Monitoring Ammonium Polyphosphate (APP) Biodegradation by Acinetobacter nosocomialis D-3 Using DAPI

Ammonium polyphosphate (APP), a pivotal constituent within environmentally friendly flame retardants, exhibits notable decomposition susceptibility and potentially engenders ecological peril. Consequently, monitoring the APP concentration to ensure product integrity and facilitate the efficacious management of wastewater from production processes is of great significance. A fluorescent assay was devised to swiftly discern APP utilizing 4′,6′-diamino-2-phenylindole (DAPI). With increasing APP concentrations, DAPI undergoes intercalation within its structure, emitting pronounced fluorescence. Notably, the flame retardant JLS-PNA220-A, predominantly comprising APP, was employed as the test substrate. Establishing a linear relationship between fluorescence intensity (F-F0) and JLS-PNA220-A concentration yielded the equation y = 76.08x + 463.2 (R2 = 0.9992), with a LOD determined to be 0.853 mg/L. The method was used to assess the degradation capacity of APP-degrading bacteria. Strain D-3 was isolated, and subsequent analysis of its 16S DNA sequence classified it as belonging to the Acinetobacter genus. Acinetobacter nosocomialis D-3 demonstrated superior APP degradation capabilities under pH 7 at 37 °C, with degradation rates exceeding 85% over a four-day cultivation period. It underscores the sensitivity and efficacy of the proposed method for APP detection. Furthermore, Acinetobacter nosocomialis D-3 exhibits promising potential for remediation of residual APP through environmental biodegradation processes.


Introduction
Flame retardants, including halogenated flame retardants (HFRs) and halogen-free flame retardants (HFFRs), have been extensively utilized to mitigate the occurrence of fires and minimize property damage [1].While HFRs present significant environmental and health hazards due to their release of toxic smoke and gaseous hydrogen halides during combustion, HFFRs have emerged as rapid alternatives, characterized by their efficiency, non-toxicity, and environmental compatibility [2,3].Ammonium polyphosphate (APP), a pivotal component of HFFRs, has garnered substantial attention for its notable biological safety profile and cost-effectiveness [4].Within many formulations of APP-based flame retardants, APP assumes a primary role, serving as both an acid and gas source, thereby determining the formulation's fire prevention efficacy based on APP quality and abundance [5].However, APP is prone to degradation, reducing its effectiveness as a flame retardant [6].Additionally, APP exhibits biological toxicity to aquatic organisms like algae and crustaceans.The discharge of APP also causes eutrophication of water sources, which leads to severe water blooms and poses serious hazards to the survival of aquatic organisms [7,8].Therefore, it is imperative to devise a rapid and straightforward method for detecting APP concentration to monitor its wastewater presence and ultimately decrease the APP concentration.
APP, a polyphosphate (PolyP) variant, is composed of PolyP and ammonium ions and has prompted the exploration of diverse detection methodologies.Techniques such as 31 P that 5 min serves as the optimal reaction duration for DAPI within the APP detection.The timeframe ensures the optimal augmentation of fluorescence intensity while preserving the swift detection pace.Subsequent experiments were conducted adhering to an incubation period of 5 min in the context of APP detection.
To investigate the sensitivity of the detection of varying concentrations of APP, a series of meticulously prepared APP solutions were subjected to gradient dilution for subsequent fluorescence analysis.As illustrated in Figure S2a, in the absence of APP, DAPI exhibits its inherent emission fluorescence peak at 475 nm.However, with the gradual increase in APP concentrations, the emission fluorescence intensities at 550 nm become markedly more pronounced than the peak at 475 nm.It delineates the emergence of a distinct peak characteristic of the DAPI/PolyP complex formation.The N-containing indole ring of DAPI intricately interacts with the O atoms of polyphosphate, resulting in the emission fluorescence change in DAPI at 550 nm [18].Subsequently, the declining intensity at 475 nm signifies a complete reaction between residual monomeric DAPI and APP, culminating in maximal fluorescence intensity at 550 nm.
The fluorescence intensity at 550 nm of DAPI exhibits a gradual increase corresponding to the escalating concentration of APP, as illustrated in Figure S2b.When APP concentrations span from 3.079 to 300 µg/L, a linear correlation emerges between the concentration of APP and the intensity of DAPI fluorescence.The relationship is encapsulated by the equation y = 4.576x + 302.6, where y represents relative fluorescence intensity (F-F0) and x signifies the concentration of APP in the solution.The commendable coefficient of determination (R 2 = 0.9906) attests to the system's adeptness in discerning and quantifying APP concentrations.Employing the 3σ/slope analysis (σ = 4.697 µg/L, slope = 4.576), the LOD for APP solution is determined to be 3.079 µg/L.Thus, it offers a practical and productive means for monitoring the concentration of APP in environmental and industrial samples.In addition, owing to the degradability of APP, the detection system can also serve as a tool for the quality assessment of several APP products, such as APP-based flame retardants.

