Advanced Treatment of High-Concentration Ammonia–Nitrogen Wastewater by Pantothenic Acid-Enhanced Photosynthetic Bacteria

Abstract

To address the slow growth rate of photosynthetic bacteria (PSB), this study introduces pantothenic acid as a biological enhancing factor. The effects of pantothenic acid on PSB proliferation and its effectiveness in treating high-concentration ammonia–nitrogen wastewater were systematically evaluated. Additionally, the effects of different culture conditions, including dark aeration, darkness, light exposure, and light aeration, on PSB growth were investigated. The results show that optimal PSB growth was achieved with 20 mg/L of pantothenic acid; however, higher concentrations of pantothenic acid inhibited bacterial growth. The addition of pantothenic acid also significantly enhanced the performance of PSB in treating high-concentration organic wastewater, increasing the removal rates of COD, ammonia nitrogen, total phosphorus, and total nitrogen to 43.0%, 94.0%, 49.7%, and 51.0%, respectively. Furthermore, a synergistic effect between dark aeration and light exposure was observed. When the time of light and dark aeration was set at 1:1, the highest PSB yield was recorded, and the removal efficiencies of COD, ammonia nitrogen, total nitrogen, and total phosphorus increased to 71.4%, 95.3%, 57.1%, and 74.7%, respectively. Through the introduction of pantothenic acid and optimization of culture mode, the rapid growth of PSB and highly efficient treatment of organic wastewater were achieved, providing a new approach for advanced wastewater treatment and resource utilization.

1. Introduction

In recent years, photosynthetic bacteria (PSB) have been widely used to treat various types of industrial wastewater [1,2]. Studies have shown that the microorganism is highly effective in removing organic pollutants and total phosphorus from wastewater. Compared to conventional activated sludge processes, PSB-based wastewater treatment offers distinct advantages: it minimizes the risk of secondary pollution from sludge disposal and produces valuable biomass rich in single-cell proteins, carotenoids, biopolymers, and food-grade pigments [3,4,5]. Industrial wastewater typically contains high concentrations of nitrogen and organic matter [6]. As natural resources become increasingly scarce, recovering resources from wastewater has gained global attention [7,8]. PSB systems not only remove nitrogen efficiently but also convert organic compounds into bioenergy carriers such as hydrogen and methane, thereby achieving waste-to-resource conversion [9,10]. Despite their potential, the slow growth rate of PSB remains a key limitation to large-scale application [11], making the enhancement of growth kinetics a priority for researchers.

To address this challenge, researchers have dedicated substantial efforts to enhancing the growth rate and biomass yield of PSB, including physical stimulation, ion supplementation, and process optimization. Zhou et al. [12] found that low-intensity ultrasonication for two minutes increased PSB yield by 110%, though longer exposure had inhibitory effects. The presence of Fe²⁺ has been demonstrated to augment the activity of dehydrogenase in PSB, which in turn facilitates microbial growth [13]. In the treatment of soybean wastewater using Rhodobacter sphaeroides, the optimal COD removal rate of 95% was achieved with the addition of 20 mg/L Fe²⁺, while the hydraulic retention time was reduced by 24 h. Under these conditions, the biomass concentration reached 5000 mg/L, which is a 1.5-fold increase compared to the control group [14]. Qin et al. [15] proposed that PSB exhibited different metabolic pathways under light and dark conditions, suggesting that growth kinetics can be improved through operational parameter optimization. Saejung et al. [16] found that Rhodopseudomonas faecalis PA2 performed best at a light intensity of 8000 lux with a light–dark ratio of 24:0, significantly increasing protein, lipid, and carbohydrate production by 121.69%, 101.69%, and 92.44%, respectively.

Pantothenic acid (Vitamin B5), widely used in pharmaceuticals, food additives, and animal feed, is essential for protein, lipid, and carbohydrate metabolism and supports growth and reproduction in animals [17,18,19]. In this study, simulated wastewater with high nitrogen and organic content was prepared to reflect actual industrial conditions. For the first time, pantothenic acid was used as a biological factor to replace Fe²⁺, investigating its effects on the yield of PSB and the efficiency of organic wastewater treatment. The impact of four culture conditions, dark aeration, darkness, light, and light aeration, on PSB growth was also investigated. Finally, two optimal culture methods were selected and combined to maximize PSB yield and treatment performance.

