# In situ-generated palladium nanoparticles promoted co-reduction of bromate and nitrate in hydrogenotrophic biofilms

Chengyang Wu, Guirong Liang, Siqing Xia, Jingzhou Zhou, Eric Lichtfouse, Xiang Cai, Suyun Xu, Hongbo Liu

► To cite this version:

Chengyang Wu, Guirong Liang, Siqing Xia, Jingzhou Zhou, Eric Lichtfouse, et al.. In situ-generated palladium nanoparticles promoted co-reduction of bromate and nitrate in hydrogenotrophic biofilms. Water Research, 2025, 286, pp.124303. 10.1016/j.watres.2025.124303. hal-05191136

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# *In situ*-generated palladium nanoparticles promoted co-reduction of bromate and nitrate in hydrogenotrophic biofilms

Chengyang Wu<sup>a,b</sup>, Guirong Liang<sup>a</sup>, Siqing Xia<sup>c</sup>, Jingzhou Zhou<sup>c</sup>, Eric Lichtfouse<sup>d,e</sup>,
Xiang Cai<sup>c,f</sup>, Suyun Xu<sup>a,b</sup>, Hongbo Liu<sup>a,b,*</sup>

## ARTICLE INFO

### Keywords:

- Bromate

- Nitrate

- Palladium nanoparticles

- Hydrogenotrophic biofilms

- Membrane biofilm reactor

## ABSTRACT

Bromate, a carcinogenic disinfection byproduct, threatens water safety due to its persistence and health risks. Although microbial reduction of bromate is a sustainable remediation approach, its efficiency is often hampered by the common co-contamination of nitrate. We addressed this issue by generating palladium nanoparticles (PdNPs) *in situ* within the biofilm matrix of a membrane biofilm reactor (MBfR), creating a Pd-MBfR. Co-reduction of bromate and nitrate by the Pd-MBfR was investigated in terms of bromate removal, reduction kinetics, and microbial functions. To evaluate the impact of catalytic hydrogenation, a non-palladized MBfR was operated in parallel. Continuous operation over 125 days demonstrated that the Pd-MBfR reduced bromate concentration (4 mg·L⁻¹) up to 70 %, versus 30 % for the conventional MBfR under competitive nitrate or nitrite conditions. Kinetic modeling revealed that extracellular Pd-catalyzed bromate reduction diverted approximately 18 % of bromate flux from intracellular NADH-dependent pathways, while biogenic PdNPs rapidly scavenged nitrite via catalytic hydrogenation, diverting 39 % of intracellular electron flux from denitrification to extracellular catalytic reduction. Under nitrate stress, biofilms in the Pd-MBfR maintained syntrophic interactions between bromate-reducing bacteria *Dechloromonas* and homoacetogens *Acetobacterium*, whereas the conventional MBfR favored autotrophic denitrifiers *Hydrogenophaga* and *Rhodoblastus* that prioritized nitrate reduction. Functional-gene profiling confirms that the intracellular electron flow from hydrogen to NADH-dependent denitrification reductases was displaced by extracellular Pd-catalyzed hydrogenation. This diversion of electron flow enhanced bromate reduction in biofilms coupled with PdNPs by minimizing competition for intracellular NADH.

## 1. Introduction

Bromate (BrO₃⁻) is a toxic disinfection byproduct generated during advanced oxidation processes when bromide (Br⁻) from natural (e.g., volcanic rock leaching, seawater intrusion) and anthropogenic (e.g., ethylene dibromide in gasoline) sources serve as a precursor for BrO₃⁻ generation (Butler et al., 2005). BrO₃⁻ is classified as a Group 2B carcinogen by the World Health Organization (IARC, 1999), stringent regulatory limits (10 µg·L⁻¹ in Europe and the United States) have been established for drinking water (EPA, 2006; IARC, 1999). Despite its

widespread detection in surface and groundwater systems, conventional removal technologies such as membrane filtration and ion exchange remain constrained by high energy demands, operational costs, and secondary brine waste generation (Xiao et al., 2017). In contrast, biological reduction strategies using gaseous electron donors such as hydrogen (H₂) and methane (CH₄), are a sustainable alternative due to their low cost, minimal residuals, and compatibility with biofilm-based systems (Shi et al., 2021; Zhang et al., 2024). For instance, delivering the gaseous donor via hollow membranes that support biofilms has been demonstrated efficacy in BrO₃⁻ reduction (Luo et al., 2017; Zuo et al.,

<footnote><sup>a</sup> School of Environment and Architecture, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai 200093, China</footnote>

<footnote><sup>b</sup> School of Intelligent Emergency Management, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai 200093, China</footnote>

<footnote><sup>c</sup> State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China</footnote>

<footnote><sup>d</sup> State Key Laboratory of Multiphase Flow in Power Engineering, Xi'an Jiaotong University, 28 Xianning West Rd, Xi'an, Shaanxi 710049 PR China</footnote>

<footnote><sup>e</sup> College of Geography and Environmental Sciences, Zhejiang Normal University, Jinhua 321004, China</footnote>

<footnote>* Corresponding author at: School of Environment and Architecture, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai 200093, China.</footnote>

<footnote>E-mail address: Liuhb@usst.edu.cn (H. Liu).</footnote>
(2024).

Nonetheless, an issue for biological reduction is that nitrate ($NO_3^−$) is often present at much higher concentrations than $BrO_3^−$. For example, groundwater from a contaminated aquifer in the United Kingdom had a $BrO_3^−$ concentration of 1.1 – 1.4 mg·L⁻¹ and a $NO_3^−$·N concentration of 8.6 – 30.7 mg·L⁻¹ (Butler et al., 2006). $NO_3^−$ could have negative effects on $BrO_3^−$ reduction such as intracellular competition for electron donors. Moreover, in hydrogenotrophic biofilms, $H_2$ oxidation by hydrogenases generates reduced nicotinamide adenine dinucleotide (NADH) (Preissler et al., 2018), which serves as the primary electron carrier for respiratory reductases targeting $BrO_3^−$, $NO_3^−$, and nitrite ($NO_2^−$). As a result, this shared reliance on NADH creates a competition where $NO_3^−$ is the preferred electron acceptor, thus suppressing $BrO_3^−$ reduction even when the electron donor is available (Wang et al., 2018a; Zhong et al., 2018).

To mitigate this competition, this study proposes a strategy using in *situ*-generated palladium nanoparticles (PdNPs) associated within biofilms (Pd-biofilm). Pd-based catalysts have been documented for their ability to activate $H_2$ into reactive atomic hydrogen (H*), which can reduce oxyanions such as $BrO_3^−$, $CrO_4^{2−}$, and $ClO_4^−$ in extracellular pathways (Li et al., 2025; Ren et al., 2021; Wu et al., 2024). By diverting electron flow from intracellular NADH-dependent pathways to extracellular Pd-catalyzed hydrogenation, this approach should alleviate competition between $BrO_3^−$ and $NO_3^−$ reduction. This hypothesis is supported by previous work, which demonstrated that microbial enzymes driving $NO_3^−$ reduction to $NO_2^−$ and PdNPs accelerating $NO_2^−$ conversion to $N_2$ (Zhou et al., 2017). Here, we test this concept for $BrO_3^−$ and $NO_3^−$ co-reduction. The first objective of this study was to document the competitive suppression of $NO_3^−$ on $BrO_3^−$ reduction in hydrogenotrophic biofilms. The second objective was to investigate the impact of biogenic PdNPs on intracellular electron competition and $BrO_3^−$ reduction. These objectives were addressed using parallel $H_2$-based membrane biofilm reactors (MBfRs): One system configured conventional biofilms and the other contained in *situ*-generated PdNPs associated with biofilms. Microbial community and functional genes were analyzed to elucidate the mechanisms underlying intracellular electron partitioning and to clarify how PdNPs diverted the electron flow.

## 2. Materials and methods

### 2.1. Configuration of the membrane biofilm reactors

The experiments were conducted in identical MBfRs in parallel (shown in Figure S1, Supporting Information), following a design adapted from our previous study (Wu et al., 2021). Each reactor comprised two interconnected tubes via Viton tubing (Saint-Gobain, USA): a main column containing a bundle of 32 nonporous polypropylene hollow-fiber membranes (Teijin, Japan) and the other contained a coupon bundle of 10 identical fibers for periodic biofilm sampling. With a total working volume of 65 mL and 54 cm² of membrane surface area, the reactors were configured to maintain consistent hydrodynamics via a recirculation loop (150 mL min⁻¹) driven by a peristaltic pump (Longer, China). The temperature was consistently maintained at 25 ± 2 °C throughout the experiments.

### 2.2. Inoculation and startup of the membrane biofilm reactors

We introduced 10 mL of anoxic sludge obtained from the Wusong wastewater treatment plant (Shanghai, China) in two MBfRs, following a standardized protocol adapted from our previous work (Wu et al., 2022a). In brief, the inoculum was allowed to attach to the exterior surface of the hollow-fiber membranes to initiate the biofilm for 48 h. Subsequently, both reactors were continuously fed with a medium containing 10 mg·L⁻¹ $NO_3^−$·N and the basal nutrients listed in Supplementary Table S1 to promote biofilm development. After two months of acclimation, mature biofilms were established on the membrane surfaces. This startup procedure ensured consistent biofilm formation

across both reactors prior to experimental interventions.

