A hydro-topological strategy enables self-regulating biofilms for sustainable wastewater treatment

Domain: biology
Published: 2026-03-13
Editorial outcome: major_revision

Abstract
The moving bed biofilm reactor (MBBR) is a cornerstone technology in modern wastewater treatment, yet its
performance is often hindered by carrier clogging, which significantly reduces overall treatment efficiency and
undermines the technology's environmental benefits. Here, we introduce a V-carrier hydro-topological design
strategy that enables biofilm self-regulation, allowing simultaneous control of biofilm thickness and continuous
hydraulic shear-induced self-cleaning. In a pure biofilm system treating real municipal wastewater for over 500
days, the V-carrier achieves stable and efficient nutrient removal even under low-temperature (9.1 °C) and high-
loading conditions. Crucially, it achieves a 3.2-fold higher unit biomass nitrification rate with a biofilm biomass
44% lower than a conventional K3 carrier, demonstrating that treatment efficiency is decoupled from biomass
quantity through optimal ecological niche design. This work establishes a paradigm for biofilm reactor design,
transforming carriers from passive substrates into active regulators of microbial ecosystems, with profound
implications for sustainable water infrastructure.

Introduction
Wastewater treatment plants (WWTPs) are critical infrastructure for protecting public health and environmental
quality, directly supporting 11 of the 17 Sustainable Development Goals (SDGs)1-4. Among advanced treatment
technologies, the moving bed biofilm reactor (MBBR) process supports SDGs 6, 11, and 12 through its ability,
enabled by suspended biofilm carriers, to deliver high nutrient removal efficiency (SDG 6), a compact physical
footprint (SDG 11), and reduced sludge production (SDG 12). Through these contributions to water security,
urban sustainability, and resource efficiency, MBBR has been established as a leading technology for new and
upgraded WWTPs worldwide5-7.

Over four decades of development have yielded numerous MBBR carrier designs for engineering applications8,9.
These efforts, even using advanced tools like 3D printing techniques, have predominantly pursued one goal:
maximizing the protected surface area to enhance biofilm attachment10. However, increased surface area has not
consistently improved treatment efficiency11, revealing a deeper issue rooted in two factors: a paradigm inherited
from suspended-growth systems (e.g., activated sludge), where more biomass implies better performance, and a
widespread lack of long-term operational data. This has led to the misapplication of the more biomass equals
more efficiency principle to biofilms—systems where controlled biofilm thickness and metabolic balance are
paramount. The design focus on maximizing protected surface area has promoted uncontrolled biofilm growth,
with carrier clogging even misinterpreted as a positive sign12,13, thereby diverting attention from the need for
biofilm managed growth. This fundamental misdirection manifests as the core limitations of conventional
MBBR systems: severe carrier clogging and scaling, drastically diminishing the effective surface area, impairing
mass transfer, and disrupting microbial community structure by fostering competition for oxygen and substrates
among unfavorable species14-16. Moreover, clogged carriers exhibit increased buoyant density, demanding higher
energy inputs for mixing and suspension, thereby elevating operational costs and environmental impacts.
Consequently, the sustainability benefits of conventional MBBR systems are compromised by these inherent
design flaws, presenting a significant challenge to achieving the SDG targets they purport to advance.

The biofilm thickness is a well-established critical parameter, governing the structure of the microbial
community, assembly processes, mass transfer dynamics, and the potential for micropollutant removal17-20.
Despite its recognized importance, a fundamental practical limitation remains: no existing MBBR carrier can
reliably control biofilm thickness under the dynamic hydraulic conditions of full-scale reactors21,22. One notable
effort to address this limitation was the Z-carrier (AnoxKaldnes), which was based on the geometric premise
that its grid height would regulate biofilm thickness17,23,24—a design that also had implications for mitigating
clogging and scaling due to its open structure. How ever, their own long-term evaluation revealed a critical flaw:
biofilms at the center of the grid cells became significantly thinner or were entirely absent25. This outcome
underscores a fundamental stalemate in sustainable carrier design, revealing a poor understanding of the
hydrodynamic mechanisms that control biofilm development. The failure of this simplistic geometric approach
highlights the necessity for a design paradigm rooted in hydraulic principles.

The propensity for clogging in conventional tubular carriers is intrinsically linked to their hydraulic design.
Carriers with large internal diameters (e.g., K3) demonstrate clogging resistance, whereas those with small
hollows (e.g., K5, Biofilm-Chip P, Biofilm-Chip M) are prone to complete clogging during long-term operation15. This phenomenon is explained by the Hagen-Poiseuille equation, which dictates that a smaller tube diameter,
under identical hydraulic conditions, results in a greater pressure drop and fluid resistance. This, in turn, leads
to a lower internal flow velocity and shear stress26-28. We therefore hypothesize that clogging in small-tube
carriers results from insufficient hydraulic shear to balance biofilm growth with detachment, allowing
uncontrolled accumulation. This principle is exemplified by fixed-bed filters, which maintain biofilm thickness
and mass transfer efficiency through periodic backwashing—a process that generates high transient shear to
scour excess biomass29-31. In an MBBR, however, the maximum continuous hydraulic shear is exerted only on
the biofilm surface directly exposed to the bulk liquid flow. This critical observation suggests that the strategic
orientation of the biofilm surface is paramount for harnessing hydraulic forces for continuous self-cleaning.

Building on this foundation, we propose a hydro-topological strategy for autonomous biofilm regulation. This
approach engineers the carrier's topology to control both biofilm thickness and its spatial orientation, thereby
enabling direct and continuous hydraulic shearing of the outer surface to maintain a stable biofilm with a
controlled, optimal thickness and high metabolic activity. We materialized this strategy by designing a V-carrier
(Fig. 1), which operates on four synergistic principles:

1. Directed Growth: The V-shaped opening guides biofilm development outward, toward the liquid phase,
ensuring efficient nutrient access for outer-layer cells and real-time clearance of metabolic products.
2. Thickness Constraint: The finite depth of the V-groove physically constrains the maximum biofilm thickness,
intrinsically preventing the mass transfer limitations associated with overgrowth.
3. Biomass Protection: The inclined surfaces of the V-groove shield the established biofilm from detachment
caused by excessive hydraulic shear forces.
4. Niche Stabilization: The continuous, staggered arrangement of V-grooves creates independent, consistent
micro-chambers that function as minimal functional ecological niches, enhancing the overall stability and
resilience of the biofilm community.

We hypothesize that this integrated V-carrier design enables stable, self-regulated biofilm maintenance, ensuring
efficient long-term mass transfer and treatment performance. To test this hypothesis, we operated a laboratory-
scale anoxic/aerobic (A/O)-MBBR system treating real municipal wastewater for over 500 days, evaluating its
potential as a sustainable solution for modern WWTPs.

Results
Design and characteristics of the V-carrier
The V-carrier used in this study was fabricated from high-density polyethylene (density 0.94–0.97 g cm⁻³) to
achieve near-neutral buoyancy for unrestricted movement in wastewater. As shown in Fig. 1a, the carrier exhibits
a square-sheet morphology with overall dimensions of 24 × 24 × 0.95 mm. Its core design consists of mirror-
symmetric, staggered V-shaped grooves that serve as the primary attachment sites for biofilms (Fig. 1b). The
groove depth is 0.72 mm, a key parameter for constraining the maximum biofilm thickness, while the opening
length is 1.0 mm, yielding an opening ratio (length/depth) of 1.39 to optimize hydraulic exposure (Fig. 1c, d and
Fig. 6a-f). The framework width is 0.7 mm, enabling each V-groove to function as an independent, consistent
ecological niche. Each carrier contains 1,536 such grooves, providing a total protected surface area of 1,885 m²
m⁻³. The effective biofilm surface area, calculated from the outer surface of the biofilm, is a more functionally
relevant 1,152 m² m⁻³ for mass transfer calculations. The carrier's bulk density and structural porosity are 95 ±
0.5 kg m⁻³ and 92%, respectively. These structural and material properties form the physical basis for testing the
proposed hydro-topological strategy.

Stable and efficient nutrient removal over long-term operation
The A/O MBBR system, packed exclusively with V-carriers, was operated for over 500 days while treating real
municipal wastewater (Supplementary Fig. 1). Sodium acetate was supplemented to provide sufficient electron
donors for denitrification, resulting in an operational C/N ratio of 4.9 ± 0.3 (Supplementary Fig. 2). The
operational timeline was strategically divided into three phases based on hydraulic retention time (HRT) and
temperature: start-up (Phase I, days 1–10), suitable temperature (Phase II-i, days 11–280; Phase II-ii, days 325–
512), and low temperature (Phase III, days 281–324) (Fig. 2a).During the start-up phase (Phase I) at 15.6 ±
0.9 °C, the HRT was successfully reduced from 24 to 8 hours within 10 days, demonstrating rapid biofilm
establishment and system acclimation. In the subsequent suitable-temperature phase (Phase II-i, HRT = 8 h), the
system achieved efficient nutrient removal, with effluent NH₄⁺-N and total inorganic nitrogen (TIN)
concentrations of 0.9 ± 0.6 mg L⁻¹ and 6.6 ± 2.3 mg L⁻¹, corresponding to removal efficiencies of 97.8 ± 1.4%
(ammonia removal efficiency, ARE) and 86.7 ± 5.2% (TIN removal efficiency, NRE), respectively (Fig. 2b,
c).When reactor temperature decreased to 10.4 ± 1.2 °C during Phase III, the HRT was increased to 9 hours to
compensate for reduced microbial activity. Despite this thermal challenge, the system maintained robust
performance, with ARE and NRE of 97.0 ± 0.5% and 85.0 ± 3.3%, respectively. Upon temperature recovery (>
14 °C) in Phase II-ii, the HRT was returned to 8 hours, resulting in further improved effluent quality (NH₄⁺-N =
0.5 ± 0.4 mg L⁻¹; TIN = 4.6 ± 1.7 mg L⁻¹) and high, stable removal efficiencies (ARE = 98.9 ± 0.9%; NRE =
90.8 ± 3.5%). Throughout the entire operational period, the system exhibited high nutrient removal rates of 0.138
± 0.022 g N g VSS⁻¹ day⁻¹ for ammonia (ammonia removal rate, ARR) and 0.587 ± 0.108 g N g VSS⁻¹ day⁻¹ for
TIN (TIN removal rate, NARR) (Fig. 2d). Notably, the nutrient removal rates remained stable even during low-
temperature operation (Supplementary Fig. 3), demonstrating minimal functional impairment and consistent
with effective microbial adaptation. In line with the inherent characteristics of pure biofilm systems featuring
extended sludge retention, phosphorus removal efficiency was variable (8–52%) (Supplementary Fig. 4),
consistent with the limited biological phosphorus removal capacity under these conditions32-34. In practical
applications, this limitation is routinely and effectively overcome by coupling the biofilm process with a
downstream chemical phosphorus removal step35, which is a standard and cost-comparable practice also
employed following conventional activated sludge processes to meet stringent effluent standards. Collectively,
these results demonstrate that the V-carrier system delivers high and stable nutrient removal performance under
varying hydraulic and temperature conditions, confirming its operational robustness.

Mechanism of biofilm regulation—Hydro-topological strategy
To elucidate the mechanisms enabling stable biofilm management, we conducted a comparative analysis of the
V-carrier against conventional tube-carriers (Mutagbiochip, K5, and K3; Supplementary Fig. 5). The V-carrier
maintained a stable volatile suspended solids-to-suspended solids (VSS/SS) ratio and consistent biofilm
thickness of 500–550 μm—a range proven to benefit mass transfer and promote the removal of diverse
micropollutants17—under both anoxic and aerobic conditions throughout long-term operation, demonstrating
effective self-cleaning coupled with thickness control (Fig. 3a, b and Supplementary Note 1). Analysis of the
Mutagbiochip carrier, designed for a ~550 μm biofilm thickness on each side (Supplementary Fig. 6), revealed
a critical limitation. Although it achieved the target thickness within 23 days—comparable to the optimal value
for mass transfer17,36—significant inorganic accumulation (ash content increased from 18% to 56%; paired t-test,
P = 2.56 × 10⁻⁶) and a substantial decline in the ARR occurred over 285 days (Fig. 3c and Supplementary Table
1). This demonstrates that geometric constraint for thickness control is insufficient without an integrated self-
cleaning capability.

We posited that the V-carrier's open structure, which guides biofilm growth outward into the liquid phase (Fig.
3d-I), is key. In contrast, biofilms grow inward in tube-carriers (Fig. 3d-II). We hypothesize that sustained
hydraulic shear on the exposed biofilm surface enables a dynamic balance between growth and decay, facilitating
real-time metabolite clearance (Fig. 3e). Support for this comes from the K5 carrier (2 mm hollows), which
clogged completely after 70 days, concomitant with a loss of hydraulic shear and a significant decrease in ARR
from 0.032 ± 0.003 to 0.010 ± 0.001 g N g VSS⁻¹ day⁻¹ (paired t-test, P = 2.53 × 10⁻⁶; Fig. 3d, f). Conversely,
the K3 carrier (5 mm hollows) maintained smooth inner-surface biofilms, a stable VSS/SS ratio (0.70 ± 0.02),
and a consistent thickness (334 ± 93 μm) over 520 days, confirming the role of preserved hydraulic shear in
sustaining mass transfer (Fig. 3d-II, g). To directly test the necessity of hydraulic exposure, we engineered a
modified K5 (MK5) carrier with both open and hollow sections (Supplementary Fig. 5). While the open sites
maintained a stable biofilm (VSS/SS = 0.72 ± 0.02) for 520 days (Fig. 3d-III), the shielded hollows accumulated
57% inorganic precipitates (Fig. 3f). This controlled experiment provides definitive evidence that direct
hydraulic shear is prerequisite for effective self-cleaning.

Finally, moving beyond simple geometric constraints, we asked whether topology alone could dictate biofilm
thickness under realistic hydraulics. The MK5 carrier revealed a critical limitation: at its open sites, excessive
hydraulic shear dominated over topological guidance, preventing biofilms from reaching the ridge height despite
available space (Fig. 3d-III). We then tested the converse scenario using a U-carrier with reduced ridge spacing
designed to attenuate shear. Here, the lack of hydraulic control led to spatial competition: biofilms preferentially
colonized the shielded ridge surfaces, eventually bridging the U-grooves and causing clogging (Fig. 3h). Thus,
neither unmanaged high shear nor excessive shielding succeeds; both fail to direct biofilm growth appropriately.
The V-carrier succeeds by resolving this spatial competition through its hydro-topological design. The oblique
grooves guide biofilm development into the liquid flow, ensuring that hydraulic shear, rather than overwhelming
or being absent, functions collaboratively with topology to maintain an optimal, stable biofilm. This synergistic
management of space and shear, which we term the hydro-topological strategy, integrates precise topological
guidance for hydraulic exposure with sustained shear for autonomous regulation to maintain an optimal, stable
biofilm.

Biofilm dynamics and functional adaptation to temperature
To elucidate the microbial basis for the observed operational stability, we investigated the physiological and
spatial dynamics of V-carrier biofilms in response to temperature fluctuations (Fig. 4). Ex situ nitrification
potential exhibited a strong positive correlation with temperature (P = 7.12 × 10⁻³⁵, R² = 0.591), confirming its
role as a primary regulator of microbial activity (Fig. 4a). Notably, the ex situ rates surpassed the in situ
performance by factors of 1.55 (< 15 °C) and 3.18 (> 15 °C), indicating that the biofilms possessed intrinsic
metabolic capacity that was not fully expressed under the reactor's substrate-limiting and dynamically fluctuating
operational conditions (e.g., temperature, pH, dissolved oxygen). Quantitative PCR revealed rapid enrichment
of ammonia-oxidizing (AOB) and nitrite-oxidizing bacteria (NOB), with their combined absolute abundance
stabilizing at (1.0 ± 0.2) × 10¹² copies per gram of biofilm VSS after 150 days of operation (Fig. 4b). This high
and stable abundance of nitrifiers confirms the effectiveness of the V-carrier's microenvironment in supporting
functional community retention. Spatial analysis provided further mechanistic insights. The relative abundance
of nitrifiers in the community was also positively correlated with temperature (P = 0.0091, R² = 0.542).
Fluorescence in situ hybridization (FISH) visualization aligned with this trend, showing stronger signals for
AOB and NOB at higher temperatures (Fig. 4c, d). More importantly, a distinct spatial reorganization was
observed: nitrifiers predominantly occupied the inner biofilm layers at higher temperatures but distributed more
uniformly throughout the biofilm matrix during seasonal cooling. This redistribution is interpreted as an adaptive
strategy to mitigate diffusion limitations and maintain metabolic activity under low-temperature stress.
Furthermore, the persistent presence of internal voids within the biofilms, irrespective of temperature
(Supplementary Fig. 7), is consistent with active cell lysis and material export37-39. This is definitively supported
by live-dead staining (Supplementary Fig. 8), which reveals a higher proportion of dead cells and lysing debris
in the inner biofilm layers, illustrating a gradient from the interior to the bulk liquid. This visual evidence
confirms that the voids are pathways formed by continuous cell lysis and subsequent export of cellular material.
This morphological feature provides physical evidence of an ongoing self-renewal process, underpinning the
dynamic self-cleaning mechanism intrinsic to the V-carrier design. Collectively, these findings demonstrate that
the V-carrier fosters a resilient biofilm ecosystem capable of physiological, spatial, and compositional adaptation
to maintain function against environmental perturbations.

Microbial community assembly and functional resilience
We analyzed the bacterial community structure and succession in aerobic V-carrier biofilms via 16S rRNA gene
amplicon sequencing to link ecosystem-level processes to system function (Fig. 5). Principal coordinates
analysis revealed that operating temperature was the primary driver of community dynamics (Adonis test, R² =
0.873, P = 0.001) (Fig. 5a). Furthermore, the strong fit to the neutral community model (R² > 0.5 across 9.1–
29.5 °C) indicated that stochastic processes (e.g., growth, apoptosis, drift) governed initial biofilm assembly and
the self-cleaning process (Supplementary Fig. 9). An iCAMP analysis quantified the ecological processes,
confirming that while stochasticity dominated assembly, temperature was the key deterministic factor
modulating succession (Fig. 5b). Microbial diversity and richness increased throughout the operation
(Supplementary Table 2). These results illustrate a dual regulatory mechanism: stochastic processes maintain a
self-cleaning, stable community foundation, while deterministic temperature shifts guide its functional
succession.

At the genus level, the core nitrifying guild consistently comprised Nitrosomonas (AOB), Nitrospira (NOB),
and Candidatus (Ca.) Nitrotoga (NOB). Their abundances shifted markedly with temperature (Fig. 5c). During
cooling, Nitrosomonas increased from 1.85% to 2.92%, while Nitrospira decreased from 4.67% to below
detection limits. Concurrently, the cold-tolerant NOB Ca. Nitrotoga increased significantly from 0.81% to 2.28%
(P = 0.006) (Fig. 5d), demonstrating a compensatory niche replacement that underpinned the functional
resilience of nitrification observed during low-temperature operation (Fig. 2). The most pronounced shift
occurred in the putative denitrifier norank_f__AKYH76740,41, which declined dramatically from 14.8% to 0.2%
(P = 0.0013) with cooling. This decline likely resulted from combined pressures of limited substrate access and
reduced enzymatic activity at low temperatures, potentially exacerbated by the outward migration of nitrifiers
within the biofilm (Fig. 4d), a shift that may be associated with elevated dissolved oxygen concentrations in the
inner biofilm layers to sustain nitrification, thereby creating a less favorable microenvironment for denitrifiers.
This could further limit nitrate diffusion to inner-layer denitrifiers. Thus, the V-carrier system maintains
functional stability through a multi-faceted strategy: it selects for beneficial, adaptive taxa (e.g., Ca. Nitrotoga),
reduces competition from less robust organisms, and its hydro-topological design ensures efficient mass transfer
even under metabolically challenging conditions.

