| The microbial-catalyzed nitrite-dependent anaerobic methane oxidation(N-DAMO)couples the anaerobic methane oxidation process and the nitrite reduction process.This bioprocess can eliminate the pollution of methane and nitrogen at the same timeand therefore is a potential process in wastewater treatment.However,the low metabolic activity-activity falls within 0.1-0.3 fmol CH4·day-1 cell-1)and slow growth rate(doubling times are as long as weeks to months)have made it difficult to obtain N-DAMO bacteria culture,which is a limit to the research of mechanism and the application in engineering application.In this work,the growth and activity of N-DAMO bacteria were investigated from aspects of mass transfer conditions,nutritional conditions and environmental conditions.Moreover,the cultivation of N-DAMO bacteria was promoted by adding second liquid to enhance the methane transfer efficiency from gas to liquid,adding growth factors to advance nutritional conditions and removing dissolved oxygen to diminish environmental stress.The major results are as follows:1.The effects of paraffin oil,C16TAB and SDS on the metabolic activity,abundance and bacterial community structure of N-DAMO bacteria were studied,and the optimal type of second liquid as well as its optimum concentration were determined.The solubility test results showed that the value of a increased with the addition of paraffin oil,C16TAB and SDS in the medium..The methane mass transfer efficiency from gas to liquid was boosted with the increase in the concentration of the second liquid phase in the test With the concentration range ofparaffin oil 0.0-12.0%v/v,C16TAB 0.0-1.0 mmol L-1 and SDS 0.0-1.0 mmol L-1,the a values are 0.8%-4.4%,0.8%-2.7%,and 0.8%-2.7%,respectively,? value increased by 1.6-4.3 times,1.2-2.3 times and 0.4-2.2 times,respectively.The results of short-term activity test showed that the addition of paraffin oil could promote the activity of N-DAMO.The optimum concentration of paraffin oil was 12.0%v/v.At this time,the nitrite conversion rate and methane consumption rate could be increased by 1.0 and 2.1 times compared to that of the control group,respectively.The addition of C16TAB and SDS could not promote the N-DAMO activity.The results of long-term paraffin oil optimized culture verification experiments showed that the addition of 12.0%v/v paraffin oil had a positive effect on the activity,quantity and relative abundance of N-DAMO bacteria.After the long-term test,the conversion rate of nitrite and methane in the paraffin oil group increased by 1.0 times and 2.6 times,compared to those of the control group respectively.The quantity of N-DAMO bacteria increased by 2.4 times compared to that in the initial period,which was 1.4 times that of the control group.The relative abundance of NC10 bacteria was increased by 2.3 times,which wasl.5 times that of the control group.2.The effects of multivitamins,nucleobases,heme and betaine on the metabolic activity,abundance and bacterial community structure of N-DAMO bacteria were studied,and the optimal growth factor combination was obtained.The results of short-term single factor test showed that vitamins could promote the activity of N-DAMO.The optimum concentration of vitamins was 15.0 ?g L-1.At this time,the conversion rate of and methane were 1.3 times that of the control group,respectively.The base pair promoted the activity of N-DAMO.When the base concentration was 5.0 ?g L-1,the N-DAMO activity was increased to the plateau stage and the nitrite reduction rate and methane consumption rate were 1.4 times and 1.6 times that of the control group,respectively..Heme had an effect on N-DAMO activity,and the optimum concentration of heme was 10.0 ?g L-1.At this time,the nitrite reduction rate and methane oxidation rate were 1.2 times and 1.9 times that of the control group,respectively.Betaine has a significant effect on N-DAMO activity During the increase of betaine concentration from 0.0 ?g L-1 to 200.0 ?g L-1,N-DAMO activity increased significantly.Simultaneously,the increase of 31.4 ?mol L-1 h-1 and 25.4 ?mol L-1 h-1 repectively in nitrite reduction rate and methane oxidation rate was observed.With the concentration of betaine continuing to go up,the increase of betaine concentration N-DAMO activity was small.of the single-factor test,the concentration range of each factor in the further orthogonal test was determined:multivitamin 2.0-18.0 ?g L-1;heme 4.0-20.0 ?g L-1;base 0.5-4.5?g L-1;betaine 40.0-200.0 ?g L-1.The results of the long-term orthogonal test showed that the optimum combination of growth factors was 2.0 ?g L-1 multivitamins,4.5 ?g L-1 nucleobases,20.0 ?g L-1 heme and 200.0 ?g L-1 betaine.In this case,the nitrite reduction rate and methane oxidation rate were 2.9 times and 1.2 times that of the control group,respectively.Taking comprehensive consideration of range analysis and single factor test results,the concentrations of nucleobase and betaine in the improved medium were set as 5 and 200 ?g L-1 respectively.The improved medium verification experiments showed that nucleobase and betaine indeed promoted the activity of N-DAMO.After 172 days operation,the ultimate nitrite conversion rate of D group(nucleobase and betaine),C group(betaine)and B group(nucleobase)was respectively 13.3 times,10.5 times and 9,0 times that of initial period.D group had the optimum promotion effect,followed by C group and B group.According to results of verification experiments,nucleobase and betaine was provided as improved medium in MGSLR reactor.The improved medium was applied in an MGSLR reactor to cultivate N-DAMO bacteria.The addition of nucleobase and betaine promoted the activity of N-DAMO as expected.After 348 days of operation,the nitrite conversion rate increased by 4.7 times,i.e.,from 12.3 mg N L-1 d-1 to 70.2 mg N L-1 d-1.The application of the improved medium also had a positive effect in the quantity and relative abundance of N-DAMO bacteria.The quantity of N-DAMO bacteria increased by 225.0 times and the relative abundance of NC10 bacteria increased by 143.0 times.Additionally,20.0-50.0 ?m dense large aggregates were gradually formed after the application of the improved medium,which reduced the bacterial loss and promoted the growth of N-DAMO bacteria3.The effect of dissolved oxygen on metabolic activity,abundance,and functional genome of N-DAMO bacteria was studied,and practical strategies of environmental regulation were put forward.Dissolved oxygen has an inhibitory effect on N-DAMO activity.In the non-deoxygenation stage,the nitrite removal rate was below 10.9 mg N L'1 d'1 and the nitrite removal efficiency was below 65.0%.In the deoxygenation stage,the nitrite removal rate was stable at 16.2 mg N L-1 d-1 and the nitrite removal efficiency was maintained at 100%.Dissolved oxygen led to the decline of quantity and relative abundance of N-DAMO bacteria.In the non-deoxygenation stage,the quantity of N-DAMO bacteria decreased by 93.2%from 2.2 × 1010 copies g-1 to 1.5 × 109 copies g-1.The relative abundance of NC10 bacteria decreased by 97.9%from 14.4%to 0.3%.In the deoxygenation stage,the quantity of N-DAMO bacteria increased by 130.4 times from 1.5 × 109 copies g-1 to 2.0 × 1011 copies g-1.The relative abundance of NC10 bacteria decreased by 53.0 times from 0.3%to 16.2%.Dissolved oxygen had an effect on the abundance of methane oxidation and nitrite reduction functional genes.The ratio of the abundance of pmo gene from aerobic methane-oxidizing bacteria to the relative abundance of pmo gene from NC10 was 2.0 and 0.7 before and after deoxygenation,and the pmo gene from NC10 bacteria was dominant in the reactor after removal of dissolved oxygen.The methane oxidation and nitrite reduction functional gene abundance associated with N-DAMO bacteria were higher in the medium deoxygenation phase than in the non-deoxygenation phase of the medium. |
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