### 2.3. In situ synthesis of biogenic Pd nanoparticles

To establish the palladized membrane biofilm reactor (Pd-MBfR), one of the MBfRs was transitioned to batch mode for PdNP synthesis. The reactor was drained and replenished with 65 mL of Pd(II)-enriched medium (160 mg·L⁻¹ Pd(II) as Na₂PdCl₄) under an $H_2$ pressure of 15 psig (2.0 atm, absolute pressure) to create PdNPs within biofilms (Pd-biofilm). The Pd(II)-deposition process lasted for 48 h until dark precipitates were observed on the fiber bundles of both reactors, and >99 % of the input Pd(II) was recovered, yielding a total Pd(II) retention of 10.4 mg and a surface loading of 1.9 g·Pd·m⁻². The unmodified reactor (designated MBfR) retained its original biofilm without PdNP.

A non-biofilm membrane Pd-film reactor (MPfR) was prepared by depositing PdNPs directly onto bare membrane fibers using identical Pd (II) concentrations and $H_2$ reduction conditions. This system achieved equivalent PdNP surface loading (1.9 g·m⁻²) but lacked microbial interactions, enabling differentiation between enzymatic and catalytic contributions.

### 2.4. Continuous operation for bromate reduction

The Pd-MBfR and the MBfR were operated in parallel under identical conditions for 125 days to evaluate bromate reduction performance. During Stage 1 (Days 1–70), reactors were fed with the basal medium (Table S1) containing $BrO_3^−$ at gradually increasing concentrations (1, 2, and 4 mg·L⁻¹) to acclimate the biofilms. In Stage 2 (Days 71–104), $NO_3^−$ was introduced by amending the influent with 4 mg·L⁻¹ $BrO_3^−$ and 1.5 mg·L⁻¹ $NO_3^−$, while Stage 3 (Days 105–125) shifted to $NO_2^−$ competition (4 mg·L⁻¹ $BrO_3^−$ + 1.5 mg·L⁻¹ $NO_2^−$). Steady-state conditions were confirmed in each stage when the effluent concentrations of $BrO_3^−$, $NO_3^−$, and $NO_2^−$ remained stable (variation < 5 %) for 3 consecutive days. The influent medium was degassed with $N_2$ for 15 min before being fed to the reactors, maintaining a dissolved oxygen (DO) concentration < 0.15 mg·L⁻¹. Hydrogen gas (99.99 %) was supplied at 20 psig and the hydraulic retention time (HRT) was maintained at 4.2-hour over 125 days of continuous operation. Key operational parameters and influent compositions for each stage are summarized in Supplementary Table S2, including the changes of $BrO_3^−$ concentration and subsequent $NO_3^−$ or $NO_2^−$ co-contaminant additions.

### 2.5. Short-term kinetics tests

Short-term tests were conducted using the Pd-MBfR, MBfR, and MPfR to systematically investigate the effects of $NO_3^−$ loading, $NO_2^−$ loading, and $BrO_3^−$ loading on $BrO_3^−$ reduction kinetics. These parallel tests were organized into three series (Supplementary Table S3), with HRT maintained at 4.2 h. Prior to each test, the three reactors were returned to the steady-state condition with an influent of 1 mg·L⁻¹ $BrO_3^−$, 1.5 mg·L⁻¹ $NO_3^−$, 20 psig $H_2$ pressure, and pH 7.0. For each short-term test, target parameters were adjusted and allowed to equilibrate over 5 HRTs before sampling commenced. Triplicate samples were collected at 6, 7, and 8 HRTs, and results are reported as mean values.

To quantify the contributions of intra- and extracellular electron flow in Pd-biofilm, kinetic modeling and comparative analysis of the three reactors were employed. Modeling for $BrO_3^−$ reduction kinetics in a steady-state continuous stirred tank reactor (CSTR) is detailed in Section S2 of Supporting Information (SI).

### 2.6. Analytical methods

Daily influent and effluent samples were collected using 10-mL syringes and immediately filtered through 0.22-µm PTFE membrane filters. The concentrations of Pd(II) were quantified by using inductively coupled plasma-mass spectrometry (ICP-MS, Agilent 7700, Agilent
Technologies, USA). And BrO<sub>3</sub><sup>-</sup>, NO<sub>3</sub><sup>-</sup>, and NO<sub>2</sub><sup>-</sup> were assayed using an ion chromatograph (Dionex Aquion, USA) with an AS19 column and AG19 precolumn, an eluent concentration of 15 mM KOH, and a 1 mL min<sup>-1</sup> flow rate. The concentration of ammonium (NH<sub>4</sub><sup>+</sup>) was determined by the standard colorimetric methods (Rice and Bridgewater, 2012). pH and DO were measured using a multi-Parameter Meter (HACH HQ40d, USA). Nitrous oxide (N<sub>2</sub>O) and nitric oxide (NO) were not analyzed as intermediates due to negligible accumulation under circumneutral pH conditions in both denitrifying biofilms and Pd-catalyzed nitrate reduction processes (Pang et al., 2022; Zhou et al., 2017).

## 2.7. Biofilm collection and solid-state characterization

At the end of Stage 3, Pd-biofilm samples from the Pd-MBfR were collected by excising 5-cm segments from coupon fibers, followed by chemical fixation and ultramicrotomy for transmission electron microscopy (TEM) analysis using transmission electron microscopy (TEM, Hitachi H-7650, Japan) and high-resolution electron microscopy (HRTEM; FEI F20, Netherlands). Biofilm solids scraped from main column fibers in both the Pd-MBfR and the MBfR were freeze-dried and analyzed via X-ray diffraction (XRD), X-ray photoelectron spectrum (XPS). Sample preparation protocols (fixation, sectioning) and instrument parameters are detailed in the published studies (Wu et al., 2022a, 2022b).

## 2.8. Analysis of microbial community and function

At the end of stages S1c, S2, and S3, biofilm samples were collected. We cut 25-cm sections from the coupon fiber in both the MBfR and the Pd-MBfR. Genomic DNA was extracted using a DNeasy PowerBiofilm Kit (QIAGEN GmbH, Germany) following the manufacturer's instructions. The quantity of the extracted DNA was determined by Picogreen on a Quantus fluorometer (Promega, USA) and through absorbance measurement using a Nanodrop 2000 (USA). The metagenomic sequencing and analysis processes are described in Section S3 of SI. Taxonomic annotations of the microbial community were carried out by comparing representative sequences of the non-redundant gene catalog against the NCBI-nr database. And functional annotation was performed using the DIAMOND alignment algorithm with an e-value cutoff of 1 × e<sup>-5</sup>, comparing against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and Clusters of Orthologous Groups of proteins (COG) databases. Gene abundance were normalized to Reads Per Kilobase Per Million mapped (RPKM) (Lawson et al., 2017).

$$RPKM_i = \frac{R_i \times 10^6}{L_i \times \sum_{i=1}^{n} R_i} \qquad (1)$$

where R<sub>i</sub> represents the number of reads of gene<sub>i</sub> (KB) in a given sample; L<sub>i</sub> represents the nucleotide length of gene<sub>i</sub>, Σ<sub>i=1</sub><sup>n</sup> R<sub>i</sub> represents the sum of all genes in the sample (KB).

To define if a certain functional gene was enriched in each biofilm compared to the bromate-reducing biofilm, we calculated the fold change as

$$FC = RPKM_i / RPKM_0 \qquad (2)$$

where RPKM<sub>i</sub> is the normalized abundance of a specific metabolic category to given genes in the Pd-biofilm (S1c, S2, or S3) or biofilm metagenomes (S2 or S3), and RPKM<sub>0</sub> is the abundance of a specific metabolic category to given genes in the biofilm metagenomes (S1c).

## 2.9. Fluxes calculations

The fluxes of the substrates (J) were calculated by

$$J = \frac{Q(C_0 - C_e)}{A} \qquad (3)$$

where J is the flux, mmol·m<sup>-2</sup>·d<sup>-1</sup>; Q is the influent flow rate, m<sup>3</sup>·d<sup>-1</sup>; C<sub>0</sub> and C<sub>e</sub> are influent and effluent concentrations of each substrate (BrO<sub>3</sub><sup>-</sup> and NO<sub>3</sub><sup>-</sup>), mmol·m<sup>-3</sup>; and A is the effective membrane surface area, m<sup>2</sup>. The reduction fluxes in electron equivalents (e<sup>-</sup>·meq·m<sup>-2</sup>·d<sup>-1</sup>) were calculated by Eq. (4) according to reaction stoichiometry:

$$BrO_{3}^{-} + 6e^{-} + 6H^{+} \rightarrow Br^{-} + 3H_{2}O \qquad (4)$$

The NO<sub>3</sub><sup>-</sup> flux in electron equivalents (e<sup>-</sup>·meq·m<sup>-2</sup>·d<sup>-1</sup>) was calculated from Eqs. (5)-(7):

$$NO_{3}^{-} + 5e^{-} + 6H^{+} \rightarrow 0.5N_{2} + 3H_{2}O \qquad (5)$$

$$NO_{2}^{-} + 3e^{-} + 4H^{+} \rightarrow 0.5N_{2} + 2H_{2}O \qquad (6)$$

$$NO_{3}^{-} + 2e^{-} + 2H^{+} \rightarrow NO_{2}^{-} + H_{2}O \qquad (7)$$

To indicate if the H<sub>2</sub> supply was limiting, the maximum H<sub>2</sub>-supply flux at applied H<sub>2</sub> pressure of 20 psig (i.e., 420 e<sup>-</sup>·meq·m<sup>-2</sup>·d<sup>-1</sup>) was calculated according to methods established by Tang et al. (2012).

