Laizhou Bay (LZB), a typical semi-enclosed bay, is situated south of the Bohai Sea and north of the Shandong Peninsula (Fig. 1). It covers an area of approximately 7000 km² with a coastline of 320 km, a mean depth of less than 10 m, and a water half-exchange time (i.e., the time for 50 % water renewal) of 55 d (Wu et al., 2023). Water quality in LZB has deteriorated due to high land-based riverine inputs, including those from the Yellow River (YR) and Xiaoqing River (XQR), as well as discharges from mariculture. Since 1980, the dissolved inorganic nitrogen (DIN) concentration has increased from 3 to 25 µmolL-1, and the ratio of DIN : PO₄-P ratio increased from 5:1 to 110:1 (Xin et al., 2019). The resulting increase in N availability, coupled with intensifying P limitation, has triggered significant ecological shifts in the bay, including more frequent harmful algal blooms and changes in dominant phytoplankton species from diatoms to dinoflagellates (Song et al., 2017; Xin et al., 2019).

To investigate the distribution of APA in LZB, four land-sea synchronous surveys were conducted on 10–12 May 2019 (spring), 18–21 August 2019 (summer), 2–14 November 2021 (autumn), and 22–27 March 2021 (winter). The land-sea synchronous survey stations covered the entire LZB and the adjacent waters of the Yellow River Estuary (YRE) (Fig. 1). Additional riverine monitoring stations were located at the mouths of ten rivers, including YR and XQR (Fig. 1). The monitored pollutant load accounted for >95 % of the total discharge load in the LZB's basin (Li et al., 2022). Surface seawater samples were collected using a 5 L Niskin bottle. For subsequent analysis of nutrients, chlorophyll a (Chl a), bacterial abundance, phytoplankton, and alkaline phosphatase activity, water was processed immediately upon collection: aliquots for nutrient analysis were filtered through acetate membranes (0.45 µmolL-1); aliquots for enzymatic activity assays were processed as either unfiltered or size-fractionated as described in Sect. 2.4.3; and samples for molecular analysis were filtered onto 0.22 µmolL-1 polycarbonate membranes. For riverine water samples collected at the mouths of ten major rivers, sampling was conducted following standard protocols for surface water monitoring in China (specifically, HJ 494-2009). An organic glass water sampler or a pre-cleaned polyethylene container was used to collect subsurface water at a depth of approximately 0.5 m. All samples were preserved according to the specific protocols detailed in Sect. 2.4.1–2.4.4 and transported on ice to the laboratory within 12 h. The August 2019 survey occurred after the landfall of the strong typhoon “Lekima” in Shandong. From late August to October 2021, the middle and lower reaches of the YR experienced a historically rare autumn flood, and the November 2021 survey took place after this flood. Samples were collected in the river and sea areas in near-synchrony.

An enclosure experiment was conducted from 23 July to 21 August 2021 to explore the effects of phosphate concentration and DIN : PO₄-P ratio on APA secretion. Two enclosed ecosystems were designed. Water samples were collected at a representative station (119.71° E, 37.54° N) at the mouth of LZB on 23 July 2021 (Fig. 1). Seawater samples were siphoned into containment bags on the shore. Incubation experiments were carried out in the nearshore sea area (120.00° E, 37.36° N) for 29 d (Fig. 1). The initial seawater nutrient environment with a DIN : PO₄-P ratio of 55 (Table S1 in the Supplement) was maintained in the control group (CG). In the treatment group (TG) the DIN : PO₄-P seawater ratio was adjusted to 28 by adding NaNO₃ and KH₂PO₄ (Tables S1 and S2). This choice (TG, DIN : PO₄-P = 28) was guided by two key considerations: (1) to establish a significant contrast (approximately halved) to the ambient ratio of 55, and (2) to approach the classical Redfield ratio of 16:1, thereby testing microbial responses under a scenario of mitigated stoichiometric imbalance. Furthermore, the higher absolute nutrient concentrations in the TG (DIN : 50.01 µmolL-1, PO₄-P: 1.78 µmolL-1) compared to the control (CG) (DIN: 1.90 µmolL-1, PO₄-P: 0.04 µmolL-1) were necessary to ensure that nutrients remained non-limiting throughout the incubation, allowing the target N:P ratio to be the primary variable influencing the system over time. In parallel, culture water samples were collected 17 times at fixed intervals. Samples were collected 17 times over the 29 d incubation to cover the full phytoplankton growth and senescence cycle. During the exponential growth phase, sampling was conducted daily. After nutrient depletion and population decline, the interval was extended to monitor the stabilized system. The detailed sampling days and weather conditions are presented in Table S2.