Methods
Reactor configuration and operation
A laboratory-scale anoxic/aerobic (A/O) MBBR system was constructed using customized polypropylene tanks.
The working volumes were 150 L (anoxic zone), 250 L (aerobic zone), and 20 L (secondary clarifier). The system
was operated in a pure biofilm mode (no sludge recirculation) for over 500 days, treating prepared municipal
wastewater. Nitrified liquor was recirculated from the clarifier to the anoxic zone at a rate of 300% relative to
the influent flow. The volumetric filling ratios of carriers were maintained at 16% and 30% in the anoxic and
aerobic reactors, respectively. The V-carriers were provided by Hangzhou Tao of Water Technology Co., Ltd
(Hangzhou, China). The methodology for determining the carrier's number, bulk density and structural porosity
is provided in Supplementary Equations. The system schematic can be found in Supplementary Fig. 1.

Wastewater characteristics and feed preparation
Raw municipal wastewater was collected daily from the septic tank effluent of Shangkun Park (Hangzhou,
China). Tap water, aerated for 24 hours to remove residual chlorine, was used to dilute this primary effluent to
simulate typical influent concentrations for a local wastewater treatment plant (Chengxi WWTP, Hangzhou,
China), resulting in a feed containing 143.8 ± 23.4 mg L⁻¹ soluble chemical oxygen demand (sCOD), 45.9 ± 6.7
mg L⁻¹ NH₄⁺-N, and 50.7 ± 7.4 mg L⁻¹ total inorganic nitrogen (TIN), with an initial C/N ratio of 2.8 ± 0.3.
Phosphorus was present predominantly as orthophosphate (> 99% of total phosphorus) at a concentration of 4.8
± 1.7 mg L⁻¹. The hydraulic retention time (HRT) was managed to comply with the discharge standards for major
water pollutants mandated by Zhejiang Province, China (see Supplementary Table 3 for detailed standards and
wastewater characteristics). To enhance denitrification and meet the designed target, sodium acetate was
supplemented into the anoxic zone, with the resultant C/N ratio being maintained at 4.9 ± 0.3 (Supplementary
Fig. 2).

Biofilm establishment and reactor startup
Prior to long-term operation, clean V-carriers were added to the reactors and fluidized to ensure uniform
distribution. Biofilm cultivation was initiated by feeding the reactors with activated sludge (mixed liquor
suspended solids concentration = 3,512 mg L⁻¹) obtained from the Chengxi Wastewater Treatment Plant
(Hangzhou, China). During the first three days, 50 L of activated sludge was introduced twice daily at an HRT
of 24 hours. The HRT was then progressively reduced by increasing the inflow rate until stable nutrient removal
performance was achieved, marking the start of the long-term experimental phase. The detailed Biofilm
Establishment and System Startup Protocol is provided in Supplementary Note 2.

Analytical methods
Water quality monitoring. Influent and effluent samples were collected daily, filtered through 0.45-μm
membrane filters (NAVIGATOR, China), and immediately analyzed. Concentrations of sCOD, NH₄⁺-N, TIN,
and PO₄³⁻-P were determined spectrophotometrically (GNST-900S, China) using commercial reagent kits
(Catalog Number G0402, Genesite®, China) according to the manufacturer's protocols. Temperature, dissolved
oxygen (DO), and pH were monitored using multiparameter probes (HC2200, USA).

Biofilm characterization. Biofilm samples were harvested from carriers by gentle brushing with a sterile brush,
followed by centrifugation. The resulting pellets were stored at –80 °C for subsequent microbial analysis.
Suspended solids (SS) and volatile suspended solids (VSS) were measured in accordance with APHA Standard
Methods57. The elemental composition (e.g., for scaling analysis) was determined using inductively coupled
plasma mass spectrometry (ICP-MS, iCAP RQ, Thermo Fisher Scientific). For morphological analysis, freshly
harvested carriers were immediately suspended in deionized water, and examined using an industrial light
microscope (GP-304K, China) to characterize structure and measure thickness. Specific removal rates for
ammonia (ARR), TIN (NRR), and sCOD (CRR) were calculated on a per-gram of biofilm VSS basis. Detailed
procedures for SS, VSS measurement, elemental analysis, and rate calculations are provided in Supplementary
Note 3 and Supplementary Equations.

Microbiological analysis
Nucleic acid extraction and quantification. Total genomic DNA was extracted from frozen biofilm biomass
samples using the E.Z.N.A.® Soil DNA Kit (Catalog Number D5625-01, Omega Bio-tek, USA) according to
the manufacturer's instructions, under aseptic conditions. The concentration and quality of the extracted DNA
were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and verified by 1.0%
agarose gel electrophoresis. Qualified DNA samples were stored at –80 °C until further analysis.

Quantitative PCR (qPCR). The absolute abundances of the 16S rRNA gene (total bacteria), and functional
genes specific to ammonia-oxidizing bacteria (AOB, amoA) and nitrite-oxidizing bacteria (NOB) were
quantified by real-time qPCR. All reactions were performed in triplicate. Primer sequences and detailed thermal
cycling conditions are provided in Supplementary Table 4.

16S rRNA gene amplicon sequencing. Bacterial community analysis was performed via high-throughput
sequencing of the 16S rRNA gene. For each selected time point, genomic DNA from three independent biofilm
samples (n = 3) was used as template. The V3–V4 hypervariable regions were amplified using primers 341F and
806R58 on a T100 Thermal Cycler (Bio-Rad, USA). Amplification and library preparation followed established
protocols. Sequencing was conducted on an Illumina MiSeq platform (Majorbio Bio-Pharm Technology Co.,
Ltd., Shanghai, China). Raw sequence data have been deposited in the China National Center for Bioinformation
(CNCB) database under BioProject accession number CRA032524. Detailed bioinformatics processing,
including quality filtering, ASV clustering, and taxonomic assignment, is described in Supplementary Note 4.

Fluorescence in situ hybridization (FISH). The spatial distributions of AOB and NOB within biofilms were
analyzed using FISH combined with confocal laser scanning microscopy (FISH-CLSM). Biofilm samples were
collected from aerobic V-carriers on day 156 (28.9 °C) and day 307 (9.7 °C). Hybridizations were performed
using Cy3-labeled oligonucleotide probes NSO190 (for most AOB) and Ntspa712E (for the genus Nitrospira)32,
with DAPI used for total cell staining. Detailed FISH protocols, including fixation, hybridization conditions, and
microscopy parameters, are provided in Supplementary Note 5.

Live-Dead Cell Staining of Biofilms. The viability and spatial distribution of cells within the biofilms were
assessed using a dual fluorescent staining method based on SYTO 9 and propidium iodide (PI), followed by
CLSM imaging. Carrier samples with attached biofilms were collected on day 409 (14.1 °C) and analyzed. The
comprehensive staining and imaging protocol is provided in Supplementary Note 6.

Ex situ assessment of nitrifying potential
The temperature-dependent nitrifying activity of biofilms grown on V-carriers was assessed periodically via ex
situ batch assays conducted every two days throughout the operational period. Assays were performed in a 3-L
transparent polypropylene batch reactor equipped with a bottom air diffuser. The reactor was pre-calibrated with
tap water to ensure complete fluidization of the carriers under aeration. The reaction medium was a synthetic
wastewater (composition detailed in Supplementary Table 5) devoid of organic carbon to selectively assess
nitrification. For each assay, 300 V-carriers were aseptically retrieved from the aerobic zone of the main A/O-
MBBR system and transferred to the batch reactor containing 3 L of the synthetic medium. The assay conditions
were rigorously controlled: temperature was maintained at the recorded in-situ value using a water bath and
monitored continuously with a multiparameter sensor (HC2200, USA); dissolved oxygen (DO) was kept at 4.5
± 0.5 mg L⁻¹; and pH was maintained at 7.5 through the continuous addition of a sodium bicarbonate solution
(NaHCO₃, 150 g L⁻¹) at a rate of 0.1 mL min⁻¹ to stabilize alkalinity. Liquid samples were collected at 0, 30, 60,
120, and 180 minutes for immediate analysis of NH₄⁺-N and NO₂⁻-N concentrations. The final NH₄⁺-N
concentration was set above 15 mg L⁻¹ to preclude substrate limitation during the assay. The specific nitrification
potential (expressed as g N g VSS⁻¹ d⁻¹) was calculated from the linear rate of ammonium depletion, as detailed
in Supplementary Equation (8). Following each assay, the carriers were promptly returned to the aerobic reactor
of the main system. Control assays (without carriers) were conducted in parallel to account for any abiotic
reactions.

Computational fluid dynamics (CFD) analysis
To provide a quantitative, mechanistic understanding of the hydraulic environments, we conducted
comprehensive CFD simulations of the flow fields and shear stresses on the carrier surfaces. The simulations
were performed using the finite volume method (FVM) implemented in the commercial software Siemens
STAR-CCM+ (version 24A06.19.04.007). The fluid flow was modelled as a three-dimensional, incompressible
system governed by the continuity equation and the Reynolds-averaged Navier–Stokes (RANS) equations, with
turbulence closure achieved using the Realisable k-ε model. All computations were executed on a high-
performance workstation (Intel Xeon Gold 6242R CPU, 3.10 GHz, 20 cores) operating in parallel mode. The
detailed geometrical models, boundary conditions, and validation of the CFD setup are provided in
Supplementary Fig. 13 and Supplementary Note 7.

