| Pleurotus ostreatus is one of the most widely cultivated edible fungi with the largest consumption in China,the growth and development of Pleurotus ostreatus were influenced by environmental conditions and nutrient composition of medium(carbon source,nitrogen source,C/N ratio,inorganic salt,growth factor and water).As an important nutrient limiting factor in mushroom cultivation,researches show that nitrogen source had important effects on the growth of mushroom hyphae,morphological transformation,fruiting body yield and nutrient composition.The physiological mechanism of nitrogen source affecting the growth and development of Pleurotus ostreatus is unknown.Extracellular enzymes(Lignin oxidase,cellulase,hemicellulase,amylase,protease)are the key enzymes for the decomposition and utilization of culture materials in edible mycelia,and extracellular enzyme activity is closely related to the growth and development of edible fungi.The results showed that the expression of extracellular enzymes in edible fungi could be influenced by different nitrogen source forms and amounts,however,there were differences in the influencing mechanism among different edible fungi.In addition,the utilization of nitrogen by organisms requires the participation of key enzymes of nitrogen metabolism,such as glutamine synthase,glutamate synthase,glutamate dehydrogenase,glutamate grass transaminase and so on.The result of research shows that the expression of key enzymes in nitrogen metabolism was affected by the nitrogen source morphology and supply level.Therefore,in order to explore the physiological mechanism of the influence of different nitrogen sources of different concentrations on the growth and development of Pleurotus ostreatus,in this study,pleurotus ostreatus was used as the experimental material,the effects of different nitrogen sources with different concentrations on mycelial growth and nitrogen source decomposition of Pleurotus ostreatus were determined by solid plate culture method;the effects of different nitrogen sources with different concentrations on extracellular enzyme activities and degradation rate of cottonseed husk were determined by liquid flask culture;the effects of different nitrogen sources at different concentrations on the activity of key enzymes in nitrogen metabolism were determined by nitrogen starvation treatment combined with nitrogen induction treatment.The main results are as follows:1.The effects of different nitrogen sources with different concentrations on mycelial growth of Pleurotus ostreatus were studied:adding Urea,glutamate,glutamine,(NH4)2HPO4 can promote the growth of mushroom hyphae obviously.Among them,when Urea concentration was 0-25 mM,the growth rate and growth potential of mushroom mycelia increased with the increase of Urea concentration;When urea concentration was 25-40 mM,there was no significant difference in the growth rate and growth potential between mushroom mycelia.When the glutamate concentration was 0-20 mM,the mycelia of pleurinus edodes increased with the increase of glutamate concentration;while the glutamate concentration was 20-40 mM,there was no significant difference in the growth rate and growth potential between mushroom mycelia.Adding low-concentration ammonium dihydrogen phosphate can significantly promote the growth of mushroom mycelia.When the concentration of ammonium dihydrogen phosphate is 5-10 mM,the mycelia grows best and grows faster;when the concentration of diammonium phosphate was higher than 15 mM,mycelial growth was significantly inhibited.When glutamine concentration was 0-35 mM,the growth rate of mushroom mycelium basically increased with the increase of glutamine concentration.Compared with the above four nitrogen sources,as two secondary sources of organic nitrogen,Pleurotus ostreatus has poor utilization of Arginine and Proline,the main performance is slow growth of mycelium.Among them,the growth rate of mushroom mycelium increased with the increase of arginine concentration,however,there was no significant difference in mycelial growth between the experimental groups.Pleurotus ostreatus hyphae has the worst utilization of proline,the mycelium growth rate of which was very slow and there was no significant difference between different experimental groups.In summary,there were significant differences in the utilization ability of mushroom mycelia to different forms of nitrogen sources and the utilization ability of pleurotus ostreatus mycelium to primary nitrogen source was significantly better than that of secondary nitrogen source.2.The color rendering results of the plate ammonium ion concentration showed that:Ammonium ions can be detected on the surface of culture medium when urea,diammonium phosphate and glutamine are used as nitrogen sources.The concentration of ammonium ions in urea medium was the highest,this shows that urea is mainly used by mycelium decomposed into ammonium;and moreover,there were significant differences in the concentrations of ammonium ions in urea plates with different concentrations.So it is speculated that the expression of pleurotus urease is regulated by the concentration of urea in the environment.A small number of ammonium ions were detected on the surface of glutamine medium,this indicated that