Denitrification by Purple Phototrophic Bacteria: A Carbon-Neutral and Resource-Efficient Route for Nitrogen Removal

Abstract

Purple phototrophic bacteria (PPB) have great potential in treating nitrogen-contaminated wastewater. Unlike conventional heterotrophic denitrification, PPB-driven denitrification utilizes light-driven metabolism, concurrently improving nitrogen removal and carbon management efficiency. This work aimed to develop a PPB denitrification process for nitrogen removal, carbon emission mitigation, and resource recovery. The PPB growth was first optimized and the most desirable light and carbon sources (i.e., incandescent light and sodium acetate) were pinpointed. PPB denitrification could reach a nitrate removal rate of 0.68 mg N/L/h, while no nitrite was detected during the process, regardless of the amount of external electron donors. This was attributed to the fact that the true reduction rate of nitrite (4.42 mg N/gVSS/h) was significantly higher than that of nitrate (1.51 mg N/gVSS/h). In the presence of a sufficient carbon source, PPB denitrification was found to be a low-carbon process, with only ~0.17% of converted nitrate being emitted as nitrous oxide. Meanwhile, PPB biomass for denitrification was rich in value-added products (e.g., protein and pigment), which potentially generated additional benefits over the biomass valued at USD 17 kg⁻¹. These results provide a theoretical basis for implementing PPB denitrification for carbon-neutral and resource-efficient wastewater treatment.

1. Introduction

Currently, nitrate (NO₃⁻) and nitrite (NO₂⁻) contamination in aquatic ecosystems has emerged as a significant global environmental concern [1,2]. This could be attributed to excess application of inorganic nitrogenous fertilizers, excessive discharge of sewage, and oxidation of nitrogenous waste products in human and animal excreta [3]. High concentrations of NO₃⁻ and NO₂⁻ in water bodies not only cause eutrophication, toxic algal blooms and hypoxia, but also increase the risk of developing methemoglobinemia and gastrointestinal cancer [4]. According to the World Health Organization (WHO), the maximum allowable concentrations of NO₃⁻ and NO₂⁻ in drinking water are set at 11.3 mg N/L and 0.91 mg N/L, respectively [5]. The NO₃⁻ concentration in nitrogen fertilizer plant wastewater exceeds ~67.74 mg N/L [6], while nitrogen source concentrations in food processing wastewater, such as brewery wastewater, are more than 100 mg N/L [7]. They necessitate further treatment to meet the discharge standards.

The biological processes for NO₃⁻ and NO₂⁻ removal are primarily driven by microbial-mediated nitrogen cycle reactions, including denitrification, anaerobic ammonia oxidation, aerobic denitrification, assimilation, and other related synergistic processes [8]. Biological denitrification is a metabolic process involving four steps, each catalyzed by specific enzymes: a nitrate reductase (NAR), a nitrite reductase (NIR), a nitric oxide reductase (NOR) and a nitrous oxide reductase (N₂OR) [9]. Due to the ability of heterotrophic denitrifying bacteria to utilize multiple carbon sources and achieve a rapid denitrification rate, they have been widely applied in wastewater treatment processes [10]. However, the implementation of heterotrophic denitrification processes is often limited by factors such as inadequate carbon sources, high operational costs, excessive sludge generation, and the potential for secondary pollution post-treatment [11]. The development and implementation of novel heterotrophic denitrification technologies for zero carbon emissions and efficient resource recovery attract intensive attention.

PPB are widely distributed prokaryotes that are capable of carrying out photosynthesis, including purple sulfur bacteria (PSB) and purple non-sulfur bacteria (PNSB) [12]. Due to their multiple pathways for energy and substance metabolism, PPB can effectively utilize various substrates, such as organic matter, ammonia, NO₃⁻ and NO₂⁻, for metabolism under the conditions of light or oxygen [13,14]. PPB can tolerate high concentrations of inhibitory substances in wastewater. For example, Hao, et al. [15] found that PPB were able to grow at higher salinities (60 g NaCl/L) without experiencing cellular lysis due to osmotic pressure imbalance. PPB cells are rich in value-added substances such as protein, carotenoid, bacteriochlorin, and polyhydroxyalkanoates (PHA) [16,17]. Recycling the residual PPB biomass can reduce the cost of residual sludge disposal and generate additional benefits by producing the above-mentioned value-added substances. Therefore, PPB-based wastewater treatment processes are expected to fulfil pollutant removal and resource recovery at the same time, which deserve a systematical and in-depth investigation.

