Algal Accumulation Decreases Sediment Nitrogen Removal By

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Algal Accumulation Decreases Sediment Nitrogen Removal by Uncoupling Nitriﬁcation-Denitriﬁcation in Shallow Eutrophic Lakes Lin Zhu,$ Wenqing Shi,$ Bryce Van Dam, Lingwei Kong, Jianghua Yu, and Boqiang Qin*

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ABSTRACT: In eutrophic lakes, the decay of settled algal biomass generates organic carbon and consumes oxygen, favoring sediment nitrogen loss via denitrification. However, persistent winds can cause algae to accumulate into dense mats, with uncertain impacts on sediment nitrogen removal. In this study, we investigated the effects of algal accumulation on sediment nitrogen removal in a shallow and eutrophic Chinese lake, Taihu. We found that experimental treatments of increased algal accumulation were associated with decreased sediment nitrogen losses, indicating the potential for a break in coupled nitrification-denitrification. Likewise, field measurements indicated similar decreases in sediment nitrogen losses when algal accumulation occurred. It is possibly caused by the decay of excess algal biomass, which likely depleted dissolved oxygen, and could have inhibited nitrification and thereby denitrification in sediments. We estimate that if such algal accumulations occurred over 20% or 10% of lake area in Taihu, sediment nitrogen removal rates decreased from 835.6 to 167.2 and 77.2 μmol N m−2h−1, respectively, during algal accumulation period. While nitrogen removal may recover later, the apparent nitrogen removal decrease may create a window for algal proliferation and intensification. This study advances our knowledge on the impacts of algal blooms on nitrogen removal in shallow eutrophic lakes.

■ INTRODUCTION organic carbon for heterotrophic denitrifiers. Furthermore, the Nitrogen plays an essential role in the balance and stability of accumulation of decaying material can physically inhibit See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. lake ecosystems.1,2 Excessive nitrogen can cause eutrophica- oxygen penetration in sediments, creating favorable conditions 12,13 Downloaded via NANJING INST OF GEOGRAPHY & LIMNOLOGY on May 12, 2020 at 08:43:08 (UTC). tion, reduce biodiversity, and degrade water quality.3,4 for denitrification. Chen et al. (2011) demonstrated that Denitrification is an important process in the nitrogen cycle, the addition of algal biomass accelerated denitrification and converting nitrate to nitrogen gas and hence permanently enhanced the loss of nitrate from lakes.7 While relatively small − removing nitrogen from lake ecosystems.5 7 It has been additions of algal biomass may enhance denitrification, the estimated that 43% of nitrogen loads are removed by impact of large algal accumulations on denitrification remains fi denitri cation in river networks before being discharged to unclear. Taihu in China.8 fi Wind-driven currents are ubiquitous hydrodynamic features In aquatic ecosystems, the processes of denitri cation and of inland waters, especially shallow lakes.14 These wind-driven nitrification often work together to achieve effective nitrogen removal.6,9,10 Nitrification is the biological oxidation of ammonia to nitrite followed by the oxidation of nitrite to Received: September 16, 2019 nitrate, which is in turn a substrate for denitrification. When Revised: February 17, 2020 coupled nitrification-denitrification is broken, effective nitro- Accepted: March 19, 2020 gen removal will be limited because denitrifiers will have to rely Published: March 19, 2020 − solely on external “new” nitrate inputs.9 11 During algal blooms in eutrophic lakes, the decay of algal biomass supplies

