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Effects of cropland conversion on OC pool in bulk soilCropland restoration identified as an efficient ecological project to promote soil C sequestration in karst erosion areas28,30. The conversion from MS to FG resulted in the total soil OC content and stock across 0–30 cm layers increasing by 46.12% and 43.73% respectively. The result was highly coincident with previous studies observed at 0–10 cm layer, which reported that FG cultivation replaced from MS cultivation could remarkably increase soil OC pool in karst region, Southwest China28. In our study, the lower OC content and stock in MS may be partially attributed to the non-returned crop residues and increased exposure of deep soil OM to oxygen under tillage disturbance, resulting in decreased soil OC accumulation through reducing the input of OM and accelerating OM decomposition28,30,37,38. Nevertheless, the conversion from MS to FG can increase the soil OC pool by increasing inputs from crops. For detail, laregly aboverground crops are harvested and removed from the fields each every year for economic production, there is thus a lack of aboverground OC input. Therefore, the root biomass became the main source of OM inputs, and even slight changes in biomass can substantially alter soil C level39. In the present study, the root biomass in FG field was approximately 6 times that in MS field (110.06 ± 17.24 kg hm−2 averagely) (Table S2). Consequently, the higher root biomass in FG are responsible for the corresponding higher C storage of fine root in FG, which is supported by the fact that higher amount of C were stored in the fine roots of FG field compared with that of MS field (Table S2). In fact, several studies have demonstrated that cultivation of perennial grasses is efficient in stimulating soil OC accumulation owing to its great amount of fine roots and underground biomass33,40. Soil disturbance (such as tillage) is one of the main causes of soil C depletion in agricultural systems, and increased tillage practice can result in greater soil C loss41,42,43. Therefore, the frequent tillage conducted in MS field resulted in lower levels of OC than that in FG field under minimal tillage disturbance.Impacts of cropland conversion on soil aggregates structure and stabilitySoil structure plays an important role in soil environment and quality, which is strongly characterized by soil aggregates and their stability43,44. In our study, soil macro-aggregates dominated the largest portion of total soil while meso-aggregates and micro-aggregates were only accounted for a small portion, indicating that cropland conversion could facilitated the formation of macro-aggregates (Table 2). These findings are in line with other studies, wherein that macro-aggregates occupied the major portion of total soil following farmland or vegetation restoration19,30. Tillage disturbance often disrupts aggregates by bringing subsurface soil to the surface, which can readily promote soil C turnover and hinder macro-aggregate formation45. Conversely, minimal tillage experienced and greater accumulation of root residues resulted in higher C accumulation in the FG field. Furthermore, fine roots improved the soil aggregate stability via the interaction with mycorrhizal fungi, which produced exudates and binding agents and promoted the formation of soil aggregates46,47. Therefore, higher inputs of root residue in the soil could enhance the capacity of aggregate re-formation. In fact, these can be supported by the higher value of root biomass and its C stock in the FG field. In addition, forage grass cultivation can enhance the formation of large and stable soil aggregates by fine roots and fungal hyphae through the production of exudates and binding agents, such as humic compounds, polymers and roots48,49. Thus, few tillage disturbance and higher inputs of root biomass in FG field resulted in soil aggregation enhanced, especially macro-aggregates.Soil aggregate stability can also be characterized by the values of MWD and GMD. Higher MWD or GMD values indicate greater aggregate stability due to more agglomerate ability. The value of MWD in the current study varied from 1.36 to 1.96, which was classified as “stable” by LeBissonnais’ categorization of aggregate stability50.Regardless of soil depth, the FG field had the greatest MWD and GMD values, indicating that its soil aggregates were more stable than those of the other three cropland use types. We may thus draw the conclusion that FG cropland conversion can improve the stability of aggregates based on MWD and GMD.Changes in OC