. For instance, in Phase 5, Reactor 2 had the lowest SMP-C and SMP-P levels, at ~54.2% and ~62.4% lower than these respective values in Phase 4. In addition, the longer SRT contributed to decreased SMP levels when comparing Reactor 2 between Phases 2 and 5. This result is consistent with previous studies35,40, which reported that the presence of QQ media reduced soluble biopolymer contents in MBRs. Another previous study also reported that increasing the SRT in MBRs alleviated biofouling42. Notably, QQ caused the more substantive and immediate decrease of SMP-C than that of SMP-P in Phase 4. This could be associated with the inhibition of protease enzyme secretion in the presence of QQ enzymes leading to reduced degradation of soluble protein43. Overall, it can be concluded that the presence of QQ media reduced biopolymer production more significantly when the SRT was extended.Effects of QQ, disturbance, and SRT on mixed liquor characteristics and biological treatment efficienciesMixed liquor characteristics, such as MLSS and floc size, were monitored over time (Fig. 2e, f). The MLSS concentration in both MBRs from Phases 1–3 varied in the range of 2100–2250 mg/L. Disturbance (starvation with shear) at the beginning of Phase 3 caused a slight decrease in biomass concentration compared to that of Phase 2. At the longer SRT (75 d) in Phases 4 and 5, the MLSS concentration increased to 2650–2900 mg/L. It is natural that a longer SRT should increase MLSS levels at the same yield. It appeared that the MLSS levels were a bit higher with QQ than without it as observed from Phase 2 through 5. Microbial growth can be promoted if QS that requires carbon sources is inhibited. A recent finding pointed out that the QQ enzyme (acylase) may increase microbial yield, converting the resource (food) to more biomass44. Operational parameters such as QQ, SRT, and disturbance did not yield significant changes in floc size during the entire study, although fluctuations were possible45. In this study, there was a slight increase in floc size with QQ in Phase 5. The microbial floc size is a function of several factors, such as QS, QQ, nutrients, and operational conditions43. A recent study reported that there was a negative correlation between floc size and EPS level, because the excessive EPS played a role in reducing the hydrophobicity of flocs and, thereby, weakening the cells’ attachment46. It is thus seen that the reduced EPS content may help enhance the floc aggregation, possibly resulting in greater floc sizes.The biological treatment efficiencies of the two MBRs were evaluated in terms of removals of chemical oxygen demand (COD), total organic carbon (TOC), total nitrogen (TN), and total phosphorus (TP) (Supplementary Fig. 3a–d). The effects of SRT and QQ on these removal efficiencies were almost negligible, although disturbance caused a slight decrease in organics removal. The result coincided with the increased SMP level with shear in Phase 3. In short, mixed liquor properties and biological treatment performances seemed to tolerate the effects of QQ and SRT, although they were slightly impacted by disturbance.Microbial community structure changeFigure 3a and Supplementary Table 2 show microbial community variations relative to phases, with clear microbial community structure shifts between phases. Two species were dominant in the seed sludge: Dokdonella immobilis (11.31%) and Sphaerotilus natans (14.91%). After inoculation in the laboratory MBRs, the dominance of these species diminished and other species, being adapted to the synthetic feed, flourished instead (Phase 1). In Phase 2, Thiothrix eikelboomii (15%–18%) and Panacibacter ginsenosidivorans (11%–14%), which were negligible in the seed sludge, became dominant in both reactors. In addition, the relative abundances of Kofleria flava and Flavitalea antarctica increased to 7.57% and 6.95%, respectively. These two species were more abundant in Reactor 1 than Reactor 2. Lastly, the