Stocking density-driven shift of atmospheric carbon dioxide source-sink functions in mussel culture systems

Stocking density-driven shift of atmospheric carbon dioxide source-sink functions in mussel culture systems | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Stocking density-driven shift of atmospheric carbon dioxide source-sink functions in mussel culture systems Shuang-Lin Dong, Miao-Jun Pan, Yu-Xi Zhao, Sheng-Jie Xu, Chang-Lin Li, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8677151/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bivalve aquaculture has long been excluded from blue carbon frameworks because bivalves release carbon dioxide (CO 2 ) into seawater during shell formation. However, this ignores the complex roles of bivalves in aquatic ecosystems. Through manipulative experiments with mesocosms, here we identify that CO 2 source-sink functions of mussel culture systems are stocking density-dependent. At a stocking density of 55.56 g m − 3 , mussels suppressed phytoplankton biomass through filter feeding (top-down effect), making bivalve respiration the dominant process governing CO 2 dynamics and turning the system into a CO 2 source. When density fell below 27.78 g m − 3 , the “bottom-up effect”from bivalve excretion prevailed, enabling net primary productivity to dominate (35.61–40.92%) and turning the system into a CO 2 sink. The actual stocking densities in coastal bivalve farms considering water renewal are below 27.78 g m − 3 , suggesting they can function as CO 2 sinks. Post-harvest monitoring further indicated that water masses flowing out from the core farming area can absorb more CO 2 than non-farming seawater. Therefore, this study recommends adopting an integrated, ecosystem-based approach to assess the role of bivalve aquaculture in global climate change. Earth and environmental sciences/Environmental sciences/Environmental impact Earth and environmental sciences/Climate sciences/Climate change/Climate-change mitigation Biological sciences/Ecology/Ecosystem ecology Earth and environmental sciences/Biogeochemistry/Carbon cycle Bivalve farming Stocking density Carbon sink Carbonate system CO2 fluxes Mesocosms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Addressing global warming and its associated environmental challenges requires not only reductions in greenhouse gas emissions but also natural and engineered approaches to capture and store atmospheric carbon dioxide (CO 2 ) 1 . Filter‑feeding bivalve aquaculture is widely considered a promising strategy for carbon sequestration and climate mitigation, since carbon is removed from the water by harvesting stable calcium carbonate shells 2 , 3 . However, this view remains highly controversial, as CO 2 is released into the water during calcification 4 , 5 , leading to the exclusion of bivalve aquaculture from the carbon trading system 6 . With global bivalve aquaculture production having exceeded 19 million tonnes 7 , it is imperative to clarify whether it can simultaneously fulfill the dual roles of providing blue food and enhancing marine carbon dioxide removal 8 – 10 . At the individual level, respiration and calcification of bivalves are the main metabolic processes affecting CO 2 concentrations in water. Respiration releases CO 2 directly, whereas calcification consumes dissolved inorganic carbon (DIC) to form calcium carbonate shells 11 . However, the precipitation of CaCO 3 also consumes more total alkalinity (TA), which shifts the carbonate system equilibrium toward CO 2 release 12 . When bivalves are introduced into an aquatic ecosystem, beside the calcification and respiration, they can impact aqueous CO 2 concentrations through multiple ecological processes, such as “top-down effects” from filter feeding, “bottom-up effects” from nutrient excretion, and biodeposition 13 – 15 . Therefore, the potential role of bivalve culture in global climate change should be assessed using an integrated ecosystem approach. Ecological processes in bivalve culture system related to CO 2 consumption or release are affected by stocking density or biomass. For example, hard clam ponds shifted from sinks of atmospheric CO 2 to sources in the mid to late stages, due to changes in photosynthesis and organic matter remineralization within the system as the clams grew 16 . Similarly, in polyculture systems, low stocking densities of razor clams can increase primary productivity and thereby enhancing the system’s CO 2 uptake, whereas high densities reduced its capacity to serve as a CO 2 sink 17 . Therefore, a comprehensive understanding of how stocking density influences the CO 2 concentrations and inorganic carbon system in water by altering ecological processes is crucial for evaluating the role of bivalve culture in the global carbon cycle. Some field surveys have shown that bivalve culture will elevate partial pressure of CO 2 ( p CO 2 ) in seawater of the coastal farm areas, thereby reducing CO 2 uptake or even converting the farm areas into net CO 2 sources 18 – 20 . However, these areas are usually open or semi-open systems, bivalves consume phytoplankton supplied by upstream waters and exert an influence on the downstream waters due to hydrodynamics 21 , 22 . This connectivity means that previous studies, which focused only on local farm areas, provide an inadequate perspective 23 . Therefore, using rigorous methods to assess the CO 2 source-sink function of the complete bivalve culture system is important to comprehensively understand its holistic ecological effects. Mesocosms systems, with clear boundaries and controllable conditions, have become a reliable approach for investigating the structure and function of aquatic ecosystems 24 , 25 . This study employed mesocosm systems to conduct manipulative experiments under different stocking densities (0-100 g m − 2 ) of filter-feeding mussels ( Mytilus galloprovincialis ). Combined with field sampling and mass balance models, the study aims to: (1) clarify the impact of stocking density on the CO 2 source-sink functions of bivalve culture ecosystems; (2) elucidate the mechanisms of CO 2 removal for bivalve culture ecosystems; (3) investigate the subsequent dynamics of the carbonate system in the waters following mussel removal. Results Metabolism of mussels The metabolic activity rates of the experimental mussels at different culture period and their effects on the carbonate system are shown in the Fig. 1 and Supplementary Fig. 2. The rates of dissolved oxygen consumption (R DO ), total ammonia nitrogen excretion (R TAN ), calcification (R cal ) and respiration (R res ) in mussels generally showed an upward trend over time, and no significant differences were observed among different density groups ( P > 0.05). After incubation, partial pressure of CO 2 ( p CO 2 ) in the water increased significantly ( P < 0.05, Fig. 1 c), with calcification and respiration contributing increases of 22.42–38.62 µatm (12.78–23.27%) and 73.60–220.00 µatm (65.65–77.53%), respectively (Fig. 1 d). Environment parameters and net primary productivity Throughout the experimental period, water temperature and salinity increased from 20.3 ℃ to 29.7 ℃ and from 30.3 to 33.0, respectively (Supplementary Fig. 3a). At the beginning of the experiment, the net primary productivity (NPP) was negative due to artificial disturbance probably and did not vary significantly across all groups ( P > 0.05, Fig. 2 a). Following the introduction of mussels, all groups except for M4 exhibited a marked upward trend in NPP. By the end of the culture stage (Day 60), the moderate stocking density (M2 and M3) of mussels significantly promoted NPP compared to the control ( P < 0.05), while the high density (M4) led to an inhibition. After mussel harvest, NPP in the M2 and M3 groups dropped sharply and returned to a level similar to the control group after 20 days. Carbonate parameters Throughout the culture stage, all groups except M4 showed an initial decline in pH, followed by a gradual rise (Supplementary Fig. 3c). In contrast, the M4 group exhibited a continuous decline. At Day 60, the high-density group showed a significantly lower pH than the control group, while the moderate-density groups were significantly higher. ( P < 0.05). Following mussel harvest, pH slightly decreased in M2 and M3 groups but increased markedly in M4 groups. By Day 80, no significant difference was observed between M2, M3 and the control groups ( P > 0.05), whereas pH in M4 group remained significantly lower ( P < 0.05). Total alkalinity (TA) in the control group increased consistently over the culture period (Fig. 2 b). In contrast, stocking mussels significantly reduced the water TA. By the end of the culture stage, TA in all mussel-stocked groups was significantly lower than in the control group ( P < 0.05), with the lowest value observed in the highest-density group. After mussel harvest, the water TA remained essentially unchanged. All groups exhibited declining trends of dissolved inorganic carbon (DIC), with mussel-stocked groups showing greater reductions than the control group (Fig. 2 c). By Day 60, DIC levels in all mussel-stocked groups were significantly lower than in control group ( P < 0.05). After mussel harvest, DIC in M4 group continued to decline noticeably, resulting in significantly lower DIC compared to the other three mussel-stocked groups by Day 80 ( P < 0.05). Partial pressure of CO in surface water and CO flux at air-water interface During the first 10 day, the partial pressure of CO 2 ( p CO 2 ) in surface water exhibited an increasing trend across all groups (Fig. 2 d). From Day 10 to Day 60, p CO 2 declined in all groups except M4, which continued to increase. By the end of the culture stage, the p CO 2 was significantly elevated in the high-density group but reduced in the moderate-density groups compared with the control group ( P 0.05). The temporal pattern of CO 2 flux at the air-water interface ( F CO 2−aw ) corresponded closely with the changes in p CO 2 (Fig. 2 e). After mussel harvest, the F CO 2−aw of mussel-stocked groups gradually converged to the level of the non-mussel control group. In addition, during the removal stage, M1-M3 groups cumulatively absorbed 4.32, 6.54, and 7.56 mol of CO 2 , respectively, which were 1.4, 2.12, and 2.45 times that of the non-culture group, while M4 group released 6.76 mol of CO 2 . The CO concentration in porewater and the CO flux at the sediment-water interface Variations in CO 2 concentration of overlying water followed a trend similar to the changes in p CO 2 in surface water (Fig. 3 a). The porewater CO₂ concentration in all groups exhibited a pronounced increasing trend, and stocking mussels at higher densities further enhanced this trend (Fig. 3 b and 3 c). By Day 60, porewater CO 2 concentrations in the M3 and M4 groups were significantly higher than those in the control and M1 groups ( P < 0.05). Notably, this upward trend continued even after the mussel were removed. Changes in CO 2 flux at the sediment-water interface ( F CO 2−sw ) closely followed the variations in porewater CO 2 concentration (Fig. 3 d). The F CO 2−sw increased throughout the experiment period in all groups, with higher mussel stocking densities leading to greater flux enhancement. Contributions of various ecological processes to the changes in carbonate parameters The contributions of various ecological processes to the changes in carbonate parameters, and their proportions in the total absolute contribution are shown in Fig. 4 and Supplementary Fig. 4. During the experiment, NPP dominated TA changes in the control group, accounting for 70.26% of the total absolute contribution and resulting in a cumulative increase of 39.03 µmol kg − ¹. In the mussel-stocked groups, calcification became the dominant process, accounting for 42.03–76.12% of the total absolute contribution. After mussel harvest, NPP became the primary contributor to TA change in all mussel-stocked groups (53.07–68.81%). As