Mechanism Of Pyrite-enhanced Anammox

Anaerobic ammonium oxidation（anammox）represents an energy-efficient process.However,the low microbial growth rate of bacteria,accumulation of nitrate by-product during the process,and the inhibition of heavy metal limited its widespread engineering applications.Pyrite,a semi-conductive iron-bearing sulfide mineral,is abundant with reducing Fe and S.It is expected to promote the application of anammox technology by enhancing the electron transfer process,reducing NO₃^-and alleviating the inhibition of heavy metals.Accordingly,we constructed the pyrite（Fe S₂）-enhanced anammox system to improve nitrogen removal performance by coupling the mineral-promoted electron transfer process,iron-and sulfur-based denitrification and detoxification of heavy metals.The mechanism was revealed.This study provides the theoretical basis for the popularization and application of anammox technology.The main contents and results are as follows:（1）The characteristics of the recovery process of the anammox reactor under long-term（144 days）starvation were clarified,and the method of improving the anammox activity by selecting pyrite as an additive was first proposed and established.It was found that abundant extracellular polymers substance（EPS）and stable expression of nir S,hzs A and hdh genes enabled the anammox bacteria to maintain cell integrity and anammox activity to resist long-term starvation.During starvation,the heterotrophic denitrifiers used EPS and bacterial lysates as carbon sources to provide CO₂ for the growth of anammox bacteria.Candidatus Kuenenia showed better resistance to starvation,whose relative abundance increased by 86%after 30 days of recovery.The effect of mediators on anammox activity was compared.As a result,pyrite(1.0 g L^-1)was selected to improve the nitrogen removal performance of the anammox reactor,SAA_ammonium and SAA_nitrite increased by 30.8%and 33.4%,respectively.（2）The study confirmed the mechanism of the Fe-N-S cycle in the pyrite-enhanced anammox process.In long-term operation,pyrite significantly increased the nitrogen removal rate of anammox by 52%.Physiological and biochemical analyses showed the improvement of electron transfer was attributed to the increase of cytochrome c and Fe-S proteins.Co-occurrence networks,nitrate-dependent Fe（II）oxidation bacteria,bacterial nutrient exchange,and Fe-,N-and S-related genes were analyzed based on multi-omics.The results showed that the nitrate removal was attributed to the Fe-N-S cycle driven by multiple nitrogen removal pathways.（3）The interaction mechanism between pyrite dissolution and anammox sludge was analyzed.The“dissolution-aggregation-promotion”mechanism of pyrite-anammox sludge was elucidated.Physicochemical and electrochemical analyses showed that the anammox sludge promoted pyrite dissolution.As a result,the roughness and specific surface area of pyrite increased by 174%and 17.5%,respectively,and its self-corrosion potential decreased from 0.127 V to-0.076 V.The dialysis bag was used to block the pyrite and explicate the enhancement of dissolved products on anammox.The dissolved product promoted sludge aggregation by EPS,i.e.,reducing its repulsive force and improving its adhesion.The results showed that the dissolved products increased the nitrogen removal rate by 21.2%.（4）The contribution of pyrite to nitrogen removal through multiple pathways in anammox system was quantified.The autotrophic denitrification,chemical denitrification and heterotrophic denitrification were enhanced in pyrite-induced complex system.The isotopic tracer results showed that pyrite not only enhanced anammox,but also promoted denitrification and Feammox,contributing 80.22%,19.53%and 0.25%to total nitrogen removal,respectively.Dosing with nitrite,pyrite addition increased the nitrogen removal by 14%.The contribution of autotrophic denitrification,chemical denitrification and heterotrophic denitrification to nitrogen removal was 34%,33%and 18%,respectively.Dosing with nitrate,pyrite addition increased the nitrogen removal by 19%.The contribution of heterotrophic denitrification to nitrogen removal increased from 5%to 18%,and the contribution of autotrophic denitrification to nitrogen removal increased from 23%to 30%.（5）The mechanism of pyrite-enhanced recovery of damaged anammox bacteria were studied.The nitrogen and Cr（Ⅵ）removal were attributed to the recovery of damaged anammox bacteria promoted by pyrite.The addition of pyrite changed the spatial and valence distribution of Cr in the anammox system,and 79%of them deposited on the sludge in the form of Cr（III）to alleviate the inhibition on anammox.Long-term operation showed that pyrite effectively prevented Cr（Ⅵ）impact,relieved Cr（Ⅵ）inhibition,and improved the anammox performance recover under Cr（Ⅵ）exposure.As a result,the nitrogen removal rate was increased by20 times.Physiological,biochemical and macro transcriptome analyses showed that,pyrite enhanced the nitrogen and Cr（Ⅵ）removal in anammox system by alleviating the damage of anammox cells,enhancing the substrate diffusion and electron transfer process,and improving the defense ability of anammox bacteria to Cr（Ⅵ）.