stocks associated –aggregates following cropland conversionCropland use change generally affects soil C sequestration through changing OM inputs and decomposition19. Our study revealed that aggregate-associated OC was significantly higher in FG field than in MS field. These increases were mainly attributed to the new C derived from root residues inputs and decreased losses of OC associated-aggregate by C mineralization in FG soil49. Generally, tillage can breakdown large aggregates into small aggregates, and thus decrease the formation of soil macro-aggregates41,42. Thus, the lower OC content and stock associated-aggregate in MS field can be attributed to the OC loss resulting from soil erosion, and OM input reduction with tillage disturbance8,30,45.In this study, the effects of cropland conversion on OC content associated-aggregate fractions occurred in the top 20 cm soil layers. In the karst region, approximate 57–89% of crop roots are concentrated in the surface soil layer, which directly affects OM inputs from underground root residues51,52. Meanwhile, tillage practices also happened on top 20 cm soil layer6,28,29. As a result, in soils below 20 cm, little or no tillage disturbance and limited OM inputs resulted in fewer or no distinctly changing levels of OC content associated with aggregate following cropland use change.Cropland use change not only affected the OC stocks in bulk soil, but also affected the OC stocks associated-aggregates (Table 1). The difference of sensitivity of OC associated-aggregate to cropland use change may affect its contribution to bulk soil OC accumulation30,38. In our study, the macro-aggregate fraction was the most important contributor to total OC stock increase, followed by meso-aggregate and micro-aggregate (Fig. 4). This is primarily due to the higher amount and OC content of macro-aggregates. Overall all cropland use types, the OC stock associated with macro-aggregate in FG field was higher than that in other three cropland types regardless of soil depth (Fig. 4). For instance, OC stocks within macro-aggregate accounted for about 85.40%, 77.72% and 97.55% of total soil OC stock at 0–10 cm, 10–20 cm and 20–30 cm, respectively, under the conversion from MS to FG. Thus, the accumulation pattern of bulk soil OC stocks could closely related with changes of OC stocks associated with macro-aggregate under cropland use change.The physical protection of OC in aggregates is regarded as one of the main mechanisms for soil OC accumulation through diminishing soil OC degradation and preventing its interaction with mineral particles53,54. In the present study, OC stock in bulk soil correlated substantially with the OC content-associated aggregate following cropland conversion (Fig. 5). Further analysised revealed that OC stocks in bulk soil was significantly correlated to OC stock associated with macro-aggregate (R2 = 0.83, p  More

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Methods for data analysis in figuresAll analyses in figures were performed using Mathematica 12.3 (Wolfram Research, Inc., Champaign, IL, USA).Analysis in Fig. 1
C&D. To calculate integrated abundances of E. huxleyi cells and EhV, we first selected days for which all the bags had a non-null value. Values were then summed up to obtain the integrated abundance.E&J. We computed a standard linear fit between the E. huxleyi total abundances and total EhV abundances for covered and uncovered bags separately. We followed the same procedure for the correlations in panel J and provide a comparison between different models in Supplementary Fig. 5.Analysis in Fig. 2
A. The ASVs that were selected appeared at a relative abundance of at least 2% in at least 4 samples for the 0.2–2 µm 16S sequences and at least in 8 samples for the 2–20 µm 18S sequences. Abundances were concatenated for each time point and normalized by row, to have maximum relative abundance of 1 across all samples. ASVs were sorted by the position of their individual center of mass ({t}_{{CM}}) defined by$${t}_{{CM}}=,frac{mathop{sum}limits_{i}{t}_{i}f({t}_{i})}{mathop{sum}limits_{i}f({t}_{i})}$$
(1)