# 3. Results and discussion

## 3.1. Characterization of biogenic PdNPs associated with biofilms

The TEM images in Fig. 1 illustrate the distribution, morphology, and size of PdNPs generated in Pd-biofilm (Fig. 1). PdNP-associated bacterial cells exhibited elliptical or circular morphologies, densely clustered within 10 µm of the fiber surface. Fig. 1b indicates that most PdNPs were well-dispersed on the surface of bacterial cells and within the extracellular polymeric substances (EPS). The particle size distribution of the PdNPs in Fig. 1c reveals that the average size is approximately 3.9 ± 0.4 nm based on 120 NPs. Lattice fringe measurements (Fig. 1d) yielded a mean spacing of 0.23 nm, corresponding to the (1 1 1) crystallographic plane of metallic Pd(0). This finding corroborated XRD patterns (Fig. 1e), which exhibit characteristic peaks at (1 1 1), 46.7° (2 0 0), 68.1° (2 2 0), and 82.1° (3 1 1), consistent with face-centered cubic Pd (0). The XPS spectrum (Fig. 1f) confirms that Pd(0) is the predominant valence state of the Pd solids.

## 3.2. Continuous co-reduction of bromate and nitrate

During stage S1, the Pd-MBfR and the conventional MBfR demonstrated distinct bromate removal patterns under incremental BrO<sub>3</sub><sup>-</sup> loading (Fig. 2). When 2 mg·L<sup>-1</sup> BrO<sub>3</sub><sup>-</sup> was introduced into the Pd-MBfR and the MBfR, both reactors realized an effluent BrO<sub>3</sub><sup>-</sup> concentration < 0.25 mg·L<sup>-1</sup> with > 90 % removal by the end of the stage S1b (Fig. 2). Especially, for the Pd-MBfR, when influent BrO<sub>3</sub><sup>-</sup> concentration was elevated from 1 to 2 mg·L<sup>-1</sup> on Day 20, the BrO<sub>3</sub><sup>-</sup> reduction flux increased proportionally from 57.2 ± 2.3 to 122.0 ± 2.5 mg·m<sup>-2</sup>·d<sup>-1</sup> in one day (Figure S2), maintaining stable removal (approximately 90 %) throughout S1a and S1b. In contrast, the MBfR required 37 days to attain comparable reduction flux and 90 % removal under identical loadings.

Under high BrO<sub>3</sub><sup>-</sup> loading (4 mg·L<sup>-1</sup> BrO<sub>3</sub><sup>-</sup> in S1c), the Pd-MBfR demonstrated superior resilience compared to the conventional MBfR, with effluent BrO<sub>3</sub><sup>-</sup> concentrations stabilizing at 1.1 ± 0.03 mg·L<sup>-1</sup> after a transient adaptation period (Fig. 2a). The MBfR exhibited a pronounced stress response: Effluent BrO<sub>3</sub><sup>-</sup> initially spiked at 3.2 mg·L<sup>-1</sup> before partially recovering to 2.5 ± 0.03 mg·L<sup>-1</sup>. This divergence was mirrored in reduction fluxes, where the Pd-MBfR achieved a sustained flux of 202.1 ± 5.2 mg·m<sup>-2</sup>·d<sup>-1</sup>—nearly double the MBfR's declining flux of 106.2 ± 4.1 mg·m<sup>-2</sup>·d<sup>-1</sup> (Figure S2). Stoichiometric Br<sup>-</sup> production (Figure S3) confirms complete BrO<sub>3</sub><sup>-</sup> reduction, with an average mass balance closure of 98 % for bromine, verifying BrO<sub>3</sub><sup>-</sup> removal via reductive transformation rather than adsorption or side reactions.

Transitioning from S1c to S2, where 1.5 mg·L<sup>-1</sup> NO<sub>3</sub><sup>-</sup>-N was introduced as a co-contaminant, the Pd-MBfR exhibited moderated
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Transmission electron microscopy (TEM) images of selected areas in cross sections of Pd-biofilm (a). Images of Pd nanoparticles deposited on the microbial cell surface of Pd-biofilm (b) and (c). The grey shapes are bacterial cells, and the dark spots are Pd(0). High resolution transmission electron microscopy (HRTEM) images of Pd(0) crystallites in Pd-biofilm (d). XRD spectra (e) and deconvoluted Pd 3d XPS spectra (f) of the solid powders collected from Pd-biofilm.</figcaption></figure>

performance decay compared to the conventional MBfR, with effluent BrO₃⁻ stabilizing at 1.6 ± 0.1 mg·L⁻¹ (corresponding to a removal decrease from 71.3 % to 60.0 %) versus the MBfR's sharp decline to 3.2 ± 0.02 mg·L⁻¹ with a removal down from 37.7 % to 20.8 %. Mono-metallic Pd(0) lacks catalytic activity for NO₃⁻ reduction, a process inherently dependent on NADH-dependent nitrate reductases. Thus, we supposed that PdNPs mitigate NADH competition via a dual pathways: 1) Accelerating extracellular BrO₃⁻ reduction via [Pd–H*]-mediated hydrogenation, bypassing intracellular NADH competition for NO₃⁻ reduction (Li et al., 2025); and 2) rapid abiotic PdNP-catalyzed conversion of accumulated NO₂⁻ to N₂ diverted electron flow from intracellular NADH-dependent denitrification to extracellular Pd-catalyzed reduction (Zhou et al., 2017). This mechanism reduced intracellular NADH demand for nitrate or nitrite metabolism, thereby enhancing biological BrO₃⁻ reduction. These pathways are supported by reduction kinetic tests and microbial function analyses (discussed in Sections 3.3 and 3.4), demonstrating that synergistic interactions within the Pd-MBfR's biofilms accounted for the 2.9-fold higher BrO₃⁻ flux (170.3 mg·m⁻²·d⁻¹) compared to the MBfR (59.3 mg·m⁻²·d⁻¹).

When the influent electron acceptor was switched from NO₃⁻ to 1.5 mg·L⁻¹ NO₂⁻-N in S3, the Pd-MBfR and the MBfR exhibited effluent BrO₃⁻ concentrations of 1.1 ± 0.03 mg·L⁻¹ and 2.6 ± 0.05 mg·L⁻¹, respectively. The minimal inhibition of BrO₃⁻ reduction suggests cross-catalytic activity of nitrite reductases toward BrO₃⁻, as supported by Lai et al. (2018), who reported a positive correlation between *nirS* gene abundance and BrO₃⁻ reduction flux. Structurally, NO₂⁻ and BrO₃⁻ share key features as compact anionic oxyanions, enabling mimicry at the active sites of *nirS*-encoded cytochrome c nitrite reductases (ccNiRs). Although NO₂⁻ exhibits a bent geometry with two oxygen atoms and *a* + 3 nitrogen, and BrO₃⁻ is trigonal pyramidal with three oxygens and *a* + 5 bromine, their close ionic radii (1.76 Å for NO₂⁻, 2.02 Å for BrO₃⁻) and charge densities (−0.04 e⁻/Å³ for NO₂⁻, −0.03 e⁻/Å³ for BrO₃⁻) facilitate

coordination to the heme-iron center of ccNiRs (Hijnen et al., 1995). Biochemically, ccNiRs catalyze the six-electron reduction of NO₂⁻ to NH₄⁺, matching the six-electron stoichiometry required for BrO₃⁻ reduction to Br⁻ (Zhu et al., 2025). This mechanistic compatibility suggests that ccNiRs leverage native multi-electron transfer pathways for BrO₃⁻ reduction, aligning with the observed correlation between *nirS* gene abundance and BrO₃⁻ flux (Lai et al., 2018).

### 3.3. Short-term kinetic tests under variable loadings

To characterize the effects of nitrate or nitrite competition on bromate reduction kinetics, short-term experiments were conducted using three reactors: the Pd-MBfR (biofilm with PdNPs), the MBfR (conventional biofilm), and the MPfR (abiotic Pd-film reactor without biofilm). These tests aimed to quantify how electron acceptor loadings influenced bromate reduction and validate the role of biogenic PdNPs in mitigating competitive inhibition. The supply of electron donors was not a limiting factor, as the reduction fluxes were significantly lower than the maximum theoretical H₂ flux (420 e⁻·meq·m⁻²·d⁻¹).

The Pd-MBfR maintained stable BrO₃⁻ removal (approximately 80 %) across influent concentrations ranging from 0.2 to 2 mg·L⁻¹, with reduction fluxes increasing linearly with BrO₃⁻ loadings (Fig. 3a). In contrast, the MBfR achieved >98 % removal when influent BrO₃⁻ concentration was 0.2 mg·L⁻¹ but declined to 71 % at ≥ 1 mg·L⁻¹. The MPfR exhibited lower removal (30–43 %), indicating that abiotic Pd-catalyzed reduction of BrO₃⁻ is slower than microbial enzymatic reduction.