Figure 1. Location of study sites and photograph of algal accumulation in Meiliang Bay region of Taihu. (A) Location of study sites. (B) Dense algal mats observed in July 2016. Red stars are sampling sites for field surveys. Intact sediment cores for incubation experiments were collected at site S3. Color pattern represents algal distribution in Taihu in Jun. 2016, which were retrieved from remote sensing data (http://www.geodata.cn) using EOF-based algorithm.25 currents occur when wind energy is transferred to the water on sediment nitrification-nitrification and thereby nitrogen and dissipated through the generation of relatively high removal in shallow eutrophic lakes, which adds to our intensity currents.15 Wind-driven currents control the spatial understanding of the impacts of algal blooms on nitrogen distribution of algae in lentic waters through the advective removal in shallow eutrophic lakes. movement of superficial water masses in a downwind direction.16 Many species of bloom-forming algae are positively ■ MATERIAL AND METHODS buoyant, containing intracellular gas vesicles which help them − Study Area. Taihu, the third largest freshwater lake by maintain a position close to the air water interface. When surface area in China, is situated along the lower reaches of the wind blows in one direction, these positively buoyant algae fl 17,18 Yangtze River oodplain in the coastal plain of China (Figure easily concentrate at the downwind shore. In the shallow 1). It has a surface area of 2338 km2, a catchment area of eutrophic Chinese lakes, Taihu and Chaohu, dense algae mats 36500 km2, and a mean water retention time of 284 days.22 have been frequently observed in the downwind northwestern The lake experiences a subtropical monsoon climate with region during the algal bloom season, particularly following 22 19−21 prevailing winds from the southeast in summer. With the persistent southeast winds. In such zones with algal rapid agricultural and industrial development of this region in accumulation, excessive algal biomass settles on the sediment recent decades, excessive nutrient loadings have driven the lake surface after algae die, and its decay can deplete dissolved to a hypereutrophic state, causing frequent severe algal blooms oxygen (DO) toward hypoxia or anoxia at the sediment-water in summer every year.23 Our study sites were located in fi interface. These conditions may inhibit sediment nitri cation Meiliang Bay, which occupies the northwestern region of (conventionally performed by obligate aerobes), possibly Taihu. This bay has a surface area of 123 km2 and a mean fi fi breaking coupled nitri cation-denitri cation and limiting depth of 1.8 m.24 Driven by southeast winds, algal sediment nitrogen removal. Moreover, in the newly algae-free accumulation often occurs in Meiliang Bay, forming dense zone (from which algae were transported), the decrease of algal mats during algal bloom seasons (Figure 1B).20 algae derived organic carbon may limit substrates for Field Surveys and Incubation Experiments. Monthly fi denitri ers, thereby potentially slowing down sediment nitrogen removal rates in Meiliang Bay sediments were fi 7 denitri cation rates. Hence, we hypothesize that the investigated at three sites (S1:31°30′53″N, 120°10′31″E; formation of wind-driven aggregations of algae can break S2:31°30′21″N, 120°10′28″E; S3:31°29′37″N, 120°12′16″E) coupled nitrification-denitrification via two related mecha- by measuring net nitrogen gas fluxes from intact sediment nisms: (1) hypoxia/anoxia limitation of sediment nitrification cores. Once per month, intact sediment cores (9 cm in in the area of algal accumulation and (2) organic carbon diameter and 50 cm in height) were collected at each site in limitation of sediment denitrification in the area from which triplicate using a Jenkin corer in 2016−2017. Nitrogen gas the algae originated. The combination of these factors may fluxes were measured in situ according to the description limit net nitrogen removal from sediments during algal bloom below. DO in the water was measured using a multisensor seasons in shallow eutrophic lakes. probe (YSI 6600, Yellow Springs Instruments, USA); nitrate In this study, sediment nitrogen removal was investigated in was measured by a flow injection analyzer (San++System, Taihu, a shallow and eutrophic lake in China. Monthly SKALAR, Netherlands), with an average RSD of < 5%; nitrogen removal rate in the sediment was monitored for one chlorophyll a (Chl-a) was determined using the hot ethanol year in the Meiliang Bay, where algal accumulation often method, respectively.26 occurs during the summer-time algal bloom season. Incubation To precisely explore how algal accumulation affects the experiments under controlled conditions were also conducted coupled nitrification-denitrification in sediments, twenty-seven to precisely study how algal accumulation affected sediment intact sediment cores were collected for additional incubation nitrification and denitrification processes by measuring nitro- experiments under controlled conditions, whereby the gen transformation and the associated microbes. The objective variations of oxygen profile, nitrifier and denitrifier abundan- of this study was to explore the impacts of algal accumulation ces, and nitrogen removal rate in the sediment were monitored