major species of the seed sludge, such as D. immobilis, S. natans, and Terrimonas lutea, became minority species ( 0.7) with SMP-C, SMP-P, and the relative abundance of four individual microbial species (i.e., F. antarctica, P. glucosidilyticus, S. piscinae, and T. carbonis). SMP had a lot stronger correlations with fouling rates than EPS, although the actual amounts of the former were a lot smaller than those of the latter. The result indicates that the soluble biopolymers present in the bulk liquid play a more important role in membrane fouling, possibly due to their direct deposition onto the membrane surface. The strong, negative correlations of fouling rates with COD and TOC removal efficiencies support the above explanation. In particular, P. glucosidilyticus and S. piscinae, which had the highly strong correlations with membrane fouling (r  > 0.87), accordingly exhibited strong correlations with SMP. Notably, T. eikelboomii, which was the most abundant in Reactor 1 of Phases 4 and 5, had a relatively weak negative correlations (r = −0.32) with membrane fouling and so not as strong as did K. flava (r = −0.73). The decrease in the relative abundance of T. eikelboomii in Reactor 2 of Phase 5 should be associated with QQ, but the species might still have been contributing to membrane fouling, as discussed above. As expected, the microbial diversity indices between OTUs and Chao1, as well as Shannon and inverse Simpson, were found to be strongly correlated. However, the fouling rate did not have strong correlations with any of the microbial diversity indices (−0.05 ≤ r ≤ 0.55), although the microbial diversity was always higher in the presence of QQ (see Table 1).Fig. 4: Correlation analysis.Spearman’s correlation coefficients between all the MBR parameters determined in the study.Full size imageOn the other hand, the content of EPS-C and EPS-P had strong, negative correlations with MLSS levels. The biomass increase was accompanied with longer SRT, so the aged sludge produced less EPS amounts leading to the floc size decline (corresponding to a negative correlation, i.e., r = −0.52). Notably, the floc size had a strong, positive correlation (r = 0.84) with TN removal, suggesting that simultaneous nitrification and denitrification possibly occurred with larger biological flocs59. In addition, D. immobilis showed a strong positive correlation (r = 0.84) with TP removal, proposing its role as a potential phosphate uptake strain60. Overall, the relationships between MBR parameters (e.g., fouling rates, mixed liquor characteristics, biological treatment efficiencies, and microbial species dominance) helped better understand the fouling patterns and biological performances in the MBRs with and without QQ.In summary, the QQ effect on MBR antifouling efficacy was clearer when the SRT was extended from 50 to 75 d, although the disturbance (starvation with shear) aggravated membrane fouling, which counteracted the positive QQ effect. QQ yielded a significant biopolymer production decrease with the longer SRT. Accordingly, organic substance removal showed relatively strong, negative correlations with MBR-fouling propensity. MBR microbial communities showed dynamic responses to the feed change, QQ, disturbance, and SRT. With disturbance, F. antarctica, S. piscinae, and P. glucosidilyticus dominated the microbial community leading to substantive membrane fouling. However, the microbial community balance between T. eikelboomii and K. flava, whose relative abundances appeared to be affected by SRT and QQ, played a key role in fouling propensity under stabilized conditions. The correlation analysis showed strong positive relationships between membrane fouling rate and the abundance of several microbial species (F. antarctica, P. glucosidilyticus, S. piscinae, and T. carbonis). However, there was no strong correlation between T. eikelboomii and membrane fouling propensity, possibly due to the antagonism by K. flava, and vice versa. More

