Bacteria-Photocatalyst Biohybrid System for Sustainable Ammonium Production

Engineering ›› 2025, Vol. 50 ›› Issue (7) :52 -59. DOI: 10.1016/j.eng.2024.08.004

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2024-08-08

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Abstract

Although the Haber–Bosch process supports the growth of modern agriculture with abundant ammonia and fertilizer production, substantial energy consumption and enormous greenhouse emissions demand an alternative and sustainable approach. Here, we report a novel approach that combines the non-photosynthetic bacterium Shewanella oneidensis MR-1 (S. oneidensis MR-1) with cadmium sulfide (CdS) nanoparticles (NPs) to enable the photosynthesis of ammonium (NH₄⁺) from nitrate (NO₃⁻) using photoexcited electrons as donors. The NO₃⁻ reduction efficiency reached almost 100%, with an NH₄⁺ production selectivity of over 90%. The maximum instantaneous quantum efficiency was 3.01% under light irradiation. The reverse metal-reducing (Mtr) pathway is responsible for the transfer of photoexcited electrons to intracellular compartments. Parallel reaction monitoring analysis illustrated that NO₃⁻ to NH₄⁺ was produced via the dissimilatory nitrate reduction to ammonium (DNRA) pathway in S. oneidensis MR-1. This study provides a facile strategy for light-driven ambient NH₄⁺ synthesis and solar-to-chemical conversion.

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Photoexcitation / Artificial photosynthesis / NH₄⁺ production / Solar energy

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Meiwei Guo, Guangfei Liu, Sen Qiao, Xie Quan. Bacteria-Photocatalyst Biohybrid System for Sustainable Ammonium Production. Engineering, 2025, 50(7): 52-59 DOI:10.1016/j.eng.2024.08.004

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1. Introduction

Nitrate (NO₃⁻), usually viewed as a contaminant, contributes to a series of environmental and public health concerns, such as eutrophication and carcinogenicity, if discharged into natural ecosystems [1], [2]. The inefficient use of anthropogenic nitrogen fertilizers and wastewater discharge are the main sources of nitrate pollution in natural ecosystems [3], [4]. Owing to its high mobility and solubility, NO₃⁻ can easily penetrate groundwater, and its transfer from surface water to the ocean accounts for up to 40–70 Tg·year⁻¹ (Tg = 10¹² g) [5]. Physical and chemical approaches can remove NO₃⁻ efficiently but have high operational costs or produce secondary pollutants [6]. Although heterotrophic denitrification enables the conversion of NO₃⁻ to harmless nitrogen (N₂), an external carbon source as an electron donor is in high demand, which increases the running costs.

The conversion of NO₃⁻ to ammonia (NH₃)/ammonium (NH₄⁺) is preferred over the reduction of NO₃⁻ to N₂, as the former can valorize the pollutant NO₃⁻ into a resource in the form of NH₄⁺. Recently, NH₃/NH₄⁺ is viewed as more than the main component of nitrogen fertilizers, but also as having great potential to play a significant role in a future hydrogen economy (as a rich hydrogen carrier) [7], [8]. Furthermore, the novel option of using ammonia as a liquid energy source rather than a hydrogen carrier has emerged, and there are several examples of direct ammonia application as an energy source, such as a fuel for heavy transport vehicles and direct ammonia fuel cells [9].

The electrochemical reduction of NO₃⁻ to NH₄⁺ (namely, electrochemical NO₃⁻ reduction (NO₃RR)) is a promising and sustainable alternative to the Haber–Bosch process, which accounts for 2% of the global energy consumption (approximately 12.1 kilowatt hour per kilogram NH₃-N) and releases 1%–2% of the global carbon dioxide to convert N₂ to NH₄⁺ [10], [11]. Additionally, the higher solubility (92 vs 2.28 × 10⁻³ gram per 100 gram water) and lower dissociation energy (204 vs 941 kJ·mol⁻¹) of NO₃⁻ compared with N₂ benefit faster NH₄⁺ production kinetics [12], [13]. Although satisfactory NO₃RR performance was consistently reported with synthetic NO₃⁻-containing wastewater, such as high faradaic efficiency (> 90%) [14] and NH₄⁺ selectivity (∼95%) [15], a high NO₃⁻ concentration (at least > 140 milligram N per liter (mg-N·L⁻¹), even up to 14 gram N per liter (g-N·L⁻¹) seemed to be a prerequisite for satisfactory achievements. The NO₃⁻ concentration in limited industrial wastewater can be sufficiently high to ensure NO₃RR performance, for example, in fertilizer industry wastewater (∼950 mg-N·L⁻¹) [16] and explosive industry wastewater (∼3000 mg-N·L⁻¹) [17]. However, NO₃⁻ concentrations in domestic wastewater, surface water, and groundwater are much more diluted than those in the above-mentioned industrial wastewater, which is a challenge for the NO₃RR. Although Kim et al. [18] designed a bifunctional electrode by coupling NO₃⁻ enrichment and electrocatalytic conversion to NH₄⁺, the challenge of low NO₃⁻ concentration was craftily detoured but not directly addressed.