B https://dx.doi.org/10.1021/acs.est.9b05549 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology pubs.acs.org/est Article after varying additions of algal biomass. The concentrations of gastight mobile rubber without air bubbles in it and left to inorganic nitrogen in the overlying water of sediment cores stand for 10 min to ensure equilibrium according to our were also measured to add the understanding of sediment preliminary experiments. Water samples were collected every 5 nitrification and denitrification processes over the incubation min over a 20 min period and stored into a 12 mL pre- period. Sediment cores were collected at site S3 in Meiliang evacuated Exetainer vial (839 W, Labco, UK) after adding 0.2 μ bay on June 15, 2016, which were collected separately from the L of saturated HgCl2 solution. The concentration of dissolved monthly samples described above. Each core contained 15 cm nitrogen gas in the water was measured using a membrane inlet of sediments and 30 cm of water column. The physiochemical mass spectrometry (MIMS) system (Bay Instruments, Easton, properties of sediment and water samples in sediment cores are MD, USA). In the field, nitrogen removal rate was immediately presented in Table S1. Cores were carefully transported to the measured in situ after sediment core collection. Anammox is laboratory in the dark within 4 h of collection and then left another contributor to nitrogen removal in lake sediment,29 overnight at ambient lake temperature (25 °C) in the dark to but it is likely slow relative to denitrification in Taihu.30 We avoid the disturbances caused by transportation. On the consider these net nitrogen gas flux measurements to be following morning, three cores were randomly selected for representative of potential denitrification rates. initial nitrogen removal rate measurements, which were Sediment Oxygen Profile Measurement. Sediment subsequently sacrificed in order to collect sediment samples oxygen profile was determined by introducing an oxygen for microbial analysis. The remaining cores were randomly microelectrode (OX-50, Unisense, Denmark) into the sedi- assigned to one of four treatments (each n = 6), which are ment at 200-μm intervals according to de la Rosa and Yu described as follows: (1) no added algal biomass (Chl-a <8μg (2005).31 The microelectrode has a tip diameter of ∼50 μm, L−1), simulating the zone where algae have left or algal bloom which is held by a Unisense MM33-2 micromanipulator and does not occur (no algal bloom, termed “NAB”); (2) run with connected to a Unisense PA2000 picoammeter coupled with a algal bloom-equivalent biomass (Chl-a =30μgL−1), which computer data-acquisition system (Figure S2). During each refers to the zone where algal bloom occurs before being measurement, the microelectrode was allowed to equilibrate aggregated to a higher density (algal bloom, termed “AB”); (3) for 5 s at each depth interval before measuring for 3 s. Prior to run with 5 times algal bloom-equivalent biomass (Chl-a = 150 use on each sampling day, the microelectrode was calibrated by μgL−1), which refers to the zone where light algal a 2-point calibration using air aerated water for 100% accumulation occurs (light algal accumulation, termed saturation and 0.1 M ascorbic acid solution for 0% saturation. “LAA”); and (4) run with 10 times algal bloom-equivalent Oxygen penetration depth refers to the depth from the biomass (Chl-a = 300 μgL−1), which refers to the zone where sediment-water interface to the first point in the tail of oxygen heavy algal accumulation occurs (heavy algal accumulation, profile where the oxygen concentration remains consistently at termed “HAA”). The amount of algal biomass in each the 0 mg L−1. The