Methane distribution , sources , and sinks in an aquaculture bay ( Sanggou Bay , China )

From 2012 to 2015, we investigated methane (CH4) distribution, air−sea fluxes, and sediment−water fluxes in an aquaculture bay (Sanggou Bay, China), and estimated the input of CH4 from potential land sources including rivers and groundwater. Surface water CH4 in the bay ranged from 3.0 to 302 nM, while bottom CH4 was usually higher due to sediment release. Water column CH4 in summer and autumn was 3 to 10 times that in spring and winter due to seasonal variation in water temperature and land source inputs. Surface CH4 was higher in kelp and scallop polyculture zones than in other culture zones and outside the bay, suggesting the influence of aquaculture activities. CH4 concentrations were 123 to 2190 nM in rivers around the bay, and 1.6 to 405 nM in groundwater along the shoreline; both showed great spatial and temporal variations. Sediment−water CH4 fluxes ranged from 0.73 to 8.26 μmol m−2 d−1, with those in bivalve culture zones higher than in polyculture zones. Sea−air CH4 fluxes ranged from 2.1 to 123.2 μmol m−2 d−1 (mean 48.2 μmol m−2 d−1) and showed seasonal variations. CH4 budget in Sanggou Bay showed that groundwater input (4.2 × 105 mol yr−1) was the largest source of CH4, followed by sediment release (2.6 × 105 mol yr−1) and riverine input (1.4 × 105 mol yr−1), while sea-to-air release (2.5 × 106 mol yr−1) and export from the bay to the Yellow Sea (8.8 × 105 mol yr−1) were the dominant CH4 sinks. Net water column production-oxidation was estimated preliminarily to produce 1.7 × 105 mol CH4 yr−1. However, there was a great imbalance of sources and sinks, with an apparent missing source of 2.4 × 106 mol yr−1 that was mostly due to an underestimate of in situ water column production and CH4 release from the sediments.


INTRODUCTION
Methane (CH 4 ), the most abundant hydrocarbon in the atmosphere, plays an important role in regulating the Earth's radiation balance and atmospheric chemistry in the troposphere (Cicerone & Oremland 1988, Lashof & Ahuja 1990).Although the current atmospheric mixing ratio of CH 4 (~1.8 ppm) is much less than that of CO 2 (~390 ppm), it is actually responsible for about 20% of the greenhouse effect (IPCC 2013).The atmospheric CH 4 mixing ratio has increased by a factor of 2.5, from 722 ppb in 1750 to 1803 ppb in 2011, as a result of human activities since the Industrial Revolution (IPCC 2013).
Oceans are a natural source of atmospheric CH 4 , and there are large spatial and temporal variations of oceanic CH 4 emissions.Typically, oligotrophic waters are only slightly supersaturated (by about 5%) in CH 4 with respect to atmospheric equilibrium (Bates et al. 1996, Bange et al. 1998, Karl et al. 2008), resulting in low sea-to-air CH 4 fluxes.High sea-to-air CH 4 emissions can occur in biologically productive regions such as estuaries and coastal and upwelling areas, which contribute to about 75% of the oceanic CH 4 emissions (Bange et al. 1994, EPA 2010).However, CH 4 emissions from coastal areas still have great uncertainties, due to poor coverage of CH 4 measurements and great spatial variations.Potential sources of CH 4 in coastal waters include riverine inputs, in situ water column production, and sediment release (Martens & Klump 1980, Kelley et al. 1990, Hornafius et al. 1999, Mau et al. 2007, Canet et al. 2010).It is generally assumed that CH 4 production and emission from coastal waters will be enhanced with the increase in human perturbations such as increased nutrient loading and intensive marine aquaculture.With the continuous decline in fishery harvests, aquaculture has become the world's fastest growing sector of food production, increasing nearly 60-fold during the last 5 decades, to meet the increasing demand for seafood (FAO 2007).However, the rapid increase in aquaculture production may also cause some environmental concerns, such as the release of greenhouse gases (Ferrón et al. 2007, Green et al. 2012).The discharge of effluent with high concentrations of organic matter and nutrients from coastal aquaculture systems to adjacent marine waters can lead to organic pollution and provide favorable conditions for the production of CH 4 .However, most studies on coastal methane emissions have focused on estuaries and coastal waters, and few studies have been conducted on coastal aquaculture systems.The aims of this study were to determine the temporal and spatial distributions of CH 4 in an intensive coastal aquaculture bay (Sanggou Bay) in China, to identify various CH 4 sources and sinks, and to evaluate CH 4 emissions from this bay and the possible impact of aquaculture activities.