Using Origin software (version 2024), the non-parametric Spearman correlation test (two-sided) was conducted to create a heat map of the correlation between APA and environmental parameters. The PO₄-P threshold for APA induction was subsequently determined using a segmented regression approach on the relevant dataset. Differences between experimental groups (e.g., CG vs. TG) were assessed for significance using t test and Mann-Whitney U test. Linear regression analysis was applied to determine whether the data were linearly correlated. The spatial distribution maps of temperature, salinity, nutrients, APA, and Chl a were generated using ordinary kriging interpolation in Surfer (version 16, Golden Software LLC), which accounts for spatial autocorrelation among sampling points to produce unbiased continuous surfaces.

Seasonal investigation of rivers emptying into LZB revealed that the average annual DIN : PO₄-P ratio of riverine loads into LZB was 39:1, with a maximum of 125:1 (summer in the Jiaolai River) and a minimum of 2:1 (spring in the Bailang River) (Fig. 2). The average annual total nitrogen to total phosphorus ratio (TN : TP) was 90:1, with a maximum of 263:1 (autumn in the Bailang River) and a minimum of 16:1 (spring in the Guangli River) (Fig. 2). The DIN : PO₄-P ratio of riverine loads was maximal (70:1) in winter (Fig. 2). For the P components of riverine loads, PP accounted for the highest percent contribution, with an average of 53±7 % (mean ± SD, standard deviation), while DOP and PO₄-P accounted for 27±17 % and 21±16 %, respectively (Fig. 2). Regarding nitrogen components, DON accounted for the highest percent contribution, with an average of 52±17 %, and DIN and PN accounted for 39±19 % and 9±9 %, respectively (Fig. 2).

The distribution of APA in LZB showed a gradual decrease from the coastline to offshore waters, with the highest values observed in nearshore coastal waters (Fig. 3). The mean value of APA_total reached 795.8±1749.8 nmolL-1h-1 in autumn and was thus 50 times higher than in winter (Fig. 3). Mean values of APA_phy, APA_bac, and APA_free in autumn were 715.1±1592.6, 55.1±286.2, and 25.7±61.1 nmolL-1h-1, respectively, and thus 78, 12, and 17 times higher, respectively, than in winter (Figs. 3, S3). Accordingly, in autumn APA_phy contributed most to APA_total while APA_free contributed the least (Fig. S4), whereas in winter APA_free and APA_bac made the highest and lowest contribution, respectively (Fig. S4). The spatial distributions of high Chl a concentrations and high APA_total values showed consistency, especially in the adjacent areas of the XQR and YR estuaries (Fig. 3).

The mean value of the AP kinetic parameter Km was 8.9±13.8×103 nmolL-1 in autumn and was thus 3.6 times lower than in winter (31.8±58.8×103 nmol L⁻¹). In contrast, the AP kinetic parameter Vmax averaged 754±1474.7 nmolL-1h-1 in autumn, approximately an order of magnitude higher than in winter (26.1±17.1 nmolL-1h-1) (Fig. 4).

In autumn, correlation analysis of field data revealed that APA_phy exhibited a significant positive correlation with the DIN/PO4-P ratio (p<0.05) (Fig. 4). However, there was no detectable correlation between APA and PO₄-P (Fig. 4). In winter, APA_total, APA_bac, and APA_free were negatively correlated with PO₄-P and Chl a, but not with the DIN/PO4-P ratio (Fig. 4). And APA_total was positively correlated with APA_phy, APA_bac, and APA_free. Additionally, APA_free was positively correlated with APA_phy (p<0.05) and APA_bac (p<0.05). APA_total, APA_bac, and APA_free were also positively correlated with the DON / TDN ratio (p<0.05) (Fig. 4).