slope of oxygen profile steadily increased treatment was adjusted by either filtering using a phytoplank- toward a maximum within the first millimeter below the initial ton net (64 μm) in the NAB treatment or by adding fresh algal concentration decrease, and the position of this maximum biomass (AB, LAA, and HAA treatments). This algal material gradient was assigned as the sediment-water interface was collected using the phytoplankton net at the same location according to Rabouille et al. (2003).32 as sediment cores. When algal accumulation occurs, light Microbial Abundance Analysis. Nitrifiers and denitrifiers hardly reaches bed sediment under dense algal canopies.27 in sediments were quantified using the quantitative polymerase Incubation experiments under dark conditions were conducted chain reaction (qPCR) method. There are many functioning indoors at room temperature (24 ± 2 °C) over a period of 30 genes involved in nitrification and denitrification processes,33 days. of which amoA, nirS, and nosZ were chosen as gene markers in On day 0 (before the prepared cores were run) and this study. The amoA is a gene marker of ammonia-oxidizing subsequent days 1, 2, 3, 4, 7, 10, 12, 15, 18, 21, 24, 27, and 30, bacteria, which are sensitive indicators of nitrification and play oxygen profiles in sediments were measured using an oxygen a more important role in the nitrification in Taihu Lake;34 the microelectrode according to the description below. Water nirS and nosZ are commonly used gene markers of denitrifiers, samples (10 mL) were carefully taken 5 cm above the sediment which encode nitric oxide reductase and nitrous oxide for dissolved inorganic nitrogen analysis (ammonium, nitrate reductase, respectively.35,36 Sediment samples were stored at and nitrite) using a 25 mL syringe. On day 10, three cylinders −80 °C for further DNA extraction using a FastDNA Power- in each treatment were randomly sacrificed to measure Max Soil DNA Isolation Kit (MP Biomedical, USA) according nitrogen removal rate and collect sediment samples for to the manufacturer’s instructions. This DNA subsequently microbial analysis. The results of these chemical analyses are served as a template for qPCR amplification. The qPCR assay presented as mean values. Water removed during sampling was was performed using the primer amoA1F/amoA2R targeting replaced with filtered (Whatman GF/C) surface water nitrifier amoA gene, the primer cd3aF/R3 cd targeting collected at the same location as the cores. Dissolved inorganic denitrifier nirS gene, and the primer nosZ2F/nosZ2R targeting nitrogen was measured by the flow injection analyzer denitrifier nosZ gene, respectively.37 Gene copies were mentioned above. Dissolved ammonium, nitrate, and nitrite amplified and quantified in a Bio-Rad cycler (CFX Manager, of water used for replacement (Table S1) were used to Bio-Rad, Hercules, CA) equipped with the iQ5 real-time calibrate dissolved inorganic nitrogen in the cylinders. fluorescence detection system and software (version 2.0, Bio- Nitrogen Removal Rate Measurement. Nitrogen Rad, USA). All reactions were completed in a total volume of removal rate was analyzed by measuring net nitrogen gas 20 μL containing 10 μL of SYBR Premix Ex TaqTM (Toyobo, fluxes using intact sediment cores (Figure S1) according to Japan), 0.5 mM of each primer, 0.8 μL of bovine serum Neiss et al. (2012),28 which was calculated based on the linear albumin (3 mg mL−1, Sigma, USA), double distilled water, and rate of nitrogen gas accumulation as a function of time. Before template DNA. The qPCR program for amoA was as follows: sample collection, the system was carefully sealed with a 95 °C for 60 s followed by 40 cycles of 95 °C for 30 s and 57