glutamine could be directly absorbed and utilized by Pleurotus ostreatus hyphae,however,free glutamine is unstable and can be easily decomposed to release ammonium.No ammonium ions were detected on the surface of glutamate,arginine and proline medium,so it is speculated that the mycelium of pleurotus edodes can directly absorb the above three amino acids from the external environment.3.The effects of different nitrogen sources with different concentrations on the activity of key enzymes in nitrogen metabolism of Pleurotus ostreatus were studied that GS enzyme activity first increased and then decreased with the increase of urea concentration,and GS enzyme activity was highest when urea concentration was 15 mM.The activity of GOT enzyme increased gradually,GOT enzyme activity was highest when urea concentration was 40 mM.The GDH enzyme activity first increased slightly and then decreased significantly,GDH enzyme activity was highest when urea concentration was 5 mM,however,there was no significant difference between the nine groups.With the increase of glutamate concentration,the activity of GOT,GDH enzyme increased gradually,therefore,the addition of glutamate can significantly induce the expression of GOT and GDH proteins.GS enzyme activity increased first and then decreased with the increase of arginine concentration,GS enzyme activity was highest when arginine concentration was 30 mM,the GOT enzyme activity increased first and then decreased,while the GOT enzyme activity was highest when the concentration of arginine was 25 mM.The GDH enzyme activity of each experimental group was significantly higher than that of the control group,however,the GDH activity decreased with the increase of arginine concentration.The GDH enzyme activity was highest at 5 mM arginine concentration.As the concentration of diammonium phosphate increased,GS enzyme activity decreased first and then increased,however,the GS enzyme activity in the experimental group was significantly higher than that in the control group when the concentration of diammonium phosphate was higher than 20 mM,while the GS enzyme activity was highest at 30 mM concentration of diammonium phosphate.With the increase of proline concentration,GS enzyme activity decreased gradually,and the enzyme activity of the experimental group was significantly lower than that of the control group.The GDH enzyme activity increased first and then decreased,however,the GDH enzyme activity of the experimental group was significantly higher than that of the control group.The GDH enzyme activity was highest at 30 mM pro line concentration.With the increase of glutamine concentration,GS enzyme activity increased first and then decreased,the GS enzyme activity of each experimental group was significantly higher than that of the control group,the GS enzyme activity was highest when glutamine concentration was 20 mM.The GOT enzyme activity showed a trend of increasing gradually,while there was no significant difference in the activity of GOT enzyme between the experimental groups with glutamine concentration more than 10 mM.It can be seen that there are significant differences in the effects of different nitrogen sources on the activities of key enzymes in nitrogen metabolism of pleurotus edodes hyphae.4.The results of liquid flask shaking experiment of cottonseed shell were shown that addition of urea,arginine,diammonium phosphate,proline and glutamine can significantly increase the concentration of ammonium ions in the fermentation broth while the ammonium ion concentration in the fermentation broth with glutamate as nitrogen source was almost constant.5.The results of liquid flask shaking experiment of cottonseed shell were shown that the degradation of cottonseed husk by Pleurotus ostreatus hyphae could be promoted by adding 6 nitrogen sources with proper concentration.The degradation rate was the highest when urea concentration was 5-15 mM;the degradation rate of cottonseed husk was the highest when glutamate concentration was 40 mM;the degradation rate of cottonseed husk was the highest when arginine concentration was 10 mM;the degradation rate of cottonseed husk was the highest when the concentration of diammonium phosphate was 35 mM;the degradation rate of cotton seed shell was the highest when the proline concentration was 15 mM.;the degradation rate of cottonseed shell was the highest when glutamine concentration was 20 mM.The degradation rates of cottonseed husks cultured with 6 nitrogen sources were shown that the effect of the preferential nitrogen source on the degradation of cottonseed husk was better than that of the secondary nitrogen source,can be ordered as Diammonium phosphate>glutamate>glutamine>urea>arginine>proline.6.The effects of different nitrogen sources with different concentrations on the extracellular enzyme(protease,amylase,cellulase,hemicellulase,lignin degradation enzyme)activity of pleurotus ostreatus hyphae were studied that the effects of six nitrogen sources with different concentrations on the specific activity of extracellular enzymes were not significant.Therefore,we speculated that the addition of nitrogen source may promote the accumulation of Pleurotus ostreatus biomass,accelerating the degradation of cottonseed husk. |
PDF Full Text Request