Effects of Dry Periods on Nitrogen and Phosphorus Removal in Runoff Infiltration Devices and Their Biological Succession Patterns

: When using runoff infiltration devices to remove nitrogen and phosphorus pollutants from urban runoff, the quality of the effluent is affected by the length of dry spells between rain events. This study presents a novel analysis of how these dry periods impact the device’s effectiveness in removing pollutants and the resulting biological succession within the filter. Our analysis examines nitrogen and phosphorus removal in a rainwater filtration context, providing new insights into how dry period duration influences infiltration system performance. The results indicate that biological processes have a significant impact on reducing total nitrogen (TN) and total phosphorus (TP) contents under different drying periods. A 3-day drying period is most effective for reducing TN through biological processes, while a 7-day period is best for TP reduction. This suggests that moderately extending the drying period improves TP removal efficiency but does not enhance TN removal. The dominant bacterial phylum responsible for denitrification and phosphorus removal is Proteobacteria , with Pseudomonas and Acinetobacter as the leading genera. As the drying period lengthens, the dominant genera shift from Pseudomonas to Massilia . At a 3-day drying period, denitrification primarily occurs through Pseudomonas on the surfaces of maifanite and zeolite. At a 7-day dry-out period, Acinetobacter is mainly responsible for phosphate removal on maifanite surfaces. However, after a 14-day dry-out period, both biomass and bioactivity of Pseudomonas and Acinetobacter decrease, leading to reduced efficiency in removing nitrogen and phosphorus pollutants from runoff infiltration devices. These results aid in developing runoff infiltration devices for specific scenarios and offer crucial guidance for regulating runoff pollution control technologies.


Introduction
The rapid urbanization process has increased the burden of non-point source pollution in urban areas, with stormwater runoff becoming a major contributor to water quality deterioration in receiving bodies.The presence of excessive nitrogen and phosphorus in runoff can lead to eutrophication and black-odorous water, underscoring the importance of removing these pollutants from runoff.Particulate matter serves as the primary carrier of nitrogen and phosphorus, accumulating on impervious surfaces during dry periods and adsorbing pollutants that are transported with rainfall.Once the rain subsides, these pollutants settle and can be released again within receiving waters or treatment facilities [1].
Water 2024, 16 Therefore, effectively removing particulate matter-carrying pollutants from runoff is crucial in addressing this issue.Runoff infiltration devices are effective and economical in controlling runoff pollution as they remove pollutants through a combination of filler matrix and microorganisms.Biological processes are the primary pathways for removing nitrogen and phosphorus pollutants [2,3].After rainfall, nitrogen and phosphorus pollutants are captured and adsorbed by the matrix.During dry periods, microorganisms attached to the matrix utilize these nutrients [4].The aerobic environment during dry periods encourages nitrifying bacteria to perform ammonification and nitrification reactions, converting organic nitrogen (ON) into ammonia nitrogen (NH 3 -N), NH 3 -N into nitrite nitrogen (NO 2 − ), and nitrate nitrogen (NO 3 − ).Phosphorus-accumulating bacteria also contribute to phosphorus enrichment [5].These reactions metabolize organic substances in the matrix, renewing adsorption sites for future pollutant removal and extending the facility's service life [6].Studies have shown that the duration of dry periods affects the efficiency of biological reduction of nitrogen and phosphorus pollutants.For nitrogen pollutants, shorter dry periods allow microorganisms to execute nitrification and denitrification alternately with higher total nitrogen (TN) removal rates.Nevertheless, as the dry period length increases, microorganisms predominantly conduct nitrification, inhibiting denitrification.This leads to an increased removal rate of NH 3 -N but significant accumulation of NO 3 − in the matrix until it is washed out during subsequent inflow events, reducing TN removal efficiency [7].To improve TN removal under lengthy dry periods, submerged zones within devices enhance denitrification effects [8].Li et al. showed that systems containing submerged zones had higher removal of NO 3 − and TN compared to systems without submerged zones [9].Studies have shown that organisms over-accumulate phosphate aerobically while releasing it anaerobically, as ample drying time favors their activity for enhancing the removal rates of total phosphorus (TP) contents [10].However, excessively long drying periods can lead to a severe shortage of water, which is detrimental to the survival of nitrifying bacteria.This may lead to lower microbial activity and loss of control over organic nitrogen and phosphates within the system [11].Therefore, further research is required to understand the impact of different drying durations on nutrient removals from stormwater filtration units and evaluate the variation trends and mechanisms regarding microorganism roles.
This study focuses on using quartz sand, maifan stone, and zeolite as substrate materials for runoff infiltration devices.These materials were chosen for their easy availability, effectiveness in adsorption, and high rates of nitrogen and phosphorus removal.The study involved adding road sediments to raw water in rainfall experiments to analyze physical-chemical actions and biological effects of substrates under different dry periods and their effects on the removal of nitrogen and phosphorus pollutants.It also examines the diversity and community structure of microorganisms in the substrate layer and their succession process during various dry periods.This research aims to explore the interaction between nitrogen-phosphorus pollutants and microbial succession and to provide valuable insights for controlling runoff pollution more effectively.