Both field and experimental data in LZB clearly showed that APA_phy, APA_bac, and APA_free were synthesized and secreted when PO₄-P <0.05 µmolL-1, and their concentrations and activity increased rapidly as PO₄-P decreased (Fig. 6). The PO₄-P threshold was consistent between the field study and enclosure experiment. Moreover, when PO₄-P was lower than its concentration threshold (0.05 µmolL-1), the enzyme kinetic parameters Km and Vmax also increased sharply (Fig. S6). There was a significant linear positive correlation (P<0.01) between APA_total and the DIN : PO₄-P ratio during both the autumn survey and the experiment (Fig. 6). During the autumn survey, this linear relationship could be further divided into two groups by K-means cluster analysis based on APA, such that the slope of the fitted straight line was significantly higher in the high APA group (P<0.01; red line in Fig. 6) relative to that of low APA group (P<0.01; black line in Fig. 6). However, no obvious linear relationship was found between APA and the DIN : PO₄-P ratio in winter (Fig. S7).

During the autumn field study, the AP regulatory gene phoD had higher read counts at stations with lower PO₄-P concentrations (S2_11 vs. S1_11). Station S2_11 also showed a significant increase in APA compared to S1_11. During the winter survey, station S4_3 had relatively low PO₄-P concentrations and high APA, along with high read counts for AP regulatory genes including phoD.

In the CG, the PO₄-P concentration gradually decreased during the 29 d experiment (Fig. 5), while the phoD and phoABAP (AP synthesis genes) and phoP (regulatory gene) increased, accompanied by an increase in APA (Fig. 7). Comparing the two groups groups with different DIN : PO₄-P ratios, the PO₄-P concentration in the CG over the 29 d was lower than that in the TG (Fig. 5). Additionally, the read counts of phoD, phoAB, and phoP were higher in the CG by the end of the experiment (day 29), and APA was significantly higher than in the TG over the same period (P<0.01; Fig. 7).

While the Yellow River and Xiaoqing River are indeed the dominant sources of nutrients to Laizhou Bay (Fig. 2), our comprehensive analysis of ten major rivers provides a complete picture of the terrigenous nutrient load spectrum. Although these smaller tributaries contribute less to the total nutrient load, their impact on local nutrient concentrations and ratios, particularly in adjacent estuarine and coastal waters, remains significant. This basin-wide analysis reveals that highly elevated N:P ratios are a regional scale of anthropogenic nutrient imbalance (Figs. 2–3). The nutrient regime of LZB has changed markedly due to anthropogenic perturbations over the past six decades, shifting from a N-limited oligotrophic state before the 1990s to a potentially P-limited eutrophic state (Xin et al., 2019). Phosphorus limitation in LZB may be influenced by both anthropogenic inputs and shifts in biogeochemical processes. From the 1980s to 2020s, the DIN/PO4-P ratio increased sharply from 200:1 to ca. 1600:1 (Xin et al., 2019), mainly due to the effects of the YR which contributes the largest river discharge in the LZB. Our study also showed that the average N and P flux ratio of the main rivers discharging into LZB is ca. 5.6× higher than the Redfield ratio (Fig. 2). The high values of the DIN : PO₄-P ratio in LZB were distributed in the nearshore area and gradually decreased moving offshore, suggesting that the terrigenous nutrient input may significantly influence the spatial distribution of the DIN : PO₄-P ratio in LZB (Fig. 3). The pronounced increase in the N:P ratio of riverine nutrient loads is driven by anthropogenic activities, especially the heavy application of N fertilizer, and industrial and domestic P restrictions (Wang et al., 2019; Liang et al., 2023). Atmospheric deposition contributes ca. 30 % to 50 % of the total DIN of LZB (Shou et al., 2018), and only about 10 % of total phosphate (Zhang et al., 2024), leading to an N:P ratio far exceeding the 16:1 Redfield ratio (Chen et al., 2021; Zhang et al., 2024). Thus, the imbalanced input of N and P from both terrigenous and atmospheric deposition is the critical factor aggravating the N/P ratio of LZB.

