Greenhouse gas fluxes in mangrove forest soil in an Amazon estuary

: Tropical mangrove forests are important carbon sinks, the soil being the main 11 carbon reservoir. Understanding the variability and the key factors that control fluxes is 12 critical to accounting for greenhouse gas (GHG) emissions, particularly in the current 13 scenario of global climate change. This study is the first to quantify methane (CH 4 ) and 14 carbon dioxide (CO 2 ) emissions using a dynamic chamber in a natural mangrove soil of 15 the Amazon. The plots for the trace gases study were allocated at contrasting 16 topographic heights. The results showed that the mangrove soil of the Amazon estuary 17 is a source of CO 2 (6.66 g CO 2 m -2 d -1 ) and CH 4 (0.13 g CH 4 m -2 d -1 ) to the atmosphere. 18 The CO 2 flux was higher in the high topography (7.858 g CO 2 m -2 d -1 ) than in the low 19 topography (4.734 g CO 2 m -2 d -1 ) in the rainy season, and CH 4 was higher in the low 20 topography (0.128 g CH 4 m -2 d -1 ) than in the high topography (0.014 g CH 4 m -2 d -1 ) in 21 the dry season. However, in the dry period, the low topography soil produced more 22 CH 4 . Soil organic matter, carbon and nitrogen ratio (C/N), and redox potential 23 influenced the annual and seasonal variation of CO 2 emissions; however, they did not 24 affect CH 4 flux. The mangrove soil of the Amazon estuary produced 35.4 Mg CO 2-eq ha - 25 1 y -1 . A total of 2.16 kg CO 2 m -2 y -1 needs to be sequestered


Introduction 28
The mangrove areas are estimated to be the main contributors to greenhouse gas 29 emissions in marine ecosystems (Allen et al., 2011; Chen et al., 2012). However, 30 mangrove forests are highly productive due to a high nutrient turnover rate (Robertson 31 et al., 1992) and have mechanisms that maximize carbon gain and minimize water loss 32 through plant transpiration (Alongi and Mukhopadhyay, 2015). A study conducted in 25 33 mangrove forests (between 30° latitude and 73° longitude) revealed that these forests 34 should change the electron flow from sulfate-reducing bacteria to methanogenesis 48 (Purvaja et al., 2004), which also results in CH 4 formation. On the other hand, an 49 ecosystem with salinity levels above 18 ppt may show an absence of CH 4 emissions 50 (Poffenbarger et al., 2011), since methane dissolved in pores is typically oxidized 51 anaerobically by sulfate (Chuang et al., 2016). Currently the uncertainty in emitted CH 4 52 values in vegetated coastal wetlands is approximately 30% (EPA, 2017). Soil flux 53 measurements from tropical mangroves revealed emissions ranging from 0.3 to 4.4 mg 54 CH 4 m -2 d -1 (Castillo et al., 2017;Chen et al., 2014;Kreuzwieser et al., 2003). 55 The production of greenhouse gases from soils is mainly driven by biogeochemical 56 processes. Microbial activities and gas production are related to soil properties, 57 including total carbon and nitrogen concentrations, moisture, porosity, salinity, and 58 redox potential (Bouillon et al., 2008;Chen et al., 2012). Due to the dynamics of tidal 59 movements, mangrove soils may become saturated and present a reduced oxygen 60 availability, or suffer total aeration caused by the ebb tide. Studies attribute soil carbon 61 flux responses to moisture perturbations because of seasonality and flooding events 62 (Banerjee et al., 2016), with fluxes being dependent on tidal extremes (high tide and low 63 tide), and flood duration (Chowdhury et al., 2018). In addition, phenolic compounds 64 inhibit microbial activity and help keep organic carbon intact, thus leading to the 65 accumulation of organic matter in mangrove forest soils (Friesen et al., 2018).