Study area
Sanggou Bay (SGB) is a semi-circular bay on the north-eastern coast of China (Fig. 1).The bay is crescent-shaped, facing the Yellow Sea in the east, and has an average water depth of approximately 7.5 m and an area of approximately 144 km 2 (Zhang et al. 2009).Water renewal between the bay and the Yellow Sea is driven by a semi-diurnal tide with the largest tide range of 3.5 m.When the tide is rising, the tidal water enters the bay from the north, rotates counterclockwise, and flows out from the south through the west coast of the bay.The ebb tide has the reverse process, and the velocity of the residual flow is slow (Chinese Gulf Compilation Committee 1991).The main rivers emptying into the bay include the Sanggan, Ba, Shili, and Gu Rivers, with the total annual water discharge ranging between 1.7 × 10 8 and 2.3 × 10 8 m 3 yr −1 (Jiang et al. 2015).The largest river, i.e. the Gu, provides almost 70% of the total water discharge.
SGB has been used for aquaculture since the mid-1980s and is among the largest aquaculture sites in China (Guo et al. 1999, Zhang et al. 2009, Jiang et al. 2015).About 2/3 of the bay area is used for farming of bivalve shellfish, seaweed, and fish, with 4 major types of culture model, i.e. the monoculture of kelps, the monoculture of scallops, the monoculture of oysters, and the polyculture of kelps and bivalves (Fig. 1) (Shi et al. 2013).The main cultivation method is longline culture, and cultivated species include kelp Saccharina japonica, scallops Chlamys farreri, and oys- ters Crassostrea gigas (Zhang et al. 2009).Kelp is tied to ropes and scallops are contained either in lantern nets or by ear-hanging (Zeng et al. 2015).Macroalgae (i.e.kelp) are grown outside the bay and only between November and May (Fang et al. 1996).During the seeding and harvesting period, kelp competes with phytoplankton for the assimilation of dissolved inorganic nitrogen.The aquaculture of bivalve shellfishes occurs from early spring to November.Shellfishes filtrate and ingest particulate matter and digest phytoplankton and particulate organic matter (POM), especially oysters when they have spawned in August, demanding more energy and stored substances (Mao et al. 2006).Most fish culture is clustered together in the southern part of the bay where water is calm and cages are within easy access from the shore (Fig. 1).

Water sampling and analysis
Eight cruises were carried out in SGB during June and September 2012, April, July, and October 2013, January and May 2014, and May 2015.The sampling locations are shown in Fig. 2. Duplicate samples of surface and bottom seawater were collected using 10 l Niskin bottles, and then filled into 116 ml glass bottles.After overflow of approximately 1.5-to 2-fold of bottle volume, 1 ml of saturated solution of HgCl 2 was added to inhibit microbial activity.The sample bottle was then immediately sealed with a butyl rubber stopper and an aluminum cap and stored upside down in a dark box (Zhang et al. 2008).All water samples were analyzed after return to the shore laboratory within 60 d of collection (Zhang et al. 2004).Salinity and seawater temperature were measured with a multi-parameter probe (WTW 350i), and wind speeds were measured with an anemometer at about 10 m above the sea surface.
To evaluate the CH 4 input from potential terrestrial sources, water samples were collected from 4 rivers (Sanggan, Ba, Shili, and Gu) and 6 groundwater wells (GW1−GW6, Fig. 2) along the shoreline of SGB in June and September 2012, April, July, and October 2013, and January 2014.River water and groundwater were collected using a 5 l plastic sampler, and the samples were processed as for seawater described above.Dissolved CH 4 in seawater was measured using a gas-stripping method described by Zhang et al. (2004).After purging with high-purity N 2 , samples were passed through a drying tube with calcium chloride to remove water vapor.CH 4 was then separated on a 3 m × 3 mm i.d.stainless steel column packed with 80/100 mesh Porapak Q and measured with a gas chromatograph (Shimadzu, model GC-14B) equipped with a flame ionization detector (FID) (Zhang et al. 2004).FID responses were calibrated using known volumes of CH 4 standards (2.05, 4.22, and 50.4 ppmV; Research Institute of China National Standard Materials).The FID response signal and CH 4 concentration had a linear relationship, so a multi-point calibration method was used to determine CH 4 concentration based on chromatographic peak areas.The precision of this method was about 3% (Zhang et al. 2004).

