The Mechanisms Of Circumneutral PH Anaerobic Nitrate Reduction Coupling To Fe(?) Oxidation And As(?) Oxidation In Flooded Paddy Soil

The paddy soil is subjected to unique anoxic-oxic alterations in different growth stages of rice due to temporal scale flooding and non-flooding,such anoxic-oxic alterations effect on geochemical properties the soil,element transformation and microbial activities.Iron?Fe?is the fourth most abundant active metal element in the earth‘s curst.Nitrogen is one of the most important elements in the biosphere,and the nitrates?NO₃^-?can be used as nutrients by plants and microorganisms.Arsenic?As?is a human carcinogen and people were exposed to arsenic mainly through As-contaminated water or food.Rice is the main source of food for people and As accumulation in rice becomes a serious disaster for human health.Previous studies verified that the biogeochemical cycling of nitrogen and iron directly influenced on immobilization and transformation of arsenic.However,it is known little about the microbial-chemical mechanism for nitrate reducing coupling Fe???oxidation and arsenite?As????oxidation.In this sthdy,the mechanism of nitrate reduction coupling to ferrous?Fe????oxidation and arsenite oxidation in paddy soil and pure strain capable of nitrate reduction and ferrous oxidation were studied using the kinetics of N/As/Fe transformation,molecular biology and the stable isotope fractionations of nitrous oxide?N₂O?.The main conclucions obtained are as follows:?1?Paddy soil was collected from Southern China and used as an inoculum in an anoxic experiment in order to investigate the process of As???oxidation and nitrate reduction and associated functional microbial communities.The results showed that As???was completely oxidized in As???+NO₃^-treatment after 6days,while no As???oxidation was observed in the As???amendment.Meanwhile,NO₃^-reduction was observed in As???+NO₃^-amendment,where nitrite?NO₂^-?and N₂O were the major products.The real-time quantitative PCR showed that the abundance of 16S rRNA gene and functional genes affiliated with As???-oxidization?aoxB?and denitrification?narG,nirS and nosZ?increased over time in both As???+NO₃^-and NO₃^-amendments,suggesting that NO₃^-may play an important role in stimulating both As???-oxidizers and denitrifiers in paddy soil.In the As???+NO₃^-amendment,the 16S rRNA-based dominant genera were Pseudogulbenkiania,Janthinobacterium,Chromobacterium?A metagenomic analysis was applied in order to gain a insight into the functional microbial community invloved into the arsenic and nitrogen cycles in Soil+As???+NO₃^-culture.Azoarcus were the dominant,role in the reducing NO₃^-and oxidizing As???;Pseudogulbenkiania were the dominant,role in the reducing NO₃^-and reducing arsenate?As?V??as well as methylating As;the dominant microorganisms for N₂O reduction to nitrogen?N₂?were Azoarcus,Flavisolibacter ginsengisoli,Rhodocyclaceae bacterium;the dominant nitrogen-fixing microorganisms were Azoarcus,Paludibacter propionicigenes,Rhodocyclaceae bacterium,Geobacter.The study provided better understanding of the functional microbial communities involved in arsenic and nitrogen cycles in anoxic paddy soil.?2?Paddy soil was collected from Southern China and used as an inoculum in an anoxic experiment in order to investigate the process of Fe???oxidation,As???oxidation,nitrate reduction and associated functional microbial communities.The results of the kinetics showed that nitrate addation stimulated to both As???oxidation and Fe???oxidation.And,As?V?produced from As???oxidation mainly existed in the culture in absorbed As?V?,suggesting that Fe???minerals produced from Fe???oxidation played an important role in immobilization of As?V?.By the benefit of the ultra-deep sequencing and the binning procedure,16near-complete genomes,included representatives of genera Azospira,Geobacter,Geothirx,Herbaspirillum and Pseudogulbenkiania were retrieved from the metagenome to evaluate the metabolic interdependencies of the most abundant organisms in the process of NO₃^-drivers Fe???and As???oxidation.The profiled organisms are involved in the network of denitrification,arsenite oxidation and iron ion metabolism.Genera affiliated with Azospira were predicted to be the major arsenite oxidizing microorganism who using nitrate as electron acceptor.The generated nitrite stimulated chemically Fe???oxidation.?3?A NO₃^-reducing Fe???oxidizer Acidovorax ebreus LS-1 isolated from red paddy soil in South China.Other species TPSY,2AN,BoFeN1,BrG1 in the Acidovorax genus identified in groundwater,sediment,lake,were also capable of Fe???oxidation and nitrate reduction,suggested that Acidovorax genus was widespread in environment and played an important role in nitrate reduction and Fe???oxidation.Acidovorax ebreus LS-1 was used to research the process of anaerobic,nitrate reduction,Fe???oxidation at neutral.The results showed that Fe???oxidation was observed in the presence of nitrate,and resulting in the formation of Fe???mineral-lepidocrocite.Meanwhile,NO₃^-could be reduced to nitrite,nitrous oxide.Nitrogen stable isotope experiments revealed that N₂O produced during NO₃^-reduction in the presence of Fe???was primarily of biotic origion,however,chemical reaction played an role in the N₂O production during NO₂^-reduction in the presence of Fe???by Acidovorax ebreus LS-1.The results of transcriptome indicated that the transcription of denitrification genes?narGH,nirS,norB,nosZ?and Fe???-oxidizing genes?cyc1,cyc2 and purA?was up-regulated under NO₃^-reduction coupled with Fe???oxidation conditions.It provided a potential mechanism about NO₃^-reduction coupled with Fe???oxidation by Acidovorax ebreus LS-1 and expands our understanding of nitrate reducing Fe???oxidizer at genes level.?4?Pseudomonas stutzeri LS-2,a denitrifying bacterium isolated from paddy soil in southern China,was used in this study to investigate the process nitrate reduction in the presence of Fe???.The results of the kinetics of Fe/N transformation revealed that the presence of Fe???resulted in a decrease of nitrate reduction rates,and an increase in the amount of nitrous oxide production.X-ray diffraction and scanning electron microscopy analyses of the precipitates after 6 days of incubation confirmed that lepidocrocite was formed due to Fe???oxidation,which fully covered the bacterial cell surfaces.Transmission electron microscopy images of the cell-mineral precipitates showed that encrustation occurred in the periplasm and on the cell surface.The stable isotope fractionations of?¹⁵N-N₂O results revealed that once NO₃^-was reduced to NO₂^-by the denitrifying bacterium P.stutzeri LS-2,the NO₂^-chemically reacted with Fe???.