2. Materials and Methods

2.1. Materials

The phototropic bacteria working solution used in this study was derived from the industrialized DL-1 strain, with dominant species including Rhodospirillum (red spirillum) and Pseudomonas (Figure 1). Figure 2 presents scanning electron microscope images that clearly show a high density of rod-shaped PSB. The water samples were prepared based on the actual quality of breeding wastewater and simulated to represent high-nitrogen and high-organic conditions. The composition of the simulated wastewater was as follows: 0.5 g/L NH₄Cl, 1 g/L NaCl, 0.5 g/L NaHCO₃, 1 g/L yeast extract, 0.1 g/L K₂HPO₄, 1 g/L protease, 8 g/L CH₃COONa, 0.1 g/L CaCl₂, 0.1 g/L MgSO₄, and 0.2 g/L KH₂PO₄. The pH was adjusted to 8. The measured concentrations of TN, COD, NH₃-N, and TP in the influent water sample were 105 mg/L, 1500 mg/L, 90 mg/L, and 52 mg/L, respectively.

2.2. Experimental Methods

In each group, 50 mL of PSB in the logarithmic growth phase were centrifuged at 10,000× g for 10 min. The resulting PSB was inoculated into 500 mL of wastewater, maintaining an OD₆₆₀ of approximately 0.37 for all groups. The conical flasks were placed in an illuminated incubator at 30 °C, with the distance from the light source adjusted to maintain a light intensity of 5000 lux. The light source was provided by a full-spectrum lamp operating at 10 W. Samples were collected every other day over a 96 h cycle to measure OD₆₆₀, OD₈₀₅, COD, NH₃-N, TN, and TP. The pantothenic acid solution used in the study had a concentration of 25%. Precise volumes of this solution were added to different conical flasks to achieve final concentrations of 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L, and 50 mg/L. Group 1 served as the control, with no pantothenic acid added. In experiments investigating the effects of different cultivation methods on the growth rate of PSB, the pantothenic acid concentration was fixed at 20 mg/L. The specific cultivation methods are detailed in

Table 1. Different cultivation methods.

Experiment Conditions	Groups
Dark aeration	R1
Light aeration	R2
Dark	R3
Light	R4
4 h dark aeration + 20 h light	R5
8 h dark aeration + 16 h light	R6
12 h dark aeration + 12 h light	R7
16 h dark aeration + 8 h light	R8
20 h dark aeration + 4 h light	R9
12 h dark + 12 h light	R10

.

The experimental setup for the treatment of high-concentration aquaculture organic wastewater using the PSB-based biological method is shown in Figure 3.

2.3. Analytical Methods

The concentration of the bacterial suspension was determined using the gravimetric method. A defined volume of the suspension was placed into centrifuge tubes, subjected to three cycles of centrifugation and washing, and then dried and weighed. The concentrations of COD, TN, TP, and NH₃-N in wastewater were measured according to Chinese national standard methods. The growth curve of PSB was measured using a 722S visible spectrophotometer (Shanghai Jinghua Technology Instrument Co., Ltd, Shanghai, China) at a wavelength of 660 nm.

3. Results and Discussions

3.1. Effects of Different Pantothenic Acid Concentrations on PSB

3.1.1. Effects of Pantothenic Acid Concentration on Photosynthetic Bacterial Biomass

As shown in Figure 4, PSB exhibited rapid growth during the first two days and reached a stable state by the third day. Within the concentration range of 0−20 mg/L, PSB biomass increased with rising pantothenic acid concentrations. However, when the concentration exceeded 30 mg/L, further increases in pantothenic acid inhibited bacterial growth. This conclusion is similar to our previous study, where the addition of 20 mg/L and 30 mg/L pantothenic acid increased the removal rate of P-Nitrophenol (350 mg/L) by halotolerant Bordetella sp. by 33% and 50%, respectively. The addition of 40 mg/L pantothenic acid resulted in a removal rate increase comparable to that of the 20 mg/L pantothenic acid treatment group [20,21]. As the bacteria proliferation progressed, the concentration of pantothenic acid in the conical flasks gradually increased. When the supplemented concentration exceeded 30 mg/L, the resulting excessive levels likely inhibited bacterial growth, leading to a decrease in biomass. Overall, low concentrations of pantothenic acid promoted photosynthetic biomass, with stimulatory effects observed within a specific concentration range. Beyond this range, the inhibitory effect became apparent.