Statistics and reproducibility
Statistical analyses were performed using OriginLab 2021 (OriginPro, OriginLab Corporation, Northampton,
MA, USA) and Microsoft Excel 2021 (Microsoft Corporation, Redmond, WA, USA). Microbiological data were
analyzed on the Majorbio Cloud Platform (https://cloud.majorbio.com). Experimental data are presented as mean
± standard deviation (s.d.). Specific statistical tests applied included the two-tailed paired t-test, Welch's two-
tailed t-test, two-tailed Pearson correlation, and the two-tailed Adonis test. A P value of less than 0.05 was
considered statistically significant, with the significance level (α) set at 0.05 for all tests. Data points below the
detection limit were assigned a value of zero for analysis purposes. No data were excluded from the analyses
unless otherwise specified. No statistical method was used to predetermine sample size. The experiments were
not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript’s main focal area entails biofilm technology development for water resource recovery, including two key\nnotions; the meaning of which needed to be defined by co-authors in the ensuing text, i.e.,\nHydro-topolgical strategy: they define it as a reactor “design strategy”, which seems to indicate the V-shape of the developed\nand tested carrier media.\nSelf-regulating biofilm: they describe it as the hydraulic behaviour of the new carrier design is intended to serve the purpose\nof more efficient biofilm sloughing from the media.\n- What are the noteworthy results? ANSWER: A new type of biofilm carrier was tested, however, the advantages of this\ncarrier – over state-of-the-art technology, e.g., zeta-carrier – were not sufficiently supported by new evidence. In fact, there is\nnot even a photograph of the carrier shown only seemingly computer-generated images.\n- Will the work be of significance to the field and related fields? ANSWER: As supported my below detailed evaluation and\nassess, my answer is NO, it will not.\n- How does it compare to the established literature? If the work is not original, please provide relevant references. ANSWER:\nthe manuscript establishes knowledge gaps based on the premises of erroneous understanding/interpretation of literature.\n- Does the work support the conclusions and claims, or is additional evidence needed? ANSWER: I could not find any\nseparate Conclusions as such.Meanwhile, some of the statements based on results do not support the e.g., high ammonia\nremoval rate – benchmarked against literature data.\n- Are there any flaws in the data analysis, interpretation and conclusions? - Do these prohibit publication or require revision?\nANSWER: I am unsure about the data analysis. To me it is mostly down to the convenient way of interpretting literature in a\nway suitable for their objectives.\n- Is the methodology sound? Does the work meet the expected standards in your field? ANSWER: It is partly sound. The\nmain issue is the lack of benchmarking against the state-of-the-art technology and the lack of clear demonstration of novelty\nand benefits of the new technology.\n- Is there enough detail provided in the methods for the work to be reproduced? ANSWER: clearly not –benchmark reference\ndata is missing.\nMore detailed critical comments:\n“inevitably clog. This clogging severely diminishes treatment efficiency” Very ambiguous and seemingly incorrect statement\n– right in the problem statement of the abstract. Do you mean biofilm sloughing and removal of sloughed off biofilm?\n“uncontrolled 29 biofilm accumulation. This results in severe carrier clogging and scaling, drastically diminishing the 30\neffective surface area, impairing mass transfer and disrupting microbial community structure” Unclear and seemingly\nerroneous set of statements. Excessive growth on e.g., K1 carrier does NOT reduce the surface area. In fact the higher the\nbiofilm thickness the lower the biofilm density, which effectively increases the effective diffusivity, i.e., f*D_water. Also,\nnitrification rate is not expected to change much, only if normalised to the total gVSS on the carrier. Example: at 50 um: 2\ngN/(d.gTSS); at 200 um: 0.6 gN/(d.gTSS).\nThere is no such thing as “disrupting community structure”. Biofilm thickness has an impact on the microbial community\nstructure, and higher thickness can select in microorganisms that e.g., harbour niche organisms with high micropollutant\nbiotransformation rate. So, it actually has advantages.\n“clogged 32 carriers exhibit increased buoyant density, demanding higher energy inputs for mixing and 33 suspension”\nNope, it is right the opposite: biofilm density decreases with increased thickness, and not the other way around. In packed-\nbed filters, back-washing is used to remove/slough off excess biofilm biomass, which is accumulating in the filter – not in\nMBBRs. MBBRs had a filter media filling of around 40%, and no such behaviour can be observed.\n“no existing MBBR carrier can reliably control biofilm thickness under the dynamic 40 hydraulic conditions” Erroneous\nidentification of knowledge gap in literature: Kruger-VEOLIA’s zeta-carrier even comes with different mesh sizes that can\ncontrol biofilm thickness between 50-500 um.\n“AnoxKaldnes introduced a carrier (the 'Z-carrier') based on a 42 geometric premise that its grid height would regulate\nbiofilm thickness13.” And it does it very effectively, as opposed to what authors claim here. Importantly, Reference 13 has\nreported the innovation of the zeta-carrier using long-term experimentation, in which no such shortcomings were observed.\nIn fact, authors of reference 13 also reported (Torresi et al., 2017, http://dx.doi.org/10.1016/j.watres.2017.06.027) on the\nsystematic behaviour of the zeta-carrier in terms of in-biofilm diffusivity and sorption-desorption, which would not have been\npossible should the zeta-carrier allowed for irregular growth of biofilm thickness.\n“However, their own 43 long-term evaluation revealed a critical flaw: biofilms at the center of the grid cells became 44\nsignificantly thinner or were entirely absent21. This outcome underscores a fundamental stalemate 45 in sustainable carrier\ndesign, revealing a poor understanding of the hydrodynamic mechanisms that 46 control biofilm development. The failure of\nthis simplistic geometric approach highlights the 47 necessity for a design paradigm rooted in hydraulic principles.” This\nseems to be a seriously flawed series of statement intended to undermine confidence in the state-of-the-art for the benefit of\nopening a knowledge gap on false premises.\n1. It greatly misinterprets what has been reported in literature. Ref. 21 states “…had a clearly defined patch in the middle of\neach cell where the biofilm was significantly thinner, as a result of the carriers scraping each other.... Since the grid\ncompartments were full of biomass up to the pre-determined wall heights, and calculations suggest that the maximum\npossible scraping depth from one Z-carrier into another Z-carrier compartment is 90 µm, the biofilm thickness on the four\ncarrier types was determined to vary between 410–500, 310–400, 210–300 and 110–200 µm, respectively.”\n2. Carriers scraping each other is not a sign of poor understanding of hydraulic behaviour.\n3. It does not indicate that these carriers would “clog” in the way authors seem to mean it.\n“The failure of this simplistic geometric approach … The propensity for clogging in conventional tubular carriers” Co-\nauthors’s may have limited understanding of the system they criticise. The zeta-carrier is NOT a tubular carrier but a three-\ndimensional wave-shaped flat surface covered with a mesh with a defined thickness.\n“system exhibited high nutrient removal rates 120 of 0.138 ± 0.022 g N g VSS⁻¹ day⁻¹ for ammonia” This observation is not\nbenchmarked against literature evidence, and does not seem to be correct. For instance, reference 13 observed much\nhigher removal rates using the zeta carrier of 50 um, i.e., around 0.7 gN/(d.gVSS), and only thicker biofilms, i.e., above 200\num showed similar but mostly higher values than that reported here.\n“131 biofilm regulation—Hydro-topological synergy” “synergy” - a possible typo. Also, authors fail to thoroughly explain the\nmeaning of the term they introduced.", "reviewer_2": "In this manuscript, the authors reported a new geometrical design for biofilm carriers used in moving bed biofilm reactor (MBBR). In contrast to the closed circular design, the authors used an open V-grove design, which was shown to lead to superior performance compared to the conventional design using various standard MBBR characterization. Microscopic analysis showed an open morphology of biofilm with holes, which the author attributed to cell lysis and contribute to biofilm self-renewal. Finally, metagenomics analysis was presented regarding the species distribution in such carrier microbial communities.\nOverall, the research is performed with high quality, and the analysis is very solid. The presented technology has a strong\npotential to improve the existing technology. I support the publication of the manuscript in Nat. Comm. My only major\nconcern is the lack of evidence of cell lysis, which is critical in the authors’ picture. Can the authors perform live-dead\nstaining to definitely prove the existence of such process in the interior of the biofilm? Also, the author can do a better job in\nexplaining some of the jargons for a broader audience. For example, in legend for Figure 3, what does VSS/SS ratio mean\nfor the performance of the device?", "reviewer_3": "The manuscript presents a novel V-carrier design and provides a thorough scientific evaluation of its performance in\nwastewater treatment. The study offers valuable insights into biofilm self-regulation and demonstrates the potential\nadvantages of the hydro-topological strategy. However, the manuscript would benefit from significant reorganization. The\noverall structure and flow of ideas need improvement to enhance clarity and readability, as some sections currently lack\nlogical sequencing despite the compelling presentation of results. Given these issues, I recommend major modifications\nbefore the manuscript can be considered for publication. Addressing the organization and ensuring a clearer narrative will\ngreatly strengthen the impact of the work.\nHERE ARE THE COMMENTS:\nIntroduction section\n1. While the manuscript states that the MBBR process aligns with SDGs 6, 11, and 12, this section does not clearly discuss\nhow MBBR operational characteristics contribute to these sustainability principles. The authors are encouraged to elaborate\non the specific mechanisms through which MBBR supports these SDGs—for example, by detailing its impacts on resource\nefficiency, energy use, sludge reduction, land footprint, or resilience of sanitation systems. Providing this clarification would\nstrengthen the justification of MBBR as a sustainable technology and improve the overall coherence of the sustainability\nnarrative.\n2. While the authors correctly identify clogging and uncontrolled biofilm accumulation as major limitations of conventional\nMBBR carriers, the manuscript does not provide any discussion of previous carrier designs reported in the literature or how\nthose studies attempted to mitigate clogging. A concise yet critical overview of earlier design approaches—such as modified\ngeometries, increased opening ratios, structured internal channels, or surface texturing—and an evaluation of their\neffectiveness in reducing fouling or improving hydrodynamics is essential. Furthermore, the manuscript should clarify the\nresidual shortcomings of these designs that directly motivated the development of the new carrier proposed in this study.\nAddressing these points will strengthen the rationale for the new design and better situate the contribution of the current\nwork within the context of prior research.\n3. The manuscript could better contextualize the rationale for the current study by discussing previous carrier design\nstrategies. Specifically, prior studies—including those employing CFD simulations—have proposed geometric or structural\nmodifications to improve biofilm distribution, mass transfer, and reduce clogging. While such studies provide valuable\nindications and guidance for experimental design, they often lack long-term operational evaluation. Highlighting both the\ninsights and the limitations of these previous approaches would strengthen the justification for developing and testing the\nnew V-carrier in a long-term experimental setting.\n4. The manuscript discusses the long-standing issue of clogging in MBBR systems but provides only a single example (the\nZ-carrier) as evidence of previous design limitations. This narrow focus unintentionally suggests that, over four decades of\ncarrier development, no other design strategies or mitigation efforts have been explored. The authors are encouraged to\nexpand this section by briefly summarizing additional carrier design attempts reported in previous studies and discussing\ntheir partial successes or remaining limitations. This broader context is essential to accurately position the Z-carrier as one\nexample rather than the sole historical effort, and to more convincingly justify the need for the new design proposed in this\nwork.\nMethods Section:\n1. Regarding the choice of using a square-shaped media instead of a cylindrical one, it would be useful to discuss the\npractical implications of this design choice. While the square shape may offer advantages in terms of maximizing surface\narea and potentially improving biofilm attachment, it is important to evaluate whether this design is as effective in real-world\napplications. Specifically, how does the square shape perform in terms of hydrodynamic behavior, ease of movement within\nthe reactor, and resistance to clogging compared to cylindrical carriers? Further clarification and comparative analysis with\ncylindrical media would help assess its practical viability in large-scale wastewater treatment systems.\n2. The authors diluted raw municipal wastewater with tap water but did not specify the dilution ratio. Additionally, residual\nchlorine and other ions in tap water may influence microbial activity, particularly nitrifiers and denitrifies, potentially affecting\nthe generalizability of the results to real WWTP conditions.\n3. The manuscript reports adding 50 L of activated sludge twice daily during biofilm establishment. While this approach can\naccelerate biofilm colonization, the authors should clarify the rationale for the chosen frequency and volume, and discuss\nwhether such repeated dosing could influence nutrient concentrations or microbial community development compared to\ntypical operational conditions.\n4. The manuscript describes the biofilm establishment protocol but does not clearly explain the rationale behind it. The\nauthors should specify the principle guiding the chosen procedure—why activated sludge was added twice daily, how this\npromotes initial biofilm attachment, and the intended objective of this approach (e.g., accelerating colonization, ensuring\nuniform coverage, or stabilizing microbial communities). Providing this explanation would clarify the purpose of the protocol\nand strengthen the methodological section.\nResults section\n1. The manuscript reports a porosity value of 92% for the V-carrier; however, this statement is scientifically ambiguous and\nrequires clarification. Because the carrier is fabricated from HDPE—a fully non-porous material—the reported porosity\ncannot correspond to material porosity. If the authors intend to describe structural or geometric porosity (i.e., the void volume\nfraction within the carrier’s overall envelope), this must be explicitly stated. Moreover, the manuscript should clearly present\nthe calculation method, including the definition of the total reference volume, how the void volume was determined (e.g.,\nCAD-based volume analysis), and the exact formula used. Without a proper definition and methodological explanation, the\nreported porosity value is misleading and cannot be accurately interpreted in relation to the carrier’s hydrodynamics or\nmass-transfer performance. The authors are encouraged to revise this section accordingly.\n2. The manuscript claims that an opening ratio (length/depth) of 1.39 “optimizes hydraulic exposure,” but no reference or\nsupporting data are provided. The authors should clarify whether this criterion is based on literature, modeling, or\nexperimental results, or remove the unsubstantiated claim.\n3. The manuscript reports maintaining a C/N ratio of 4.9 ± 0.3 by supplementing sodium acetate to ensure sufficient electron\ndonors for denitrification. The authors should clarify whether this supplementation reflects typical operational conditions for\nmunicipal wastewater treatment or was solely applied to optimize denitrification. Furthermore, a reference to prior studies\nsupporting this C/N range should be provided. The potential impact of acetate addition on microbial community structure and\nprocess performance should also be briefly discussed, as it may affect the generalizability of the results to real-world\nWWTPs.\n4. The manuscript reports phosphorus removal efficiency up to 52% under fully aerobic biofilm conditions. This value\nappears high given that conventional enhanced biological phosphorus removal typically requires alternating anaerobic and\naerobic phases. The authors should clarify whether the V-carrier design created localized micro-anoxic or anaerobic zones\nwithin the biofilm or provide an alternative explanation for the observed phosphorus removal. This clarification is important to\naccurately interpret the results and the functional contribution of the carrier design.\n5. The manuscript attributes the decline in ammonium removal rate (ARR) on the Mutag-biochip to biofilm accumulation and\ninorganic buildup. However, it is unclear how biofilm accumulation was directly measured or confirmed. The authors should\nclarify whether this conclusion is based on biofilm thickness measurements over time, microscopy observations,\nVSS/SS ratio tracking, or solely on ash content. Providing explicit evidence would strengthen the link between biofilm\naccumulation and the observed decrease in ARR.", "reviewer_4": "This manuscript presents an innovative “hydro-topological” design strategy for MBBR carriers, exemplified by the V-carrier,\nwhich enables self-regulation of biofilm thickness and continuous self-cleaning. The study demonstrates superior pollutant\nremoval performance and stability over more than 500 days of operation with real wastewater, including under low-\ntemperature conditions. The experimental design is rigorous, with comprehensive data on performance, mechanisms, and\nmicrobial ecology. The integration of hydraulic principles with biofilm dynamics represents a meaningful contribution to the\nfield of wastewater treatment technologies. However, several aspects require clarification and enhancement to strengthen\nthe manuscript’s scientific rigor and clarity. The work is promising but would benefit from revisions to address the points\nbelow.\n1. The “hydro-topological” strategy integrates carrier geometry, hydraulic shear, and microbial ecology into a unified\nframework. Consider expanding the explanation of its physical and biological principles in the discussion section. A\nsimplified diagram or model could be added to illustrate relationships, such as those between opening ratio, groove depth,\nand shear forces.\n2. The inclusion of over 500 days of real wastewater treatment data is noted. However, direct measurements of hydraulic\nshear forces are lacking, with reliance on inferences from the Hagen-Poiseuille equation and carrier structure. Are there any\nmethods such as computational fluid dynamics simulations or experimental measurements could provide quantitative data\non carrier surface shear forces?\n3. The explanation for variability in phosphorus removal performance (lines 125-126) is brief. A discussion could address\nwhether this reflects inherent limitations of pure biofilm systems and if additional chemical phosphorus removal measures\nmight be relevant in practical applications.\n4. The claim in lines 15-16 that treatment efficiency decouples from biomass quantity may refer to the unit biomass\nnitrification rate comparison between the V-carrier and K3 carrier. Additionally, is it possible that the observation in lines\n186-189 regarding spatial redistribution of nitrifying bacteria under low temperatures could potentially relate to changes in\ndissolved oxygen gradients?\n5. Precise geometric parameters for the V-carrier are provided (depth 0.72 mm, opening 1.0 mm, opening ratio 1.39). Clarify\nhow this specific opening ratio was determined? Please provide a brief description in the methods or results section.\n6. Ensure consistent terminology throughout, such as “V-carrier” and “hydro-topological strategy.”\n7. The supplier details for carriers (beyond the V-type) and catalog numbers for reagent kits are omitted in methods section.\n8. Engineering parameters, such as effective biofilm surface area and bulk density, are reported and useful for applications.\nThe discussion could briefly address potential challenges in scaling to pilot or full-scale operations, including large-scale\nproduction costs and long-term mechanical wear.\n9. The finding in lines 175-177 that ex situ nitrification potential exceeds in situ activity is attributed to substrate limitations.\nThe authors could consider discussing additional possible factors.\n10. The V-carrier maintains a stable biofilm thickness of 0.72 mm with reported performance benefits. The discussion could\ninclude an explanation of why approximately 0.72 mm falls within a theoretical optimal range, potentially referencing\nliterature on substrate penetration depths (e.g., oxygen and ammonia) to support how this thickness balances active layer\nmaximization and mass transfer resistance minimization.\n11. Over 500 days operation, indicate whether any physical wear, deformation, or gradual performance decline was\nobserved in the carriers."}, "author_response": "Response to Comments\nWe thank the editor and the reviewers for their thoughtful and constructive comments on\nour manuscript (Manuscript ID: NCOMMS-25-91216), which have significantly helped\nus improve the quality of this work. We have carefully considered all points raised and\nhave revised the manuscript and supplementary materials accordingly.\nBelow, we provide detailed point-by-point responses. Our responses are presented in blue,\nand revised manuscript or supplementary material text is shown in red. In the revised\nmain manuscript and supplementary files, all changes are highlighted in red font for ease\nof review.\nEDITOR COMMENTS\nAs you will see from the reports copied below, the reviewers raise important concerns. We find\nthat these concerns limit the strength of the study, and therefore we ask you to address them\nwith additional work. Without substantial revisions, we will be unlikely to send the paper back\nto review. We are also convinced that providing robust responses to robust responses to all the reviewers’ concerns\ncould significantly improve the manuscript, but specifically we suggest you to provide proper\nrestructuring and enhance the clarity of the manuscript, as suggested by all 4 reviewers.\nMoreover, inclusion of CFD supporting biofilm temporal evolution as a function of fluid\ndynamic clues and presentation of proper performance benchmarking vs. all the published\ncarriers are envisioned to enhance the work quality.\nResponse:\nWe sincerely thank you for the opportunity to revise our manuscript and for the constructive\nfeedback from the reviewers. We have carefully addressed all the concerns raised, making\nsubstantial revisions to the manuscript and providing robust responses to each point, as detailed\nin the point-by-point replies below.\nIn particular, we have taken your specific suggestions to heart and performed the following\nmajor enhancements:\n1. Inclusion of CFD Analysis Supporting Biofilm Evolution: In direct response to your\nsuggestion to include CFD analysis that links fluid dynamic clues to biofilm temporal\nevolution, we have conducted comprehensive CFD simulations. These new results are\nnow presented in Fig. 6 (see Fig. R1) of the revised manuscript. The simulations vividly\nillustrate the hydrodynamic mechanisms behind the V-carrier's effective self-cleaning\ncapability and, crucially, provide a quantitative comparison of flow fields and shear\nstresses across different carrier geometries, directly linking hydraulic conditions to\nbiofilm development patterns.