Analysis of APP in JLS-PNA220-A by DAPI
To assess the method's feasibility in detecting APP-based flame-retardant qualities, we investigated the correlation between fluorescence intensity and APP concentration of the APP-based flame retardant JLS-PNA220-A.Considering the microbial requirement for APP degradation within JLS-PNA220-A, the JLS-PNA220-A sample was dissolved in an R2A liquid medium.As anticipated, the APP-based flame retardant JLS-PNA220-A elicited alterations in DAPI fluorescence in Figure 1a, mirroring similar fluorescence emission spectra at 550 nm as observed for the APP/PolyP complex in Figure S2a.In the absence of JLS-PNA220-A, there was a characteristic peak at 475 nm caused by the DAPI fluorescence signal, consistently exhibiting a blue shift and decay with increasing JLS-PNA220-A concentration.Upon reaching an excess of JLS-PNA220-A, a robust fluorescence signal at 550 nm emerged, reaching maximal intensity as the JLS-PNA220-A concentration reached 12 mg/L.It was simultaneously accompanied by a fluorescence signal at 420 nm due to the R2A medium.It suggests that the APP of JLS-PNA220-A can bind with DAPI to form DAPI/PolyP structures, offering a pathway for assessing APP quality in JLS-PNA220-A.
Regarding APP detection in JLS-PNA220-A, the maximal emission intensity at 550 nm exhibited a proportional increase with the elevation in JLS-PNA220-A concentration, as depicted in Figure 1b.Across the JLS-PNA220-A concentration range of 0-12 mg/L, the change in fluorescence intensity (F-F0) displayed a linear relationship with the JLS-PNA220-A concentration.The concentration of JLS-PNA220-A can be ascertained using the equation y = 76.08x+ 463.2, where y represents fluorescence intensity and x represents JLS-PNA220-A concentration.The coefficient of determination (R 2 ) calculated for this equation was 0.9992, indicating a high correlation between the relative fluorescence intensity (F-F0) and JLS-PNA220-A concentration.Moreover, it exhibited a LOD of 0.856 mg/L based on the 3σ/slope analysis; σ is 21.71 mg/L, and the slope is 76.08.
the equation y = 76.08x+ 463.2, where y represents fluorescence intensity and x represen JLS-PNA220-A concentration.The coefficient of determination (R 2 ) calculated for th equation was 0.9992, indicating a high correlation between the relative fluorescen intensity (F-F0) and JLS-PNA220-A concentration.Moreover, it exhibited a LOD of 0.8 mg/L based on the 3σ/slope analysis; σ is 21.71 mg/L, and the slope is 76.08.Since flame retardants constitute a uniform mixture, the proportion of APP with the flame retardant remains consistent, establishing a direct correlation between A concentration and flame retardant concentration.The concentration of the flame retarda can be accurately calculated by utilizing the linear relationship derived from t interaction between APP and DAPI, coupled with the fluorescence intensity variation.T approach enables the assessment of the efficiency and integrity of APP flame-retarda products.Significantly, leveraging fluorescence detection technology, the fluoresce agent DAPI forms linear relationships with the concentrations of APP-based JLS-PNA22 A, demonstrating remarkable detection sensitivity.Consequently, it holds promise f environmental or industrial APP detection in wastewater.Therefore, this method w used as an efficient APP concentration monitoring method for selecting APP-domina degrading bacteria to detect and remediate polluted water sources.