Soil characteristics 259
Silt concentration was higher at the low topography (LSD: 14.763; p= 0.007) and clay 260 concentration was higher at the high topography plots (LSD: 12.463; p= 0.005), in both 261 seasons studied (Table 1). Soil particle size analysis did not differ statistically (p > 0.05) 262 between the two seasons (Table 1). Soil moisture did not vary significantly (p > 0.05) 263 between topographies at each season, or between seasonal periods at the same 264 topography (Table 1). The pH varied statistically (LSD: 5.950; p= 0.006) only at the 265 low topography when the two seasons were compared, being more acidic in the dry 266 period (Table 1). The pH values were significantly (LSD: 0.559; p= 0.008) higher in the 267 dry season (Table 1). No variation in Eh was identified between topographies and 268 seasons (Table 1), although it was higher in the dry season than in the rainy season. 269 However, Sal values were higher (LSD: 3.444; p = 0.010) at the high topography than at 270 the low topography in the dry season (Table 1). In addition, Sal was significantly higher 271 in the dry season than in the rainy season, in both high (LSD: 2.916; p < 0.001) and low 272 (LSD: 3.003; p < 0.001) topographies (Table 1). (Table 1). 273 The C mic did not differ between topographies in the two seasons (Table 2). However, T C 279 was significantly higher in the low topography in the dry season (LSD: 5.589; p < 280 0.000) and in the rainy season (LSD: 5.777; p = 0.024). In addition, C mic was higher in 281 the dry season in both the high (LSD: 11.325; p < 0.010) and low (LSD: 9.345; p < 282 0.000) topographies (Table 2). N mic did not vary between topographies seasonally. 283 However, N mic in the high (LSD: 9.059; p = 0.013) and low topographies (LSD: 4.447; 284 p = 0.001) was higher during the dry season ( Table 2). The C/N ratio (Table 2) was 285 higher in the low than in the high topography in both the dry (LSD: 3.142; p < 0.000) 286 and rainy seasons (LSD: 3.675; p = 0.033). However, only in the low topography was 287 the C/N ratio higher (LSD: 1.863; p < 0.000) in the dry season than in the rainy season 288 (Table 2). Soil OM was higher at the low topography in the rainy (LSD: 9.950; p = 289 0.024) and in the dry seasons (LSD: 9.630; p < 0.000). However, only in the lowland 290 topography was the OM concentration higher in the dry season than in the rainy season 291 (Table 2). 292 Table 2. Seasonal and topographic variation in microbial Carbon (C mic ; mg kg -1 ), microbial Nitrogen (N mic , mg kg -1 ), Total Carbon (T C ; g kg -1 ), 293 Total Nitrogen (N T ; g kg -1 ), Carbon/Nitrogen ratio (C/N) and Soil Organic Matter (OM; g kg -1 ). Numbers represent the mean (±standard error

Vegetation structure and biomass 297
Only the species R. mangle and A. germinans were found in the floristic survey carried 298 out. The DBH did not vary significantly between the topographies for either species 299 (Table 3). However, R. mangle had a higher DBH than A. germinaris at both high 300 (LSD: 139.304; p = 0.037) and low topographies (LSD: 131.307; p = 0.001). The basal 301 area (BA) and AGB did not show significant variation (Table 3). A total aboveground 302 biomass of 322.1 ± 49.6 Mg ha -1 was estimated. 303 304

Drivers of greenhouse gas fluxes 311
In the rainy season, CO 2 efflux was correlated with T air (Pearson = 0.23, p = 0.03), RH 312 (Pearson = -0.32, p < 0.00) and T s (Pearson = 0.21, p = 0.04) only at the low 313 topography. In the dry season CO 2 flux was correlated with T s (Pearson = 0.39, p < 314 0.00) at the low topography. The dry season was the period in which we found the 315 greatest amount of significant correlations between CO 2 efflux and soil chemical 316 parameters, while the C:N ratio, OM, and Eh were correlated with CO 2 efflux in both 317 seasons ( Table 4). The negative correlation between T C , N T , C/N, and OM, along with 318 the positive correlation of N mic with soil CO 2 flux, in the dry period, indicates that 319 microbial activity is a decisive factor for CO 2 efflux (Table 4). Soil moisture in the 320 Mojuim River mangrove forest negatively influenced CO 2 flux in both seasons (Table  321 4). However, soil moisture was not correlated with CH 4 flux. No significant correlations 322 were found between CH 4 efflux and the chemical properties of the soil in the mangrove 323 of the Mojuim River estuary (Table 4)  Total Carbon (T C ; g kg -1 ); Total Nitrogen (T N ; g kg -1 ); Microbial Carbon (Cmic, g kg -1 ); Microbial Nitrogen (N mic , g kg -1 ); Carbon and Nitrogen 331 ratio (C/N); Organic Matter (OM; g kg -1 ); Salinity (Sal; ppt); Redox Potential (Eh; mV); Soil Moisture (Moisture, %). 332 NS= not significant; * significant effects at p ≤ 0.05; ** significant effects at p ≤ 0.01 333 334 20 4 Discussion 335