the main driver of DIC changes, NPP accounted for 48.04% of the variation in the control group throughout the experiment. In M1-M3 groups, NPP was still the largest contributor during the culture stage (41.15–47.74%), cumulatively consuming 217.92-636.86 µmol kg − ¹ of DIC, which was higher than the amount released by respiration (63.68–248.10 µmol kg − ¹). This pattern shifted in the high-density group, where bivalve respiration became the dominant process (37.62%). Moreover, F CO 2‑aw (27.80%) and calcification (14.60%) replaced NPP as important DIC‑consuming processes in this group. After mussel harvest, NPP (34.11–59.97%) and F CO 2−aw (10.09–40.90%) jointly governed DIC changes in all mussel-stocking groups. Notably, F CO 2−aw contributed negatively in M4 group but positively in the other groups. During the culture stage, NPP was the primary driver in the control group (41.66%), reducing p CO 2 by 257.26 µatm. Its role was enhanced in M1-M3 groups, where the p CO 2 reductions (372.76 to 957.95 µatm) were substantial enough to offset the concurrent p CO 2 increases from respiration and calcification (312.93 to 888.49 µatm). In contrast, bivalve respiration dominated (39.76%) in M4 group, cumulatively increasing p CO 2 by 1984.98 µatm during the culture stage, far exceeding the NPP effect (367.68 µatm). After mussel harvest, NPP (27.27–51.48%) and F CO 2−aw (12.01–29.83%) became the main drivers of p CO 2 change in all mussel-stocking groups. However, their combined cumulative contribution to p CO 2 was − 302.06 µatm in M4 group, but only − 2.01 µatm and 2.34 µatm in M2 and M3 groups, respectively. Discussion Effect of mussel stocking density on p CO 2 in surface water The p CO 2 in the water increased significantly after incubation (Fig. 1 c), and more than 65% of the CO 2 was contributed by mussel respiration (Fig. 1 d). Similar conclusions were also drawn from previous studies in small-volume incubation chambers with relatively high bivalve biomass 26 , 27 . Therefore, at the individual level, the metabolic activity of bivalves is undoubtedly a source of atmospheric CO 2 . At the ecosystem level, the long-term mesocosm experiment in the present study showed that the decisive effect of stocking density on CO 2 source-sink functions of bivalve culture systems. The systems with the highest mussel stocking density (100.0 g m − 2 ) exhibited significantly elevated p CO 2 compared to the no-mussel control and other stocking densities (Fig. 2 d). This may indicate that mussel respiration dominated and established it as a net CO 2 source (Fig. 2 e), as observed in incubation experiments. In contrast, systems with moderate stocking densities (25.0 g m − 2 and 50.0 g m − 2 ) had significantly lower p CO 2 than the control and functioned as a sink of atmospheric CO 2 during the late culture stage. This may suggest that ecological processes other than respiration play a more important role in moderate‑density mussel culture systems 28 . Bivalve culture in coastal seas is the dominant form of global bivalve production 29 and the stocking density varied greatly among areas (Table S1 ). A comparison with data from typical coastal farms worldwide reveals that most farms operate at stocking densities exceeding that of the M4 in the present study (55.56 g m − ³), particularly in small-volume lagoons (Fig. 5 ). This implies that, under static water conditions, these farms would likewise function as net CO 2 sources. However, coastal environments are characterized by frequent water renewal driven by tidal exchange, riverine input, and wind-induced mixing 30 – 32 . As a result, the effective water volume available to support the cultured bivalves often substantially exceeds the apparent volume of the farm area. When the water renewal is considered, the actual or adjusted stocking densities in the farms fall below that of the M3 group (27.78 g m − 3 ), which functioned as a CO 2 sink in our study. Therefore, from a holistic farm ecosystem perspective, bivalve culture in dynamic coastal areas is expected to serve as a net sink for atmospheric CO 2 . Effects of stocking density on ecological processes in mussel culture systems The carbonate system in bivalve culture ecosystems is regulated by multiple ecological processes 33 . In this study, the contributions of different processes to the change in water p CO 2 were quantified using mass budget models. Results showed that NPP (7.61–40.92%), respiration (20.56–39.76%), F CO 2‑aw (13.18–17.13%), temperature (4.07–10.86%) and calcification (2.36–7.85%) were influential processes or factors driving p CO 2 changes in mussel culture system (Supplementary Fig. 4l-o). However, the fundamental role of F CO 2‑aw is to equilibrate water p CO 2 with atmosphere 34 , meaning it does not alter the system’s inherent status as a CO 2 source or sink. As for temperature, its effect did not differ among systems due to minimal temperature variation. Consequently, NPP, respiration and calcification emerge as the key processes determining whether the system functions as a CO 2 source or sink. Respiration and calcification push the system toward a CO 2 source, increasing with higher bivalve biomass. In contrast, NPP promotes a CO 2 sink, and its magnitude tied to phytoplankton biomass. Filter‑feeding bivalves exert a dual regulatory effect on phytoplankton communities 35 . On the one hand, they suppress primary production through the direct consumption of phytoplankton (top‑down effect) 13 , 28 . On the other hand, nutrient excretion by the bivalves promotes phytoplankton growth and primary production (bottom‑up effect) 13 , 22 . In our study, when bivalves were stocked at high density (M4), “top-down effects” prevailed and primary productivity was suppressed. As a result, CO 2 uptake by NPP was lower than the CO 2 released by respiration and calcification, rendering the system a net source of atmospheric CO 2 . Conversely, at low and moderate stocking densities (M2 and M3), “bottom‑up effects” prevailed and the system acts as a CO 2 sink. In coastal bivalve farming waters, key processes exhibit significant spatial heterogeneity 36 . The core farming area resembles a high‑density system dominated by bivalve respiration and calcification, functioning as a local CO 2 source. In contrast, adjacent waters, although not directly influenced by bivalve’s filter-feeding, experience a “bottom-up effect” driven by nutrient release from the core farm area, turning these waters into a strong CO 2 sink (Chen et al., 2025). Although some field surveys reported higher p CO 2 within core farming areas compared to non-farmed surrounding areas 18 – 20 , when the actual stocking densities resulting from water renewal are taken into account, their bivalve culture systems are still a net sink for atmospheric CO 2 at a holistic ecosystem level (Fig. 5 ). Legacy effects of mussel culture on the carbonate system The TA in the mussel-stocked systems exhibited a marked decline during the culture stage (Fig. 2 b). Calcification was the main driver of this decline, accounting for 42.03–76.12% of the total absolute contribution (Supplementary Fig. 4b-e). Following mussel harvest, NPP replaced calcification as the dominant process (53.07–68.81%). Since the effect of NPP on TA is relatively weak (Yin et al., 2024), water TA remained stable during the post-harvest period (Fig. 2 b). During the culture stage, DIC decreased in all mussel-stocked systems (Fig. 2 c). However, the dominant processes responsible for the DIC decrease varied with stocking density. In the low- and moderate-density systems (M1-M3), phytoplankton growth was promoted by "bottom-up effects", NPP removed more DIC than bivalve respiration released (Fig. 4 g-i), resulting in a net decrease in DIC. In the high-density system (M4), primary productivity was suppressed by the “top-down effect” and NPP was no longer the dominant process. Instead, the F CO 2−aw and calcification became the primary drivers of the DIC decrease (Fig. 4 j). After mussel harvest, the dynamics of DIC diverged. In the low- and moderate-density systems, the absorption of CO 2 from atmosphere largely offset DIC consumption by photosynthesis, leading to only minor net changes in DIC (Fig. 2 c). In contrast, the release of CO 2 to atmosphere and uptake of CO 2 by photosynthesis resulted in a continuous decrease in DIC in the high‑density system. As mentioned above, stocking mussels at moderate densities (M2 and M3) reduces surface water p CO 2 , while high densities (M4) lead to an increase. After mussel removal, the trend of change in p CO 2 also varies depending on the initial stocking density. Specifically, an initial decline of p CO 2 was observed in the moderate-density systems. However, the sharp decrease in NPP following the disappearance of "bottom-up effects" (Fig. 2 a), coupled with the decomposition of phytoplankton detritus (Lan et al., 2024), consequently drove a rebound in p CO 2 in the systems (Fig. 2 d). By contrast, in the high-density system, the cessation of mussel respiration and calcification, coupled with a gradual recovery in NPP as “top-down effects” disappeared, collectively drove a sustained decrease in p CO 2 . Overall, the harvest caused significant shifts in bivalve related ecological process, driving the p CO 2 in mussel-stocked systems converging toward the level of the non‑culture system. In summary, bivalve culture can significantly impact carbonate system in the water. After bivalve removal, p CO 2 will rapidly return to natural seawater levels, but the reductions in TA and DIC persist in the short term. In fact, this effect has already been observed in previous studies 19 , 37 . TA is the key factor in maintaining the buffering capacity of seawater 38 , and DIC serves as the primary form of marine carbon storage and transport, supporting multiple marine biogeochemical processes 39 , 40 . Therefore, to achieve sustainable bivalve aquaculture and fully realize its potential as a CO 2 sink, it is recommended to compensate for the loss of carbonate components by returning calcium carbonate shells to the aquaculture areas 41 , 42 . Furthermore, the systems after mussel removal simulated the water mass flowing out of the core farming area. Over the 20 days after harvesting, M1-M3 systems cumulatively absorbed 4.32, 6.54, and 7.56 mol of CO 2 , respectively, which were 1.4, 2.12, and 2.45 times that of the non-culture system. This confirms the strong CO 2 sink capacity of the water masses flowing out of the core culture area and further underscores the importance of adopting a holistic perspective when studying open or semi-open bivalve culture systems. Conclusion This study employed mesocosm experiments to assess the influence of filter-feeding mussels on the carbonate system and CO₂ flux in culture system. The results indicate that although the metabolic activity of bivalve releases CO 2 at the individual level, bivalve culture ecosystems at appropriate stocking densities (below 27.78 g m − 3 ) act as sinks for atmospheric CO 2 . In practice, the stocking densities in typical coastal bivalve farms that account for water exchange are lower than the reference density in this study, suggesting that these systems can be identified as CO 2 sinks. Further analysis using a mass-balance model reveals that net primary production, respiration, and calcification are the key ecological processes determining whether the system functions as a CO 2 source or sink. And the relative contributions of these processes are closely linked to stocking density: in high-density systems, shellfish respiration predominates (39.76%), whereas in medium- and low-density systems, net primary production is dominant (35.61–40.92%). In open or semi-open coastal bivalve farming areas, these three processes may exhibit significant spatial heterogeneity due to hydrodynamics. Post-harvest monitoring experiments further indicate that water masses flowing out of the core farming area have a