The Novel Biology Mechanism And Reduction Of Ammonia Inhibition In Anaerobic Digestion And The Deep Removal Of Ammonia

With the growth of population and the acceleration of urbanization,the discharge of organic solid waste has increased rapidly.Anaerobic digestion is widely used for treating organic waste because it can simultaneously achieve organic contamination control and energy recovery.The biochemical mechanism of anaerobic digestion must include the entry of organic matter from the outside of active microbial cell membrane,and the enzyme synthesis by the intracellular process of transcription and translation,and the conversion of the organic matrices into methane via the catalyzation of enzymes.In view of the common thorny problem of high ammonia inhibiting anaerobic digestion,the novel biology mechanism of ammonia influencing the community of active microorganisms,cellular membrane,intracellular enzyme synthesis and key enzymes in anaerobic digestion was firstly elucidated by the integrated metagenomic and metaproteomic analyses in this thesis.Based on this,a new method of in-situ reducing ammonia inhibition to anaerobic digestion based on the regulation of microbial biology was proposed.Finally,a new combined process of"recovery and reuse-selective oxidation"was established to achieve the deep removal of ammonia in anaerobic digestion liquor.This thesis firstly reported a novel biology mechanism of the active microbial community,cellular membrane,intracellular enzyme synthesis and key enzymes in anaerobic digestion.The integrated metagenomic and metaproteomic analyses showed that high ammonia（2100 mg/L）obviously changed the population structure of active microorganisms.The obligate acetotrophic methanogens（e.g.,Methanosaeta）and the syntrophic propionate and butyrate oxidation bacteria（such as Pelotomaculum）along with their assistant taxa（e.g.,Desulfovibrio）was significantly reduced.The total population of hydrolyzing and acidifying microbes change insignificantly,but the hydrolysis-acidification bacteria with ammonia resistance were largely enriched,which became the dominant bacteria capable of hydrolysis and acidification.The reasons for the enrichment may be related to the up-regulation of key genes and proteins that regulate microbial carbohydrate metabolism,glycan biosynthesis and metabolism,genetic information processing and membrane transport.In addition,high ammonia nitrogen impaired membrane integrity and increased permeability of the representative methanogen（Methanosarcina barkei）,thereby probably damaging cells and interfering normal intracellular substance transformation and metabolism processes.Further studies revealed that the expressions of proteins that regulate the synthesis of intracellular enzymes is significantly inhibited,i.e.,the expressions of the RNA polymerase（subunits A’and D）for transcription and the ribosomal large subunits（L3,L12,L13,L22 and L25）and small subunits（S3,S3Ae,and S7）and the aminoacyl-t RNA synthetase such as aspartate-t RNA synthetase for translation were obviously down-regulated.Meanwhile,it was found that the expressions of key enzymes regulating propionate and butyrate oxidation such as methylmalonyl-Co A mutase and the key enzymes of acetate methanogenesis such as CH₃-Co M reductase were significantly down-regulated under ammonia stress.According to the biological mechanism obtained above,this study proposed a new method to reduce ammonia toxicity in anaerobic digestion based on the regulation of microbial biology,i.e.,the in-situ addition of trace reduced-glutathione significantly decreased the inhibition of ammonia to anaerobic digestion.The results showed that the dosage of 5 mg/g VS of glutathione was suitable.At this dose,the methane production in the ammonia-suppressing anaerobic digestion reactor increased by 42.0%,which was almost the same as the methane yield in the non-ammonia-inhibiting group.Further studies suggested that although glutathione itself can be converted to a few methane,this contributes negligibly to the total methane yield during the reduction of ammonia toxicity by glutathione.And the detoxification effect is neither due to the alleviation of ammonia-induced intracellular proton imbalance and potassium loss,nor the traditional function of glutathione quenching of reactive oxygen species.This study has found that the detoxification effect was ascribed to the abundant enrichment of active vulnerable microorganisms under ammonia pressure,i.e.,acetotrophic methanogens such as Methanosaeta,propionic acid and butyric acid oxidizing bacteria such as Pelotomaculum and their assistant genera Desulfovibrio.And glutathione protected the integrity and permeability of the cell membrane under ammonia stress,which favored the normal transformation and metabolism of intracellular substances.Moreover,it was found that in the process of detoxification,the expressions of key proteins that drive intracellular enzyme synthesis such as RNA polymerase,ribosomal protein and aminoacyl-t RNA synthesis were significantly upregulated.Meanwhile,the intracellular iron level that is conducive to enzyme synthesis was significantly increased,which was related to the up-regulation of the expression of iron transmembrane transporter,and all the above conditions were beneficial to reduce the inhibition of high ammonia on enzyme synthetase of microbial cells.Especially,the expressions of vital enzymes which catalyze propionate and butyrate oxidation（methylmalonyl-Co A mutase,succinyl-Co A synthase（ADP-forming）,succinate dehydrogenase（menaquinone）,pyruvate oxidoreductase and acetyl-Co A C-acetyltransferase）and acetate methanogenesis（CO dehydrogenase-acetyl-Co A synthase complex and tetrahydromethanopterin methyltransferase）were obviously up-regulated.In order to deeply remove the high ammonia nitrogen in the digested liquid after anaerobic digestion,a new combined process of"recovery and reuse-selective oxidation"was established in this thesis.Firstly,the crystallization method of magnesium ammonium phosphate（the molar ratio of Mg:NH₄⁺-N:P=1.8:1:1）was to recover most ammonia.Then,photosynthetic bacteria were used to further recover ammonia nitrogen,and remove most of the COD at the same time,and synthesize microbial protein.Finally,the above-mentioned digestive solution is further processed by the method of"ultraviolet-chlorine-persulfate",so that residual ammonia is selectively oxidized to nitrogen gas.The final ammonia concentration was decreased to 1.6 mg/L,the concentrations of nitrate and nitrite were 5.6 and 0.1 mg/L,respectively,and the proportion of target product（N₂）reached 75.3%.With the treatment of this new technology,the major water quality index such as total nitrogen,COD and total phosphorus in the digested effluent all reached the Class A standard of"Pollutant Discharge Standards for Urban Sewage Treatment Plants"（GB18918-2002）.It can be seen that the"recovery and reuse-selective oxidation"process can obtain struvite and microbial protein products while achieving the deep removal of ammonia in the digested effluent,and the terminal nitrogen emissions are mostly environment-friendly N₂ with small secondary pollution.