Sediment sampling and incubation experiments
CH 4 emission from the sediments was measured by the closed chamber incubation method previously described by Sun et al. (2015), which was modified from Barnes & Owens (1999).Sediment samples were collected by a box corer at different sampling stations (Fig. 1), and only samples with undisturbed sediment surfaces were used.At each station, 15 sediment cores were collected using plexiglass tubes (i.d.= 5 cm, height = 30 cm) and sealed using air-tight rubber bungs.After ambient bottom water was added carefully with no gas headspace, the core was capped with a plexiglass top with 2 sampling ports.All cores were placed in a water-filled tank held at ambient room temperature, and the overlying water was stirred by magnetic stirrers rotated at 60 rpm.Ten glass bottles filled with ambient bottom water were placed in the same tank as water column controls.Cores and bottled waters were incubated in the dark for ~24 to 48 h.Overlying water samples (56.5 ml) from 3 cores were collected each time at intervals of 4 to 8 h to measure CH 4 concentration.At the same time, 2 bottled water samples were also treated with 0.5 ml HgCl 2 as a water column control.The CH 4 concentrations of all samples were measured by the gas-stripping method described above.Sediment− water CH 4 flux was estimated from the slope of the CH 4 increase in the overlying water versus time.The discrepancy in the CH 4 emission rate that resulted from differences between incubation and in situ temperatures was calibrated by the Arrhenius empirical equation as described by Aller et al. (1985) and Song et al. (2016).

Water incubation experiments
Time series incubation experiments were conducted to determine net CH 4 production-oxidation rates and understand the potential production mechanism in April, July, and October 2013 and January, May, and September 2014.To test for the effects of methylated compounds on CH 4 production, surface water samples were incubated with or without the addition of dimethylsulfoniopropionate (DMSP; final concentration 50 µM), or trimethylamine (final concentration 1 µM), or with added 2-bromoethane sulfonic acid (BES; final concentration 10 mM) to inhibit methanogenesis, and the CH 4 concentrations were monitored for more than 9 d in October 2013.Seawater for incubation experiments was transferred from the 10 l polyvinylchloride sampling bottles into clean polycarbonate carboys before the start of each experiment.DMSP, trimethylamine, or BES were added to the final concentration, and subsamples were transferred into 56.5 ml glass serum bottles that were capped with gas-tight Teflon-lined silicone stoppers and crimp-sealed with aluminum caps.Subsamples with no added reagents were used as controls.The incubations were conducted at approximately in situ water temperatures (3°C for January, 10°C for April, 15°C for May, 26°C for July, 23°C for September, 18°C for October) under an approximately 12:12 h light:dark cycle.Duplicate water samples were poisoned by addition of 0.5 ml of saturated HgCl 2 solution at 1 to 2 d intervals and analyzed for CH 4 as described above.Two additional samples for dissolved oxygen (DO) were collected and measured using the Winkler titration method (Bryan et al. 1976).Net CH 4 production-oxidation rates were estimated from the initial slope of the increase of CH 4 over time.

Saturation and flux calculation
The saturation (R, %) and sea-to-air fluxes of CH 4 (F, µmol m −2 d −1 ) were calculated using the following equations: R = C obs /C eq × 100% (1) where C obs is the observed concentration of dissolved CH 4 , and C eq is the air-equilibrated seawater CH 4 concentration calculated from the in situ temperature and salinity using the equation of Wiesenburg & Guinasso (1979) esrl.noaa.gov/gmd), were used for calculations.We found that the variation of assumed atmospheric CH 4 concentrations in the range of 1.85 to 1.95 ppmv make differences less than ± 2% in the computed air−sea CH 4 fluxes.Hence use of the annual mean atmospheric CH 4 concentration from monitoring networks for the sea−air flux calculation will not introduce significant errors.k w is the gas transfer coefficient in cm h −1 , which is a function of wind speed and Schmidt number (Sc).Various empirical equations were employed to estimate k, among which the equations from Liss & Merlivat (1986) and Wanninkhof (1992) were used most frequently and represent the estimation in a lower and higher level, respectively.Nightingale et al. (2000) proposed a gas exchange relationship that shows a dependence on wind speed, and the corresponding value lies near the median of extensive methods and models.Wanninkhof (2014) recently updated the most frequently used method of Wanninkhof (1992), and this update reflects advances that have occurred over the last 2 decades in quantifying the gas transfer coefficient.Hence the methods from Nightingale et al. (2000) and Wanninkhof (2014) (hereafter N2000 and W2014) were chosen to calculate air−sea fluxes in this study.