Isolation and Identification of the APP-Degradation Strain
To effectively address the management of wastewater generated in APP-based flam retardant production and the residual presence of APP in the environment, microb degradation emerges as a promising environmental remediation method.In isolati efficient bacteria, six colonies (D-1~D-6) were isolated and purified from R2A cultu media.Subsequently, their abilities to degrade APP in JLS-PNA220-A were studi individually, leveraging the equation y = 76.08x+ 463.2 developed in Figure 2b to calcula the APP concentration in JLS-PNA220-A and APP degradation rate (Degradation rate (C(JLS-PNA220-A initial) − C(JLS-PNA220-A test))/C(JLS-PNA220-A initial)).During the 24-h monitoring assa strain D-3 exhibited the highest degradation capacity, as illustrated in Figure 2a.With the initial two days of incubation, the fluorescence intensity of DAPI decreased rapid with over 75% of the APP in the medium being degraded by strain D-3 (Figure 2b).Since flame retardants constitute a uniform mixture, the proportion of APP within the flame retardant remains consistent, establishing a direct correlation between APP concentration and flame retardant concentration.The concentration of the flame retardant can be accurately calculated by utilizing the linear relationship derived from the interaction between APP and DAPI, coupled with the fluorescence intensity variation.The approach enables the assessment of the efficiency and integrity of APP flame-retardant products.Significantly, leveraging fluorescence detection technology, the fluorescent agent DAPI forms linear relationships with the concentrations of APP-based JLS-PNA220-A, demonstrating remarkable detection sensitivity.Consequently, it holds promise for environmental or industrial APP detection in wastewater.Therefore, this method was used as an efficient APP concentration monitoring method for selecting APP-dominant degrading bacteria to detect and remediate polluted water sources.

Isolation and Identification of the APP-Degradation Strain
To effectively address the management of wastewater generated in APP-based flame retardant production and the residual presence of APP in the environment, microbial degradation emerges as a promising environmental remediation method.In isolating efficient bacteria, six colonies (D-1~D-6) were isolated and purified from R2A culture media.Subsequently, their abilities to degrade APP in JLS-PNA220-A were studied individually, leveraging the equation y = 76.08x+ 463.2 developed in Figure 2b to calculate the APP concentration in JLS-PNA220-A and APP degradation rate (Degradation rate = (C (JLS-PNA220-A initial) − C (JLS-PNA220-A test) )/C (JLS-PNA220-A initial) ).During the 24-h monitoring assays, strain D-3 exhibited the highest degradation capacity, as illustrated in Figure 2a.Within the initial two days of incubation, the fluorescence intensity of DAPI decreased rapidly, with over 75% of the APP in the medium being degraded by strain D-3 (Figure 2b).It suggests rapid growth in the bacterial population, efficiently utilizing a significant quantity of APP as a phosphate resource for growth and development.In the subsequent two days of incubation, the degradation rates remained stable, indicating a sustained level of APP concentration and slower bacterial propagation.Overall, strain D-3 demonstrated an impressive capacity for APP degradation within a short-term incubation period, highlighting its significance for achieving high-efficiency eco-recovery.
quantity of APP as a phosphate resource for growth and development.In the subseque two days of incubation, the degradation rates remained stable, indicating a sustained le of APP concentration and slower bacterial propagation.Overall, strain D-3 demonstrat an impressive capacity for APP degradation within a short-term incubation perio highlighting its significance for achieving high-efficiency eco-recovery.Certain microorganisms, known as phosphate-accumulating organisms (PAOs), c degrade polyphosphate and accumulate phosphorus for growth.These microorganis include Accumulibacter, Acinetobacter, Dechloromonas, and Zoogloea.As depicted in Figu 3a, strain D-3 was streaked on R2A agar medium for one day in a 37 °C incubator, formi round, moist, and white colonies.The Gram stain exhibited in Figure S3 indicates th strain D-3 is a Gram-negative strain.The cell morphologies of strain D-3 were observ using TEM, as illustrated in Figure 3b.It revealed characteristic features of a ball-ro shaped morphology with a capsule, without flagellum, and arranged singly or in pai Upon comparing these morphology characteristics, it was found that the traits of stra D-3 align with the typical features of the Acinetobacter genus [29,30].Certain microorganisms, known as phosphate-accumulating organisms (PAOs), can degrade polyphosphate and accumulate phosphorus for growth.These microorganisms include Accumulibacter, Acinetobacter, Dechloromonas, and Zoogloea.As depicted in Figure 3a, strain D-3 was streaked on R2A agar medium for one day in a 37 • C incubator, forming round, moist, and white colonies.The Gram stain exhibited in Figure S3 indicates that strain D-3 is a Gram-negative strain.The cell morphologies of strain D-3 were observed using TEM, as illustrated in Figure 3b.It revealed characteristic features of a ball-rod-shaped morphology with a capsule, without flagellum, and arranged singly or in pairs.Upon comparing these morphology characteristics, it was found that the traits of strain D-3 align with the typical features of the Acinetobacter genus [29,30].
The analysis compared the 16S rDNA sequence of strain D-3 with DNA sequences using BLAST.The results revealed the highest homology between strain D-3 and Acinetobacter nosocomialis.Genetic sequences of similar strains were then selected and downloaded to construct a phylogenetic tree using the neighbor-joining method, as illustrated in Figure 3c.The tree demonstrated a clear linkage relationship, placing the isolated strain D-3 within the genus Acinetobacter.Based on these findings, it can be concluded that strain D-3 belongs to the Acinetobacter nosocomialis species.The 16S rDNA sequence of strain D-3 has been deposited in the NCBI database with the GenBank accession number PP565354.Acinetobacter, as part of the PAOs group, exhibits a robust metabolism of polyphosphates.In aerobic conditions, Acinetobacter absorbs phosphorus from the environment and stores it as PolyP for energy storage.PolyPs are hydrolyzed to ATP without oxygen, serving as an energy source [31,32].Since the 1980s, Acinetobacter has been recognized as a highly efficient bacterium for phosphorus removal and has played a significant role in wastewater treatment processes [33].Acinetobacter's release of organic acids and phosphatases facilitates the breakdown of PolyP, releasing soluble phosphate [34,35].Thus, Acinetobacter nosocomialis D-3 holds promise as an efficient bacterium for degrading APP and can be utilized to remedy APP-based flame retardants.The analysis compared the 16S rDNA sequence of strain D-3 with DNA sequences using BLAST.The results revealed the highest homology between strain D-3 and Acinetobacter nosocomialis.Genetic sequences of similar strains were then selected and downloaded to construct a phylogenetic tree using the neighbor-joining method, as illustrated in Figure 3c.The tree demonstrated a clear linkage relationship, placing the isolated strain D-3 within the genus Acinetobacter.Based on these findings, it can be

