| Anaerobic ammonium oxidation(Anammox)process has been considered as an environment-friendly alternative for nitrogen removal due to its high removal efficiency and low energy consumption.Based on the physiological characteristics of anammox bacteria,low/normal/high nitrogen loading rate(NLR)and salt-tolerance typical anammox process can be developed.Up to now,the anammox process has been successfully implication in special cases,but it is still facing severe challenges in the operation of typic anammox process:1)The effluent total nitrogen of anammox process under low NLR is difficult to meet the discharge standard;2)The emission characteristics and generation mechanism of N2O in anammox process under normal NLR are not well understood;3)The performance of anammox process under high NLR remained to be demonstrated after being inhibited with the substrate concentration decreasing;4)The performance of salt-tolerance anammox process under mixed salt remains poor described.This study aimed to study the characteristics and microbial mechanism of low/normal/high nitrogen loading rate(NLR)and salt-tolerance typical anammox process using the environmental engineering methods combined with the microbiome technology.The major results are as follows:1)The mechanism of high effluent nitrate at low NLR anammox process was revealed,and the enhancement strategy was studied.The performance of low NLR anammox granular sludge reactor was investigated.The results showed that the production of nitrate was significantly higher than the predicted value from anammox reaction stoichiometry.The total nitrogen(TN)removal efficiency was only 57.9±8.0%.which resulted in the TN in the effluent being up to 21.4 mg·L-1.which was beyond the TN value of 15 mg·L-1 in grade A of discharge standard(GB18918-2002).Metagenomic analysis and in-situ batch assays revealed that the higher nitrate production was not solely due to obligate NOB,but other nxr-containing bacteria(diverse NOB)are important contributors as well.The integrated granular sludge and fixed-biofilm(iGB)reactor to carry out anammox/partial denitrification(A/PD)was developed to enhance the total nitrogen removal ability of low NLR anammox process.Excellent effluent quality of～3 mg·L-1 of total effluent nitrogen was obtained,which even meets the TN value of 10 mg·L-1 in the strictest integrated wastewater discharge standard in China(DB31/199-2018).Combined metatranscriptomic and metagenomic analyses showed that DNRA,anammox,and partial denitrification(PD)were coupled in both the granules and fixed-biofilm portions of the reactor.The batch assays and mass balance evaluation found that PD and DNRA were responsible for 50%and 25%of nitrate reduction,respectively,in the reactor which is critical for polishing TN in the effluent.Kinetic assays and mass balance evaluation found the majority of the TN(approximately 78%)was removed by anammox granules,while the fixed-biofilm was responsible for the removal of an additional approximately 16%of the total TN.This suggested that the granules with high nitrogen removal activity are the dominant site of the NRR in the reactor while the fixed-biofilm is critical to the improvement of effluent quality.2)The ecological and physiological mechanism of N2O emission at normal NLR anammox process was revealed,and the enhancement strategy of "carbon" emission reduction was studied.The performance of nitrogen removal and N2O production of low NLR anammox granular sludge reactor was investigated.Results showed that the N2O emission factor(EF_N2O)of normal NLR anammox granular sludge reactor was up to 0.6±0.1%.The batch experiments found that the granule was the main contributor to nitrogen removal(～95%),while the flocs contributed only～5%nitrogen removal and contributed～60%of the total N2O production.metagenomics and fed-batch experiments found that NO,which accumulated in the reactor,could lead to N2O production from both granules and flocs.However,since anammox bacteria,which were shown to be in higher abundance in granules than in flocs,have the capacity to scavenge NO this provides a mechanism by which its inhibitory effects can be mitigated.limiting N2O release from the granules.The strategy of optimizing the biomass ratio of flocs and granules in a normal NLR anammox granular sludge reactor to enhance the "carbon"emission reduction was studied.Results showed that when the biomass of flocs decreased to～2%,the EF_N2O was significantly decreased to 0.1%at a stable nitrogen removal rate,which was lower than the reported values.3)The physiological mechanism of substrate inhibition at high NLR anammox process was revealed,and the restored strategy of nitrogen removal was studied.The performance of high NLR anammox granular sludge reactor was investigated.Results showed that the threshold substrate concentration of high NLR anammox granular sludge reactor is 400 mg·L-1 NH4+-N and 480 mg·L-1NO2--N,the NLR is 14.4±0.6 kg·m-3.d-1.The sludge nitrogen removal rate calculation found that the sludge activity can’t be restored after high substrate concentration inhibition even if the substrate concentration decreased back.Nano-CT scanning predicted that the anammox granular sludge has effective stability and mass transfer.Microforce measurement and kinetic assays confirmed that the high substate concentration inhibition has no impact on the stability and mass transfer of granules.Combined metatranscriptomic and metagenomic analyses found that the expression of hydrazine synthetase γ subunit gene(hzsC),which is encoded by the dominate anammox bacteria(Ca.Kuenenia stuttgartiensis),was significantly decreased in inhibited sludge.While the expression of the key genes for energy production,such as the hydrazine dehydrogenase gene(hdh)and nitrite oxidoreductase gene(nxr),were exceptionally increased,indicating the bacteria was energy starved.The strategy of exogenously adding NH2OH in the inhibited reactor to enhance the activity restored was studied.Results showed that 1 mg-N·L-1 NH2OH addition can restore the efficiency of the reactor in 15 days while the control is still inhibited.Moreover,the performance is kept stable when there is no exogenously NH2OH addition.4)The new anammox species affiliated with the Ca.Jettenia was identified at a salttolerance anammox reactor with chlorides and sulfates,and the salt-tolerance mechanism of anammox bacteria was revealed.The performance of salt-tolerance anammox granular sludge reactor under mixed salt was investigated.Results showed that the FAB-Anammox reactor could tolerate the 3%salinity with mixed chlorides and sulfates,reaching the highest salinity in previous report.In the middle salinity stage(1.0%-3.0%),the performance of the reactor was declined first and restored gradually.Combined metatranscriptomic and metagenomic analyses found that a new anammox species affiliated with Ca.Jettenia,who encoded trehalose synthesis genes(otsB and treS)and reactive oxygen species(ROS)defensive genes(sodB and kat).JC-1 staining confirmed that salt stress can cause anammox bacteria biofilm damage and ROS production,and induce oxidative stress.Batch experiments confirmed that the anammox bacteria could defend against the osmotic stress and ROS stress induced by salt through increasing the ROS defensive level and the intracellular trehalose. |