\n2. Comprehensive Performance Benchmarking: As you recommended, we have\nsignificantly strengthened the presentation of proper performance benchmarking\nagainst published carrier designs. The revised discussion now includes a systematic\ncomparison of our V-carrier not only with the carriers tested in this study but also places\nits performance (e.g., nitrification rates, volumetric removal rates) squarely within the\ncontext of the broader literature on advanced biofilm carriers. This provides a clear and\ncompelling benchmark for its efficiency and potential.\n3. Manuscript Restructuring and Enhanced Clarity: As suggested by all reviewers and\nemphasized by you, we have thoroughly restructured the manuscript to improve its\nlogical flow and clarity. This includes refining the Introduction to better frame the\nresearch gap, streamlining the results narrative to focus on key comparisons, and\nsignificantly expanding the discussion to articulate the novel \"hydro-topological\"\ndesign paradigm and its implications more clearly.\nWe believe that these substantial revisions have fully addressed the reviewers' concerns and,\nfollowing your guidance, have significantly enhanced the quality, clarity, and impact of our\nwork. The revised manuscript now presents a more robust, comprehensive, and clearly\narticulated study. We are grateful for the feedback that has helped us achieve this improvement\nand hope that the changes meet with your approval.\nFig. R1 (Fig. 6) | CFD-simulated flow fields and biofilm morphology of carriers after 405\ndays of operation. a–b, Flow fields around a V-carrier with an opening ratio of 1.39 (1.0/0.72)\nunder (a) parallel and (b) 45° flow incidence. c, Biofilm morphology on the stably-operated V-\ncarrier with opening ratio 1.39. d–e, Flow fields around a V-carrier with an opening ratio of\n2.08 (1.5/0.72) under (d) parallel and (e) 45° flow incidence. f, Biofilm morphology on the\nstably-operated V-carrier with opening ratio 2.08. g–h, Flow fields (parallel flow) around\ncarriers with cell dimensions of 2.3 × 2.3 mm and ridge heights of (g) 1.0 mm and (h) 0.5 mm.\ni, Biofilm morphology on the MK5 carrier. j–k, (j) Flow field (parallel flow) and (k) biofilm\nmorphology of the U-carrier. l–m, (l) Flow field (parallel flow) and (m) biofilm morphology of\nthe K3 carrier. n–o, (n) Flow field (parallel flow) and (o) biofilm morphology of the K5 carrier.\nREVIEWER COMMENTS\nReviewer #1:\nThe manuscript’s main focal area entails biofilm technology development for water resource\nrecovery, including two key notions; the meaning of which needed to be defined by co-authors\nin the ensuing text, i.e.,\nHydro-topological strategy: they define it as a reactor “design strategy”, which seems to\nindicate the V-shape of the developed and tested carrier media.\nSelf-regulating biofilm: they describe it as the hydraulic behavior of the new carrier design is\nintended to serve the purpose of more efficient biofilm sloughing from the media.\n- What are the noteworthy results? ANSWER: A new type of biofilm carrier was tested, however,\nthe advantages of this carrier – over state-of-the-art technology, e.g., zeta-carrier – were not\nsufficiently supported by new evidence. In fact, there is not even a photograph of the carrier\nshown only seemingly computer-generated images.\n- Will the work be of significance to the field and related fields? ANSWER: As supported my\nbelow detailed evaluation and assess, my answer is NO, it will not.\n- How does it compare to the established literature? If the work is not original, please provide\nrelevant references. ANSWER: the manuscript establishes knowledge gaps based on the\npremises of erroneous understanding/interpretation of literature.\n- Does the work support the conclusions and claims, or is additional evidence needed?\nANSWER: I could not find any separate Conclusions as such. Meanwhile, some of the\nstatements based on results do not support the e.g., high ammonia removal rate – benchmarked\nagainst literature data.\n- Are there any flaws in the data analysis, interpretation and conclusions? - Do these prohibit\npublication or require revision? ANSWER: I am unsure about the data analysis. To me it is\nmostly down to the convenient way of interpreting literature in a way suitable for their\nobjectives.\n- Is the methodology sound? Does the work meet the expected standards in your field?\nANSWER: It is partly sound. The main issue is the lack of benchmarking against the state-of-\nthe-art technology and the lack of clear demonstration of novelty and benefits of the new\ntechnology.\n- Is there enough detail provided in the methods for the work to be reproduced? ANSWER:\nclearly not –benchmark reference data is missing.\nResponse:\nWe sincerely thank you for your thorough evaluation and for the challenging questions that\nhave prompted us to substantially strengthen the clarity, evidence, and logical foundation of our\nmanuscript. We have undertaken a major revision to address your overarching concern\nregarding the novelty, benchmarking, and mechanistic evidence for our hydro-topological\nstrategy.\n1. Directly Addressing the Core Concerns: Novelty and Benchmarking\nYour primary critique was that the advantages of the V-carrier over state-of-the-art technology\nwere not sufficiently supported. We have addressed this in three key ways:\n• Providing Direct Visual Evidence: You noted the lack of a photograph. We have now\nincluded high-resolution images of both clean and biofilm-attached V-carriers after\nlong-term operation in the supplementary materials (Supplementary Fig. 12; see Fig.\nR2), providing clear visual documentation of the carrier and its performance.\n• Clarifying the Benchmarking Strategy: Our goal was not a simple side-by-side\ncomparison but a fundamental test of the prevailing design paradigm of geometric\nconfinement. We engineered the U-carrier and MK5 carrier as critical test cases to\ndemonstrate that passive geometric features alone are insufficient for reliable biofilm\ncontrol under dynamic conditions. Their failure (Fig. 3) underscores the limitation of\nthe paradigm exemplified by the Z-carrier. In contrast, our hydro-topological strategy\nactively leverages hydraulic shear for continuous self-cleaning. The superior and stable\nperformance of the V-carrier, validated over 500 days, demonstrates the success of this\nnew paradigm.\n• Enhanced Mechanistic Evidence with CFD: As you suggested, we have conducted\ncomprehensive Computational Fluid Dynamics (CFD) simulations to move beyond\ninference. The new Fig. 6 provides quantitative, visual evidence of the flow fields and\nshear stresses, directly linking the V-carrier's geometry to its hydrodynamic efficiency\nand self-cleaning mechanism, and offering a rigorous comparison with conventional\ncarriers.\nIn summary, we acknowledge the contribution of the Z-carrier in mitigating clogging through\nits geometric design. However, our work reveals a more fundamental paradigm shift in carrier\ndesign: passive geometric confinement versus active hydraulic regulation. The Z-carrier's\napproach sets a theoretical maximum thickness but fails to ensure its uniformity and stability\n(as evidenced by the thinning patches). In contrast, our hydro-topological strategy, by\ntopologically guiding biofilm growth into the hydraulic flow, achieves autonomous, continuous,\nand uniform control over biofilm thickness through sustained shear. This not only resolves the\nclogging issue but, more importantly, enables a dynamic self-balancing of the biofilm\necosystem. This represents a progressive direction for the next generation of biofilm carrier\ndesign.\n2. Substantive Revisions to Specific Contentious Points\nWe have carefully revised the manuscript to address the potential misinterpretations you\nhighlighted:\n• Clogging and Surface Area (Comment #2): We have restructured the introduction to\nclarify that the reduction in \"effective surface area\" refers to the physical blockage of\na carrier's internal spaces due to clogging and scaling, not to the intrinsic properties of\na thick biofilm. We have added a citation with photographic evidence of severely\nclogged K1 carriers from the literature to support this claim.\n• Carrier Buoyancy (Comment #3): We have clarified that the increased buoyant density\nof clogged carriers is due to the accumulation of dense inorganic precipitates (scaling),\nnot the biofilm itself, and have referenced our data on ash content (Supplementary\nTable 1).\n• The Z-Carrier and the Knowledge Gap (Comments #4, #5, #6): We stand by our\ninterpretation of the Z-carrier literature. The long-term study we cite (Ref. 25 in the\nrevised manuscript) empirically documents non-uniform biofilm thickness, which\ndemonstrates a failure of reliable control. We argue that the authors' attribution of this\nto \"scraping\" is an unverified hypothesis, and our CFD data suggest a hydrodynamic\ncause. This observed non-uniformity is the specific limitation our hydro-topological\nstrategy is designed to overcome by ensuring uniform hydraulic exposure.\n• Nitrification Rate Benchmarking (Comment #7): We have clarified that a direct\ncomparison of specific rates (per gVSS) with studies using much thinner biofilms is\ninvalid, as biofilm thickness is a primary determinant of this metric. We now provide a\nmore meaningful comparison at similar thicknesses and emphasize that our carrier's\ninnovation lies in maintaining stable, high performance at an optimal thickness under\nreal-world conditions, not in achieving an arbitrary maximum rate.\n3. Overall Impact on the Manuscript\nYour rigorous critique was instrumental in driving these significant improvements. The revised\nmanuscript now:\n• Presents robust visual and quantitative evidence (CFD, pilot-scale results) to support\nits claims.\n• Articulates a clearer and more compelling narrative regarding the novelty of the hydro-\ntopological strategy over geometric confinement.\n• Uses more precise and defensible language throughout, eliminating ambiguity.\nWe believe the manuscript is now substantially stronger and fully addresses your concerns.\nDetailed point-by-point replies to your specific comments are provided below. We deeply\nappreciate your constructive critique, which has significantly improved this work.\nFig. R2 (Supplementary Fig. 12) | Pilot plant of the anoxic/aerobic-moving bed biofilm\nreactor (A/O-MBBR) process and overall morphology of V-carriers after long-term\noperation. a, Installation site and overall view of the pilot plant. b-c, Field views of the anoxic\n(b) and aerobic (c) reactors, respectively. d, Carriers during complete fluidization. e, Clean and\ndried V-carriers. f-g, Morphology of biofilm-attached anoxic (f) and aerobic (g) carriers after\n409 days of operation. h-i, Morphology of biofilm-detached anoxic (h) and aerobic (i) carriers\nafter 409 days of operation. The pilot system was commissioned on November 12, 2024, and\nremains operational. It has a total working volume of 24 m³, comprising a 9 m³ anoxic reactor\nand a 15 m³ aerobic reactor. It operates in a pure biofilm mode (no sludge recirculation) with a\n300% internal nitrified liquor recycle ratio. The system treats municipal wastewater with\ninfluent characteristics similar to those used in this study (see Supplementary Table 3 for\nconcentrations). Operating at a 9-hour hydraulic retention time, it achieves nitrification and\ndenitrification rates comparable to the laboratory-scale results. No carrier clogging has been\nobserved, and the operational stability of the system has been fully validated.\nMore detailed critical comments:\n“inevitably clog. This clogging severely diminishes treatment efficiency” Very ambiguous and\nseemingly incorrect statement – right in the problem statement of the abstract. Do you mean\nbiofilm sloughing and removal of sloughed off biofilm?\nResponse:\nWe thank you for this insightful comment, which has allowed us to clarify an important point.\nThe phrase \"diminishes treatment efficiency\" refers specifically to the overall performance of\nthe MBBR system.\nCarrier clogging leads to a reduction in the effective surface area available for biofilm growth,\nimpairs mass transfer of substrates and oxygen, and can increase energy consumption for\nmixing and suspension. Collectively, these factors diminish the system's macro-scale treatment\nefficiency, resulting in poorer effluent quality (e.g., higher effluent nutrient concentrations) or\nrequiring a longer hydraulic retention time.\nYou correctly infer the connection to biofilm sloughing. The impaired hydraulic conditions\nwithin a clogged carrier indeed disrupt the natural balance between biofilm growth and\ndetachment (sloughing), preventing effective self-cleaning. Our hydro-topological strategy is\ndesigned precisely to restore this balance by leveraging controlled hydraulic shear, thereby\nenabling continuous self-regulation (sloughing) of the biofilm to maintain optimal system-level\ntreatment efficiency. We have revised the text in the abstract (Lines 7-8) to clarify this point,\nwhich now reads:\n\"This clogging severely diminishes the overall treatment efficiency of the MBBR system\".\n“uncontrolled 29 biofilm accumulation. This results in severe carrier clogging and scaling,\ndrastically diminishing the 30 effective surface area, impairing mass transfer and disrupting\nmicrobial community structure” Unclear and seemingly erroneous set of statements. Excessive\ngrowth on e.g., K1 carrier does NOT reduce the surface area. In fact the higher the biofilm\nthickness the lower the biofilm density, which effectively increases the effective diffusivity, i.e.,\nf*D_water. Also, nitrification rate is not expected to change much, only if normalised to the\ntotal gVSS on the carrier. Example: at 50 um: 2 gN/(d.gTSS); at 200 um: 0.6 gN/(d.gTSS).\nThere is no such thing as “disrupting community structure”. Biofilm thickness has an impact\non the microbial community structure, and higher thickness can select in microorganisms that\ne.g., harbour niche organisms with high micropollutant biotransformation rate. So, it actually\nhas advantages.\nResponse:\nWe thank you for this critical comment, which allows us to clarify the distinction between two\nimportant concepts: 1) the intrinsic effects of biofilm thickness on microbial ecology and\nphysiology, and 2) the detrimental consequences of carrier clogging due to uncontrolled biofilm\naccumulation. Our statements specifically refer to the latter scenario.\n1. Clarification on Surface Area Reduction and Mass Transfer Impairment\nYou correctly note that an increase in biofilm thickness itself does not reduce the physical\nsurface area of a clean carrier. However, our point concerns the situation of carrier clogging. To\npreempt potential misunderstandings and enhance the clarity of our narrative, we have\nrestructured the introduction to provide a critical overview of this issue. The revised text (Lines\n38-41) now explicitly states:\n“The design focus on maximizing protected surface area has promoted uncontrolled biofilm\ngrowth, with carrier clogging even misinterpreted as a positive sign12,13, thereby diverting\nattention from the need for biofilm managed growth. This fundamental misdirection manifests\nas the core limitations of conventional MBBR systems: severe carrier clogging and scaling,\ndrastically diminishing the effective surface area, impairing mass transfer, and disrupting\nmicrobial community structure by fostering competition for oxygen and substrates among\nunfavorable species14-16”\nThis revision clarifies that the drastic reduction in effective surface area is a direct consequence\nof clogging and scaling, not of the biofilm thickness per se. In a clogged carrier, biofilm\novergrowth physically blocks the internal spaces (e.g., channels or hollows), rendering a\nsignificant portion of the protected surface area inaccessible for biofilm establishment and mass\ntransfer. This phenomenon, while often overlooked, is visually evident in the literature. For\ninstance, a study investigating the effect of organic loading rates on different media presents an\nimage that clearly shows a severely clogged K1 carrier (Fig. R3; Figure S4 and Figure S7 in\nthat publication)1. This visual evidence directly supports our assertion that uncontrolled\naccumulation can drastically reduce the effective surface area. We have added this reference to\nthe manuscript (Ref. 12 in the revised manuscript) to support our claim.\nRegarding mass transfer, we agree that a thicker, less dense biofilm can exhibit higher effective\ndiffusivity (f×D_water). However, in a clogged carrier, the primary issue is not diffusivity\nwithin the biofilm matrix but the severely restricted convective flow through the carrier's\ninternal structure. This restriction creates stagnant zones where substrates are depleted and\nmetabolites accumulate, leading to an overall impairment of mass transfer at the carrier scale,\nwhich is fundamentally distinct from the intrinsic diffusivity at the biofilm scale.\n2. Clarification on Nitrification Rate Normalization\nWe appreciate your point about the specific nitrification rate (per gVSS) decreasing with\nbiofilm thickness due to diffusion limitations. Our argument is fully consistent with this\nfundamental principle. We normalized the nitrification rate to the total biofilm VSS precisely\nto reveal this intrinsic mass transfer limitation. As shown in our Fig. 3, carriers prone to\nclogging (e.g., K5, Mutagbiochip) accumulated substantial biomass but exhibited low specific\nnitrification rates. This observation directly supports our statement that clogging impairs mass\ntransfer, as the large amount of biomass becomes physiologically inefficient. The volumetric\nremoval rate is indeed a critical performance metric for reactors, which our system also\nachieved at a high level (as discussed in the manuscript). The use of specific rate here was\nintentional to decouple and highlight the mass transfer efficiency of the biofilm ecosystem itself.\n3. Clarification on Microbial Community Structure\nWe agree that biofilm thickness is a key determinant of microbial community structure and can\nfoster beneficial niche stratification, such as for micropollutant removal2. This is a well-\nestablished ecological principle. However, our statement refers specifically to the community\ndisruption caused by carrier clogging. The extreme conditions within a clogged carrier—\ncharacterized by severe substrate and oxygen gradients, metabolite accumulation, and potential\nfor cell lysis—can lead to the dominance of non-productive or fermentative organisms, thereby\n\"disrupting\" the functional microbial structure optimal for nutrient removal. This is different\nfrom the structured, stratified community that develops in a well-controlled, thick biofilm,\nwhich is an optimal community structure indicative of a healthy ecosystem.\nWe believe these clarifications directly address your concerns. Thank you again for your\ncomments, which have been instrumental in helping us improve the clarity of our arguments.\nFig. R3 | Severe clogging of a K1 carrier, depicted in Supplementary Fig. S4 and S7 of Ref.\n12.\nReference:\n1. Bassin, J. P. et al. Effect of increasing organic loading rates on the performance of moving-\nbed biofilm reactors filled with different support media: Assessing the activity of\nsuspended and attached biomass fractions. Process Saf. Environ. Protect. 100, 131-141\n(2016).\n2. Torresi, E. et al. Biofilm Thickness Influences Biodiversity in Nitrifying MBBRs—\nImplications on Micropollutant Removal. Environ. Sci. Technol. 50, 9279-9288 (2016).\n“clogged 32 carriers exhibit increased buoyant density, demanding higher energy inputs for\nmixing and 33 suspension” Nope, it is right the opposite: biofilm density decreases with\nincreased thickness, and not the other way around. In packed-bed filters, back-washing is used\nto remove/slough off excess biofilm biomass, which is accumulating in the filter – not in\nMBBRs. MBBRs had a filter media filling of around 40%, and no such behaviour can be\nobserved.\nResponse:\nWe thank you for this comment, which provides an opportunity to clarify two key points\nregarding the physical properties of clogged carriers and the intended analogy to fixed-bed\nfilters.\n1. Clarification on the Buoyant Density of Clogged Carriers\nYou are correct that the density of a biofilm itself typically decreases as its thickness increases\ndue to a higher water content and a more porous structure. However, our statement specifically\naddresses the overall buoyant density of the entire carrier unit when it becomes clogged.\nThe key mechanism leading to increased buoyant density is not the biofilm's intrinsic density,\nbut the significant accumulation of inorganic precipitates (scaling) within the clogged biofilm\nmatrix. In a clogged carrier, the stagnant hydraulic conditions promote the precipitation of\nminerals, such as phosphates. As we show in Supplementary Table 1, carriers prone to clogging\n(e.g., K5, Mutagbiochip) accumulated substantial inorganic content (up to 56% ash content),\nwhich has a much higher density than water or organic biomass. This incorporation of dense\ninorganic material into the biofilm-carrier composite is the primary reason for the observed\nincrease in overall buoyant density, which in turn demands higher energy inputs for mixing and\nsuspension.\n2. Clarification on the Fixed-Bed Filter Analogy\nWe agree that fixed-bed filters (or packed-bed filters) and MBBRs are distinct technologies\nwith different operational modes (e.g., fixed vs. suspended media, backwashing vs. continuous\nshear). Our intention was not to equate them but to use the well-established principle of\nbackwashing in fixed-bed filters as a clear example to illustrate the critical role of transient,\nhigh hydraulic shear in controlling biofilm thickness.\nThe sentence in question reads: \"This principle is exemplified by fixed-bed filters...\". The\n\"principle\" we refer to is the one stated immediately before: that clogging results from an\nimbalance between biofilm growth and detachment due to insufficient hydraulic shear. Fixed-\nbed filters actively manage this balance through backwashing. We then contrast this with the\npassive, continuous shear environment of an MBBR to highlight the design challenge and\nintroduce our solution.\nWe believe these clarifications directly address your concerns. Thank you again for helping us\nimprove the precision of our manuscript.\n“no existing MBBR carrier can reliably control biofilm thickness under the dynamic 40\nhydraulic conditions” Erroneous identification of knowledge gap in literature: Kruger-\nVEOLIA’s zeta-carrier even comes with different mesh sizes that can control biofilm thickness\nbetween 50-500 um.\nResponse:\nWe thank you for this critical comment, which allows us to substantiate the identified\nknowledge gap regarding reliable biofilm thickness control. We respectfully disagree that the\nZ-carrier invalidates this gap, as the long-term performance data from its own developers\nconfirms its inability to maintain uniform biofilm thickness, which aligns with our central\nargument.\nOur statement that \"no existing MBBR carrier can reliably control biofilm thickness under\ndynamic hydraulic conditions\" is supported by the very reference we cite (originally Ref. 21,\nnow Ref. 25 in the revised manuscript), which is the foundational study on the Z-carrier. While\nthe Z-carrier's geometric design aims to limit maximum thickness, the study reveals a critical\nfailure in reliable control—defined as the ability to maintain uniformity under dynamic\nconditions: \"After running the reactors for over 300 days... carriers from reactor D (Z-200) had\na clearly defined patch in the middle of each cell where the biofilm was significantly thinner\"\n(Fig. R4; Figure 4 in originally Ref. 21, now Ref. 25). This observation directly demonstrates\na lack of uniform control.\nThe authors of this study (originally Ref. 21, now Ref. 25) attribute this thinning to \"carriers\nscraping each other.\" However, this mechanism remains a hypothesis and was not\nexperimentally verified. Our analysis challenges this explanation based on two lines of\nevidence:\n1. Computational Fluid Dynamics (CFD) Analysis: We conducted CFD simulations (Fig.