Research On Iron Compounds For Enhancing Anaerobic Digestion Of Swine Manure And Resistance Gene Reduction And Its Microbiological Mechanisms

The enhancement of anaerobic digestion by iron compounds is currently a hot research topic in the word,and its reduction of antibiotic resistance genes（ARGs）has also attracted widespread attention.However,the mechanism of enhanced anaerobic digestion by different forms/valence iron compounds is unclear,and the effect on anaerobic digestion has not attracted enough attention.There is no systematic study on the enhancement of anaerobic digestion by different iron compounds,and the enhancement mechanisms of anaerobic digestion by different forms/valences of iron compounds were different.At present,most of the substrates related to the iron compounds enhanced anaerobic digestion were simulated substrates,sludge,etc.,and there are few studies on swine manure.Therefore,the aim of this study was to investigate the effects of different forms/valences of iron compounds（Fe⁰,FeCl₂,FeCl₃,Fe₂O₃,Fe₃O₄,Fe⁰,nano-Fe₂O₃,nano-Fe₃O₄）addition on methane production during AD of swine manure while also evaluating optimal iron compounds addition levels.In addition,the effects on ARGs,heavy metal resistance genes（MRGs）and transfer elements（MGEs）during the mesophilic anaerobic digestion of swine manure will be investigated.We try to compare and optimize the enhancement of the efficiency of swine manure anaerobic digestion through iron compounds addition in different forms and valences,and the main phase and microbial mechanisms of swine manure anaerobic digestion（hydrolysis phase,acidification phase,hydrogenesis and acetogenesis phase and methanogenesis phase）affected by the addition are clarified.And find out the major drivers of antibiotic resistance evolution during swine manure anaerobic digestion due to iron compounds addition.The batch experiments of adding Fe⁰ indicated that Fe⁰ improved the methane production by maximum 23.9%at 150 mmol/L through the chemical reaction and direct interspecies electron transfer（DIET）.Nonetheless,Fe⁰specially reduced the antibiotic inactivation of the aminoglycoside resistance genes and antibiotic target protection of tetracycline resistance genes.The batch experiments of adding nano-Fe⁰ indicated that nano-Fe⁰ improved the methane production by maximum 23.9%at 75mmol/L.And the appropriate concentration of nano-Fe⁰（75-150 mmol/L）could promote the degradation of propionic acid during the anaerobic digestion of swine manure.However,the high concentration of nano-Fe⁰（350 mmol/L）may damage cell membrane of the microorganisms,preventing the material exchange of microorganisms,and cause the suppression of methane production.The difference between the optimal concentration of the Fe⁰ and nano-Fe⁰ may have a great relationship with the particle size of the material.The bioavailability of metals is also important for microorganisms.The important role of microbial community on the dynamic changes of ARGs in the AD system response to Fe⁰ and nano-Fe⁰.Fe₂O₃ addition can improve the accumulative methane production in swine manure AD by 11.04%,as evinced by the 31.9%increase in maximum methane production rate in response to the addition 75 mmol of Fe₂O₃.And Fe2O3 did promote higher mcr A copy numbers and promote the reduction of ARGs.The addition of nano-Fe₂O₃ inhibits the methanogenesis process,but it can promote the reduction of ARGs.The inhibitory effect is mainly due to the attached nanoparticles on cell surface or nanoparticles internalization would direclty cause cell physical deformation,perforation and membrane or internal content disorganization,resulting in a decrease in methane production.The batch experiments of adding FeCl₂ and FeCl₃ indicated that they improved the methane production by maximum 23.9%and 7.3%respectively both at 5 mmol/L.FeCl₂ intensified the utilization of acetate and propionate by considerably enhancing H₂ utilization in microorganisms and DIET.And FeCl₃mainly enhances methanogenesis by strengthening key functional genes and increasing the abundance of key functional microorganisms.And the addition of FeCl₂ and FeCl₃ can strengthen the reduction of ARGs.The bacterial community played an important role in the evolution of ARGs（68.26%）,which were also affected by MRGs,mobile genetic elements（MGEs）,and environmental factors.Through the batch experiment of adding Fe₃O₄ and nano-Fe₃O₄,it was found that the optimal concentrations of the two iron oxides were 350 mmol/L and 75mmol/L,respectively,which increased the methane production by 16.1 and 6%.Improvement of the methane production could be attributed to the DIET enhancement of the propionate degradation and syntrophic acetate oxidation（SAO）pathway by adding Fe₃O₄.The functional genes and microbial community analysis indicated that magnetite enhanced the acetoclastic methanogenesis not hydrogenotrophic methanogenesis.Microbial community changes caused by magnetite well explained the ARGs fate.Nano-magnetite could improve the methane production due to the enhancement of DIET and Fe-S precipitation.Dynamics of microbial community was not significantly influenced by nano-magnetite.Microbial community dynamics contributed most to the fate of ARGs,which well explained the limited effects of nano-magnetite on the ARGs fate.The last batch experiment show that the different iron compounds can both enhanced the methanogenesis,and the increasing order of methanogenesis accumulation were:nano-Fe⁰（22.4%）>FeCl₂（20.7%）>Fe₂O₃（20.4%）>FeCl₃（19.7%）>Fe₃O₄（19.3%）>nano-Fe⁰（19.0%）>nano-Fe₃O₄（12.4%）.Analysis of the results of ARGs found that the addition of iron compounds can strengthen the reduction of ARGs,and the enhancement order were:Fe₂O₃>Fe₃O₄>Fe⁰NPs>FeCl₃>FeCl₂>Fe⁰.The reduction of MRGs and MGEs was also facilitated.This also shows that the changes of substrate and inoculum during the anaerobic digestion of swine manure may have a great influence on the changes of ARGs.In short,the addition of iron compounds is a feasible solution to enhance the anaerobic digestion of swine manure.The addition of iron compounds can not only increase methane production,but also promote the reduction of ARGs during anaerobic digestion.It also reveals that the succession of microbial community structure was the leading factor in the change of ARGs.The addition of iron compounds is feasible for the simultaneous strengthening of anaerobic digestion and the reduction of ARGs.