3.1.2. Effects of Pantothenic Acid Addition on Wastewater Treatment

Figure 5 illustrates the impact of different pantothenic acid concentrations on the removal efficiencies of COD, NH₃-N, TP, and TN in wastewater. As shown in Figure 5a, the COD removal followed a pattern similar to that of bacterial biomass. When the biomass reached its maximum, the COD removal efficiency also stabilized, suggesting that COD degradation primarily depends on PSB growth. The influent COD concentration was relatively high (approximately 1500 mg/L), and the initial degradation rate was low. After two days, the COD removal efficiency slightly increased, indicating enhanced bacteria metabolic activity. By day four, the COD removal efficiency reached 40.4%, with an effluent concentration of 894.5 mg/L. Among the different pantothenic acid concentrations, the highest COD removal efficiency (43.1%) and lowest effluent COD concentration (853.2 mg/L) were achieved at 20 mg/L, highlighting the bacteria’s effectiveness in treating high-concentration organic pollutants. Overall, pantothenic acid improved COD removal within the range of 0−40 mg/L. However, concentrations above 40 mg/L had a negative effect on COD removal.

As shown in Figure 5b, the addition of pantothenic acid significantly enhanced NH₃-N removal by PSB. At concentrations of 10−20 mg/L, the NH₃-N removal efficiency reached 94.0%, with the effluent concentration decreasing to approximately 5 mg/L, representing a 14% improvement compared to the control group (80.0%). During the initial stage, the NH₃-N removal rate was relatively slow, likely due to low bacterial biomass and weak nitrification activity. As the PSB population increased, the removal efficiency improved. However, after the second day, although the NH₃-N concentration continued to decline, the removal efficiency slowed, possibly as a result of the slower growth rate of PSB biomass.

As shown in Figure 5c,d, the highest removal efficiencies for TP and TN were 49.7% and 51.0%, respectively, at a pantothenic acid concentration of 20 mg/L, with corresponding effluent concentrations decreasing to 26.2 mg/L and 51.5 mg/L for TP and TN, respectively. The removal of TP and TN closely followed the trend in the biomass of PSB, with efficiency increasing up to a certain biomass level before plateauing or declining. Overall, the data from Figure 4 and Figure 5 consistently show that pantothenic acid concentrations between 0 and 20 mg/L enhance both bacterial growth and the removal efficiencies of COD, NH₃-N, TN, and TP. Beyond this range, these benefits declined.

3.2. Effects of Different Cultivation Methods on Photosynthetic Bacterial Growth

Light and aeration significantly influence the growth and proliferation of PSB [22]. As indicated in Table 1, conditions R1, R2, R3, and R4 represent four cultivation methods: dark aeration, light aeration, dark, and light, respectively. The growth curves of PSB under these different cultivation methods (Figure 6) reveal that PSB exhibited higher growth rates and biomass yields under light and dark aeration conditions compared to light aeration and dark conditions. Notably, light conditions yielded higher biomass than dark aeration. In contrast to previous studies that focused on pure cultures of Rhodobacter sphaeroides [23], the application of mixed bacterial strains in this study demonstrated enhanced activity under illuminated conditions, which suggests a higher degree of feasibility for future industrial-scale operations.

3.3. Effects of Different Cultivation Method Ratios on PSB

As shown in Table 1, conditions R5, R6, R7, R8, and R9 represent five cultivation methods with dark aeration to light cultivation time ratios of 1:5, 1:2, 1:1, 2:1, and 5:1, respectively. A comparison of Figure 6 and Figure 7 reveals that the growth dynamics of PSB closely follow the trend of their biomass. Figure 6 shows that, under dark aeration conditions, photosynthetic bacterial biomass began to decline after two days. When the dark-to-light time ratio exceeded 1:1, the biomass progressively decreased with increasing ratios, eventually approaching the levels observed under dark aerobic conditions. This trend is likely due to the dominance of Rhodopseudomonas spp. in the bacterial consortium, which exhibit optimal growth under light–anaerobic conditions.

