| At present,the annual discharge of high salinity organic wastewater in China has exceeded 10 billion tons,and has become the main industrial pollution source.Aerobic granular sludge has the characteristics of dense structure,high salinity tolerance,diverse microbial flora,efficient retention of functional bacteria,and strong tolerance in adverse environment such as heavy metals and antibiotics,which has been proved to be the the effective technologies for the treatment of hypersaline organic wastewater.Microorganisms in aerobic granular sludge can grow under high salt environment,mainly due to the presence of some salt-tolerant bacteria and halophiles in aerobic granular sludge.Compared with common microorganisms,these salt-tolerant bacteria have special cellular structure and physiological functions.Moreover,aerobic granular sludge has a stable three-dimensional structure,which can protect internal microorganisms to a certain extent and enable them to survive in different salinity.Therefore,the salt-tolerant aerobic granular sludge has great potential in treating high salinity organic wastewater.However,there are few studies on the microstructure,formation mechanism and structural stabilization mechanism of salt-tolerant aerobic granular sludge,and the molecular regulation mechanism of aerobic granular sludge under hypersaline stress is still unclear.These problems need further exploration.In this paper,the formation mechanism,the structure,the performance enhancement,and the molecular mechanism of salt-tolerant aerobic granular sludge analysis based on the macro-genome technology were studied in depth.The main findings are as follows:Activated sludge was used as seed sludge to cultivate aerobic granular sludge which could tolerate 6%salinity and had good removal and sedimentation properties.At 7%salinity,the granule disintegrated and the salt-tolerant sludge system collapsed.High salinity stimulated bacteria secrete more extracellular polymers(EPS).Total protein(PN)was highly correlated with the total EPS(R=0.951),and PN concentration was positively correlated with the increase of salinity,which had improved the whole performance of the aerobic granular sludge under hepersaline stress.The relative abundance of Salinicola in aerobic granular sludge was positively correlated with the increase of salinity(R=0.953).At 6%salinity,the relative abundance of Salinicola was as high as 91%as the dominant and functional bacteria,and the salt-tolerant aerobic granular sludge dominated by Salinicola,a moderately halophilic bacteria,was successfully cultivated.The addition of anaerobic granular sludge accelerated the formation process of salt-tolerant aerobic granular sludge and shortened the time for granulation by 26 days.The salt tolerance of the sludge system was enhanced.At the salinity level of 7%,the salt-tolerant granular sludge did not appear the phenomenon of disintegration and collapse,and maintained good sedimentation and removal performance at the salinity level of 9%.The formation process of salt-tolerant aerobic granular sludge in SBR reactor presented three phases.Phase ?(start-up):under the low salinity the aerobic activated sludge gradually adapted the salinity stress.The reactor operation was optimized and the water parameters were adjusted to promote the formation of aerobic granular sludge;Phase ?(acclimation under saline stress):the salinity increased step by step,under this salt stress,the non-salt-resistant bacteria in aerobic granular sludge was gradually eliminated,salt-tolerant aerobic granular sludge gradually formed;Phase ?(elevated salt concentration):under this high salinity conditions,the salt-tolerant aerobic granular sludge occupied a dominant position in SBR reactor.The crystal nucleus hypothesis,selective pressure hypothesis and DLVO theory were used to analyze the formation mechanism of salt-tolerant aerobic granular sludge,that is,anaerobic particles as strong supporting materials became the beginning of the formation of young particles.Under the double selective pressure of sedimentation time and salinity,young particles gradually accumulated in the SBR system.Meanwhile,halophilic microorganisms corresponding to high salt stress were enriched.Under the combined action of electrostatic force and hydrophobic force,aerobic granular sludge gradually transited from young stage to mature stage.Finally,salt-tolerant aerobic granular sludge with moderate halophilic bacteria was successfully cultivated under high salt stress.The structure features from the macroscopic scale and micro scale for salt-tolerance aerobic granular sludge were investigated based on a variety of microscope observation techniques coupled with multiple fluorescent staining techniques.Under the salt stress environment,the formation of granular sludge needed a period of time,and its internal responsive ecosystem of high salt stress also needed a process.With the formation and mature of granular sludge,the establishment of the internal new ecosystem,the enrichment of the donimadted bacterium,the salt resistance of granular sludge system constantly improved.The regulation measures to promote the formation and stability of salt-tolerant aerobic granular sludge were found.One measure was to create enough niches for salt-tolerant bacteria to colonize in large quantities at the initial stage of the formation of granular sludge.The other measure was to purposefully stimulate microorganisms to secrete more EPS in the view of the role of EPS in the formation and stabilization of granular sludge.The culture strategy of increasing salt concentration by gradient was adopted.On the one hand,the bacteria,which could not adapt to salt stress,was washed out from the SBR reactor,so as to achieve the goal of accumulating a large number of salt-tolerant bacteria and secreting sufficient amount of EPS.On the other hand,the impact of salinity could be weakened by gradient increase,and the purpose of directional cultivation of salt-tolerant aerobic granular sludge could be achieved under the premise of maintaining the stability of the reactor system.High-throughput sequencing technology based on macrogenomics was used to study the molecular mechanism of salt-tolerant aerobic granular sludge under salt stress from the perspectives of microbial community structure,functional genes and metabolic pathways.It was found that during the directional cultivation of salt-tolerant aerobic granular sludge,the structure of the microbial community showed the obvious temporal and spatial succession,which is that the microbial community dominated by Proteobacteria gradually developed to the microbial community dominated by Ascomycota.Aerobic granular sludge system dominated by salt-tolerant bacteria actively regulated the expression pattern of its metabolic pathways to adapt to the gradually increased salt stress,among which carbohydrate metabolism pathway,amino acid metabolism and energy metabolism pathway were most significantly regulated,indicating that these physiological and biochemical processes were more susceptible to hypersaline stress.At 0%salinity level,the abundance of Cell motility related proteins was 5 times compared with 9%salinity.However,at the 9%salinity level,the expression of proteins associated with Extracellular structures was more active,and the abundance was 3.6 times compared with 0%salinity,which is likely to be resistant to salt granular sludge microbial response to high salt stress in a strategy.Under the low salinity levels,cellular functions mainly focused on maintaining their physiological and biochemical process.Meanwhile,the cell secreted compatible solutes or absorbed K+ to maintain osmotic pressure balance,stimulated and produced more EPS to promote the formation of granular sludge under the hypersaline stress.The cellular activity was enhanced at the same time.After the formation and maturity of granular sludge,proteins related to cytoskeleton,extracellular structure,nuclear structure and biosynthesis of secondary metabolites were expressed more at a higher salinity level,with more emphasis on maintaining the stability of the internal structure of granular sludge. |
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