Experimental Materials and Equipment
The experimental device comprises two main components: a leachate packing column and a rainfall apparatus.The leachate packing column, made of plexiglass, has a diameter of 300 mm and a height of 800 mm.It is equipped with six sampling ports for the packing material (100 mm in diameter), three water sampling ports, and one overflow port.The rainfall apparatus consists of a sprinkler, stirring water tank, and peristaltic pump.The sprinkler, with a diameter of 28 mm, is connected to the outlet of the stirring water tank via the peristaltic pump.The water tank stores experimental water prepared as per simulation requirements and is fitted with an internal stirring mechanism.Water is pumped from the tank through the peristaltic pump into the filtration-packing column.Figure 1 illustrates the experimental setup.Chlorine-free tap water was utilized as experimental water, while sediment was obtained from asphalt pavement, dried, sieved, mixed in the appropriate proportions, and made ready for use.as per simulation requirements and is fitted with an internal stirring mechanism.Water is pumped from the tank through the peristaltic pump into the filtration-packing column.Figure 1 illustrates the experimental setup.Chlorine-free tap water was utilized as experimental water, while sediment was obtained from asphalt pavement, dried, sieved, mixed in the appropriate proportions, and made ready for use.In this experiment, several cost-effective and convenient media for adsorption have been chosen based on their superior removal effects on particulate nitrogen and phosphorus pollutants.Such media include quartz sand, maifan stone, and zeolite.The experimental device has been filled from top to bottom in a coarse-to-fine manner with quartz sand (10-15 mm), maifan stone (6-8 mm), and zeolite (3-4 mm).To ensure that no salts or organic matter adsorbed in the filler would affect the results, the filler was washed with deionized water and then dried in an oven at 105 °C.After thorough cooling and drying, the filler was stored in sealed bags for future use.

Experimental Design
The experiment in this work utilized a uniform rainfall pattern with an intensity of 23.5 mm/h and a duration of 60 min.To assess the impact of physical-chemical and biological actions on removing nitrogen and phosphorus pollutants in runoff water filters, two types of substrate devices have been implemented: one with added fungicide (physical-chemical action) called the JZ group and one without fungicide (both physical-chemical and biological actions) named the CY group.The key component of the fungicide is isothiazolinone, which sterilizes by disrupting microbial protein bonds, is biodegradable and has minimal impact on the substrate environment [12].Specific design parameters are outlined in Table 1.The rainfall experiment took place from January 20 to February 20, 2024.Continuous simulated rainfall was initially conducted to stabilize the device, followed by experiments at intervals of 1 day, 2 days, 3 days, 7 days, and 14 days, respectively.In this experiment, several cost-effective and convenient media for adsorption have been chosen based on their superior removal effects on particulate nitrogen and phosphorus pollutants.Such media include quartz sand, maifan stone, and zeolite.The experimental device has been filled from top to bottom in a coarse-to-fine manner with quartz sand (10-15 mm), maifan stone (6-8 mm), and zeolite (3-4 mm).To ensure that no salts or organic matter adsorbed in the filler would affect the results, the filler was washed with deionized water and then dried in an oven at 105 • C.After thorough cooling and drying, the filler was stored in sealed bags for future use.