Compared to chemical catalysts, microorganisms as biocatalysts have the advantages of high selectivity and mild reaction conditions, in addition to being nontoxic and reproducible [19]. Shewanella oneidensis MR-1 (S. oneidensis MR-1) was reported to have NrfA genes [20], indicating that it is able to reduce NO₃⁻ to NH₄⁺ via the dissimilatory nitrate reduction to ammonium (DNRA) pathway with accessible electron donors. Furthermore, it is well known that S. oneidensis MR-1, the most well-understood exoelectrogen, has extracellular electron transfer capabilities [21]. Therefore, S. oneidensis MR-1 was able to perform the DNRA process using cathodes as the sole electron donors [22]. Recently, Sakimoto et al. [23] developed a pioneering semiconductor and non-photosynthetic bacterial hybrid system that implemented CO₂ reduction to acetate with an acetate yield of about 90%.

Therefore, we hypothesized that Shewanella bacteria can capture and utilize the photoexcited electrons produced by semiconductors (e.g., CdS NPs) for NO₃⁻ reduction to NH₄⁺ via the DNRA pathway. Consequently, in this study, we constructed a CdS NPs-coated S. oneidensis MR-1 biohybrid system for NO₃⁻ reduction to NH₄⁺. A series of experiments were conducted to verify the above hypothesis. The constructed S. oneidensis MR-1/CdS NPs biohybrid system demonstrated a high NH₄⁺ selectivity of higher than 90% and a maximum instantaneous quantum efficiency of 3.01%. Finally, the possible mechanism by which Shewanella bacteria utilize photoexcited electrons was extensively evaluated.

2. Experimental section

**2.1. Culture medium and conditions of S. oneidensis MR-1**

Bacterial activation was completed in Luria–Bertani (LB) medium (5 g·L⁻¹ yeast extract, 10 g·L⁻¹ tryptone, and 10 g·L⁻¹ NaCl, pH 7). Agar (2 g) was added to 100 mL LB medium to solidify the medium. The contents of Medium A for CdS biosynthesis by S. oneidensis MR-1 were as follows: 2.38 g·L⁻¹ C₈H₁₈N₂O₄S, 0.62 g·L⁻¹ NaHCO₃, 3.73 mL·L⁻¹ sodium lactate, 0.12 g·L⁻¹ MgSO₄·7H₂O, 0.46 g·L⁻¹ NaCl, 1.0 g·L⁻¹ β-glycerophosphate·2Na·xH₂O, and 0.23 g·L⁻¹ (NH₄)₂SO₄. All media were sterilized prior to application.

The composition of Medium B was almost the same as Medium A only without organic carbon components addition: 2.25 g·L⁻¹ NaHCO₃, 0.12 g·L⁻¹ MgSO₄·7H₂O, 0.46 g·L⁻¹ NaCl, 0.80 g·L⁻¹ KH₂PO₄ supplemented with 1 mL of trace element solution (0.5 g·L⁻¹ ethylene diamine tetraacetie acid (EDTA)−2Na, 0.3 g·L⁻¹ CoCl₂·6H₂O, 0.32 g·L⁻¹ CuSO₄·5H₂O, 0.24 g·L⁻¹ NiCl₂·6H₂O, 0.018 g·L⁻¹ H₃BO₃, 1.24 g·L⁻¹ MnCl₂·4H₂O, 0.28 g·L⁻¹ NaMoO₄·2H₂O, and 0.1 g·L⁻¹ Na₂WO₄·2H₂O), 1 mL of vitamin nutrient solution (2 mg·L⁻¹ D-vitamin, 2 mg·L⁻¹ folic acid, 10 mg·L⁻¹ pyridoxine-HCl, 5 mg·L⁻¹ thiamine-HCl, 5 mg·L⁻¹ riboflavin, 5 mg·L⁻¹ niacin, 5 mg·L⁻¹ thbrthdrexvbdr, 0.1 mg·L⁻¹ mecobalamin, 5 mg·L⁻¹ p-aminobenzoic acid, and 5 mg·L⁻¹ lipoic acid), and 0.3 g·L⁻¹ L-cysteine-HCl·H₂O.

