Dark Carbon Fixation Process And Influencing Factors In Seawater Of The Estuary And Adjacent Coastal Areas

Ocean is important carbon sink area,which absorbs a large amount of CO₂ emitted by human beings every year.Net primary productivity is the decisive factor of carbon sink.The main contributor of net primary productivity is concentrated in marine phytoplankton,but most studies ignore the contribution of dark carbon fixation（chemoautotrophic）microorganisms to the net primary productivity of seawater.At the same time,the ammonia oxidation process（NH₄⁺oxidation to NO₂^–）can provide energy for the dark carbon fixation process.The oxidation and reduction reaction of NH₄⁺in the nearshore water is strong,which can promote the primary productivity of the marine area to a certain extent and the nitrogen and carbon balance of the estuary ecosystem.At present,the study on dark carbon fixation mainly concentrate on the ocean,deep sea and extreme heat flow regions,while the study on estuaries and adjacent sea areas are relatively small.In addition,the process and driving factors of dark carbon fixation in estuaries and adjacent seawater are still unclear.In this study,the Bohai Sea and Yangtze River Estuary were slected to study the dark carbon fixation（DCF）rate,ammonia oxidation（AO）rate,dark carbon fixation driven by ammonia oxidation bacteria(DCF_AOB)rate,ammonia oxidation rate（AOBr）driven by ammonia–oxidizing bacteria,functional gene abundance and community structure of the whole bacterial community in Bohai Sea using ¹⁴C(NaH¹⁴CO₃)labeling method and ¹⁵N isotope tracer method combined with allylthiourea（ATU）inhibitor method and molecular biological technology to reveal the process and influencing factors of dark carbon fixation in Bohai Sea;at the same time,the temperature adaptability of the dark carbon fixation rates in the Yangtze River Estuary were studied.The results are helpful to improve the understanding of the dark carbon fixation in the estuary and adjacent sea ecosystems.The main study results are as follows:（1）The dark carbon fixation rates in the Bohai seawater were between 0.21 and 1.12μmol C L^–1 d^–1in summer and 0.10～0.24μmol C L^–1 d^–1in winter,which showed significant temporal difference.At the same time,the dark carbon fixation rates of the bottom layer were about 35%higher than that of the surface layer in summer,but the variations were not obvious in winter.In addition,the dark carbon fixation rates of water were affected by temperature,NO₂^–,TN,ammonia oxidation rate and cbb L gene abundance.According to the inhibitor method,it was preliminarily estimated that the range of dark carbon fixation rates driven by ammonia oxidation were 0.01～0.38μmol C L^–1 d^–1,accounting for about 18%of the total,indicating that the ammonia oxidation process plays an important role in the autotrophic CO₂ fixation process.In addition,we roughly estimated that dark carbon fixation contributed 1.88 TgC yr^–1(0.91～2.85 TgC yr^–1),accounting for 20%of the total carbon fixation value in the Bohai Sea(8.66±3.63 TgC yr^–1)and equivalent to the carbon flux deposited in the region（～2.0 TgC yr^–1）.（2）The abundance of cbb L gene was between 4.37×10⁶～1.84×10⁸ copies L^–1,and the abundance of amo A-AOB was between 2.81×10⁴～3.83×10⁶ copies L^–1,which were significantly positively correlated with the dark carbon fixation rate of the seawater.The abundance of the cbb M gene(4.91×10⁴～2.34×10⁶ copies L^–1)has no significant temporal and spatial differences,and the variations were mainly affected by the physical and chemical factors such as water temperature,dissolved oxygen and inorganic nitrogen.The sequencing results showed that Proteobacteria dominated the bacterial community in winter（34%）,and Cyanobacterium dominated the bacterial community in summer（43%）.The seasonal composition of the community was significantly different.The abundance of the main AOB community Nitrosopira and Nitrosomonas showed significantly temporal variations,indicating that temperature was an important factor affecting the AOB community.（3）Based on the laboratory temperature control experiment,the dark carbon fixation rates in the Yangtze River Estuary seawater were between 0.23 and 0.33μmol C L^–1 d^–1,in which the dark carbon fixation driven by ammonia oxidation microorganism accounted for about 4.13～43.61%（0.01～0.20%）of the total dark carbon fixation.In the optimum temperature range,the dark carbon fixation rate increased significantly with the increase of the ambient temperature,but the dark carbon fixation rates in the low–salinity near the estuary increased more significantly with the variations of the ambient temperature,and the maximum values of the dark carbon fixation rate in the low–salinity and the high–salinity appeared at 30℃and 25℃,respectively.Under this temperature condition,adding ammonia nitrogen can significantly promote the increase of the dark carbon fixation rates in the seawater.