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Shape analysis of 3038 daily flow patterns (2016–2020) for WWTP1 and WWTP2
An example effluent flow pattern for a 10-day period at WWTP1 is shown in Fig. 1. EDM detected spilling intervals are overlaid to demonstrate the flattening effect of spilling on the profile of the flow pattern.
Fig. 1: WWTP1: example effluent flow pattern for 10 days annotated with EDM confirmed spilling intervals.

A 24-h (midnight to midnight) daily flow pattern of 96 15-min-interval average flow rates (litres/second) of treated effluent is shown in blue. The black horizontal, linear annotations represent EDM recorded intervals denoting a discharge from a storm tank (i.e., consented spill or potentially unconsented spill of untreated sewage), the shortest being 15 min and the longest over 24-h. Total daily rainfall (mm/d) is provided in green. The first two days, with no detected spills, show diurnal patterns of low flow between midnight (previous day) and the first peak after mid-morning, followed by a lull until a second, smaller peak in the evening. The next seven days (15/12/18 through 21/12/18) involve spill intervals of various length (black EDM line), showing a flattening of flow, which is typical of storm discharge during heavy rainfall. The last day shows elevated flows and a partial return to a diurnal flow pattern with no spills reported.

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For WWTP1 (resp. WWTP2), EDM data were available for 446 (resp. 471) consecutive days for 2018–2020 during which untreated sewage spill intervals of varying lengths had been recorded. For each day, spill intervals were aggregated to the total number of hours of discharge. Of the days used for machine learning for WWTP1 (resp. WWTP2), 339 (resp. 346) involved no EDM recorded spilling incidents and 107 (resp. 125) days had spills with various lengths of which over a third were for 24-h. For WWTP1 (resp. WWTP2), 97 (resp. 117) days with an aggregated ‘spill’ length of at least 3-h were labelled as ‘spill’ and 349 (resp. 354) with an aggregated ‘spill’ length of below 3-h as ‘normal’. A 3-h aggregation period was selected because it guaranteed a reasonable number of ‘spill’ days on which to base the supervised learning and preliminary attempts to predict spilling hours per day were weakest for aggregated daily spills under 3-h. Where no EDM data was available, days were labelled as ‘unknown’. The average ‘normal’ (blue line) and ‘spill’ (black line) daily flow patterns as a proportion of storm overflow rates (red line) are shown in Fig. 2 for each WWTP. The storm overflow rates mark the minimum flow that should be treated before untreated sewage spills can be made in compliance with EA permits to discharge to watercourses.
Fig. 2: Average daily flow patterns.

a WWTP1: black curve for ‘spill’ days (n = 97) and blue curve for ‘normal’ days (n = 349); b WWTP2: black curve for ‘spill’ days (n = 117) and blue curve for ‘normal’ days (n = 354).

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Separate shape models were generated for flow patterns from 2016 to 2020 for WWTP1 (n = 1511) and WWTP2 (n = 1527). The first principle component of shape variation, PCA1, in both models, is associated with magnitude, and temporal shifting of morning flow peak (see Supplementary Video 1.mp4) as well as “seasonal” changes related to daylight saving, public holidays and vacation periods (Supplementary Fig. 1). Despite differences in the population served by WWTP1 and WWTP2, Fig. 3a, b shows similar distributions for scatter plots of PCA1 vs PCA2 for 2121 flows for 2016–2018 without EDM data. Analogous plots of PCA1 vs PCA2 for 917 flows for 2018–2020 with EDM data (Fig. 3c, d) suggest that, for both WWTPs, PCA2 is correlated with shape difference between ‘normal’ flow (open circles) and ‘spill’ affected flow (filled triangles). This spill-related flattening is illustrated by morphing the overall average daily flow pattern for WWTP1 between −1 and +1 standard deviations of PCA2 (Supplementary Video 2.mp4). Interestingly, the area under the receiver-operating characteristics curve associated with using PCA2 alone for ‘normal’/’spill’ discrimination is 0.88 and 0.91 for WTTP1 and WWTP2, respectively (this is the estimated probability of correctly classifying a pair of flow patterns selected randomly, one each, from the ‘normal’ and ‘spill’ labelled subsets).
Fig. 3: PCA1 vs PCA2 for daily flow patterns.

Unknown spill status (grey filled circles); spill confirmed by EDM (filled black triangles); confirmed as normal by EDM (unfilled grey circle) 2016–2018 without EDM data a WWTP1 (n = 1065); b WWTP2 (n = 1056); 2018–2020 with EDM data c WWTP1 (n = 466); d WWTP2 (n = 471).

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Supervised learning of the effect of sewage spills on 917 effluent flow patterns
The performance of 20-folded cross-validation of supervised learning for labelled flow patterns for WWTP1 and WTTP2 is shown in Supplementary Tables 3 and 4 for 20 support vector machine (SVM) variations while retaining up to 15 PCA modes for flow pattern synthesis. The number of PCA modes retained for shape synthesis affects the validity of the reconstruction of each daily flow pattern and hence classification accuracy. For the three best-performing algorithms, Supplementary Fig. 2 shows the variation in classification accuracy of daily flow patterns for different numbers of retained PCA modes estimated as the average area under the 20 receiver-operating characteristic curves associated with the cross-validation folds. For the optimal classifiers, the average area under the receiver-operating characteristic curve was 0.97 for WTTP1 and 0.96 for WWTP2.
For verification, prior to wider application, the optimal ML classifiers defined for each WWTP were used to reclassify the flow patterns used in their derivation. Figure 4 shows these flow patterns in contiguous temporal sequence with annotations for each day reflecting EDM detected spill intervals (horizontal black segments) and ML confirmation of ‘spill’ (unfilled gold circles). During this period there were 97 (resp. 117) days with an EDM confirmed aggregated spill of at least 3-h at WWTP1 (resp. WWTP2). The agreement between optimal ML classification and spill day labels derived from EDM data was extremely high (WTTP1: sensitivity = 0.91, specificity = 0.95; WTTP2: sensitivity = 0.98, specificity = 0.98), as would be expected for such “training” data.
Fig. 4: Daily effluent flow patterns and event duration monitor (EDM) detected spill intervals at WTTP1 and WWTP2 used as training data (Dec’2018–Mar’2020).

