Influence Mechanism Of Small-molecule Carbon Sources On The Heterotrophic Nitrification-aerobic Denitrification Process

As a novel biological nitrogen removal pathway,the driven mechanisms of heterotrophic nitrification-aerobic denitrification（HNAD）is still unclear.In this study,a strain with the capability of HNAD,named ZHL01,was isolated from the activated sludge of Jiang Cun Gou landfill leachate wastewater treatment plant（Xi’an City,Shaanxi Province,China）.The strain was identified as Acinetobacter johnsonii by 16S r DNA.The effects of C/N ratio,p H value,dissolved oxygen（DO）concentration,temperature,nitrogen source and carbon source on the growth and nitrogen removal performance of Acinetobacter johnsonii ZHL01 were investigated by the single factor experiment.Besides,the final destinations of nitrogen in the HNAD process catalized by Acinetobacter johnsonii ZHL01 were analyzed with the nitrogen balance experiment.Eventually,the influencing mechanisms of small molecular carbon sources on the nitrogen removal performance of Acinetobacter johnsonii ZHL01 was analyzed from the perspective of enzyme activity,electron transport and genomics.The following conclusions are drawn:（1）When the C/N ratio was 20,p H value was 8,the shaker speed was 180 rpm,and the temperature was 30℃,Acinetobacter johnsonii ZHL01 achieved the optimum nitrogen removal performance.Under these conditions,the maximum OD₆₀₀ was 0.982±0.011,and the maximum TN removal efficiency was 89.48±1.04%.The denitrification capability of Acinetobacter johnsonii ZHL01 was weak,however,Acinetobacter johnsonii ZHL01 had a relatively strong assimilation capability,and the assimilation efficiency was 58.29±1.05%.（2）When ammonia nitrogen（NH₄⁺-N）was employed as the sole nitrogen source,the maximum nitrogen removal efficiency was 79.21±0.49%,and the maximum removal rate was5.48±0.05 mg/（L·h）.Meanwhile,there was no obvious accumulation of hydroxylamine（NH₂OH）,nitrite（NO₂^-）,and nitrate（NO₃^-）.When nitrite nitrogen（NO₂^--N）or nitrate nitrogen（NO₃^--N）was utilized as the sole nitrogen source,the corresponding maximum NO₂^--N and NO₃^--N removal efficiencies were 41.81±0.30%and 40.16±0.58%respectively,and the maximum removal rates of NO₂^--N and NO₃^--N were 2.85±0.20 mg/（L·h）and 3.04±0.15mg/（L·h）.（3）When sodium citrate（SC）,sodium acetate（SA）,sodium fumarate（SF）and sodium succinate（SS）were used as the carbon source,the sequences of the averaged degradation rates of NH₄⁺-N and COD was SA>SS>SF>SC at the beginning 12 h,however,the sequences of the averaged degradation rates of NH₄⁺-N and COD was SS>SF>SA>SC in 24 h.（4）When SC was used as the carbon source,Acinetobacter johnsonii ZHL01 tended to metabolize the carbon source with the glyoxylate cycle.When SA was employed as the carbon source,Acinetobacter johnsonii ZHL01 tended to metabolize the carbon source with the glyoxylate cycle and the tricarboxylic acid（TCA）cycle.However,when the carbon sources were SF and SS respectively,Acinetobacter johnsonii ZHL01 tended to metabolize the carbon source with the TCA cycle.Moreover,when metabolizing SS,the activity of Isocitric dehydrogenase（ICDH）was higher than that when the carbon source was SF,and this may be the main reason why the metabolic performance of SS was better than that of SF.Furthermore,the metabolic differences between different carbon sources resulted in the variation of intracellular reduced nicotinamide adenine dinucleotide（NADH）concentration and NADH/nicotinamide adenine dinucleotide（NAD⁺）ratio,and the overhigh NADH concentration and NADH/NAD⁺ratio were probably responsible for the occurrence of HNAD process.（5）The nitrogen metabolic pathway of Acinetobacter johnsonii ZHL01,NH₄⁺-N→NH₂OH-N→NO was quite unique.NO could be oxidized to NO₃^--N by nitric oxide dioxygenase（NOD）,and NO₃^-was reduced to NH₄⁺by nitrate assimilation reductase（NAS）and nitrite dissimilatory reductase（NIR）.The unique nitrogen metabolic pathway may be the main reason why Acinetobacter johnsonii ZHL01 had a relatively strong assimilation capability.