\nR5; included in the revised manuscript as Fig. 6g-h) which quantitatively demonstrate\nthat the central thinning in grid-like structures is a direct result of localized hydraulic\nconditions, not physical scraping. The simulations revealed a 56–91% higher hydraulic\nshear stress in the center of the grid cell compared to the area near the ridges. This\nsignificant mechanical force imbalance directly explains the observed failure to\nmaintain a stable target biofilm thickness, as the elevated shear stress in the central\nregion preferentially removes biomass, leading to the characteristic patchy thinning.\nThis hydrodynamic mechanism provides a more fundamental explanation for the\nphenomenon than the previously hypothesized scraping mechanism.\n2. Controlled Experimental Comparison: If inter-carrier scraping were the dominant\nmechanism, our V-carrier—with its sharp, square-sheet morphology—should\nexperience far more severe scraping and unpredictable biofilm loss than the rounded\nZ-carrier. Contrary to this expectation, our V-carrier maintained a stable, uniform\nbiofilm thickness over 500 days (Fig. 3a, b). This critical observation suggests that the\nhydro-topologically guided exposure to hydraulic shear, not the avoidance of scraping,\nis the key to stable thickness control.\nTherefore, the Z-carrier's failure to prevent non-uniform biofilm distribution under long-term\noperation, as documented in its own evaluation, precisely validates the knowledge gap we\nidentified. The carrier's geometric constraint sets a theoretical maximum thickness but fails to\nensure a stable, uniform biofilm due to uncontrolled hydrodynamic effects. This outcome\nunderscores a fundamental difference in design philosophy. It is not that the Z-carrier's\ngeometric design is entirely without merit in preventing clogging, but that our hydro-\ntopological strategy achieves a higher level of control: moving from passive geometric\nconfinement to active hydraulic regulation, thereby ensuring both clogging prevention and\noptimal biofilm function through continuous self-cleaning.\nWe believe the addition of the CFD analysis and the clarifications provided in this response\nrobustly support our position. Thank you for prompting this crucial discussion.\nFig. R4 | This figure, reproduced from originally Ref. 21 (now Ref. 25), shows the key\nobservation from the long-term evaluation of the Z-carrier. Comparing biofilm growth at day\n83 (left) and day 293 (right) reveals the development of a clearly defined patch in the middle\nof each grid cell where the biofilm became significantly thinner or entirely absent over time.\nFig. R5 (Fig. 6) | CFD-simulated flow fields and biofilm morphology of carriers after 405\ndays of operation. a–b, Flow fields around a V-carrier with an opening ratio of 1.39 (1.0/0.72)\nunder (a) parallel and (b) 45° flow incidence. c, Biofilm morphology on the stably-operated V-\ncarrier with opening ratio 1.39. d–e, Flow fields around a V-carrier with an opening ratio of\n2.08 (1.5/0.72) under (d) parallel and (e) 45° flow incidence. f, Biofilm morphology on the\nstably-operated V-carrier with opening ratio 2.08. g–h, Flow fields (parallel flow) around\ncarriers with cell dimensions of 2.3 × 2.3 mm and ridge heights of (g) 1.0 mm and (h) 0.5 mm.\ni, Biofilm morphology on the MK5 carrier. j–k, (j) Flow field (parallel flow) and (k) biofilm\nmorphology of the U-carrier. l–m, (l) Flow field (parallel flow) and (m) biofilm morphology of\nthe K3 carrier. n–o, (n) Flow field (parallel flow) and (o) biofilm morphology of the K5 carrier.\n“AnoxKaldnes introduced a carrier (the 'Z-carrier') based on a 42 geometric premise that its\ngrid height would regulate biofilm thickness13.” And it does it very effectively, as opposed to\nwhat authors claim here. Importantly, Reference 13 has reported the innovation of the zeta-\ncarrier using long-term experimentation, in which no such shortcomings were observed.\nIn fact, authors of reference 13 also reported (Torresi et al., 2017,\nhttp://dx.doi.org/10.1016/j.watres.2017.06.027) on the systematic behaviour of the zeta-carrier\nin terms of in-biofilm diffusivity and sorption-desorption, which would not have been possible\nshould the zeta-carrier allowed for irregular growth of biofilm thickness.\nResponse:\nWe thank you for emphasizing the significant contributions of the Z-carrier literature. We agree\nthat References 13 (now Ref. 17 in the revised manuscript) and Torresi et al., 2017, represent\nimportant advancements, particularly in demonstrating operational stability and in measuring\nbiofilm properties under the assumption of controlled thickness.\nHowever, we respectfully maintain that the strong performance reported in these studies does\nnot contradict our statement regarding the limitation of reliably controlling uniform biofilm\nthickness. Our critique focuses on a specific, unverified aspect of the Z-carrier's performance\nthat is separate from its overall treatment efficacy.\n1. The distinction between stable operation and thickness control: The Z-carrier's open\ndesign successfully mitigates clogging by exposing biofilm to hydraulic shear, which\nleads to stable reactor performance—a benefit we acknowledge. Similarly, large-\nhollow carriers like K3 also achieve stability (Fig. 3g). However, stable operation is\nnot synonymous with precise control over biofilm thickness uniformity. The study we\ncite (originally Ref. 21, now Ref. 25 in the revised manuscript), which is a long-term\nevaluation by the developers themselves, directly observed non-uniform biofilm\nthickness (\"a clearly defined patch... where the biofilm was significantly thinner\"). This\nfinding reveals a limitation in the geometric control paradigm that is not addressed in\nstudies focusing solely on bulk performance parameters like nitrification rate or\ndiffusivity.\n2. The need for direct validation: The systematic measurements of in-biofilm diffusivity\nand sorption by Torresi et al. (2017) were conducted under the premise that thickness\nwas controlled by the grid. However, that study did not include a spatial analysis of\nbiofilm thickness uniformity to validate this premise over long-term operation. The\nsubsequent findings of originally Ref. 21 (now Ref. 25) suggest that this underlying\npremise may be flawed. Therefore, while the data from Torresi et al. are valuable, they\ndo not constitute direct evidence against the occurrence of irregular biofilm growth, as\nrevealed by later, more focused long-term examinations.\nIn summary, our argument is not that the Z-carrier is ineffective, but that its design principle\nhas a fundamental limitation in ensuring reliable and uniform thickness control under dynamic\nconditions, a limitation that was uncovered by long-term, specific observation (originally Ref.\n21, now Ref. 25). The strong performance in other studies demonstrates the carrier's ability to\navoid clogging and maintain function, but it does not invalidate the specific shortcoming related\nto thickness uniformity that we highlight. Our work addresses this very limitation by\nintroducing a hydro-topological strategy that actively guides biofilm growth to maintain\nuniformity.\n“However, their own 43 long-term evaluation revealed a critical flaw: biofilms at the center of\nthe grid cells became 44 significantly thinner or were entirely absent21. This outcome\nunderscores a fundamental stalemate 45 in sustainable carrier design, revealing a poor\nunderstanding of the hydrodynamic mechanisms that 46 control biofilm development. The failure of\nthis simplistic geometric approach highlights the 47 necessity for a design paradigm rooted in\nhydraulic principles.” This seems to be a seriously flawed series of statement intended to\nundermine confidence in the state-of-the-art for the benefit of opening a knowledge gap on\nfalse premises.\n1. It greatly misinterprets what has been reported in literature. Ref. 21 states “…had a clearly\ndefined patch in the middle of each cell where the biofilm was significantly thinner, as a result\nof the carriers scraping each other.... Since the grid compartments were full of biomass up to\nthe pre-determined wall heights, and calculations suggest that the maximum possible scraping\ndepth from one Z-carrier into another Z-carrier compartment is 90 µm, the biofilm thickness\non the four carrier types was determined to vary between 410–500, 310–400, 210–300 and 110–\n200 µm, respectively.”\n2. Carriers scraping each other is not a sign of poor understanding of hydraulic behaviour.\n3. It does not indicate that these carriers would “clog” in the way authors seem to mean it.\nResponse:\nWe thank you for pushing us to clarify the foundation of our argument. We respectfully disagree\nthat our interpretation is flawed or intended to undermine the state-of-the-art on false premises.\nOur analysis directly engages with the empirical findings reported in the literature to highlight\na specific, not fully resolved.\n1. On the Interpretation of Ref. 21 (now Ref. 25) and the \"Scraping\" Mechanism:\nYou quote Ref. 21 (now Ref. 25 in the revised manuscript) to assert that the biofilm thinning\nwas due to \"carriers scraping each other,\" implying this is a settled matter. However, we draw\na critical distinction between the observation reported in Ref. 21 (now Ref. 25) and the\ninterpretation offered by its authors.\n• Observation (Fact): The study clearly documents a \"clearly defined patch... where the\nbiofilm was significantly thinner.\" This is an empirical finding that demonstrates a\nfailure to maintain uniform biofilm thickness.\n• Interpretation (Hypothesis): The authors' attribution of this thinning to \"carriers\nscraping each other\" is a post-hoc hypothesis that was not experimentally verified in\nthat study.\nWe contend that this interpretation reflects a \"poor understanding of hydrodynamic\nmechanisms\" because it overlooks a more fundamental hydraulic explanation. Our CFD\nanalysis (Fig. 6, see Comment #4 above) demonstrates that such localized thinning in confined\nspaces is a predictable consequence of hydrodynamic conditions (e.g., localized shear patterns),\nnot necessarily physical contact. By proposing an untested mechanical explanation, the original\nstudy missed an opportunity to identify the underlying hydraulic principle. Our work addresses\nthis gap by prioritizing hydraulic design.\n2. On Clogging and the Z-Carrier's Contribution:\nYou rightly point out the need for precision regarding the Z-carrier's design objective and its\neffects. We fully recognize that its open-grid design successfully prevents the severe clogging\ntypical of small-tube carriers. This is a key advancement. However, our argument is that\novercoming clogging is a necessary but not sufficient condition for optimal biofilm control.\nThus, while the Z-carrier avoids clogging due to its design, it introduces a new challenge:\nunpredictable biofilm uniformity due to uncontrolled hydrodynamic effects (as evidenced by\nthe thinning patches). Our hydro-topological strategy aims to solve both problems\nsimultaneously by design.\n3. Separating the Issues:\nThe core of our disagreement may stem from a conflation of two separate issues:\n• Issue A (Effectively prevented by the Z-carrier's design): Preventing catastrophic\ncarrier clogging through an open structure.\n• Issue B (Defined and addressed in our work): Ensuring reliable, uniform biofilm\nthickness control through active hydraulic management, which the Z-carrier's\ngeometric approach does not achieve.\nOur citation of Ref. 21 (now Ref. 25) is not to deny its contribution to Issue A, but to use its\nown data to highlight the persistence of Issue B, which our strategy is designed to solve.\nIn summary, our statements are not flawed but are based on a critical analysis of the literature.\nWe use the empirical evidence from the Z-carrier's own long-term evaluation to highlight a\nlimitation that calls for a new design paradigm rooted in hydraulic principles, which we have\ndeveloped and validated.\n“The failure of this simplistic geometric approach … The propensity for clogging in\nconventional tubular carriers” Co-authors’s may have limited understanding of the system they\ncriticise. The zeta-carrier is NOT a tubular carrier but a three-dimensional wave-shaped flat\nsurface covered with a mesh with a defined thickness.\nResponse:\nWe thank you for this comment, which allows us to clarify a potential misunderstanding in our\ntext structure. We fully agree that the Z-carrier is not a tubular carrier but a three-dimensional\nwave-shaped surface with a mesh. Our writing did not intend to imply otherwise, and we\napologize if the sequence of paragraphs led to this interpretation.\nOur argument follows a clear logical progression:\n1. First, we introduce the Z-carrier as a notable effort to address the fundamental\nlimitation of biofilm thickness control, whose open structure also successfully prevents\nthe clogging typical of conventional carriers.\n2. Then, in a separate and subsequent paragraph, we discuss the hydraulic principles\ngoverning \"conventional tubular carriers\" (such as K3 and K5) to explain the root cause\nof clogging, which the Z-carrier's open design successfully overcomes.\nTherefore, the statement \"The failure of this simplistic geometric approach\" refers solely to the\nZ-carrier's inability to ensure uniform biofilm thickness (as documented in originally Ref. 21,\nnow Ref. 25 in the revised manuscript), which we had just discussed. The subsequent sentence,\n\"The propensity for clogging in conventional tubular carriers...\", begins a new topic to provide\nthe hydraulic background that justifies the need for the kind of open design that the Z-carrier\nexemplifies.\nIn summary, we do not criticize the Z-carrier by misclassifying it as a tubular carrier. Instead,\nwe:\n• Acknowledge it as an advanced design that effectively prevents the clogging issues\nassociated with tubular carriers.\n• Identify a separate limitation in its geometric approach to uniform biofilm thickness\ncontrol.\n• Use the hydraulic principles of tubular carriers to build the foundational rationale for\nour own hydro-topological strategy.\nWe will ensure that this logical distinction is made unequivocally clear in any future\npresentation of this work. Thank you for prompting this important clarification.\n“system exhibited high nutrient removal rates 120 of 0.138 ± 0.022 g N g VSS⁻¹ day⁻¹ for\nammonia” This observation is not benchmarked against literature evidence, and does not seem\nto be correct. For instance, reference 13 observed much higher removal rates using the zeta\ncarrier of 50 um, i.e., around 0.7 gN/(d.gVSS), and only thicker biofilms, i.e., above 200\num showed similar but mostly higher values than that reported here.\nResponse:\nWe thank you for this comment regarding the benchmarking of our reported nitrification rate.\nWe agree that comparing performance metrics is essential, but we respectfully argue that a\ndirect comparison of specific nitrification rates (per gVSS) between studies with fundamentally\ndifferent biofilm thicknesses and operating conditions is not scientifically valid and misses the\nkey innovation of our work.\nThe core of the issue lies in the purpose of controlling biofilm thickness. As Reference 13 (now\nRef. 17 in the revised manuscript) correctly demonstrates, biofilm thickness is a primary\ndeterminant of specific nitrification rate, with thinner biofilms yielding higher rates due to\nreduced mass transfer limitations. Therefore, comparing a thick biofilm from one system to a\nthin biofilm from another does not benchmark carrier performance; it merely confirms the well-\nestablished effect of biofilm thickness.\nA meaningful comparison of carrier efficacy must be made under comparable conditions. We\npropose two more appropriate benchmarks:\n1. Comparison at similar biofilm thickness: When compared at a similar thickness (~500\nμm), the specific nitrification rate of our V-carrier (0.138 gN gVSS⁻¹ d⁻¹) is notably\nhigher than that of a Z-carrier at 500 μm (approximately 0.12 gN gVSS⁻¹ d⁻¹, estimated\nfrom Ref. 13's, now Ref. 17's data using a VSS/TSS ratio of 0.85). More importantly,\nour V-carrier maintained biofilm thickness stably, while the Z-carrier, as discussed,\nsuffers from non-uniform thickness.\n2. Comparison of intrinsic potential under standardized conditions: Our ex-situ\nnitrification potential assays, conducted under optimal, non-limiting substrate\nconditions analogous to the batch tests in Ref. 13 (now Ref. 17), revealed a rate of 0.65\ngN gVSS⁻¹ d⁻¹. This intrinsic potential is significantly higher than the rates reported in\nRef. 13 (now Ref. 17) and underscores the high metabolic capacity of the biofilm\ncultivated by our carrier. The lower in situ rate (0.138 gN gVSS⁻¹ d⁻¹) reflects the\nsubstrate-limiting conditions of our real wastewater system, not a limitation of the\ncarrier technology itself.\nFurthermore, critical operational differences preclude a direct comparison:\n• Wastewater composition: Our system treated real municipal wastewater, while Ref. 13\n(now Ref. 17) used synthetic wastewater. Real wastewater introduces complexity and\nvariability that typically results in lower observed rates.\n• Reactor design and loading: Differences in carrier filling ratio, hydraulic regime, and\nnitrogen loading rates significantly influence the specific nitrification rate normalized\nto biomass.\nIn conclusion, the novelty of our work is not in achieving an arbitrarily high nitrification rate,\nbut in enabling stable, efficient nutrient removal with a self-regulating biofilm at an optimal\nthickness under challenging, real-world conditions. The value of our V-carrier is its ability to\nautonomously maintain this optimal thickness for high performance under real-world\nconditions, a capability that we demonstrate is lacking in geometric confinement-based designs.\n“131 biofilm regulation—Hydro-topological synergy” “synergy” - a possible typo. Also,\nauthors fail to thoroughly explain the meaning of the term they introduced.\nResponse:\nWe thank you for this attentive comment regarding terminology and conceptual explanation.\nYou are correct to point out the inconsistency in the heading. \"Hydro-topological strategy\" is\nthe central term introduced in our work. The use of \"synergy\" in the subheading was an error;\nwe have corrected it to \"Mechanism of biofilm regulation—Hydro-topological strategy\"\nthroughout the manuscript to maintain consistency.\nRegarding the request to thoroughly explain the term, we appreciate the opportunity to clarify.\nThe concept of the \"hydro-topological strategy\" is developed and explained incrementally\nthrough the mechanistic experiments in this section. It is not merely the V-shape itself, but the\nprincipled interaction between carrier topology and hydraulic forces that it enables. Our results\ndemonstrate that this strategy operates on two synergistic principles:\n1. Topological Guidance for Hydraulic Exposure: The V-groove topology is designed to\nphysically guide biofilm growth outward, into the liquid flow path, ensuring the biofilm\nsurface is continuously exposed to hydraulic shear. This is contrasted with carriers\nwhere biofilm grows inward, shielded from shear (e.g., tube-carriers), or where\ntopology fails to ensure exposure (e.g., U-carrier).\n2. Hydraulic Shear for Autonomous Regulation: The sustained hydraulic shear on the\nexposed surface creates a dynamic balance between biofilm growth and decay, enabling\ncontinuous self-cleaning and preventing uncontrolled accumulation. This is\ndefinitively shown by the contrasting fates of the open versus shielded sections of the\nMK5 carrier.\nThe failure of carriers that rely on only one aspect—excessive shear without guidance (MK5\nopen sites) or shielding without shear (U-carrier, clogged tubes)—highlights that neither\ntopology nor hydraulics alone is sufficient. The \"hydro-topological strategy\" is therefore\ndefined by the synergistic integration of topological guidance and hydraulic regulation to\nachieve stable, self-cleaning biofilms.\nTo incorporate this clarification directly into the manuscript, we have revised the final sentence\nof the 'Mechanism of biofilm regulation' section (currently on Lines 187-189) to read as follows:\n\"This synergistic management of space and shear, which we term the hydro-topological strategy,\nintegrates precise topological guidance for hydraulic exposure with sustained shear for\nautonomous regulation to maintain an optimal, stable biofilm.\"\nThis revision provides a concise, consolidated definition based on the experimental evidence\npresented in the section.\nReviewer #2:\nIn this manuscript, the authors reported a new geometrical design for biofilm carriers used in\nmoving bed biofilm reactor (MBBR). In contrast to the closed circular design, the authors used\nan open V-grove design, which was shown to lead to superior performance compared to the\nconventional design using various standard MBBR characterization. Microscopic analysis\nshowed an open morphology of biofilm with holes, which the author attributed to cell lysis and\ncontribute to biofilm self-renewal. Finally, metagenomics analysis was presented regarding the\nspecies distribution in such carrier microbial communities.\nResponse:\nWe sincerely thank you for your highly positive assessment of our work and for your strong\nsupport for publication in Nature Communications. We are greatly encouraged by your\ncomments that the research is of high quality, the analysis is solid, and the technology has strong\npotential.\nWe have carefully considered the two constructive points you raised and provide the following\nresponses and commitments to address them, which we believe will further enhance the clarity\nand impact of our manuscript.\nOverall, the research is performed with high quality, and the analysis is very solid. The\npresented technology has a strong potential to improve the existing technology. I support the\npublication of the manuscript in Nat. Comm. My only major concern is the lack of evidence of\ncell lysis, which is critical in the authors’ picture. Can the authors perform live-dead\nstaining to definitely prove the existence of such process in the interior of the biofilm? Also, the author can do a better job in\nexplaining some of the jargons for a broader audience. For example, in legend for Figure 3, what does VSS/SS ratio mean\nfor the performance of the device?\nResponse:\n1. Evidence for cell lysis\nWe thank you for this excellent suggestion. As you recommended, we have performed live-\ndead staining to directly visualize and quantify cell viability within the biofilm. The results,\nnow included as Supplementary Fig. 8 (Fig. R6), provide definitive evidence for the cell lysis\nand self-renewal process.\nThe new images clearly show a higher proportion of dead cells in the inner layers of the biofilm,\nwith a visible gradient of cell debris and lysed material suggesting an outward migration path\nfrom the interior to the bulk liquid. This observation directly supports our initial interpretation\nthat the internal voids (holes) observed via microscopy are indeed pathways created by active\ncell lysis and material export, facilitating continuous biofilm renewal.\nTo incorporate this new evidence and clarify the mechanistic pathway, we have strengthened\nthe corresponding description in the main text (Lines 213-216) as follows:\n“Furthermore, the persistent presence of internal voids within the biofilms, irrespective of\ntemperature (Supplementary Fig. 7), is consistent with active cell lysis and material export37-39.\nThis is definitively supported by live-dead staining (Supplementary Fig. 8), which reveals a\nhigher proportion of dead cells and lysing debris in the inner biofilm layers, illustrating a\ngradient from the interior to the bulk liquid. This visual evidence confirms that the voids are\npathways formed by continuous cell lysis and subsequent export of cellular material. This\nmorphological feature provides physical evidence of an ongoing self-renewal process,\nunderpinning the dynamic self-cleaning mechanism intrinsic to the V-carrier design.”\n2. Clarification of jargon (e.g., VSS/SS ratio)\nWe appreciate this valuable suggestion to enhance the manuscript's accessibility for a broader\naudience. As you rightly inferred, the VSS/SS ratio serves as a key integrated indicator of\nbiofilm health and the occurrence of clogging or scaling. The core interpretation is as follows:\n• VSS (Volatile Suspended Solids) primarily represents the active microbial biomass.