The Pd-MBfR exhibited resilience to nitrate interference (Fig. 3b). Under increasing NO₃⁻-N loadings, the Pd-MBfR maintained more stable BrO₃⁻ removal (declined from 80 % to 70 %), outperforming the MBfR which collapsed at 2.5 mg·L⁻¹ NO₃⁻-N (BrO₃⁻ removal < 30 %)—accompanied by residual NO₃⁻-N (1–1.7 mg·L⁻¹ in effluent, Figure S4). This indicates electron acceptor saturation and intense NADH
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Reduction performance for BrO<sub>3</sub><sup>-</sup> and NO<sub>2</sub><sup>-</sup> in the pallidized membrane biofilm reactor (Pd-MBfR) (a) and the membrane biofilm reactor (MBfR) (b). No accumulation of NO<sub>2</sub><sup>-</sup> and NH<sub>4</sub><sup>+</sup> detected in the effluent of the MBfRs during the continuous operation.</figcaption></figure>

competition in the enzymatic system. The MPfR showed no NO₃⁻ reduction (Figure S4), confirming that abiotic PdNPs cannot catalyze NO₃⁻ removal, leaving BrO₃⁻ reduction unaffected. This contrast implies that the [Pd–H*]-mediated extracellular hydrogenation of BrO₃⁻ bypassed intracellular NADH competition, whereas the enzymatic pathways were limited by electron carrier saturation under high NO₃⁻ stress.

For NO₂⁻ co-reduction, the Pd-MBfR sustained 73–80 % BrO₃⁻ removal across 0–5 mg·L⁻¹ NO₂⁻-N (Fig. 3c), driven by rapid abiotic NO₂⁻ scavenging over PdNPs. The MBfR displayed dual-phase behavior: Low NO₂⁻ concentration (≤ 0.5 mg·L⁻¹) enhanced BrO₃⁻ reduction from 75 % to 86 % by inducing NO₂⁻ reductase expression (Lai et al., 2018), while higher loadings (> 2.5 mg·L⁻¹) caused inhibition from enzymatic competition, and BrO₃⁻ removal dropped to 69 % due to substrate saturation. The BrO₃⁻ removal in the MPfR collapsed from 31 % to 12 % at influent NO₂⁻-N > 1.5 mg·L⁻¹, indicating direct catalytic competition for catalytic sites.

Results of electron flux analysis (Table S4) reveal that Pd-MBfR allocated a higher proportion of electrons to BrO₃⁻ compared to the MBfR as loading increased. These results suggest that the Pd-MBfR prioritized BrO₃⁻ reduction, maintaining stable electron allocation to BrO₃⁻ even under escalating NO₃⁻ or NO₂⁻ loadings, whereas the MBfR exhibited significant electron diversion away from BrO₃⁻ as competing acceptors accumulated. This divergence could stem from the Pd-MBfR's dual-pathway design: biogenic PdNPs rapidly cleared NO₂⁻ via catalytic hydrogenation, diverting electron flow away from intracellular NADH consumption for NO₂⁻ reduction to extracellular Pd-catalyzed reduction and thereby liberating electron flux for enzymatic BrO₃⁻ reduction. The observed nitrate-inhibition hierarchy (MBfR > MPfR > Pd-MBfR) underscores the inherent vulnerability of biotic systems electron

competition, while coupling of PdNPs and biofilm enabled efficient multi-oxyanions co-remediation.

Kinetic modeling validated pseudo-first-order kinetics for BrO₃⁻ and NO₂⁻ reduction (all r²>0.98, Section S2 of SI), consistent with published findings (Downing and Nerenberg, 2007; Zhou et al., 2017). Fitting effluent data to a steady-state CSTR model (Equation S3, Section S2) yielded rate constants (k) revealing additive contributions from extracellular Pd catalysis and intracellular enzymatic reduction in the Pd-MBfR (k<sub>MBfR</sub>+k<sub>MPfR</sub> ≈ k<sub>Pd-MBfR</sub>). For BrO₃⁻ reduction, the Pd-MBfR (k = 0.92 h⁻¹) outperformed the MBfR (0.70 h⁻¹) and the MPfR (0.17 h⁻¹), and Pd-catalyzed reduction accounted for approximately 18 % of total BrO₃⁻ reduction flux in the Pd-MBfR (k<sub>MPfR</sub>/k<sub>Pd-MBfR</sub>). For NO₂⁻ reduction, the rate constant in the Pd-MBfR (15.91 h⁻¹) was approximately 1.5-fold and 3-fold greater than the rates in the MBfR (10.32 h⁻¹) and the MPfR (6.39 h⁻¹), respectively, indicating that PdNPs contributing approximately 65 % of NO₂⁻ reduction. Since NO₃⁻ reduction to NO₂⁻ is enzymatic processes, the bioPd-enhanced NO₂⁻ reduction diverted approximately 39 % of intracellular electron flow from denitrification to extracellular catalysis. Under a high NO₃⁻ loading (Series B5, Table S4), the electron consumption flux of the Pd-MBfR (125.7 e⁻·meq·m⁻²·d⁻¹) was 1.5-fold higher than that of the MBfR (85.5 e⁻·meq·m⁻²·d⁻¹), with a difference of 40.2 e⁻·meq·m⁻²·d⁻¹, closely matching the calculated extracellular electron flux (48.5 e⁻·meq·m⁻²·d⁻¹). This electron diversion explains the Pd-MBfR's enhanced reduction flux and preferential selectivity for BrO₃⁻ during NO₃⁻ co-reduction.

### 3.4. Characterization of the bacterial communities and their functional genes

#### 3.4.1. Microbes involved in bromate reduction

To identify microbes involved in bromate reduction, biofilm samples from the Pd-MBfR and the MBfR were collected at the end of Stages S1c, S2, and S3 for 16S rRNA gene-targeted amplicon sequencing. Fig. 4 shows the relative abundance of dominant bacteria at the genus level for the biofilm samples. In S1c, *Dechloromonas* dominated both reactors (40.9 % in the Pd-MBfR, 48.7 % in the MBfR), which is a denitrifying bacterium capable of reducing bromate using volatile fatty acids (VFAs) and H₂ (Luo et al., 2017; Wu et al., 2024). *Acidovorax* is another mixotrophic denitrifying bacterium (DB) that can also utilize acetate and H₂ as electron donor (Wang et al., 2023), occupied 17.2 % in the Pd-MBfR when BrO₃⁻ was sole electron acceptor. *Acetobacterium*, a homoacetogenic bacterium (9.3 % in the Pd-MBfR, 7.3 % in the MBfR), indicated potential syntrophic interactions between fermentative and bromate-reducing bacteria, as VFAs produced by *Acetobacterium* could enhance bromate removal (Merli et al., 2021; Morinaga and Kawada, 1990). Zuo et al. (2024) demonstrated that VFAs contributed > 90 % to bromate reduction in hybrid electron donor (VFAs and H₂) condition.

#### 3.4.2. Microbial community shaped by nitrate

In S2, introduction of NO₃⁻ shaped communities differently in the two reactors. *Hydrogenophaga*, a hydrogen-oxidizing DB (Ryan et al., 2008), became dominant in both systems (23.6 % in the Pd-MBfR, 53.4 % in the MBfR). Notably, *Rhodoblastus* (22.7 % in the MBfR), a hydrogenotrophic bacterium mediating dissimilatory nitrate reduction to ammonium (DNRA) (Chen et al., 2022), enriched in the MBfR, likely diverting electron flow from bromate reduction to ammonium assimilation. Concurrently, *Acetobacterium* disappeared in the MBfR, and the abundance of mixotrophic *Dechloromonas* or *Acidovorax* declined. The calculated fₛ value (the portion of the electrons converted to biomass) of autotrophic denitrification (fₛ= 0.24) is much greater than that of autotrophic acetogen (fₛ=0.04), and DB has a higher biomass yield and specific growth rate than does acetogens (Rittmann and McCarty, 2020). This shift reflected competitive exclusion: autotrophic denitrifiers outcompeted homoacetogens and disrupted the syntrophic partnerships. In contrast, the Pd-MBfR showed suppressed *Hydrogenophaga* and absent *Rhodoblastus*, suggesting PdNPs inhibited DNRA process and autotrophic
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 3.** The effect of influent concentrations of bromate (a), nitrate (b), and nitrite (c) on bromate reduction in the palladized biofilm reactor (Pd-MBfR), the biofilm reactor (MBfR), and the Pd-film reactor (MPfR).</figcaption></figure>

denitrification by catalyzing rapid $NO_2^-$ reduction, limiting electron acceptor availability for these pathways. This preservation of synergistic interactions between acetogens and mixotrophic reducers in the Pd-MBfR maintained stable $BrO_3^-$ reduction under $NO_2^-$ stress.

Introduction of $NO_2^-$ (in S3) led to recovery of *Dechloromonas* and *Acetobacterium* but reduced *Hydrogenophaga* in the MBfR (Fig. 4). These observations suggest that *Dechloromonas* and *Acetobacterium* provided metabolic intermediates that prioritized $BrO_3^-$ reduction, while autotrophic denitrifiers *Hydrogenophaga* were outcompeted due to their stricter substrate specificity. The inverse correlation between *Hydrogenophaga* abundance and $BrO_3^-$ removal further supports this functional adaptation.