Carbon dioxide and methane flux 336
It is important to consider that the year under study was rainier in the dry season (2017)  the CO 2 flux from the mangrove soil ranged from -5.06 to 68.96 g CO 2 m -2 d -1 (mean 342 6.66 g CO 2 m -2 d -1 ), while the CH 4 flux ranged from -5.07 to 11.08 g CH 4 m -2 d -1 (mean 343 0.13 g CH 4 m -2 d -1 ), resulting in a total carbon rate of 1.92 g C m -2 d -1 or 7.00 Mg C ha -1 344 y -1 (Figure 2). The negative CO 2 flux is apparently a consequence of the increased CO 2 345 solubility in tidal waters or of the increased sulfate reduction, as described in the other tropical mangrove areas (2.57 to 11.00 g CO 2 m -2 d -1 ; Shiau and Chiu, 2020). 353 However, the mean flux of 6.2 mmol CO 2 m -2 h -1 recorded in this Amazonian mangrove 354 was much higher than the mean efflux of 2.9 mmol CO 2 m -2 h -1 recorded in 75 355 mangroves during low tide periods (Alongi, 2009). 356 An emission of 0.010 Tg CH 4 y -1 , 0.64 g CH 4 m -2 d -1 (Rosentreter et al., 2018a), or 26.7 357 mg CH 4 m -2 h -1 has been reported for tropical latitudes (0 and 5°). In our study, the 358 monthly average of CH 4 flux was higher at the low (7.3 ± 8.0 mg CH 4 m -2 h -1 ) than at 359 the high topography (0.9 ± 0.6 mg C m -2 h -1 ), resulting in 0.13 g CH 4 m -2 d -1 or 0.48 Mg 360 CH 4 ha -1 y -1 (Figure 2). Therefore, the CH 4 -C fluxes from the mangrove soil in the 361 Mojuim River estuary were much lower than expected. It is known that there is a 362 microbial functional module for CH 4 production and consumption (Xu et al., 2015) and  Davidson et al., 2000;Ehrenfeld, 1995). In the two climatic periods of the year, the high 380 topography produced more CO 2 (7.869 ± 1.873 g CO 2 m -2 d -1 ) than the low topography 381 (5.212 ± 1.225 g CO 2 m -2 d -1 ) (Figure 2; SI 1). No significant influence on CO 2 flux was 382 observed due to the low variation in high tide level throughout the year (0.19 m) ( Figure  383 2), although it was numerically higher at the high topography. However, tidal height 384 and the rainy season resulted in a higher CO 2 flux (rate high/low =1.7) at the high 385 topography (7.858 ± 0.039 g CO 2 m -2 d -1 ) than at the low topography (4.734 ± 0.335 g 386 CO 2 m -2 d -1 ) (Figure 2; SI 1). This result is because the root systems of most flood-387 tolerant plants remain active when flooded (Angelov et al., 1996). Still, the high 388 topography has longer flood-free periods, which only happens when the tides are 389 syzygy or when the rains are torrential. 390 CO 2 efflux was higher in the high topography than in the low topography in the rainy 391 season (when soils are more subject to inundation), i.e., 39.8% lower in the forest soil 392 exposed to the atmosphere for less time. Measurements performed on 62 mangrove 393 forest soils showed an average flux of 2.87 mmol CO 2 m -2 h -1 when the soil was 394 our study showed a range of -0.01 to 31.88 mg C m -2 h -1 (mean of 4.70 ± 5.00 mg C m -2 414 h -1 ). The monthly CH 4 fluxes were generally higher at the low (0.232 ± 0.256) than at 415 the high (0.026 ± 0.018) topography, especially during the rainy season when the tides 416 were higher (Figure 2). Only in the dry season was there a significantly higher 417 production at the low than at the high topography (Figure 2; SI 1). The low topography 418 produced 0.0249 g C m -2 h -1 more to the atmosphere in the rainy season than in the dry 419 season (Figure 2), and a similar seasonal pattern was recorded in other studies 420 (Cameron et al., 2021). 421 The mangrove soil in the Mojuim River estuary is rich in silt and clay (Table 1) (Table 2). The finer 434 soil texture at the low topography (Table 1)  Amazonian mangroves, despite being much lower than that found in other Brazilian 452 mangroves. The estimated primary production for tropical mangrove forests is 218 ± 72 453 Tg C y (Bouillon et al., 2008). 454

