Optimization Of Fermentation Conditions And Magnification For Cellulose Production By A Marine Aspergillus Niger

Cellulase is a compound enzyme, consisted of endoglucanase, exoglucanase and β-glucosidase. Through the synergy of these three enzymes, cellulose can be degraded to available sugar. Thus, it is widely used in many industries. Marine Aspergillus niger described here can produce a salt tolerant cellulase. And because of its salt tolerance characteristics, it can efficiently degrade cellulose under high salt conditions. So the salt tolerant cellulase has great prospects in papermaking wastewater treatment, development and utilization of marine cellulose waste, development use of marine resources and cellulosic feedstock pretreatment of cellulose wastewater.In this paper, we optimize fermentation of producing cellulase in the shake flask level, then have been achieved from the shake flask level to the fermentor level. Then the submerged fermentation culture conditions are optimized, and then the crude enzyme solution is preliminary concentrated and purified. And finally, the salt tolerance and enzymatic properties are researched and compared by an ordinary cellulase. The main results are as follows:Firstly, we optimize fermentation of producing cellulase in the shake flask level. In order to produce cellulase in high efficiency using a marine Asergillus niger, the response surface analysis and variable temperature culture were used to optimize the fermentation conditions. Firstly, the single factor test were carried out to ascertain carbon source, nitrogen source, temperature, surface active agent, inoculum concentration, liquid volume in flask and initial pH. Then carbon source and Tween-80 were identified by Plackett-Burman(PB) design as two main factors on FPA, and their concentration were ascertained by steepest ascent path. Finally, using response surface analysis and variable temperature culture, the optimal fermentation medium was determined as 40 mL of medium consisting of 5.53% wheat bran,0.5% corn steep liquor,0.2% KH2PO4,0.197% Tween-80 in a 150 mL flask, and the optimal fermentation conditions were initial pH of 5, inoculum concentration of 8%, culture temperature 37℃ for 2 days and 28℃ for 2 days.The maximum FPA under the optimized conditions was 0.566 U/mL, which was increased by 94.5% compared with 0.283 U/mL obtained under the normal conditions.Secondly, we have been achieved from the shake flask level to the fermentor level, fermentation tanks and the submerged fermentation culture conditions are optimized. Firstly we use fermentation optimal culture conditions as the foundation, set the appropriate ventilation ratio and speed of agitator to obtain a final dry weight of mycelium 9 g/L, the filter paper activity 0.42 U/mL. After the optimization of temperature and pH and DO respectively, we choose the appropriate subdivision control of temperature and pH in the culture fermentation, and finally the dry weight of mycelium reachs 15.7g/L, increase by 74%, the filter paper activity reached 0.72 U/mL, improved 71.4%.Thirdly, the crude enzyme solution is preliminary concentrated and purified. Firstly, we concentrate crude cellulase enzyme by ammonium sulfate fractionation.And find its highest enzyme activity in ammonium sulfate saturation of 55% of the time, and which have the highest specific activity. However, because there are also many cellulase protein at 60% saturation of ammonium sulfate precipitation, so 60% should be selected as the optimal saturation in the industry. Then fermentation broth is concentrated by ammonium sulfate precipitation or ultrafiltration, we find that ultrafiltration is better than the ammonium sulfate precipitation, but in industry, people pay more attention to the cost, ammonium sulfate precipitation may be more popular.Finally, we select cellulase producted by Normal Aspergillus niger ATCC 16888 as the contrast, finding that salt tolerant cellulase is significantly better than ordinary cellulase in either salt tolerance or heat tolerance. And the enzyme compositions of them are similar, and they all belong to the acidic cellulase, but in terms of adaptability of pH, salt tolerant cellulase is relatively narrow, nearly lost more than a half of enzyme activity in near neutral pH.