The increasing DIN concentration in coastal waters, driven by anthropogenic N inputs, promotes phytoplankton growth, leading to elevated Chl a levels (Fig. 3). Prior studies demonstrate that phytoplankton nutrient uptake under such conditions does not adhere to the classical 16:1 Redfield ratio but rather exhibits higher DIN : PO₄-P uptake ratios (Fransner et al., 2018; Macias et al., 2019). This pattern is also observed in our study where phytoplankton culture groups, with high DIN : PO₄-P ratios showed correspondingly high nutrient absorption ratios (Fig. 5). Under high DIN : PO₄-P ratio conditions, phytoplankton upregulate high-affinity phosphate transport systems (e.g., pstSCAB), whose Km value (Michaelis-Menten constant, reflecting the affinity of the transport system for phosphate, with lower Km indicating higher affinity) can be as low as 0.1 µmolL-1 (Dyhrman and Haley, 2006). This enables efficient dissolved phosphate uptake even under extremely low P concentrations (<1 µmolL-1). When P is sufficient, a delayed negative feedback mechanism in the PhoB / PhoR two-component system allows continuous P uptake, with rapid conversion to storage forms such as polyphosphate (polyP) (Martin et al., 2011). Concurrently, phytoplankton secrete AP to mineralize DOP, further enhancing P acquisition (Lin et al., 2016). This highly efficient P competition strategy not only exacerbates P limitation for non-storing species but also widens the range of the N:P ratio of residual dissolved inorganic nutrients due to their high N:P uptake demands (Fransner et al., 2018), creating a positive feedback loop that ultimately favors the dominance of P-storing species (Klausmeier et al., 2004).

Therefore, rather than improving the DIN : PO₄-P imbalance, biological adaptations to anthropogenic high DIN : PO₄-P inputs appear to amplify and perpetuate elevated DIN : PO₄-P conditions in coastal ecosystems. This creates an ecological “trap” where the ecosystem becomes increasingly dominated by high adapted species that reinforce the imbalance through their nutrient acquisition and storage strategies. Management strategies must prioritize the reduction of nitrogen inputs at their source to mitigate the N:P imbalance and restore ecological balance, rather than relying on intrinsic biological compensation within the coastal zone.

APA is regulated by multiple environmental factors, such as PO₄-P concentration, the DIN : PO₄-P ratio and temperature. The PO₄-P threshold (0.05 µmolL-1) identified in this study aligns with reported values from both coastal and open-ocean waters, including the semi-enclosed Bohai and Yellow Seas (Jin et al., 2024), the Sargasso Sea (Lomas et al., 2010), the eastern subtropical Atlantic (Mahaffey et al., 2014), and the subtropical Pacific (Suzumura et al., 2012). In the present study, both data from field surveys and enclosure experiment data confirmed this concentration threshold, with the field data demonstrating a significant increase in APA and substrate affinity below this concentration. Moreover, enzyme kinetics data from field investigations in LZB further validated the phosphate concentration threshold, showing that AP activity and substrate affinity increased significantly when phosphate concentration dropped below the threshold (Fig. S6). The Km in autumn was lower than in winter, while Vmax was much higher in autumn than in winter. The lower Km (indicating higher substrate affinity) and much higher Vmax (indicating higher maximum hydrolysis capacity) in autumn compared to winter collectively resulted in higher APA (Fig. 3).

When PO₄-P concentrations fell below the threshold in autumn, a strong positive APA-DIN : PO₄-P ratio correlation was found (P<0.01), consistent with observations in the Bohai Sea (Jin et al., 2024) and NW Mediterranean (Bogé et al., 2017). In winter, however, this relationship was undetectable in LZB. The absence of a significant APA response to low PO₄-P or high DIN : PO₄-P ratios in winter may be attributed to several factors. Low temperatures likely directly suppress microbial metabolic rates and enzyme production, including APA. Additionally, seasonal shifts in phytoplankton community composition – such as a predominance of diatoms in winter, which may exhibit an inherently lower responsiveness to phosphorus stress compared to the autumn assemblages – could also contribute to the observed seasonal discrepancy in APA regulation (Ivancic et al., 2016; Ou et al., 2024). Furthermore, genetic analysis showed no significant AP gene upregulation at high DIN : PO₄-P ratios when PO₄-P remained above the identified threshold (station S3_3, Fig. 7), confirming that sub-threshold PO₄-P concentrations are essential for APA induction. Below this threshold, APA increases proportionately with increasing DIN : PO₄-P ratios.