Water column CH 4 and other parameters
Table 1 shows the temperature, salinity, and CH 4 concentration in surface and bottom waters of SGB during the 8 cruises.Water temperature ranged from 3.1 to 23.2°C, with the extremes in January and September.Salinity varied slightly, from 29.3 to 31.6 psu, with the lowest in September.DO in the water column ranged from 4.6 to 12.3 mg l −1 with an average of 10. 2 ± 2.6 for June 2012, 6.3 ± 0.8 for September  2012, 9.8 ± 0.8 for April 2013, 9.1 ± 1.3 for July 2013,  10.5 ± 1.9 for January 2014, and 6.2 ± 0.4 mg l −1 for May 2015.DO in the surface water is usually comparable or slightly higher than at the bottom.Suspended particulate matter (SPM) in surface and bottom waters, respectively, was 18.6 ± 2.8 (mean ± SD) and 26.9 ± 13.9 mg l −1 for September 2012, 13.6 ± 9.1 and 14.9 ± 11.6 for April 2013, 11.3 ± 6.9 and 37.6 ± 26.8 for July 2013, 14.1 ± 5.7 and 27.6 ± 15.0 for October 2013, and 15.9 ± 14.1 and 13.5 ± 12.6 mg l −1 for January 2014.Obvious high bottom SPM was observed during the period from July to October.CH 4 concentrations in the water column ranged between 3.0 and 356 nM, and showed clear seasonal variation, with higher levels occurring in summer and autumn and lower levels in winter and early spring.CH 4 concentrations in autumn were comparable to those in summer and about 7-to 8-fold higher than those in winter and spring.Bottom CH 4 concentrations were higher than those at the surface during all cruises except in September 2012, during which surface CH 4 was 50% higher than in bottom water together with the lowest salinity among all cruises.

Geographical distributions of CH 4 in SGB
Fig. 3 shows the geographical distributions of temperature, salinity, and CH 4 in surface waters of SGB measured during this study.Two cruises were carried out in spring (April 2013 andMay 2015), during which the kelp thrived and the long kelp enhanced frictional effects in the upper layers and influenced the water exchange between the bay and the Yellow Sea (Zeng et al. 2015).Surface water temperature was higher in May than in April and presented a similar trend, which decreased gradually from nearshore to offshore, and showed an obvious gradient.In contrast, surface salinity increased from nearshore to offshore in May, while it decreased gradually from the northeast to the southwest in April.Dissolved CH 4 in decreased gradually from the southwest and the northeast to the central bay, with concentrations ranging between 6 and 8 nM.In May, CH 4 concentrations in the inner bay were higher than those in outer bay, with highest dissolved CH 4 occurring in the northern part of the bay.
During summer cruises (June 2012 and July 2013), kelps had already been harvested, and water exchange with the Yellow Sea was not influenced by suspended kelp.Water temperature had a similar trend as in spring, i.e. decreasing from nearshore to offshore.Surface salinity varied in a narrow range (30.4−31.0) in June and increased slightly from the coast to the center of the bay, while in July, salinity increased gradually from the inner to the outer bay with low salinity (< 30) in nearly half of the bay.CH 4 concentrations were much lower in June (mean ± SD: 38.3 ± 21.9 nM) than in July (53.0 ± 17.3 nM), and decreased gradually from the inner to the outer bay.In July, dissolved CH 4 concentrations increased from about 50 nM in the inner bay to > 70 nM at the mouth of the bay, then decreased to < 40 nM in the outer bay.
During autumn (September 2012 and October 2013), water temperature increased gradually from the inner to the outer bay, but the gradient was less pronounced.Salinity was relatively low (< 30) compared to other seasons due to heavy rainfall and freshwater input.In September, low salinity (< 29) together with high CH 4 (> 90 nM) was observed in the southern part of the bay.However, CH 4 concentrations presented an opposite trend in October, with the highest value (>140 nM) measured in the northeastern part of the bay, while salinity increased gradually from inner to outer bay.
During winter (January 2014), the Bohai South Coast Current enters the bay from the north and flows out from the south through the west coast of the bay (Sun et al. 2007).Surface seawater temperature decreased gradually from the outer to the inner bay, while surface salinity (30.2−30.6)showed little variation over the whole bay.Dissolved CH 4 concentrations in January were the lowest during the whole year, with highest CH 4 (8 nM) occurring near the center of the bay.It then decreased rapidly seaward to < 4 nM at the bay mouth.