The daily flow and EDM spill data are measured at 15 min intervals. Flow is coloured (orange/blue/pink) to distinguish different years. Black horizontal lines delimit EDM detected spill intervals. Daily flows of aggregated spill length of at least/less than 3-h are labelled as ‘spill’/‘normal’ prior to the supervised learning. Gold circles indicate days classified as ‘spill’ following the training of the machine learning (ML) algorithms to produce an optimal classifier for each WWTP. The grey dashed line represents the storm overflow which defines the minimum sewage flow that should be treated even during storm filling or overflow. Additional annotations are telemetry alarms provided by the operator. These alarms have the potential to corroborate ML predictions of ‘spill’ days for the unseen flow patterns from 2009 to 2018 for which there is no EDM data. Similar charts showing the unseen ML classification of the 2009–2018 daily flow patterns overlaid with rainfall and river level data are provided in Supplementary Figs 5–10.

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Figure 4 also includes data from other alarms related to untreated sewage discharges that have the potential to corroborate ML flow pattern classification for historical periods without EDM data. For WWTP1, there is near-perfect agreement (Cohen’s kappa: 0.81–1.00) between the EDM, STO (Storm Tank Overflow) and COL (Consented Overflow Level) alarms and ML classification for Feb ‘19–Feb ‘20 (Fig. 4 and Table 2). For just two months, Dec ‘18 and Jan ‘19, the EDM and COL devices concur with near-perfect agreement (Cohen’s kappa = 0.95), the STO device was largely at odds (Cohen’s kappa ≤ 0), and the ML classifier flagged incidents detected by all three. These results suggest that the STO is a good candidate and the COL alarm is an excellent candidate for corroborating ML detected putative spills at WWTP1 when EDM data is unavailable.
Table 2 Agreement of ML classification, EDM, COL and STO alarms for the supervised learning.
Full size table

For WWTP2, there is almost perfect agreement between EDM and COL alarms (Cohen’s kappa = 0.87) and with ML classification (Cohen’s kappa = 0.78) (Fig. 4 and Table 2). No STO alarm data were provided for 2020 and between Dec ‘18 and Dec ‘19 STO showed only chance agreement with other devices and the ML classifier (Cohen’s kappa  More

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In Lake Hallwil, the contribution of oxic methanogenesis to overall diffusive CH4 emissions has been estimated to be 90%6 or 63–83%5, but we show here that NOMC ~ 17%.
In the mass balance of the SML extending from 0 to 5 m water depth5,6, Günthel et al.5 used an average sediment flux of Fsed = 1.75 mmol m−2 day−1, averaging flux estimates of Donis et al.6 from two sediment cores, one collected at 3 m and the other at 7 m water depth. The δ13C of the CH4 in the pore water of these two cores differ substantially6, indicating differences in production and oxidation of CH4 between the sediments in the SML and at 7 m water depth. The estimate of Fsed in the SML should therefore be based on the core collected at 3 m water depth. Using the approach of Donis et al.6, the correct Fsed derived from the data of this core is Fsed = 2.8 mmol m−2 day−1 (Peeters et al.9, see Supplementary Note 2.1 for details).
Günthel et al.5 and Donis et al.6 apparently have erroneously used gas transfer coefficients instead of proper CH4 fluxes to calculate emissions. This conclusion is demonstrated by the perfect agreement between the values published erroneously as CH4 fluxes, Fsurf, by Günthel et al.5 and the values of the gas transfer coefficients of CH4 at 20 °C, kCH4, calculated by us (Table 1). The values published by Donis et al.6 as CH4 fluxes are very similar to these kCH4 and therefore also do not represent CH4 fluxes but gas transfer coefficients (for details, see Supplementary Note 2.2).
Table 1 Average CH4 surface fluxes from Lake Hallwil and gas transfer velocities.
Full size table