PPB showed great potential in treating wastewater containing NO₃⁻ and NO₂⁻. According to Idi, et al. [18], PPB successfully removed 60–70% of nitrates from synthetic wastewater containing different gradient concentrations of nitrate. Zhu, et al. [8] isolated a PPB strain and observed that it could completely eliminate NO₂⁻ (about 60 mg N/L). The genome sequencing results also suggested the presence of a denitrification pathway. Meanwhile, Yang, et al. [14] and Yang, et al. [19] found that nitrite was not detected during the efficient removal of nitrate by PPB. In these studies on denitrification by PPB, the light source and carbon source are two essential conditions. Different wavelengths of light may influence the photosynthetic efficiency of PPB, as well as the activity of relevant intracellular enzymes [20], indirectly affecting their capacity to carry out denitrification. Furthermore, the carbon source not only provides building blocks for the cells but also serves as an electron donor participating in the denitrification process [21]. Given the above, the PPB denitrification process can be a carbon-neutral and cost-effective nitrogen removal technology, which holds the promise of achieving nitrate elimination and resource recovery simultaneously. However, the metabolic kinetics of PPB denitrification under varying conditions remain poorly characterized, hindering its further implementation in wastewater treatment.

This study investigates the denitrification metabolic mechanisms of PPB, providing key insights into their potential application in large-scale microbial denitrification, with a focus on their effectiveness in enhancing nitrogen removal and supporting sustainable wastewater treatment processes. In this regard, the relevant parameters affecting PPB denitrification and the denitrification removal rate kinetics have been examined. The objectives of the present research are as follows: (1) optimize the light and carbon sources for the growth of PPB; (2) investigate the effects of different C/N ratios on the performance of PPB denitrification; (3) analyze kinetic rate at each step of the PPB denitrification process; and (4) evaluate implications for the PPB denitrification process.

2. Materials and Methods

2.1. PPB Enrichment

The PPB culture in this research was enriched from the anaerobic sludge of an industrial park in Wuhan, China. The collected sludge samples were inoculated into 500 mL Schott bottles for incubation. The details of the enrichment medium are shown in Table S1. The enrichment was performed on a stirrer at 150 rpm in duplicate, with each Schott bottle containing 400 mL of inoculum culture (10%, v/v). The system was sparged with N₂ for 7 min to establish and maintain an anaerobic environment. An amount of 10 mL of sterile liquid paraffin was layered on top of the solution, and the bottle was sealed with a screw cap equipped with a silicone rubber septum to ensure anaerobic conditions. The culture was subjected to five distinct light sources, including an incandescent lamp (IL) (40 W, Beineng Lighting Co., Ltd., China), infrared light (IFL) (consisting of an incandescent lamp with an infrared filter) and light-emitting diodes (LEDs) in blue, green and yellow (20 W, Forward Lighting Technology Co., Ltd., Rizhao, China). The temperature, pH and light intensity were set to 30 ± 1 °C, 7.0 ± 0.2 and 3000 ± 100 lux, respectively, and were kept constant across all experiments.

COD and NH₄⁺ in the medium were monitored at 24 h intervals under different light sources. The absorbance between 300 and 1000 nm was measured using a UV–vis spectrophotometer (UV–5500PC, Shanghai Metash Instruments Co., Ltd., Shanghai, China) when the enrichment culture turned reddish (Figure S1) to detect the presence of pigment absorption peaks. The enrichment cultures with the possible presence of PPB were identified by high-throughput 16S rRNA sequencing (see Section 3.1). The culture enriched under an incandescent lamp was used for further growth experiments.

