Optimization And Mechanism Of Alleviating Ammonia Inhibition Through Bioaugmentation In Anaerobic Digestion Process

Anaerobic digestion(AD)has gained extreme attention since many forms of renewable energy can be produced through biological treatment of organic wastes.Ammonia toxicity is regularly reported as the primary cause of limiting operation efficiency and energy conversion efficiency of digestion when fed with nitrogen-rich substrates,resulting in a "steady state of inhibition".In order to alleviate ammonia inhibition,bioaugmentation can be used as a potential solution.However,further exploration is needed on the selection of microbial strains or flora.Moreover,the effects of bioaugmentation on methane production,methanogenic pathway and microbial communities are still unclear.In addition,the modified kinetic model can be used to simulate the AD process,which can provide theoretical support for optimizating operation conditions.Firstly,ammonia inhibition concentration was determined.Secondly,the strains which could alleviate the ammonia inhibition were selected.Thirdly,the long-term evaluation of bioaugmentation in AD process was investigated to determine the effect on methanogenic pathway shift.Fourthly,based on the AD with actual complex substrates,the practical application potential of bioaugmentation was evaluated,and the effect on the microbial communities and functional genes was clarified.Finally,according to the experimental data,the dynamic model was modified to simulate the "bioaugmentation-AD"system,and methanogenic pathway was further quantitatively evaluated through the analysis of kinetic parameters.(1)The effects of ammonia on methane production,methanogenesis pathway,microbial community,and reactor performance were investigated in this study.More than 20%of methane production loss was observed in mesophilic and thermophilic reactors when the ammonia level exceeded 2 and 5 g NH4+-N L-1,respectively.The volatile fatty acid data indicated that acetogenesis and methanogenesis are strongly influenced by ammonia inhibition under the mesophilic condition,while methanogenesis was mainly affected under thermophilic condition.No evident methanogenesis pathway shift was found in the mesophilic reactor,whereas the increase in NH4+-N concentration to more than 5 g L-1 led to a clear shift from aceticlastic to complex and flexible pathways,thereby significantly enriching the hydrogenotrophic pathway in the thermophilic reactor.The function and response of the microbial community to ammonia inhibition were consistent despite the difference in population and diversity under mesophilic and thermophilic conditions.(2)The effect of bioaugmentation with different microorganisms on AD to mitigate the ammonia inhibition problem was investigated.Seven pure strains of microorganisms(including obligate aceticlastic methanogen,facultative aceticlastic methanogen,hydrogenotrophic methanogen,syntrophic acetate oxidizing bacteria(SAOB)were selected and thirteen bioaugmentation approaches were tested.Bioaugmentation with hydrogenotrophic methanogen Methanobrevibacter smithii(MBS)and SAOB Syntrophaceticu schinkii together was the optimal choice,methane production(MP)was 71.1%higher than that in Blank,the activity of hydrogenotrophic methanogenesis was greatly heightened according to specific methanogenic activity(SMA)analysis.Bioaugmentation with facultative aceticlastic methanogen Methanosarcina barkeri(MSB)alone without SAOB addition was also proven efficient(MP was 59.7%higher than that in Blank),both aceticlastic and hydrogenotrophic methanogenesis were enhanced.Further evaluation with carbon isotope fractionations analysis indicated that balancing the activities of the aceticlastic and hydrogenotrophic methanogenic pathways is of great importance.16s rRNA gene sequencing results showed that Methanobacterium spp.and Methanosaeta spp.were the dominant archaea in all 14 reactors.Nevertheless,bioaugmentation with Methanosaeta spp.did not result in a positive effect on MP.On the other hand,Methanobrevibacter spp.and Methanosarcina spp.were non-dominant archaea(even after bioaugmentation with MBS or MSB,the relative abundances were still poor(<2%)),but displayed pivotal roles in determining the overall microbial consortium and,in turn,improved the overall performance.