Effect of the Phosphorus Resource
K 2 HPO 4 is commonly employed as a phosphorus source in bacterial culture media.To investigate the phosphate resources utilized by Acinetobacter nosocomialis D-3, an incubation process was conducted in the absence of K 2 HPO 4 .This allowed for the evaluation of Acinetobacter nosocomialis D-3's ability to use alternative phosphate sources.In Figure 4a, when the R2A medium lacked both Acinetobacter nosocomialis D-3 and K 2 HPO 4 , the degradation rates of APP over four days were approximately 10%, suggesting that APP cannot be substantially hydrolyzed under short-term incubation without efficient APP degradation strain D-3.Moreover, the initial average fluorescence intensity indicates that the absence of K 2 HPO 4 does not affect R2A fluorescence intensity.In Figure 4b,c, both groups of Acinetobacter nosocomialis D-3 exhibited a high ability to degrade APP.However, in the absence of K 2 HPO 4 , phosphate acquisition was limited, affecting the degradation system of Acinetobacter nosocomialis D-3.After four days of incubation, the APP degradation rates in the absence and presence of K 2 HPO 4 were 74% and 81%, respectively.It indicates that when a directly accessible phosphorus source is absent from the environment, Acinetobacter nosocomialis D-3 decomposes APP for its life activities as a phosphorus source, limiting its growth.
bacterium for degrading APP and can be utilized to remedy APP-based flame retardants.

Effect of the Phosphorus Resource
K2HPO4 is commonly employed as a phosphorus source in bacterial culture media.To investigate the phosphate resources utilized by Acinetobacter nosocomialis D-3, an incubation process was conducted in the absence of K2HPO4.This allowed for the evaluation of Acinetobacter nosocomialis D-3's ability to use alternative phosphate sources.In Figure 4a, when the R2A medium lacked both Acinetobacter nosocomialis D-3 and K2HPO4, the degradation rates of APP over four days were approximately 10%, suggesting that APP cannot be substantially hydrolyzed under short-term incubation without efficient APP degradation strain D-3.Moreover, the initial average fluorescence intensity indicates that the absence of K2HPO4 does not affect R2A fluorescence intensity.In Figure 4b,c, both groups of Acinetobacter nosocomialis D-3 exhibited a high ability to degrade APP.However, in the absence of K2HPO4, phosphate acquisition was limited, affecting the degradation system of Acinetobacter nosocomialis D-3.After four days of incubation, the APP degradation rates in the absence and presence of K2HPO4 were 74% and 81%, respectively.It indicates that when a directly accessible phosphorus source is absent from the environment, Acinetobacter nosocomialis D-3 decomposes APP for its life activities as a phosphorus source, limiting its growth.