\n• SS (Suspended Solids) includes both organic (VSS) and inorganic components.\n• Therefore, a high and stable VSS/SS ratio indicates a healthy, active biofilm with\nminimal inorganic accumulation.\n• Conversely, a declining VSS/SS ratio signals the onset of clogging/scaling, as inorganic\nprecipitates accumulate within the biofilm matrix, increasing the SS without\ncontributing to activity. This is precisely what we observed in carriers like K5 and\nMutagbiochip (Supplementary Table 1), where a dropping VSS/SS ratio correlated with\nperformance failure.\nTo make this interpretation immediately clear to the reader, we have revised the figure legend\nfor Fig. 3 (appearing on lines 491-495 in the revised manuscript) to include direct explanatory\nnotes. The key additions are:\n“a, b, ... The VSS/SS ratio is a key indicator of biofilm health; a high, stable ratio signifies a\nbiofilm with minimal inorganic scaling and high metabolic activity. c, f–h, ... The declining\nVSS/SS ratio in panels c, f and h indicates inorganic accumulation and clogging.”\nA more comprehensive explanation is also provided in the Supplementary Note 1. By clarifying\nthis terminology directly in the figure legend and the supplementary information, we ensure\nthat the functional significance of the VSS/SS ratio is immediately apparent, making it clear\nthat it is a vital diagnostic tool for assessing the biofilm's state and the system's operational\nstability.\nFig. R6 (Supplementary Fig. 8) | Evidence of cell lysis and self-renewal in V-carrier\nbiofilms revealed by live-dead staining. V-carrier biofilms were sampled after 409 days of\noperation and analyzed by confocal laser scanning microscopy (CLSM). a, Three-dimensional\nreconstruction showing the spatial distribution of live and dead cells. b–c, Representative two-\ndimensional optical sections at different focal planes. Cells were stained with SYTO 9\n(excitation/emission: 495 nm / 519 nm) and propidium iodide (PI) (excitation/emission: 305\nnm / 617 nm). For visualization, the SYTO 9 signal (all cells) is pseudo-coloured blue, and the\nPI signal (dead cells) is shown in red. The images reveal a higher proportion of dead cells (red)\nand lysing debris in the inner layers, illustrating a viability gradient from the interior to the bulk\nliquid. This provides direct visual evidence that internal voids serve as pathways for cellular\nmaterial export, confirming an ongoing self-cleaning process.\nReviewer #3:\nThe manuscript presents a novel V-carrier design and provides a thorough scientific evaluation\nof its performance in wastewater treatment. The study offers valuable insights into biofilm self-\nregulation and demonstrates the potential advantages of the hydro-topological strategy.\nHowever, the manuscript would benefit from significant reorganization. The overall structure\nand flow of ideas need improvement to enhance clarity and readability, as some sections\ncurrently lack logical sequencing despite the compelling presentation of results. Given these\nissues, I recommend major modifications before the manuscript can be considered for\npublication. Addressing the organization and ensuring a clearer narrative will greatly strengthen\nthe impact of the work.\nResponse:\nWe sincerely thank you for your positive assessment of our work and for the overarching\nrecommendation to improve the manuscript's organization and narrative flow. We agree that\nenhancing the logical structure and clarity is crucial for maximizing the impact of the study.\nIn direct response to your feedback, we have undertaken a comprehensive restructuring and\nrewriting of the manuscript. The revisions are substantial and are detailed in our point-by-point\nresponses to each of your specific comments below. The key organizational improvements\ninclude:\n1. Strengthened Introduction: We have significantly revised the introduction to provide a\nmore critical and coherent narrative. This includes a clearer explanation of how MBBR\ntechnology supports the Sustainable Development Goals (Comment #1) and a critical\noverview of prior carrier design strategies and their limitations, which now effectively\nsets the stage for introducing our novel V-carrier design (Comments #2, #3, #4).\n2. Clarified Methodological Rationale: The Methods section has been enhanced with\nclearer justifications for key experimental choices, such as the biofilm establishment\nprotocol (Comments #3, #4) and the wastewater dilution methodology (Comment #2).\nThis provides a stronger foundation for the reported results.\n3. Precise Terminology and Robust Results Presentation: In the Results section, we have\nclarified critical terminology (e.g., specifying \"structural porosity\" versus material\nporosity, Comment #1) and provided robust evidence to support key claims, such as\nthe optimal opening ratio, by integrating new experimental data and CFD simulations\n(Comments #2, #5). This ensures that all assertions are well-substantiated and\nunambiguous.\n4. Enhanced Discussion with Practical Implications: The Discussion section has been\nexpanded to better integrate our findings with the broader context of the field. Most\nimportantly, as suggested in your Comment #1 on the Methods section, we have added\na new discussion on the practical implications of our findings, directly linking the V-\ncarrier's design to its hydrodynamic advantages and long-term stability demonstrated\nin pilot-scale operations.\nWe are confident that these extensive revisions have successfully addressed your primary\nconcern regarding the manuscript's organization. The narrative now flows more logically from\na well-motivated introduction, through clearly presented methods and robust results, to a\ndiscussion that effectively highlights the significance and practical potential of our findings.\nWe believe the manuscript is now substantially stronger and clearer, and we are grateful for\nyour guidance in achieving this improvement.\nHERE ARE THE COMMENTS:\nIntroduction section\n1. While the manuscript states that the MBBR process aligns with SDGs 6, 11, and 12, this\nsection does not clearly discuss how MBBR operational characteristics contribute to these\nsustainability principles. The authors are encouraged to elaborate on the specific mechanisms\nthrough which MBBR supports these SDGs—for example, by detailing its impacts on resource\nefficiency, energy use, sludge reduction, land footprint, or resilience of sanitation systems.\nProviding this clarification would strengthen the justification of MBBR as a sustainable\ntechnology and improve the overall coherence of the sustainability narrative.\nResponse:\nWe thank you for the valuable suggestion to strengthen the link between MBBR operational\ncharacteristics and the SDGs. We agree that a clearer explanation of the underlying mechanisms\nwould enhance the sustainability narrative. In response, we have revised the relevant section in\nthe introduction to explicitly elaborate on how the key features of the MBBR process contribute\nto the specific SDGs. The revised text (Lines 24-29) now reads:\n“Among advanced treatment technologies, the moving bed biofilm reactor (MBBR) process\nsupports SDGs 6, 11, and 12 through its ability, enabled by suspended biofilm carriers, to\ndeliver high nutrient removal efficiency (SDG 6), a compact physical footprint (SDG 11), and\nreduced sludge production (SDG 12). Through these contributions to water security, urban\nsustainability, and resource efficiency, MBBR has been established as a leading technology for\nnew and upgraded WWTPs worldwide.”\nThis revision directly addresses your point by detailing the specific pathways—high treatment\nefficiency, compact design, and lower resource intensity—through which MBBR technology\nsupports the principles of clean water, sustainable cities, and responsible consumption. We\nbelieve this clarification significantly strengthens the justification for MBBR as a sustainable\ntechnology and improves the coherence of our introduction.\n2. While the authors correctly identify clogging and uncontrolled biofilm accumulation as major\nlimitations of conventional MBBR carriers, the manuscript does not provide any discussion of\nprevious carrier designs reported in the literature or how those studies attempted to mitigate\nclogging. A concise yet critical overview of earlier design approaches—such as modified\ngeometries, increased opening ratios, structured internal channels, or surface texturing—and an\nevaluation of their effectiveness in reducing fouling or improving hydrodynamics is essential.\nFurthermore, the manuscript should clarify the residual shortcomings of these designs that\ndirectly motivated the development of the new carrier proposed in this study. Addressing these\npoints will strengthen the rationale for the new design and better situate the contribution of the\ncurrent work within the context of prior research.\nResponse:\nWe thank you for this critical suggestion. We agree that providing a broader context of prior\ncarrier design strategies and their limitations is essential to strengthen the rationale for our new\ndesign. In response, we have significantly revised the introduction to include a concise yet\ncritical overview of previous design approaches, as you suggested.\nSpecifically, we have expanded the discussion on the historical development of MBBR carriers\nto explicitly address the points you raised. The revised text no longer presents conventional\ncarriers' limitations in isolation but instead critically examines the prevailing design paradigm\nthat has guided most previous efforts, including those utilizing advanced tools like 3D printing.\nThis new narrative clarifies that the core issue is not a lack of attempts but a widespread\nstrategic focus on maximizing protected surface area—a goal rooted in a paradigm inherited\nfrom suspended-growth systems (e.g., activated sludge) and often pursued without sufficient\nlong-term validation. This focus, while successful in increasing surface area, has not\nconsistently improved treatment efficiency and has inadvertently promoted uncontrolled\nbiofilm growth, diverting attention from the need for biofilm managed growth based on\nhydraulic principles.\nThe revised text now reads as follows (new text appeared on lines 31-41):\n“Over four decades of development have yielded numerous MBBR carrier designs for\nengineering applications8,9. These efforts, even using advanced tools like 3D printing\ntechniques, have predominantly pursued one goal: maximizing the protected surface area to\nenhance biofilm attachment10. However, increased surface area has not consistently improved\ntreatment efficiency11, revealing a deeper issue rooted in two factors: a paradigm inherited from\nsuspended-growth systems (e.g., activated sludge), where more biomass implies better\nperformance, and a widespread lack of long-term operational data. This has led to the\nmisapplication of the ‘more biomass equals more efficiency’ principle to biofilms—systems\nwhere controlled biofilm thickness and metabolic balance are paramount. The design focus on\nmaximizing protected surface area has promoted uncontrolled biofilm growth, with carrier\nclogging even misinterpreted as a positive sign12,13, thereby diverting attention from the need\nfor biofilm managed growth. This fundamental misdirection manifests as the core limitations\nof conventional MBBR systems: severe carrier clogging and scaling, drastically diminishing\nthe effective surface area, impairing mass transfer, and disrupting microbial community\nstructure by fostering competition for oxygen and substrates among unfavorable species14-16.”\nThis revision directly addresses your comment by:\n1. Providing a critical overview: It characterizes the primary goal of previous designs as\n\"maximizing protected surface area,\" which encompasses strategies like modified\ngeometries.\n2. Evaluating effectiveness: It states that this strategy \"has not consistently improved\ntreatment efficiency,\" directly addressing its limitations.\n3. Clarifying residual shortcomings: It identifies the root causes as a \"misapplied\nparadigm\" and a \"lack of long-term operational data,\" which are the residual\nshortcomings that directly motivate our hydro-topological approach.\nWe believe this new framework effectively situates our contribution within the context of prior\nresearch by clearly defining the conceptual limitation we aim to overcome. Thank you for this\nsuggestion, which has greatly improved the motivation and clarity of our introduction.\n3. The manuscript could better contextualize the rationale for the current study by discussing\nprevious carrier design strategies. Specifically, prior studies—including those employing CFD\nsimulations—have proposed geometric or structural modifications to improve biofilm\ndistribution, mass transfer, and reduce clogging. While such studies provide valuable\nindications and guidance for experimental design, they often lack long-term operational\nevaluation. Highlighting both the insights and the limitations of these previous approaches\nwould strengthen the justification for developing and testing the new V-carrier in a long-term\nexperimental setting.\nResponse:\nWe thank you for this comment, which further underscores the importance of situating our work\nwithin the context of prior research strategies.\nWe agree that highlighting both the insights and limitations of previous design approaches is\ncrucial. As detailed in our response to Comment #2 above, we have thoroughly revised the\nintroduction to address this point. The new text provides a critical examination of the prevailing\ndesign paradigm, which we explicitly identify as being hampered by a “widespread lack of\nlong-term operational data,” a limitation you rightly emphasized.\nThis revision establishes that previous strategies were pursued without sufficient long-term\nvalidation, thereby directly creating the rationale for our long-term experimental study.\nImportantly, the introduction's critical insight—that the \"more biomass equals more\nefficiency\" paradigm is misapplied to biofilms—is strongly supported by our long-term results.\nAs presented in the Discussion, the V-carrier maintained 44% less biomass than a conventional\ncarrier yet achieved a 3.2-fold higher nitrification rate, demonstrating that treatment efficiency\nis decoupled from sheer biomass quantity and is instead governed by the efficacy of the biofilm\necosystem.\nBy stating these limitations as a root cause, our revision effectively contextualizes our study\nand justifies the need for a long-term, hydraulically-informed design. Thank you for reinforcing\nthis essential point.\n4. The manuscript discusses the long-standing issue of clogging in MBBR systems but provides\nonly a single example (the Z-carrier) as evidence of previous design limitations. This narrow\nfocus unintentionally suggests that, over four decades of carrier development, no other design\nstrategies or mitigation efforts have been explored. The authors are encouraged to expand this\nsection by briefly summarizing additional carrier design attempts reported in previous studies\nand discussing their partial successes or remaining limitations. This broader context is essential\nto accurately position the Z-carrier as one example rather than the sole historical effort, and to\nmore convincingly justify the need for the new design proposed in this work.\nResponse:\nWe thank you for this comment. We apologize for the lack of clarity in our original manuscript,\nwhich may have unintentionally given the impression that the Z-carrier was an isolated example.\nThe core issue was a misstatement regarding the Z-carrier's primary design objective. We have\ncorrected this in the revised introduction (Lines 53-56). The text now accurately presents the\nZ-carrier not as a singular effort to address clogging, but as one notable effort to address the\nfundamental limitation of biofilm thickness control—a challenge that sits within the broader\ncontext of prior design strategies that we now critically overview in the preceding paragraph.\nThe revised passage reads:\n\"One notable effort to address this limitation was the 'Z-carrier' (AnoxKaldnes), which was\nbased on the geometric premise that its grid height would regulate biofilm thickness17,23,24—a\ndesign that also had implications for mitigating clogging and scaling due to its open structure.\"\nThis revision, combined with the comprehensive critical overview of previous design\nparadigms added earlier in the introduction (as detailed in our response to Comments #2 and\n#3 above), effectively addresses your concern. The Z-carrier is now positioned as a\nrepresentative case within the wider historical effort, rather than as an isolated example. This\nprovides the broader context needed to accurately situate its contribution and limitations.\nWe believe this correction, integrated with the expanded discussion of prior design strategies,\nmore convincingly justifies the need for our new design. Thank you for prompting this\nimportant clarification.\nMethods Section:\n1. Regarding the choice of using a square-shaped media instead of a cylindrical one, it would\nbe useful to discuss the practical implications of this design choice. While the square shape may\noffer advantages in terms of maximizing surface area and potentially improving biofilm\nattachment, it is important to evaluate whether this design is as effective in real-world\napplications. Specifically, how does the square shape perform in terms of hydrodynamic\nbehavior, ease of movement within the reactor, and resistance to clogging compared to\ncylindrical carriers? Further clarification and comparative analysis with cylindrical media\nwould help assess its practical viability in large-scale wastewater treatment systems.\nResponse:\nWe sincerely thank you for this insightful comment regarding the practical implications of the\nsquare-shaped V-carrier design compared to conventional cylindrical media. We agree that\nevaluating hydrodynamic behavior, mobility, and clogging resistance is crucial for assessing\nreal-world viability. In response, we have conducted additional comparative experiments and\nadded a detailed discussion to address these points directly.\n1. Hydrodynamic Behavior and Movement Efficiency:\nTo quantitatively assess the hydrodynamic performance, we conducted fluidization tests in a 2\nm³ pilot-scale bioreactor. The results, now included as Supplementary Fig. 11 (Fig. R7),\ndemonstrate that the V-carrier requires over 30% less aeration intensity than cylindrical carriers\n(K3 and K5) to achieve complete fluidization at the same filling ratio. This advantage stems\nfrom its unique design:\n• V-carrier: The square-sheet morphology of the V-carrier is subjected to asymmetric\nflow around its structure. The V-grooves generate high drag, creating a significant\ntorque that efficiently converts hydraulic energy into full carrier motion (translation\nand rotation).\n• K3/K5 carriers: In contrast, the cylindrical geometry of K3/K5 carriers, with their\nhollow structure, allows complete cross-flow. This results in a balanced pressure\ndistribution around the carrier, minimizing torque and leading to less efficient,\npredominantly translational motion.\nThis comparative analysis confirms that the square-shaped, V-grooved design exhibits superior\nhydrodynamic efficiency, directly reducing the energy required for mixing and suspension—a\ncritical factor for large-scale operational costs.\n2. Anti-clogging Performance and Practical Validation:\nRegarding long-term stability and resistance to clogging, the V-carrier's open-topology design\ninherently prevents the internal pore blockage typical of cylindrical carriers. More importantly,\nwe have included preliminary data from a long-term pilot study (now in Supplementary Fig.\n12) treating real municipal wastewater in Chengxi WWTP (Hangzhou, China). The results show\nstable operation of the V-carrier system over 400 days of operation with no signs of clogging\nor performance decay, demonstrating its strong potential for real-world applications.\n3. Discussion Integration:\nFurthermore, as you suggested, we have integrated a discussion on the practical implications of\nthese findings into the revised manuscript (Lines 345-357). The added discussion reads as\nfollows:\n\"From an engineering perspective, the hydro-topological design of the V-carrier translates into\nsignificant practical advantages for large-scale implementation. Pilot-scale fluidization tests\ndemonstrated the V-carrier's superior hydrodynamic efficiency: it requires over 30% less\naeration intensity than conventional cylindrical carriers for complete mixing, a result of the\nasymmetric flow around its grooves generating high drag for efficient motion, unlike the\nbalanced pressure and minimal torque from the cross-flow in cylindrical carriers which leads\nto less efficient movement (Supplementary Fig. 11). This confirms that the V-carrier’s design\ndirectly addresses a major operational cost factor. Furthermore, long-term pilot operation\ntreating real municipal wastewater over 400 days confirmed the system's robust stability, with\nno carrier clogging or performance decay observed (Supplementary Fig. 12). The V-carriers\nmaintained excellent physical integrity, with only slight wear at the corners of the anoxic\ncarriers attributed to mechanical stirring—an issue being addressed by redesigning the blades.\nThis successful validation underscores the V-carrier's potential for energy-efficient and reliable\nlarge-scale application.\"\nThis section explicitly discusses the hydrodynamic advantages of the V-shaped design and its\ndirect contribution to energy efficiency and operational stability, thereby evaluating its potential\nfor large-scale wastewater treatment systems.\nWe believe that this additional experimental data and discussion directly address your concerns\nand robustly support the practical viability of the V-carrier design. Thank you for prompting\nthese important improvements.\nFig. R7 (Supplementary Fig. 11) | Hydrodynamic behavior and fluidization performance\nin pilot-scale reactors. a, Overall view of conventional cylindrical carriers. b-c, Flow pattern\naround a cylindrical carrier (b) and the corresponding CFD-simulated flow field (c). d, Overall\nview of the V-carriers. e-f, Flow pattern around the square-shaped V-carrier (e) and the\ncorresponding CFD-simulated flow field (f). g, Quantitative assessment of aeration intensity\nrequired for the square-shaped V-carrier and the cylindrical carriers (K3 and K5). Fluidization\ntests were conducted in a 2 m³ pilot-scale bioreactor.\n2. The authors diluted raw municipal wastewater with tap water but did not specify the dilution\nratio. Additionally, residual chlorine and other ions in tap water may influence microbial activity,\nparticularly nitrifiers and denitrifies, potentially affecting the generalizability of the results to\nreal WWTP conditions.\nResponse:\nWe sincerely thank you for this important comment regarding the dilution ratio and the potential\neffects of tap water constituents. We have addressed these concerns as follows:\nThe dilution ratio was not fixed because the raw municipal wastewater collected from the septic\ntank exhibited natural variability due to weather conditions (primarily rainfall). It is important\nto note that this wastewater source is the same as that feeding the local Chengxi WWTP\n(Supplementary Fig. 1). The final diluted influent was specifically designed to simulate the\ntypical influent concentrations of Chengxi WWTP, and the effluent targets were also set\naccording to this plant's design standards.\nWe apologize for omitting the dechlorination procedure. Before dilution, tap water was aerated\nfor 24 hours to remove residual chlorine. After this treatment, no residual chlorine was detected\n(< 0.01 mg L⁻¹), and other ions were below detection limits.\nTo incorporate these clarifications, we have revised the manuscript (Lines 372-374). The text\nnow explicitly states the dechlorination process and the purpose of dilution:\n\"Tap water, aerated for 24 hours to remove residual chlorine, was used to dilute this primary\neffluent to simulate typical influent concentrations for a local wastewater treatment plant\n(Chengxi WWTP, Hangzhou, China), ...