strong capacity to absorb CO 2 . Therefore, it is necessary to adopt an integrated ecosystem approach to assess the potential role of bivalve aquaculture in global climate change. Furthermore, filter-feeding bivalve culture will significantly decrease the levels of TA and DIC in seawater, and this reduction does not recover in the short term even after the bivalve are removed. Therefore, returning shells to the farming waters to compensate for the loss of carbonate components can help support the sustainable development of bivalve aquaculture. Materials and methods Experimental mesocosms The experimental site was located in an aquaculture farm on the southern shore of Bohai Bay, China (Supplementary Fig. 1a; 37.0736° − 37.0747° N, 19.4819° − 19.4836° E). Fifteen mesocosms were established within a seawater pond measuring 100 m × 150 m. Each mesocosms covered an area of 100 m 2 (10 m × 10 m) and was constructed using wooden stakes and a waterproof high-density polyethylene tarpaulin (Supplementary Fig. 1b and 1c). The tarpaulin was buried 0.5 m into the sediment to isolate the mesocosm from the external pond water. A U-shaped tube, buried below the tarpaulin, was installed at the bottom of each mesocosm to maintain hydraulic equilibrium by allowing minimal water exchange. One end of the tube was equipped with a 180-µm mesh sieve to prevent seston from entering the mesocosms. Experimental design The experimental mussels ( Mytilus galloprovincialis ) were obtained from a mariculture farm in Shandong province, China. To minimize biological variability, individuals of uniform size and morphology and in good health were selected. Prior to the experiment, the mussels were acclimated for two weeks in the experimental pond. The experiment comprised five groups with three replicates: a control group without mussels (Ctl) and four groups with different mussel stocking densities (M1: 12.5 g m − 2 ; M2: 25.0 g m − 2 ; M3: 50.0 g m − 2 ; M4: 100.0 g m − 2 ). Before the experiment, seawater was pumped into the pond and each mesocosm via the U-shaped tube until the water depth reached 1.8 m. At the beginning of the experiment, pre‑acclimated mussels were randomly distributed into net cages within the mesocosms at the designated densities. Throughout the experimental period, water exchange between the mesocosms and pond was minimal, with only occasional supplementation to offset evaporation and leakage. Fouling organisms (such as algae and barnacles) on the inside tarpaulin surfaces were cleaned every other day. Sample collection and analysis The experiment consisted of two stages over 80 days, from May 1 to July 20, 2024 (Supplementary Fig. 1d). In the first stage (culture stage), intensive sampling was carried out on Day 0, 1, 3, 6 and 10 to closely monitor changes in indicators after the introduction of mussels. From Day 10 to 60, sampling occurred at 10-day intervals. After Day 60, the experiment entered the second stage (removal stage). During this stage, all mussel cages were removed from the mesocosms to investigate the legacy effects of mussel culture on the carbonate system. Subsequent sampling was conducted on Day 61, 63, 66, 70, 75, and 80. All sampling activities were performed between 07:00 and 10:00 a.m. During each sampling event, subsurface water samples (0.2 m depth) were collected from all mesocosms using a 1.0 L plexiglass water sampler. The water was filtered through 0.7 µm Whatman GF/F membranes, which had been acid-washed and burned at 450°C for 4 hours before use. One aliquot of the filtrate was analyzed immediately on site for total alkalinity (TA), while another was preserved in borosilicate glass bottles by adding saturated HgCl 2 solution for subsequent dissolved inorganic carbon (DIC) analysis. TA was determined by acid-base titration method with an accuracy of ± 4 µmol kg − 1 , and DIC was analyzed using a TOC analyzer (multi N/C 2100, Jena, Germany) with an accuracy of ± 3 µmol kg − 1 . Environmental parameters were analyzed in situ during each sampling event. Salinity (± 0.1) and water temperature (± 0.2 ℃) were measured with a water quality analyzer (YSI-EC300A, Xylem Analytics, USA). pH (± 0.02) was determined using a portable multi-parameter pH meter (QH40d, HACH, USA). Meteorological data, including air temperature, atmospheric pressure and wind speed, were recorded using a portable weather station. At each sampling event, primary productivity was measured using the light-dark bottle method 43 , 44 from both the surface and bottom layers of each mesocosm. The Dissolved oxygen (DO) concentration in the initial bottles was measured immediately using a portable DO meter (Multi 3510IDS, WTW GmbH, Germany). The dark and light bottles were incubated for 24 hours, after which their DO concentration was determined. Net primary productivity (NPP, mgO 2 L − 1 d − 1 ) for each layer was calculated as the difference in DO between the light and initial bottles. The gross NPP of water column (gO 2 m − 2 d − 1 ) was estimated using the cumulative arithmetic mean method based on the NPP in each water layer 45 . Partial pressure of CO 2 ( p CO 2) in surface water was calculated using the CO2SYS program (v2.5, running in Excel 2024) based on measured temperature, salinity, TA and DIC 46 . To assess the accuracy of CO2SYS program outputs, measured pH and calculated pH were compared. As shown in Supplementary Fig. 3b, they are significantly correlated ( R 2 = 0.9734, P < 0.01), indicating that the p CO 2 values estimated in this study reliably represent the actual p CO 2 in surface water. The CO 2 flux at the air-water interface ( F CO 2−aw ) was estimated using the thin boundary layer model 47 , detailed calculation procedures are provided in the supplementary method 1. Dissolved CO 2 concentrations in the overlying water and porewater were determined using the headspace gas chromatography method 48 , 49 . The CO 2 flux at the sediment-water interface ( F CO 2−sw ) was estimated using Fick’s first law, which models molecular diffusion based on the concentration gradient between porewater and overlying water 50 , 51 . The complete methodology is provided in the supplementary method 2. Mussel incubation experiment To assess the metabolic activity rates of bivalves, mussels were sampled from the mesocosms at 10-day intervals. Six cleaned mussels were placed in each 5 L plexiglass incubation chambers filled with filtered seawater. Following a 15 minutes acclimation, the chambers were sealed and suspended within the mesocosms for a 3-hour incubation. Each incubation comprised three mussel-containing chambers and three mussel-free control chambers. Water temperature, salinity and DO were measured before and after incubation. Concurrent water samples were collected for the analysis of TA, DIC and total ammonia nitrogen (TAN). TAN was determined using the indophenol blue method 52 . The DO consumption rate (R O2 , µmol g − 1 h − 1 ), TAN excretion rate (R TAN , µmol g − 1 h − 1 ), calcification rate (R Cal , µmol g − 1 h − 1 ) and respiration rates of mussels are calculated (See Supplementary Method 3 for details). Quantify the contributions of various processes to changes in the carbonate parameters Based on the 1-D mass balance model 20 , 53 , the contributions of temperature, salinity, F CO 2−aw , F CO 2−sw , net primary productivity (NPP), and metabolic activities of mussels (calcification and respiration) to the changes in carbonate parameters (TA, DIC, and p CO 2 ) in surface waters were quantified for each 10-day interval (See Supplementary Method 4 for details). Data Analysis and Visualization After assessing normality with the Shapiro-Wilk test, differences in NPP, carbonate parameters, CO 2 concentrations and CO 2 fluxes were analyzed using one‑way ANOVA (for homogeneous variances) or Welch’s ANOVA (for heterogeneous variances), with Tukey’s HSD or Tamhane’s T2 for post hoc pairwise comparisons. All statistical analyses were performed in SPSS software (version 26.0), and figures were generated using GraphPad Prism (version 10.5) and Adobe Illustrator (version 2025). Declarations Competing interests The authors declare no competing interests. Author contributions Miao-Jun Pan contributed to experimental design, mesocosm construction, sampling, data analysis, and manuscript drafting and revision. Yu-Xi Zhao, Sheng-Jie Xu, Chang-Lin Li, Zhou Zhang and Shuan-Jie Tian assisted with mesocosm construction and sampling. Xiang-Li Tian, Yan-Gen Zhou and Yun-Wei Dong helped secure the experimental site and provided methodological advice. Li Li and Shuang-Lin Dong conceived and designed the study, and reviewed the manuscript. Acknowledgements The study was funded by the Natural Science Foundation of China (grant number: 32373105). We sincerely appreciate Shandong Dehe Aquaculture Co., Ltd., Weifang, China, for the support provided to the study. References Lehmann, N. & Bach, L. T. Global carbonate chemistry gradients reveal a negative feedback on ocean alkalinity enhancement. Nat. Geosci. 18, 232–238 (2025). Zhang, H., Cheong, K. L. & Tan, K. R. 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M. et al. Expanding ocean food production under climate change. Nature 605, 490–496 (2022). IPCC. Climate Change 2022 - Mitigation of Climate Change: Working Group III Contribution to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change . (Cambridge University Press, 2023). Wolf-Gladrow, D. A., Zeebe, R. E., Klaas, C., Körtzinger, A. & Dickson, A. G. Total alkalinity: The explicit conservative expression and its application to biogeochemical processes. Mar. Chem. 106, 287–300 (2007). Humphreys, M. P., Daniels, C. J., Wolf-Gladrow, D. A., Tyrrell, T. & Achterberg, E. P. On the influence of marine biogeochemical processes over CO 2 exchange between the atmosphere and ocean. Mar. Chem. 199, 1–11 (2018). Karpowicz, M. et al. Top-down and bottom-up control of phytoplankton communities by zebra mussels Dreissena polymorpha (Pallas, 1771). Sci. Total Environ. 877, 162899 (2023). Giles, H. & Pilditch, C. A. Effects of mussel ( Perna canaliculus ) biodeposit decomposition on benthic respiration and nutrient fluxes. Mar. Biol. 150, 261–271 (2006). Kim, S. H. et al. Aquaculture farming effect on benthic respiration and nutrient flux in semi-enclosed coastal waters of Korea. J. Mar. Sci. Eng. 9, 554 (2021). Weerathunga, V. et al. Temporal variability of air-water gas exchange of carbon dioxide in clam and fish aquaculture ponds. Sci. Total Environ. 917, 170090 (2024). Li, S. et al. Influencing mechanism of farming clams on the CO 2 flux from aquaculture ponds: Insights from ecosystem carbon metabolism. Agric. Ecosyst. Environ. 374, 109166 (2024). Han, T. T., Shi, R. J., Qi, Z. H., Huang, H. H. & Gong, X. Y. Impacts of large-scale aquaculture activities on the seawater carbonate system and air-sea CO 2 flux in a subtropical mariculture bay, southern China. Aquac. Environ. Interact. 13, 199–210 (2021). Li, J. Q. et al. Effect of large-scale kelp and bivalve farming on seawater carbonate system variations in the semi-enclosed Sanggou Bay. Sci. Total Environ. 753, 142065 (2021). Yang, B. et al. Massive shellfish farming might accelerate coastal acidification: A case study on carbonate system dynamics in a bay scallop ( Argopecten irradians ) farming area, North Yellow Sea. Sci. Total Environ. 798, 149214 (2021). Grizzle, R. E., Greene, J. K. & Coen, L. D. Seston removal by natural and constructed intertidal eastern oyster ( Crassostrea virginica ) reefs: a comparison with previous laboratory studies, and the value of in situ methods. Estuaries Coasts 31, 1208–1220 (2008). Dugdale, R. C., Wilkerson, F. P. & Parker, A. E. The effect of clam grazing on phytoplankton spring blooms in the low-salinity zone of the San Francisco Estuary: A modelling approach. Ecol. Model. 