Study On The Conditions And Differentiation Mechanism Of Chlamydospore Formation Of Phanerochaete Chrysosporium

Phanerochaete chrysosporium is a typical white rot fungus that not only has nonspecific ability to degrade lignin,but also produces chlamydospore that has strong resistance.Once the fungi have formed chlamydospores,they can have a longer shelf life.Therefore,this experiment describes a fermentation process suitable for the industrial production of P.chrysosporium chlamydospore.At the same time,in order to guid the production,we also explore its formation mechanism.The research of this topic is divided into the following five parts:The first part was to provide a single-factor experiment in fed-batch fermentation.The technology of producing chlamydospore,which was suitable for industrial fermentation,was set up through the single factor experiment of optimizing the chlamydospore induction medium(feed)nutrient composition and fermentation conditions.The optimized dosage formulas were 3.8 g/L of inducer a,2.5 g/L of magnesium sulfate heptahydrate,3.0 g/L of sulfuric acid,30.0 g/L of glucose,0.50 mL/L of benzyl alcohol,2.0 g/L of potassium dihydrogen phosphate,0.50 g/L of calcium chloride,0.50 mL/L of Tween 80,0.001 g/L of vitamin B1 and 70.0 mL/L of trace element solution.The optimal culture conditions were as follows: 100 mL four-fold-enriched feed(pH 4.5)was fed into medium with hyphae that had been incubated 48 h,then fermenting at 37 ? and160 r/min for 3 d.The dry weight of mycelium pellet and the number of chlamydospores were3.97 g/L and 2.687 × 109 chlamydospore/L,respectively.The second part was to apply the above optimized conditions to 50 L fermentor.The optimum conditions for the production were that mycelium was cultured for 36 hours and theninducing medium was supplemented.After incubation for 60 hours,the fermentation broth was harvested.Compared with shake flask,the whole fermentation period was shortened by 24 h and the number of chlamydochlamydosporeswere 5.710 × 109 chlamydospore/L.About 300.0 g of product was obtained.At the same time,the residue of reducing sugar was only 0.18 g/L.The germination rate of the product was 72% by wet film culture technique.The third part was through the screening of the solid culture medium and the inoculation status of P.chrysosporium coming to a conclusion that inoculating 1.0 g mycelium pellet on the equal proportion of straw wheat bran culture medium and fermenting for a week could produce a large number of chlamydospores,and formed a fungal mattress of about 2.5 mm thick;the air contact surface was smooth with a small amount of conidias;about 1.336 g of straw bran was left,with a utilization rate of 86.64%.The cell wall of the chlamydospores produced by solid fermentation was generally thicker and larger than that of liquid fermentation.For a chlamydospore with diameter of 40 ?m,thickness of the cell wall was about 6.7 ?m.The maximum the chlamydospore measured was about 70 ?m in diameter.The fourth part was to study the composition of chlamydospores.The cells were stainde by polysaccharide fluorescent dye.The result showed that chlamydospores consisted of a thin outer wall and a thick inner wall.The composition of thickening part probably consisted of cellulose,chitin,?-glucan,or a mixture of them in different proportions.The cell contained large amounts of neutral fat drops.In the cellulase digestion experiment,the same 20 mg hyphae and chlamydospore cell walls were digested and the final product concentrations were 0.244 g/L and0.215 g/L respectively,thus cellulose was not the main component of thickening of the chlamydospore cell wall.In the ?-glucan enzymolysis experiment,the same 20 mg hyphae andchlamydospore cell walls were digested and the final product concentrations were 0.480 g/L and0.598 g/L respectively,therefor compared with hyphae,the content of plastid dextran of chlamydospore was higher.In the chitin digestion experiment,5 mg mycelium and chlamydospore cell walls were digested and the final product concentrations were 0.313 g/L and 0.290 g/L,respectively,thus there was no significant difference between the two contents.The fifth part focused on three major metabolic pathways-starch and sucrose metabolic pathways,MAPK metabolic pathways and fatty acid degradation pathways,and the pathways of glucan synthesis,fatty acid metabolic pathway and MAPK regulatory pathway of P.chrysosporium were deduced.It was suggested that the cell wall composition of thick subcoat might be related to glucan.Fatty acids were synthesized and accumulated at the same time in cell to provide energy to maintain the normal growth.Meanwhile,we selected six genes to validate by Real-time PCR.In view of the overall level,this transcriptome sequencing results were credible.