Effect of Culture Temperature on the Degrading Ability of Acinetobacter nosocomialis D-3
The incubation temperatures for the three groups were set at 27 °C, 37 °C, and 47 °C, respectively.Figure 5 illustrates a divergence in the degradation trend and final concentration after four days of culture, highlighting the significant impact of temperature on the characteristics of Acinetobacter nosocomialis D-3.During the initial two days, APP degradation in the culture medium amounted to 31%, 69%, and 24%, respectively, with increasing culture temperature, indicating a notable discrepancy in the APP degradation capability.Figure 5d

Effect of Culture Temperature on the Degrading Ability of Acinetobacter nosocomialis D-3
The incubation temperatures for the three groups were set at 27 • C, 37 • C, and 47 • C, respectively.Figure 5 illustrates a divergence in the degradation trend and final concentration after four days of culture, highlighting the significant impact of temperature on the characteristics of Acinetobacter nosocomialis D-3.During the initial two days, APP degradation in the culture medium amounted to 31%, 69%, and 24%, respectively, with increasing culture temperature, indicating a notable discrepancy in the APP degradation capability.Figure 5d depicts the degradation of APP on the final day of culture.Notably, Acinetobacter nosocomialis D-3 incubated at 37 • C exhibited the highest APP degradation ability, approximately 82%.It underscores 37 • C as the optimal incubation temperature.Deviation from the optimal temperature restricts the cellular activities of Acinetobacter nosocomialis D-3 and affects the activities of phosphatases within Acinetobacter nosocomialis D-3.Collectively, these factors contribute to a decrease in the rate of APP degradation as the temperature strays from the optimum.
ability, approximately 82%.It underscores 37 °C as the optimal incubation temperatu Deviation from the optimal temperature restricts the cellular activities of Acinetobac nosocomialis D-3 and affects the activities of phosphatases within Acinetobacter nosocomia D-3.Collectively, these factors contribute to a decrease in the rate of APP degradation the temperature strays from the optimum.

Effect of Initial pH Values on the Degrading Ability of Acinetobacter nosocomialis D-3
pH represents another pivotal factor influencing microbial behavior.As depicted Figure 6, varying pH levels notably impact the APP degradation rate.Across pH rang of 6 to 8, the degradation rates of APP after four days of incubation reached 81%, 85%, a 82%, respectively.However, compared to the groups with pH levels of 6 and Acinetobacter nosocomialis D-3 required more time to degrade an equivalent amount of A when the incubation pH was 8. Given that the secretion of organic acids, such as gluco acid [34], is one of the fundamental mechanisms by which Acinetobacter decompos PolyP, fluctuations in incubation pH significantly influence the efficiency of A degradation.In an alkaline environment, organic acids are consumed in neutralizi reactions, resulting in a lower rate of APP degradation.Conversely, as acidity increas the hydrolysis of APP intensifies.Coordinated with organic acids, it amplifies A degradation.Moreover, a neutral environment is more conducive to Acinetobacter grow [36].Thus, the results suggest that the pH value of the incubating environment equal t is favorable for Acinetobacter nosocomialis D-3 in degrading APP.

Effect of Initial pH Values on the Degrading Ability of Acinetobacter nosocomialis D-3
pH represents another pivotal factor influencing microbial behavior.As depicted in Figure 6, varying pH levels notably impact the APP degradation rate.Across pH ranges of 6 to 8, the degradation rates of APP after four days of incubation reached 81%, 85%, and 82%, respectively.However, compared to the groups with pH levels of 6 and 7, Acinetobacter nosocomialis D-3 required more time to degrade an equivalent amount of APP when the incubation pH was 8. Given that the secretion of organic acids, such as gluconic acid [34], is one of the fundamental mechanisms by which Acinetobacter decomposes PolyP, fluctuations in incubation pH significantly influence the efficiency of APP degradation.In an alkaline environment, organic acids are consumed in neutralizing reactions, resulting in a lower rate of APP degradation.Conversely, as acidity increases, the hydrolysis of APP intensifies.Coordinated with organic acids, it amplifies APP degradation.Moreover, a neutral environment is more conducive to Acinetobacter growth [36].Thus, the results suggest that the pH value of the incubating environment equal to 7 is favorable for Acinetobacter nosocomialis D-3 in degrading APP.
To accurately obtain the optimal culture conditions for Acinetobacter nosocomialis D-3 to degrade APP, orthogonal experiments were conducted on the two conditions of temperature and pH.From Table S1, the effect of temperature on the degradation rate of Acinetobacter nosocomialis D-3 was more significant.At four days of incubation, the 37 • C group generally exhibited the highest degradation efficiency.Comparing the effects of pH, the best degradation effect was shown in group pH 7. In the experiment, the highest APP degradation effect of Acinetobacter nosocomialis D-3 was obtained at pH 7 and 37 • C. To accurately obtain the optimal culture conditions for Acinetobacter nosocomialis D to degrade APP, orthogonal experiments were conducted on the two conditions temperature and pH.From Table S1, the effect of temperature on the degradation rate Acinetobacter nosocomialis D-3 was more significant.At four days of incubation, the 37 group generally exhibited the highest degradation efficiency.Comparing the effects pH, the best degradation effect was shown in group pH 7. In the experiment, the high APP degradation effect of Acinetobacter nosocomialis D-3 was obtained at pH 7 and 37 °C

