The Carbon And Nitrogen Cycling Characteristic Of Intensitive Pond And The Influence Of Organic Carbon Sources On The Nitrogen Removal Of Biofloc

The characteristic microbial communities in the water and sediment layers of an aquaculture pond have adapted to inhabit each type of environment and display specific functions.Studying the microbial community structure and its ecological functions can help elucidate the biogeochemical cycles of the nutrients and their interactions in situ.Because of the substantial mass of allochthonous organic carbon?C?and nitrogen?N?input,C and N nutrient cycles are particularly important in this type of ecosystem.To improve our understanding of the geochemical cycling of nitrogen,reduce the harm of nitrogen pollutants for aquaculture organisms and regulate a more flow of nitrogen nutrients to aquatic products,we establish the complete C and N cycling functional and microbial characteristic in the vertical stratification of pond.The results prompted us to propose hypotheses concerning the potential relevance of the pathways among populations that promote carbonic and nitrogenous nutrient cycling in a pond ecosystem.In the subsequent research of biofloc system,we have explored the effect and mechanism of nitrogen removal in aquaculture water by the additon of organic carbon.The major results are as follows:1.The identities and functional capacities of the microbial populations within surface water?SW?,bottom water?BW?,surface sediment?SS?and deep sediment?DS?samples from a typical grass carp?Ctenopharyngodon idellus?culturing pond in central China were explored using metagenomics.In total,the community structure and microbial processes of the water columns was distinct from that of the SS and DS layers.Alphaproteobacteria,Actinobacteria,Cyanobacteria and Planctomycetes were abundant in the water,while Deltaproteobacteria,Gammaproteobacteria,Euryarchaeota?Archaea?and Nitrospirae were more abundant in the sediment?P<0.05?.The functional potential and microorganisms responsible for the N nutrient cycles were also reconstructed in silico.The high functional potential for N assimilation,protein synthesis and cell proliferation in pond water should be responsible for low ammonia concentration detected.Ammonia oxidation functional genes were present in very low abundances,and were mostly detected in the water columns and related to Nitrosomonas?100%?.Denitrification were observed mostly in SS and the main taxon involved was Rhodocyclales.The potential for N fixation?nif genes?and dissimilatory nitrate reduction to ammonium was also observed mostly in sediment,which is a disadvantage for ammonia reduction in the pond ecosystem.Overall,these results offer a more detailed perspective on the microbial functional ecology of the aquaculture pond.2.The functional potential and microorganisms responsible for the C nutrient cycles in aquaculture pond ecosystem were explored using metagenomics.Compared to the photoautotrophs?Cyanobacteria and Crytophyta?,heterotrophs?Actinobacteria and Betaproteobacteria?were the dominant microorganisms driving spiration in the pond.The chemosynthesis of Methanomicrobials?strictly carbon dioxide-reducing methanogens?and Syntrophobacterales?strictly anaerobic sulfate-reducing bacteria?in the sediment was the main carbon fixation process in pond.The hydrogenotrophic methanogenesis?Methanomicrobiales?wasthedominantmethanogenic microorganisms in pond sediment because of the selective inhibitory effect of methanogens by free ammonia.In the pond water,the AOB can oxidize CH₄ to CO₂,whereas Methylococcaceae?type ? methanotrophs?use the ribulose monophosphate?RuMP?pathway and Methylocystaceae?type ? methanotrophs?utilize the serine pathway for CH₄ assimilation.In addition,our results implied the aerobic respiration of allochthonous organic C in the aquaculture pond provided the energy required for N assimilation and denitrification.In addition to nitrification,Nitrosomonadales fulfilled various other functions in the pond water,including N mineralization and methane oxidation.Methanomicrobiales were the dominant microorganisms carrying out methanogenesis,CO₂ fixation and N₂ fixation in the sediment.Overall,these results indicate the