Figure 8 show that when subjected to 12 h of dark aeration followed by 12 h of light, the removal efficiencies of NH₃-N, TP, COD, and TN were significantly improved. This suggests that dark aeration and light cultivation conditions can synergistically enhance the removal of specific pollutants. As shown in Figure 8a, under a dark-to-light time ratio of 1:1, the COD removal efficiency reached 71.4%, with the effluent COD concentration of 428.6 mg/L. The highest removal efficiency occurred between the second and third days, likely due to the notable contribution of PSB to COD degradation under dark aeration. However, as shown in Figure 7, the bacterial biomass remained relatively low under dark aeration. At the 1:1 time ratio, the bacterial biomass increased after the second day, resulting in improved COD removal during the 12 h dark aeration phase compared to continuous dark aeration.

TN removal is typically achieved through aerobic nitrification and anaerobic denitrification, while NH₃-N removal depends on anaerobic denitrification. Conventional activated sludge systems utilize autotrophic nitrifying bacteria and heterotrophic denitrifying bacteria for TN removal [24,25,26]. As facultative anaerobes, PSB can perform both nitrification and denitrification in a single reactor by adjusting environmental conditions, enabling simultaneous nitrogen removal and bacterial biomass recovery. As shown in Figure 8b, NH₃-N removal under light conditions significantly exceeded that under dark aeration. Higher NH₃-N removal efficiencies were associated with greater proportions of light–anaerobic phases. At a dark-to-light ratio of 1:2, NH₃-N removal reached a maximum of 97.2%, while a 1:1 ratio achieved 95.3% removal, yielding an effluent NH₃-N concentration of 4.2 mg/L. Lai et al. [27] demonstrated that PSB assimilate phosphorus from wastewater to synthesize polyphosphates under light conditions. Consistent with this finding, Figure 8c reveals that TP removal mainly occurred during light phases, with maximal efficiency (57.1%) observed at a 1:1 ratio, exceeding the performance under continuous light conditions and resulting in an effluent TP concentration of 22.3 mg/L. Figure 8d demonstrates that TN removal efficiency remained below 50.0% under pure light or dark aeration. However, at a 1:1 ratio, TN removal reached 74.7%, with an effluent concentration of 26.6 mg/L. These results suggest that optimizing cultivation methods for PSB can enable effective TN removal.

4. Comparison of Biomass Accumulation Among Different PSB

Inorganic salt ions, ultrasonication, and intermittent aeration significantly influence biomass accumulation and the synthesis of high-value products in PSB [28] (

Table 2. Biomass accumulation, pollutant removal, and hydrogen production in various photosynthetic bacteria.

