Study On The Enzymatic Hydrolysis And Utilization Of Organophosphorus Of Microcystis Aeruginosa And Its Influencing Factors

Cyanobacteria blooms caused by lake eutrophication are an increasingly serious environmental problem facing the world.Microorganisms(phytoplankton and bacteria)induce alkaline phosphatases(APases)when orthophosphate is deficient,and utilize dissolved organic phosphorus(DOP)as substrate by mineralizing them to dissolve inorganic phosphorus(DIP).In lakes,the content of enzymolyzable phosphorus,which the main form of endogenous phosphorus,is much higher than that of DIP.It provides an important driving factor for the continuous cyanobacteria blooms.In marine ecosystems,the intracellular enzymatic hydrolysis of DOP is an important mechanism for bacteria to obtain phosphorus from the ocean surface.Although APases have been proved to exist both inside and outside the cell,so far,no studies have systematically evaluated the intracellular and extracellular effects of APases and the enzymatic utilization pathways of DOP in freshwater lake ecosystems.In this study,Microcystis aeruginosa(M.aeruginosa)PCC7806,a dominant cyanobacteria in eutrophic freshwater lakes,was taken as the research object and designed simulation experiments.The effects of attached bacteria,DOP source types and concentrations,and initial cell density on the growth and enzymatic utilization of DOP by M.aeruginosa are studied.Meanwhile,the utilization pathways of different DOP sources by M.aeruginosa are further explored.The main research results are as follows:(1)The attached bacteria inhibition test showed that the attached bacteria had some effects on the growth and the enzymatic utilization of DOP by M.aeruginosa.Adding antibiotic(D-cycloserine)and protecting from light for 24 h could reduce the number of attached bacteria,and the proportion of 16S r RNA gene copies of attached bacteria decreased from 84.2%to 18.7%.The presence of attached bacteria could help M.aeruginosa adapt to the new culture environment and promote its growth,and promote the increase of extracellular dissolved alkaline phosphatase activity(EAPA)and intracellular alkaline phosphatase activity(IAPA).Among them,the promotion of EAPA is more lasting,so M.aeruginosa's enzymatic hydrolysis of biological phosphorus from attached bacteria was mainly occurred outside the cell.(2)DOP source types(orthophosphate,glycerophospholipid(GP)and deoxyribonucleic acid(DNA))and concentrations(0.05,0.5 and 2.0 mg/L)influence experiments revealed that the DOP type and concentration have an impact on the growth and enzymatic utilization of DOP by M.aeruginosa.Our results suggest that M.aeruginosa is more affected by the phosphorus concentration than the source type.The higher the phosphorus concentration,the better growth of M.aeruginosa under different phosphorus source cultures.Phosphorus-starved M.aeruginosa can quickly transport and utilize inorganic and organic phosphorus sources in the environment during a short time(2 d).Phosphorus concentration affects the utilization rate of each phosphorus sources by M.aeruginosa,and the utilization rate increases with the increase of phosphorus concentration.The utilization rates of 0.05,0.5and 2.0 mg/L orthophosphate(0.003,0.041 and 0.166 mg/L/h)by M.aeruginosa were higher than those of DOP,and the utilization rate of DNA(0.003,0.024 and 0.064 mg/L/h)was higher than that of GP(0.003,0.012 and 0.022 mg/L/h),and there has a buffer period of4?12 h for utilization of GP.M.aeruginosa has different utilization pathways of GP and DNA,and the enzymatic hydrolysis of organophosphorus are affected by the phosphorus concentration.When the phosphorus concentration reached 2.0 mg/L,IAPA in GP treatment group was significantly increased,suggesting that GP was mainly transported to the cytoplasm and/or periplasm for enzymatic hydrolysis.With the increase of phosphorus concentration,the total alkaline phosphatase activity(TAPA),IAPA and EAPA in DNA treatment group significantly decreased,suggesting that in addition to APases hydrolysis,nucleases and/or nucleotidases hydrolysis may be an important pathway for M.aeruginosa to utilize DNA,except alkaline phosphatase hydrolysis.(3)The results of intracellular,extracellular and total alkaline phosphatase activity showed that the TAPA was one order of magnitude higher than IAPA and three orders of magnitude higher than EAPA,These suggest that in addition to EAPA,cytoplasmic and periplasmic APA,there are also a large number of of cell-surface-bound APA attached to the polysaccharide regions on the outer surface of cell membrane that have not been individually quantified.The abundant presence of cell-surface-bound APases indicate that they have a potentially important role.Our results also showed that the IAPA was two orders of magnitude higher than EAPA,revealing the existence and importance of enzymatic hydrolysis within cell membrane of M.aeruginosa.(4)The initial cell densities(0.02×10⁶,0.5×10⁶ and 3.5×10⁶cells·m L^-1)influence experiments suggested that the initial cell density affects the adaptation period,maximum algal density,growth rate and total specific growth rate of M.aeruginosa.With the increase of the initial cell density,the adaptation period is shorter,the maximum algal density and growth rate are greater,and the differences of total specific growth rates between different phosphorus treatment groups are smaller.The maximum algal density in the 3.5×10⁶cells·m L^-1 group was increased by 2.3 to 2.8 times and 5.6 to 28.4 times as compared with that in 0.5×10⁶and 0.02×10⁶cells·m L^-1 groups,respectively;The growth rates in 3.5×10⁶and 0.5×10⁶ cells·m L^-1 groups were was about 9.1 and 3.8 times higher than that of the0.02×10⁶cells·m L^-1 group,respectively.With the decrease of the initial cell density,the total specific growth rate of M.aeruginosa in each phosphorus treatment are greater,and the total specific growth rate of the orthophosphate group(0.41 d^-1)was significantly higher than that of DOP group(0.38 d^-1).Our results indicate that M.aeruginosa in a state of severe bloom(?0.5×10⁶cells·m L^-1)can maintain continuous severe bloom rely on the biological phosphorus and other nutrients released by its own decayed algal cells,even in the environment without exogenous phosphorus load.Therefore,the effective prevention of cyanobacteria blooms should 1)strictly control the total phosphorus concentration in lakes to 0.05 mg/L that required by the third-class water quality standards or even lower,to prevent the occurrence of cyanobacteria blooms;2)When the degree of cyanobacteria blooms is lower,priority should be given to controlling the DIP content.