Experimental Design
The experiment in this work utilized a uniform rainfall pattern with an intensity of 23.5 mm/h and a duration of 60 min.To assess the impact of physical-chemical and biological actions on removing nitrogen and phosphorus pollutants in runoff water filters, two types of substrate devices have been implemented: one with added fungicide (physicalchemical action) called the JZ group and one without fungicide (both physical-chemical and biological actions) named the CY group.The key component of the fungicide is isothiazolinone, which sterilizes by disrupting microbial protein bonds, is biodegradable and has minimal impact on the substrate environment [12].Specific design parameters are outlined in Table 1.The rainfall experiment took place from 20 January to 20 February 2024.Continuous simulated rainfall was initially conducted to stabilize the device, followed by experiments at intervals of 1 day, 2 days, 3 days, 7 days, and 14 days, respectively.

Sample Collection Method
In each rainfall experiment, water samples were collected from both the JZ and CY groups, including inflow and outflow samples.Initially, 450 mL of water was taken using a sampling bottle before starting the formal inflow process.Throughout the experiment, 50 mL of outflow water every 5 min was collected at the sampling port.After 30 min, the outflow water was stored every 10 min for nine times.At the end of the rainfall, fungicide was added to the JZ group's device.The nine water samples from group JZ, collected during the experiment, were combined into a single batch for subsequent testing; group CY was treated in the same manner.
Equal volumes of quartz sand, maifan stone, and zeolite substrate samples were extracted after 3 days, 7 days, and 14 days of the drying period, respectively.These samples were sequentially collected through three filler sampling ports on the side wall of the CY group's experimental device.Each type of substrate sample was placed into ultrapure water, shaken, and then centrifuged to remove the supernatant before being sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China).for microbial metagenomic analysis by high-throughput sequencing.

Sample Index Determination Method
Water quality testing indicators include TN and TP.TN is measured using the alkaline persulfate oxidation UV spectrophotometry method, while TP is measured using the molybdate spectrophotometry method.All tests have been conducted in strict accordance with the "Methods for Monitoring and Analysis of Water and Wastewater (Fourth Edition)".

Pollutant Total Reduction Rate
The total pollutant reduction rate in each rainfall experiment was according to Formula (1).
where R L is the total pollutant reduction rate (%), C in is the inlet water pollutant concentration (mg/L), V in is the inlet water volume (L), C out is the outlet water pollutant concentration (mg/L), and V out is the outlet water volume (L).

Material Balance Equation
The runoff infiltration devices can be considered as an ideal reactor V.According to the law of conservation of mass, the nitrogen and phosphorus pollutants entering V equal those exiting V plus those transformed within V through physical-chemical and biological actions.The material balance equation was calculated according to Formula (2).
where M R is the mass of nitrogen and phosphorus pollutants entering V (mg), M C is the mass of nitrogen and phosphorus pollutants exiting V (mg), M T is the mass of nitrogen and phosphorus pollutants transformed by substrate action in V(mg), and M W is the mass of nitrogen and phosphorus pollutants transformed by biological action in V (mg).
The fungicide was added to the JZ unit to inhibit biological activity, resulting in an M W value of 0. The M T value can be calculated using the mass of pollutants entering and exiting the JZ unit, as shown in Formula (3).
where M C ′ is the mass of nitrogen and phosphorus pollutants transformed by biological action in V (mg).Substituting Equation ( 3) into (2) leads to the Equation shown below.The M W value can be calculated using the effluent data from both the JZ and CY groups, as shown in Formula (4). M where M W is the mass of nitrogen and phosphorus pollutants transformed by biological action in V (mg). Figure 2 illustrates the trend of TN removal over varying dry periods.Runoff infiltration devices primarily use biological processes to eliminate nitrogen pollutants.Initially, the cumulative TN (by mass) in the device increased with longer dry periods, but then it decreased.Biological removal of TN increased at 1, 2, and 3 days of dry periods but dropped at 7 and 14 days.The biggest reduction in subsequent rainfall runoff occurred after a 3-day dry period, reaching up to 86.1 mg, while it declined to 35.4 mg after a 14-day dry period.Longer dry periods were less effective for biological TN removal; around three days can be termed as an optimal duration for this process.Studies indicated that extended dry periods led to a lack of organic matter, causing some denitrifying microorganisms to enter endogenous respiration or decay phases, thereby reducing microbial efficiency in removing TN [13].Additionally, prolonged drying times hindered microbial denitrification processes, and significant NO 2 − and NO 3 − accumulation in the substrate can also decrease biological activity [14].where MC′ is the mass of nitrogen and phosphorus pollutants transformed by biological action in V (mg).Substituting Equation ( 3) into (2) leads to the Equation shown below.The MW value can be calculated using the effluent data from both the JZ and CY groups, as shown in Formula (4).