340, 1–16 (2016). McKindsey, C. W., Thetmeyer, H., Landry, T. & Silvert, W. Review of recent carrying capacity models for bivalve culture and recommendations for research and management. Aquaculture 261, 451–462 (2006). Lv, C. C. et al. Quantifying critical thresholds of submerged macrophyte coverage to buffer climate-amplified ammonium pulses and stabilize clear-water states. Environ. Sci. Technol. 59, 17070–17083 (2025). Chown, S. L. Marine food webs destabilized. Science 369, 770–771 (2020). Han, T. T. et al. Carbon dioxide fixation by the seaweed Gracilaria lemaneiformis in integrated multi-trophic aquaculture with the scallop Chlamys farreri in Sanggou Bay, China. Aquac. Int. 21, 1035–1043 (2013). Xie, Y. M. et al. Evaluating the attribution of bivalve-macroalgae polyculture as a carbon source or sink from an ecosystem perspective: A case study of kelp-oyster IMTA. Aquaculture 599, 742165 (2025). Filgueira, R., Guyondet, T., Comeau, L. A. & Tremblay, R. Bivalve aquaculture-environment interactions in the context of climate change. Glob. Change Biol. 22, 3901–3913 (2016). FAO. The State of World Fisheries and Aquaculture 2024 – Blue Transformation in action . (FAO, 2024). Maicu, F., De Pascalis, F., Ferrarin, C. & Umgiesser, G. Hydrodynamics of the Po River-Delta-Sea System. J. Geophys. Res.-Oceans 123, 6349–6372 (2018). Jiang, L., Soetaert, K. & Gerkema, T. Decomposing the intra-annual variability of flushing characteristics in a tidal bay along the North Sea. J. Sea Res. 155, 101821 (2019). Defne, Z. & Ganju, N. K. Quantifying the residence time and flushing characteristics of a shallow, back-barrier estuary: Application of hydrodynamic and particle tracking models. Estuaries Coasts 38, 1719–1734 (2015). Tomasetti, S. J., Doall, M. H., Hallinan, B. D., Kraemer, J. R. & Gobler, C. J. Oyster reefs' control of carbonate chemistry-Implications for oyster reef restoration in estuaries subject to coastal ocean acidification. Glob. Change Biol. 29, 6572–6590 (2023). McGillis, W. R. & Wanninkhof, R. Aqueous CO 2 gradients for air-sea flux estimates. Mar. Chem. 98, 100–108 (2006). Asmus, R. M. & Asmus, H. Mussel beds: limiting or promoting phytoplankton? J. Exp. Mar. Biol. Ecol. 148, 215–232 (1991). Chen, X. W. J. et al. Oyster farming acts as a marine carbon dioxide removal (mCDR) hotspot for climate change mitigation. Proc. Natl. Acad. Sci. U. S. A. 122, e2504004122 (2025). Li, J. Q. et al. Large-scale oyster farming accelerates the removal of dissolved inorganic carbon from seawater in Sanggou Bay. Mar. Environ. Res. 202, 106798 (2024). Middelburg, J. J., Soetaert, K. & Hagens, M. Ocean alkalinity, buffering and biogeochemical processes. Rev. Geophys. 58, e2019RG000681 (2020). Yin, H., Jin, L. & Hu, X. P. Interpreting biogeochemical processes through the relationship between total alkalinity and dissolved inorganic carbon: Theoretical basis and limitations. Limnol. Oceanogr. Meth. 22, 311–320 (2024). Falkowski, P. et al. The global carbon cycle: A test of our knowledge of earth as a system. Science 290, 291–296 (2000). Namikawa, Y. & Suzuki, M. Atmospheric CO 2 sequestration in seawater enhanced by molluscan shell powders. Environ. Sci. Technol. 58, 2404–2412 (2024). Wang, H. J., Teevan-Kamhawi, F. & Rebernik, O. Harnessing nature's buffer: Assessing the role of bivalve shells in coastal alkalinity regeneration. Limnol. Oceanogr. Lett. 10, 774–781 (2025). García-Martín, E. E., Serret, P. & Pérez-Lorenzo, M. Testing potential bias in marine plankton respiration rates by dark bottle incubations in the NW Iberian shelf: incubation time and bottle volume. Cont. Shelf Res. 31, 496–506 (2011). Ostrom, N. E., Carrick, H. J., Twiss, M. R. & Piwinski, L. Evaluation of primary production in Lake Erie by multiple proxies. Oecologia 144, 115–124 (2005). Wei, J., Ji, X. N. & Hu, W. Characteristics of phytoplankton productivity in three typical lake zones of Taihu, China. Sustainability-Basel 16, 2376 (2024). Pierrot, D., Wallace, D. & Lewis, E. MS Excel program developed for CO 2 system calculations . (Carbon dioxide information analysis center, 2006). Liss, P. S. & Slater, P. G. Flux of Gases across the Air-Sea Interface. Nature 247, 181–184 (1974). Yuan, D. N. et al. Nitrogen addition effect overrides warming effect on dissolved CO 2 and phytoplankton structure in shallow lakes. Water Res. 244, 120437 (2023). Donis, D. et al. Full-scale evaluation of methane production under oxic conditions in a mesotrophic lake. Nat. Commun. 8, 1661 (2017). D'Ambrosio, S. L. & Harrison, J. A. Measuring CH 4 fluxes from lake and reservoir sediments: Methodologies and needs. Front. Environ. Sci. 10, 850070 (2022). Sun, H. Y. et al. Drivers of spatial and seasonal variations of CO 2 and CH 4 fluxes at the sediment water interface in a shallow eutrophic lake. Water Res. 222, 118916 (2022). Ivančič, I. & Degobbis, D. An optimal manual procedure for ammonia analysis in natural waters by the indophenol blue method. Water Res. 18, 1143–1147 (1984). Xue, L. et al. Sea surface carbon dioxide at the Georgia time series site (2006–2007): Air-sea flux and controlling processes. Prog. Oceanogr. 140, 14–26 (2016). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMaterials.pdf Supplemental Material Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8677151","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":581997324,"identity":"5482c66d-4ddc-47bc-abee-93aaa45928d8","order_by":0,"name":"Shuang-Lin Dong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYDCCAyCiwAbC4SFei0Ea6VoOk6CF73jv4Zc/DM7LGRw/wPjgbRuDvDkhLZJnzqVZ8xjcNjY4k8BsOLeNwXBnAwEtBjdyzIwZDG4nbrjBwCbN28aQYHCACC2GPwzOgbSw/yZWi/EDHoMDYFuYidIieeaMGTOPQbKx5JnEZsk55yQMNxDSwne8x/jjjwo7Ob7jhw9+eFNmI0/QFiBgk4DQjA1AQoKweiBg/kCUslEwCkbBKBi5AAA0JkBXWsDoxQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0320-9136","institution":"Ocean University of China","correspondingAuthor":true,"prefix":"","firstName":"Shuang-Lin","middleName":"","lastName":"Dong","suffix":""},{"id":581997325,"identity":"04d48896-4004-4c85-a50c-578687eb16d7","order_by":1,"name":"Miao-Jun Pan","email":"","orcid":"","institution":"Ocean University of China","correspondingAuthor":false,"prefix":"","firstName":"Miao-Jun","middleName":"","lastName":"Pan","suffix":""},{"id":581997326,"identity":"89a9c687-efaf-4b17-9bd9-9b0c710c906e","order_by":2,"name":"Yu-Xi Zhao","email":"","orcid":"","institution":"Ocean University of China","correspondingAuthor":false,"prefix":"","firstName":"Yu-Xi","middleName":"","lastName":"Zhao","suffix":""},{"id":581997327,"identity":"27575217-b3e0-4538-8d83-bdcd35c48c68","order_by":3,"name":"Sheng-Jie Xu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sheng-Jie","middleName":"","lastName":"Xu","suffix":""},{"id":581997328,"identity":"b28c1f99-ab68-4f4d-b9bd-531e4f2a05be","order_by":4,"name":"Chang-Lin Li","email":"","orcid":"","institution":"Key Laboratory of Mariculture of Ministry of Education, Ocean University of China","correspondingAuthor":false,"prefix":"","firstName":"Chang-Lin","middleName":"","lastName":"Li","suffix":""},{"id":581997329,"identity":"210f9694-3e1d-4cda-92c3-eeccc6f7615c","order_by":5,"name":"Zhou Zhang","email":"","orcid":"","institution":"Key Laboratory of Mariculture of Ministry of Education, Ocean University of China","correspondingAuthor":false,"prefix":"","firstName":"Zhou","middleName":"","lastName":"Zhang","suffix":""},{"id":581997330,"identity":"92c03a92-1a33-44d9-8e1a-d6975e2f10cf","order_by":6,"name":"Shuang-Jie Tian","email":"","orcid":"https://orcid.org/0009-0005-9704-4118","institution":"Key Laboratory of Mariculture of Ministry of Education, Ocean University of China","correspondingAuthor":false,"prefix":"","firstName":"Shuang-Jie","middleName":"","lastName":"Tian","suffix":""},{"id":581997331,"identity":"6d0f4050-e35c-4e24-b351-b968bcaeea09","order_by":7,"name":"Xiang-Li Tian","email":"","orcid":"","institution":"Key Laboratory of Mariculture of Ministry of Education, Ocean University of China","correspondingAuthor":false,"prefix":"","firstName":"Xiang-Li","middleName":"","lastName":"Tian","suffix":""},{"id":581997332,"identity":"402ac88b-c04d-4330-9ed3-33f25b5a0420","order_by":8,"name":"Yan-Gen Zhou","email":"","orcid":"","institution":"Ocean University of China","correspondingAuthor":false,"prefix":"","firstName":"Yan-Gen","middleName":"","lastName":"Zhou","suffix":""},{"id":581997333,"identity":"b683a0d5-5366-48d3-a5ae-35408a0c8e89","order_by":9,"name":"Yun-wei Dong","email":"","orcid":"https://orcid.org/0000-0003-4550-2322","institution":"Ocean University of China","correspondingAuthor":false,"prefix":"","firstName":"Yun-wei","middleName":"","lastName":"Dong","suffix":""},{"id":581997334,"identity":"74f9c513-4666-4906-8ecc-cf92b7a0a81b","order_by":10,"name":"Li Li","email":"","orcid":"","institution":"Ocean University of China","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-01-23 09:01:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8677151/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8677151/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101513336,"identity":"d42919e7-0819-48cb-8952-ec0897a4cbea","added_by":"auto","created_at":"2026-01-30 15:31:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3107884,"visible":true,"origin":"","legend":"\u003cp\u003eThe metabolic activity rates of mussels and their effects on \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e in water. (a) The calcification rate (R\u003csub\u003ecal\u003c/sub\u003e) of mussels at different culture period. (b) The respiration rate (R\u003csub\u003eres\u003c/sub\u003e) of mussels at different culture period. (c) The partial pressure of CO₂ (\u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e) in the water before and after incubation. “*” represents significant differences between data from different groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). (d) Contribution of mussel metabolic activity to the change in \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8677151/v1/e7bc8cc564fe3b9dd14957ae.png"},{"id":101513338,"identity":"2236b628-f83a-463f-9d45-3d7ac9dc3492","added_by":"auto","created_at":"2026-01-30 15:31:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5902706,"visible":true,"origin":"","legend":"\u003cp\u003eNet primary productivity (NPP), carbonate parameters and CO\u003csub\u003e2\u003c/sub\u003e flux (\u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2-aw\u003c/sub\u003e) at the air-water interface of mussel culture systems during the experiment. (a) Variations in NPP of water column. (b) Variations in total alkalinity (TA) of the surface water. (c) Variations in dissolved inorganic carbon (DIC) of the surface water. (d) Variations in the partial pressure of CO\u003csub\u003e2\u003c/sub\u003e (\u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e) in the surface water. (e) Variations in CO\u003csub\u003e2\u003c/sub\u003e flux at the air-water interface (\u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2-aw\u003c/sub\u003e). The error bars represent the standard deviation. Different letters indicate significant differences between data from different groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8677151/v1/009283b8456262414d1ec283.png"},{"id":101752239,"identity":"779c385d-6800-4713-9fef-7df020947ba8","added_by":"auto","created_at":"2026-02-03 10:26:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4351432,"visible":true,"origin":"","legend":"\u003cp\u003eCO\u003csub\u003e2\u003c/sub\u003e concentration in the overlying water and porewater, and CO\u003csub\u003e2\u003c/sub\u003e flux at the sediment-water interface (\u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2-sw\u003c/sub\u003e) of mussel culture systems during the experiment. (a) Variations in CO\u003csub\u003e2\u003c/sub\u003e concentration in the\u0026nbsp;overlying water. (b) Variations in porewater CO\u003csub\u003e2\u003c/sub\u003e concentration within the 0-2 cm sediment layer. (c) Variations in porewater CO\u003csub\u003e2\u003c/sub\u003e concentration within the 2-4 cm sediment layer. (d) Variations in CO\u003csub\u003e2\u003c/sub\u003e flux at the sediment-water interface (\u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2-sw\u003c/sub\u003e). The error bars represent the standard deviation. Different letters indicate significant differences between data from different groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8677151/v1/a2ac81db7631a841e4f4b1bd.png"},{"id":101513339,"identity":"57df5f6b-22d8-453f-9bbe-7fd60dca4b10","added_by":"auto","created_at":"2026-01-30 15:31:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3095926,"visible":true,"origin":"","legend":"\u003cp\u003eThe contributions of various ecological processes to the changes in carbonate parameters for mussel culture systems during the experiment. (a-e) Contributions to TA changes in the surface water. (f-j) Contributions to dissolved inorganic carbon (DIC) changes in the surface water. (k-p) Contributions to changes in the partial pressure of CO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;(\u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e) in the surface water.