### 3.4.3. Functional-gene responses under competitive electron acceptors

Functional gene abundance (Fig. 5) reflects stage-specific responses related to electron transfer and oxyanions reduction. Genes encoding hydrogen dehydrogenase and ATPase remained stable, indicating

consistent electron donor ($H_2$) utilization across stages. During S2, dissimilatory nitrate reduction genes (*narG* and *nrfA*) surged in the MBfR, coinciding with *Rhodoblastus* enrichment and 45 % decline in $BrO_3^-$ reduction fluxes (from 106.2 ± 4.1 to 58.6 ± 3.4 mg·m$^2$.d$^{-1}$)—a shift reflecting prioritized $NO_3^-$ reduction over $BrO_3^-$. Genes involved in the reduction of $NO_2^-$ to $N_2$ (*nirS*, *norB*, and *nosZ*) decreased in S2 but rebounded in S3 upon $NO_2^-$ introduction. The positive correlation between the abundance of nitrite reductase genes and $BrO_3^-$ reduction flux suggests that nitrite reductases allocated electrons between $NO_2^-$ and $BrO_3^-$ based on substrate availability. This aligns with the observed substrate-dependent regulation: high $NO_2^-$ concentrations (≥ 2.5 mg·L$^{-1}$) minimally inhibited $BrO_3^-$ reduction, whereas low $NO_2^-$ concentrations (≤ 0.5 mg·L$^{-1}$) enhanced $BrO_3^-$ reduction (Fig. 3c).

In the Pd-MBfR, introduction of $NO_3^-$ induced *narG* ($NO_3^-$ to $NO_2^-$ reduction) but left *nirS*, *norB*, and *nosZ* unchanged, because extracellular Pd-catalyzed $NO_2^-$ reduction bypassed intracellular $NO_2^-$ reduction. This extracellular pathway explains the higher abundance of genes related to
<figure><img src="image_10.png" /><figcaption>Fig. 4. Relative abundance of bacterial genera during stages in the Pd-MBfR and the MBfR at the genus level, < 3 % phylotypes in the biofilms are not shown.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>Fig. 5. Fold change (FC) of functional-gene abundance in the Pd-MBfR compared to those in the MBfR, with S1c biofilm set as the reference. Red highlight indicates an increase, while blue highlight indicates a decrease.</figcaption></figure>

homoacetogenesis in the Pd-MBfR (2.4 folds compared to the MBfR, data shown in Table S5), as reduced competition for intracellular NADH facilitated syntrophic interactions. NO₂⁻ was reported to suppress CO dehydrogenase activity via competitive inhibition and toxicity (Moon et al., 2021; Wang et al., 2018b). The minimal effect of NO₂⁻ on Pd-biofilm's functional genes for homoacetogenesis indicated its catalytic detoxification, minimizing metabolic interference with bromate reduction.

### 3.5. Proposed mechanisms of bromate and nitrate co-reduction in the Pd-MBfR

Coupling biogenic PdNPs with hydrogenotrophic biofilms established a synergistic system that rerouted electron flow to mitigate competition inhibition between BrO₃⁻ and NO₃⁻ reduction. This mechanism operates through three interconnected processes (Fig. 6),

integrating intracellular enzymatic catalysis, extracellular Pd-catalyzed hydrogenation, and microbial community adaptation. Biogenic PdNPs activate hydrogen into reactive atomic hydrogen, which shifts about 18 % of BrO₃⁻ reduction flux from intracellular NADH-dependent enzymatic pathways to extracellular catalytic reduction. This decoupled BrO₃⁻ reduction from the microbial electron transport chain allows electron flow to prioritize BrO₃⁻ reduction even as NO₃⁻ competes for NADH. Meanwhile, unlike abiotic Pd systems, NO₃⁻ is first reduced to NO₂⁻ over nitrate reductases, and biogenic PdNPs rapidly catalyze NO₂⁻ hydrogenation to N₂, eliminating its accumulation and diverting 39 % of intracellular electron flux from denitrification to extracellular catalysis. This extracellular NO₂⁻ scavenging preserves NADH for enzymatic BrO₃⁻ reduction and improves BrO₃⁻ overall removal efficiency. Additionally, PdNPs mitigate metabolic toxicity induced by NO₃⁻, which facilitates synergistic interactions between homoacetogens *Acetobacterium* and mixotrophic denitrifiers *Dechloromonas*. VFAs produced by
<figure><img src="image_12.png" /><figcaption>Fig. 6. Schematic pathways of bromate and nitrate co-reduction in biofilm coupled with palladium nanoparticles (PdNP).</figcaption></figure>

*Acetobacterium* serve as supplementary electron donors, a syntrophic pathway absent in conventional biofilms under $NO_3^-$ stress, where autotrophs outcompete syntrophic microbes. Therefore, mixotrophic genera capable of reducing both $BrO_3^-$ and $NO_3^-$ gain a competitive advantage over autotrophic denitrifiers within the Pd-biofilm and further improving $BrO_3^-$ reduction through integrated catalytic and enzymatic pathways.

## 4. Conclusion

The Pd-MBfR exhibited sustained $BrO_3^-$ reduction during $NO_3^-$ co-reduction, outperforming conventional biofilms by diverting electron flow from intracellular NADH-dependent metabolic pathways to extracellular Pd-catalyzed hydrogenation. Key mechanisms include Pd-catalyzed reduction of $BrO_3^-$ and $NO_2^-$, which preserved NADH for enzymatic $BrO_3^-$ reduction alongside enhanced microbial synergies. Microbial community structure of Pd-biofilm remained stable under $NO_3^-$ stress, which maintained syntrophic interactions between bromate-reducing denitrifiers *Dechloromonas* and homoacetogens *Acetobacterium*, whereas conventional biofilms shifted toward autotrophic denitrifiers. Functional gene analysis revealed increased abundance of acetogenesis-related genes and lower abundance of genes involved in dissimilatory nitrate reduction (nrfA) in Pd-biofilms, which favored metabolic pathways prioritizing $BrO_3^-$ reduction. This study underscores the potential of Pd-MBfR systems for multi-oxyanion remediation, offering a sustainable and low-carbon alternative to address oxyanion co-contamination challenges.

### CRediT authorship contribution statement

**Chengyang Wu:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Guirong Liang:** Investigation, Data curation. **Siqing Xia:** Supervision, Conceptualization. **Jingzhou Zhou:** Methodology. **Eric Lichtfouse:** Writing – review & editing. **Xiang Cai:** Writing – review & editing. **Suyun Xu:** Writing – review & editing. **Hongbo Liu:** Validation, Supervision, Project administration, Funding acquisition.

### Declaration of competing interest

The authors declare that they have no known competing financial

interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

The authors would like to acknowledge the co-funding of this work by the National Natural Science Foundation of China (No.52400008) and National Natural Science Foundation of China (No.52470049).

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.watres.2025.124303.

### Data availability

Data will be made available on request.

### References

Butler, R., Ehrenberg, S., Godley, A.R., Lake, R., Lytton, L., Cartmell, E., 2006. Remediation of bromate-contaminated groundwater in an ex situ fixed-film bioreactor. Sci. Total Environ. 366 (1), 12–20.

Butler, R.A.Y., Andrew, G., Lucy, L., Cartmell, E., 2005. Bromate environmental contamination: review of impact and possible treatment. Crit. Rev. Env. Sci. Technol. 35 (3), 193–217.

Chen, Y.-Z., Zhang, L.-J., Ding, L.-Y., Zhang, Y.-Y., Wang, X.-S., Qiao, X.-J., Pan, B.-Z., Wang, Z.-W., Xu, N., Tao, H.-C., 2022. Sustainable treatment of nitrate-containing wastewater by an autotrophic hydrogen-oxidizing bacterium. Environ. Sci. Ecotechnology 9, 100146.

Downing, L.S., Nerenberg, R., 2007. Kinetics of microbial bromate reduction in a hydrogen-oxidizing, denitrifying biofilm reactor. Biotechnol. Bioeng. 98 (3), 543–550.

EPA, U.S., 2006. National primary drinking water regulations: stage 2 disinfectants and disinfection byproducts rule. Fed. Regist. 71, 388–493.

Hijnen, W., Voogt, R., Veenendaal, H.R., van der Jagt, H., van der Kooij, D., 1995. Bromate reduction by denitrifying bacteria. Appl. Env. Microbiol. 61 (1), 239–244.

IARC, 1999. Potassium Bromate (Summary of Data Reported and Evaluation). France.

Lai, C.-Y., Lv, P.-L., Dong, Q.-Y., Yeo, S.L., Rittmann, B.E., Zhao, H.-P., 2018. Bromate and nitrate bioreduction coupled with poly-β-hydroxybutyrate production in a methane-based membrane biofilm reactor. Env. Sci. Technol. 52 (12), 7024–7031.

Lawson, C.E., Wu, S., Bhattacharjee, A.S., Hamilton, J.J., McMahon, K.D., Goel, R., Noguera, D.R., 2017. Metabolic network analysis reveals microbial community interactions in anammox granules. Nat. Commun. 8 (1), 15416.

Li, A., Yu, F., Jia, C., Sun, L., Gao, J., Zhang, J., Zhu, X., 2025. Continuous atomic hydrogen for eminent bromate reduction via palladium coordination manipulation. ACS ES&T Water 5 (3), 1399–1405.

Luo, J.-H., Wu, M., Yuan, Z., Guo, J., 2017. Biological bromate reduction driven by methane in a. Membr. Biofilm React. Environ. Sci. Technol. Lett. 4 (12), 562–566.
Merli, G., Becci, A., Amato, A., Beolchini, F., 2021. Acetic acid bioproduction: the technological innovation change. Sci. Total Environ. 798, 149292.

Moon, J., Dönig, J., Kramer, S., Poehlein, A., Daniel, R., Müller, V., 2021. Formate metabolism in the acetogenic bacterium *cetobacterium woodii*. *Env. Microbiol.* 23 (8), 4214–4227.

