Regulation Mechanism Of High Salinity Wastewater Biotreatment Colonies Developed From Marine Sediments

The industrial production and utilization of seawater resources generate a large amount of high salinity wastewater rich in organic matter,nitrogen,phosphorus,and high concentration of inorganic salts.When using biological process to treat organic matter,nitrogen and phosphorus in high salinity wastewater,the high concentration of inorganic salt ions and salt ion fluctuations inhibit the biological treatment effect,making it difficult to meet the discharge standards of high salinity wastewater.The construction of efficient and stable halophilic treatment colonies based on halophilic biomass is the key to break through the bottleneck of difficult biological treatment of high salinity wastewater.Due to different sources of halophilic inoculation,there are differences in nutrient metabolism potential,optimized regulation strategy and environmental adaptability.It is very important to select appropriate inoculants and determine optimal regulatory strategies for the rapid construction of efficient halophilic treatment colonies.Marine sediments are rich in natural halophilic/salt-tolerant microorganisms.many of which are involved in the biogeochemical cycle of carbon,nitrogen and phosphorus,which are reliable sources of inoculation for the construction of halophilic treatment colonies.However,the directional construction,optimized regulation and environmental adaptability of halophilic treatment colonies based on marine sediments have not been systematically studied.In view of the above problems,this study constructed halophilic biofilm and aerobic granular sludge by inoculating marine sediments and adding inert carriers,which verified the application potential of marine microorganism community in high salinity wastewater treatment.Selecting substrate COD/N as the key parameter,exploring and revealing the regulatory mechanism of COD/N on the nitrogen transformation of halophilic treatment colonies and microbial community structure,and clarifying the key threshold for regulating nitrification and ammonia assimilation pathways.The robustness and adaptability of constructing halophilic treatment colonies under environmental fluctuation conditions were evaluated,and their engineering application prospects were verified.The main results of this study are as follows:（1）A halophilic biofilm system was constructed based on marine sediments,and 95%COD and 98%ammonia removal efficiency were achieved in simulated high salinity wastewater treatment.The abundance of ammonia oxidation gene amoA and denitrification genes napA and nirS in the biofilm system gradually increased with the operation of the system,while the abundance of nitrite oxidation gene nxrA sharply decreased.The native ammonia oxidizing bacteria Nitrosomonas halophila in marine sediments was enriched to 0.2%-0.7%in biofilms.In order to enhance the biomass retention and adverse impact tolerance of halophilic treatment colonies,the granulation process was accelerated by adding granular activated carbon（GAC）.The addition of GAC shortened the granulation time by 41.6%and increased theicrobial activity and the abundance of almost all nitrogen transformation related genes.HAGS with GAC addition still achieved an ammonia removal efficiency of over 82%when the temperature drops to 13 ℃,significantly higher than the control system（p<0.05）.The addition of GAC enriched microorganisms with EPS production and signal molecule release functions,significantly upregulated the "signal molecule and interaction" metabolic pathway,promoted the synthesis of protein in EPS,and thus accelerated sludge granulation.The constructed halophilic biofilm and halophilic granular sludge have verified the application potential of halophilic treatment colonies developed from marine sediments in the treatment of high salinity wastewater.（2）According to the regulation theory of biological nitrogen conversion pathway,influent COD/N was taken as the key parameter,and the nitrogen conversion pathway and microbial community of saline treated colonies were regulated by increasing COD/N and decreasing COD/N,Increasing COD/N increased the removal efficiency of total inorganic nitrogen（TIN）and phosphorus,the contribution rate of ammonia assimilation and the activity of heterotrophic bacteria,strengthened the pathway of heterotrophic ammonia assimilation,and decreasing COD/N strengthened the activity of autotrophic nitrifying bacteria and the ammonia oxidation pathway.When COD/N exceeded 15,the activity of heterotrophic bacteria was superior to that of autotrophic bacteria,and the main pathway for ammonia removal was from ammonia