with i representing the different time points and f(({t}_{i})) the relative abundance of the ASV. The same figure for the individual bags in shown in Supplementary Fig. 14 and Supplementary Fig. 15.B. We selected 18S ASVs with a maximum relative abundance of at least 2% and observed in at least five samples. We averaged relative abundance across bags and then smoothed the time series with a moving average filter (width 2). Then, we grouped all ASVs into clusters based on their cosine distance using Mathematica’s FindClusters function and the KMeans method. The number of possible clusters ranged from 2 to 12, and the final number of clusters was decided using the silhouette method71. Only silhouette scores for 2 and 6 clusters were positive (between-cluster distance minus within-cluster distance).D. We subset reads that map to either Flavobacteriales or Fhodobacterales, then renormalized within each class, taking the mean over bags. Results per bag are shown in Supplementary Fig. 9.F. The turnover time was defined by the exponential rate k at which the Bray-Curtis similarity ({BC}(t)) declined over time. To this end, for a given bag, we computed the Bray–Curtis similarity between the composition vector at a starting day t’ with all following days t, giving a curve that declined roughly exponentially. For earlier starting days (for which the similarity curves declined the furthest), we found that the Bray–Curtis similarity never reached 0 but instead leveled out around ({{BC}}_{infty }=0.05) (due to ASVs that are constantly present in all the samples and maintain a minimal level of similarity between bags). Thus, we imposed an offset at(,{{BC}}_{infty }) for all fits (using Mathematica’s FindFit function) with the function:$${BC}(t)=(1-{{BC}}_{{{infty }}}) times {e}^{-kleft({t}^{{prime} }-tright)}+{{BC}}_{{{infty }}}$$
(2)
The turnover is averaged over bags, showing the standard deviation as error bars in the figure.G. To find differentially abundant ASVs, we first selected a subset of ASVs that had a maximum abundance of at least 10%, and performed Mann–Whitney U-Tests between the relative abundance values of a given ASV in the focal bag and all the other bags over all timepoints of the bloom’s demise. Correcting for multiple testing, we found four 16S ASVs that were differentially abundant in any of the bags, three of which were specific to bag 7, shown in Fig. 2g; and five 18S ASVs, two specific to bags 5 and 6 (Rhizosolenia delicatula and Aplanochytrium), one specific to bag 4 (Pterosperma), and two specific to bag 7 (MAST-1C and Woloszynskia halophila, shown in Fig. 2g).H. The divergence between bags was calculated as follows: we first measured, for each bag, the Bray–Curtis distance between this given bag and all the other bags at the end of the experiment (Supplementary Fig. 13). In order to control for the existing differences between bags at the beginning of the bloom, Bray–Curtis distances were normalized according to the differences between bags at the starting day of the E. huxleyi bloom. As the exact starting days of the bloom is not clear, we normalized for starting days 11, 12, or 13. The plot shows averages with the standard deviation as error bars. For the 18S microbiome, we first removed reads that map to E. huxleyi to reduce bias toward bag 7 (which had by far the lowest E. huxleyi abundance, Fig. 1c).Analysis in Fig. 3
A. Functional annotation of dominant 18S ASVs was based on manual literature search for the 100 most abundant 18S ASVs. Automatic annotation using the functional database created by72 gave qualitatively identical results but contained fewer organisms (covering about 50% of reads). The relative abundance of each trait was obtained by summing up the relative abundance of all the species harboring a specific trait. We used the annotations from72 to further subdivide heterotrophs into osmotrophs, saprotrophs, and other types of heterotrophy (e.g., grazing), ignoring ASVs with missing annotations.D. Growth rates were computed by fitting a linear model to the log-transformed absolute abundances. For thraustochytrids, we measured growth rates until the abundances reached their maximum, i.e., for days indicated by solid lines in Fig. 3b. For bacteria in the 0.2–2 micron fraction, we measured growth rates during the bloom and demise of E. huxleyi, i.e., for the time period after day 15 until the final day, except for bag 4 (until day 22) and bag 7 (until day 18) to account for their different bloom and demise dynamics. For bacteria in the 2–20 micron fraction, we measured growth rates similarly, starting after day 10 until the final day, except bags 4 and 7 (until day 22).E. To quantify the rate of change k of the biomass ratio of thraustochytrids to bacteria we fit a linear function to the log of biomass ratio from day 10 to the time point t where the ratio was maximal; for bag 7, this was day 18, for all others, day 23. We thus have:$$,{{log }},{BR},(t)={kt},+,{{log }},{BR},(0)$$
(3)
Analysis in Fig. 4