Morinaga, T., Kawada, N., 1990. The production of acetic acid from carbon dioxide and hydrogen by an anaerobic bacterium. J. Biotechnol. 14 (2), 187–194.

Pang, S., Rittmann, B.E., Wu, C., Yang, L., Zhou, J., Xia, S., 2022. Synergistic inorganic carbon and denitrification genes contributed to nitrite accumulation in a hydrogen-based membrane biofilm reactor. Bioengineering 9 (5), 222.

Preissler, J., Wahlefeld, S., Lorent, C., Teutloff, C., Horch, M., Lauterbach, L., Cramer, S. P., Zebger, I., Lenz, O., 2018. Enzymatic and spectroscopic properties of a thermostable [NiFe]-hydrogenase performing H2-driven NAD⁺-reduction in the presence of O2. Biochim. Biophys. Acta (BBA) - Bioenerg. 1859 (1), 8–18.

Ren, C., Yang, P., Sun, J., Bi, E.Y., Gao, J., Palmer, J., Zhu, M., Wu, Y., Liu, J., 2021. A bioinspired molybdenum catalyst for aqueous perchlorate reduction. J. Am. Chem. Soc. 143 (21), 7891–7896.

Rice, E.W., Bridgewater, L., 2012. Standard Methods for the Examination of Water and Wastewater. American public health association, Washington, DC. Vol. 10.

Rittmann, B.E., McCarty, P.L., 2020. Environmental Biotechnology: Principles and Applications, 2nd ed. McGraw-Hill Book Co, New York.

Ryan, R.P., Germaine, K., Franks, A., Ryan, D.J., Dowling, D.N., 2008. Bacterial endophytes: recent developments and applications. FEMS Microbiol. Lett. 278 (1), 1–9.

Shi, L.-D., Wang, Z., Liu, T., Wu, M., Lai, C.-Y., Rittmann, B.E., Guo, J., Zhao, H.-P., 2021. Making good use of methane to remove oxidized contaminants from wastewater. Water Res. 197, 117082.

Tang, Y., Zhou, C., Van Ginkel, S.W., Ontiveros-Valencia, A., Shin, J., Rittmann, B.E., 2012. Hydrogen permeability of the hollow fibers used in H2-based membrane biofilm reactors. J. Memb. Sci. 407-408, 176–183.

Wang, F., van Halem, D., Ding, L., Bai, Y., Lekkerkerker-Teunissen, K., van der Hoek, J. P., 2018a. Effective removal of bromate in nitrate-reducing anoxic zones during managed aquifer recharge for drinking water treatment: laboratory-scale simulations. Water Res. 130, 88–97.

Wang, H.-M., Hu, G.-R., Luo, W.-Y., Li, F.-L., 2023. The horizontal gene transfer of perchlorate reduction genomic island in three bacteria from an ecological niche. Appl. Microbiol. Biotechnol. 108 (1), 22.

Wang, Y., Li, P., Zuo, J., Gong, Y., Wang, S., Shi, X., Zhang, M., 2018b. Inhibition by free nitrous acid (FNA) and the electron competition of nitrite in nitrous oxide (N2O) reduction during hydrogenotrophic denitrification. Chemosphere 213, 1–10.

Wu, C., Zhou, J., Pang, S., Yang, L., Lichtfouse, E., Liu, H., Xia, S., Rittmann, B.E., 2024. Reduction and precipitation of chromium(VI) using a pallidized membrane biofilm reactor. Water Res. 249, 120878.

Wu, C., Zhou, L., Zhou, C., Zhou, Y., Xia, S., Rittmann, B.E., 2022a. Co-removal of 2,4-dichlorophenol and nitrate using a pallidized biofilm: denitrification-promoted microbial mineralization following catalytic dechlorination. J. Hazard. Mater. 422, 126916.

Wu, C., Zhou, L., Zhou, C., Zhou, Y., Zhou, J., Xia, S., Rittmann, B.E., 2022b. A kinetic model for 2,4-dichlorophenol adsorption and hydrodechlorination over a pallidized biofilm. Water Res. 214, 118201.

Wu, C., Zhou, L., Zhou, Y., Zhou, C., Xia, S., Rittmann, B.E., 2021. Dechlorination of 2,4-dichlorophenol in a hydrogen-based membrane palladium-film reactor: performance, mechanisms, and model development. Water Res. 188, 116465.

Xiao, Q., Yu, S., Li, L., Wang, T., Liao, X., Ye, Y., 2017. An overview of advanced reduction processes for bromate removal from drinking water: reducing agents, activation methods, applications and mechanisms. J. Hazard. Mater. 324, 230–240.

Zhang, W., Wu, Y., Chen, Y., Zheng, X., 2024. Hydrogen-driven autotrophic degradation of halogenated organic pollutants in a membrane biofilm reactor: advances and challenges. Chem. Eng. J. 479, 147754.

Zhong, Y., Yang, Q., Fu, G., Xu, Y., Cheng, Y., Chen, C., Xiang, R., Wen, T., Li, X., Zeng, G., 2018. Denitrifying microbial community with the ability to bromate reduction in a rotating biofilm-electrode reactor. J. Hazard. Mater. 342, 150–157.

Zhou, C., Wang, Z., Ontiveros-Valencia, A., Long, M., Lai, C.-y., Zhao, H.-p., Xia, S., Rittmann, B.E., 2017. Coupling of Pd nanoparticles and denitrifying biofilm promotes H2-based nitrate removal with greater selectivity towards N2. Appl. Catal. B: Environ. 206, 461–470.

Zhu, S., Liu, Z., Hu, B., Feng, Y., Pan, G., 2025. Nitrite reductases in biomedicine: from natural enzymes to artificial mimics. Res. (Wash D C) 8, 0710.

Zuo, Z., Niu, C., Zhao, X., Lai, C.-Y., Zheng, M., Guo, J., Hu, S., Liu, T., 2024. Biological bromate reduction coupled with in situ gas fermentation in H2/CO2-based membrane biofilm reactor. Water Res. 254, 121402.
# In situ-generated palladium nanoparticles promoted co-reduction of bromate and nitrate in hydrogenotrophic biofilms

Chengyang Wu<sup>a,b</sup>, Guirong Liang<sup>a</sup>, Siqing Xia<sup>c</sup>, Jingzhou Zhou<sup>c</sup>, Eric Lichtfouse<sup>d</sup>, Xiang Cai<sup>e</sup>

Suyun Xu<sup>a,b</sup>, Hongbo Liu<sup>a,b *</sup>

## Summary:

Pages: 11

Figures: 5

Tables: 5

<footnote><sup>a</sup> School of Environment and Architecture, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai 200093, China</footnote>

<footnote><sup>b</sup> School of Intelligent Emergency Management, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai 200093, China</footnote>

<footnote><sup>c</sup> State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China</footnote>

<footnote><sup>d</sup> State Key Laboratory of Multiphase Flow in Power Engineering, Xi'an Jiaotong University, 28 Xianning West Rd, Xi'an, Shaanxi 710049 P.R. China</footnote>

<footnote><sup>e</sup> College of Geography and Environmental Sciences, Zhejiang Normal University, Jinhua 321004, China</footnote>

<footnote>* Corresponding author</footnote>

<footnote>E-mail address: Liuhb@usst.edu.cn (H. Liu)</footnote>
<figure><img src="image_13.png" /></figure>
Section S1. Supplementary Figures

<figure><img src="image_14.png" /><figcaption>**Figure S1.** Schematic of the Pd-MBfR system: ① pure-H₂ gas tank to feed the fiber bundles; ② pure-N₂ gas to feed the headspace of the influent medium bottle; ③ gas-pressure regulator; ④ influent-medium bottle; ⑤ effluent bottle; ⑥ influent pump; ⑦ Recirculation configuration (the blue arrow indicates the flow direction); ⑧ main bundle with 32 fibers; ⑨ sampling coupon bundle with 10 fibers; and ⑩ recirculation pump.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Figure S2.** Reduction fluxes of $BrO_3^-$ in the Pd-MBfR and the MBfR.</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>**Figure S3.** The concentrations of released $Br^-$ during continuous operation. Measured released $Br^-$ is the difference between the concentrations of free $Br^-$ in the effluent and influent; calculated release $Br^-$ is the loss of Br concentrations in $BrO_3^-$ between the influent and effluent. Mass balance percentage is the ratio of measured released $Br^-$ and calculated released $Br^-$.</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>**Figure S4.** The effluent concentrations of nitrate in the palladized biofilm reactor (Pd-MBfR), the biofilm reactor (MBfR), and the Pd-film reactor (MPfR) during Series B (Table S3).</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>**Figure S5.** The effluent concentrations of nitrite in the palladized biofilm reactor (Pd-MBfR), the biofilm reactor (MBfR), and the Pd-film reactor (MPfR) during Series C (Table S3).</figcaption></figure>
## Section S2. Kinetic Model Solution

The reactors were modeled as continuous stirred tank reactors (CSTR) with an immobilized catalyst. The mass balance on a solute is:

$$V \frac{dC_i}{dt} = QC_0 - QC_e + Vr \quad (S1)$$

in which

$$r = -kC_i \quad (S2)$$

where $C_0$, $C_i$, and $C_e$ (mg L⁻¹) are solute concentrations in the influent, the bulk in reactor, and the effluent, respectively; $Q$ (L·h⁻¹) is influent flow rate of the reactor; $t$ is time (h); $V$ is the working volume of reactor (65 mL); and $k$ is a first-order reaction rate constant for the solute (h⁻¹)

Because in a continuous reactor at steady state, $dC_i/dt=0$. Substitution of Eq. (S2) into Eq. (S1) and rearrangement yields

$$\frac{Q}{V}(C_0 - C_e) = kC_e \quad (S3)$$

To quantify the contributions of intra- and extracellular electron transfer in the bioPd system, we leveraged kinetic modeling and comparative analysis of three reactors. Bromate and nitrite reduction followed pseudo-first-order kinetics, as validated by Downing et al., who used the Monod kinetic model to characterize bromate reduction in hydrogen-based biofilms, reporting a half-saturation concentration of 15 mg L⁻¹ [a]. Given the influent $BrO_3^-$ concentrations (0.2–4 mg L⁻¹) in this study, biological bromate reduction can be treated as pseudo-first-order kinetics. Previous studies have shown that nitrite reduction in Pd-biofilm, biofilm, and Pd-film systems also follows pseudo-first-order kinetics [b]. This was further validated by fitting effluent concentrations to a steady-state continuous stirred tank reactor (CSTR) model.