2.2. Growth of the biohybrid system

Therefore, the optimal Cd concentration for S. oneidensis MR-1 biosynthesis in CdS needs to be determined. To avoid cadmium phosphate precipitation during CdS nanoparticles formation, β-glycerophosphate·2Na·xH₂O was used instead of K₂HPO₄. The S. oneidensis MR-1 (ATCC 700550) was obtained from the American Type Culture Collection (USA) and stored in the −80 °C refrigerator. First, S. oneidensis MR-1 was activated in LB medium and allowed to reach logarithmic phase. Next, S. oneidensis MR-1 cells were washed with phosphate buffered saline (PBS) and cultured strictly anaerobically in Medium A. The experimental groups with 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, and 0.2 mmol·L⁻¹ CdCl₂ were applied to generate CdS nanoparticles, compared to the control groups without Cd²⁺ supplementation. The reaction systems were incubated at 30 °C with a speed of 180 r·min⁻¹. The optical density (OD₆₀₀) of the medium was recorded using a ultraviolet–visible spectrophotometer (UV–Vis; UH5300, Hitachi, Japan).

To determine the optimal cadmium concentration for CdS generation, the S. oneidensis MR-1 was cultured strictly anaerobically in Medium A containing 0.10 mmol·L⁻¹ of CdCl₂ and 0.30 mmol·L⁻¹ of cysteine (30 °C, 180 r·min⁻¹). S. oneidensis MR-1 can synthesize CdS by microbes themselves to form the S. oneidensis MR-1/CdS biohybrid system. The synthesized biohybrid system was centrifuged at 11 000 r·min⁻¹ for 10 min at 4 °C and transferred to Medium B supplemented with cysteine (0.3 g·L⁻¹) and nitrate (about 10 mg·L⁻¹) and cultured under light conditions (30 °C, 180 r·min⁻¹).

**2.3. Characterization of S. oneidensis MR-1/CdS biohybrid system**

The absorption spectrum of the biohybrid system was detected using UV–Vis spectroscopy in the range of 450–700 nm. The biohybrid system suspension bacteria was removed by sterilization, and the CdS nanoparticles were measured using X-ray diffraction (XRD; D8 Advance, Bruker, Germany) with a Co Kα source to identify crystalline properties.

S. oneidensis MR-1/CdS samples were observed via scanning electron microscopy (SEM; JSM-7610 Plus, JEOL, Japan) using the procedure described by Sakimoto et al. [23]. The samples were fixed overnight in a 50:50 ratio of Medium B (45 v%)/glutaraldehyde (5 v%):S. oneidensis MR-1/CdS suspension (50 v%). The fixed samples were then filtered onto a 0.1 μm filter membrane by a vacuum pump and subsequently washed several times with ethanol solution with increasing concentrations (0, 25%, 50%, 75%, 90%, and 100%). Finally, the washed samples on the filter membrane were transferred to hexamethyldisilazane for 1 h and dried overnight.

To prepare the samples for high-angle annular dark field transmission electron microscopy (HAADF TEM; Talos F200S, Thermo Fisher Scientific, USA), the collected pellets were washed with PBS and freeze-dried to obtain a powder. The dried powders were dissolved in 1 mL of ethanol solution (100 v%) and then transferred onto the surface of a copper grid with a Formvar coating. After the samples were fixed on the grid, HAADF TEM imaging and energy dispersive X-ray spectroscopy (EDS; SUPER X, USA) analysis were performed.

**2.4. Photo-electrochemical characterization of S. oneidensis MR-1/CdS biohybrid system**

Photocurrent response tests were performed using a CHI650E electrochemical workstation (Chenhua Co., Ltd., China). The 100 μL suspension of S. oneidensis MR-1/CdS biohybrid system was dropped onto the surface of the screen-printed electrode (SPE) containing a carbon working electrode, a carbon auxiliary electrode, and an Ag/AgCl reference electrode, and then the photocurrent was measured in the chronoamperometry mode. Steady-state photoluminescence (PL) decay spectra were obtained using an FLS1000 PL spectrometer (FLS1000, Edinburgh, UK).