C https://dx.doi.org/10.1021/acs.est.9b05549 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology pubs.acs.org/est Article

Figure 2. Changes of oxygen penetration depth in sediments in incubation experiments. (A) NAB; (B) AB; (C) LAA; (D) HAA.

°C for 45 s and 72 °C for 60 s. The qPCR program for nirS was relatively stable throughout the experiments, despite slight and nosZ commenced with 95 °C for 60 s, followed by 40 increases in nitrate and nitrite concentrations early on. The cycles of 95 °C for 30 s and 57/60 °C(nirS/nosZ) for 45 s and concentrations of ammonium, nitrate, and nitrite were 0.06− 72 °C for 60 s. A standard curve was established by serial 0.31, 0.08−0.67, and 0−0.02 mg L−1, respectively. In the − − dilution (10 2 to 10 8) of known concentration plasmid DNA treatments with added (AB) or aggregated algal biomass (LAA with the target fragment. All PCRs were run in triplicate on 96- and HAA), ammonium accumulation and nitrate increase were well plates (Bio-Rad, USA) sealed with optical-quality sealing observed within initial incubation days, with ammonium tape (Bio-Rad, USA). Three negative controls without the accumulation becoming larger with more algal biomass. DNA template were included for each PCR run. The Ammonium concentration reached a maximum of 1.55 mg amplification efficiency of 90%−100% and R2 of > 0.99 for L−1 and lasted for 7 days in the AB treatment. Similarly, each qPCR run were accepted for further calculation. ammonium reached a maximum of 2.01 mg L−1 and lasted 15 Statistical Analysis. One-way analysis of variance days in the LAA treatment, and reached a maximum of 2.17 (ANOVA) was used to test the statistical significance of mg L−1 and lasted 24 days in the HAA treatment. Moreover, in ff di erences between treatments. Posthoc multiple comparisons the presence of excessive algal biomass, there were notable ’ of treatment means were performed using the Tukey s least increases in nitrate and nitrite at the end of incubation fi ff signi cant di erence procedure. Prior to analysis of variance, experiments. On day 30, nitrate and nitrite increased to 1.67 − − the normality test using Kolmogorov Smirnov and variance and 0.02 mg L 1 in the LAA treatment, and 1.73 and 0.09 mg homogeneity test were conducted. Logarithmic transformation L−1 in the HAA treatment, a factor of 9.8 rise relative to the was also conducted and the transformed data were examined average surface water concentration. to ensure satisfaction of ANOVA assumptions. All statistical Nitrogen Removal Rate. The nitrogen removal rate was calculations were performed using SPSS 22.0 (SPSS Inc., 66.4 μmol N m−2h−1 in the NAB treatment and increased to fi − − North Chicago, IL, USA), and the level of signi cance was P < 835.6 μmol N m 2h 1 in the AB treatment. However, nitrogen 0.05 for all tests. removal rate decreased when algal biomass was excessive. Nitrogen removal rate was only 570.3 μmol N m−2h−1 in the ■ RESULTS LAA treatment and even lower in the HAA treatment, at 173.9 μ −2 −1 Sediment Oxygen Profiles. Over the 30-day incubation mol N m h . fi fi period, oxygen penetration depth in sediments was relatively Nitri er and Denitri er Abundance. Compared with the × 6 −1 consistent at 7.6−9.0 mm in the NAB treatment but changed control NAB treatment (amoA gene = 1.61 10 copies g greatly in the treatments with added algal biomass throughout sediment), nitrifier abundance was relatively high in the AB the incubation experiments. In the AB treatment, oxygen treatment and relatively low with dense algal aggregations (LAA and HAA). The amoA gene was 2.28 × 106, 0.46 × 106, penetration depth sharply decreased from 8.2 to 0.4 mm on − day 1 and immediately began to recover. Severe anoxia (DO ≈ and 0.99 × 106 copies g 1 sediment in the AB, LAA, and HAA 0mgL−1) occurred in treatments with excessive algal biomass, treatments, respectively. Unlike nitrifiers, denitrifier abundance and the length of this hypoxia was greater with increased algal in treatments with algal biomass (nirS: 2.21 ± 0.51 × 109; accumulation. In the presence of excessive algal biomass nosZ: 0.92 ± 0.18 × 109) was significantly greater (P < 0.05) (treatments LAA and HAA), oxygen penetration depth quickly than those with no algal bloom (nirS: 0.85 ± 0.39 × 109; nosZ: decreased to 0 mm; the oxygen penetration depth began to 0.51 ± 0.15 × 109). However, denitrifier abundance in the recover on days 10 and 15 in the LAA and HAA treatment, treatments with dense algal aggregations (LAA and HAA) was respectively (Figure 2). lower than the treatment with algal bloom-equivalent biomass Water Ammonium, Nitrate, and Nitrite. In the NAB additions (AB). The nirS and nosZ gene reached 0.85 × 109 treatment, dissolved inorganic nitrogen in the overlying water and 0.51 × 109 copies g−1 sediment in the NAB treatment, 2.69

D https://dx.doi.org/10.1021/acs.est.9b05549 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology pubs.acs.org/est Article

Figure 3. Changes of dissolved inorganic nitrogen in the overlying water in simulation experiments. (A) Ammonium; (B) Nitrate; (C) Nitrite. Error bars indicate standard deviations.