For bromate reduction in Pd-MBfR, the reaction can be decomposed into:

$$\text{Pd-catalyzed reduction: } BrO_3^- + 6e^- + 6H^+ \xrightarrow{k_i} \underset{\text{PdNPs}}{Br^-} + 3H_2O$$
$$ \text{Enzymatic reduction: } BrO_3^- + 6e^- + 6H^+ \xrightarrow{k_2 \text{ enzyme}} Br^- + 3H_2O $$

with the overall rate constant $k=k_1+k_2$

$$ \text{NO}_3^- \text{ reduction to NO}_2^- \text{ is enzymatically driven: } NO_3^- + 2e^- + 2H^+ \xrightarrow{\text{enzyme}} NO_2^- + H_2O $$

NO₂⁻ reduction to N₂ in the Pd-MBfR involves both Pd-catalyzed and enzymatic pathways:

$$ NO_2^- + 3e^- + 4H^+ \xrightarrow{k_1 \text{ PdNPs}} 0.5N_2 + 2H_2O $$

$$ NO_2^- + 3e^- + 4H^+ \xrightarrow{k_2 \text{ enzyme}} 0.5N_2 + 2H_2O $$

with the overall rate constant $k=k_1+k_2$

Due to the dispersive deposition of PdNPs within the biofilms, direct quantification of catalytic and enzymatic bromate reduction is infeasible. Instead, we estimated intra- and extracellular contributions by analyzing reduction kinetics in the MPfR (same Pd loading as the Pd-MBfR) and the MBfR (same biofilm area as the Pd-MBfR). Fitting Eq. (S3) to bromate (Series A, Figure 3a) and nitrite (Series C, Figure S5) concentration data yielded the pseudo-first-order rate constants below:

<table><caption>Table. Pseudo-first-order reduction rate constants in the reactors.</caption><thead><tr><th rowspan="2">Reactors</th><th colspan="2">BrO<sub>3</sub><sup>-</sup></th><th colspan="2">NO<sub>2</sub><sup>-</sup></th></tr><tr><th>K(h<sup>-1</sup>)</th><th>r<sup>2</sup></th><th>k/(h<sup>-1</sup>)</th><th>r<sup>2</sup></th></tr></thead><tbody><tr><td>Pd-MBfR</td><td>0.92</td><td>0.981</td><td>15.91</td><td>0.995</td></tr><tr><td>MBfR</td><td>0.70</td><td>0.991</td><td>10.32</td><td>0.997</td></tr><tr><td>MPfR</td><td>0.17</td><td>0.982</td><td>6.39</td><td>0.997</td></tr></tbody></table>

[a] Downing, L.S. and Nerenberg, R. 2007. Kinetics of microbial bromate reduction in a hydrogen-oxidizing, denitrifying biofilm reactor. Biotechnology and Bioengineering 98(3), 543-550.

[b] Zhou, C., Wang, Z., Ontiveros-Valencia, A., Long, M., Lai, C.-y., Zhao, H.-p., Xia, S. and Rittmann, B.E. 2017. Coupling of Pd nanoparticles and denitrifying biofilm promotes H2-based nitrate removal with greater selectivity towards N2. Applied Catalysis B: Environmental 206, 461-470.
## Section S3. Metagenomics Sequencing and Analysis Procedures

The extracted DNA was fragmented into ~400 bp segments using a Covaris M220 (Gene Company Limited, China). A paired-end sequencing library was subsequently constructed with the NEXTFLEX™ Rapid DNA-Seq Kit (Bioo Scientific, USA). HiSeq X Reagent Kits (Illumina, USA) were used to ligate adapters containing full sequencing primer hybridization sites to the blunt ends of the fragmented DNA. Library sequencing was performed on the Hiseq X-ten platform (Illumina, USA). For sequence quality control, raw reads were filtered to remove low-quality data, yielding clean reads. These clean reads were assembled into contigs (≥ 300 bp) using MEGAHIT (https://github.com/voutcn/megahit). Open reading frame (ORF) prediction for the assembled contigs was conducted via MetaGene (http://metagene.cb.ky.tokyo.ac.jp/). Predicted genes with ≥95% similarity (90% coverage) were clustered using CD-HIT software (http://www.bioinformatics.org/cd-hit/), with the longest sequence in each cluster selected as the representative to construct non-redundant gene sets. Gene abundances for each sample were calculated by aligning clean reads to these non-redundant gene sets using SOAPaligner (http://soap.genomics.org.cn/).
## Section S4. Supplementary Tables

<table><caption>**Table S1.** Composition of the mineral-salts medium fed to the reactors.</caption><thead><tr><th>Component</th><th>Concentration (mg·L<sup>-1</sup>)</th><th>Contents</th><th>Concentration (mg·L<sup>-1</sup>)</th></tr></thead><tbody><tr><td>NaHCO<sub>3</sub><sup>*</sup></td><td>50</td><td>ZnCl<sub>2</sub></td><td>0.070</td></tr><tr><td>KH<sub>2</sub>PO<sub>4</sub></td><td>20</td><td>Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O</td><td>0.036</td></tr><tr><td>MgCl<sub>2</sub>·6H<sub>2</sub>O</td><td>2.0</td><td>NiCl<sub>2</sub>·6H<sub>2</sub>O</td><td>0.024</td></tr><tr><td>FeCl<sub>2</sub>·4H<sub>2</sub>O</td><td>0.10</td><td>Na<sub>2</sub>SeO<sub>3</sub></td><td>0.009</td></tr><tr><td>CaCl<sub>2</sub>·2H<sub>2</sub>O</td><td>0.10</td><td>Na<sub>2</sub>WO<sub>4</sub>·2H<sub>2</sub>O</td><td>0.008</td></tr><tr><td>CoCl<sub>2</sub>·6H<sub>2</sub>O</td><td>0.19</td><td>H<sub>3</sub>BO<sub>3</sub></td><td>0.006</td></tr><tr><td>MnCl<sub>2</sub>·4H<sub>2</sub>O</td><td>0.10</td><td>CuCl<sub>2</sub>·2H<sub>2</sub>O</td><td>0.002</td></tr></tbody></table>