Studies indicate that interspecific differences in APA expression capacity, induction thresholds, and regulatory patterns may influence phytoplankton adaptation to phosphorus limitation and community succession. Laboratory cultures show that APA induction thresholds vary widely (10.1–16.4 µmolL-1), with the dinoflagellate P. minimum requiring significantly higher phosphate concentrations than diatoms (Meseck et al., 2009). In natural systems, diatom taxa that dominate under phosphorus limitation commonly express APA, whereas few dinoflagellates do so, and their APA expression is not linked to dominance (Ivancic et al., 2016). Furthermore, diatoms exhibit phosphate-regulated APA, while dinoflagellate APA appears less responsive to phosphate levels, possibly regulated by other factors (Dyhrman and Ruttenberg, 2006). Such heterogeneity in APA strategies suggests that species with lower induction thresholds or stronger expression may gain a competitive advantage under fluctuating phosphorus supply, potentially influencing succession (Fig. 5). However, APA represents only one of multiple phosphorus-acquisition strategies, and its role is likely a contributing factor rather than the sole driver of community shifts.

The phenomenon of APA surge in coastal phytoplankton reveals complex regulatory mechanisms and ecological adaptation strategies. When ambient PO₄-P concentrations fall below the 0.05–0.2 µmolL-1 threshold (Dyhrman and Ruttenberg, 2007), phytoplankton activate the pho regulon (a gene suite responsible for phosphorus scavenging) operon (a gene cluster containing the pstSCAB phosphate transport system and phoA/phoX alkaline phosphatase genes) through the PhoB/PhoR two-component system (a P-sensing system consisting of histidine kinase PhoR and the response regulator PhoB) (Lin et al., 2016). Notably, our findings demonstrate a significant positive correlation between APA and the DIN : PO₄-P ratio when PO₄-P concentrations fall below the threshold. This result may stem from the following fundamental mechanisms: (1) High DIN : PO₄-P conditions induce an enhanced capacity for P acquisition to maintain elemental stoichiometric balance, manifested as increased APA with rising DIN : PO₄-P ratios; (2) Elevated DIN : PO₄-P ratios may indirectly modulate APA through N-sensitive regulators (e.g., PII protein) in the pho signaling pathway (Ustick et al., 2021).

It is noteworthy that prior studies have found that under the same N:P ratio and active phosphate concentration, phytoplankton APA in DON-enriched cultures was significantly higher than that in cultures without DON (Fitzsimons et al., 2020). Our winter survey data revealed a significant positive correlation between DON and APA_bac (Fig. 4), similar to findings of a correlation between APA_phy and DON concentration (Ou et al., 2024), indicating that not only PO₄-P but also nutrient composition and ratios, can affect APA. Regulation of APA by DON may potentially operate through specific DON components or their degradation products influencing P metabolic pathways, or through DON serving as an alternative N source that stimulates phytoplankton growth and P demand (Ma et al., 2018; Forchhammer et al., 2022). Meanwhile, DON serving as alternative nitrogen source may enhance phytoplankton growth and phosphorus demand.

Human activities have significantly altered coastal nutrient dynamics via multi-level ecological feedback mechanisms that regulate APA activity patterns (Fig. 8). The “anthropogenic N pump” driven by riverine discharge and atmospheric deposition (Jin et al., 2024) has induced a disproportionate increase in DIN inputs, leading to relative PO₄-P depletion and elevated DIN : PO₄-P ratios (Figs. 2 and 3). This nutrient imbalance triggers phytoplankton adaptive responses, i.e., when PO₄-P concentrations fall below the identified threshold, the pho regulon and other APA-related gene clusters are activated (Fig. 7), while the combined effects of high DIN : PO₄-P ratios and P limitation significantly enhance APA expression and DOP mineralization capacity (Fig. 6). These biochemical responses initiate ecological cascades that favor phytoplankton with high APA activity and superior DOP utilization capabilities (Fig. 3; Hackett et al., 2005; Nicholson et al., 2006; Ivancic et al., 2016; Ou et al., 2024). Variation in seasonal temperature regulates this process both through direct suppression of APA activity (via reduced enzymatic kinetics and microbial metabolism) and indirect modulation of community composition (reflecting thermal niche differentiation among phytoplankton groups) (Toseland et al., 2013; Thomas et al., 2017).

The above system ultimately establishes a positive feedback loop: APA-mediated DOP utilization increases phytoplankton biomass (as reflected in elevated Chl a), which accelerates PO₄-P depletion and exacerbates the DIN : PO₄-P imbalance, thereby continuously selecting for APA-enhanced genotypes. This conceptual framework demonstrates how anthropogenic alteration of DIN : PO₄-P stoichiometry triggers cross-scale responses from molecular to ecosystem levels, creating self-reinforcing ecological restructuring.