C&D. Since TEP accumulates over time, it cannot be expressed as a weighted sum of phytoplankton abundances. Instead, we formulate the model as a recursive relation where TEP can be produced by E. huxleyi, naked nanophytoplankton, and picophytoplankton, and degraded or lost through sinking:$${TEP}left(tright)=left(1-dright){TEP}left(t-1right)+{a}_{E}Eleft(tright)+{a}_{N}Nleft(tright)+{a}_{P}Pleft(tright),$$
(4)
The amount of TEP at time t is given by the fraction (1-d) of TEP at time t-1, where d corresponds to the fraction of TEP that is degraded between time points, plus the amount of TEP produced by the phytoplankton cells present at time t (or time t-1, which gives equivalent results). E, N, and P correspond to E. huxleyi, naked nanophytoplankton, and picophytoplankton, respectively. The parameter ({a}_{E}) corresponds to the amount of TEP produced per E. huxleyi cell, reported in panel D. ({a}_{E}) is set to be fixed through time, and different for each bag. This recursion can be solved to give an explicit expression for TEP(t):$${TEP}left(tright)=mathop{sum }limits_{{t}^{{prime} }=0}^{t}{left(1-dright)}^{t-{t}^{{prime} }}[{a}_{E}Eleft({t}^{{prime} }right)+{a}_{N}Nleft({t}^{{prime} }right)+{a}_{P}Pleft({t}^{{prime} }right)].$$
(5)
This functional form was then used to perform a linear model fitting with the constraint ({a}_{i}ge 0) for various values of the parameter d. The best fit, defined by maximum ({R}^{2}) over the resulting linear model, was used to fix d = 0.12. Our model considers that the fraction of non-calcified E. huxleyi cells in the nanophytoplankton counts is small.Larger phytoplankton cells ( >40 μm) filtered out from flow-cytometry measurements can also be a major source of TEP, despite low cell density. In order to verify this, FlowCam data was analyzed. None of the identified classes of larger phytoplankton (such as Phaeocystis or Dinobryon) increased in a systematic manner toward later stages of the bloom, explaining why larger phytoplankton were not included in the TEP model (Supplementary Fig. 24 and Supplementary Fig. 25).E. Using the smFISH method that reports the proportion of infected E. huxleyi cells, we estimated the amount of TEP produced from infected cells. We first used the least infected uncovered bags (bags 1 and 3) as a baseline to fix model parameters such as how much TEP does a non-infected cell produce. We then split the E. huxleyi abundance into an uninfected subpopulation producing T TEP/cell as in the uninfected bags, and an infected subpopulation producing I×T TEP/cells. To define I, we combined the fixed model parameters (i.e., amount of TEP produced per cell from Fig. 4d for bags 1 and 3) with the measured fraction of infected cells. We adjusted the factor I = 4 to minimize deviation of the measure total TEP concentration from the model prediction including the two subpopulations. The same procedure was used for panel H, using the corresponding model for PIC.F&G. To model the amount of PIC produced per cell we assume that the measured PIC only increases via new E. huxleyi coccoliths. The equivalent model for PIC reads$${PIC}left(tright)=left(1-dright){PIC}left(t-1right)+{a}_{E}{{max }}left(Eleft(tright)-Eleft(t-1right)right).$$
(6)
Where ({a}_{E}) is the amount of PIC produced per cell, and displayed in panel G. Using the same procedure as for TEP, we obtain the best fit for d = 0.0075. Our PIC model assumes that all PIC production comes from E. huxleyi, supported by large occurrence of E. huxleyi cells observed in scanning electron microscopy (Supplementary Fig. 1).Methods for data collectionMesocosm core setupThe mesocosm experiment AQUACOSM VIMS-Ehux was carried out for 24 days between 24th May (day 0) and 16th June (day 23) 2018 in Raunefjorden at the University of Bergen’s Marine Biological Station Espegrend, Norway (60°16′11 N; 5°13′07E). The experiment consisted of seven enclosure bags made of transparent polyethylene (11 m3, 4 m deep and 2 m wide, permeable to 90% photosynthetically active radiation) mounted on floating frames and moored to a raft in the middle of the fjord. The bags were filled with surrounding fjord water (day −1; pumped from 5 m depth) and continuously mixed by aeration (from day 0 onwards). Each bag was supplemented with nutrients at a nitrogen to phosphorus ratio of 16:1 according to the optimal Redfield Ratio (1.6 µM NaNO3 and 0.1 µM KH2PO4 final concentration) on days 0–5 and 14–17, whereas on days 6, 7 and 13 only nitrogen was added to limit the growth of pico-eukaryotes and favor the growth of E. huxleyi that is more resistant to phosphate limited conditions. Silica was not added as a nutrient source in order to suppress the growth of diatoms and to enhance E. huxleyi proliferation. Bags 5, 6, 7 were