Results and Discussion
where MW is the mass of nitrogen and phosphorus pollutants transformed by biological action in V (mg). Figure 2 illustrates the trend of TN removal over varying dry periods.Runoff infiltration devices primarily use biological processes to eliminate nitrogen pollutants.Initially, the cumulative TN (by mass) in the device increased with longer dry periods, but then it decreased.Biological removal of TN increased at 1, 2, and 3 days of dry periods but dropped at 7 and 14 days.The biggest reduction in subsequent rainfall runoff occurred after a 3-day dry period, reaching up to 86.1 mg, while it declined to 35.4 mg after a 14day dry period.Longer dry periods were less effective for biological TN removal; around three days can be termed as an optimal duration for this process.Studies indicated that extended dry periods led to a lack of organic matter, causing some denitrifying microorganisms to enter endogenous respiration or decay phases, thereby reducing microbial efficiency in removing TN [13].Additionally, prolonged drying times hindered microbial denitrification processes, and significant NO2 − and NO3 − accumulation in the substrate can also decrease biological activity [14].

The Removal Trend of Total Phosphorus by Runoff Infiltration Devices
Figure 3 depicts how the removal of TP changes during different dry periods.The runoff stormwater filter works mainly through the physical and chemical properties of the substrate to eliminate phosphorus pollutants.It was observed that with an increase in the duration of the dry period, the cumulative TP mass in the device decreased correspondingly.In terms of TP removal, the biological action was the most effective during dry periods of 1, 2, 3, and 7 days but less so at 14 days.During a 7-day dry period, biological action achieved the maximum TP reduction from subsequent rainfall runoff by up to 15 mg, dropping further to only 9 mg during a 14-day dry period.In comparison with the shorter dry periods, longer drying periods resulted in better microbial efficiency in removing TP, which is consistent with the findings of Wang Xiaoping [11].Studies have revealed that aerobic microorganisms known as polyphosphate-accumulating organisms play a role in converting organic phosphorus retained by substrates into usable phosphates under sufficient oxygen levels.These organisms thrive and carry out these processes more effectively during drying periods [6].However, excessively long drying periods cause severe water deficiency in the filler layer, harming the survival of polyphosphate microorganisms and reducing the effectiveness of phosphate control as the drying time increases [11].
the duration of the dry period, the cumulative TP mass in the device decreased correspondingly.In terms of TP removal, the biological action was the most effective during dry periods of 1, 2, 3, and 7 days but less so at 14 days.During a 7-day dry period, biological action achieved the maximum TP reduction from subsequent rainfall runoff by up to 15 mg, dropping further to only 9 mg during a 14-day dry period.In comparison with the shorter dry periods, longer drying periods resulted in better microbial efficiency in removing TP, which is consistent with the findings of Wang Xiaoping [11].Studies have revealed that aerobic microorganisms known as polyphosphate-accumulating organisms play a role in converting organic phosphorus retained by substrates into usable phosphates under sufficient oxygen levels.These organisms thrive and carry out these processes more effectively during drying periods [6].However, excessively long drying periods cause severe water deficiency in the filler layer, harming the survival of polyphosphate microorganisms and reducing the effectiveness of phosphate control as the drying time increases [11].

Microbial Succession Patterns on the Surface of Runoff Infiltration Devices Media during
Various Dry Periods

Microbial Sequences and Alpha Diversity on Substrate Surfaces
The Alpha diversity of microbial communities characterizes their distribution on the substrate surface.Table 2 tabulates the diversity and richness of microorganisms on three different substrates during different drying periods.In all the sequencing results for substrate surface microorganisms during each drying period, the coverage was above 0.99, indicating that the sequencing accurately represented the samples [15].As drying periods lengthened, community diversity increased in both maifan stone and zeolite layers, while species richness initially decreased before increasing again.
The findings from the previous section, which analyzed the removal of nitrogen and phosphorus pollutants in runoff water filters under different drying periods, suggest that specific types of bacteria responsible for denitrification and dephosphorization were present on the substrate layer during these times.During a 3-day drying period, there was a high species richness, and mainly nitrification-denitrification reactions were observed.Biological activity improved as the number of species increased.In contrast, during a 7-day drying period, the species richness decreased due to the decline of denitrifying microbes and the proliferation of dephosphorizing microbes.Furthermore, at a 14-day drying period, both species richness and community diversity increased significantly in major filler

