Effects Of Nitrogen And Phosphorus Deficiency On Metabolic Changes Of Microcystis Aeruginosa PCC7806

Microcystis aeruginosa PCC7806 has a complex life cycle and efficient carbon enrichment ability.Coupled with the release of microcystins as a defense,it can cope with the stress of natural and make it one of the dominant cyanobacterial bloom causing species.At present,the natural stress of cyanobacteria is mainly concentrated on nutrient stress,especially nitrogen and phosphorus as the key nutrient elements to limit the growth of cyanobacteria by directly affecting the physiological and ecological characteristics of algae cells.However,the current studies on nutrient stress generally focus on physiological changes,yet the regulation of the molecular mechanism remains unclear.The purpose of this study was to explore the physiological and metabolic responses of M.aeruginosa PCC7806 to nitrogen deficiency and phosphorus deficiency.Through isotope tagging for relative and absolute protein quantitation(iTRAQ)proteomic differential expression analysis,the metabolic transformation of M.aeruginosa PCC7806 under nitrogen deficiency and phosphorus deficiency was determined,and the pathway of carbon sink to poly-β-hydroxybutyrate(PHB)synthesis was found.PHB as a common energy storage substance in bacteria under environmental stress has been widely applied to materials,medicine and environment because of its thermoplasticity,biocompatibility and degradability.Studies have found that some cyanobacteria can also accumulate plenty of PHB under nitrogen and phosphorus stress but this study first discovered that M.aeruginosa PCC7806 could accumulate mass PHB under nitrogen deficiency and phosphorus deficiency.Therefore,in this study,the ability of M.aeruginosa PCC7806 to accumulate PHB was evaluated,and the mechanical properties,thermal stability and degradability of PHB were analyzed,which showed the potential of using M.aeruginosa as a producer to produce PHB and the feasibility of practical application of PHB.The specific results are as follows:(1)The growth rate of M.aeruginosa slowed down,the exponential growth period shortened obviously,and the biomass decreased during the stable period,which indicated that the algae cells growth was inhibited under nitrogen deficiency and phosphorus deficiency.Further analysis using iTRAQ proteomics revealed that 417 proteins with differentially expressed proteins in algae cells cultured under nitrogen deficiency and phosphorus deficiency.Based on GO and KEGG annotations,these differentially expressed proteins are mainly involved in energy generation and transformation,ribosome transcription and translation,and amino acid transport and metabolism.Combining proteomics with physiological results,it was found that the stress of nitrogen deficiency and phosphorus deficiency induced algae cells to produce microcystins(MCs)to cope with adverse environment.The expression levels of enzymes related to gluconeogenesis and starch synthesis in energy metabolism were up-regulated,indicating that energy was stored in the form of starch in algal cells.In the process of amino acid metabolism,glutamate and arginine metabolism is active,resulting in an increase in the content of acetyl-CoA,and finally promote the synthesis of PHB.In addition,the levels of light trapping proteins,photosynthetic pigments,photoreaction center proteins and key enzymes in Calvin cycle were all down-regulated expressed,resulting in a decrease in the rate of photosynthesis.Level of proteins associated with ribosomal transcription and translation was also inhibited.In summary,M.aeruginosa PCC7806 regulate intracellular metabolism in response to nitrogen deficiency and phosphorus deficiency mainly through accumulation of starch,microcystins and PHB,and decrease of photosynthesis and protein synthesis.(2)The PHB content and the expression of PHB synthesis related gene(phaA,phaB,phaC,phaE)were measured in algae cells after 30 days of nitrogen deficiency and phosphorus deficiency culture.The results showed that nitrogen deficiency and phosphorus deficiency stress increased PHB content by 18.98 times and 6.41 times respectively compared with the control,and PHB synthesis related genes were significantly up-regulated by 2.7-4.6 times,which indicated that nitrogen deficiency treatment was more beneficial to the accumulation of PHB in algal cells.Then,the functional groups,crystal configuration and hydrocarbon structure of PHB produced in algae cells were identified by infrared spectroscopy,X-ray diffraction and nuclear magnetic resonance,and compared with the standard PHB.The results showed that PHB-MA was poly-β-hydroxybutyrate.Finally,the mechanical properties,thermal properties,hydrophilicity and degradability of the PHB-MA were analyzed and compared with conventional plastics.The results show that the tensile strength and tensile fracture rate of PHB-MA are 57.65%and 84.71%lower than those of conventional plastics,respectively,while the Young’s modulus is 169.46%higher than that of conventional plastics,indicating that PHB-MA has low toughness and high hardness.The hydrophilic Angle of PHB-MA is 97.4°,which is higher than that of plastic 95.7°,indicating that PHB is highly hydrophobic.The temperature of the processability window of PHB-MA is only between 154.2-273℃,which is far less than that of plastics at 124.9-465℃,which shows that PHB-MA has strong crystallization ability and high hardness。The Tc of PHB-MA is 87.3℃,which is 20.89%lower than that of plastics,which shows that PHB-MA has strong crystallization ability and high hardness.The degradation rate of PHB-MA reached 9.55%after 50 days,while the plastic film barely degraded,indicating that PHB-MA has the ability to degrade.In summary,PHB-MA has hard and brittle properties,low processing temperature requirements,good hydrophobicity,strong crystallization ability,and excellent degradation performance,so it is more suitable for use as engineering plastics.