Biogeochemical parameters 455
During the seasonal and annual periods, CH 4 efflux was not significantly correlated 456 with chemical parameters (Table 5), which is similar to the observed in another study 457 (Chen et al., 2010). Flooded soils present reduced gas diffusion rates, which directly 458 affects the physiological state and activity of microbes, by limiting the supply of the 459 dominant electron acceptors (e.g., oxygen), and gases (e.g., CH 4 ) (Blagodatsky and 460 Smith, 2012). The importance of soil moisture was evident in the richness and diversity 461 of bacterial communities in a study that compared the different pore spaces filled with 462 water (Banerjee et al., 2016). Furthermore, sulfate reduction in flooded soils (another 463 pathway of organic matter metabolism) is dependent on the redox potential of the soil. 464 However, no sulfate reduction occurs when the redox potential has values are above -465 24 150 mv (Connell and Patrick, 1968). In our study, Eh was above 36.0 mV indicating 466 that sulfate reduction probably did not influence the OM metabolism. 467 On the other hand, increasing soil moisture provides the microorganisms with essential 468 substrates such as ammonium, nitrate, and soluble organic carbon, and increases gas 469 diffusion rates in the water (Blagodatsky and Smith, 2012). Biologically available 470 nitrogen often limit marine productivity (Bertics et al., 2010), and thus can affect CO 2 471 fluxes to the atmosphere. However, a mangrove fertilization experiment showed that 472 CH 4 emission rates were not affected by N addition (Kreuzwieser et al., 2003). A higher 473 concentration of C mic and N mic in the dry period (Table 2), both in the high and low 474 topographies, indicated that microorganisms are more active when the soil spends more 475 time aerated in the dry period (Table 2) The high OM concentration at the two topographic heights (Table 2), at the two seasons 482 studied, and the respective negative correlation with CO 2 flux (Table 5)  The higher water salinity influenced by the tidal movement in the dry season (Table 1)  it to CO 2 (Coyne, 1999;Segarra et al., 2015), increasing the efflux of CO 2 and reduced 498 25 CH 4 (Megonigal and Schlesinger, 2002;Roslev and King, 1996). This may explain the 499 high CO 2 efflux found throughout the year at the high and, especially, at the low 500 topographies (Figure 3). 501 Studies in coastal ecosystems in Taiwan have reported that methanotrophic bacteria can 502 be sensitive to soil pH, and reported an optimal growth at pH ranging from 6.5 to 7.5 503 (Shiau et al., 2018). The higher soil acidity in the Mojuim River wetland (Table 1) (Table 1), hinder CH 4 emission. Soil Eh above -512 150 mV has been considered limiting for CH 4 production (Yang and Chang, 1998). 513 Increases in CH 4 efflux with reduced salinity were found as a consequence of intense 514 oxidation or reduced competition from the more energetically efficient SO 4 2and NO 3-515 reducing bacteria when compared to the methanogenic bacteria (Biswas et al., 2007). 516 This fact can be observed in the CH 4 efflux in the mangrove of the Mojuim River, 517 because there was an increased CH 4 production especially in the low topography in the 518 rainy season (Figure 3), when water salinity is reduced (Table 1) due to the increased  519 precipitation. However, we did not find a correlation between CH 4 efflux and salinity, 520 as previously reported (Purvaja and Ramesh, 2001) 521

Conclusions 522
The most recent estimate between latitude 0° to 23.5° S shows an emission of 2.3 g CO 2 523 m -2 d -1 (Rosentreter et al., 2018b). However, the efflux in the mangrove of the Mojuim 524 River estuary was 6.7 g CO 2 m -2 d -1 . For the same latitudinal range, Rosentreter et al. 525 (2018c) estimated an emission of 0.64 g CH 4 m -2 d -1 , and we found an efflux of 0.13 g 526 CH 4 m -2 d -1 . Seasonality was important for CH 4 efflux but did not influence CO 2 efflux. 527 The differences in fluxes may be an effect of global climate changes on the terrestrial 528 biogeochemistry at the plant-soil-atmosphere interface, as indicated by the deviation in 529 precipitation values from the climatology normal, making it necessary to extend this 530 study for more years. Using the factor of 23 to convert the global warming potential of 531 26 CH 4 to CO 2 (IPCC, 2001), the CO 2 equivalent emission was 35.4 Mg CO 2-eq ha -1 yr -1 . 532 Over a 100-year time period, a radiative forcing due to the continuous emission of 0.05 533 kg CH 4 m -2 y -1 found in this study, would be offset if CO 2 sequestration rates were 2.16 534 kg CO 2 m -2 y -1 (Neubauer and Megonigal, 2015). 535 Microtopography should be considered when determining the efflux of CO 2 and CH 4 in 536 mangrove forests in an Amazon estuary. The low topography in the mangrove forest of 537 Mojuim River had a higher concentration of organic carbon in the soil. However, it did 538 not produce a higher CO 2 efflux because it was negatively influenced by soil moisture, 539 which was indifferent to CH 4 efflux. MO, C/N ratio, and Eh were critical in soil 540 microbial activity, which resulted in a variation in CO 2 flux during the year and 541 seasonal periods. Thus, the physicochemical properties of the soil are important for CO 2 542 flux, especially in the rainy season. Still, they did not influence CH 4 fluxes. 543