Microbial Sequences and Alpha Diversity on Substrate Surfaces
The Alpha diversity of microbial communities characterizes their distribution on the substrate surface.Table 2 tabulates the diversity and richness of microorganisms on three different substrates during different drying periods.In all the sequencing results for substrate surface microorganisms during each drying period, the coverage was above 0.99, indicating that the sequencing accurately represented the samples [15].As drying periods lengthened, community diversity increased in both maifan stone and zeolite layers, while species richness initially decreased before increasing again.The findings from the previous section, which analyzed the removal of nitrogen and phosphorus pollutants in runoff water filters under different drying periods, suggest that specific types of bacteria responsible for denitrification and dephosphorization were present on the substrate layer during these times.During a 3-day drying period, there was a high species richness, and mainly nitrification-denitrification reactions were observed.Biological activity improved as the number of species increased.In contrast, during a 7-day drying period, the species richness decreased due to the decline of denitrifying microbes and the proliferation of dephosphorizing microbes.Furthermore, at a 14-day drying period, both species richness and community diversity increased significantly in major filler layers, but the biological denitrification-dephosphorization effects were lower.This was likely because environmental factors contributed to the succession towards more adaptive microbial populations, while the dominant populations experienced stress, reducing their biological activity and thus decreasing nitrogen-phosphorus pollutant removal efficiency.Quartz sand at the topmost layer, with larger particle size, was more susceptible to environmental impacts, resulting in significant fluctuations in biodiversity and species richness data.

Composition of Microbial Community Structure at the Phylum Levels
As shown in Figure 4, after a 3-day drying period, the phyla with relative abundance >1% on the substrate included Proteobacteria, Cyanobacteria, Actinobacteriota, Deinococcota, Firmicutes, Bacteroidota, Acidobacteriota, and Chloroflexi.During 7 days of drying, only Proteobacteria, Bacteroidota, Actinobacteriota, and Cyanobacteria remained above 1% relative abundance.Similarly, at 14 days of drying, additional phyla such as Deinococcota, Acidobacteriota, and Chloroflexi were detected compared to 7 days duration.Throughout the entire drying process, Proteobacteria, Bacteroidota, Actinobacteriota, and Cyanobacteria consistently appeared as common phyla in the substrate.

Composition of Microbial Community Structure at the Genus Levels
The top 30 genera, based on the relative abundance of microbial populations from the detection results, were analyzed as the main genera.Figure 5 exhibits the primary denitrifying and phosphorus-removing genera detected on the substrate surface at 3 days, 7 days, and 14 days during the drying period.These genera include Massilia, Sphingomonas, Arthrobacter, Pseudomonas, Rheinheimera, Acinetobacter, and Flavobacterium.Among these, Pseudomonas, Massilia, Sphingomonas, Rheinheimera, and Acinetobacter belonged to the Proteobacteria class, whereas Arthrobacter and Flavobacterium were affiliated with Actinobacteria species.
Pseudomonas was the most common heterotrophic nitrifying-aerobic denitrifying bacterium that effectively degraded nitrogen and phosphorus pollutants [21].Massilia, a Gram-negative aerobic bacterium, also excelled in removing nitrogen and phosphorus It was found that the Proteobacteria were the dominant group of microorganisms on the substrate, encompassing most nitrifying and denitrifying bacteria as well as polyphosphateaccumulating organisms, hence playing a key role in removing nitrogen and phosphorus pollutants [16,17].Some microorganisms belonging to the Bacteroidetes were also involved in the denitrification process [18], while Actinobacteria hosted certain nitrifying bacteria.Cyanobacteria primarily contributed to single-cycle processes through nitrogen fixation [19].Based on the data analysis of microbial diversity and abundance in Section 2.1, at 7 days into the dry period, nitrogen-removing microorganisms on the substrate are significantly impacted by environmental changes, while phosphorus-removing microorganisms are less affected.The number of Firmicutes and other microorganisms decreases, leading to a decline in the device's nitrogen removal efficiency.At 14 days into the dry period, more environmentally adaptive populations thrive on the substrate surface; however, dominant populations experience stress effects and reduced biological activity, resulting in decreased nitrogen and phosphorus removal efficiency of the device.
The analysis of microbial community structure at the phylum level in substrate samples during different drying periods revealed that Proteobacteria had the highest relative abundance in all filler layers.They are the main bacteria involved in removing nitrogen and phosphorus.This is consistent with the findings of Zhang et al. in constructed wetland systems [20].The relative abundance of Proteobacteria varied with drying periods as follows: 7 days > 3 days > 14 days.As mentioned in Section 3.1, during a 3-day drying period, most microorganisms on the substrate surface were denitrifying bacteria performing nitrification and denitrification reactions.During 7 days, a reduction in denitrifying bacteria within Proteobacteria was observed, while phosphate-accumulating bacteria increased, focusing on polyphosphate accumulation activities.During a 14-day period, although the relative abundance of Proteobacteria remained above 50%, their biological activity for nitrogen and phosphorus removal decreased.This indicated that prolonged drought conditions promoted microbial succession within Proteobacteria towards more environmentally adaptive populations, reducing both the numbers and activity levels of primary nitrogen-and phosphorus-removing bacterial communities.