Apparatus
The solid media and liquid media were respectively incubated in the water-jacket constant temperature incubator GSP-9270MBE (Boxun Co., Ltd., Shanghai, China) and the shaker incubator TS-200B (Tiancheng Experimental Instrument Manufacturing Co., Ltd., Shanghai, China).The morphological characteristics of the isolated strain were observed under a transmission electron microscope (TEM).The fluorescence intensity was assessed using the fluorescence spectrometer F-4600 (Hitachi, Tokyo, Japan).Measurements were conducted under reaction conditions maintained at 30 • C in darkness.Emission spectral data were recorded within the 360-640 nm range, with an excitation wavelength of 330 nm.The maximum emission (Em max ) peak was observed at 550 nm, where fluorescence intensities were recorded.Both excitation and emission slit widths were set to 5 nm.The TMP voltage was maintained at 600 V for optimal performance.

Analysis of APP
To evaluate the optimal reaction time, 1 mL of 3 mg/L APP and 5 µL of 100 µM DAPI were combined in 1.5 mL centrifuge tubes.The reaction mixtures were thoroughly mixed and then incubated at 30 • C in darkness.Incubation times ranging from 1, 2, 3, 4, 5, 6, 7, and 8 min were examined.To assess APP detection sensitivity, 1 mL of various mixtures containing different concentrations of APP and 5 µL of 100 µM DAPI were prepared.The final APP concentrations ranged from 0, 30, 90, 150, 240, 300, 900, 1500, 2400, and 3000 µg/L, while the final DAPI concentration was maintained at 0.5 µM.The mixtures were vigorously shaken and then incubated in darkness for 5 min.Fluorescence intensity at 550 nm was detected by the fluorescence spectrometer F-4600 under a 330 nm excitation wavelength.Each sample underwent analysis at least three times.

JLS-PNA220-A Detection
Considering the biodegradation potential of JLS-PNA220-A, it was solubilized in the R2A liquid medium.A total of 3 mg of JLS-PNA220-A flame retardant was introduced into 250 mL of R2A liquid medium, where it underwent complete dissolution through agitation in a shaker at 30 • C and 220 rpm, resulting in a final concentration of 12 mg/L.Various JLS-PNA220-A solutions were prepared, spanning concentrations of 0, 1, 3, 6, 10, 12, 16, 24, and 36 mg/L.Subsequently, 1 mL of the JLS-PNA220-A solution was combined with 5 µL of 100 µM DAPI and mixed.The mixture was incubated for 5 min in darkness, followed by fluorescence analysis.Fluorescence intensity at 550 nm was detected by the fluorescence spectrometer F-4600 under a 330 nm excitation wavelength.Each sample underwent analysis at least three times.