The gas transfer coefficient of CH4 must be multiplied by the difference between the surface concentration (0.3 mmol m−3, ref. 6) and the atmospheric equilibrium concentration of CH4 (CH4,equ = 0.003 mmol m−3 at 20 °C9), i.e. by ~0.3 mmol m−3, to obtain Fsurf. Fsurf is therefore ~3.3 times smaller than the values of the gas transfer coefficients erroneously taken by Günthel et al.5 and Donis et al.6 as CH4 fluxes (Table 1 and details in Supplementary Note 2.2).
Donis et al.6 and Günthel et al.5 used values obtained from measurements with floating chambers to calculate emissions, but these values claimed to represent Fsurf appear to be in fact values for transfer coefficients, suggesting the same mistake as in the case of the wind models. Donis et al.6 stated: “Average flux (April–August 2016) is equal to 0.8 ± 0.2 mmol m−2 d−1 from MacIntyre relationship for positive buoyancy and to 0.6 ± 0.3 mmol m−2 d−1 from chamber measurements. The latter, not significantly different from the wind-based relationship, was used for the mass balance”. Günthel et al.5, co-authored by D. Donis, claim that the “MacIntyre relationship for positive buoyancy”10 provides an average value of 0.7 for Fsurf, but in fact 0.7 is the average value for kCH4 in unit m day−1 (0.7 m d−1, see Table 1) and Fsurf for this model is 3.3 times smaller (0.21 mmol m−2 d−1, see Table 1). The value by Donis et al.6 for the MacIntyre relationship10 is even slightly larger than 0.7 and therefore clearly incompatible with Fsurf but is rather a gas transfer coefficient as is obvious in the case of Günthel et al.5. The good agreement between the value for the gas transfer coefficient obtained from the MacIntyre model for positive buoyancy flux10 and the values from the chamber measurements suggests that the values from the chamber measurements are not gas fluxes but also gas transfer coefficients (see Supplementary Note 2.2 for more details).
Donis et al.6 derived from their chamber measurements the wind-based model “Hallwil relationship” specifically for Lake Hallwil. The establishment of this Hallwil relationship required that Donis et al.6 calculated gas transfer coefficients from their chamber measurements. In their Supplementary Fig. 4, Donis et al.6 show that the values from their chamber measurements agree well with those from the Hallwil relationship (Supplementary Fig. 2 and Supplementary Note 2.2). Note, however, that the values for the Hallwil relationship are in fact gas transfer coefficients and not Fsurf, supporting that also the values from the chamber measurements represent gas transfer coefficients and not Fsurf (Supplementary Fig. 2 and Supplementary Note 2.2 for more details). This conclusion implies that the values from the chamber measurements by Donis et al.6 must be multiplied by ~0.3 mmol m−3 to give proper CH4 fluxes, which are then ~3.3 times smaller than the CH4 fluxes used in the mass balances of refs. 5,6.
Because there are only four chamber measurements available for 2016 and one of them was exceptionally low (see ref. 6 and Supplementary Note 2.2), the Hallwil relationship is considered here to provide the most reliable estimate of the average k600 in Lake Hallwil and therefore applied to calculate the average surface CH4 flux for April to August 2016, i.e., Fsurf = 0.24 mmol m−2 d−1 (see Table 1 and Supplementary Note 2.2). The reliability of the Hallwil relationship was confirmed by Günthel et al.5 and by Hartmann et al.11 comparing different estimates of surface fluxes in the South Basin of Lake Stechlin.
With Fsed = 2.8 mmol m2 day−1 and Fsurf = 0.24 mmol m2 day−1, NOM = 416 mol day−1 and the contribution of NOM to total emissions is NOMC = 17% (Supplementary Table 1 in Supplementary Note 2.3 includes also additional estimates of NOMC). The low value of NOMC suggests that most of CH4 in the SML originates from the sediments, which is consistent with the δ13C isotopic composition of CH4 in Lake Hallwil9. The uppermost CH4 in the sediment core from the SML is characterized by δ13C about –59‰, which corresponds very closely to the δ13C of the CH4 in the open water of the SML ranging from −62‰ to −58‰ (Figs. 4 and 5 both in ref. 6). Thus the δ13C values suggest that the CH4 from the uppermost pore water in the sediment of the SML is the source of the CH4 in the open water and do not indicate a reduction of the δ13C expected in case of substantial CH4 production. More