Riverine and groundwater input
Rivers and groundwater are potential sources for dissolved CH 4 in SGB.CH 4 concentrations in the main rivers around the bay are shown in Fig. 4, which ranged from 123 to 2190 nM and were 1 to 2 orders of magnitude higher than those (3−356 nM) observed in the water column of the bay.Riverine CH 4 also presented obvious spatial and seasonal variations.For example, lowest CH 4 values usually occurred in winter, and the highest values occurred in summer and early fall for the smaller rivers (i.e.Sanggan, Shili, and Ba Rivers), while higher CH 4 values occurred in winter and summer, and lower CH 4 occurred in spring and late fall in the Gu River.
Considering that the runoff from the Sanggan, Shili, and Ba Rivers is limited and we lack discrete flow rate data for each river, we attributed 70% of the total runoff to the Gu River and 30% to the other rivers (Jiang et al. 2015).Riverine CH 4 flux to the SGB was estimated to be 1.4 × 10 5 mol yr −1 , using the average CH 4 concentrations in the Gu River (665 nM) and other small rivers (857 nM), and mean annual runoff (2.0 × 10 8 m 3 ).
Dissolved CH 4 concentrations in groundwater ranged from 1.6 to 405 nM and showed large spatial and temporal variations (Fig. 5).Groundwater near the mouth of the bay (GW4) had the highest CH 4 concentrations (26.2-255 nM, mean 98.6 nM), and Stn GW1 had medium values (6.7-109 nM, mean 40.7 nM), while those in the other areas usually had low CH 4 (<10 nM).For each region, lowest groundwater CH 4 usually occurred in winter (January) and early spring (April), while the highest values all occurred in late summer (September).In general, CH 4 concentrations in groundwater were much lower than those in rivers, but the submarine groundwater discharge to the SGB was ~50 times larger than river (bivalve culture zone) and 1.48 µmol m −2 d −1 (polyculture zone), and assuming that the area ratio of the 2 culture zones is 1:1, annual CH 4 emission from sediments of SGB was estimated to be about 2.6 × 10 5 mol.

Water column methane production-oxidation
Net water column CH 4 production-oxidation rates (CH 4 formation-CH 4 oxidation) were estimated to be 0.25, 0.41, 0.19, 0.39, and 0.31 nM d −1 for Stn MC in April and July 2013 and January, May, and September 2014, which showed significant seasonal variation and correlated well with temperature (R = 0.01T + 0.19, n = 5, r 2 = 0.70).Net water column CH 4 production-oxidation rates were 0.28 and 0.67 nM d −1 for Stns ST2 and SG-3 in September 2014.If we take the mean value of 0.42 nM d −1 at these stations as the net CH 4 production rate in the water column, together with the area of 144 km 2 and a mean water depth of 7.5 m, total net CH 4 productionoxidation in the water column was estimated to be 1.7 × 10 5 mol yr −1 .

Surface CH 4 saturation and sea-to-air fluxes
CH 4 saturation in the surface waters of SGB ranged from 202 to 2734% (Fig. 7), with great spatial and temporal variation.Average saturation was higher in autumn and summer than in spring and winter.In general, the surface waters of SGB were all over-saturated with CH 4 , except for a few stations during winter.Thus, SGB is a net source of atmospheric CH 4 .
Sea-to-air CH 4 fluxes calculated with the W2014 equation ranged from 2.1 to 123 µmol m −2 d −1 with a mean of 48.2 µmol m −2 d −1 , which was comparable to the results from the N2000 equation ( 2 m -2 d -1 with a mean of 50.3 µmol m −2 d −1 ; Fig. 8).CH 4 fluxes showed clear seasonal variation, with those in early autumn (September) comparable to those in summer (July and June), and more than 8-fold higher than those in spring (April) and autumn (October); the lowest values occurred in winter.In addition, we estimated the CH 4 emission from SGB to be 2.5 × 10 6 mol yr −1 based on the annual mean atmospheric CH 4 flux (48.2 µmol m −2 d −1 ) from this study and the area of SGB (144 km 2 ).

CH 4 exchange with the Yellow Sea
Considering that dissolved CH 4 in SGB was much higher than in the adjacent Yellow Sea, water exchange with the Yellow Sea should cause net loss of CH 4 from the bay.Aquaculture activities may influence water exchange between SGB and the Yellow Sea; however, suspended kelp culture mainly changed the spatial pattern of the tidal flux but not the tidal prism (Zeng et al. 2015).Jiang et al. (2015) estimated the annual water exchange volume to be about 8.3 × 10 10 m 3 .CH 4 concentrations gradients between the inner and outer bay were 3.6 nM for September 2012, 1.5, 11.9, and 40.7 nM for April, July, and October 2013, and 0.9 nM for January 2014, respectively.During the kelp seeding to harvesting period (November to May), the CH 4 concentration gradient (mean: 1.2 nM) was lower than during the non-aquaculture period (mean: 18.7 nM).Hence, based on the annual mean observed CH 4 concentration gradient between the inner and outer bay (10.0 nM), and the annual water exchange volume of the bay (8.3 × 10 10 m 3 ), we estimated the CH 4 flux exported out of the bay to be 8.8 × 10 5 mol yr −1 .
The estimated atmospheric CH 4 fluxes from the SGB in this study were close to those from Jiaozhou Bay (Zhang et al. 2007) and Dalian Bay (Wang et al. 2011), and slightly higher than those from some estuaries, e.g. the Changjiang estuary (Zhang et al. 2008).However, CH 4 fluxes from SGB were far higher than those from shelf areas, e.g.2-fold higher than those from the adjacent North Yellow Sea during Spring (April), and 5 times higher than during winter (January) and summer (July for SGB, August for North Yellow Sea), respectively, but comparable to those from the adjacent North Yellow Sea during October (Yang et al. 2010).Hence SGB is a hot spot of CH 4 emissions to the atmosphere.