Composition of Microbial Community Structure at the Genus Levels
The top 30 genera, based on the relative abundance of microbial populations from the detection results, were analyzed as the main genera.Figure 5 exhibits the primary denitrifying and phosphorus-removing genera detected on the substrate surface at 3 days, 7 days, and 14 days during the drying period.These genera include Massilia, Sphingomonas, Arthrobacter, Pseudomonas, Rheinheimera, Acinetobacter, and Flavobacterium.Among these, Pseudomonas, Massilia, Sphingomonas, Rheinheimera, and Acinetobacter belonged to the Proteobacteria class, whereas Arthrobacter and Flavobacterium were affiliated with Actinobacteria species.
Pseudomonas was the most common heterotrophic nitrifying-aerobic denitrifying bacterium that effectively degraded nitrogen and phosphorus pollutants [21].Massilia, a Gram-negative aerobic bacterium, also excelled in removing nitrogen and phosphorus [22].Sphingomonas participated in the decomposition of organic nitrogen, ammonia nitrogen, and nitrite nitrogen [23].Arthrobacter acts as an aerobic denitrifier for raw water with high nitrite content and also aids in removing nitrogen and phosphorus from substrates using polyphosphate-accumulating organisms (PAO) [24].Acinetobacter has been recognized for its high capacity for phosphorus removal and is often identified as a PAO [25].Studies have shown that microorganisms from Rheinheimera and Flavobacterium possess both heterotrophic nitrification-aerobic denitrification capabilities along with polyphosphate accumulation functions [26].
During the 3-day drying period, Pseudomonas was the dominant bacterial genus on the substrate layer, primarily attaching to maifan stone and zeolite layers with relative abundances of 87.76% and 57.84%, respectively.These bacteria are mainly responsible for nitrification and denitrification processes, significantly contributing to TN reduction through biological action, as validated by the experimental results in Section 3.1.During the 7-day drying period, Massilia became dominant on quartz sand, while Acinetobacter replaced Pseudomonas on maifan stone.Together, Massilia and Acinetobacter facilitated phosphorus enrichment and transformation processes.The cumulative TP mass increased due to biological activity during this period, indicating that TP accumulation peaked at a 7-day drying period, which is consistent with the findings obtained in Section 3.1.These results demonstrated that excessive uptake of phosphorus by PAOs during dry periods can populations responsive to drought stress.Biodiversity increases while the biomass and activity of dominant populations decrease, leading to reduced efficiency in nitrogen and phosphorus pollutant removal.Previous studies have shown that decreased organic matter metabolism and substrate moisture content also impaired aerobic microbial activity, further reducing the removal efficiency of nitrogen and phosphorus [28].For an entire drying period, Pseudomonas remained predominant on zeolite layers, which can be attributed to the smallest particle size among all layers with good water retention properties.Consequently, microorganisms attached to it were less affected by environmental changes compared to those on quartz sand or maifan stone.After 14 days of drying, Massilia became the dominant genus on quartz sand and maifan stone, while Pseudomonas prevailed on zeolite but with a lower relative abundance compared to 3 and 7 days.Flavobacterium emerged as the third most abundant microbial population across the studied substrates.Studies have shown that Massilia exhibited better environmental tolerance compared to Pseudomonas under drought stress, and Flavobacterium was found to be more resilient in severe drought conditions [27].This suggests that during extended drying periods, surface microorganisms on substrates adapt to form populations responsive to drought stress.Biodiversity increases while the biomass and activity of dominant populations decrease, leading to reduced efficiency in nitrogen and phosphorus pollutant removal.Previous studies have shown that decreased organic matter metabolism and substrate moisture content also impaired aerobic microbial activity, further reducing the removal efficiency of nitrogen and phosphorus [28].For an entire drying period, Pseudomonas remained predominant on zeolite layers, which can be attributed to the smallest particle size among all layers with good water retention properties.Consequently, microorganisms attached to it were less affected by environmental changes compared to those on quartz sand or maifan stone.