<table><caption>**Table S2.** Parameters for operations for each stage of the palladized membrane biofilm reactor (Pd-MBfR) and the membrane biofilm reactor (MBfR).</caption><thead><tr><th>stage</th><th>days (d)</th><th>BrO<sub>3</sub><sup>-</sup><br>(mg·L<sup>-1</sup>)</th><th>NO<sub>3</sub><sup>-</sup>-N<br>(mg·L<sup>-1</sup>)</th><th>NO<sub>2</sub><sup>-</sup>-N<br>(mg·L<sup>-1</sup>)</th><th>HRT<br>(h)</th><th>H<sub>2</sub> pressure<br>(psig)</th><th>pH</th></tr></thead><tbody><tr><td>1a</td><td>1~19</td><td>1</td><td>0</td><td>0</td><td></td><td></td><td></td></tr><tr><td>1b</td><td>20-45</td><td>2</td><td>0</td><td>0</td><td></td><td></td><td></td></tr><tr><td>1c</td><td>46-70</td><td>4</td><td>0</td><td>0</td><td>4.2</td><td>20</td><td>7.0</td></tr><tr><td>2</td><td>71-104</td><td>4</td><td>1.5</td><td>0</td><td></td><td></td><td></td></tr><tr><td>3</td><td>105-125</td><td>4</td><td>0</td><td>1.5</td><td></td><td></td><td></td></tr></tbody></table>
<table><caption>**Table S3.** Variable system conditions for the short-term kinetic tests.</caption><thead><tr><th rowspan="2">Series</th><th colspan="3">Influent concentration (mg·L<sup>-1</sup>)</th><th rowspan="2">H<sub>2</sub> pressure (psig)</th><th rowspan="2">Influent pH</th></tr><tr><th>BrO<sub>3</sub><sup>-</sup></th><th>NO<sub>3</sub><sup>-</sup>-N</th><th>NO<sub>2</sub><sup>-</sup>-N</th></tr></thead><tbody><tr><td>Steady state</td><td>1</td><td>1.5</td><td>0</td><td>20</td><td>7.0</td></tr><tr><td>A1</td><td>0.2</td><td></td><td></td><td></td><td></td></tr><tr><td>A2</td><td>0.5</td><td></td><td></td><td></td><td></td></tr><tr><td>A3</td><td>1</td><td>1.5</td><td>0</td><td>20</td><td>7.0</td></tr><tr><td>A4</td><td>1.5</td><td></td><td></td><td></td><td></td></tr><tr><td>A5</td><td>2</td><td></td><td></td><td></td><td></td></tr><tr><td>B1</td><td></td><td>0</td><td></td><td></td><td></td></tr><tr><td>B2</td><td></td><td>0.5</td><td></td><td></td><td></td></tr><tr><td>B3</td><td>1</td><td>1.5</td><td>0</td><td>20</td><td>7.0</td></tr><tr><td>B4</td><td></td><td>2.5</td><td></td><td></td><td></td></tr><tr><td>B5</td><td></td><td>5</td><td></td><td></td><td></td></tr><tr><td>C1</td><td></td><td></td><td>0</td><td></td><td></td></tr><tr><td>C2</td><td></td><td></td><td>0.5</td><td></td><td></td></tr><tr><td>C3</td><td>1</td><td>0</td><td>1.5</td><td>20</td><td>7.0</td></tr><tr><td>C4</td><td></td><td></td><td>2.5</td><td></td><td></td></tr><tr><td>C5</td><td></td><td></td><td>5</td><td></td><td></td></tr></tbody></table>
<table><caption>**Table S4.** The electron consumption flux and distribution of pollutants under different series in the short-term kinetic tests.</caption><thead><tr><th rowspan="3">Series</th><th colspan="4">Pd-MBfR</th><th colspan="4">MBfR</th></tr><tr><th colspan="2">BrO<sub>3</sub><sup>-</sup></th><th colspan="2">NO<sub>3</sub><sup>-</sup>/NO<sub>2</sub><sup>-</sup></th><th rowspan="2">Electron<br>consumption flux<br>(e<sup>-</sup>-meq·m<sup>-2</sup>·d<sup>-1</sup>)</th><th colspan="2">BrO<sub>3</sub><sup>-</sup></th><th colspan="2">NO<sub>3</sub><sup>-</sup>/NO<sub>2</sub><sup>-</sup></th><th rowspan="2">Electron<br>consumption flux<br>(e<sup>-</sup>-meq·m<sup>-2</sup>·d<sup>-1</sup>)</th></tr><tr><th>Reduction flux<br>(e<sup>-</sup>-meq·m<sup>-2</sup>·d<sup>-1</sup>)</th><th>Proportion<br>(%)</th><th>Reduction flux<br>(e<sup>-</sup>-meq·m<sup>-2</sup>·d<sup>-1</sup>)</th><th>Proportion<br>(%)</th><th>Reduction flux<br>(e<sup>-</sup>-meq·m<sup>-2</sup>·d<sup>-1</sup>)</th><th>Proportion<br>(%)</th><th>Reduction flux<br>(e<sup>-</sup>-meq·m<sup>-2</sup>·d<sup>-1</sup>)</th><th>Proportion<br>(%)</th></tr></thead><tbody><tr><td>A1</td><td>0.6</td><td>1.6</td><td>34.6</td><td>98.4</td><td>35.1</td><td>0.7</td><td>1.7</td><td>37.9</td><td>98.3</td><td>38.6</td></tr><tr><td>A2</td><td>1.4</td><td>3.8</td><td>34.1</td><td>96.2</td><td>35.5</td><td>1.1</td><td>2.9</td><td>37.5</td><td>97.1</td><td>38.7</td></tr><tr><td>A3</td><td>2.7</td><td>7.1</td><td>35.0</td><td>92.9</td><td>37.7</td><td>2.4</td><td>5.9</td><td>37.9</td><td>94.1</td><td>40.3</td></tr><tr><td>A4</td><td>4.1</td><td>10.3</td><td>35.4</td><td>89.7</td><td>39.5</td><td>3.6</td><td>8.6</td><td>37.9</td><td>91.4</td><td>41.5</td></tr><tr><td>A5</td><td>5.4</td><td>13.7</td><td>34.2</td><td>86.3</td><td>39.6</td><td>4.7</td><td>11.2</td><td>37.6</td><td>88.8</td><td>42.4</td></tr><tr><td>B1</td><td>2.8</td><td>100</td><td>0.0</td><td>0.0</td><td>2.8</td><td>2.3</td><td>100.0</td><td>0.0</td><td>0.0</td><td>2.3</td></tr><tr><td>B2</td><td>2.6</td><td>17.7</td><td>12.1</td><td>82.3</td><td>14.7</td><td>2.0</td><td>14.2</td><td>12.1</td><td>85.8</td><td>14.1</td></tr><tr><td>B3</td><td>2.6</td><td>6.5</td><td>37.4</td><td>93.5</td><td>40.0</td><td>2.3</td><td>7.0</td><td>31.0</td><td>93.0</td><td>33.3</td></tr><tr><td>B4</td><td>2.1</td><td>3.3</td><td>61.1</td><td>96.7</td><td>63.2</td><td>1.0</td><td>2.7</td><td>35.8</td><td>97.3</td><td>36.8</td></tr><tr><td>B5</td><td>2.3</td><td>1.8</td><td>123.4</td><td>98.2</td><td>125.7</td><td>0.7</td><td>0.8</td><td>84.8</td><td>99.2</td><td>85.5</td></tr><tr><td>C1</td><td>2.6</td><td>100.0</td><td>0.0</td><td>0.0</td><td>2.6</td><td>2.3</td><td>100.0</td><td>0.0</td><td>0.0</td><td>2.3</td></tr><tr><td>C2</td><td>2.7</td><td>25.7</td><td>7.8</td><td>74.3</td><td>10.5</td><td>2.9</td><td>27.6</td><td>7.6</td><td>72.4</td><td>10.5</td></tr><tr><td>C3</td><td>2.5</td><td>9.2</td><td>24.8</td><td>90.8</td><td>27.3</td><td>2.6</td><td>9.8</td><td>24.0</td><td>90.2</td><td>26.6</td></tr><tr><td>C4</td><td>2.6</td><td>6.5</td><td>37.6</td><td>93.5</td><td>40.2</td><td>2.3</td><td>5.9</td><td>36.8</td><td>94.1</td><td>39.1</td></tr><tr><td>C5</td><td>2.5</td><td>3.3</td><td>74.5</td><td>96.7</td><td>77.0</td><td>2.3</td><td>3.1</td><td>72.9</td><td>96.9</td><td>75.2</td></tr></tbody></table>
<table><caption>**Table S5.** Results for the abundance of gene sets</caption><thead><tr><th rowspan="2">Category</th><th rowspan="2">Entry</th><th rowspan="2">Description</th><th rowspan="2">abbreviation</th><th colspan="6">Reads Per Kilobase Per Million mapped</th></tr><tr><th colspan="3">Pd-biofilm</th><th colspan="3">Biofilm</th></tr><tr><th></th><th></th><th></th><th></th><th>S1c</th><th>S2</th><th>S3</th><th>S1c</th><th>S2</th><th>S3</th></tr></thead><tbody><tr><td rowspan="2">Electron transfer</td><td>M00157</td><td>F-type ATPase</td><td></td><td>2156</td><td>2567</td><td>2357</td><td>2575</td><td>2957</td><td>2882</td></tr><tr><td>EC:1.12.1.2</td><td>Hydrogen dehydrogenase; H<sub>2</sub> + NAD+ = H<sup>+</sup> + NADH</td><td></td><td>569</td><td>783</td><td>602</td><td>650</td><td>734</td><td>816</td></tr><tr><td rowspan="5">Denitrification</td><td>K00370</td><td>nitrate reductase; nitrate → nitrite</td><td>narG</td><td>4.45</td><td>17.2</td><td>7.32</td><td>3.56</td><td>12.34</td><td>5.32</td></tr><tr><td>K15864</td><td>nitrite reductase (NO-forming); nitrite → nitric oxide</td><td>nirS</td><td>0.36</td><td>0.35</td><td>0.38</td><td>0.45</td><td>0.4</td><td>0.76</td></tr><tr><td>K04561</td><td>nitric oxide reductase subunit B; nitric oxide → nitrous oxide</td><td>norB</td><td>0.92</td><td>0.37</td><td>1.06</td><td>2.72</td><td>1.56</td><td>5.32</td></tr><tr><td>K00376</td><td>nitrous-oxide reductase; nitrous oxide → nitrogen</td><td>nosZ</td><td>0.32</td><td>0.15</td><td>0.36</td><td>1.19</td><td>0.97</td><td>2.56</td></tr><tr><td>K00370</td><td>nitrate reductase; nitrate → nitrite</td><td>narG</td><td>2.45</td><td>17.2</td><td>4.32</td><td>3.56</td><td>12.34</td><td>6.32</td></tr><tr><td rowspan="2">Dissimilatory nitrate reduction to ammonium</td><td>K03385</td><td>nitrite reductase (cytochrome c-552); nitrite → ammonia</td><td>nrfA</td><td>0.81</td><td>2.23</td><td>1.65</td><td>3.12</td><td>12.3</td><td>4.78</td></tr><tr><td>M00422</td><td>Acetyl-CoA pathway, CO<sub>2</sub> → acetyl-CoA</td><td></td><td>321</td><td>134</td><td>287</td><td>163</td><td>22</td><td>124</td></tr></tbody></table>