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8677151/v1/5657deba0e66d78b481232dc.png"},{"id":101751795,"identity":"eeb99569-b536-4848-a147-afc6e6519520","added_by":"auto","created_at":"2026-02-03 10:23:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2060420,"visible":true,"origin":"","legend":"\u003cp\u003eStocking densities under static water conditions and the adjusted stocking densities under water-exchange conditions in coastal bivalve culture systems.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8677151/v1/d457ab216574facc6b196aee.png"},{"id":107487474,"identity":"a38d7bcf-633d-4d5d-9454-e9d6b9e80766","added_by":"auto","created_at":"2026-04-22 02:41:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18821791,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8677151/v1/e2d1c83f-d405-41c9-93ce-c5d5fbcb5fe7.pdf"},{"id":101513341,"identity":"db561cc3-1420-474c-b0b7-a75514295e4c","added_by":"auto","created_at":"2026-01-30 15:31:32","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":935501,"visible":true,"origin":"","legend":"Supplemental Material","description":"","filename":"SupplementaryMaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8677151/v1/d50c5bad1ae0d242131f2aaa.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Stocking density-driven shift of atmospheric carbon dioxide source-sink functions in mussel culture systems","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAddressing global warming and its associated environmental challenges requires not only reductions in greenhouse gas emissions but also natural and engineered approaches to capture and store atmospheric carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Filter‑feeding bivalve aquaculture is widely considered a promising strategy for carbon sequestration and climate mitigation, since carbon is removed from the water by harvesting stable calcium carbonate shells\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, this view remains highly controversial, as CO\u003csub\u003e2\u003c/sub\u003e is released into the water during calcification\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, leading to the exclusion of bivalve aquaculture from the carbon trading system\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. With global bivalve aquaculture production having exceeded 19\u0026nbsp;million tonnes\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, it is imperative to clarify whether it can simultaneously fulfill the dual roles of providing blue food and enhancing marine carbon dioxide removal\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt the individual level, respiration and calcification of bivalves are the main metabolic processes affecting CO\u003csub\u003e2\u003c/sub\u003e concentrations in water. Respiration releases CO\u003csub\u003e2\u003c/sub\u003e directly, whereas calcification consumes dissolved inorganic carbon (DIC) to form calcium carbonate shells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, the precipitation of CaCO\u003csub\u003e3\u003c/sub\u003e also consumes more total alkalinity (TA), which shifts the carbonate system equilibrium toward CO\u003csub\u003e2\u003c/sub\u003e release\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. When bivalves are introduced into an aquatic ecosystem, beside the calcification and respiration, they can impact aqueous CO\u003csub\u003e2\u003c/sub\u003e concentrations through multiple ecological processes, such as \u0026ldquo;top-down effects\u0026rdquo; from filter feeding, \u0026ldquo;bottom-up effects\u0026rdquo; from nutrient excretion, and biodeposition\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Therefore, the potential role of bivalve culture in global climate change should be assessed using an integrated ecosystem approach.\u003c/p\u003e \u003cp\u003eEcological processes in bivalve culture system related to CO\u003csub\u003e2\u003c/sub\u003e consumption or release are affected by stocking density or biomass. For example, hard clam ponds shifted from sinks of atmospheric CO\u003csub\u003e2\u003c/sub\u003e to sources in the mid to late stages, due to changes in photosynthesis and organic matter remineralization within the system as the clams grew\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Similarly, in polyculture systems, low stocking densities of razor clams can increase primary productivity and thereby enhancing the system\u0026rsquo;s CO\u003csub\u003e2\u003c/sub\u003e uptake, whereas high densities reduced its capacity to serve as a CO\u003csub\u003e2\u003c/sub\u003e sink\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Therefore, a comprehensive understanding of how stocking density influences the CO\u003csub\u003e2\u003c/sub\u003e concentrations and inorganic carbon system in water by altering ecological processes is crucial for evaluating the role of bivalve culture in the global carbon cycle.\u003c/p\u003e \u003cp\u003eSome field surveys have shown that bivalve culture will elevate partial pressure of CO\u003csub\u003e2\u003c/sub\u003e (\u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e) in seawater of the coastal farm areas, thereby reducing CO\u003csub\u003e2\u003c/sub\u003e uptake or even converting the farm areas into net CO\u003csub\u003e2\u003c/sub\u003e sources \u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, these areas are usually open or semi-open systems, bivalves consume phytoplankton supplied by upstream waters and exert an influence on the downstream waters due to hydrodynamics\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This connectivity means that previous studies, which focused only on local farm areas, provide an inadequate perspective\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Therefore, using rigorous methods to assess the CO\u003csub\u003e2\u003c/sub\u003e source-sink function of the complete bivalve culture system is important to comprehensively understand its holistic ecological effects.\u003c/p\u003e \u003cp\u003eMesocosms systems, with clear boundaries and controllable conditions, have become a reliable approach for investigating the structure and function of aquatic ecosystems \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. This study employed mesocosm systems to conduct manipulative experiments under different stocking densities (0-100 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) of filter-feeding mussels (\u003cem\u003eMytilus galloprovincialis\u003c/em\u003e). Combined with field sampling and mass balance models, the study aims to: (1) clarify the impact of stocking density on the CO\u003csub\u003e2\u003c/sub\u003e source-sink functions of bivalve culture ecosystems; (2) elucidate the mechanisms of CO\u003csub\u003e2\u003c/sub\u003e removal for bivalve culture ecosystems; (3) investigate the subsequent dynamics of the carbonate system in the waters following mussel removal.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMetabolism of mussels\u003c/h2\u003e \u003cp\u003eThe metabolic activity rates of the experimental mussels at different culture period and their effects on the carbonate system are shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Fig.\u0026nbsp;2. The rates of dissolved oxygen consumption (R\u003csub\u003eDO\u003c/sub\u003e), total ammonia nitrogen excretion (R\u003csub\u003eTAN\u003c/sub\u003e), calcification (R\u003csub\u003ecal\u003c/sub\u003e) and respiration (R\u003csub\u003eres\u003c/sub\u003e) in mussels generally showed an upward trend over time, and no significant differences were observed among different density groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). After incubation, partial pressure of CO\u003csub\u003e2\u003c/sub\u003e (\u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e) in the water increased significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), with calcification and respiration contributing increases of 22.42\u0026ndash;38.62 \u0026micro;atm (12.78\u0026ndash;23.27%) and 73.60\u0026ndash;220.00 \u0026micro;atm (65.65\u0026ndash;77.53%), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEnvironment parameters and net primary productivity\u003c/h3\u003e\n\u003cp\u003eThroughout the experimental period, water temperature and salinity increased from 20.3 ℃ to 29.7 ℃ and from 30.3 to 33.0, respectively (Supplementary Fig.\u0026nbsp;3a). At the beginning of the experiment, the net primary productivity (NPP) was negative due to artificial disturbance probably and did not vary significantly across all groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Following the introduction of mussels, all groups except for M4 exhibited a marked upward trend in NPP. By the end of the culture stage (Day 60), the moderate stocking density (M2 and M3) of mussels significantly promoted NPP compared to the control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the high density (M4) led to an inhibition. After mussel harvest, NPP in the M2 and M3 groups dropped sharply and returned to a level similar to the control group after 20 days.\u003c/p\u003e\n\u003ch3\u003eCarbonate parameters\u003c/h3\u003e\n\u003cp\u003eThroughout the culture stage, all groups except M4 showed an initial decline in pH, followed by a gradual rise (Supplementary Fig.\u0026nbsp;3c). In contrast, the M4 group exhibited a continuous decline. At Day 60, the high-density group showed a significantly lower pH than the control group, while the moderate-density groups were significantly higher. (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Following mussel harvest, pH slightly decreased in M2 and M3 groups but increased markedly in M4 groups. By Day 80, no significant difference was observed between M2, M3 and the control groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), whereas pH in M4 group remained significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eTotal alkalinity (TA) in the control group increased consistently over the culture period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In contrast, stocking mussels significantly reduced the water TA. By the end of the culture stage, TA in all mussel-stocked groups was significantly lower than in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the lowest value observed in the highest-density group. After mussel harvest, the water TA remained essentially unchanged.