Response of Substrate Microbial Communities to Nitrogen and Phosphorus Pollutants Under Different Drying Periods
Research shows that TN and TP significantly affect microbial community structures [29].As runoff infiltration devices remove runoff pollution, nitrogen and phosphorus pollutants accumulate in the substrate, promoting the growth of microorganisms involved in nitrogen and phosphorus removal.These microorganisms gradually engage in denitrification and dephosphorization processes.Redundancy analysis was performed to investigate the dominant microorganisms and the accumulated nitrogen and phosphorus pollutants on substrate surfaces during different drying periods.The study compared the effects of TN and TP on Pseudomonas, Acinetobacter, and Massilia attached to the substrates.The results, shown in Figure 6, revealed a strong correlation between nitrogen-phosphorus pollutants and microbial communities.After 3 days of drying, the main denitrifying bacteria produced on the substrate are Pseudomonas.TN was found to have a positive correlation with Pseudomonas on maifan stone and zeolite surfaces, suggesting that nitrificationdenitrification by Pseudomonas enhanced TN removal efficiency through biological action.After 7 days, TP was positively correlated with Acinetobacter on maifan stone, while TN exhibited an association with Pseudomonas on zeolite; the main phosphorus-removing bacteria produced on the substrate are Acinetobacter.During this period, polyphosphate accumulation by Acinetobacter occurred on maifan stone while denitrification by Pseudomonas happened on zeolite, significantly increasing TP removal but decreasing TN removal efficiency.When the drying period was extended to 14 days, a negative correlation between nitrogen-phosphorus pollutants and Massilia across all substrates was observed.This can be attributed to the prolonged duration of drying, which promoted succession towards more environmentally tolerant microbial species in substrates, thereby reducing biological denitrification-dephosphorization efficiency.

Response of Substrate Microbial Communities to Nitrogen and Phosphorus Pollutants under Different Drying Periods
Research shows that TN and TP significantly affect microbial community structures [29].As runoff infiltration devices remove runoff pollution, nitrogen and phosphorus pollutants accumulate in the substrate, promoting the growth of microorganisms involved in nitrogen and phosphorus removal.These microorganisms gradually engage in denitrification and dephosphorization processes.Redundancy analysis was performed to investigate the dominant microorganisms and the accumulated nitrogen and phosphorus pollutants on substrate surfaces during different drying periods.The study compared the effects of TN and TP on Pseudomonas, Acinetobacter, and Massilia attached to the substrates.The results, shown in Figure 6, revealed a strong correlation between nitrogen-phosphorus pollutants and microbial communities.After 3 days of drying, the main denitrifying bacteria produced on the substrate are Pseudomonas.TN was found to have a positive correlation with Pseudomonas on maifan stone and zeolite surfaces, suggesting that nitrification-denitrification by Pseudomonas enhanced TN removal efficiency through biological action.After 7 days, TP was positively correlated with Acinetobacter on maifan stone, while TN exhibited an association with Pseudomonas on zeolite; the main phosphorus-removing bacteria produced on the substrate are Acinetobacter.During this period, polyphosphate accumulation by Acinetobacter occurred on maifan stone while denitrification by Pseudomonas happened on zeolite, significantly increasing TP removal but decreasing TN removal efficiency.When the drying period was extended to 14 days, a negative correlation between nitrogen-phosphorus pollutants and Massilia across all substrates was observed.This can be attributed to the prolonged duration of drying, which promoted succession towards more environmentally tolerant microbial species in substrates, thereby reducing biological denitrification-dephosphorization efficiency.