Factors influencing spatial and temporal distribution of CH 4 in SGB
The concentration, saturation, water column production, and sediment−water fluxes of CH 4 in SGB all had obvious seasonal variation and were closely related to water temperature.Mean water column CH 4 concentrations and saturations during different cruises correlated positively with mean water temperature (CH 4 conc.= 2.33T − 4.70, r 2 = 0.93, n = 11, p < 0.0001; R(CH 4 ) = 124.8T− 380.4,r 2 = 0.8 n = 7, p < 0.008).This is consistent with the positive correlations observed for net water column CH 4 productionoxidation rates at Stn MC (R = 0.01T + 0.19, n = 5, r 2 = 0.82, p < 0.09) and sediment−water CH 4 fluxes at Stn ST1 (F = 0.039T + 0.66, n = 5, r 2 = 0.82, p < 0.09), which showed that CH 4 production rates in both water column and sediments increase with rising temperature.Temperature mainly controls the organic matter decomposition and the activity of methanogenesis, and the rising temperature may increase the relative abundance and diversity of methanogenic communities (Metje & Frenzel 2005, Høj et al. 2008).Yvon-Durocher et al. (2014) also reported seasonal variation in CH 4 emissions from diverse ecosystems using meta-analysis, and showed that CH 4 emissions increased significantly with seasonal increases in temperature.Hence our results suggest that water temperature plays a significant role in regulating the seasonal variation of CH 4 in SGB.
Although the salinity of SGB only showed a slight fluctuation throughout the year, mean water column CH 4 concentrations during different cruises correlated negatively with mean salinity (S) (CH 4 conc.= −0.33S+ 5.43, r 2 = 0.94, n = 11, p < 0.001), suggesting that terrestrial input (i.e.rivers and groundwater) also play a role in the seasonal variation of CH 4 in the bay.In general, rivers and groundwater are primary routes for delivery of dissolved and particulate carbon and nutrients from land to coastal areas, and they are usually supersaturated with CH 4 (Taniguchi et al. 2002, Striegl et al. 2012).Observed CH 4 (123.3 to 2189.7 nM) in rivers around SGB are within the CH 4 range (5−5000 nM) reported for rivers worldwide (de Angelis & Lilley 1987, Upstill- Goddard et al. 2000) and much higher than those in the water column of the bay.Especially during the wet seasons (summer and early autumn), high discharges of river water and groundwater with rich CH 4 enter the bay, affecting its spatial distribution.For example, observed surface CH 4 in the bay was 50% higher than that in bottom water in September 2012 together  Data are mean ± SD with the lowest salinity (Table 1), while CH 4 concentrations were usually higher at the bottom than at the surface during other cruises.CH 4 distribution in SGB may also be influenced by aquaculture activities.The bay is extensively used for culture of macroalgae and shellfish.Previous studies showed that CH 4 can be produced in anaerobic microenvironments of SPM and digestive tracts of zooplankton and fish in oxygenic surface water (Marty 1993, Karl &Tilbrook 1994).Obvious high bottom SPM was observed during the shellfish culture period from July to October.Dense populations of bivalve shellfish (i.e.scallop and oyster) in shallow water can produce a large amount of feces and pseudo-feces, hence the digestive tract of shellfish and their waste provide favorable environments for potential water column methanogenesis.Suspended shellfish culture also accelerates biodeposition and results in sediments with rich organic matter and high microbial activity (Green et al. 2012), which in turn enhances rates of anaerobic decomposition of organic matter and lead to high CH 4 production in the sediment (Nizzoli et al. 2006, Jiang et al. 2015).Due to the sediment release, bottom CH 4 concentrations in SGB were usually higher than those at the surface, especially in the culture zones with bivalve shellfish in summer (Fig. 9).For example, we observed CH 4 concentrations at Stn ST2 in the oyster culture zone to be 4.0 nM at the surface (0 m), 3.8 nM at 2 m, 5.9 nM at 4 m, and 12.0 nM at the bottom (6 m) in May 2014.We also observed significant high sediment−water CH 4 fluxes in the oyster culture zone (8.26 µmol m −2 d −1 ) compared to the polyculture zone (1.09 µmol m −2 d −1 ) in June 2012.Green et al. (2012) observed greater CH 4 emissions from the sediment in areas that had the highest cover of oysters compared to areas with medium cover.They attributed this to more CH 4 produced by the reduction of CO 2 and the stimulation by the 'priming effect,' whereby the addition of fresh labile organic matter (such as from oyster biodeposits) temporarily stimulates microbial decomposition, including that of older, buried, recalcitrant organic matter (Green et al. 2012).
Recent research has shown that under certain nutrient-limited conditions, a variety of methyl-rich organic phosphorus or sulfur compounds are likely to be utilized by microorganism and serve as precursors of CH 4 production in aerobic surface waters (Damm et al. 2008, Karl et al. 2008, Zindler et al. 2013).Integrated multi-trophic aquaculture in SGB can enhance the recycling of organic matter and nutrients and provide favorable conditions for aerobic CH 4 production.Bivalve shellfish ingest algae, POM, bac-teria, and other micro-organisms, while kelp absorbs organic and inorganic wastes from the shellfish (Fang et al. 1996).Commonly cultivated seaweeds (Gracilaria lemaneiformis and Laminaria japonica) in SGB have a high nutrient uptake efficiency (Mao et al. 2009, Xu et al. 2011) and may lead to nitrogen and phosphorus limitation in spring as well as phosphorus limitation in summer (Zhang et al. 2010), which in turn implies a potential formation of CH 4 from methyl-rich organic phosphorus or sulfur compounds (Damm et al. 2008, Karl et al. 2008, Zindler et al. 2013).DMSP can be produced by macroalgae, and grazing by bivalves appears to facilitate the release of DMSP from kelp (Smit et al. 2007).Hence the water column in polyculture zones may contain higher levels of DMSP and enhance the production of CH 4 .Seasonal variation in mean CH 4 in different mariculture areas of SGB support this hypothesis and show that higher surface CH 4 concentrations usually occur in the polyculture areas of kelp and bivalve shellfish (Fig. 9).We also observed a highly significant and rapid increase in CH 4 during incubations with DMSP spikes in October 2013 (Fig. 10).During the incubations, CH 4 concentration increased sharply by more than 60-fold and reached 460 nM on Day 3.5, then decreased sharply to the initial concentration around 7 nM.The addition of 10 µM BES reduced the CH 4 production by more than half, while the addition of 50 µM DMSP and 1 µM trimethylamine significantly enhanced CH 4 production.During all of these incubations, DO in the bottles ranged from 5 to 8 mg l −1 , suggesting that CH 4 might be produced under aerobic conditions with the degradation of methylated compounds such as DMSP and trimethylamine in the water column in SGB.