\"\nWe believe these revisions fully address your concerns regarding the dilution methodology and\npotential effects of tap water constituents on microbial activity, ensuring the generalizability of\nour results to real WWTP conditions.\n3. The manuscript reports adding 50 L of activated sludge twice daily during biofilm\nestablishment. While this approach can accelerate biofilm colonization, the authors should\nclarify the rationale for the chosen frequency and volume, and discuss whether such repeated\ndosing could influence nutrient concentrations or microbial community development compared\nto typical operational conditions.\nResponse:\nWe sincerely thank you for this insightful comment regarding the rationale behind our biofilm\nestablishment protocol. We appreciate the opportunity to clarify our strategy.\n1. Rationale for Dosing Frequency and Volume:\nThe protocol of adding 50 L of activated sludge twice daily was the result of a systematic\noptimization to accelerate biofilm colonization, specifically the growth of an autotrophic\nnitrifying biofilm for rapid system startup. The chosen volume and frequency were determined\nby comparing the time required to achieve the target hydraulic retention time (HRT) under\ndifferent dosing strategies:\n• No inoculation (natural colonization): 21 days\n• Once-daily dosing: 14 days\n• Twice-daily dosing (our chosen protocol): 10 days\n• Thrice-daily dosing: 9 days\nWe selected the twice-daily protocol as it offered an optimal balance between startup\nacceleration and operational practicality. This approach maintains a substantial initial microbial\npopulation while continuous inflow provides nutrients, accelerating the formation of an initial\nheterotrophic biofilm layer on the carrier surface. The growth of this heterotrophic layer is\nknown to enhance extracellular polymeric substances (EPS) production, which promotes\nbiomass-carrier adhesion and has been proven to significantly accelerate the subsequent\ndevelopment of the nitrifying biofilm1.\n2. Influence on Microbial Community Development:\nRegarding the potential long-term influence of the initial sludge inoculation on the microbial\ncommunity, our results indicate that its effect is transient. As demonstrated in the manuscript,\nthe primary factors shaping the mature biofilm community structure over the long term are\noperational temperature (Fig. 4a) and the intrinsic biofilm self-assembly process (Fig. 5b,\nSupplementary Fig. 9). The initial inoculum serves to jump-start the process, but the final,\nstable community is selected by the ongoing environmental conditions and carrier ecology.\n3. Revision for Clarity:\nAs suggested, we have provided a detailed description of this startup strategy and its\noptimization in the Supplementary Information (Supplementary Note 2) to enhance the clarity\nand reproducibility of our methodological description.\nWe believe these clarifications fully address your concerns regarding the startup protocol.\nThank you for this valuable suggestion, which has helped us improve the manuscript.\nReference:\n1. Bassin, J. P., Kleerebezem, R., Rosado, A. S., van Loosdrecht, M. C. M. & Dezotti, M.\nEffect of Different Operational Conditions on Biofilm Development, Nitrification, and\nNitrifying Microbial Population in Moving-Bed Biofilm Reactors. Environ. Sci. Technol.\n46, 1546-1555 (2012).\n4. The manuscript describes the biofilm establishment protocol but does not clearly explain the\nrationale behind it. The authors should specify the principle guiding the chosen procedure—\nwhy activated sludge was added twice daily, how this promotes initial biofilm attachment, and\nthe intended objective of this approach (e.g., accelerating colonization, ensuring\nuniform coverage, or stabilizing microbial communities). Providing this explanation would clarify the\npurpose of the protocol and strengthen the methodological section.\nResponse:\nWe thank you for this comment, which underscores the importance of clarifying the rationale\nbehind our biofilm establishment protocol.\nWe agree that a clear explanation of the guiding principles—the objective, the mechanism of\npromotion, and the reasoning behind the specific dosing strategy—is essential. We have\nprovided a comprehensive response to this exact point in our reply to Comment #3 above.\nTo avoid redundancy, we respectfully refer you to the full explanation provided there. We\nbelieve it fully addresses the concerns you have raised and strengthens the methodological\nsection as suggested.\nResults section\n1. The manuscript reports a porosity value of 92% for the V-carrier; however, this statement is\nscientifically ambiguous and requires clarification. Because the carrier is fabricated from\nHDPE—a fully non-porous material—the reported porosity cannot correspond to material\nporosity. If the authors intend to describe structural or geometric porosity (i.e., the void volume\nfraction within the carrier’s overall envelope), this must be explicitly stated. Moreover, the\nmanuscript should clearly present the calculation method, including the definition of the total\nreference volume, how the void volume was determined (e.g., CAD-based volume analysis),\nand the exact formula used. Without a proper definition and methodological explanation, the\nreported porosity value is misleading and cannot be accurately interpreted in relation to the\ncarrier’s hydrodynamics or mass-transfer performance. The authors are encouraged to revise\nthis section accordingly.\nResponse:\nWe sincerely thank you for this critical and insightful comment regarding the ambiguity of the\nreported porosity value. You are absolutely correct that the term \"porosity\" requires precise\ndefinition in the context of a non-porous HDPE carrier.\nWe have revised the manuscript to fully address your concerns as follows:\n1. Clarification of Terminology:\nWe have explicitly specified that the reported value refers to the structural (geometric)\nporosity of the carrier, not the material porosity of HDPE. The revised text in the Results\nsection (Line 111) now reads:\n\"The carrier's bulk density and structural porosity are 95 ± 0.5 kg m⁻³ and 92%, respectively.\"\n2. Provision of Calculation Methodology:\nAs you suggested, we have provided a detailed and explicit description of the calculation\nmethods in the Supplementary Information (Supplementary Equations 1.1.2 and 1.1.3). The\nmethodology is described as follows:\n• Bulk Density (Supplementary Equation 2): Clean, dry carriers were thoroughly mixed\nand placed into a standard 1 m³ container. The mass of carriers was measured for five\nreplicate containers, and the bulk density was calculated as the average mass per unit\nvolume.\n• Structural Porosity (Supplementary Equation 3): Clean, dry carriers were placed into a\n20-L graduated container and filled with water. After removing the carriers, the volume\nof the remaining water was measured. The structural porosity was calculated as the\nratio of this water-filled void volume to the total container volume, averaged over five\nreplicates. This method explicitly defines the total reference volume and the procedure\nfor determining the void volume.\nA reference to this methodology has been added to the Methods section (Line 367-368):\n\"The methodology for determining the carrier's number, bulk density and structural porosity is\nprovided in Supplementary Equations.\"\nWe believe these revisions have eliminated the ambiguity, provided the necessary\nmethodological rigor, and ensured that the reported structural porosity value can be accurately\ninterpreted in relation to the carrier's hydrodynamic and mass transfer properties. Thank you\nfor prompting this essential clarification.\n2. The manuscript claims that an opening ratio (length/depth) of 1.39 “optimizes hydraulic\nexposure,” but no reference or supporting data are provided. The authors should clarify whether\nthis criterion is based on literature, modeling, or experimental results, or remove the\nunsubstantiated claim.\nResponse:\nWe sincerely thank you for this important comment regarding the basis for the opening ratio of\n1.39. We apologize for the lack of clarity in our original statement.\nThe claim that an opening ratio of 1.39 optimizes hydraulic exposure is based on our\nexperimental results and subsequent CFD modeling validation, not merely on a theoretical\npresumption. This conclusion was drawn from a comparative analysis of V-carriers with\ndifferent opening ratios.\nTo substantiate this claim, we have revised the manuscript and provided the following\nsupporting evidence:\n1. Experimental Validation: We tested carriers with opening ratios of 2.08 (1.5/0.72) and\n1.39 (1.0/0.72). The results showed that the larger opening ratio (2.08) led to excessive\nhydraulic shear, resulting in a significantly thinner biofilm (~240 μm), as visually\ndocumented in the new experimental photos provided in Fig. 6f.\n2. CFD Modeling Corroboration: CFD simulations of the flow fields for these carriers\nrevealed a mechanistic explanation: the larger opening ratio (2.08) caused a ~44%\ndownward shift of the iso-velocity contours within the groove compared to the 1.39\ndesign, directly correlating with the observed over-thinning of the biofilm (Fig. 6a-f;\nFig. R8).\nThis comprehensive experimental and modeling data demonstrates that an opening ratio of 1.39\nprovides a balanced hydraulic environment, avoiding the pitfalls of both insufficient and\nexcessive shear. The supporting discussion and data have been added to the manuscript (Lines\n293-298):\n\"Validation tests using a V-carrier with a larger opening ratio (1.5/0.72 = 2.08) confirmed a\nsignificantly thinner biofilm (~240 μm) (Fig. 6f). Crucially, CFD simulations revealed a ~44%\ndownward shift of the iso-velocity contours within the groove, which correlated with the\nobserved biofilm reduction, demonstrating that hydro-topological parameters (hydraulic shear,\nopening ratio, and groove depth) exert absolute control over biofilm thickness by dictating the\nhydraulic shear environment (Fig. 6a-f).\"\nWe believe these revisions, supported by the new figures, adequately justify the designation of\nthe 1.39 opening ratio as optimal based on our systematic evaluation.\nFig. R8 (Fig. 6) | CFD-simulated flow fields and biofilm morphology of carriers after 405\ndays of operation. a–b, Flow fields around a V-carrier with an opening ratio of 1.39 (1.0/0.72)\nunder (a) parallel and (b) 45° flow incidence. c, Biofilm morphology on the stably-operated V-\ncarrier with opening ratio 1.39. d–e, Flow fields around a V-carrier with an opening ratio of\n2.08 (1.5/0.72) under (d) parallel and (e) 45° flow incidence. f, Biofilm morphology on the\nstably-operated V-carrier with opening ratio 2.08. g–h, Flow fields (parallel flow) around\ncarriers with cell dimensions of 2.3 × 2.3 mm and ridge heights of (g) 1.0 mm and (h) 0.5 mm.\ni, Biofilm morphology on the MK5 carrier. j–k, (j) Flow field (parallel flow) and (k) biofilm\nmorphology of the U-carrier. l–m, (l) Flow field (parallel flow) and (m) biofilm morphology of\nthe K3 carrier. n–o, (n) Flow field (parallel flow) and (o) biofilm morphology of the K5 carrier.\n3. The manuscript reports maintaining a C/N ratio of 4.9 ± 0.3 by supplementing sodium acetate\nto ensure sufficient electron donors for denitrification. The authors should clarify whether this\nsupplementation reflects typical operational conditions for municipal wastewater treatment or\nwas solely applied to optimize denitrification. Furthermore, a reference to prior studies\nsupporting this C/N range should be provided. The potential impact of acetate addition on\nmicrobial community structure and process performance should also be briefly discussed, as it\nmay affect the generalizability of the results to real-world WWTPs.\nResponse:\nWe sincerely thank you for this important comment regarding the rationale and implications of\nsodium acetate supplementation. We apologize for the lack of clarity in our original manuscript,\nwhich has been revised to address your concerns.\n1. Typicality of the Operational Condition:\nThe supplementation of sodium acetate directly reflects a typical operational condition for\nmunicipal wastewater treatment plants (WWTPs) that receive low-carbon wastewater. The raw\nwastewater used in our study had a low C/N ratio of 2.8 ± 0.3 (Supplementary Table 3),\nwhich is representative of the typical influent characteristic of the local Chengxi WWTP\n(Hangzhou, China). At this and many other full-scale plants, adding an external carbon source is a standard\npractice to ensure complete denitrification and comply with effluent standards. Therefore, our\napproach was not merely for experimental optimization but simulated a common real-world\nscenario.\n2. Clarification of the C/N Ratio and Practical Validation:\nWe have clarified the relationship between carbon dosing and the reported C/N ratio in the\nrevised manuscript. The sentences now read:\n\"Sodium acetate was supplemented to provide sufficient electron donors for denitrification,\nresulting in an operational C/N ratio of 4.9 ± 0.3 (Supplementary Fig. 2).\" (Lines 116-118)\n\"To enhance denitrification and meet the designed target, sodium acetate was supplemented\ninto the anoxic zone, with the resultant C/N ratio being maintained at 4.9 ± 0.3 (Supplementary\nFig. 2).\" (Lines 380-382)\nThis revision makes it clear that the C/N ratio of 4.9 was the operational result of the carbon\ndosing strategy required to achieve sufficient denitrification and meet the discharge standards,\nnot a predetermined target. This empirically determined ratio, while specific to our system's\nobjective, falls within the range documented in prior studies for effective denitrification1,\nconfirming its practical viability.\n3. Impact on Microbial Community and Generalizability:\nThe addition of acetate enriches denitrifying bacteria, which is the intended functional outcome.\nA low C/N ratio would cause denitrification to fail, which in turn would impair the nitrification\nprocess due to metabolite accumulation, disrupting the metabolic balance of the A/O system.\nThe microbial community structure over the long term is ultimately shaped by environmental\nfactors (e.g., temperature, carrier type). Since carbon addition is standard practice in full-scale\nplants facing low C/N influent, the microbial communities and high performance achieved in\nour study are directly relevant and generalizable to such real-world applications.\nWe believe these revisions and clarifications fully address your concerns.\nReference:\n1. Li, J. et al. Quantify the contribution of anammox for enhanced nitrogen removal through\nmetagenomic analysis and mass balance in an anoxic moving bed biofilm reactor. Water\nRes. 160, 178-187 (2019).\n4. The manuscript reports phosphorus removal efficiency up to 52% under fully aerobic biofilm\nconditions. This value appears high given that conventional enhanced biological phosphorus\nremoval typically requires alternating anaerobic and aerobic phases. The authors should clarify\nwhether the V-carrier design created localized micro-anoxic or anaerobic zones within the\nbiofilm or provide an alternative explanation for the observed phosphorus removal. This\nclarification is important to accurately interpret the results and the functional contribution of\nthe carrier design.\nResponse:\nWe sincerely thank you for this insightful comment regarding the observed phosphorus\nremoval and its potential mechanism. We appreciate the opportunity to clarify this point.\nYou are correct that conventional enhanced biological phosphorus removal (EBPR) requires\nalternating anaerobic/aerobic conditions, which were not present in our fully aerobic system.\nThe phosphorus removal efficiency of up to 52% should be interpreted with important context:\n1. Context of the Reported Value:\nThe value of 52% represents the upper end of a wide performance range (8–52%), with an\naverage removal efficiency of 19% (Supplementary Fig. 4), which is consistent with the limited\nphosphorus removal capacity typical of pure biofilm systems relying solely on aerobic\nphosphorus assimilation for cell growth. The higher removal rates were primarily observed\nduring periods of lower influent phosphorus concentration, rather than indicating an enhanced\nintrinsic removal capability.\n2. Explanation for Removal Mechanism:\nThe observed removal is attributed to standard aerobic phosphorus assimilation for microbial\ncell synthesis, not to EBPR. The extended sludge retention time (SRT) of the biofilm allows for\na stable microbial biomass, but the efficiency of this assimilation process is inherently low and\nvariable, as reflected in the wide performance range. There is no evidence that the V-carrier\ndesign created localized anaerobic conditions sufficient to support EBPR.\n3. Practical Implication and Revision:\nThe variability and general limitation of biological phosphorus removal in biofilm systems are\nwell-recognized in practice. To address this point, we have added the following discussion in\nthe manuscript (Lines 140-143):\n\"In practical applications, this limitation is routinely and effectively overcome by coupling the\nbiofilm process with a downstream chemical phosphorus removal step35, which is a standard\nand cost-comparable practice also employed following conventional activated sludge processes\nto meet stringent effluent standards.\"\nTherefore, the results confirm that the V-carrier system, like other pure biofilm systems, does\nnot overcome the inherent limitation of low biological phosphorus removal, and the occasional\nhigher removal rates are linked to operational conditions rather than to a unique design feature.\nWe believe this clarification accurately interprets the results and confirms the functional role of\nthe carrier design within the expected boundaries of pure biofilm system performance.\n5. The manuscript attributes the decline in ammonium removal rate (ARR) on the Mutag-\nbiochip to biofilm accumulation and inorganic buildup. However, it is unclear how biofilm\naccumulation was directly measured or confirmed. The authors should clarify whether this\nconclusion is based on biofilm thickness measurements over time, microscopy observations,\nVSS/SS ratio tracking, or solely on ash content. Providing explicit evidence would strengthen\nthe link between biofilm accumulation and the observed decrease in ARR.\nResponse:\nWe sincerely thank you for this critical comment regarding the evidence linking the decline in\nammonium removal rate (ARR) to biofilm accumulation and inorganic scaling on the Mutag-\nbiochip carrier. We agree that providing explicit evidence is essential.\nYou are correct in noting that the direct measurement of biofilm accumulation over time is\ncrucial. Our conclusion was primarily based on tracking the Volatile Suspended Solids to\nSuspended Solids (VSS/SS) ratio in the biofilm, which serves as a key indicator of biofilm\nhealth and inorganic accumulation.\n1. Evidence from VSS/SS Ratio:\n• The VSS fraction primarily represents the active microbial biomass.\n• The SS fraction includes both organic (VSS) and inorganic components.\n• Therefore, a declining VSS/SS ratio signals that inorganic precipitates are\naccumulating within the biofilm matrix, increasing the total solids (SS) without\ncontributing to metabolic activity. This is a direct indicator of clogging and scaling.\nIn the case of the Mutag-biochip carrier, we observed a clear decline in the VSS/SS ratio over\nthe operational period (Fig. 3c). This provides direct evidence that the biofilm was experiencing\nsignificant inorganic accumulation (e.g., scaling, Supplementary Table 1), which physically\nclogged the carrier, impaired mass transfer, and led to the decrease in ARR.\nTo make this mechanistic interpretation immediately clear to the reader, we have revised the\nfigure legend for Fig. 3 (Lines 491-495) to include the following explanatory note:\n“a, b, ... The VSS/SS ratio is a key indicator of biofilm health; a high, stable ratio signifies a\nbiofilm with minimal inorganic scaling and high metabolic activity. c, f–h, ... The declining\nVSS/SS ratio in panels c, f and h indicates inorganic accumulation and clogging.”\nA more comprehensive explanation of the VSS/SS ratio as a diagnostic tool is provided in\nSupplementary Note 1.\n2. Broader Implications and Validation:\nThis observation strongly supports a fundamental insight of our study: that treatment efficiency\nis decoupled from sheer biomass quantity. A carrier may accumulate a large amount of biomass\n(high SS), but if a significant portion is inactive inorganic material (low VSS/SS ratio), its\nactivity per unit of biomass becomes very low. This is further validated by the contrasting\nperformance of our V-carrier, which, as presented in the Discussion, maintained 44% less\nbiomass (VSS) than a conventional carrier yet achieved a 3.2-fold higher nitrification rate,\ndemonstrating that a healthy, efficient biofilm ecosystem is paramount.\nWe believe this evidence, along with the clarifications added to the manuscript, robustly\nsupports the link between inorganic buildup, biofilm dysfunction, and the observed\nperformance decline.\nReviewer #4:\nThis manuscript presents an innovative “hydro-topological” design strategy for MBBR carriers,\nexemplified by the V-carrier, which enables self-regulation of biofilm thickness and continuous\nself-cleaning. The study demonstrates superior pollutant removal performance and stability\nover more than 500 days of operation with real wastewater, including under low-\ntemperature conditions. The experimental design is rigorous, with comprehensive data on performance,\nmechanisms, and microbial ecology. The integration of hydraulic principles with biofilm\ndynamics represents a meaningful contribution to the field of wastewater treatment\ntechnologies. However, several aspects require clarification and enhancement to strengthen the\nmanuscript’s scientific rigor and clarity. The work is promising but would benefit from\nrevisions to address the points below.\nResponse:\nWe sincerely thank you for your positive assessment of our work and for the constructive\nfeedback aimed at strengthening the scientific rigor and clarity of our manuscript. We are\ngrateful for your recognition of the study's innovation, rigorous experimental design, and\ncontribution to the field.\nIn direct response to your overarching comments and specific points, we have undertaken a\ncomprehensive revision of the manuscript. The key enhancements, detailed in our point-by-\npoint responses below, include:\n1. Enhanced Explanation of Principles: We have significantly expanded the discussion to\nelucidate the physical and biological principles of the \"hydro-topological\" strategy.\nThis includes a clearer exposition of the relationships between carrier geometry,\nhydraulic shear, and biofilm dynamics, supported by new CFD analyses and\nexperimental data (Response to Comments #1, #2, #10).\n2. Strengthened Mechanistic Evidence: As you suggested, we have integrated quantitative\nComputational Fluid Dynamics (CFD) simulations to move beyond inference and\nprovide direct, visual evidence of the shear forces and flow fields acting on different\ncarrier designs. This provides a solid, mechanistic foundation for discussing the hydro-\ntopological strategy (Response to Comments #2, #5).\n3. Improved Context and Discussion: We have enriched the discussion to better\ncontextualize our findings. This includes addressing the practical implications of\nperformance variability (e.g., phosphorus removal, Comment #3), clarifying key\nconcepts such as the decoupling of efficiency from biomass (Comment #4), and\ndiscussing the theoretical rationale behind observed optimal parameters (e.g., biofilm\nthickness, Comment #10).\n4. Methodological Precision and Completeness: We have ensured the precise use of\nterminology (e.g., \"structural porosity,\" Comment #5; consistent use of \"V-carrier\" and\n\"hydro-topological strategy,\" Comment #6) and provided all necessary methodological\ndetails, such as supplier information and catalog numbers, to enhance reproducibility\n(Comment #7).\n5. Explicit Address of Scale-up Challenges: A significant addition to the discussion\nexplicitly addresses the practical challenges of scaling up, including operational costs\nand long-term mechanical wear, based on the results of our pilot-scale study (Response\nto Comments #8, #11).