Mechanism Of The Natural Pyrite-Based Extracellular Electron Transfer Enhancing Constructed Wetland-Microbial Fuel Cell

Constructed wetlands are a environmental friendly,green ecological wastewater treatment technology,and have been widely applied in decentrialized wastewater treatment.To solve the problems of the limited oxidation-reduction processes in the hypoxic environment of the system,and the consequently poor treatment performance,this thesis proposed a novel technology,namely the natural pyrite-based extracellular electron transfer enhancing constructed wetland-microbial fuel cell（CW-MFC）.The treatment performance and the electricity recovery of the system were examined in the first place.Then,the mechanism of organics transformation regulated by the external circuit was explored.Moreover,the underlying magnification of electroactive scope of anode,as well as the iron redox cycling was revealed.Besides,the functional microbe of long distance electron transfer in the anodic zone was identified.Furthermore,the sulfur metabolism mediated by the bioelectrochemical processes was illustrated by the metagenomic analysis,with lights sheded on the elemental inter-cycling of carbon,nitrogen,and sulfur.Lastly,the removal patterns of nitrogen and phosphorous under the functions of wetland plant-microbes was discussed.The major conclusions were as follows.（i）Anodic pyrite filling material can enhance the treatment performance and electricity recovery for the CW-MFC.The experiment group filling pyrite in the anodic zone（Py Ano）showed higher removal of COD,NH₄⁺-N,TN,and TP（86.7±7.2%,32.5±2.7%,36.7±3.6%,and 20.1±6.3%,respectively）than the group filling pyrite in both the anodic and cathodic zone（PS;59.9±7.4%,25.7±2.1%,30.2±3.4%,and 46.6±5.8%,respectively）and that filling quart sand（QC;72.6±6.5%,20.1±2.7%,25.0±4.0%,and 7.0±5.1%,respectively）.Meanwhile,the Py Ano showed superior electricity traits than QC;its average voltage output,coulombic efficiency,maximum power density,were 416.0±65.4 m V,0.42%,22.0 m W/m²,respectively.Though the PS showed higher coulombic efficiency（0.46%）and maximum power density（27.8 m W/m²）,the divided current in the inner system and the competitive inhibition to the aerobic COD degradation denied its overall performance.The Py Ano CW-MFCs showed adventages to some distance when comparing with other strengthening constructed wetlands and other CW-MFC.（ii）The external circuit might function as a regulator for the organics transformation in the CW-MFC.It was found that the connection of the external circuit could enhance the COD removal in different systems,particularly during the first 12 h,when the external circuit increased the COD removal by 9–13.5%.Though the Py Ano showed retarded COD removal during the first hours,it surpassed that of the QC in the later hours.The consumption of influent acetate was in a similar pattern with the COD diminishment;at 24 h,the influent acetate was depleted,the humic-like and protein/amino acid-like dissolved organic metters appared in the meantime.The CO₂flux of the systems showed obvious day-and-night pattern,while the CH₄ accumulated gradually.The connection of the external circuit would increase the CO₂ flux with decreasing CH₄ accumulation.Meanwhile,the CO₂ and CH₄ flux in the Py Ano was higher than that in the QC.Insterestingly,the gaseous carbon metabolites was far less than the consumption of the organic substrate;according to the accumulation of the poly-β-hydroxybutyricacid,the connection of the external circuit might facilitate the microbial assimilation of organics,which might explain the low coulombic efficiency that has been documented in many studies.（iii）The utilization of pyrite filling material magnified the anodic zone,and closes the iron redox cycle.Based on the MFC culturing experiments,the pyrite filling material collected from the Py Ano showed higher electron donating potential than the quartz sand from the QC,indicating that the pyrite could enlange the electroactive scope of anode.The comparison with the autoclaved group further evidenced the possible electrochemical and bioelectrochemical oxidation of pyrite,demonstrating that the occurrence of the oxidation of pyrite in the anodic zone of Py Ano.Nevertheless,the results of microbial dissimilatory iron reducing potential indicated that the microorganisms in the Py Ano might have higher iron reducing potential,this closed the iron redox cycle in the system.According to the XPS results,the pyrite filling material in the anodic and cathodic zones showed different oxidation states,with the anodic one being moderately and the cathode one being thoroughly oxidized.This supplementarily evidence the iron redox cycle in the system.The results of high throughput sequencing of the 16 S r RNA showed that an exoelectrogens,Geobacter,was enriched by the anodic pyrite,which suggested that the pyrite could facilitate the bioelectrochemical process,either by anode magnification or by iron redox cycling,and explained its higher treatment performance and electricity recovery.（iv）The anodic pyrite filling material induced the sulfur metabolism within the microbial long distance electron transfer,as well as the elemental intercycling between the carbon,nitrogen,and sulfur.By utilizing the stereoscopic microscope,scaning electron microscope,and laser scaning confocal microscopy,the apparence and morphology of the filamentous bacerteria potentially executing the microbial long distance electron transfer was observed.Therein,a fluorescence Desulfobulbaceae-specific oligonucleotide probe was used to carry out the fluorescence in-situ hybridization,and it was found a direct evidence for the cable bacteria.According the the analyses of the composition and difference of the microbial community,it was found that the candidate families of long distance electron transfer（Desulfobulbaceae）and extracellular electron transfer（Geobacteraceae）,were enriched in the anodic zone of the Py Ano.According the interspecies network,the cable bacteria might co-exist with electrogens/dissimilatory iron reducer（e.g.Geobacteria）,sulfate reducer（e.g.Desulfuromonas）,sulfur oxidizer（e.g.Sedimenticola）,syntrophic acidogens（e.g.unclassified Candidatus Aminicenantes）,heterotrophic organics oxidizer（e.g.unclassified Anaerolineales）,and methanogens（e.g.Methanosarcina）.The sulfur metabolism pathways was revealed by the metagenomic analyses,and the results suggested that the anodic pyrite in the Py Ano enhanced the redox processes between the sulfide and elemental sulfur,the reduction of thiosulfate,and the oxidation of the sulfite.（v）The pirite filling material facilitated the growth of the wetland plants,and consequently inproved the removal of nitrogen and phosphorous by the plant-microorganism system.As revealed by the differences of plant biomass and their nitrogen and phosphorous contents,the Py Ano was found to enhance the the plant growth and their uptake of nutrient contaminants.The photosynthetic pigments were measured,but there was no difference between Py Ano and QC.However,the Py Ano plants were found to have higher radius oxygen loss than the QC,which might account for the higher ammonium oxidation in the cathode zone.A DCB extracted method was adopted to evaluate the iron plaque on the roots.The results showed that the plants in the Py Ano immobilized higher contents of iron and phosphorous on the root surface as consequence of iron plaque.This explained the higher TP removal and minor iron ions detected in the effluent.To conclude,this thesis put forward a Py Ano CW-MFC configuration,which can achieve significant treatment performance and energy recovery.The underlying mechanisms,including i)the regulation of organic carbon by external circuit,ii)the iron redox cycle governed by the microbial extracellular electran transfer,and iii)the long-distance-electron-transfer mediated microbial sulfur cycle,will contribute to the understanding of indepth elemental cycles interaction.