Preliminary CH 4 budget and its implication
In order to understand the contributions of different sources and sinks to dissolved CH 4 in SGB, a preliminary CH 4 budget was constructed, although there are still great uncertainties in the estimate of each term.Considering that the sea−air flux values from W2014 and N2000 were quite similar, we took the results estimated by W2014 for the budget estimation.From the budget (Fig. 11), we can see that the groundwater input (4.2 × 10 5 mol yr −1 ) was the largest quantified CH 4 source, followed by sediment release (2.6 × 10 5 mol yr −1 ) and riverine input (1.4 × 10 5 mol yr −1 ), while sea-to-air release (2.5 × 10 6 mol yr −1 ) and export from the bay to the Yellow Sea (8.8 × 10 5 mol yr −1 ) were the dominant sinks for CH 4 in SGB.Net water column production-oxidation was estimated preliminarily to produce 1.7 × 10 5 mol CH 4 yr −1 .However, there is still a large imbalance between the sources and sinks of methane in the water column of SGB, with an apparent missing source of 2.4 × 10 6 mol yr −1 needed to balance the budget, although this value might be overestimated due to the propagation of the errors in the other terms in the budget.
Because of the large spatial and temporal variations in CH 4 concentrations in the groundwater samples, our ability to provide an accurate estimate for the CH 4 flux via submarine groundwater discharge is rather limited due to the small number of groundwater end-member samples used in the calculation (n = 6) and lack of seasonal variation in groundwater fluxes.Hence the source item of groundwater may have large uncertainties.
Previous studies have demonstrated that sediments are a significant CH 4 source for bays and coastal waters (Sansone et al. 1998, Ferrón et al. 2010). Ferrón et al. (2010) found that benthic CH 4 fluxes from the shelf of the Gulf of Cádiz ranged from 0.5 to 24.1 µmol m −2 d −1 with an average of 5 ± 6 µmol m −2 d −1 using benthic chambers.Sansone et al. (1998) reported that CH 4 benthic fluxes from Tomales Bay ranged from 0.4 to 16 µmol m −2 d −1 with an average of 5.5 and 2.5 µmol m −2 d −1 for summer and winter, respectively.These results are comparable to or slightly higher than our results (0.7−8.3 µmol m −2 d −1 ) for SGB.However, given the high rates of labile organic matter loading in SGB, this strongly indicates that the fluxes assessed during the sediment incubations in this study might be underestimated.The most influential factors for this underestimation are likely to be an insufficient number of sampling stations and the ex situ sediment incubation method we used.Sediment−water CH 4 fluxes from SGB showed large spatial variation, although we only measured benthic fluxes at 3 stations and did not measure benthic fluxes from eelgrass beds in the southern region of SGB near Chudao.Previous studies showed that CH 4 benthic fluxes from eelgrass beds may be about 10-fold higher than those from unvegetated areas in Tomales Bay (Sansone et al. 1998).Due to lack of a benthic chamber, the emission of CH 4 from sediments was measured by a modified closed chamber incubation method (Barnes & Owens 1999).This method changes the environment (i.e.pressure and  temperature) and cannot simulate real in situ conditions such as resuspension, deposit feeding, burrowing, and irrigation, which often significantly change the geochemical characteristics of sediments and overlying water and increase the benthic fluxes (Sansone et al. 1998, Upstill-Goddard et al. 2000).Hence, an underestimation of the CH 4 released from the sediments may account for part of the missing source.
Underestimating in situ water column production is also likely to contribute to the missing source.As discussed above, CH 4 might be produced in aerobic water columns of the bay with the degradation of methylated compounds such as DMSP produced from the macroalgae, and grazing by bivalves appeared to facilitate the release of DMSP from kelp (Smit et al. 2007).However, the time series incubation method employed in this study only focuses on the microbial activity in the water column itself and neglects the interaction of algae, shellfish, and microbes, which may result in great underestimation of potential contributions from in situ production in the water column.If the in situ production in the water column is indeed the only missing source for CH 4 budget in the bay, the net water column production-oxidation rate is estimated to be about 7 nM d −1 .This is reasonable based on our incubation results in October 2013, which showed a net water column CH 4 increase of ~40 nM d −1 with a DMSP spike.However, our incubation results also showed that CH 4 produced might be oxidized rapidly.Hence it is difficult to evaluate how much CH 4 is accumulated in the water column over time.Considering the complicated interactions between macroalgae, shellfish, and microbes, mesocosm experiments should be carried out in the future to further understand the in situ water column CH 4 production and consumption and to understand the CH 4 budget in multi-trophic aquaculture systems like that in SGB.