**2.5. Photocatalytic NO₃⁻ reduction in S. oneidensis MR-1/CdS biohybrid system**

The system used in the photocatalytic experiment was the same as that used for growth of the biohybrid system. The biohybrid system was centrifuged to collect cell pellets. The collected pellets were cultured in Medium B. The experiment was carried out under anaerobic conditions, and the applied light source was an light-emitting diode (LED) lamp of (395 ± 5) nm. The concentrations of NO₂⁻-N, NO₃⁻-N, and NH₄⁺-N were detected using a spectrophotometer (V-560, JASCO, Japan) and ion chromatograph (DIONEX ICS, Thermo Fisher Scientific, USA), and the concentrations of N₂ and N₂O were detected using a gas chromatograph (GC7900, Tianmei, China).

2.6. Colony forming unit (CFU) assay

The solution of the constructed biohybrid system was diluted step by step, and 100 μL of the dilution was spread evenly on the solid medium. The smeared plates were placed upside down in a 30 °C incubator for 24 h. Visible white and circular colonies were counted to determine the CFU as a measure of cell number and viability.

2.7. Calculation of the instantaneous quantum efficiency

The instantaneous quantum efficiency (IQE) was determined by comparing the number of electrons received by the NO₂⁻-N and NH₄⁺-N with the number of incident photons. The reduction of NO₃⁻-N to NO₂⁻-N and NH₄⁺-N requires two and eight electrons, respectively, resulting in the following IQE equation:

I Q E = \frac{T h e n u m b e r o f e l e c t r o n s r e c e i v e d b y t h e r e d u c t i o n p r o d u c t s}{T h e n u m b e r o f i n c i d e n t p h o t o n s} \times 100 % = \frac{[2 \times n_{{N O}_{2}^{-}} + 8 \times n_{{N H}_{4}^{+}}] \times N_{A}}{\frac{I_{l} \times S \times t \times λ}{h \times c}} \times 100 %

where n_NO₂⁻ and n_NH₄⁺ (mol) were the amount of substance of NO₂⁻-N and NH₄⁺-N; N_A was the Avogadro constant; I_l (W·cm⁻²) was the light intensity of 395 nm of the LED light; S (cm²) was the effective area of light irradiation; t (s) was the light-driven experiment time; λ (m) was the wavelength of incident light; h (J·S) was the Planck constant; and c (m·s⁻¹) was the light speed.

2.8. Parallel reaction monitoring (PRM) analysis

Samples from the S. oneidensis MR-1/CdS biohybrid system after photocatalytic NO₃⁻ reduction (experimental groups) and the biohybrid system without NO₃⁻-N (control groups) were washed with PBS several times. Bacterial samples were collected and frozen in liquid nitrogen. The proteins were extracted from the frozen samples, treated by reductive alkylation, purified by trypsin enzymatic hydrolysis, and then the peptides after enzymatic hydrolysis were analyzed using a liquid chromatograph mass spectrometer (LC-MS; Thermo Fisher Scientific, USA). Peak extraction was performed on the raw data using skyline, and 3–4 ions with higher abundances in the product ions were selected for quantitative analysis and manually checked and corrected. The target peptide sequences are listed in Table S1 in Appendix A.

3. Results and discussion

3.1. Characterization of S. oneidensis MR-1/CdS biohybrid system

After Cd²⁺ and cysteine were added to Medium A containing S. oneidensis MR-1 in the logarithmic phase, the color of the solution changed from transparent to yellow, indicating the formation of CdS. Subsequently, SEM demonstrated that the particles were tightly bound to the bacterial cells (Fig. 1(a), Figs S1 and S2). HAADF TEM and EDS mapping images (Figs. 1(b)–(d)) illustrated that the nanoparticles were mainly aggregated on the cell surface and consisted of Cd and S. C, N, and O were derived from S. oneidensis MR-1 (Figs. 1(e)–(g)). According to the UV–Vis absorption spectrum, the formed biohybrid system absorbed light and had a bandgap of (2.37 ± 0.05) eV (Fig. S3 in Appendix A). XRD observation (Fig. S4 in Appendix A) confirmed that the nanoparticles in the bacterial solution were CdS with a cubic structure (Joint Committee on Powder Diffraction Standards (JCPDS) card: 10-0454).