oxidation to ammonia assimilation.The relative abundance of ammonia oxidizing bacteria Nitrosomonas reached a maximum of 3.5%at COD/N of 1,but gradually decreased with the increase of COD/N and decreased to 0%over 300 days of long-term operation.The relative abundance of the ammonia oxidation gene amoA was highest at COD/N of 5,and gradually decreases by 0%with the increase of COD/N.Regulating heterotrophic ammonia assimilation（HAA）colonies by increasing COD/N requires high time and operating costs.Four specialized ammonia assimilation microbiomes were rapidly constructed by inoculating marine sediments containing obligate ammonia assimilation bacteria under COD/N conditions of 5,10,15,and 20.The removal efficiency of ammonia and TN increased linearly with the increase of COD/N.and ammonia was almost completely converted into biomass nitrogen through the HAA pathway.There is no significant nitrogen loss or accumulation of NO₂^--N和NO₃^--N in the system.The abundance of ammonia oxidation gene amoA can be almost ignored,and COD/N exceeding 15 was beneficial for the enrichment of ammonia assimilation glnA gene.The original nitrifying bacteria in the inoculated marine sediments were completely eliminated within the first 5 days.Phenotypic,genetic,and microbial evidence collectively confirmed that the HAA process dominated ammonia conversion in the microbiomes.The microbial community results indicated that COD/N retained or enriched microorganisms adapted to or preferred to certain nutritional conditions through selective pressure,while regulated the cooperation and competition between microorganisms,thereby regulating the self-assembly process of the microbiomes.（3）In practical engineering applications,biological treatment systems may experience long-term system stagnation.Assessing the robustness and resilience of saline treated bacterial colonies after system stagnation is crucial for rapid start-up and risk prevention.After 28 days of system stagnation,the fluctuations of treatment performance and settleability of the HAA colony were smaller than those of the AN colony,and the required time of recovery was shorter than that of the AN colony,indicating that the HAA colony have higher robustness.The abundance of ammonia oxidizing bacteria Nitrosomonas in the AN colony sharply decreased after system stagnation,while Nitrosomonas was almost undetectable in the HAA colony.The ammonia removal of the HAA system mainly relied on the assimilation of various heterotrophic bacteria such as Vibrio,Paracocus,Stappia,Halomonas,Microbacterium,Muricauda,and Marinobacterium.The cooperation of these heterotrophic bacteria ensured the strong resilience and efficient ammonia assimilation performance of the HAA system.Considering the differences in inorganic salt composition and concentration of actual high salinity wastewater from different sources,five concentration ratios of Cl-and SO₄^2-were set by adjusting the concentrations of sodium sulfate to sodium chloride to evaluate the robustness of halophilic treatment colonies.The results indicated that when the proportion of SO42increased,the fluctuation of nitrogen removal performance in AN system is greater than that in HAA system,and the adaptation time is longer.The increase in SO₄^2-proportion increased the settleability,hydrophobicity,total EPS content,and richness of microbial community of sludge,promoted the activity of ammonia oxidizing bacteria in the AN system,and inhibited the activities of heterotrophic bacteria and nitrite oxidizing bacteria.The relative abundance of ammonia oxidizing bacteria Nitrosomonas increased from 2.3%to 10.4%with the increase of SO₄^2-concentration in the AN colony,while it remained below 0.1%in the HAA colony.With the increase of SO₄^2-concentration,the relative abundance of ammonia assimilation genes glnA and gdhA decreased by 3%-11%,while the relative abundance of ammonia oxidation genes hao,amoA,and nxrA increased by 37%-363%.The halophilic treatment colonies constructed based on marine sediments can quickly recover performance during both system fluctuations and salt ion fluctuations,and the HAA colony have stronger robustness and resilience than the AN colony.In summary,this study provided a reliable inoculation source and construction strategy for the biological treatment of high salinity wastewater,revealed the regulatory mechanism and key threshold of COD/N on the nitrogen conversion pathway and microbial community structure of halophilic treatment colonies,evaluated the engineering application prospects of constructed halophilic treatment colonies,providing alternative strategies and theoretical basis for the biological treatment of high salinity wastewater.