× 109 and 1.15 × 109 copies g−1 sediment in the AB treatment, 1.50 × 109 and 0.71 × 109 copies g−1 sediment in the LAA treatment, and 2.44 × 105 and 0.90 × 105 copies g−1 sediment in the HAA treatment, respectively. Monthly Sediment Nitrogen Removal Rate and Water Characteristics in Meiliang Bay, Taihu. In Meiliang Bay, annual nitrogen removal in sediment was estimated to 1.6 × 103 ton in 2016−2017; however, monthly nitrogen removal rate in sediments exhibited a clear seasonal trend. The removal rate was 186.5−226.0 μmol N m−2h−1 during March−May and then sharply decreased to below 10 μmol N m−2h−1 in the summer, after algal accumulation began in June (Chl-a = 240.5 μgL−1). The low removal rate lasted throughout the summer and began to recover post algal accumulation before rising to Figure 4. Net nitrogen gas fluxes in simulation experiments. Error 151.7 μmol N m−2h−1 in February of the following year bars indicate standard deviations. (Figure 6A). Similarly, nitrate was 0.31−0.34 mg N L−1 in the water during March−April, then sharply decreased to below In aquatic systems, the synergy of nitrification and 0.05 mg N L−1 during algal accumulation, and finally recovered denitrification achieves effective nitrogen removal. Nitrification to 0.43 mg N L−1 in February of the following year. In contrast, converts ammonium to nitrate in oxic surface sediments, DO in the water did not exhibit remarkable fluctuations (7.4− supplying substrates for nitrogen removal by denitrification in 12.7 mg L−1), with the lowest levels during algal accumulation. anoxic subsurface sediments. When algal blooms occur in shallow eutrophic lakes, the decay of settled algal biomass ■ DISCUSSION supplies organic carbon and decreases oxygen penetration depth into sediments, favoring sediment denitrification for In shallow lakes, surface sediments are often oxic due to nitrogen removal.11 Accordingly, a higher abundance of oxygen penetration from oxygen-rich overlying water with sediment denitrifiers and rapider nitrogen removal were − 38,39 rapid air water gas exchanges. In the present study, we detected in treatments with added algal bloom biomass when observed deep oxygen penetration in sediments for treatments compared with those in the control treatment without algae. In without added algal bloom material (Figure 2). Following algal the presence of excessive algal biomass, the decay of algal biomass additions, rapid algal decay consumed oxygen and biomass created favorable conditions for denitrification decreased the oxygen penetration depth in sediments (Figure (replete organic carbon, anoxia). However, these anoxic 40 2). In our treatments with dense algae accumulations, the conditions may have also inhibited nitrification, potentially decay of excessive algal biomass depleted dissolved oxygen, limiting the supply of nitrate for subsequent denitrification. causing severe anoxia in sediments (Figure 2C,D). As algal This may partially explain the low nitrogen removal rate found biomass was gradually consumed, algae-induced anoxia slowly in the HAA treatment, relative to LAA and AB (Figure 4). receded with oxygen delivery from the air. However, the Accordingly, we observed rapid ammonium accumulation duration of this anoxia varied with the amount of algal biomass (Figure 3A) and decreased nitrifier and denitrifier abundances added. In the presence of excess algal biomass (LAA, HAA), (Figure 5) in the treatment with excessive algal biomass (LAA, anoxia persisted for a longer time (Figure 2C). In fact, this HAA) relative to those in the treatment with algal bloom- anoxic duration nearly doubled when algal biomass was equivalent biomass (AB). Hence, this study modified the doubled (Figure 2C,D). This redox variation will affect present views that algal blooms can enhance denitrification and redox-sensitive microbes and can alter biogeochemical thereby nitrogen removal in eutrophic lakes, and algal processes in lake sediments.41,42 For instance, Shi et al. accumulation may inhibit nitrogen removal during algal (2018) found that redox-sensitive methanogens/methano- bloom seasons, which is concerning in the restoration of trophs were inhibited in sediments when anoxia/hypoxia was eutrophic lakes. reduced, causing a decrease in methane emission from a Our monthly time-series measurements in Meiliang Bay eutrophic lake.42 See Figures 3 and 4. show a sharp decrease of nitrogen removal rate during the

