Study On Nitrification Efficiency And Glyoxylate Cycle By Heterotrophic Nitrication Bacteria

With the rapid development of coking industry,a large number of coking wastewater was discharged into the water,and which caused a series of increasingly serious problems on the water environment.Coking wastewater is a typical refractory organic wastewater,which with stable quality and complex components,and contains a variety of refractory organic pollutants,such as polycyclic aromatic compounds,phenol,benzene,pyridine,indole,quindine and sulfur-containing heterocyclic compounds,etc.Coking wastewater contains high concentration of ammonia nitrogen,which could cause water eutrophication and threat the ecological environment and human health.So ammoniam treatment is an essential key step of coking wastewater treatment.The new biological nitrogen removal technology would make up for the shortcomings of traditional nitrogen removal process,which is a more economic and efficient treatment of coking wastewater.Heterotrophic nitrification and aerobic denitrification further enrich the theory of biological nitrogen removal,thus the study of nitrogen removal performance and growth characteristics of heterotrophic nitrification bacteria would provide theoretical support and reference frame for the practical application of new biological nitrogen removal process.The two strains of heterotrophic nitrification bacteria for this experiment were screen separation and purification by laboratory,Alcaligenes faecalis C16 and Acinetobacter sp.Yl.By drawing the growth curve and ammonia oxidation curves of these two strains,we found that the growth and nitrogen removal performance were different between different heterotrophic nitrification bacteria.The growth of strain Y1 was more rapid and exuberant than strain C16,the two strains would get full growth in 24h and efficient degradation of ammonia nitrogen.The ammonium removal rate of strain C16 and strain Y1 in 48h reached 94.7%and 99%,respectively.In the ammonia oxidation process,strain C16 accumulated nitrite（NO₂^--N 14.442mg/L）and hydroxylamine（NH2OH-N 18.496mg/L）,while strain Y1 almost without accumulation of nitrite or hydroxylamine.The two strains both reflected aerobic denitrification,and the denitrification ability of strain Y1 was more capable than strain C16.In order to explore the characteristics of the key enzymes of glyoxylate cycle,the activities of iscocitrate lyase and malate synthase by strain C16 were determined with different conditions of physical and chemical.The results of the optimum temperature,pH and substrate concentration for the two enzymes would provide a theoretical basis for follow-up experiments.The optimum pH of isocitrate lyase is 7.7,optimum temperature is 30℃,and optimum substrate concentration is 1.8mmol/L isocitric acid.While the optimum pH of malate synthase is 8.0,optimum temperature is 30 C,and optimum substrate concentration is 3mmol/L glyoxylic acid.Both the two enzymes were influenced by Mg²⁺.To obtain a better insight into the carbon sources dependency of heterophic nitrification bacterium,the growth,ammonium removal ability and the two key enzymes of glyoxylate cycle of strain C16 and Y1 grew with different carbon sources（glucose,glycerol,sucrose,succinic acid,ethanol,fumaric acid,malic acid,pyruvate,sodium acetate and sodium citrate）were examined.The results showed that strain C16 failed to grow on the medium with glucose,glycerol,sucrose or ethanol,and strain C16 got the best growth with fumaric acid as carbon source,OD₆₀₀ was 0.920,and the nitrification was accompanied by the growth of strain C16.Strain Y1 failed to grow on the medium with glucose or ethanol,and got the best growth with pyruvate as carbon source,OD₆₀₀ was 1.146,and grew but on nitrify in the medium with sucrose and glycerol as sole carbon source.With different carbon sources,the two strains both showed high activities of isocitrate lyase and malate synthase when the ammonium removal rate reached highest,and no activities of the two enzymes were detected when the strain grew without nitrification phase.The positive correlation relationship was found between the glyoxylate cycle and nitrification.In order to analysis the effects of metal ions on the growth,nitrification and glyoxylate cycle of heterotrophic nitrification bacteria,the culture medium were added with different concentrations of Mg²⁺,Fe²⁺,Mn²⁺,Cu²⁺ and Zn²⁺.The results showed that the growth and nitrification of heterotrophic nitrification bacteria were strongly depended on the type and concentration of metal ions.Mg²⁺ and Fe+2 promoted the growth and ammonium removal ability of strain C16 and Y1;while Cu²⁺ and Zn²⁺ inhibited the growth and nitrification of the two strains,and higher the concentration of Cu²⁺ and Zn²⁺,more significant the inhibitory action;low concentration of Mn²⁺ promoted the growth and nitrification of strain C16 but showed little effect on the strain Yl.To find the effects of metal ions on the glyoxylate cycle of heterotrophic nitrification bacteria,the ICL reaction and MS reaction were added into 0.1 mmol/L and 1 mmol/L Mg²⁺,Fe²⁺,Mn²⁺,Cu²⁺ and Zn²⁺,respectively,the activity of isocitrate lyase and malate synthase were determined.The results showed that Mg²⁺,Fe²⁺ and Mn²⁺ promoted the activity of key enzymes of the glyoxylate cycle by strain C16,and Cu²⁺,Zn inhibited the enzyme activity of strain C16;while Mg²⁺,Fe²⁺ promoted the activity of key enzymes of the glyoxylate cycle by strain Y1,Cu²⁺,Zn²⁺ and Mn²⁺ inhibited the enzyme activity of strain Y1 in varying degrees.To obtain a further study on the relationship between the glyoxylate cycle and nitrification of heterotrophic nitrification bacteria,the culture medium were consisted 1 mmol/L Mg²⁺,Fe²⁺,Mn²⁺,Cu²⁺and Zn²⁺,respectively,and with the original culture medium as the control.The ammonium removal rete and the activity of key enzymes of glyoxylate cycle were detected by strain C16 and Y1 at logarithmic growth phase.The results showed that the promoting effect of Mg²⁺ and Fe²⁺ were most significant,while Cu²⁺ inhibited the nitrification and glyoxylate cycle of the two strains.The two strains both showed higher activities of isocitrate lyase and malate synthase when the ammonium removal rate reached higher.The positive correlation relationship was found between the glyoxylate cycle and nitrification by heterotrophic nitrification bacteria.