Diversity And Nitrogen Removal Functions Of The Aerobic Denitrifying Bacteria From Aquaculture Water

Excessive inorganic nitrogen in aquaculture water not only poses a health threat to aquatic animals but also causes severe environmental pollution to the surrounding natural ecosystem.Nitrogen pollution is a common major issue in aquaculture production.Aerobic denitrifying bacteria that can convert nitrate or nitrite to gaseous nitrogen under aerobic or micro-aerobic conditions possess a great value of application in the nitrogen removal of aquaculture water.In this study,high-throughput sequencing(HTS)of the 16S r RNA gene was used to analyze the bacterial diversity and community composition of the aquaculture water from five different aquaculture systems and investigate the effect of environmental factors on bacterial community structures.Enrichment and direct screening strategies were used for the isolation of aerobic denitrifying bacteria from the different aquaculture water.And the aerobic denitrifying bacteria possessing excellent nitrogen removal ability were obtained by using the secondary screening method.Four potentially novel species were identified by using the polyphasic taxonomic approach and the nitrogen removal characteristics of the two high-efficient denitrifiers were investigated simultaneously.The synthetic bacterial consortia with high-efficient nitrogen removal capability were pairwise formulated and obtained.Furthermore,the comparison of genome-scale metabolic reconstruction of the two strains contained in the bacterial consortia was analyzed to investigate the synergistic denitrification mechanism of the two strains.1.Water samples were collected from five different aquaculture systems including the traditional freshwater aquaculture system(t PAS＿fa),the freshwater semi-recirculating aquaculture system(s RAS＿fa),the freshwater recirculating aquaculture system(RAS＿fa),the higher-place ponds of marine aquaculture system(h PAS＿ma)and the marine recirculating aquaculture system(RAS＿ma).Results showed that the nitrate concentrations in RAS＿f and RAS＿m were much higher than that in the other aquaculture systems.Phyla Proteobacteria and Bacteroidetes were the dominant phyla in all the aquaculture systems,and Proteobacteria was to be significantly more abundant in the recirculating aquaculture systems compared with the non-recirculating aquaculture systems.Furthermore,Actinobacteria and Cyanobacteria were also the dominant phyla in t PAS＿f and h PAS＿m.The dominant genera were significantly different among various aquaculture systems.Pseudoalteromonas,Nautella and Vibrio were the dominant genera in RAS＿m;Aeromonas,Cetobacterium,Acinetobacter,Acidovorax,Rheinheimera,Gemmobacter,Pseudomonas and Hydrogenophaga were the dominant genera in RAS＿f;Novosphingobium and Rhodobacter were the dominant genera in s RAS＿f;Ruegeria,Marivita,Muricauda and Salinihabitans were the dominant genera in h PAS＿m;Fluviicola and Burkholderiaceae were the dominant taxa in t PAS＿f.Salinity was the main factor affecting the bacterial community structures,followed by total phosphorus,ammonium,nitrate,temperature and oxidation-reduction potential.2.A total of 714 bacteria with the potential aerobic denitrification capability were isolated from different aquaculture water.These bacteria were classified into the phyla Proteobacteria,Firmicutes,Actinobacteria and Bacteriodetes,and Pseudomonas strains accounted for 33.8%of the total bacteria.The bacterial species richness and number of the novel bacteria obtained by using the direct screening strategy were both higher than those obtained by the enrichment screening strategy.The high-efficient denitrifiers obtained by using the secondary screening method were classified into genera Pseudomonas,Achromobacter,Alcaligenes,Acinetobacter,Ensifer,Hydrogenophaga and Gemmobacter.Conjoint analysis of the potential aerobic denitrifying bacteria and the results of HTS implied that sixteen taxa(genera or families)of the potential aerobic denitrifying bacteria were widespread and abundant in aquaculture water.Co-occurrence potential network of the sixteen taxa indicated that Hydrogenophaga and Gemmobacter were the highly-connected nodes,and these two genera were positively correlated with the genus Pseudomonas.3.This study subsequently identified the strains JM10B5a~T,JB02H27~T,DN00404~T and DN04309~T,and named them Pseudomonas oligotrophica,Marinobacter denitrificans,Sphingobacterium micropteri and Sphingobacterium litopenaei,respectively.The functional analysis based on the genome revealed that strain JM10B5a~T possessed the denitrification genes including nap A,nar G,nir S,nor B and nos Z and strain JB02H27~T possessed the denitrification genes including nar G,nap A,nir K,nir S,nor B,qnor and nos Z.These results suggested that strains JM10B5a~T and JB02H27~T both possessed the denitrification capability of reducing nitrate or nitrite to dinitrogen gas.Moreover,strain JM10B5a~T showed the high-efficient denitrification ability when sodium acetate was used as a sole carbon source and this strain also exhibited the complete nitrate removal under the carbon-nitrogen ratio of 3.And strain JB02H27~T showed the high-efficient denitrification ability under the high salinity condition.4.Nineteen synthetic bacterial consortia with high-efficient nitrogen removal capability were obtained,including eleven consortia comprised of strain Hydrogenophaga bisanensis JM15B35c1 and Pseudomonas strains,six consortia comprised of strain Gemmobacter caeruleus JM14B9b and Pseudomonas strains,one consortium comprised of strain Hydrogenophaga sp.JM15B46 and a Pseudomonas strain,one consortium comprised of strain Gemmobacter sp.JM10B15 and a Pseudomonas strain.These findings revealed the interspecies synergistic denitrification between Pseudomonas and Hydrogenophaga,Pseudomonas and Hydrogenophaga,respectively,which may verfied the positive interactions of the two species in the co-occurrence potential network.Comparative analysis of metabolic reconstruction showed that a large proportion of Pseudomonas strains,which were contained in the synthetic bacterial consortia with high-efficient nitrogen removal capability,lacked the key gene for nitrite reductase,but strains JM15B35c1 and JM14B9b could perform the conversion of nitrate to gaseous nitrogen.Moreover,Pseudomonas strains possessed more genes related to amino acids and derivatives,cofactors and vitamins,cell wall and capsule,iron acquisition and metabolism.Therefore,we speculated that these substances might play an important role in the synergistic nitrogen removal between Pseudomonas and Hydrogenophaga strains or Pseudomonas and Gemmobacter strains.Further study on the synthetic bacterial consortium containing Pseudomonas geniculata DN02703 and Hydrogenophaga bisanensis JM15B35c1indicated that the cell-free bacterial supernatant of strain DN02703 could significantly enhance the denitrification and electron transport system activities in the nitrogen removal process of strain JM15B35c1.Moreover,we speculated that biotin exchange plays a vital role in the synergistic nitrogen removal of these two strains.