Study On Light Response Characteristics Of Nitrifying Bacterial Populations And Their Oxidative Stress Mechanisms

Partial nitrification,as a new biological denitrification technology,shows strong advantages in reducing costs and simplifying the denitrification process in wastewater treatment,while it can be further combined with the anaerobic ammonia oxidation process（Anammox）to achieve autotrophic deamination,so it has broad development and practical application prospects.The enrichment of ammonia oxidizing bacteria（AOB）and maximum inhibition of nitrite oxidizing bacteria（NOB）in the system as much as possible,so that AOB becomes the dominant species in the whole reaction stage and NOB is eluted out of the system is the key to achieve partial nitrification.Although various NOB selective inhibition strategies have been shown to be feasible in achieving partial nitrification,the emergence of NOB adaptation has led to their poor long-term inhibition of NOB.Moreover,most of the mechanisms of NOB inhibition by inhibition strategies stop at the effects on NOB transcriptional genes and enzyme activities,and there is a lack of further investigation into the causes of these phenomena.In this paper,we investigated the photoresponse characteristics and inhibition kinetics of two typical nitrifying bacteria to low-dose ultraviolet（UVA）irradiation,and revealed the oxidative stress effect and response mechanism of AOB and NOB induced by UVA irradiation,and further applied oxidative stress theory to analyze the inhibition mechanism of free nitrite（FNA）on nitrifying bacteria.The mechanism of free nitrite（FNA）inhibition on nitrifying bacteria was further analyzed by applying oxidative stress theory,aiming to provide theoretical guidance to reveal the mechanism of NOB selective inhibition strategy from the perspective of oxidative stress.The main conclusions are as follows.（1）With the continuous feed of substrate flow,free ammonia（FA）and FNA inhibition combined with low dissolved oxygen control strategy could successfully enrich the AOB enrichment culture with Nitrosomonas as the dominant genus（50.89%relative abundance）,in which NOB was effectively eluted out of the system（less than 0.01%）.The same continuous water feeding method of substrate flow addition was adopted to control the relatively low concentration of nitrite in the reactor,and the enriched culture of Nitrosomonas spp.（Nitrospira）was achieved（39.05%relative abundance）,which was the absolutely dominant species in the reactor system.（2）The effects of UVA irradiation intensity and irradiation time on AOB activity were relatively small and were able to significantly promote AOB activity at a shorter exposure time（15 min）.On the contrary,the increase of both UVA irradiation intensity and irradiation time significantly intensified the inhibitory effect on NOB.Under UVA irradiation conditions,NOB exhibited a more sensitive decay rate（b-value）than AOB,and increasing the UVA irradiation intensity showed an increasing trend in b-value for both bacteria.When the UVA irradiation intensity was increased from 8 to 46μmol·m^-2·s^-1,the decay rate of AOB increased from 0.0174 to 0.0213 min^-1and that of NOB from 0.0234 to 0.0599 min^-1.It was also found that the actual factors affecting the decay rates of AOB and NOB were the average light intensity in the system rather than the UVA irradiation intensity.（3）The inhibition mechanism of UVA irradiation on nitrifying bacteria is mainly to induce oxidative stress effect on nitrifying bacteria,i.e.,to stimulate the elevation of intracellular reactive oxygen species（ROS）in nitrifying bacteria,which causes oxidative damage to the cell membrane,prompting sludge morphological destruction and intracellular material release,and eventually leading to bacterial death.Under UVA stress,NOB tended to produce more ROS than AOB,resulting in a stronger photoinhibition response than AOB under the same light conditions.（4）After exposure to UVA,catalase（CAT）of AOB functioned normally,while CAT of NOB showed little activity,resulting in the inability of NOB to metabolize the overproduced H2O2 and accumulate intracellularly,exposing them to severe oxidative stress stress hazards,which is an important reason for the selective inhibition of NOB.In addition,under UVA irradiation stress,nitrobacteria would initiate protective mechanisms,including the secretion of more extracellular polymers（EPS）,especially proteins,to promote bacterial aggregation to form a shading effect,and the upregulation of superoxide dismutase（SOD）activity to reduce the damage of AOB and NOB by oxidative stress.（5）FNA can also induce oxidative stress in nitrobacteria,resulting in a surge of intracellular ROS,damage to cell membrane integrity,release of intracellular substances,and reduction of key enzyme activities（AMO and NXR）,etc.AOB can maintain intracellular H₂O₂levels through SOD and CAT,while NOB cannot further convert H₂O₂through CAT leading to its continuous accumulation in the cell and contributing to NOB’s demise.The inhibition of NOB activity was more pronounced than that of AOB,which exhibited a greater increase in b-value.