2.2. Growth Tests

Five carbon sources (sodium acetate, glucose, sucrose, sodium citrate and sodium malate), which are five small-molecule organic carbon compounds listed in ascending order of molecular weight, were tested at an equal COD concentration of 3 g/L based on the growth medium (Table S1) for the mixed PPB culture. The growth medium was pre-autoclaved at 121 °C for 20 min prior to inoculation. The light source was an incandescent lamp. The other conditions were the same as in the enrichment experiment. The optical density (OD) at 660 nm was monitored on a daily basis. COD was monitored at the beginning and end states of the samples during the tests. Two parallels were made for each carbon source condition. The growth tests lasted six days.

2.3. Batch Denitrification Experiments

Synthetic wastewater was used as the growth substrate for PPB in batch experiments, which contained NaHCO₃ (0.8 g/L), CaCl₂·2H₂O (0.012 g/L), MgCl₂·6H₂O (0.18 g/L), NaCl (0.5 g/L), KCl (0.78 g/L) and trace elements (Table S1). Sodium acetate and incandescent lamps were chosen as carbon and light sources, respectively. NaNO₃, NaNO₂ and N₂O were selected as nitrogen sources. NaOH solution was used to adjust the pH to 7.0 ± 0.1. For these experiments, 500 mL Schott bottles were employed as bioreactors, containing a working volume of 400 mL. The PPB were inoculated into synthetic wastewater in the Schott bottle at 30 ± 1 °C with an initial inoculation ratio of 25% (v/v). The Microbial Live Volatile Suspended Solids (MLVSS) concentration was about 180~200 mg/L. Each test lasted for 3 days, after which PPB tended to cease growing due to the limitation of the growth substrate. Light plays a crucial role in influencing the energy metabolic pathways in PPB. Therefore, while maintaining the anaerobic conditions of light, a dark-anaerobic experimental group was set up to eliminate the interference of other factors. The C/N ratio is set based on the requirements for complete microbial response of heterotrophic denitrifying microorganisms to different nitrogen sources, corresponding to sufficient, theoretical and deficient ratios. The nitrogen sources and their associated conditions were established as follows:

(a)

NaNO₃ was used as the sole nitrogen source (50 mg N/L). C/N was set to 10:1, 2.86:1, 1:1;

(b)

NaNO₂ was as the sole nitrogen source (50 mg N/L). C/N was set to 10:1, 1.71:1, 1:1;

(c)

N₂O was as the sole nitrogen source (0.38 ± 0.02 mg N/L). C/N was set to 10:1, 0.57:1, 0.1:1 (to minimize the potential hazards to experimental personnel due to the toxicity and low solubility of nitrous oxide, a low concentration of N₂O was used for nitrogen source exploration. Meanwhile, experimental conditions such as biomass and reaction volume were scaled down accordingly).

Two light conditions were applied in all three experiments with single nitrogen sources to investigate the effect of light on the experimental process. Light-anaerobic condition: the light intensity was maintained at 3000 ± 100 lux by regulating the distance between the fluorescent lamps and the bioreactors; dark-anaerobic condition: the light intensity was kept below 2 lux by covering the bioreactors with tinfoil. This anaerobic condition was kept by flushing with nitrogen and sealing the bioreactors with a rubber plug. The MLVSS concentration was measured in triplicate at the end of each experiment. Samples were collected at multiple time points to monitor changes in NO₃⁻, NO₂⁻ and N₂O levels in the experiment. The variations in protein and pigment content in the samples were assessed both at the onset and at the termination of the experiment.

In addition, the PPB strain Rhodobacter sphaeroides (Rb. sphaeroides), which was present in the laboratory and isolated from a local pond in Wuhan, China, was also compared and investigated [22]. Its denitrification performance experimental design was consistent with that of the above strains.

2.4. Analytical Methods

The OD at 660 nm was monitored by taking 5 mL of samples under different carbon sources at regular intervals throughout the day. The pH was measured using an FE20 pH meter (FE20 pH meter, Mettler Toledo, Shanghai, China) with a resolution of 0.01 pH units [23]. Light intensity was determined with a lux meter (TES 1330A, TES Electrical Electronic Corp., Taiwan, China). Samples were harvested from the bioreactors and centrifuged at 11,000 rpm for 10 min to separate the supernatant, which was subsequently analyzed for NO₃⁻, NO₂⁻ and COD. The NO₃⁻, NO₂⁻ and COD were tested according to APHA standard methods [24] (detection limit: 0.1 mg/L), with calibration curves validated daily. Protein content was measured using the modified Lowry method as outlined by Hulsen, et al. [25] (detection limit: 1 µg/L). Carotenoids and bacteriochlorophylls were determined by Spectrophotometry according to Peng, et al. [22]. The N₂O concentration was analyzed following the method reported, using a gas chromatograph (7890 plus GC, Lunan Ruihong Chemical Instrument, Shandong, China) [26].