Additives	Photosynthetic Bacteria (Dominant Strain)	Wastewater	Condition	Performance	Ref.
Fe3O4 nanoparticles (1 g/L)	Rhodopseudomonas faecalis PA2	Oil wastewater (1% cooking oil)	Anoxygenic–light, 24 h	The biomass was elevated by 2.28 times, while the protein production rose by 1.7 times.	[13]
Zero-valent iron nanoparticles (20 mg/L)	Ectothiorhodospira	Sugar wastewater (COD 6000 mg/L, TN 423 mg/L)	Anoxygenic–light, 24 h	The biomass and COD removal rate were enhanced by 122% and 164.3%, respectively.	[30]
Fe2+ (20 mg/L)	Rhodobacter sphaeroides	Soybean wastewater (BOD 4370 mg/L, COD 9940 mg/L, TN 590 mg/L)	Aerobic–dark	The biomass production achieved 5000 mg/L. The maximum COD removal rate was 95%, and the HRT was decreased to 24 h.	[14]
Alginate-embedded magnetic biochar	Rhodopseudomonas palustris	Secondary effluent (TOC 22 mg/L, TN 50 mg/L, Sulfadiazine 1 mg/L)	Anoxygenic–light, 24 h	The removal rates of sulfadiazine and NH3-N were enhanced by 21.23% and 38%, respectively.	[29]
ZnO/ZnS30 (100 mg/L)	Rhodobacter capsulatus	MedA medium (Photo-fermentative)	Anoxygenic–light, 24 h	The hydrogen production increased by 30%.	[31]
Graphitic carbon nitride nanosheets (16.5 mg/L)	Purple non-sulfur bacteria	Acetate yeast extract basil medium	Anoxygenic–light, 24 h	Biohydrogen production was four times that of the control group, exceeding 15,000 mL.	[32]
Low-intensity ultrasound	Rhodopseudomonas	Artificial wastewater (NaAC 3 g/L, TN 212 mg/L)	Micro-aerobic and natural light, 0.3 W/cm2 with 40 kHz frequency	The cell yield and biomass production rose by 110% and 93%, respectively.	[12]
Electrical stimulation	Rhodopseudomonas palustris	Van Niel’s yeast agar medium	Anoxygenic-light, periodic power-on/power-off mode	The CO2 removal rate increased by 10%, and the activity of the key CO2 fixation enzyme Rubisco was enhanced by 16%.	[33]
Periodic oxygen supplementation	Ectothiorhodospira	Sugar wastewater (COD 4455 mg/L, TN 272 mg/L, TP 45 mg/L)	Periodic oxygen supplementation with oxygen off/on for every 12 h	The biomass concentration achieved 1338.5 mg/L, while the removal rates for COD and NH3-N were 91.4% and 78.6%, respectively.	[34]
Flashing light	Rhodopseudomonas palustris	Sugar wastewater (COD 3100-3500 mg/L, NH4+-N 197-223 mg/L, TP 130-139 mg/L)	Ia 88 μmol/m2/s, I0 147 μmol/m2/s, F 10 Hz, φ 0.6	The concentrations of biomass and protein achieved 2815.2 mg/L and 1944.3 mg/L, respectively. These values were 23.2–31.1% and 27.4–56.4% higher than the continuous light groups.	[35]
Pantothenic acid (20 mg/L)	Rhodospirillum, Pseudomonas	High-concentration organic wastewater	Aerobic–light, 24 h	The biomass was 2500 mg/L. The removal rates for COD, NH3-N, TN, and TP were increased to 71.4%, 95.3%, 57.1%, and 74.7%, respectively.	This study

). In this study, pantothenic acid was used as an additive. Compared to additives containing zinc and iron, the effluent of the reaction system did not pose a risk of heavy metal ion exceedance. Chaiyarat, A et al. [18] added 1 g/L of Fe₃O₄ to treat oily wastewater, which increased the biomass production from 1.19 g/L to 1.92 g/L. In contrast, the current research demonstrates that the addition of only 20 mg/L of pantothenic acid can achieve a biomass concentration of 2.5 g/L. Additionally, compared to graphitic carbon nitride nanosheets, pantothenic acid offered lower operational costs. The bacterial biomass achieved a maximum value exceeding 2500 mg/L. The removal efficiencies of COD, NH₃-N, TN, and TP were raised to 71.4%, 95.3%, 57.1%, and 74.7%, respectively. The composite material, created by integrating Fe₃O₄ with the conventional microbial immobilization carrier biochar, was employed for immobilizing Rhodopseudomonas palustris. The removal efficiencies of sulfadiazine and NH₃-N were increased by 21.23% and 38%, respectively. This enhancement was credited to the increased bacterial activity due to Fe₃O₄ and the adsorptive properties of biochar for pollutants [29]. The integration of PSB with microbial immobilization materials loaded with additives holds broad prospects for enhancing wastewater treatment efficiency and the accumulation of high-value products.

5. Conclusions

This research proposes a technique that involves adding the growth factor pantothenic acid and optimizing the culture mode to stimulate PSB proliferation and elevate the treatment of organic wastewater. The highest PSB proliferation rate was noted with 20 mg/L pantothenic acid, while higher concentrations were not favorable for pollutant removal. Comparative analysis of different cultivation modes revealed that PSB exhibited superior growth rates and biomass yields under light and dark aeration conditions, with light conditions yielding higher biomass than dark aeration. Notably, a 1:1 ratio of light-to-dark aeration phases not only maximized bacterial biomass but also improved removal efficiencies for COD, NH₃-N, TN, and TP to 71.4%, 95.3%, 57.1%, and 74.7%, respectively. These findings provide novel insights and methodologies for rapid, efficient cultivation of PSB and advanced treatment of high-concentration organic wastewater.