CONCLUSIONS
CH 4 concentrations in SGB showed obvious seasonal and spatial variation.CH 4 concentrations were 3 to 10 times higher in summer and autumn than in spring and winter.Bottom CH 4 concentrations were obviously higher than those in the surface water due to sediment release.Higher surface CH 4 concentrations occurred in the polyculture areas of kelp and bivalves.Seasonal variation in water temperature, terrestrial freshwater input, and aquaculture activities play significant roles in regulating the spatial and temporal variation of CH 4 in the bay.Ground -water input (4.2 × 10 5 mol yr −1 ) was the largest quantified source of CH 4 , followed by sediment release (2.6 × 10 5 mol yr −1 ), and riverine input (1.4 × 10 5 mol yr −1 ), while sea-to-air release (2.5 × 10 6 mol yr −1 ) and export from the bay to the Yellow Sea (8.8 × 10 5 mol yr −1 ) were the dominant CH 4 sinks.Net water column production-oxidation was estimated preliminarily to produce 1.7 × 10 5 mol CH 4 yr −1 ; however, this value may have been underestimated due to the neglect of interactions between algae, shellfish, and microbes.There was a great imbalance of sources and sinks, with an apparent missing source of 2.4 × 10 6 mol yr −1 , most of which might be attributed to underestimates of in situ water column production and CH 4 released from the sediments.Benthic chamber measurements and mesocosm experiments should be carried out in the future to further understand the CH 4 budget in multi-trophic aquaculture systems like that in SGB.

Fig. 1 .
Fig. 1.Location of Sanggou Bay, China, and the main aquaculture practices

k
w was estimated by the LM86 equation (Liss & Herlivat 1986) b k w was estimated by the W92 equation (Wanninkhoft 1992) c k w was estimated by the W2014 equation (see 'Materials and methods' for details) d k w was estimated by the N2000 equation (see 'Materials and methods' for details) Table 2. Compilation of surface concentrations and sea-to-air fluxes of CH 4 in different coastal areas of China (see Fig. 2 for station locations).

Fig. 9 .
Fig. 9. Comparison of seasonal mean (± SD) CH 4 concentrations in surface and bottom waters in different culture areas of Sanggou Bay . Atmospheric CH 4 was not measured during these cruises.Therefore, mean atmo spheric CH 4 mixing ratios of 1.896, 1.901, and 1.929 ppm by volume (ppmv) at 3 observation stations near the coastal seas of China (NOAA Stns LLN, TAP, and SDZ) for 2012, 2013, and 2014, from the NOAA/ESRL Global Monitoring Division in situ program (www.