The maximum consumption rates of NO₃⁻, NO₂⁻ and N₂O were calculated by performing linear regression on their respective concentration profiles, with accumulation indicated as negative consumption. The apparent biomass-specific NO₃⁻, NO₂⁻ and N₂O consumption rates (R_1,a, R_2,a, R_4,a) were determined by dividing the rate data determined above by the MLVSS concentration measured. The true reduction rate of each nitrogen oxide was calculated using the following formula [27,28]:

$${\mathit{R}}_{\mathbf{1}}\mathbf{=}{\mathit{R}}_{\mathbf{1}\mathbf{,}\mathit{a}}$$

(1)

$${\mathit{R}}_{\mathbf{2}}\mathbf{=}{\mathit{R}}_{\mathbf{2}\mathbf{,}\mathit{a}}\mathbf{+}{\mathit{R}}_{\mathbf{1}}$$

(2)

$${\mathit{R}}_{\mathbf{3}}\mathbf{=}{\mathit{R}}_{\mathbf{3}\mathbf{,}\mathit{a}}\mathbf{+}{\mathit{R}}_{\mathbf{2}}$$

(3)

$${\mathit{R}}_{\mathbf{4}}\mathbf{=}{\mathit{R}}_{\mathbf{4}\mathbf{,}\mathit{a}}\mathbf{+}{\mathit{R}}_{\mathbf{3}}$$

(4)

where R_1,a, R_2,a, R_3,a and R_4,a (mg N/gVSS/h) are the true reduction rates of nitrate, nitrite, NO and N₂O, respectively. The apparent rate of reduction is a macroscopic measurement that may be affected by a variety of factors and may not accurately reflect the intrinsic rate of interaction between reactant molecules [29]. Compared to the apparent reduction rate, the true rate of reduction is the rate at which the reduction reaction proceeds based solely on the intrinsic mechanism of the chemical reaction itself, which is a more accurate representation of the rate of reaction at each step in biological denitrification [28].

To ensure data accuracy, all tests were performed in triplicate, and the final values reported represent the averages. The error bars indicate the standard deviations.

2.5. Microbial Community Analysis

Sampling for microbial community analysis was conducted when the OD of the culture remained stable during the enrichment process. Genomic DNA from the enriched culture was extracted using the DNA extraction kit (DL2000, Magigene Biotechnology, Shanghai, China) and the 16S rRNA gene was amplified using a pair of universal bacterial primers, 338F and 806R. The detailed operational procedure was adapted from Yu, et al. [30]. The polymerase chain reaction (PCR) products were analyzed by electrophoresis on agarose gel (1%, w/v), followed by purification and sequencing (Magigene Biotechnology, China). Based on these findings, the microorganisms enriched under both the incandescent lamp and infrared light were classified into genus of PNSB Rhodopseudomonas palustris (Rps. palustris).

3. Results and Discussion

3.1. PPB Enrichment and Growth Performance

Figure S1 shows that the mediums under the incandescent lamp and infrared light turned red after five days, while the color of the mediums under the other light sources were visually unchanged (pale yellow). Consistently, the distinct carotenoid and bacteriorhodopsin characteristic peaks were detected in samples under IL and IFL (Figure S2), which are typical features of purple non-sulfur bacteria, but not in those under other light sources [18]. The COD removal efficiency was about 90% under IL and IFL after five days, which was almost twice as high as the other experimental groups. The Rps. palustris contained carotenoids such as lycopene and rhodopin [31], which displayed a reddish color when present in large numbers. The infrared region is one of the major pigment absorption peaks of PPB [32], while about 90~95% of the input energy of incandescent lamps will be released in the form of heat, including a large amount of infrared radiation [33]. It is clearly shown that IFL and IL would favor PPB growth over other microbes. More evidence from the microbial community analyses is shown in Figure 1. It was confirmed that Rps. palustris was the dominant genus under IFL and IL, the abundance of which accounted for 68.5% and 67.7%, respectively. Other microorganisms, such as Streptococcus and Lactobacillus, accounted for less than 5% of the total number of microorganisms and were not part of the photosynthetic bacterial population.

