| Ammonia nitrogen is one of the main pollutants in water bodies.The research on the theory and technology of water nitrogen removal is of great significance.Among many nitrogen removal technologies,simultaneous heterotrophic nitrification and aerobic denitrification(SND)has significant advantages such as high nitrogen removal efficiency,mild conditions,simple process equipment and no secondary pollution.Certain industries produce high-salt(>20 g/L)nitrogen-containing wastewater.Because high salt inhibits the growth and metabolism of microorganisms,the current SND nitrogen removal efficiency of this type of wastewater is low.The research on the theory and technology of microbial SND nitrogen removal under high salt conditions is one of the current research hotspots in this field.The compatible solute Ectoine(1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid)synthesized by Halomonas in the moderately halophilic bacteria under salt stress.Ectoine has the function of balancing the osmotic pressure inside and outside the cell.Some Halomonas strains also have SND nitrogen removal ability.Investigating the nitrogen removal methods of these Halomonas strains,elaborating the salt-tolerant nitrogen removal mechanism are important for improving the nitrogen removal efficiency of SND under high salt conditions.In addition,there is a type of Ectoine-secreting Halomonas strains,which can secrete part of the Ectoine synthesized by the cell to the outside of the cell with reverse osmotic pressure gradient,which can build a kind of based on Ectoine "synthetic secretion-absorption transmission-population salt tolerance" in the microbial flora.The salt tolerance enhancement system provides an efficient and convenient technical strategy and method for the microbial high salt nitrogen removal and even the salt tolerance enhancement of the microbial degradation system.In this paper,the Ectoine-secreting Halomonas strain which could removed nitrogen by SND method was isolated and screened from the salt pond of a salt factory in Dalian.The SND nitrogen removal ability,Ectoine synthesis and secretion were investigated.The enzyme genes related to nitrogen removal of Halomonas strain were cloned and the molecular basis of SND nitrogen removal was revealed.A strain B01 with higher SND nitrogen removal rate and highest Ectoine secretion rate was selected for morphological,growth,physiological and biochemical identification,and named Halomonas sp.B01.The nitrogen removal characteristics of SND by Halomonas sp.B01 were investigated.The optimized SND nitrogen removal conditions were sodium succinate as carbon source,C/N of 5,pH of 8,shaker speed of 90 rpm,and NaCl concentration of 60 g/L.The SND nitrogen removal process under the optimized conditions of this strain showed that under the conditions of 60 g/L NaCl and 4000 mg/L initial NH4+-N,the nitrogen removal rate reached 99.2%and the nitrogen removal efficiency reached 15.2 mg/(L·h)for 180 h.There was almost no accumulation of intermediate products(NH2OH-N,NO2--N,NO3--N)during the nitrogen removal process.By limiting the supply of sub-appropriate amount of inorganic phosphorus in the nitrogen removal matrix to control the state of resting cells in the non-growth stage,resting cells had a higher SND nitrogen removal efficiency than growing cells.This feature was of special significance for the nitrogen removal of immobilized cells or membrane bioreactors.Halomonas sp.B01 was a highly efficient SND nitrogen removal strain that could tolerate both high salt and high concentration of NH4+-N.The salt-tolerant growth and nitrogen removal mechanism of Halomonas sp.B01 were studied.The stress effect of high salt on the strain was mainly to inhibit growth and metabolism and cause lipid peroxidation,and the response of this strain to the effect of high salt stress was the cooperative salt tolerance mechanism by Ectoine protection and superoxide dismutase(SOD)repair.Within the concentration range of 30-120 g/L NaCl,the intracellular Ectoine concentration increased with the increase of NaCl concentration,and cells balanced extracellular high salt stress by synthesizing compatible solute.Extreme salt concentration(120 g/L NaCl)caused cell lipid peroxidation and also initiated the SOD repair mechanism.The synergistic salt tolerance mechanism by Ectoine protection and SOD repair endowed Halomonas sp.B01 with significant salt tolerance.The related enzymes in the growth core metabolic pathway(glucokinase,GK;dehydrogenase,DHA;succinate dehydrogenase,SDH)and nitrogen removal related enzymes(ammonia monooxygenase,AMO;nitrite reductase,NIR;nitrate reductase,NAR)in Halomonas sp.B01 all showed the highest or higher(DHA)enzyme activities in culture medium containing 60 g/L NaCI.Systematic studied on the properties of nitrate reductase,a key enzyme in nitrogen removal pathway,showed that the enzyme was identified as a dissimilar nitrate reductase type NAR;expression of membrane-bound inducible enzyme NAR under NO3--N induction culture;the optimal pH of the NAR enzyme reaction system was 8,the optimal reaction temperature was 30?,and the 7 mM Ectoine added to the NAR in vitro enzyme reaction system could maintain the enzyme activity at high concentrations of NaCl.In the aerobic denitrification of this strain with 60 g/L NaCl and NO3--N as the substrate,the 24 h denitrification rate reached 97.1%,and the NAR of this strain was not sensitive to oxygen molecules.RT-PCR results showed that the coding genes of Halomonas sp.B01 AMO and NAR(amoA,narH)had the highest relative expression abundance when cultured in 60 g/L NaCl medium.The mechanism of salt-tolerant SND nitrogen removal of Halomonas sp.B01 was revealed from cell growth,enzyme properties and gene expression levels.Compared with the traditional salt tolerance acclimation method,the salt tolerance enhancement system based on the compatible solute Ectoine" synthetic secretion-absorption transmission-population salt tolerance" constructed in this paper had increased the nitrogen removal rate and nitrogen removal efficiency by 50%and 90%,respectively.Under the optimized conditions,the nitrogen removal based on the salt tolerance enhancement system in high ammonia nitrogen seawater,removed nitrogen 144 h,the nitrogen removal rate reached 95.3%,and the nitrogen removal efficiency reached 19.9 mg/(L·h),which are 53.0%and 11.1 mg/(L·h)higher than those without salt tolerance enhancement.The process of nitrogen removal was accompanied by the production,secretion and absorption of Ectoine.The salt tolerance enhancement system based on Ectoine "synthetic secretion-absorption transmission-population salt tolerance" showed a strong salt tolerance and nitrogen removal efficiency.The microbial diversity analysis of the salt tolerance enhancement system based on 16S rDNA high-throughput sequencing technology showed that the number of activated sludge microbial communities increased at the level of phylum,class,order,family,and genus under high salt conditions after salt tolerance enhancement.Among them,the abundance.of Halomonas genus increased from 0.6%to 8.6%.It can be inferred that Halomonas sp.B01,as a salt tolerant enhancement strain,became the dominant genus in enhancement activated sludge under high salinity.The nitrogen removal function of the microbial flora after the enhancement of salt tolerance was improved with the change of the abundance of the microbial community.16S rDNA diversity analysis revealed the microbial ecological mechanism of nitrogen removal efficiency improved by Halomonas sp.B01 salt tolerant enhancement activated sludge under high salt concentration. |
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