| As a by-product of antibiotic industry, a large percentage of Penicillium chrysogenum mycelia are disposed off as waste. The development of a process integrative using this biomass to produce ergosterol,(1â†'3)-α-D-glucan and chitosan exhibits not only commercial advantage but also ecological benefit. At the same time, the technology of this process can be used as the reference or direction of other waste mycelia utilization.In the study, the structure of alkali-soluble polysaccharide of Penicillium chrysogenum mycelia has been identified as linear (1â†'3)-α-D-glucan with a molecular weight of180kDa. The main influence factors for the separation of the (1â†'3)-α-D-glucan include the temperature of reaction, the time of extraction, the concentration of NaOH and the ratio of solid and liquid of reaction system. Based on the result of optimizing experiment of single influence factor, the condition of the extraction should be set as:the temperature70～90℃, the extraction time120～150min, the concentration of NaOH2mol/L, the alkali liquid volume4-6times of wet mycelia. Then the yield of (1â†'3)-α-D-glucan is6.5%of dry mycelia weight.Isolation of ergosterol from Penicillium chrysogenum mycelia was carried out by the saponification reaction at80℃. The other optimal conditions were:the NaOH concentration2.0mol/L, the reaction time2h, the concentration of alcohol20%, the alkali liquid volume3times of wet mycelia. The highest yield of ergosterol of5.8mg of per gram of dry mycelia was achieved under the other optimal conditions. In the procedure of extracting ergosterol with petroleum ether, the optimal temperature is50℃, and the volume of petroleum ether is80ml/100g wet mycelia. The resulting ergosterol crystal showed ultraviolet absorption peaks at294nm,282nm,272nm and261nm in petroleum ether. The ergosterol was harvest from the evaporate-dried solid by crystallization。After crystallization, the primrose crystals was found to be0.1226g with recovery of52.1%. The white crystals had been gained in recrystallization with weight0.0806g, recovery65.7%and purity95.7%, The total recovery was34.4%.The deacetylation reaction condition for the extraction of chitosan from Penicillium chrysogenum mycelia is optimized as:temperature80℃, NaOH concentration2mol/L, time2.5h. The concentration of hydrochloric acid is2%. Then the deacetylation of chitosan is87%with the yield of48mg per garmm dry mycelia weight. The molecular weight of deacetylated chitosans around10kDa,30kDa and50kDa with yields are71.2%,18.4%and10.4%, respectively.It can be concluded that the key steps in ergosterol, chitosan and (1â†'3)-α-D-glucan extraction processes are saponification, deacetylation and dissolving, respectively. In these steps, the amount of alkali profoundly effect the yields, cost and productions of waste water. Based on those experiments, a process had been designed to integratively extract (1-+3)-α-D-glucan, chitosan and ergosterol from Penicillium chrysogenum mycelia. The extraction conditions had been studied and followed by scaling-up. The saline organic waste water produced from total process had been used for anaerobic fermention, and culturing Rhodotorula glutinis to product β-carroten. The results indicated that the concentration of NaOH influences the yield of chitosan and ergosterol, and the ethanol also facilitated the reaction of saponification. Under the condition of85℃,2M NaOH,20%ethanol and1.5h, the yields of chitosan, ergosterol and (1â†'3)-α-D-glucan are6.3%,0.585%and6.7%of dry mycelia. Considering the complexity of the process system, the Back-Propagation Artificial Neural Networks (BPANN) method had been used to study the condition of process. A BPANN with two layers had been built. The tan-sigmoid and linear transfer function had been adopted in hidden layer and output layer, respectively. At beginning four and two nerve centers had been set up in hidden layer and output layer, respectively. The arithmetic of Levengerg-Marquardt had been used in network training. The result indicated that the number of crunode in hidden layer has effect on the training error and nine was optimal. There has linear relationship between training data and experiment data. The slops and the correlation coefficients were totally as1. The validating experiments show that the data from computer modeling and experiment are tallying.According to the result of BPANN and cost of materials, the condition was confirmed as:85℃,2M NaOH,20%ethanol,120min and2.5times alkali liquid volume of wet mycelia. The simulation output of yield of chitosan was6.5791%, and ergosterol was0.5902%. The real yield of chitosan was6.43%, and ergosterol was0.58%. Based upon those studies, four times of primary enlarging experiments had been carried out with10kg wet mycelia each time. The average yields of (1â†'3)-α-D-glucan, chitosan and ergosterol were7.76%,4.37%and0.665%, respectively. After twice crystallization, the total harvest of crystal was36.2%with purity of93.5%. For chitosan, its purity was95.4%.For the treatment of saline organic waste water produced from total process, the anaerobic fermention reactor which contains domesticated sludge for the production of methane got balance after40days running. Waste water100ml could be treated and methane70ml could be produced a day. The COD value of output water had been declined below3000mg/kg. After six days culturing using wastewater, the biomass of Rhodotorula glutinis was9g/L, and the COD removal efficiency was50%. If1%glucose was added in the waste water, the biomass of Rhodotorula glutinis got to13g/L, and the COD removal efficiency got to65%after seven days culturing. The β-carroten produced from Rhodotorula glutinis was0.25mg/g of biomass and3.3mg/L of waste water.For the utilization of (1â†'3)-α-D-glucan, the works of preparation activated glucan microspheres for lipase immobilization and of synthesis quaternary ammonium-glucan for antimicrobial had been studied. The total protein loading was found to be1.41mg/g of supports, with a loading yield of31.3%and an enzyme activity of30.5U, a specific activity of26.8U.mg-1protein and an activity yield of83.8%at the optimum condition. The thermal stability of the immobilized enzyme in the temperature range of45-65℃was also improved. After two hours incubation of in45℃, the residual activity of immobilized lipases still remained higher than80%of the origin, and in55℃, remained higher than70%. Even in65℃, after0.5h, the residual activity still remained higher than50%of the origin. The immobilized lipase could be stored at room temperature for at least two months without significant loss of catalytic activity. After10cycles of consecutive operations, the residual activity of immobilized lipases still remained higher than80%of the originUnder alkali condition, glycidyl trimethylammonium chloride (GTMAC) reacted with (1â†'3)-α-D-glucan to form quaternary ammonium-glucan which has stronger antibacterial. The MIC(minimal inhibitory concentrations) for E.coli and St.aureus was1.0mg/ml and2.0mg/ml, respectively. The quaternary ammonium-glucan effects on bacteria membrane.Oligo-chitosan is bio-pestcide prepared from chitosan. The field experiment of resistance on watermelon Fusarium wilt indicated that during early days, by the way of irrigated root,200～600times diluted5%oligo-chitosan can play effectively. From comparison of different zones and different time, it can be shown that the prevention and cure efficiencies were stable.For resistance on cotton Fusarium wilt, oligo-chitosan showed stimulative effect on seedling growing. After treated with oligo-chitosan, the high of individual plant and the length of root were more then control experiment. And the ratio of died was lower then control experiment. The prevention and cure efficiencies obtained to70%. |
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