Conclusions
This paper examined the impact of matrix and biological functions on the treatment of nitrogen and phosphorus pollutants in runoff infiltration devices.It summarized the patterns in the biological removal of these pollutants under varying drying periods, analyzed microbial community characteristics and succession patterns at both phylum and genus levels within substrate communities, and examined their responses to different drying periods.It was found that a 3-day drying period was the most effective for reducing TN through biological action in runoff infiltration devices, while a 7-day period was determined to be the best for reducing TP.Longer drying periods favored TP removal but hindered TN removal.Pseudomonas (Proteobacteria) were identified as the main denitrifying bacteria, while Acinetobacter (Proteobacteria) primarily facilitated the dephosphorization process.It was also observed that extended drying caused dominant genera to shift from Pseudomonas to Massilia.During 3 days, Pseudomonas species was responsible for the denitrification process on maifan stone and zeolite surfaces, while during a 7-day period, Acinetobacter species contributed predominantly to the dephosphorization action on maifan stone surfaces.After 14 days, biomass and bioactivity of both Pseudomonas and Acinetobacter decreased across all substrates' surfaces, reducing pollutant removal efficiency by runoff infiltration devices.
The impact of drying periods on microorganisms before and after rainfall is complex.Future research should refine the experimental design to better analyze the interactions between substrate, microorganisms, and drying time within runoff infiltration devices.This will offer valuable guidance for urban runoff pollution control technology.

Figure 1 .
Figure 1.Experimental device diagram.(a) Experimental device design diagram; (b) photo of the actual experimental device.

Figure 1 .
Figure 1.Experimental device diagram.(a) Experimental device design diagram; (b) photo of the actual experimental device.

3 . 1 .
Trends in Nitrogen and Phosphorus Removal from Runoff Infiltration Devices during Various Dry Periods 3.1.1.The Removal Trend of Total Nitrogen by Runoff Infiltration Devices

Water 2024 ,
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3. 1 .
Trends in Nitrogen and Phosphorus Removal from Runoff Infiltration Devices during Various Dry Periods 3.1.1.The Removal Trend of Total Nitrogen by Runoff Infiltration Devices

Figure 2 .
Figure 2. The trend of TN accumulation changes with the drying period.(a) The amount of TN removed by the device, biological action, and substrate action under different drying periods; (b) the proportional distribution of TN transformation by biological action and substrate action.

3. 1 . 2 .
Figure 3 depicts how the removal of TP changes during different dry periods.The runoff stormwater filter works mainly through the physical and chemical properties of the substrate to eliminate phosphorus pollutants.It was observed that with an increase in

Figure 2 .
Figure 2. The trend of TN accumulation changes with the drying period.(a) The amount of TN removed by the device, biological action, and substrate action under different drying periods; (b) the proportional distribution of TN transformation by biological action and substrate action.

Figure 3 .
Figure 3.The trend of TP accumulation changes with the drying period.(a) The amount of TP removed by the device, biological action, and substrate action under different drying periods; (b) the proportional distribution of TP transformation by biological action and substrate action.

Figure 3 .
Figure 3.The trend of TP accumulation changes with the drying period.(a) The amount of TP removed by the device, biological action, and substrate action under different drying periods; (b) the proportional distribution of TP transformation by biological action and substrate action.

Water 2024 , 12 within
16,  x FOR PEER REVIEW 8 of Proteobacteria was observed, phosphate-accumulating bacteria increased, focusing on polyphosphate accumulation activities.During a 14-day period, although the relative abundance of Proteobacteria remained above 50%, their biological activity for nitrogen and phosphorus removal decreased.This indicated that prolonged drought conditions promoted microbial succession within Proteobacteria towards more environmentally adaptive populations, reducing both the numbers and activity levels of primary nitrogenand phosphorus-removing bacterial communities.
on substrates.Substrates with biofilm have a higher phosphorus adsorption capacity compared to those without biofilm during inflow periods.

Figure 6 .
Figure 6.RDA results of dominant microbial communities in the substrate and nitrogen-phosphorus pollutants under different drying periods.

Figure 6 .
Figure 6.RDA results of dominant microbial communities in the substrate and nitrogen-phosphorus pollutants under different drying periods.

Table 1 .
Biological control experiment design parameters.

Table 1 .
Biological control experiment design parameters.

Table 2 .
Richness and evenness of biodiversity in filtration layer samples during different drying periods.