The Multi-path Coupled Technologies And Mechanisms In Nitrogen Removal Process Treating Saline Wastewater

The nitrogen removal process of high-strength organic industrial wastewater under high salt stress,has problems of low treatment efficiency,long construction time and unclear mechanism.At the same time,the structure distribution and succession of denitrification and nitrification functional microorganisims in response to high-salt were unknown.Moreover,by preliminary study of the research group,the nitrogen removal pathways were not through single process.The unclear diversified migration pathways and coupling mechanisms in the system are urgent issues to be studied.Aimming at solving the problems,nitrogen removal process in sequencing batch biofilm reactors（SBBR）were established to treat high carbon and nitrogen saline/hypersaline wastewater.By modern molecular biological techniques,the bacteria,archaeal population structure,distribution of the functional microorganisims were investigated.The effect of carbon source,temperature and salinity on system efficiency and succession of functional microorganism were studied.The diversification nitrogen removal pathways responsed to environmental factors were identified further.Based on those results,a multipath coupling nitrogen removal system based on enhanced heterotrophic nitrification-aerobic denitrification treating saline wastewater,and a simultaneous nitrogen and sulfur removal system based on enhanced sulfur cycle treating hypersaline wastewater,were constructed respectively.The key control parameters for the system operation were obtained.Using molecular biology techniques such as Illumina MiSeq high throughput sequencing and Q-PCR,the microbial mechanisms of the multi-path coupled system of saline/hypersaline wastewater were revealed.The main results are as follows:The simultaneous nitrification and denitrification system treating saline wastewater was constructed,its COD and TN removal efficiency was 86.48%±1.7%and94.37%±3.15%,respectively.Under high salt stress,the abundance and species of normal denitrification functional flora decreased significantly,and the abundance of halophilic functional bacteria increased.The fluorescence quantitative PCR results showed that compared with the low salt（1%）control group,the copy number of the amoAgeneof AOA andammonia-oxidizingbacteriaAOB decreased by 37.80%and 60.94%,respectively,andthe AOA/AOB copynumber ratio was 1.20×10³,which was 1.59 times of low salt group,indicating archaea ammoxidation became an important part of nitrification under saline condition.The full-length 16s rRNA sequencing and the llumina MiSeq archaea sequencing showed that the conventional nitrifying bacteria such as Nitrospirae disappeared compared to the control group,and the conventional denitrifying bacterial population was replaced by halophilic functional bacteria.Autotrophic ammonia-oxidizing bacteria（AOB）Nitrosomonas and ammonia-oxidizing archaea（AOA）Candidatus Nitrososphaera both contributed to ammonium oxidation.Pseudomonas/Denitromonas were the main aerobic denitrification bacterium,Thauera was the main anoxia denitrification bacterium.Carbon source had significant effects on system performance,microbial structure and nitrogen removal pathways.Acetate,glucose and composite carbon source SBBRs,achieving average total nitrogen removal efficiency of 97.15%,63.94%and 94.99%,respectively,the sodium acetate carbon source system had a higher removal efficiency.At the same time,the proportion of functional bacteria in the sodium acetate reactor was the highest,and the denitrification species was the most abundant,sodium acetate played a significant role in promoting nitrogen removal.Illumina MiSeq16S rDNA sequencing showed that in sodium acetate reactor,the main nitrification flora were Pseudomonas_stutzeri（heterotrophic nitrification and aerobic denitrification）,the main denitrification flora were Thauera,Denitromonas,Pseudomonas and Halomonas.In the sodium acetate and composite carbon source reactors,autotrophic（AOA-dominated）and heterotrophic nitrification coexisted,and aerobic/anoxic denitrification were responsible for the denitrification process;Glucose was not conducive to the growth of AOA,and the reactor only had heterotrophic nitrification,aerobic/anoxic denitrification mechanism.Temperature had significant effects on system performance,microbial structure and nitrogen removal pathways.When the temperature were 10,15,20,25,30 and 35℃,the TN removal efficiencies of SBBRs were 61.96%,80.40%,85.19%,95.85%,98.15%and 97.50%,respectively.Under the condition of 25℃³5℃,the system mainly conducted synchronous partial nitrification（AOA+AOB）and denitrification.The ratio of AOA ammoxidation rate/total ammoxidation rate was 48.08%⁶8.71%,and ammonia oxidation rate was 5.03¹3.54 times of nitrous oxide oxidation rate,indicating AOA and AOB jointly undertook the system ammonia oxidation task and the nitrogen removal path of the system is mainly short range nitrification denitrification.In addition,aerobic denitrification was relatively dominant at the temperature range of 20℃³0℃.Under the condition of 10℃²0℃,the system was dominated by synchronous nitrification（AOA dominant）and denitrification.The ratio of AOA ammoxidation rate/total ammoxidation rate was 87.61%⁹4.89%,and ammonia oxidation rate was 0.51⁰.77times of nitrous