(3)The effect of different bioaugmentation strategies on AD related to the alleviation of ammonia inhibition was investigated in a long-term operation.The long-term operation confirmed that bioaugmentation is a stable method.A 35%increase in MP was observed in bottles bioaugmented with Methanosarcina barkeri(MSB)or Syntrophaceticu schinkii(SS)+Methanobrevibacter smithii(MBS),and a 49%increment was obtained from the bottles bioaugmented with Methanosaeta harundinacea(MSH)+SS+MBS.Results suggest that the enhancement in both aceticlastic and hydrogenotrophic methanogenic pathways should be considered,and bioaugmentation stains should be properly selected to achieve a synergistic effect.The microbial community analysis indicated Methanosarcina spp.was the dominant archaea in all the bottles,combined with the results on SMA and carbon isotope fractionation,it was suggested that Methanosarcina spp.performed differently in methanogenic pathways in the bottles.The abundance of COG and total enzymes in the bottles with high MP(MSB and MSH+SS+MBS)was higher than that in the other bottles.The ratio of the functional enzyme EC 2.1.1.86 to EC 1.2.99.5 and the relative abundance of EC 1.2.1.2 confirmed the aceticlastic methanogenic pathway of MSB and the pathway enhancement balance in MSH+SS+MBS.(4)Bioaugmentation feed seeds were added in AD reactors fed with diary manure(DM)and pig manure(PM)to investigate the effect of bioaugmentation.Bioaugmentation with feed seeds could obviously increase the MP.After bioaugmentation,the MPs of DM reactors and PM reactors were increased by 26-39%and 25-50%,respectively.The results of VFA profile experiment and SMA indicated that bioaugmentation with feed seeds from the reactor fed with same substrates could mainly enhance acetogenesis and methanogenesis steps.Bioaugmentation with S-C could enhance acidogenesis,acetogenesis,and methanogenesis steps.According to microbial communities,the relative abundance of dominant microbial strains in the feed seeds could be increased after bioaugmentation in the original AD system.From the perspective of functional enzymes and functional genes,the abundance of key functional enzymes and functional genes in different methanogenic pathways increased after bioaugmentation,indicating that the activities of both aceticlastic methanogenic pathway and hydrogenotrophic methanogenic pathway were enhanced.At the same time,functional enzyme EC 2.1.2.1 might be a breakthrough to further strengthen aceticlastic methanogenic pathway.Based on the analysis of the contribution of "microbial-COG functions",compared with Methanosaeta,Methanosarcina expressed more comprehensive functions or stronger activity in different functions.(5)Based on the experimental results,the Anaerobic Digestion Model No.1(ADM1)was modified with ammonia inhibition,bioaugmentation and syntrophic acetate oxidation step to simulate "bioaugmentation-AD" system.A well simulation was obtained from the modified ADM1(R2>0.96).In the modified ADM1,kinetic parameters km,which means the maximum specific degradation rate,performed the highest sensitivity index.After bioaugmentation,all km values increased,indicating that bioaugmentation mainly enhanced the degradation rate of substrates by microorganisms.At the same time,the values of kmsu,kmbu,kmpro,kmac and kmh2 were all increased after bioaugmentation with feed seeds,indicating that bioaugmentation with feed seeds could strengthen all degradation steps during AD process.Simulated contribution rate data indicated that the hydrogenotrophic methanogenic pathway was dominant while both two pathways got strengthened,a result that is consistent with the findings of the microbial analysis.From the perspective of dynamics,it was found that in the reactor with complex substrates,the enhancement of aceticlastic methanogenic pathway could be the key bottleneck to further optimize the MP in AD process.In this study,after the ammonia inhibition concentration was defined,the AD reactors under high ammonia level were bioaugmented to realize the transformation from "steady state of inhibition" to "steady state of high efficiency".From the aspects of actual operation performance,microbial ecology,reaction dynamics,etc,the theoretical basis and technical support for high-efficiency AD process with high ammonia concentration are provided.