Study On Effects Of Inorganic Nitrogen Compounds On The Activity And Microbial Community Of Anaerobic Granular Sludge

With the development of industry and wastewater treatment technology,increasing reports on wastewater contained three kinds of inorganic nitrogen compounds(nitrate nitrogen,nitrite nitrogen and ammonia nitrogen)flowing into anaerobic zone were found.The three kinds of inorganic nitrogen compounds generally brought adverse impact to anaerobic process because of their inhibitiory effect on anaerobic microbes.So far researches on toxic effects of the three inorganic compounds to the activity of anaerobic microbes and microorganism communities are absent,and furthermore explanation on inhibitory mechanism is in need.As a consequence,it is important to research the inhibitiory effects of the three inorganic nitrogen compounds on normal operation of anaerobic process.In this thesis,three inorganic nitrogen compounds(ammonia nitrogen,nitrate nitrogen and nitrite nitrogen)were selected to explore its inhibitory effect on anaerobic microorganisms in mesophilic anaerobic granular sludge system.The specific methanogenic activity(SMA)and performance of anaerobic granular sludge was studied and analyzed systematically.The categories and variation of phospholipid fatty acid(PLFA)in anaerobic granular sludge microorganisms were also investigated.The variation of community structure in anaerobic microorganisms was studied in the end.The main conclusions are as follows:1.The inhibitory effect caused by nitrite nitrogen was apparent to the SMA of anaerobic granular sludge and would get more serious with increasing concentration.The EC50 of SMA was about 12 mg N·L-1.Nitrite nitrogen in high concentration(>30 mg N·L-1)could affect energy metabolism process and cytomembrane structure of microorganisms simultaneously while nitrite nitrogen in low concentration(<30 mg N·L-1)decreased ATP slightly.Co-enzyme F420 and Co-enzyme M in the sludge would be inhibited if the concentration of nitrite nitrogen was over 12 mg N·L-1.With the inhibition of nitrite nitrogen,C16:00,C18:00,C12:00 exhibited better flexibility than other PLFAs in microbes.The percentage of anaerobic bacteria and gram-positive bacterium marked by PLFA decreased with the increasing concentration of nitrite nitrogen.In addition,nitrite nitrogen could inhibit the diversity of PLFA in anaerobic bacteria.With the inhibition effect of nitrite nitrogen,dominant phylum in anaerobic bacteria changed from Actinobacteria to Bacteroidetes while dominant genus of methanogens had no variation.Bacteria genera with higher nitrite endurance and adaptability were Atopobium,Thauera,Sulfuricurvum,Geobacter,Arcobacter,Petrimonas,Bacteroides and Vulcanibacillus.Corresponding archaea genera were Methanosaeta,Methanobacterium,Methanolinea.Methanospirillum,Methanoculleus,Methanosphaera,Methanoregula.2.The inhibitory effect caused by nitrate nitrogen to SMA of anaerobic granular sludge exhibited similar model with nitrite nitrogen.EC50 of nitrate nitrogen to SMA was about 30 mg N·L-1.Nitrate nitrogen in high concentration(>60 mg N·L-1)could affect energy metabolism process and cytomembrane structure of microorganisms simultaneously.Nitrite nitrogen in moderate concentration(60 mg N·L-1)could destroy cytomembrane structure of microorganisms apparently with little ATP variation.Nitrite nitrogen in low concentration(<30 mg N·L-1)had slightly effect on the content of ATP merely.Nitrate nitrogen could inhibit the content of co-enzyme M but not co-enzyme F420.With the inhibition of nitrate nitrogen,some saturated fatty acids(C16:00,C17:00,C18:00,C19:00)and branched chain fatty acid(C14:0i,C16:0i,C7:0a)exhibited better flexibility than other PLFAs in microbes.The percentage of anaerobic bacteria and gram-positive bacterium marked by PLFA decreased with the increasing concentration of nitrate nitrogen.In addition,nitrate nitrogen above 30 mg N·L-1 could inhibit the diversity of PLFA in anaerobic bacteria.With the inhibition of nitrate nitrogen,dominant phylum of anaerobic bacteria and genera of methenogenesis bacteria remained unchanged whereas relative content changed.Bacteria genera with higher nitrate endurance and adaptability were Paludibacter,Bacteroides,Desulfomonile,Thauera,Pseudomonas,Sphingomonas,Desulfuromonas,Wolinella,Levilinea,Bellilinea.Corresponding archaea genera were Methanosaeta,Methanobacterium,Methanocorpusculum.3.Effect of ammonia nitrogen to SMA exhibited a tendency of decline with increasing concentration.EC50 of ammonia nitrogen to SMA was about 3000 mg N·L-1.The inhibitory effect of ammonia nitrogen to anaerobic microorganisms only affected energy metabolism process of microorganism with a steady live bacteria proportion.EC50 of ammonia nitrogen to ATP was about 1200 mg N·L-1.Ammonia nitrogen could inhibit co-enzyme F420 and co-enzyme M in anaerobic sludge simultaneously,but the inhibitory effect to co-enzyme M only happened when the concentration of ammonia nitrogen was above 600 mg N·L-1.With the inhibition of ammonia nitrogen,some saturated fatty acids(C16:00,C17:00,C18:00,C19:00)exhibited better flexibility than other PLFAs in microbes.The percentage of gram-negative bacterium marked by PLFA decreased with the increasing concentration of ammonia nitrogen.In addition,ammonia nitrogen had no effect on the diversity of PLFA in anaerobic bacteria.With the inhibition of ammonia nitrogen,dominant phylum of anaerobic bacteria and genera of methenogenesis bacteria remained unchanged whereas relative content changed.Bacteria genera with higher nitrate endurance and adaptability were Brooklawnia,Kosmotoga,Pseudoramibacter,Trichococcus,Saccharofermentans,Bacillus,Aminobacterium,Aminiphilus,Levilinea,Caldilinea,Leptolinea,Bellilinea.Corresponding archaea genera were Methanosaeta,Methanobacterium,Methanofollis.4.All of the three toxic compounds exhibited inhibitory effect to the SMA of anaerobic granular sludge.Effect caused by nitrite nitrogen to SMA was the most apparent according to EC50.Generally the inhibitory effect of nitrite nitrogen and nitrate nitrogen on anaerobic granular sludge exhibited some similarity,which indicated the two compounds affected energy metabolism process and cytomembrane structure of anaerobic microbes simultaneously in high concentration.Whereas ammonia nitrogen only affected energy metabolism process in anaerobic microbes which lead to a decrease of ATP volume.Three toxic compounds inhibited co-enzyme M while only nitrite nitrogen and ammonia nitrogen inhibited co-enzyme F420 with different concentration.Two saturated fatty acids(C16:00 and C18:00)exhibited better flexibility to toxic compounds than other PLFAs in microbes.Bacteria genera with higher nitrite and nitrate endurance were Thauera and Bacteroides,while archaea genera with better endurance and adaptability to all three compounds were Methanosaeta and Methanobacterium.