The photoactivity of S. oneidensis MR-1/CdS was evaluated by recording photocurrent generation in a photo-electrochemical cell. Fig. 2(a) shows a satisfactory photocurrent production capability of the S. oneidensis MR-1/CdS biohybrid system under light irradiation compared with that of the same system without light irradiation. The S. oneidensis MR-1/CdS biohybrid system had a higher photocurrent response intensity than that of S. oneidensis MR-1 (dead)/CdS, indicating the photogenerated electron transfer properties in the S. oneidensis MR-1/CdS biohybrid system. To probe the deep mechanisms of the photoexcited electron transfer, steady-state PL decay spectra were obtained (Fig. 2(b)). The fluorescence intensity of S. oneidensis MR-1/CdS decayed faster than that of S. oneidensis MR-1 (dead)/CdS, reflecting the more available proximal electron acceptors from live S. oneidensis MR-1 [24].

3.2. Photocatalytic reduction of NO₃⁻ in the biohybrid system

Photocatalytic NO₃⁻ reduction was carried out with the same Medium B but divided into four parallel groups without light, without active S. oneidensis MR-1, without CdS NPs, and with all these three components. Fig. 3(a) showed that the NO₃⁻-N concentrations in the vials without light, without active S. oneidensis MR-1, and without CdS NPs were almost stable at the initial concentration, and no obvious NO₂⁻/NH₄⁺ generation was observed in these cases. While in the vials with light, active S. oneidensis MR-1, and CdS NPs, NO₃⁻ was clearly reduced over time and completely consumed at the 36 h, with a reduction rate of 0.24 mg NO₃⁻-N·h⁻¹·g⁻¹. Consequently, light-active S. oneidensis MR-1 and CdS NPs are essential for light-driven NO₃⁻ reduction. Furthermore, CFU assays illustrated that S. oneidensis MR-1 concentrations (CFU values) almost doubled (versus the control) in the biohybrid system by converting NO₃⁻ to NH₄⁺ with photoexcited electrons as donors. In contrast, the CFU values of S. oneidensis MR-1 in vials without light and without CdS NPs decreased by 38.71% and 75.54%, respectively, compared to the control, as shown in Fig. 3(b). The above results supported that S. oneidensis MR-1 (although a non-photosynthetic microbe) could grow by harnessing energy directly from photocatalytic NO₃⁻ reduction.

After confirming light-driven NO₃⁻ reduction, the products were further analyzed to explore possible degradation pathways, for example, denitrification or the DNRA pathway. Fig. 4(a) shows that NH₄⁺ was the main end product (around 93.0% of all the end nitrogen products) and no other gaseous nitrogen-containing compounds were monitored when NO₃⁻ was consumed within 36 h. Moreover, NO₂⁻ accumulation was also observed during the whole period, which indirectly implied that the DNRA pathway should be the main route for light-driven NO₃⁻ reduction by the S. oneidensis MR-1/CdS NPs biohybrid system. The NH₄⁺ production rate was calculated to be 0.25 mg-N·h⁻¹·g⁻¹. The NH₄⁺ + NO₂⁻-N production rate was almost in balance with the NO₃⁻-N reduction rate; therefore, this light-driven NO₃⁻ reduction could be deduced via the DNRA pathway based on the above experimental results. The IQE reached a maximum value of 3.01% at 24 h.

Subsequently, day/night cycle conditions (3 h light vs 3 h dark) were simulated to test their effect on the S. oneidensis MR-1/CdS biohybrid system (Fig. 4(b)). The concentration of NH₄⁺-N continuously increased not only under light conditions but also during dark periods. A possible reason for this is the accumulation of excess biosynthetic intermediates, such as nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH). All the reducing substances produced during light periods can be used in the dark to reduce NO₃⁻ [23]. Generally, NO₃⁻ reduction, NO₂⁻ variation, and NH₄⁺ generation were identical to those under continuous light illumination conditions. An NO₃⁻ removal efficiency of 93.7%, NH₄⁺-N generation rate of 0.25 mg-N·h⁻¹·g⁻¹, and NH₄⁺ selectivity of 90.5% were achieved simultaneously, which was nearly identical to the performance under continuous light illumination.

Subsequently, the effects of different concentrations of S. oneidensis MR-1 on light-driven NO₃⁻ reduction were investigated (Fig. 5). When the OD₆₀₀ of S. oneidensis MR-1 decreased to 1.5 from 0.7, the final NO₃⁻ reduction efficiency was 56.4%, and NO₂⁻ was the main end-product. When the OD₆₀₀ increased to greater than 2, no NO₃⁻ reduction was observed. This could be ascribed to the high bacterial concentration, which unquestionably enhanced the turbidity in the experimental vials and further blocked the transmission and utilization of light. The results implied that an OD₆₀₀ of approximately 1.5 was the most suitable option in this study.