\nWe are confident that these revisions have fully addressed your concerns and significantly\nenhanced the manuscript's clarity, depth, and scientific rigor. Thank you for the insightful\nsuggestions that have helped us improve the quality and impact of our work.\n1. The “hydro-topological” strategy integrates carrier geometry, hydraulic shear, and microbial\necology into a unified framework. Consider expanding the explanation of its physical and\nbiological principles in the discussion section. A simplified diagram or model could be added\nto illustrate relationships, such as those between opening ratio, groove depth, and shear forces.\nResponse:\nWe sincerely thank you for this valuable suggestion to enhance the explanation of the hydro-\ntopological strategy's principles. We agree that a clearer illustration of the relationships between\nkey parameters is essential.\nIn response, we have significantly expanded the discussion in the manuscript (Lines 293-298)\nto elucidate the physical principles linking carrier geometry to hydraulic conditions and biofilm\ndevelopment. The added text explicitly demonstrates how hydro-topological parameters govern\nbiofilm thickness:\n\"Validation tests using a V-carrier with a larger opening ratio (1.5/0.72 = 2.08) confirmed a\nsignificantly thinner biofilm (~240 μm) (Fig. 6f). Crucially, CFD simulations revealed a ~44%\ndownward shift of the iso-velocity contours within the groove, which correlated with the\nobserved biofilm reduction, demonstrating that hydro-topological parameters (hydraulic shear,\nopening ratio and groove depth) exert absolute control over biofilm thickness by dictating the\nhydraulic shear environment (Fig. 6a-f).\"\nThis addition, supported by the new experimental data and CFD analysis in Fig. 6 (Fig. R9),\nprovides a clear cause-and-effect explanation: adjusting the opening ratio directly alters the\nhydraulic shear profile, which in turn dictates the resulting biofilm thickness. We believe this\nrevision effectively illustrates the core physical and biological principles of the hydro-\ntopological framework, as you recommended, by visually and quantitatively linking design\nparameters to performance outcomes.\nFig. R9 (Fig. 6) | CFD-simulated flow fields and biofilm morphology of carriers after 405\ndays of operation. a–b, Flow fields around a V-carrier with an opening ratio of 1.39 (1.0/0.72)\nunder (a) parallel and (b) 45° flow incidence. c, Biofilm morphology on the stably-operated V-\ncarrier with opening ratio 1.39. d–e, Flow fields around a V-carrier with an opening ratio of\n2.08 (1.5/0.72) under (d) parallel and (e) 45° flow incidence. f, Biofilm morphology on the\nstably-operated V-carrier with opening ratio 2.08. g–h, Flow fields (parallel flow) around\ncarriers with cell dimensions of 2.3 × 2.3 mm and ridge heights of (g) 1.0 mm and (h) 0.5 mm.\ni, Biofilm morphology on the MK5 carrier. j–k, (j) Flow field (parallel flow) and (k) biofilm\nmorphology of the U-carrier. l–m, (l) Flow field (parallel flow) and (m) biofilm morphology of\nthe K3 carrier. n–o, (n) Flow field (parallel flow) and (o) biofilm morphology of the K5 carrier.\n2. The inclusion of over 500 days of real wastewater treatment data is noted. However, direct\nmeasurements of hydraulic shear forces are lacking, with reliance on inferences from the\nHagen-Poiseuille equation and carrier structure. Are there any methods such as computational\nfluid dynamics simulations or experimental measurements could provide quantitative data on\ncarrier surface shear forces?\nResponse:\nWe sincerely thank you for this insightful comment regarding the quantitative assessment of\nhydraulic shear forces. We agree that direct measurements or simulations are crucial for\nstrengthening the mechanistic interpretation.\nIn response to your suggestion, we have conducted comprehensive CFD simulations to\nquantitatively analyze the flow fields and shear stresses on the carrier surfaces. While we\nobtained specific shear stress values, we note that the absolute magnitude is highly sensitive to\nsimulation setup and hydraulic conditions. Therefore, we focused our analysis on the relative\ndifferences in the flow field patterns (e.g., iso-velocity contours), which provide a more robust\nand generalizable indicator of hydraulic shear effects.\nThe key findings from the CFD simulations have been integrated into the revised manuscript:\n1. For the V-carrier: As detailed in our response to Comment #1 above, we have added\ntext in Lines 294-299 demonstrating how hydro-topological parameters control biofilm\nthickness through hydraulic shear regulation.\n2. For conventional tubular carriers (Lines 283-286): “CFD simulations under identical\nhydraulic conditions revealed that a smaller tube diameter results in lower internal flow\nvelocity, which in turn leads to reduced shear stress (Fig. 6l-o), a phenomenon directly\nsupported by the Hagen-Poiseuille equation.”\nBy supplementing the long-term operational data with these quantitative CFD analyses, we\nhave moved beyond inference to provide direct evidence of the relationship between carrier\ngeometry and hydraulic conditions. We believe these additions successfully address your\nconcern by providing a more rigorous foundation for discussing the hydro-topological strategy.\n3. The explanation for variability in phosphorus removal performance (lines 125-126) is brief.\nA discussion could address whether this reflects inherent limitations of pure biofilm systems\nand if additional chemical phosphorus removal measures might be relevant in practical\napplications.\nResponse:\nWe sincerely thank you for this insightful comment regarding the variability in phosphorus\nremoval and its practical implications.\nYou are correct that the observed variability in phosphorus removal (8–52%) is an inherent\ncharacteristic of pure biofilm systems, which lack the anaerobic-aerobic cycling necessary for\nenhanced biological phosphorus removal (EBPR) and rely on the limited phosphorus\nassimilation for cell growth.\nTo address your suggestion and strengthen the discussion, we have revised the manuscript in\nthe Results section (Lines 140-143). The text now explicitly discusses the practical context:\n\"In practical applications, this limitation is routinely and effectively overcome by coupling the\nbiofilm process with a downstream chemical phosphorus removal step35, which is a standard\nand cost-comparable practice also employed following conventional activated sludge processes\nto meet stringent effluent standards.\"\nThis addition clarifies that the inherent limitation of biofilm systems is well-understood in\nengineering practice and is mitigated by a standard, cost-effective solution, thereby\ncontextualizing the performance variability and affirming the practical viability of the\ntechnology.\nWe believe this revision directly addresses your concern by linking the observed performance\nto established engineering solutions.\n4. The claim in lines 15-16 that treatment efficiency decouples from biomass quantity may refer\nto the unit biomass nitrification rate comparison between the V-carrier and K3 carrier.\nAdditionally, is it possible that the observation in lines 186-189 regarding spatial redistribution\nof nitrifying bacteria under low temperatures could potentially relate to changes in\ndissolved oxygen gradients?\nResponse:\nWe sincerely thank you for this insightful comment regarding the interpretation of our key\nfindings. We appreciate the opportunity to clarify these points.\n1. Clarification on the Decoupling of Efficiency from Biomass:\nYou are correct in your interpretation. The claim that “treatment efficiency decouples from\nbiomass quantity” indeed refers to the comparison of the unit biomass nitrification rate (i.e.,\nnitrification rate per gram of volatile suspended solids, g N g VSS⁻¹ day⁻¹) between the V-carrier\nand the conventional K3 carrier.\nTo make this comparison explicit and avoid any potential misunderstanding, we have revised\nthe sentence in the manuscript (Lines 15-17) to read:\n\"Crucially, the V-carrier achieved a 3.2-fold higher unit biomass nitrification rate with a biofilm\nbiomass 44% lower than a conventional K3 carrier, …\"\n2. Discussion on Dissolved Oxygen Gradients and Microbial Redistribution:\nWe thank you for the excellent suggestion regarding the potential role of dissolved oxygen (DO)\ngradients in the observed spatial redistribution of nitrifying bacteria at low temperatures. We\nagree that this is a plausible and important mechanistic factor.\nThe decline of the putative denitrifier norank_f__AKYH767likely results from a combination\nof factors, including limited substrate access and reduced enzymatic activity at low\ntemperatures. As you suggested, changes in the DO gradient could be a key contributor. The\noutward migration of nitrifiers (Fig. 4d) may be associated with an elevated DO concentration\nin the inner biofilm layers to maintain their metabolic activity, which would create a more\naerobic microenvironment less favorable for denitrifiers like norank_f__AKYH767.\nTo incorporate this insightful perspective, we have added a discussion of this potential\nmechanism in the revised manuscript (Lines 245-248). The text now reads:\n\"This decline likely resulted from combined pressures of limited substrate access and reduced\nenzymatic activity at low temperatures, potentially exacerbated by the outward migration of\nnitrifiers within the biofilm (Fig. 4d), a shift that may be associated with elevated dissolved\noxygen concentrations in the inner biofilm layers to sustain nitrification, thereby creating a less\nfavorable microenvironment for denitrifiers. This could further limit nitrate diffusion to inner-\nlayer denitrifiers.\"\nWe believe this addition significantly enriches the mechanistic discussion and will investigate\nthe dynamics of DO gradients within the biofilm in our future work, as you recommended.\nThank you for these valuable suggestions, which have helped us strengthen the clarity and depth\nof our manuscript.\n5. Precise geometric parameters for the V-carrier are provided (depth 0.72 mm, opening 1.0\nmm, opening ratio 1.39). Clarify how this specific opening ratio was determined? Please\nprovide a brief description in the methods or results section.\nResponse:\nWe sincerely thank you for this important question regarding the determination of the specific\nopening ratio (1.39) for the V-carrier.\nThe selection of this opening ratio was not arbitrary but was determined through comparative\nexperimental optimization and subsequent validation via CFD modeling. The process and\njustification are as follows:\n1. Experimental Optimization: We designed and tested V-carriers with different opening\nratios, specifically comparing a ratio of 2.08 (1.5/0.72) against 1.39 (1.0/0.72). The\nexperimental results clearly showed that the larger opening ratio (2.08) led to excessive\nhydraulic shear, resulting in a significantly thinner biofilm (~240 μm), which is\nsuboptimal for maintaining a stable and active biomass inventory. This comparative\ndata is now provided in Fig. 6f (see Comment #1 above).\n2. CFD Modeling Validation: To understand the hydrodynamic mechanism behind these\nexperimental results, we conducted CFD simulations. These simulations revealed that\nthe larger opening ratio (2.08) caused a ~44% downward shift of the iso-velocity\ncontours within the groove compared to the 1.39 design, directly correlating with the\nobserved over-thinning of the biofilm. This provides a quantitative, mechanistic\nexplanation for why the 1.39 ratio provides a more balanced hydraulic environment\n(Fig. 6a-f, see Comment #1 above).\nThe comprehensive explanation of how this opening ratio was determined and validated has\nbeen added to the manuscript in the discussion section (Lines 293-298), as detailed in our\nresponse to Comment #1 above.\nIn summary, the opening ratio of 1.39 was empirically identified as optimal because it provides\nsufficient hydraulic exposure to maintain biofilm activity and prevent clogging, while avoiding\nthe excessive shear that leads to an overly thin and potentially unstable biofilm. We believe this\nexperimental and modeling evidence fully clarifies the basis for this specific geometric\nparameter.\n6. Ensure consistent terminology throughout, such as “V-carrier” and “hydro-topological\nstrategy.”\nResponse:\nWe thank you for this important reminder regarding the consistency of terminology throughout\nthe manuscript.\nWe have carefully reviewed the entire text to ensure consistent use of key terms, such as “V-\ncarrier” and “hydro-topological strategy.” As a specific example, we have revised the section\ntitle in the manuscript (now at line 148) from “Mechanism of biofilm regulation—Hydro-\ntopological synergy” to “Mechanism of biofilm regulation—Hydro-topological strategy” to\nalign with the terminology used elsewhere.\nWe appreciate this suggestion, which has helped improve the overall clarity and professionalism\nof the presentation.\n7. The supplier details for carriers (beyond the V-type) and catalog numbers for reagent kits are\nomitted in methods section.\nResponse:\nWe thank you for this important reminder to ensure the completeness of our methodological\ndescription.\nWe have addressed the omissions as follows:\n1. The catalog numbers for all reagent kits have been added to the Methods section at\nLines 396 and 412.\n2. As you suggested, the supplier details and photographic documentation for all carriers\nused in the comparative study (beyond the V-carrier) have been provided in\nSupplementary Fig. 5 to give a clear and systematic overview.\nWe believe these additions provide the necessary details for reproducibility and thank you for\nhelping us improve the manuscript.\n8. Engineering parameters, such as effective biofilm surface area and bulk density, are reported\nand useful for applications. The discussion could briefly address potential challenges in scaling\nto pilot or full-scale operations, including large-scale production costs and long-term\nmechanical wear.\nResponse:\nWe sincerely thank you for this valuable comment regarding the engineering parameters and\nthe practical challenges of scaling up the V-carrier technology.\nWe have addressed your concerns as follows:\n1. Clarification of Engineering Parameters: As you noted, the key engineering parameters\nfor the V-carrier, including the effective biofilm surface area, protected surface area,\nbulk density, and structural porosity, are reported in the Results section (Lines 106-111).\nFurthermore, the detailed calculation methodologies for these parameters are provided\nin the Supplementary Equations to ensure clarity and reproducibility.\n2. Discussion of Scale-up Challenges and Validation: In direct response to your\nsuggestion to address scaling challenges, we have significantly expanded the\nDiscussion section (Lines 345-357). The added text explicitly discusses the practical\nimplications based on pilot-scale data, covering the key points you raised. The revised\nparagraph reads:\n\"From an engineering perspective, the hydro-topological design of the V-carrier translates into\nsignificant practical advantages for large-scale implementation. Pilot-scale fluidization tests\ndemonstrated the V-carrier's superior hydrodynamic efficiency: it requires over 30% less\naeration intensity than conventional cylindrical carriers for complete mixing, a result of the\nasymmetric flow around its grooves generating high drag for efficient motion, unlike the\nbalanced pressure and minimal torque from the cross-flow in cylindrical carriers which leads\nto less efficient movement (Supplementary Fig. 11). This confirms that the V-carrier’s design\ndirectly addresses a major operational cost factor. Furthermore, long-term pilot operation\ntreating real municipal wastewater over 400 days confirmed the system's robust stability, with\nno carrier clogging or performance decay observed (Supplementary Fig. 12). The V-carriers\nmaintained excellent physical integrity, with only slight wear at the corners of the anoxic\ncarriers attributed to mechanical stirring—an issue being addressed by redesigning the blades.\nThis successful validation underscores the V-carrier's potential for energy-efficient and reliable\nlarge-scale application.\"\nThis addition directly tackles:\nEconomic Viability (Operational Cost): by quantifying the reduction in aeration energy.\no\nLong-term Mechanical Wear and Stability: by presenting results from a 400-day pilot\no\nstudy and addressing the observed wear with an engineering solution.\nBy integrating this pilot-scale validation and discussion, we move from theoretical parameters\nto demonstrated practical performance. We believe this provides a strong and realistic\nfoundation for assessing the V-carrier's potential for full-scale application.\n9. The finding in lines 175-177 that ex situ nitrification potential exceeds in situ activity is\nattributed to substrate limitations. The authors could consider discussing additional possible\nfactors.\nResponse:\nWe sincerely thank you for this insightful comment, which encourages a deeper discussion of\nthe factors contributing to the observed difference between ex situ and in situ nitrification\nactivity.\nYou are correct that while substrate limitation is a key factor, other operational conditions may\nalso play a role. In the ex situ assays, temperature, pH, and dissolved oxygen (DO) were strictly\ncontrolled at optimal levels, whereas in the actual reactor, these parameters fluctuated naturally\nwithin a certain range over daily operation cycles.\nTo incorporate this perspective, we have expanded the discussion in the revised manuscript\n(Lines 198-199). The relevant sentence now reads:\n\"Notably, the ex situ rates surpassed the in situ performance by factors of 1.55 (< 15 °C) and\n3.18 (> 15 °C), indicating that the biofilms possessed significant intrinsic metabolic capacity\nthat was not fully expressed under the reactor's substrate-limiting and dynamically fluctuating\noperational conditions (e.g., temperature, pH, dissolved oxygen).\"\nWe believe this addition provides a more comprehensive explanation and thank you for the\nsuggestion, which has strengthened the interpretation of our results.\n10. The V-carrier maintains a stable biofilm thickness of 0.72 mm with reported performance\nbenefits. The discussion could include an explanation of why approximately 0.72 mm falls\nwithin a theoretical optimal range, potentially referencing literature on substrate penetration\ndepths (e.g., oxygen and ammonia) to support how this thickness balances active layer\nmaximization and mass transfer resistance minimization.\nResponse:\nWe sincerely thank you for this insightful comment regarding the theoretical basis for the\nobserved optimal biofilm thickness (~500-550 μm) maintained by the V-carrier. We agree that\nexplaining how this thickness balances active layer maximization and mass transfer resistance\nis crucial.\nIn response, we have expanded the discussion in the manuscript (Lines 152-153) to provide a\nclear rationale. The revised text now reads:\n\"The V-carrier maintained a stable volatile suspended solids-to-suspended solids (VSS/SS)\nratio and consistent biofilm thickness of 500–550 μm—a range proven to benefit mass transfer\nand promote the removal of diverse micropollutants17—under both anoxic and aerobic\nconditions throughout long-term operation, …\"\nThis addition directly links the maintained biofilm thickness to its demonstrated functional\nadvantages—improved mass transfer and enhanced micropollutant removal—thereby\naddressing the \"why\" behind the specific thickness.\nFurthermore, the comparison with the Mutagbiochip carrier—which was similarly designed for\na ~550 μm biofilm thickness but failed due to clogging—demonstrates that geometric constraint\nalone is insufficient without an integrated self-cleaning capability. This contrast highlights that\nthe V-carrier's success lies not merely in targeting a specific thickness, but in its ability to\nactively maintain it through hydrodynamic regulation.\nWe acknowledge that the effectiveness of a given biofilm thickness is context-dependent. This\naligns with the principle, noted in the Introduction (Lines 49-51), that biofilm thickness is a\ncritical parameter governing microbial community structure and mass transfer dynamics. The\nstrength of the V-carrier lies in its hydro-topological design, which provides a versatile\nframework to achieve and stabilize a target biofilm thickness that is appropriate for the intended\napplication, rather than merely imposing a fixed geometric constraint.\nWe believe these revisions successfully address your comment by clarifying the rationale\nbehind the observed thickness and emphasizing the importance of active thickness control over\npassive geometric confinement.\n11. Over 500 days operation, indicate whether any physical wear, deformation, or gradual\nperformance decline was observed in the carriers.\nResponse:\nWe thank you for this important question regarding the physical stability of the carriers over\nthe long-term operation, which is indeed critical for practical application feasibility.\nIn this study, after more than 500 days of operation, both the aerobic and anoxic phase carriers\nmaintained their original morphology, with no visible wear observed, and their performance\nshowed high stability (Fig. 2). Furthermore, our pilot-scale results (Supplementary Fig. 12; Fig.\nR10) demonstrate the excellent physical stability of the carriers under real-world conditions.\nAfter over 400 days of operation, the aerobic phase carriers retained their original appearance\nwith no visible wear. The anoxic phase carriers showed slight wear at the four corners, which\nwas attributed to direct impact from the mechanical stirring blades during long-term operation.\nTo address this, we have redesigned the stirring blades with larger curvature in our ongoing\nwork to minimize such long-term wear issues. Importantly, the pilot-scale results confirm that\nthe V-carrier system operated stably for 400 days without clogging or performance decline,\ndemonstrating its strong potential for real-world applications.\nFor detailed information on the pilot study, please refer to our response to Comment #8 above.\nFinally, we sincerely thank you for all your valuable comments, which have significantly\nimproved the quality of our manuscript.\nFig. R10 (Supplementary Fig. 12) | Pilot plant of the anoxic/aerobic-moving bed biofilm\nreactor (A/O-MBBR) process and overall morphology of V-carriers after long-term\noperation. a, Installation site and overall view of the pilot plant. b-c, Field views of the anoxic\n(b) and aerobic (c) reactors, respectively. d, Carriers during complete fluidization. e, Clean and\ndried V-carriers. f-g, Morphology of biofilm-attached anoxic (f) and aerobic (g) carriers after\n409 days of operation. h-i, Morphology of biofilm-detached anoxic (h) and aerobic (i) carriers\nafter 409 days of operation. The pilot system was commissioned on November 12, 2024, and\nremains operational. It has a total working volume of 24 m³, comprising a 9 m³ anoxic reactor\nand a 15 m³ aerobic reactor. It operates in a pure biofilm mode (no sludge recirculation) with a\n300% internal nitrified liquor recycle ratio. The system treats municipal wastewater with\ninfluent characteristics similar to those used in this study (see Supplementary Table 3 for\nconcentrations). Operating at a 9-hour hydraulic retention time, it achieves nitrification and\ndenitrification rates comparable to the laboratory-scale results. No carrier clogging has been\nobserved, and the operational stability of the system has been fully validated."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have addressed my concerns.", "reviewer_3": "Thank you for submitting the revised version of the manuscript.\nI have carefully reviewed the updated paper and would like to acknowledge the considerable effort, made in addressing the\nreviewers’ comments.\nThe revisions have been satisfactorily implemented, and the manuscript has significantly improved in clarity and technical\nquality.\nBased on the revised version, the requested modifications are considered acceptable, and the manuscript is approved for\npublication.", "reviewer_4": "The authors have satisfactorily resolved all the issues I raised. I have no additional concerns and support the publication of\nthe manuscript in its current form."}, "author_response": ""}]

Decision letter