E https://dx.doi.org/10.1021/acs.est.9b05549 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology pubs.acs.org/est Article

Figure 5. Average nitriﬁer and denitriﬁer abundance in each experimental treatment. (A) amoA gene; (B) nirS gene; (C) nosZ gene. Error bars indicate standard deviations.

Figure 6. Sediment nitrogen removal rate and water characteristics in Meiliang Bay, Taihu. (A) Sediment nitrogen removal rate and water Chl-a; (B) water DO and nitrate. Annual nitrogen removal was estimated by the trapezoidal method using MATLAB (MathWorks, USA). The data were collected in 2016−2017. Error bars indicate standard deviations. summer algal bloom and accumulation period. At the same anoxia was not detected in the water during algal accumulation time, wind-driven algal accumulations also generate large areas (Figure 6B), since dead algae cells often settle on sediment of relatively low algal biomass, where decreased organic carbon surface and decay. However, denitrification possibly occurs supplies and strong oxygen penetration may also hamper inside suspended particles, contributing to nitrogen removal,46 fi denitri cation. In support of this hypothesis, we found which needs further studies to quantify the contribution. relatively low nitrogen removal rates in the control treatment As anoxia receded (Figure 2), a decrease in ammonium without added algal biomass (Figure 4). Nitrates from (Figure 3A) and subsequent increases in nitrate (Figure 3B) anthropogenic sources and the zone without algal accumulation may be transported to the zone with algal accumulation and nitrite (Figure 3C) were observed at the end of incubation and subsequently support its denitrification. However, nitrate experiments in the treatments with dense algal aggregations. in the overlying water maintained extremely low levels, and We suggest that this was caused by an “awakening” of the nitrogen removal recovery was not detected during the algal nitrifying community, helping to restart coupled nitrification- accumulation period in our field surveys (Figure 6), indicating denitrification. In our monthly field measurements, we the limited contribution of external nitrate inputs during the detected a gradual increase in nitrogen removal rate post severe algal accumulation. During algal blooms in Taihu, if algal accumulation (Figure 6). The negative impacts of algal algae are aggregated by wind to a region that is 20% of the total accumulation on nitrogen removal can weaken or disappear lake surface area, 80% of the lake area will be relatively poor in later, as seen in the increased nitrogen gas flux in the fall algal biomass. In this case, nitrogen removal rate is estimated − − months in Meiliang Bay (Figure 6); and the decreased nitrogen to decrease from 835.6 to 167.2 μmol N m 2h 1 by 80.0% according to nitrogen removal rates in our simulation loss in the algal accumulation period may be just the experiments; if algal biomass accumulates to 10% of lake redistribution of nitrogen removal amount over the whole area, creating 90% of lake area poor in algal biomass, the year. However, we suggest that frequent algal accumulations in nitrogen removal rate is estimated to further decrease to 77.2 shallow lakes may cause considerable negative impacts on − − μmol N m 2h 1 by 90.8% (Figure S3). The measured nitrogen nitrogen removal and hence lake restoration. The decreased gas fluxes in the field (Figure 6) were generally much less than nitrogen removal can make more nutrients available for algal those in the simulation experiments (Figure 4). It is plausible proliferation, thereby intensifying existing algal blooms. As the fi fi 43 that nitrogen gas xation could have been greater in the eld saying goes, “United we stand; divided we fall”. During blooms, fl but inhibited in the cores, causing nitrogen gas uxes in the it appears that algae united by wind-driven currents can algal accumulation treatments to be relatively high. Moreover, generate conditions favorable for nutrient retention and while our study focused on microbial abundance, it is likely that the activity of nitrifiers and denitrifiers may have also subsequent proliferation. On the contrary, algal blooms that changed under the shift of redox conditions caused by algal are evenly distributed during quiescent conditions may create biomass, further altering nitrogen removal rate.44 This deserves denitrification-favorable conditions, encouraging the loss of further study using other molecular biology techniques, such as nutrients vital for further bloom development. This may be a DNA/RNA-based stable isotope probing.45 In fields, hypoxia/ new law of algal survival in shallow eutrophic lakes.