covered to collect aerosols and guarantee minimal contamination while sampling for core variables. Bags 1, 2, 3, 4 were sampled for additional assays such as metabolomics, polysaccharides profiling, and vesicles, which increase sampling time and potential for contamination.Measurement of dissolved inorganic nutrientsUnfiltered seawater aliquots (10 mL) were collected from each bag and the surrounding fjord water in 12 mL polypropylene tubes and stored frozen at −20 °C. Dissolved inorganic nutrients were measured with standard segmented flow analysis with colorimetric detection73, using a Bran & Luebe autoanalyser. Data are available in ref. 74 and values for individual bags are plotted in Supplementary Fig. 26.Measurement of water temperature and salinityWater temperature and salinity were measured in each bag and the surrounding fjord water using a SD204 CTD/STD (SAIV A/S, Laksevag, Norway). Data points were averaged for 1–3 m depth (descending only). When this depth was not available, the available data points were taken. Data are missing for the fjord in days 0–1. Outliers were removed for the following samples: bag 1 at days 0, 4, 15; bag 7 at day 15. Data are available in ref. 74.Flow cytometry measurementsSamples for flow cytometric counts were collected twice a day, in the morning (7:00 a.m.) and evening (8:00–9:00 p.m.) from each bag and the surrounding fjord, which served as an environmental reference. Water samples were collected in 50 mL centrifugal tubes from 1 m depth, pre-filtered using 40 µm cell strainers, and immediately analyzed with an Eclipse iCyt (Sony Biotechology, Champaign, IL, USA) flow cytometer. A total volume of 300 µL with a flow rate of 150 µL/min was analyzed with the machine’s software ec800 v1.3.7. A threshold was applied based on the forward scatter signal to reduce the background noise.Phytoplankton populations were identified by plotting the autofluorescence of chlorophyll versus phycoerythrin and side scatter: calcified E. huxleyi (high side scatter and high chlorophyll), Synechococcus (high phycoerythrin and low chlorophyll), nano- and picophytoplankton (high and low chlorophyll, respectively). Chlorophyll fluorescence was detected by FL4 (excitation (ex): 488 nm and emission (em): 663–737 nm). Phycoerythrin was detected by FL3 (ex: 488 nm and em: 570–620 nm). Raw.fcs files were extracted and analyzed in R using ‘flowCore’ and ‘ggcyto’ packages and all data are available on Dryad74. In particular, the gating strategy was adapted to each day and each bag and individual plots for each days and each bag can be found in the Dryad link.For bacteria and viral counts, 200 µL of sample were fixed with 4 µL of 20% glutaraldehyde (final concentration of 0.5%) for 1 h at 4 °C and flash frozen. They were thawed and stained with SYBR gold (Invitrogen) that was diluted 1:10,000 in Tris-EDTA buffer, incubated for 20 min at 80 °C and cooled to room temperature. Bacteria and viruses were counted and analyzed using a Cytoflex and identified based on the Violet SSC-A versus FITC-A by comparing to reference samples containing fixed bacteria and viruses from lab cultures. A total volume of 60 µL with a flow rate of 10 µL/min was analyzed. A threshold was applied based on the forward scatter signal to reduce the background noise. For plotting bacteria (Fig. 1h), a moving average of three successive days was used.Enumeration of extracellular EhV abundance by qPCRDNA extracts from filters from the core sampling (see above) were diluted 100 times, and 1 µL was then used for qPCR analysis. EhV abundance was determined by qPCR for the major capsid protein (mcp) gene: 5′-acgcaccctcaatgtatggaagg-3′ (mcp1F) and 5′-rtscrgccaactcagcagtcgt -3′ (mcp94Rv). All reactions were carried out in technical triplicates using water as a negative control. For all reactions, Platinum SYBER Green qPCR SuperMix-UDG with ROX (Invitrogen, Carlsbad, CA, USA) was used as described by the manufacturer. Reactions were performed on a QuantStudio 5 Real-Time PCR System equipped with the QuantStudio Design and Analysis Software version 1.5.1 (Applied Biosystems, Foster City, CA, USA) as follows: 50 °C for 2 min, 95 °C for 5 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. Results were calibrated against serial dilutions of EhV201 DNA at known concentrations, enabling exact enumeration of viruses. Samples showing multiple peaks in melting curve analysis or peaks that were not corresponding to the standard curves were omitted. Data are available in ref. 74. A comparison of viral counts based on flow-cytometry and qPCR is shown in Supplementary Fig. 2.FlowCam analysisSamples