Isolation, Enrichment, and Identification of Acinetobacter nosocomialis D-3
Activated sewage sludge samples, intended for isolating specific degrading APP strains, were procured from the sewage pool of JLS Chemical Flame Retardant Co., Ltd.These samples were solubilized in ddH 2 O in a 1:1 ratio by vigorous shaking and precipitated for 30 min.Subsequently, 0.5 mL of the supernatant gradient was subjected to dilution (10 −1 -10 −4 ) and evenly spread onto R2A solid media, which were then incubated at 37 • C for 2 days.Following the incubation period, media exhibiting moderate colony density were selected, and individual colonies (D-1~D-6) were transferred to a sterile R2A solid medium to purify single colonies at 37 • C.This purification process was iterated at least three times to ensure the isolation of pure strains.The purified strains were systematically labeled and expanded in R2A liquid media.
To detect the APP degradation ability of isolated strains, a pre-sterilized R2A medium was employed to dissolve JLS-PNA220-A.The mixture was achieved by shaking at 220 rpm and 30 • C. Subsequently, the mixture was filtered using filters (0.22 µm).A total of 1 mL of the expanded strain solutions was added to the 30 mL sterilized R2A liquid media that contained 12 mg/L JLS-PNA220-A, and the mixtures were incubated at 37 • C and 220 rpm for 4 days.Every 24 h, 1 mL of the mixture was sampled and then centrifuged in a Centrifuge MiniSpin ® plus (Eppendorf, Hamburg, Germany) at 8000 rpm for 90 s.A total of 1 mL of samples were stored in a 4 • C refrigerator until the fourth day.Based on the detection system developed in Section 3.4, a uniform fluorescence assay was performed on the last day to calculate the remaining concentration of JLS-PNA220-A for stored samples.
Based on this, the degradation rate of APP over four days can be reckoned, targeting high-APP degradation efficiency strains.Each sample was measured three times.
Strain D-3 was isolated with an efficient degradation capacity, streaked on R2A agar plates, and incubated at 37 • C for 1 day to observe the morphological characteristics of terminal single colonies.TEM was used to characterize the morphological attributes of strain D-3 cells; the magnification was ×10,000.For species identification, the purified colonies were sent to Sangon Biotech Co., Ltd.(Shanghai, China) and amplified 16S rDNA sequence by bacterial primers 27F and 1492R for sequencing.The alignment of the result was compared with other 16S rDNA sequences in the GenBank database (https://www.ncbi.nlm.nih.gov,accessed on 20 May 2024) using the Basic Local Alignment Search Tool program (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi,accessed on 20 May 2024).The phylogenetic tree was developed in MEGA11 based on the 16S rDNA sequence by the neighbor-joining method, and the bootstrap replications were 1000 in the conduction.

APP Degradation Conditions
For the study of phosphate resources, the initial concentrations of K 2 HPO 4 in the R2A medium were set to 0 and 0.3 g/L.Two single-factor experiments were conducted to study the APP degradation characteristics of Acinetobacter nosocomialis D-3 by changing the culture temperature and initial pH of the R2A medium.For the temperature factor, different groups of Acinetobacter nosocomialis D-3 were incubated at 27 • C, 37 • C, and 47 • C environments in incubators.For the pH factor, the initial pH of the media was accommodated to 6, 7, and 8 using 2 M NaCl and 1 M NaOH.Orthogonal experiments with temperature and pH were performed.When the two factors were not subjects of the experiment, the initial JLS-PNA220-A concentration, pH, and culture temperature were set to 12 mg/L, 7.3, and 37 • C, respectively.In contrast, the other cultural factors remained consistent.

Conclusions
This study presents a pioneering approach for assessing APP concentration, leveraging the symbiotic interplay between DAPI and the PolyP structure.Specifically, the N-containing indole ring of DAPI intricately interacts with the O atoms of phosphate, culminating in the formation of DAPI/PolyP complexes.These complexes emit characteristic fluorescence with a discernible peak at 550 nm upon excitation at 330 nm, directly mirroring the concentration of APP.It showcases remarkable sensitivity and an impressively low LOD of 3.079 µg/L, rendering it a swift and precise method for APP detection.Moreover, its potential extends to monitoring the APP content in flame-retardant products, such as JLS-PNA220-A, thereby facilitating comprehensive evaluations of flame-retardant efficacy.Furthermore, the above assay provides an accurate and efficient method for assessing the biodegradation of APP flame retardants and establishes a feasible environmental remediation strategy.An adept APP-degrading strain, named D-3, was meticulously isolated from sewage sludge.Through meticulous morphological characterization and 16S rDNA sequencing, strain D-3 was taxonomically classified within the Nosocomialis genus.A detailed examination of strain D-3's APP degradation prowess under diverse incubation conditions revealed optimal parameters: an incubation temperature of 37 • C and a pH of 7.Under these conducive conditions, a rapid degradation phase was observed over two days, culminating in approximately 85% degradation within four days.The exemplary proficiency displayed by Acinetobacter nosocomialis D-3 in APP degradation underscores its potential as an eco-friendly agent for remediating residual APP through environmentally benign biodegradation processes.Moreover, its utilization is promising for optimizing wastewater treatment in industrial settings, fostering sustainable environmental management practices.

