Rapid Cultivation Of Anammox Bacteria And Its Adaptation To High NO₂^–-N Concentration Inhibition

Anaerobic ammonia oxidation（anammox）is a novel technology of biological nitrogen removal,which save energy consumption of aeration and the cost of additional organic carbon sources,while simultaneously realizing advanced nitrogen removal.It was considered to be the most promising low-energy wastewater technology.However,anammox bacteria is typically autotrophic and grow slowly,making it difficult to rapidly enrich and retain anammox bacteria during the start-up period.The low growth rate has emerged as a main obstacle to its wide implementation in sewage treatment.The substrate of anammox is nitrite,but anammox was inhibited by high nitrite concentration.The anammox activity was decreased due to high nitrite concentration.It leads to the further increase of NO₂^–-N concentration in the system,resulting in stronger inhibition,which is one of the troubles in the stable operation of anammox system.To solve the above problems,the advantages of fast growth rate of free cells were used to culture the free-living anammox bacteria.The doubling time of anammox bacteria was shortened by cultivating free anammox bacteria the rapid cultivation of anammox bacteria was realized.Then,using the adaptive characteristics of microorganisms,the high NO₂^–-N concentration environment was setted up artificially to realize the rapid adaptation of anammox bacteria to the high NO₂^–-N concentration.Through this method,the system improved the resistance under the fluctuation of nitrite concentration,the operation stability of anammox system was improved.Based on the above research,the main conclusions are as follows:（1）Rapid cultivation of anammox bacteria by forming free cellsIn membrane bioreactor（MBR）,anammox bacteria was inoculated to start MBR successfully.The nitrogen removal performance of anammox MBR,characteristics of sludge morphology and growth of anammox bacteria were investigated.The results showed that free anammox bacteria could be rapidly cultivated by inoculating floc anammox bacteria.When the influent ammonia/nitrite ratio was 1:1,the nitrogen removal rate（NRR）increased to 0.116 g-N·L^-1·d^-1,and a good nitrogen removal performance was achieved.Free-living anammox bacteria were successfully cultured in MBR,and more than 95%of them were less than 50μm in diameter.The results of quantitative polymerase chain reaction showed that the maximum specific growth rate of free-living anammox bacteria was reduced to 0.12d^-1,and the doubling time was shortened to 5.68 d.The rapid growth of anammox bacteria was realized through the cultivation of free-living bacteria.The key functional genes of free-living anammox bacteria were identified and expressed using metagenomic and metaproteomic analyses.The four candidate genera,Candidatus Kuenenia,Candidatus Scalindua,Candidatus Brocadia,and Candidatus Jettenia,were found in the free-living anammox sludge.Among them,the relative abundance of Candidatus Brocadia in the free-living anammox was 8.83%.At the species level,Candidatus Brocadia sinica is the main component of anammox bacteria,with a relative abundance of 8.18%.This outcome may be because Candidatus Brocadia sinica grows more effectively in free living cells compared to other species.Through metagenome and metaproteome analysis,the key functional genes and proteins in anammox reaction,including hydrazine synthesis,hydrazine dehydrogenase and hydrazine oxidatoreductase,were encoded and expressed.（2）Adaptation of anammox bacteria cultured by nitrite treatment in side-stream unitIn order to enhance the stability of the system when the nitrite concentration fluctuated,anammox up-flow anaerobic sludge bed（UASB）reactor was operated with a side-stream unit,part of the sludge in the reactor was treated with nitrite in the side-stream unit t and then returned to the reactor.The effect of nitrite treatment on the stable operation of UASB reactor was investigated by increasing nitrite concentration in the side-stream unit.The results showed that the activity of anammox bacteria with nitrite treatment in the side-stream unit was 7-8.5 times higher than that without nitrite treatment in the environment of free nitrous acid was 2.25μg HNO₂^–-N·L^-1.The influent nitrite concentration of UASB suddenly increased from 80 mg·L^-1 to 200 mg·L^-1.After five hours of continuous monitoring,it was found that the decline rates of NRR in UASB without side-stream unit was 2.1-2.7 times that of UASB with side-stream unit.Nitrite treatment improved the adaptability of anammox bacteria to high nitrite concentration,so that the operation of the reactor was more stable.Through the analysis of population distribution and diversity,the nitrite treatment in the side-stream unit changed the dominant population,and Candidatus Jettenia showed a stronger adaptability to high concentration of nitrite.