oxide oxidation rate,indicating AOA contributed relatively more to ammoniaoxidationandthesystemgraduallyshiftedfrompartial nitrification-denitrificationtosynchronous nitrification-denitrification.Whenthe temperature was 20℃,aerobic denitrification was relatively dominant.However,anoxic denitrification was relatively dominant at 15℃.Salinity has significant effects on system performance,microbial structure and nitrogen removal pathways.With salinity raised from high salt（3%）to ultra-high salt（7%）,the TN removal efficiency decreased from 94.37%to 89.89%.When the salinity was 7%,the ammoxidation functional microorganism AOA/AOB severely inhibited,but AOA was less inhibited than AOB,gene copy number ratio of AOA/AOB rose from 2.47×10³ to 1.20×10³.Along with salinity increase,ordinary halotolerant/marine strains such as Oceanimonas,Phaeodactylibacter were eliminated,the abundance of halophilic bacteria such as Halomonas increased,and the sulfate-oxidation and sulfate-reducing bacteria were increased.The dominant denitrification functions in ultra high salt samples belonged to Halomonas（74.71%）,Phaeodactylibacter（aerobic denitrification,1.15%）,Arcobacter（sulfur autotrophic denitrification,1.20%）.Compared tothehighsaltsystem,the multi-pathofammoniaoxidation（AOB/AOA/sulfur-involved ammoxidation）ensured the ammonia oxidation effect of the system under ultra-high salt conditions.Under the conditions of ultra-high salt,in addition to anoxic/aerobic denitrification,there was also sulphur autotrophic denitrification.Using acetate as carbon source,a heterotrophic nitrification-aerobic denitrification system for saline wastewater was successfully constructed.The C/N ratio,DO and salinity had significant effects on system removal efficiency and pathways.The enrichment conditions（acetate as carbon source,C/N:25/1,salinity:1%,DO:6.0mg/L）of heterotrophic nitrification-aerobic denitrification bacteria were summarized,under this condition,the dominant heterotrophic nitrification-aerobic denitrification species of the system were Pseudomonas（4.05%）,Bacillus（1.87%）and Paracoccus（1.69%）.With addition of heterotrophic nitrification-aerobic denitrification bacterium mixed liquid,a multipath coupling nitrogen removal system based on enhanced heterotrophic nitrification-aerobic denitrification treating saline wastewate was constructed within 2 days,and the TN removal effciency reached 97.51%.The nitrogen removal rate was greatly increased.The nitrification rate and the aerobic denitrification rate were 2.0 and 1.6 times that of the non-enhanced system,respectively,and the heterotrophic/autotrophic nitrification rate ratio increased from 0.81 to 2.15.Illumina Mi Seq high-throughput sequencing studies showed that the enriched system successfully enriched abundance of heterotrophic nitrification-aerobic denitrification species,such as Pseudomonas（3.42%）,Vibrio（1.40%）.And compared to non-enhanced system,denitrifying bacteria increased from 3 kinds of species to 9 species,which was Oceanimona,Halomona,Nitrincola,Flavobacterium,Proteiniclasticum,Phyllobacteria-ceae,Pseudomonas,Nitratireductor,Vibrio;the system achieved enhanced heterotrophic-autotrophic nitrification,aerobic-anoxic denitrification coupling multi-pathways.Through the enhancement of the sulfur cycle,a simultaneous nitrogen and sulfur removal system for ultra-high salt wastewater was successfully constructed in a single-stage system.Organic loading rate and sulphate loading rate had significant effects on the performance of the hypersaline system.Under the optimum conditions(temperature:30°C,salinity:7%,organic loading rate:0.5kgCOD/（m³·.d）,sulphate loading rate:0.06kgSO₄^2-/（m³·.d）),the average removal effciency of TN,COD and SO₄^2-were 98.40%,84.69%and 50.97%,respectively.Compared with the conventional system,the removal effciency of TN and SO₄^2-were increased by 8.79%and 22.38%,respectively.Meanwhile,the sulfur balance study showed that SO₄ ^2-of inflow was mainly converse to gaseous hydrogen sulfide（12.50 mg/d）,partly converted to recyclable sulfur（2.56mg/d）,a very small part converted to sodium thiosulfate and sulfides.Applying the diversified carbon/nitrogen/sulfur conversion and transfer pathways in the same unit,a high-efficiency carbon,nitrogen and sulfur removal coupling processe based on sulfate reduction,nitrification,and mixed denitrification was proposed.FISH results showed that the abundances of sulfate-reducing bacteria and autotrophic denitrifyingbacteriareached 26.54%and 16.38%;IlluminaMiSeq16S rDNA sequencing showed that the functional bacteria involved in the sulfate reduction process were（3.95%）,Desulfovibrio（2.47%）and Desulfatitalea（1.39%）;Nitrosococcus（AOB,1.23%）,Nitrosomonas_halophila（AOB,0.01%）and Nitrospira（NOB,1.24%）were the main nitrifying strains in the system;the heterotrophic nitrifying bacteria Halomonas（anaerobic-denitrification,22.91%）,Phaeodactylibact-er（aerobic-denitrificati on,2.75%）,Defluviicoccus（denitrification with internal carbon source,3.18%）,autotrophic denitrifiers were Thiohalobacter（6.49%）,Thioalkalivibrio（1.75%）and Arcobacter（1.71%）.Proper proportions of carbon,nitrogen,and sulfur functional bacteria contributed to the transformation of competing functional microbes in the system into mutually beneficial and complementary ecological relationships,which was the key factor in achieving multi-path coupling in the system.