3.3. PRM Analysis

In order to clarify the possible photoexcited electron transfer mechanism, the mutants of metal reductase C and outer membrane cytochrome c protein A (△MtrC-△OmcA) and cytochrome c menaquinol oxidoreductase A (△CymA) were applied to construct the identical biohybrid systems coupled with CdS NPs. As shown in Fig. S5 in Appendix A, NH₄⁺ was detected in very small amounts in both △MtrC–△OmcA/△CymA/CdS NPs biohybrid systems. Furthermore, PRM results showed that the FccA protein was upregulated by approximately 7 fold compared to the control, as shown in Fig. 6. These results illustrate that MtrC (metal reductase C) , OmcA (outer membrane cytochrome c protein A), MtrA (metal reductase A), MtrB (metal reductase B), FccA (fumarate reductase) and CymA (cytochrome c menaquinol oxidoreductase A) proteins are undeniably involved in extracellular photoexcited electron transfer from CdS NPs to the intracellular compartment of Shewanella. The PRM analysis results also showed that the NapA and NrfA proteins were upregulated, which further verified that NO₃⁻ reduction to NH₄⁺ occurred via the DNRA pathway in this biohybrid system. The proposed electron transport mechanism is illustrated in Fig. 7(a). Based on these results, we proposed the reaction equations for photocatalytic NO₃⁻ reduction by S. oneidensis MR-1 (Text S2 in Appendix A).

Moreover, the S. oneidensis MR-1/CdS biohybrid system exhibited superior NH₄⁺ synthesis performance. The NH₄⁺ generation rate of our biohybrid system reached 0.25 mg-N·h⁻¹·g⁻¹, which was much higher than the rates obtained via conventional heterotrophic (organic carbon) [31] or autotrophic (Fe²⁺ and S²⁻) DNRA processes [32], [33]. Furthermore, the product rates and maximum IQE in this study outperformed those of other identical microbe/semiconductor biohybrid systems, which were employed to carry out conversions such as CO₂-to-acetate [26], NO₃⁻-to-N₂O [27], CO₂-to-methane [28], [29], and so forth [30], as shown in Fig. 7(b). This bacterial-semiconductor biohybrid system demonstrated exceptional survivability and excellent feasibility, which further provided a solid basis for its potential application in solar light and waste nitrogen sources. The strategy reported in this scenario can also expand the employment range of light energy from photosynthetic to non-photosynthetic bacteria.

In addition, several types of industrial wastewater contain extremely high levels of NO₃⁻ (300–2000 mg·L⁻¹) [16], [34], [35], [36], [37]. Conventional denitrification technology, although effective in removing nitrate-containing wastewater, inevitably increases costs (2.458 USD·kg-N⁻¹) because of the requirement of an additional carbon source [38]. Therefore, converting pollutant nitrate into high value-added ammonia is more beneficial for environmental sustainability than using N₂ as the end product. For the downstream process of ammonium recovery, struvite precipitation is a good alternative that has already been applied in practice at the Amsterdam West Wastewater Treatment Plant [39]. Our S. oneidensis MR-1/CdS biohybrid system could convert NO₃⁻ to NH₄⁺ only under light illumination, laying a theoretical foundation for the recycling of industrial NO₃⁻ containing wastewater. However, the bacteria-semiconductor biohybrid system is only in its infancy, and much more effort is required for real-world implementation.

4. Conclusions

In summary, the S. oneidensis MR-1/CdS biohybrid system enabled ammonia photosynthesis from nitrate using photoexcited electrons as donors. The nitrate reduction efficiency reached almost 100%, with an ammonia production selectivity of over 90%. The maximum instantaneous quantum efficiency was 3.01% under light irradiation. The reverse Mtr pathway is responsible for the transfer of photoexcited electrons to intracellular compartments. PRM analysis illustrated that NO₃⁻ to NH₄⁺ was produced via the DNRA pathway in S. oneidensis MR-1.

CRediT authorship contribution statement

Meiwei Guo: Resources, Methodology, Investigation, Data curation. Guangfei Liu: Supervision, Investigation, Formal analysis, Conceptualization. Sen Qiao: Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Xie Quan: Supervision, Project administration, Investigation, Formal analysis, Conceptualization.

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

This work was supported by the National Key Research and Development Project (2019YFA0705804). The authors deeply appreciate Prof. Kenneth Nealson (University of Southern California) for kindly providing us MR-1 and related mutant strains.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.eng.2024.08.004.

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