Application Of A Strain Denitrifying Halomonas Sp. And Environmental Conditions On Denitrificationof A Strain Denitrifying Pseudomonas Sp.

1.Comparison of denitrification performance of 5 strains of nitrogen-denitrifying bacteriaFive bacteria strains including Halomonas 04,Halomonas 02,Halomonas 20,Acinetobacter baumannii 03,and Bacillus baekryungensis 04 with potential for denitrification were selected and inoculated into nitrifying medium and nitrite medium,both with a nitrogen concentration of 20 mg/L respectively,the effect of denitrification of ammonia nitrogen and nitroso nitrogen were analyzed.The results showed that the removal rate of ammonia nitrogen can reach 100%by both Halomonas 04 and Halomonas 02 in 20 h.The time required for Halomonas 04 and Halomonas 02 to remove 100%of the nitroso nitrogen in the medium was 60h and 72h.Further comparison of activity of ammonia monooxygenase?AMO?,hydroxylamin oxidase?HAO?and nitrite reductase?NIR?of strain Halomonas 04 and Halomonas 02 cultured in nitrifying medium and nitrite medium after the stable period,the results showed that activities of the three denitrification enzymes of Halomonas 04 were higher than those of Halomonas 02.Comprehensive analysis gave the result that candidate strain Halomonas 04 is an efficient denitrification strain that can metabolize both ammonia nitrogen and nitroso nitrogen,and has potential development value in aquaculture.2.Removal effect of strain Halomonas 04 on high concentration of harmful nitrogenIn order to study the application potential of strain Halomonas 04,four concentration groups of strain Halomonas 04 were set at 0,10⁴,10⁵and 10⁶CFU/m L in the shrimp stocking water,and 10 mg/L ammonia nitrogen,5mg/L nitroso nitrogen and mixture of 10mg/L ammonia nitrogen and 5mg/L nitroso were added to each group every day,also sucrose were added to the experimental groups and the control group according to the concentration of C:N=15 to supply carbon source.The denitrification ability of Holomonas 04 was determined by analyzing the concentration of ammonia nitrogen and nitroso nitrogen and the survival rate of shrimp.The results show that the addition of Halomonas 04 in the stocking system could significantly reduce the concentration of ammonia nitrogen and nitroso nitrogen in the water body,and improve the survival rate of shrimp.The highest nitroso nitrogen removal rate was 84.8%,which was in group of strain Holomonas 04 at concentration of 10⁶CFU/ml with sucrose.Adding sucrose and strain Holomonas 04 together to the culture environment to cultivate bio-floc is beneficial for the removal of ammonia nitrogen and nitroso nitrogen,and improving the survival rate of shrimp under ammonia nitrogen and nitrite stress,and promoting the colonization of strain halomonas 043.Colonization effect of strain Halomonas 04 in simulated aquaculture environmentIn order to study the colonization effect of Halomonas 04 in the aquaculture system,a simulated aquaculture system was constructed by adding environmental strains of Pseudoalteromonas sp.Bacillus licheniformis,Vibrio parahaemolyticus and Shewanella.sp isolated from the shrimp pond and adding shrimp feed according to the shrimp production of 2.5kg/m².An experiment on simulating colonization effect of Halomonas 04 in the first 10 days in early period and 10 days in later period of shrimp culture was carried out.The results showed that in the early stage of cultivation with low residual concentration of shrimp feed and no other environmental bacteria existing in the water body,the concentration of Halomonas 04 could be maintained at the order of 10⁴CFU/ml.Increasing of residual concentration of shrimp feed water,Halomonas04 could reach the order of 10⁵CFU/ml.When there were kinds of bacteria in the environment,the colonization of Halobacterium 04 would be affected,no matter whether there is shrimp feed or not,the concentration of bacteria can only be maintained at the order of 10³CFU/ml.4.Isolation and identification of a strain of Pseudoalteromonas with denitrification and its denitrification effectIn this experiment,a strain with high efficiency of nitrogen removal?06Pseudoalteromonas?was isolated from the shrimp culture pond,and then the carbon source,C/N,p H,salinity and nitrogen concentration of the nitrification medium used for the culture of the bacteria were changed.The effect of environmental conditions on its nitrogen removal efficiency.The results showed that among the seven carbon sources selected in the experiment,sodium citrate was the best carbon source,and it had the best denitrification effect in the medium with sodium citrate as the carbon source;in the medium with different salinity The lower the salinity,the higher the OD value of the bacterial liquid at the same time,indicating that the strain is a low salt tolerance strain;the strain grows best in the medium with C/N of 20,and has the best denitrification effect.,indicating that C/N is 20 is the best C/N;in different c?NH₄⁺?medium,c?NH₄⁺?=20mg/l medium has the highest OD value,and the denitrification efficiency is also the highest,c?The increase or decrease of NH₄⁺?inhibited the growth of the strain and reduced the efficiency of nitrogen removal.In the different c?NO₂^-?medium,the growth effect of each group was not obvious;after 96 hours of culture,the p H=8 group nitrogen removal efficiency Significantly higher than p H=5 and p H=6 groups;the identification of 06 Pseudoalteromonas by 16S r DNA sequence alignment analysis showed that it was a strain of Alternaria alternata;the strain was inoculated to infect P.vannamei for biosafety.Experiments,the results show that 06 Pseudoalteromonas has better safety for Penaeus vannamei.This study can provide theoretical support for the development and utilization of biological nitrogen removal technology.