The species composition and abundance of the mixed cultures obtained under both IL and IFL were similar in the experiment. However, IFL (USD 0.81 unit⁻¹) in engineering applications requires higher initial costs and sometimes necessitates specialized optical equipment for light adjustment, while IL (USD 0.32 unit⁻¹) is not only cost-effective but also easier to replace and maintain [34]. Thus, IL was chosen as the light source in the subsequent experimental investigation.

Compared to small-molecule carbon sources, the uptake of large-molecule organic matter by PPB is much slower. Wastewater containing highly polymerized or structurally complex organic matter is generally subjected to pretreatment such as fermentation before further treatment [35]. Thus, only small-molecule carbon sources are discussed in this Section 3.1. Figure 2 shows the growth curves of PPB supplied with sodium acetate, glucose, sucrose, sodium citrate and sodium malate. The PPB grown with all carbon sources showed a similar growth trend over 24 h, presumably due to the fact that PPB were still acclimating to their environment and were in a period of stabilization. However, when sodium acetate was used as the carbon source, the growth of PPB was much superior to that of the other groups after 24 h. Volatile fatty acids (VFAs) can stimulate the glyoxylate cycle pathway in PPB and enhance the metabolism of organic matter and the synthesis of more biomass in PPB [36,37], which could explain the variability in the PPB growth curves after 24 h described above. And methanogenic archaea that may be present in the bacterial solution could convert acetate [38], produced by the glyoxylate cycle and not assimilated by PPB, into methane, reducing the inhibitory effects of substrate accumulation on PPB. The released H₂ provides reducing equivalents for PPB’s photosynthetic electron transport chain [39], contributing to the rapid growth of PPB. Moreover, smaller molecules of sodium acetate were more readily absorbed by PPB compared to other carbon sources.

Among the aforementioned carbon sources, sodium acetate (USD 7.44 kg⁻¹), glucose (USD 7.34 kg⁻¹), and sucrose (USD 8.59 kg⁻¹) have similar prices, while the prices of sodium citrate and sodium malate are comparatively higher, recorded at USD 36.48 kg⁻¹ and USD 67.35 kg⁻¹, respectively [40]. The promoting effect of sucrose and glucose on the growth of PPB was less significant than that of sodium acetate (Figure 2), resulting in lower nitrogen removal that may not meet the requirement of treating high-load wastewater. Sodium acetate can be derived from the anaerobic fermentation of organic wastewater [41]. Therefore, sodium acetate was chosen as the sole organic carbon source in subsequent denitrification experimental studies.

3.2. The Impact of Varying Nitrogen Sources and C/N Ratios on PPB Denitrification

The essential conditions for PPB to perform heterotrophic denitrification include a light source, an organic carbon source, and an anaerobic environment [8]. The nitrogen removal was not observed under dark-anaerobic conditions for all three nitrogen sources (Figure S3). Non-purple photosynthetic bacteria that could denitrify were not detected in the PPB enrichments (Figure 1). These could exclude the interference from other heterotrophic denitrifying strains during the experiment. The utilization of different nitrogen sources by PPB under light-anaerobic conditions with different C/N applied is shown in Figure 3. When C/N was set at 1:1 or 0.1:1, none of the three nitrogen source contents changed significantly. This suggested that the denitrification performance of PPB was inhibited due to a lack of electron donors. The theoretical C/N ratio for NO₃⁻, NO₂⁻ and N₂O to be removed completely was 2.86:1, 1.71:1 and 0.57:1, respectively. However, under these conditions, only ~30% of the three nitrogen sources was reduced. This was because the experimental conditions were set to be suitable for the survival of PPB, but did not completely inhibit the activities of other microorganisms, such as Lactobacillus. Therefore, some of the carbon source would be utilized by these microorganisms as well, which made PPB denitrification incomplete. The removal efficiencies for NO₃⁻, NO₂⁻ and N₂O were then enhanced to 100%, 100% and 71%, respectively, under sufficient carbon sources (C/N:10:1). Under carbon-sufficient conditions that are inherently conducive to PPB growth, PPB would compete for much more electron donors compared to those in the carbon-insufficient groups. Nitrogen removal is enhanced when there are enough electron donors present [42].