for automated flow imaging microcopy were collected once a day in the morning (7:00 a.m.) from each bag and the surrounding fjord, which served as an environmental reference. Water samples were collected in 50 mL centrifugal tubes from 1 m depth, kept at 12 °C in darkness, and analyzed within 2 h of sampling, using a FlowCAM II (Fluid Imaging Technologies Inc., Scarborough, ME, USA) fitted with a 300 µm path length flow cell and a 4× microscope objective. Images were collected using auto-image mode at a rate of 7 frames/second. A sample volume of 10 mL was processed at a flow rate of 0.7 mL/min. Individual objects within each sample were clustered and annotated using the Ecotaxa platform75. Absolute counts for major groups, including the most abundant ciliate category Ciliophora U04, were then exported and normalized by the individual amount of water volume processed for each sample.Data are available under “Flowcam Composite Aquacosm_2018_VIMS-Ehux” project on Ecotaxa.Scanning electron microscopy50 ml of water samples from bags or fjord were collected on polycarbonate filters (0.2 µm pore size, 47 mm diameter, Millipore). The filters were air dried and stored on petri-slides (Millipore) at room temperature. Prior to observation, a small fraction of the filter was cut and coated with 2 nm of iridium using a Safematic CCU-010 coater (Safematic GMBH, Switzerland). Samples were observed on a Zeiss Ultra SEM that was set at a working distance of 6.2 ± 0.1 mm, an acceleration voltage of 3.0 kV and an aperture size of 30 mm. The secondary electron detector was used for image acquisition.Paired dilution experimentPhytoplankton growth and microzooplankton grazing rates were estimated using the dilution method76,77. A slightly modified version of the method was used with only one low dilution level (20%) and an undiluted treatment used78. Rates calculated using this method are considered conservative but accurate when compared with those using multiple dilution levels and a linear regression. Water from bags 1–4 was collected using a peristaltic pump at ~1 m depth and mixed into a 20 L clean carboy. Water was screened through a 200 µm mesh to remove larger mesozooplankton. The collected water was shaded with black plastic and returned to shore. Dilution experiments were set-up in a temperature-controlled room, set to ambient water temperature (±2 °C). Particle-free diluent (FSW) was prepared by gravity filtering whole seawater (WSW) through a 0.45 µm inline filter (PALL Acropak™ Membrane capsule) into a clean carboy. To the FSW, WSW was gently siphoned at a proportion of 20%. The 20% dilution and 100% WSW treatments were prepared in single carboys and then siphoned into triplicate 1.2 L Nalgene™ incubation bottles. To control for nutrient limitation, additional triplicate bottles of 100% WSW were incubated without added nutrients (10 µM nitrate and 1 µM phosphate). The incubation bottles were incubated for 24 h in an outdoor tank maintained at in-situ water temperatures by a flow-through system of ambient seawater. Bottles could float freely, and the seawater inflow caused gentle agitation throughout the 24 h period. A screen was used to mimic light conditions experienced within the mesocosm bags.To quantify viral mortality, we used the paired dilution method79 which involves setting up an extra low dilution level (20%) containing water filtered through a tangential flow filter (TFF) of 100 kDå to remove viral particles. During this experiment, TFF water was produced 1–2 days prior to the dilution experiment, to ensure the chemical composition of the water was as similar as possible, and experiments could be set up in a timely manner.At T0 hours and T24 hours from all dilution experiments, sub-samples were taken for the determination of chlorophyll-a and flow cytometry. For chlorophyll-a, 100–150 mL of seawater was filtered under low vacuum pressure through a 47 mm Whatman GF/F filters (effective pore size 0.7 µm), and then extracted in 7 mL of 97% methanol at 4 °C in the dark for 12 h. All chlorophyll readings were conducted on a Turner TD700 fluorometer80. Methanol blanks were included, and all samples were corrected for phaeophytin using a drop of 10% hydrochloric acid and then reading the sample again81.Water samples (2 × 1 mL) for flow cytometry were taken at T0 and T24 of dilution experiments for the determination of phytoplankton abundances. Water samples were taken in triplicate from T0, and from each bottle at T24. Samples were immediately fixed in 20 µL of glutaraldehyde (final concentration More

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