F https://dx.doi.org/10.1021/acs.est.9b05549 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology pubs.acs.org/est Article ■ ASSOCIATED CONTENT ■ REFERENCES *sı Supporting Information (1) Houser, J. N.; Richardson, W. B. Nitrogen and phosphorus in The Supporting Information is available free of charge at the Upper Mississippi River: transport, processing, and effects on the river ecosystem. Hydrobiologia 2010, 640 (1), 71−88. https://pubs.acs.org/doi/10.1021/acs.est.9b05549. (2) Heffernan, J. B.; Cohen, M. J. Direct and indirect coupling of fl primary production and diel nitrate dynamics in a subtropical spring- Experimental designs for nitrogen gas uxe and sediment − fi fed river. Limnol. Oceanogr. 2010, 55 (2), 677 688. oxygen pro le analyses, estimations of nitrogen removal (3) Lewis, W. M., Jr; Wurtsbaugh, W. A.; Paerl, H. W. 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Key Laboratory of Atmospheric Environmental Monitoring & Three-dimensional numerical modeling of cohesive sediment trans- Pollution Control, School of Environmental Science & port and wind wave impact in a shallow oxbow lake. Adv. Water − Engineering, Nanjing University of Information Science & Resour. 2008, 31 (7), 1004 1014. Technology, Nanjing 210044, China (15) G.-Toth, L.; Parpala, L.; Balogh, C.; Tatrai, I.; Baranyai, E. Zooplankton community response to enhanced turbulence generated Complete contact information is available at: by water-level decrease in Lake Balaton, the largest shallow lake in https://pubs.acs.org/10.1021/acs.est.9b05549 Central Europe. Limnol. Oceanogr. 2011, 56 (6), 2211−2222. (16) MARCE, R.; FEIJOO, C.; NAVARRO, E.; ORDONEZ, J.; Author Contributions GOMA, J.; ARMENGOL, J. Interaction between wind-induced $These authors contributed equally to the paper. seiches and convective cooling governs algal distribution in a canyon-shaped reservoir. Freshwater Biol. 2007, 52 (7), 1336−1352. Notes (17) Cyr, H. Winds and the distribution of nearshore phytoplankton The authors declare no competing financial interest. in a stratified lake. Water Res. 2017, 122, 114−127. (18) George, D. G.; Edwards, R. W. The Effect of Wind on the Distribution of Chlorophyll A and Crustacean Plankton in a Shallow ■ ACKNOWLEDGMENTS Eutrophic Reservoir. J. Appl. Ecol. 1976, 13 (3), 667. This study was supported by the National Natural Science (19) Zhang, Y.; Ma, R.; Min, Z.; Duan, H.; Loiselle, S.; Xu, J. Fourteen-Year Record (2000−2013) of the Spatial and Temporal Foundation of China (No. 41621002, 41701112, 51709181, Dynamics of Floating Algae Blooms in Lake Chaohu, Observed from 51979171), Starting Research Fund of Nanjing University of Time Series of MODIS Images. Remote Sensing 2015, 7 (8), 10523− Information Engineering (No. 20181015). 10542.

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