\u003c/p\u003e \u003cp\u003eAll groups exhibited declining trends of dissolved inorganic carbon (DIC), with mussel-stocked groups showing greater reductions than the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). By Day 60, DIC levels in all mussel-stocked groups were significantly lower than in control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After mussel harvest, DIC in M4 group continued to decline noticeably, resulting in significantly lower DIC compared to the other three mussel-stocked groups by Day 80 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePartial pressure of CO in surface water and CO flux at air-water interface\u003c/h3\u003e\n\u003cp\u003eDuring the first 10 day, the partial pressure of CO\u003csub\u003e2\u003c/sub\u003e (\u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e) in surface water exhibited an increasing trend across all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). From Day 10 to Day 60, \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e declined in all groups except M4, which continued to increase. By the end of the culture stage, the \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e was significantly elevated in the high-density group but reduced in the moderate-density groups compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Following mussel harvest, \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e levels converged across all groups, and no significant differences were observed by Day 80 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe temporal pattern of CO\u003csub\u003e2\u003c/sub\u003e flux at the air-water interface (\u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;aw\u003c/sub\u003e) corresponded closely with the changes in \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). After mussel harvest, the \u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;aw\u003c/sub\u003e of mussel-stocked groups gradually converged to the level of the non-mussel control group. In addition, during the removal stage, M1-M3 groups cumulatively absorbed 4.32, 6.54, and 7.56 mol of CO\u003csub\u003e2\u003c/sub\u003e, respectively, which were 1.4, 2.12, and 2.45 times that of the non-culture group, while M4 group released 6.76 mol of CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003eThe CO concentration in porewater and the CO flux at the sediment-water interface\u003c/h3\u003e\n\u003cp\u003eVariations in CO\u003csub\u003e2\u003c/sub\u003e concentration of overlying water followed a trend similar to the changes in \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e in surface water (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The porewater CO₂ concentration in all groups exhibited a pronounced increasing trend, and stocking mussels at higher densities further enhanced this trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). By Day 60, porewater CO\u003csub\u003e2\u003c/sub\u003e concentrations in the M3 and M4 groups were significantly higher than those in the control and M1 groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, this upward trend continued even after the mussel were removed.\u003c/p\u003e \u003cp\u003eChanges in CO\u003csub\u003e2\u003c/sub\u003e flux at the sediment-water interface (\u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;sw\u003c/sub\u003e) closely followed the variations in porewater CO\u003csub\u003e2\u003c/sub\u003e concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The \u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;sw\u003c/sub\u003e increased throughout the experiment period in all groups, with higher mussel stocking densities leading to greater flux enhancement.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eContributions of various ecological processes to the changes in carbonate parameters\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe contributions of various ecological processes to the changes in carbonate parameters, and their proportions in the total absolute contribution are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Fig.\u0026nbsp;4. During the experiment, NPP dominated TA changes in the control group, accounting for 70.26% of the total absolute contribution and resulting in a cumulative increase of 39.03 \u0026micro;mol kg\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;. In the mussel-stocked groups, calcification became the dominant process, accounting for 42.03\u0026ndash;76.12% of the total absolute contribution. After mussel harvest, NPP became the primary contributor to TA change in all mussel-stocked groups (53.07\u0026ndash;68.81%).\u003c/p\u003e \u003cp\u003eAs the main driver of DIC changes, NPP accounted for 48.04% of the variation in the control group throughout the experiment. In M1-M3 groups, NPP was still the largest contributor during the culture stage (41.15\u0026ndash;47.74%), cumulatively consuming 217.92-636.86 \u0026micro;mol kg\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1; of DIC, which was higher than the amount released by respiration (63.68\u0026ndash;248.10 \u0026micro;mol kg\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;). This pattern shifted in the high-density group, where bivalve respiration became the dominant process (37.62%). Moreover, \u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2‑aw\u003c/sub\u003e (27.80%) and calcification (14.60%) replaced NPP as important DIC‑consuming processes in this group. After mussel harvest, NPP (34.11\u0026ndash;59.97%) and \u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;aw\u003c/sub\u003e (10.09\u0026ndash;40.90%) jointly governed DIC changes in all mussel-stocking groups. Notably, \u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;aw\u003c/sub\u003e contributed negatively in M4 group but positively in the other groups.\u003c/p\u003e \u003cp\u003eDuring the culture stage, NPP was the primary driver in the control group (41.66%), reducing \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e by 257.26 \u0026micro;atm. Its role was enhanced in M1-M3 groups, where the \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e reductions (372.76 to 957.95 \u0026micro;atm) were substantial enough to offset the concurrent \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e increases from respiration and calcification (312.93 to 888.49 \u0026micro;atm). In contrast, bivalve respiration dominated (39.76%) in M4 group, cumulatively increasing \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e by 1984.98 \u0026micro;atm during the culture stage, far exceeding the NPP effect (367.68 \u0026micro;atm). After mussel harvest, NPP (27.27\u0026ndash;51.48%) and \u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;aw\u003c/sub\u003e (12.01\u0026ndash;29.83%) became the main drivers of \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e change in all mussel-stocking groups. However, their combined cumulative contribution to \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e was \u0026minus;\u0026thinsp;302.06 \u0026micro;atm in M4 group, but only\u0026thinsp;\u0026minus;\u0026thinsp;2.01 \u0026micro;atm and 2.34 \u0026micro;atm in M2 and M3 groups, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eEffect of mussel stocking density on\u003c/b\u003e \u003cb\u003ep\u003c/b\u003e\u003cb\u003eCO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003ein surface water\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e in the water increased significantly after incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), and more than 65% of the CO\u003csub\u003e2\u003c/sub\u003e was contributed by mussel respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Similar conclusions were also drawn from previous studies in small-volume incubation chambers with relatively high bivalve biomass\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Therefore, at the individual level, the metabolic activity of bivalves is undoubtedly a source of atmospheric CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eAt the ecosystem level, the long-term mesocosm experiment in the present study showed that the decisive effect of stocking density on CO\u003csub\u003e2\u003c/sub\u003e source-sink functions of bivalve culture systems. The systems with the highest mussel stocking density (100.0 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) exhibited significantly elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e compared to the no-mussel control and other stocking densities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). This may indicate that mussel respiration dominated and established it as a net CO\u003csub\u003e2\u003c/sub\u003e source (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), as observed in incubation experiments. In contrast, systems with moderate stocking densities (25.0 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and 50.0 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) had significantly lower \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e than the control and functioned as a sink of atmospheric CO\u003csub\u003e2\u003c/sub\u003e during the late culture stage. This may suggest that ecological processes other than respiration play a more important role in moderate‑density mussel culture systems\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBivalve culture in coastal seas is the dominant form of global bivalve production\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and the stocking density varied greatly among areas (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A comparison with data from typical coastal farms worldwide reveals that most farms operate at stocking densities exceeding that of the M4 in the present study (55.56 g m\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup3;), particularly in small-volume lagoons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This implies that, under static water conditions, these farms would likewise function as net CO\u003csub\u003e2\u003c/sub\u003e sources. However, coastal environments are characterized by frequent water renewal driven by tidal exchange, riverine input, and wind-induced mixing\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. As a result, the effective water volume available to support the cultured bivalves often substantially exceeds the apparent volume of the farm area. When the water renewal is considered, the actual or adjusted stocking densities in the farms fall below that of the M3 group (27.78 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e), which functioned as a CO\u003csub\u003e2\u003c/sub\u003e sink in our study. Therefore, from a holistic farm ecosystem perspective, bivalve culture in dynamic coastal areas is expected to serve as a net sink for atmospheric CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEffects of stocking density on ecological processes in mussel culture systems\u003c/h3\u003e\n\u003cp\u003eThe carbonate system in bivalve culture ecosystems is regulated by multiple ecological processes\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In this study, the contributions of different processes to the change in water \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e were quantified using mass budget models. Results showed that NPP (7.61\u0026ndash;40.92%), respiration (20.56\u0026ndash;39.76%), \u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2‑aw\u003c/sub\u003e (13.18\u0026ndash;17.13%), temperature (4.07\u0026ndash;10.86%) and calcification (2.36\u0026ndash;7.85%) were influential processes or factors driving \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e changes in mussel culture system (Supplementary Fig.\u0026nbsp;4l-o). However, the fundamental role of \u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2‑aw\u003c/sub\u003e is to equilibrate water \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e with atmosphere\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, meaning it does not alter the system\u0026rsquo;s inherent status as a CO\u003csub\u003e2\u003c/sub\u003e source or sink. As for temperature, its effect did not differ among systems due to minimal temperature variation. Consequently, NPP, respiration and calcification emerge as the key processes determining whether the system functions as a CO\u003csub\u003e2\u003c/sub\u003e source or sink. Respiration and calcification push the system toward a CO\u003csub\u003e2\u003c/sub\u003e source, increasing with higher bivalve biomass. In contrast, NPP promotes a CO\u003csub\u003e2\u003c/sub\u003e sink, and its magnitude tied to phytoplankton biomass.