Microbial community analysis
An overall microbial community analysis is presented as PCA plots and a dendrogram in Fig. 1. The cluster analysis showed good agreement between sample replicates, which clustered most closely. WWTP influent (i.e., untreated sewage) collected in the post-monsoon season clustered most closely with water samples from S4, S5 and S6 collected at same time, while WWTP influent from the monsoon season clustered with water samples from S6 collected in the same season. The WWTP effluent from both monsoon and post-monsoon season clustered together. The PCA plot with data from all the sampling times (Fig. 1b) generally showed a separation of downstream and WWTP influent water samples from the upstream and WWTP effluent samples along principal component 1, with only a few exceptions. Genera mostly found in the human gut microbiome15,26 like Streptococcus, Trichococcus, Lactobacillus, Enterococcus, Prevotella and Arcobacter, were highly prevalent in downstream and WWTP influent water samples, which separated these samples from the upstream water samples in the PCA. Among the three factors analysed (i.e., location, sampling time and water sample types), locations and sampling time had a significant effect on the similarity of the samples in the ANOSIM, although with relatively low R values (ANOSIM; Location: R = 0.29, p value = 0.001 and Sampling time: R = 0.16, p value = 0.01). ANOSIM further indicated no statistically significant differences between the microbial communities in water from locations S4, S5 and S6 and the wastewater influent (ANOSIM; (1) S4 and Inf: R = 0.0309, p value = 0.357: (2) S5 and Inf: R = 0.0617, p value = 0.369 and (3) S6 and Inf: R = 0.0123, p value = 0.3690).
Fig. 1: Cluster and PCA analysis at rank genus for 16S rRNA gene sequencing reads.

a Cluster analysis [all seasons], PCA plot b all seasons, c monsoon [June 2019] and d post monsoon [August 2019]. Arrows in the PCA plots indicate the ten variables with the highest loadings (vector lengths) in the PC1 and 2 space.

Full size image

An interesting picture emerged when a separate PCA (Fig. 1c, d) and cluster analysis (Supplementary Fig. 1) was conducted for water samples from the monsoon and the post-monsoon season. In both seasons, there were substantial, but seasonally distinct, contributions of genera found in the human gut microbiome to the variance among water samples along principal component 1: in the monsoon season Arcobacter, Aeromonas, Streptococcus and Prevotella had significant PC1 loadings; in the post-monsoon season Enterococcus, Acinetobacter, Streptococcus and Trichococcus had significant PC1 loadings. Separation of wastewater treatment plant effluent (WWTP Effluent) samples along PC1 away from the WWTP influent (WWTP Influent) samples in both sampling events signified the benefits of wastewater treatment, because human gut-associated genera became less predominant in treated wastewater microbiomes, as expected27. Accordingly, there was a clear separation of the most upstream water samples from the most downstream water samples along PC1 in both events, with the downstream water samples becoming more similar to WWTP Influent (Fig. 1c, d). Evidently, as the Bagmati River flowed into more densely populated areas, the characteristics of its water microbiome changed from a composition more similar to treated, to a composition more similar to untreated urban sewage, but the composition of the urban sewage was variable for the monsoon and post-monsoon season.
Abundance of human gut and putative pathogenic bacteria in the water microbiomes
A more detailed breakdown of the microbial community composition in the Bagmati River for the monsoon and post-monsoon season is reported in Table 1, and Supplementary Tables 1 and 2, which compare the total percentage relative abundance of putative human gut28 and pathogenic29 bacteria at genus and species level for different sampling sites in the Bagmati River, and the WWTP influent and effluent (Refer to Supplementary Tables 3–5 for more detailed lists of bacteria). Based on our previous findings24, species identities are not always reliable due to the limited read accuracy of the MinION sequencing reads, but the overall trends are nonetheless indicative of changes in microbial composition. For all sampling events, the water collected at the most upstream site S1 and S2 showed the lowest relative abundance for both human gut and putative pathogenic bacteria, whereas the highest relative abundance was observed in the water collected at the most downstream sites S4–S6 (Table 1, Supplementary Tables 1 and 2). The microbial water quality of water samples collected at site S1 can be considered as baseline data, as this watershed is distant from the densely populated Kathmandu Valley and has the minimal influence of human and urbanisation activities. Figure 2, and additional figures in Supplementary Information (Supplementary Fig. 2–9) show how the abundance of human gut and putative pathogenic genera changed in space and time along the Bagmati River. As the river flowed downstream, the abundance of some of these groups of bacteria increased, and the most drastic and significant increase was observed at the sites S4, S5 and S6 downstream of the Pashupatinath Temple as compared to site S1 (Two-sample t test, p value  More