accumulation of ammonia in pond sediment has an effect on methanogenesis.Conversely,organic carbon and the methane cycle also have an significant influence on N cycling processes.3.As the organic carbon in aquaculture sysment can affect nitrogen cycling through multiple metabolic pathways,we have investigated how different carbon sources affect microbial diversity of water in the biofloc system.The experiment consisted of the control and three types of biofloc systems using different carbon sources,tapioca starch?TS?,plant cellulose?PC?and the combination of tapioca starch and plant cellulose?TS+PC?,and conducted for 42 days in twelve 300 L Fiber Reinforce Plastic?FRP?circular tanks.Pelteabaggrus vachelli?n=72?of mean weight?38.1±5.9 g?were randomly distributed into twelve tanks.Community characterization of total bacteria and ammonia-oxidizing bacteria in all treatments were respectively investigated by Illumina Miseq analysis using PCR-amplified 16 S rRNA gene fragments?primer 27F-338 R for total bacteria and primer CTO189F-CTO654 R for ammonia-oxidizing bacteria?.The ammonia-nitrogen concentration in control?3.6±4.6 mg/L?was significantly higher than that in all biofloc systems?2.4±2.9 mg/L,1.8±2.4 mg/L and 2.2±2.5 mg/L for TS,PC and TS+PC,respectively??P<0.05?.Weight gain ratio in TS was significantly higher than the control?P<0.05?,while there is no significant difference between the control and treatment groups added plant cellulose?PC and TS+PC??P>0.05?.The Illumina MiSeq sequencing results showed that biofloc systems incorporating plant cellulose treatment groups had a higher total bacterial diversity and greater microbial richness than those who no plant cellulose treatment groups?Control and TS??P<0.05?.There was significant increase of Betaproteobacteria in the control and TS treatments,whereas Alphaproteobacteria was higher in treatment groups added plant cellulose?P<0.05?.Illumina sequencing analysis of ammonia-oxidizing bacteria V3 region also detected a higher community diversity in biofloc systems?Shannon index,>1.21;Simpson index,<0.3949?compared to control?Shannon index,0.84;Simpson index,0.6149?.We conclude that organic carbon sources in biofloc system can decrease ammonia-nitrogen concentration and improve the community diversities of overall and ammonia-oxidizing bacteria.4.For a detailed understanding of the addition of organic carbon on microbial community in biofloc system,we have investigated the time dependent community composition evolution of bacteria in mixed-population suspended growth reactors during the growth period?GP?and the maintenance period?MP?.The effect of different C/N ratios feeds solutions on nitrifiers and potential ammonia oxidizing rate were further detected during the treatment period?TP?.All biofloc reactor?BR?systems have excellent NH₄⁺-N removal efficiency?>95%?after 15-days GP.In total,1 191 895 effective reads?average 418 bp?were obtained from 18 samples using Illumina MiSeq sequencing.The PCA analysis indicated bacterial community in seeding pond water?S0?was separated from that in BR,and?-diversity indexes of bacteria in S0 was significantly lower than that in BR systems.Most of increasing bacteria during GP were heterotrophs,including many aerobic denitrifiers,such as Zoogloea,Rhodobacter,Pseudomonas,Methyloversatilis and Terrimonas.Autotrophic nitrifying bacteria were also weighty accumulated during GP and that caused a peaking of NO₃^--N on day 28.Aerobic or facultative aerobic denitrifiers,mainly Rhodobacter,Hydrogenphaga,and Sphaerotilus,were significant increased during MP as a supplement to removal the increased NO₃^--N.The different C/N ratios experiment indicated Rhodobacter,Hydrogenphaga and Sphaerotilus were positive corrected with organic carbon,while autotrophic nitirifiers was negative corrected with organic carbon.The significantly higher potential ammonia oxidizing rate in high C/N ratio group implied the presence of heterotrophic nitrification in Pseudomonas in BR.Many pathogens,such as Aeromonas,Flavobacterium,Cytophaga,and Flectobacillus,were significantly decreased in BR.Our results indicate aerobic denitrification plays an important role in nitrogen removal in suspended growth reactor using biofloc technology.