\u003c/p\u003e \u003cp\u003eFilter‑feeding bivalves exert a dual regulatory effect on phytoplankton communities\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. On the one hand, they suppress primary production through the direct consumption of phytoplankton (top‑down effect)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. On the other hand, nutrient excretion by the bivalves promotes phytoplankton growth and primary production (bottom‑up effect)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In our study, when bivalves were stocked at high density (M4), \u0026ldquo;top-down effects\u0026rdquo; prevailed and primary productivity was suppressed. As a result, CO\u003csub\u003e2\u003c/sub\u003e uptake by NPP was lower than the CO\u003csub\u003e2\u003c/sub\u003e released by respiration and calcification, rendering the system a net source of atmospheric CO\u003csub\u003e2\u003c/sub\u003e. Conversely, at low and moderate stocking densities (M2 and M3), \u0026ldquo;bottom‑up effects\u0026rdquo; prevailed and the system acts as a CO\u003csub\u003e2\u003c/sub\u003e sink.\u003c/p\u003e \u003cp\u003eIn coastal bivalve farming waters, key processes exhibit significant spatial heterogeneity\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The core farming area resembles a high‑density system dominated by bivalve respiration and calcification, functioning as a local CO\u003csub\u003e2\u003c/sub\u003e source. In contrast, adjacent waters, although not directly influenced by bivalve\u0026rsquo;s filter-feeding, experience a \u0026ldquo;bottom-up effect\u0026rdquo; driven by nutrient release from the core farm area, turning these waters into a strong CO\u003csub\u003e2\u003c/sub\u003e sink (Chen et al., 2025). Although some field surveys reported higher \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e within core farming areas compared to non-farmed surrounding areas\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, when the actual stocking densities resulting from water renewal are taken into account, their bivalve culture systems are still a net sink for atmospheric CO\u003csub\u003e2\u003c/sub\u003e at a holistic ecosystem level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLegacy effects of mussel culture on the carbonate system\u003c/h2\u003e \u003cp\u003eThe TA in the mussel-stocked systems exhibited a marked decline during the culture stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Calcification was the main driver of this decline, accounting for 42.03\u0026ndash;76.12% of the total absolute contribution (Supplementary Fig.\u0026nbsp;4b-e). Following mussel harvest, NPP replaced calcification as the dominant process (53.07\u0026ndash;68.81%). Since the effect of NPP on TA is relatively weak (Yin et al., 2024), water TA remained stable during the post-harvest period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eDuring the culture stage, DIC decreased in all mussel-stocked systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). However, the dominant processes responsible for the DIC decrease varied with stocking density. In the low- and moderate-density systems (M1-M3), phytoplankton growth was promoted by \"bottom-up effects\", NPP removed more DIC than bivalve respiration released (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-i), resulting in a net decrease in DIC. In the high-density system (M4), primary productivity was suppressed by the \u0026ldquo;top-down effect\u0026rdquo; and NPP was no longer the dominant process. Instead, the \u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;aw\u003c/sub\u003e and calcification became the primary drivers of the DIC decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). After mussel harvest, the dynamics of DIC diverged. In the low- and moderate-density systems, the absorption of CO\u003csub\u003e2\u003c/sub\u003e from atmosphere largely offset DIC consumption by photosynthesis, leading to only minor net changes in DIC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In contrast, the release of CO\u003csub\u003e2\u003c/sub\u003e to atmosphere and uptake of CO\u003csub\u003e2\u003c/sub\u003e by photosynthesis resulted in a continuous decrease in DIC in the high‑density system.\u003c/p\u003e \u003cp\u003eAs mentioned above, stocking mussels at moderate densities (M2 and M3) reduces surface water \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e, while high densities (M4) lead to an increase. After mussel removal, the trend of change in \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e also varies depending on the initial stocking density. Specifically, an initial decline of \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e was observed in the moderate-density systems. However, the sharp decrease in NPP following the disappearance of \"bottom-up effects\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), coupled with the decomposition of phytoplankton detritus (Lan et al., 2024), consequently drove a rebound in \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e in the systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). By contrast, in the high-density system, the cessation of mussel respiration and calcification, coupled with a gradual recovery in NPP as \u0026ldquo;top-down effects\u0026rdquo; disappeared, collectively drove a sustained decrease in \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e. Overall, the harvest caused significant shifts in bivalve related ecological process, driving the \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e in mussel-stocked systems converging toward the level of the non‑culture system.\u003c/p\u003e \u003cp\u003eIn summary, bivalve culture can significantly impact carbonate system in the water. After bivalve removal, \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e will rapidly return to natural seawater levels, but the reductions in TA and DIC persist in the short term. In fact, this effect has already been observed in previous studies\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. TA is the key factor in maintaining the buffering capacity of seawater\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and DIC serves as the primary form of marine carbon storage and transport, supporting multiple marine biogeochemical processes\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Therefore, to achieve sustainable bivalve aquaculture and fully realize its potential as a CO\u003csub\u003e2\u003c/sub\u003e sink, it is recommended to compensate for the loss of carbonate components by returning calcium carbonate shells to the aquaculture areas\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, the systems after mussel removal simulated the water mass flowing out of the core farming area. Over the 20 days after harvesting, M1-M3 systems cumulatively absorbed 4.32, 6.54, and 7.56 mol of CO\u003csub\u003e2\u003c/sub\u003e, respectively, which were 1.4, 2.12, and 2.45 times that of the non-culture system. This confirms the strong CO\u003csub\u003e2\u003c/sub\u003e sink capacity of the water masses flowing out of the core culture area and further underscores the importance of adopting a holistic perspective when studying open or semi-open bivalve culture systems.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study employed mesocosm experiments to assess the influence of filter-feeding mussels on the carbonate system and CO₂ flux in culture system. The results indicate that although the metabolic activity of bivalve releases CO\u003csub\u003e2\u003c/sub\u003e at the individual level, bivalve culture ecosystems at appropriate stocking densities (below 27.78 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) act as sinks for atmospheric CO\u003csub\u003e2\u003c/sub\u003e. In practice, the stocking densities in typical coastal bivalve farms that account for water exchange are lower than the reference density in this study, suggesting that these systems can be identified as CO\u003csub\u003e2\u003c/sub\u003e sinks.\u003c/p\u003e \u003cp\u003eFurther analysis using a mass-balance model reveals that net primary production, respiration, and calcification are the key ecological processes determining whether the system functions as a CO\u003csub\u003e2\u003c/sub\u003e source or sink. And the relative contributions of these processes are closely linked to stocking density: in high-density systems, shellfish respiration predominates (39.76%), whereas in medium- and low-density systems, net primary production is dominant (35.61\u0026ndash;40.92%). In open or semi-open coastal bivalve farming areas, these three processes may exhibit significant spatial heterogeneity due to hydrodynamics. Post-harvest monitoring experiments further indicate that water masses flowing out of the core farming area have a strong capacity to absorb CO\u003csub\u003e2\u003c/sub\u003e. Therefore, it is necessary to adopt an integrated ecosystem approach to assess the potential role of bivalve aquaculture in global climate change.\u003c/p\u003e \u003cp\u003eFurthermore, filter-feeding bivalve culture will significantly decrease the levels of TA and DIC in seawater, and this reduction does not recover in the short term even after the bivalve are removed. Therefore, returning shells to the farming waters to compensate for the loss of carbonate components can help support the sustainable development of bivalve aquaculture.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eExperimental mesocosms\u003c/h2\u003e \u003cp\u003eThe experimental site was located in an aquaculture farm on the southern shore of Bohai Bay, China (Supplementary Fig.\u0026nbsp;1a; 37.0736\u0026deg; \u0026minus;\u0026thinsp;37.0747\u0026deg; N, 19.4819\u0026deg; \u0026minus;\u0026thinsp;19.4836\u0026deg; E). Fifteen mesocosms were established within a seawater pond measuring 100 m \u0026times; 150 m. Each mesocosms covered an area of 100 m\u003csup\u003e2\u003c/sup\u003e (10 m \u0026times; 10 m) and was constructed using wooden stakes and a waterproof high-density polyethylene tarpaulin (Supplementary Fig.\u0026nbsp;1b and 1c). The tarpaulin was buried 0.5 m into the sediment to isolate the mesocosm from the external pond water. A U-shaped tube, buried below the tarpaulin, was installed at the bottom of each mesocosm to maintain hydraulic equilibrium by allowing minimal water exchange. One end of the tube was equipped with a 180-\u0026micro;m mesh sieve to prevent seston from entering the mesocosms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eThe experimental mussels (\u003cem\u003eMytilus galloprovincialis\u003c/em\u003e) were obtained from a mariculture farm in Shandong province, China. To minimize biological variability, individuals of uniform size and morphology and in good health were selected. Prior to the experiment, the mussels were acclimated for two weeks in the experimental pond. The experiment comprised five groups with three replicates: a control group without mussels (Ctl) and four groups with different mussel stocking densities (M1: 12.5 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e; M2: 25.0 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e; M3: 50.0 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e; M4: 100.0 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eBefore the experiment, seawater was pumped into the pond and each mesocosm via the U-shaped tube until the water depth reached 1.8 m. At the beginning of the experiment, pre‑acclimated mussels were randomly distributed into net cages within the mesocosms at the designated densities. Throughout the experimental period, water exchange between the mesocosms and pond was minimal, with only occasional supplementation to offset evaporation and leakage. Fouling organisms (such as algae and barnacles) on the inside tarpaulin surfaces were cleaned every other day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSample collection and analysis\u003c/h2\u003e \u003cp\u003eThe experiment consisted of two stages over 80 days, from May 1 to July 20, 2024 (Supplementary Fig.