The pollutant reduction rate reflects the metabolic activity of microorganisms from a kinetic perspective [19]. Figure 4 shows the apparent rate of reduction and true rate of reduction for different nitrogen sources during denitrification. The true reduction rate of NO₂⁻ was 4.42 mg N/gVSS/h, while the true reduction rate of NO₃⁻ was only 1.51 mg N/gVSS/h. It explained the fact that in three sets of experiments, where NO₃⁻ was used as the nitrogen source, no NO₂⁻ was detected during the experiments regardless of the amount of external electron donors. NO₂⁻ accumulation is one of the main challenges for NO₃⁻ conversion during denitrification, which could inhibit cell growth and increase the denitrification time [43]. Thus, the PPB denitrification overcomes the problem of nitrite accumulation, which facilitates efficient biological nitrate removal. Poh, et al. [44] reported that the true reduction rates of NO₃⁻ and NO₂⁻ by traditional heterotrophic denitrifying microorganisms exceed ~5.8 mg N/gVSS/h and ~7.2 mg N/gVSS/h, respectively, significantly higher than the reduction rates of nitrogen sources by PPB. This difference may be attributed to the fact that the activity, specificity and substrate affinity of the relevant nitrogen source reductases could vary among microbial species and may also be influenced by experimental environmental conditions.

The change in N₂O content in the experiments with NO₃⁻ and NO₂⁻ as nitrogen sources is shown in Figure 5a. It can be observed that the accumulation of N₂O decreased gradually as the reaction progressed, due to the fact that the amount of N₂O consumed by PPB during the denitrification process is greater than the amount of N₂O produced by PPB. The N₂O emission factor is the ratio of N₂O produced during denitrification to the total amount of nitrogen source consumed. Regardless of the C/N ratio, the N₂O emission factor using NO₂- as the nitrogen source was higher than that using NO₃⁻ as the sole nitrogen source (as shown in Figure 5b). This may be due to the fact that nitrite can reduce the expression of the N₂O reductase (NosZ) gene by influencing the activity of transcription factors and inhibiting the NosZ activity [45]. Under such conditions, NosZ is unable to fully utilize adequate electron donors to completely reduce all nitrous oxide, resulting in N₂O accumulation.

3.3. Implications for Theory and Practice

Previous studies have shown that some PPBs have multiple nitrogen metabolism pathways, such as complete reduction of nitrate through assimilation and through nitrogen fixation [46,47]. This paper explored the denitrification kinetic rates of Rps. palustris under different C/N and nitrogen source conditions, which helps to provide some references regarding the possible industrial application of PPB denitrification technology in the future. In addition, Rb. sphaeroides and Rps. palustris are both PNSBs but differ significantly in their denitrification capabilities. Figure S3 shows that the removal efficiencies of another PPB strain, Rb. sphaeroides, on three nitrogen sources under the same experimental conditions were all zero, suggesting that Rb. sphaeroides is unable to remove nitrogen via the denitrification pathway. According to Zhu, et al. [8], some PPB strains lack the genes encoding NAR, NIR, NOR and N₂OR, which prevents them from carrying out denitrification metabolism. Therefore, it is important to the select desirable PPB strains in the future industrial application of PPB denitrification technology.