Figure 2 .
Figure 2. (a) APP degradation ability of different isolated strains, from strain D-1 to strain D-6.The variation in fluorescence intensity (F-F0) at 550 nm and APP degradation as the strain D-3 w incubated for 4 days.The initial concentration of JLS-PNA220-A was 12 mg/L, and the fi concentration of DAPI was 0.5 μM.Ex = 330 nm, Em = 550 nm.The blue line was the fluoresce intensity.The orange line was the degradation rate.

Figure 2 .
Figure 2. (a) APP degradation ability of different isolated strains, from strain D-1 to strain D-6.(b) The variation in fluorescence intensity (F-F0) at 550 nm and APP degradation as the strain D-3 was incubated for 4 days.The initial concentration of JLS-PNA220-A was 12 mg/L, and the final concentration of DAPI was 0.5 µM.Ex = 330 nm, Em = 550 nm.The blue line was the fluorescence intensity.The orange line was the degradation rate.

Figure 3 .
Figure 3. Morphological structure of the strain D-3 in R2A agar medium: (a) Characters of colonies after one-day culture; (b) Characteristics of strain D-3 cells through the TEM; the magnification is ×10,000; (c) Neighbor-joining tree of Acinetobacter nosocomialis D-3 developed based on the 16S rDNA sequence in MEGA 11.

Figure 3 .
Figure 3. Morphological structure of the strain D-3 in R2A agar medium: (a) Characters of colonies after one-day culture; (b) Characteristics of strain D-3 cells through the TEM; the magnification is ×10,000; (c) Neighbor-joining tree of Acinetobacter nosocomialis D-3 developed based on the 16S rDNA sequence in MEGA 11.

Figure 4 .
Figure 4. (a) The fluorescence intensity (F-F0) change in the R2A medium in the absence of K2HPO4 and Acinetobacter nosocomialis D-3 during the four-day culture; the effect of phosphorus resource on DAPI fluorescence intensity (F-F0) and degradation rate when (b) K2HPO4 is absent and (c) K2HPO4 is present.The blue line is the fluorescence intensity.The orange line is the degradation rate of APP calculated based on fluorescence intensity.

Figure 4 .
Figure 4. (a) The fluorescence intensity (F-F0) change in the R2A medium in the absence of K 2 HPO 4 and Acinetobacter nosocomialis D-3 during the four-day culture; the effect of phosphorus resource on DAPI fluorescence intensity (F-F0) and degradation rate when (b) K 2 HPO 4 is absent and (c) K 2 HPO 4 is present.The blue line is the fluorescence intensity.The orange line is the degradation rate of APP calculated based on fluorescence intensity.

Figure 5 .
Figure 5.Effect of temperature on DAPI fluorescence intensity (F-F0) at 550 nm and A degradation rate when the environment temperature was (a) 27 °C, (b) 37 °C, and (c) 47 °C.The b line is the fluorescence intensity.The orange line is the degradation rate of APP calculated based fluorescence intensity.(d) The degradation rate of Acinetobacter nosocomialis D-3 when cultured 27 °C, 37 °C, and 47 °C on the fourth day.

Figure 5 .
Figure 5.Effect of temperature on DAPI fluorescence intensity (F-F0) at 550 nm and APP degradation rate when the environment temperature was (a) 27 • C, (b) 37 • C, and (c) 47 • C. The blue line is the fluorescence intensity.The orange line is the degradation rate of APP calculated based on fluorescence intensity.(d) The degradation rate of Acinetobacter nosocomialis D-3 when cultured at 27 • C, 37 • C, and 47 • C on the fourth day.

cules 2024 ,Figure 6 .
Figure 6.Effect of pH on DAPI fluorescence intensity (F-F0) at 550 nm and APP degradation r when (a) pH = 6, (b) pH = 7, and (c) pH = 8.The blue line is the fluorescence.The orange line is degradation rate.(d) The degradation rate of pH = 6, 7, 8 on the fourth day.

Figure 6 .
Figure 6.Effect of pH on DAPI fluorescence intensity (F-F0) at 550 nm and APP degradation rate when (a) pH = 6, (b) pH = 7, and (c) pH = 8.The blue line is the fluorescence.The orange line is the degradation rate.(d) The degradation rate of pH = 6, 7, 8 on the fourth day.