\u0026nbsp;1d). In the first stage (culture stage), intensive sampling was carried out on Day 0, 1, 3, 6 and 10 to closely monitor changes in indicators after the introduction of mussels. From Day 10 to 60, sampling occurred at 10-day intervals. After Day 60, the experiment entered the second stage (removal stage). During this stage, all mussel cages were removed from the mesocosms to investigate the legacy effects of mussel culture on the carbonate system. Subsequent sampling was conducted on Day 61, 63, 66, 70, 75, and 80. All sampling activities were performed between 07:00 and 10:00 a.m.\u003c/p\u003e \u003cp\u003eDuring each sampling event, subsurface water samples (0.2 m depth) were collected from all mesocosms using a 1.0 L plexiglass water sampler. The water was filtered through 0.7 \u0026micro;m Whatman GF/F membranes, which had been acid-washed and burned at 450\u0026deg;C for 4 hours before use. One aliquot of the filtrate was analyzed immediately on site for total alkalinity (TA), while another was preserved in borosilicate glass bottles by adding saturated HgCl\u003csub\u003e2\u003c/sub\u003e solution for subsequent dissolved inorganic carbon (DIC) analysis. TA was determined by acid-base titration method with an accuracy of \u0026plusmn;\u0026thinsp;4 \u0026micro;mol kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and DIC was analyzed using a TOC analyzer (multi N/C 2100, Jena, Germany) with an accuracy of \u0026plusmn;\u0026thinsp;3 \u0026micro;mol kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEnvironmental parameters were analyzed in situ during each sampling event. Salinity (\u0026plusmn;\u0026thinsp;0.1) and water temperature (\u0026plusmn;\u0026thinsp;0.2 ℃) were measured with a water quality analyzer (YSI-EC300A, Xylem Analytics, USA). pH (\u0026plusmn;\u0026thinsp;0.02) was determined using a portable multi-parameter pH meter (QH40d, HACH, USA). Meteorological data, including air temperature, atmospheric pressure and wind speed, were recorded using a portable weather station.\u003c/p\u003e \u003cp\u003eAt each sampling event, primary productivity was measured using the light-dark bottle method\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e from both the surface and bottom layers of each mesocosm. The Dissolved oxygen (DO) concentration in the initial bottles was measured immediately using a portable DO meter (Multi 3510IDS, WTW GmbH, Germany). The dark and light bottles were incubated for 24 hours, after which their DO concentration was determined. Net primary productivity (NPP, mgO\u003csub\u003e2\u003c/sub\u003e L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for each layer was calculated as the difference in DO between the light and initial bottles. The gross NPP of water column (gO\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was estimated using the cumulative arithmetic mean method based on the NPP in each water layer\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePartial pressure of CO\u003csub\u003e2\u003c/sub\u003e (\u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2)\u003c/sub\u003e in surface water was calculated using the CO2SYS program (v2.5, running in Excel 2024) based on measured temperature, salinity, TA and DIC\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. To assess the accuracy of CO2SYS program outputs, measured pH and calculated pH were compared. As shown in Supplementary Fig.\u0026nbsp;3b, they are significantly correlated (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9734, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that the \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e values estimated in this study reliably represent the actual \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e in surface water. The CO\u003csub\u003e2\u003c/sub\u003e flux at the air-water interface (\u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;aw\u003c/sub\u003e) was estimated using the thin boundary layer model\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, detailed calculation procedures are provided in the supplementary method 1.\u003c/p\u003e \u003cp\u003eDissolved CO\u003csub\u003e2\u003c/sub\u003e concentrations in the overlying water and porewater were determined using the headspace gas chromatography method\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The CO\u003csub\u003e2\u003c/sub\u003e flux at the sediment-water interface (\u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;sw\u003c/sub\u003e) was estimated using Fick\u0026rsquo;s first law, which models molecular diffusion based on the concentration gradient between porewater and overlying water\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The complete methodology is provided in the supplementary method 2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMussel incubation experiment\u003c/h2\u003e \u003cp\u003eTo assess the metabolic activity rates of bivalves, mussels were sampled from the mesocosms at 10-day intervals. Six cleaned mussels were placed in each 5 L plexiglass incubation chambers filled with filtered seawater. Following a 15 minutes acclimation, the chambers were sealed and suspended within the mesocosms for a 3-hour incubation. Each incubation comprised three mussel-containing chambers and three mussel-free control chambers. Water temperature, salinity and DO were measured before and after incubation. Concurrent water samples were collected for the analysis of TA, DIC and total ammonia nitrogen (TAN). TAN was determined using the indophenol blue method\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The DO consumption rate (R\u003csub\u003eO2\u003c/sub\u003e, \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), TAN excretion rate (R\u003csub\u003eTAN\u003c/sub\u003e, \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), calcification rate (R\u003csub\u003eCal\u003c/sub\u003e, \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and respiration rates of mussels are calculated (See Supplementary Method 3 for details).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eQuantify the contributions of various processes to changes in the carbonate parameters\u003c/h2\u003e \u003cp\u003eBased on the 1-D mass balance model\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, the contributions of temperature, salinity, \u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;aw\u003c/sub\u003e, \u003cem\u003eF\u003c/em\u003eCO\u003csub\u003e2\u0026minus;sw\u003c/sub\u003e, net primary productivity (NPP), and metabolic activities of mussels (calcification and respiration) to the changes in carbonate parameters (TA, DIC, and \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e) in surface waters were quantified for each 10-day interval (See Supplementary Method 4 for details).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis and Visualization\u003c/h2\u003e \u003cp\u003eAfter assessing normality with the Shapiro-Wilk test, differences in NPP, carbonate parameters, CO\u003csub\u003e2\u003c/sub\u003e concentrations and CO\u003csub\u003e2\u003c/sub\u003e fluxes were analyzed using one‑way ANOVA (for homogeneous variances) or Welch\u0026rsquo;s ANOVA (for heterogeneous variances), with Tukey\u0026rsquo;s HSD or Tamhane\u0026rsquo;s T2 for post hoc pairwise comparisons. All statistical analyses were performed in SPSS software (version 26.0), and figures were generated using GraphPad Prism (version 10.5) and Adobe Illustrator (version 2025).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eMiao-Jun Pan contributed to experimental design, mesocosm construction, sampling, data analysis, and manuscript drafting and revision. Yu-Xi Zhao, Sheng-Jie Xu, Chang-Lin Li, Zhou Zhang and Shuan-Jie Tian assisted with mesocosm construction and sampling. Xiang-Li Tian, Yan-Gen Zhou and Yun-Wei Dong helped secure the experimental site and provided methodological advice. Li Li and Shuang-Lin Dong conceived and designed the study, and reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe study was funded by the Natural Science Foundation of China (grant number: 32373105). We sincerely appreciate Shandong Dehe Aquaculture Co., Ltd., Weifang, China, for the support provided to the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLehmann, N. \u0026amp; Bach, L. T. Global carbonate chemistry gradients reveal a negative feedback on ocean alkalinity enhancement. \u003cem\u003eNat. Geosci.\u003c/em\u003e 18, 232\u0026ndash;238 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, H., Cheong, K. L. \u0026amp; Tan, K. R. Bivalves as climate-friendly high quality animal protein: a comprehensive review. \u003cem\u003eFood Secur.\u003c/em\u003e 17, 739\u0026ndash;748 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSea, M. 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Oceanogr.\u003c/em\u003e 140, 14\u0026ndash;26 (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bivalve farming, Stocking density, Carbon sink, Carbonate system, CO2 fluxes, Mesocosms","lastPublishedDoi":"10.21203/rs.3.rs-8677151/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8677151/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBivalve aquaculture has long been excluded from blue carbon frameworks because bivalves release carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) into seawater during shell formation. However, this ignores the complex roles of bivalves in aquatic ecosystems. Through manipulative experiments with mesocosms, here we identify that CO\u003csub\u003e2\u003c/sub\u003e source-sink functions of mussel culture systems are stocking density-dependent. At a stocking density of 55.56 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, mussels suppressed phytoplankton biomass through filter feeding (top-down effect), making bivalve respiration the dominant process governing CO\u003csub\u003e2\u003c/sub\u003e dynamics and turning the system into a CO\u003csub\u003e2\u003c/sub\u003e source. When density fell below 27.78 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, the \u0026ldquo;bottom-up effect\u0026rdquo;from bivalve excretion prevailed, enabling net primary productivity to dominate (35.61\u0026ndash;40.92%) and turning the system into a CO\u003csub\u003e2\u003c/sub\u003e sink. The actual stocking densities in coastal bivalve farms considering water renewal are below 27.78 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, suggesting they can function as CO\u003csub\u003e2\u003c/sub\u003e sinks. Post-harvest monitoring further indicated that water masses flowing out from the core farming area can absorb more CO\u003csub\u003e2\u003c/sub\u003e than non-farming seawater. Therefore, this study recommends adopting an integrated, ecosystem-based approach to assess the role of bivalve aquaculture in global climate change.\u003c/p\u003e","manuscriptTitle":"Stocking density-driven shift of atmospheric carbon dioxide source-sink functions in mussel culture systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 15:31:27","doi":"10.21203/rs.3.rs-8677151/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"91a631fa-bcc5-4fb3-b1a6-aa75f721ec73","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61920963,"name":"Earth and environmental sciences/Environmental sciences/Environmental impact"},{"id":61920964,"name":"Earth and environmental sciences/Climate sciences/Climate change/Climate-change mitigation"},{"id":61920965,"name":"Biological sciences/Ecology/Ecosystem ecology"},{"id":61920966,"name":"Earth and environmental sciences/Biogeochemistry/Carbon cycle"}],"tags":[],"updatedAt":"2026-04-20T19:45:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-30 15:31:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8677151","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8677151","identity":"rs-8677151","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}