At present, the need for greenhouse gas emission reduction in the wastewater treatment process is increasingly urgent. The N₂O emission factor in conventional denitrification could fluctuate between 1% and 90% in practice [48,49,50]. Under conditions of sufficient carbon, the N₂O emission factor by PPB remains at a low level when utilizing different nitrogen sources. For example, the N₂O emission factor was ~0.17% in the NO₃⁻ removal process by PPB in the presence of a sufficient carbon source (Figure 5b), which can reduce the carbon emissions by about a factor of 4 compared to conventional heterotrophic denitrification [51,52]. When the carbon source was insufficient, the N₂O emission factor in the denitrification process of PPB significantly increased, which was similar to the N₂O emission factor (between 20% and 30%) observed in some traditional heterotrophic denitrifying microorganisms under the same conditions [53,54]. It is important to control the carbon source during the denitrification process in PPB to prevent the excessive production of N₂O. Furthermore, PPB has been extensively examined due to its high concentration of value-added compounds such as proteins and pigments in comparison to other bacteria [13]. Table S2 shows the changes in the content of value-added substances in PPB biomass before and after the removal of nitrogen sources. It can be seen that intracellular proteins and pigments were elevated by more than 10% after nitrogen source removal by PPB under sufficient carbon supply. Due to the short doubling time of PPB [55], value-added substances could be obtained from the excess PPB biomass in the denitrification process. The simple estimation of the value-added product of PPB denitrification yielded a benefit of USD 17.63 kg⁻¹ biomass (Table S3), which could effectively address the issue of sludge disposal difficulties encountered in the conventional heterotrophic denitrifying microbial treatment process. However, the economic feasibility of high-value product production from PPB microorganisms is limited by light energy consumption and technological bottlenecks in scaling up [56]. In-depth research is needed to explore the stability of mixed cultivation, product separation processes, and performance optimization under real wastewater matrices to enable industrial-scale implementation. The production of PPB value-added substances was affected by various factors, such as light intensity and salinity [57,58]. In comparison to conventional heterotrophic denitrification, the additional cost of light in PPB denitrification needs to be taken into account. It is possible to optimize the experimental conditions for PPB denitrification to balance the costs and benefits by reducing PPB denitrification costs through light intensity optimization (e.g., intermittent illumination) and reactor design (e.g., LED arrays).

Nevertheless, there are still technical challenges to overcome in the application of PPB denitrification technology. First, the denitrification rates of PPB were more than 38.6% lower compared to those of heterotrophic denitrifying bacteria. This dilemma may be solved by screening and breeding PPB strains with a higher denitrification capacity. Simultaneously, the competitive interactions between PPB and heterotrophic bacteria must be considered. Future research should focus on strategically modulating the C/N ratio and optimizing the light–dark cycle to sustain the ecological dominance of PPB. Second, adequate carbon source supplementation was required to prevent the excessive accumulation of N₂O in the PPB denitrification process. This inevitably increases the operational costs of the process. It can be achieved by adding high-concentration organic wastewater as the carbon source during the denitrification process [59,60]. Third, the collection of PPBs poses a significant challenge due to their small particle size and poor sedimentation properties. Collecting concentrated PPB products may lead to an increase in overall costs [61]. Stegman, et al. [62] showed that the formation of granular sludge from PPBs within ~50 days could primarily be achieved by controlling the upward flow velocity, but using PPB granular sludge to treat realistic wastewater requires more investigation. Fourth, the risk and market value of the products extracted from PPB biomass should be further evaluated. Finally, in wastewater anaerobic treatment, batch experiments elucidate microbial mechanisms and pollutant removal potential, while continuous reactors simulate real-world hydraulic patterns, validate process stability and establish critical scale-up design parameters [63]. Although PPB batch studies yield fundamental metabolic insights, their limitations in replicating engineering realities necessitate continuous reactor validation for technology transfer. This integration requires multidisciplinary strategies encompassing biofilm engineering, light-transfer optimization and intelligent control systems.

4. Conclusions

This study investigated the denitrification processes in PPBs, providing both theoretical foundations and engineering implications for their scalable implementation in sustainable wastewater treatment. The key results are summarized as follows:

• Incandescent light and sodium acetate were the ideal light source and carbon source, respectively, for PPB (Rps. palustris) growth.
• The true nitrite reduction rate of PPB denitrification (4.42 mg N/gVSS/h) was significantly higher than its nitrate reduction rate (1.51 mg N/gVSS/h), eliminating the possibility of nitrite accumulation.
• Under conditions of sufficient carbon, the N₂O emission factor by PPB denitrification was ~0.17%, about 4 times lower than that of some conventional heterotrophic denitrification.
• The PPB biomass generated during the denitrification process could bring an additional revenue of USD 17.63 per kg⁻¹ of biomass.