| A bioflocculant is a kind of green wastewater treatment agents which has arouse attention for recent years due to its innocuity, no secondary pollution and biodegradability, and also has vast applied foreground in flocculation procedure of wastewater treatment for potential economic benefits. The limits of industrial production of bioflocculant are mainly high cost and oneflod method of fermentation. Therefore, in order to reduce fermention cost using low-cost wastewater comprising of sugars and improve fermentation method by immobilization technccutology, this dissertation is to analyze the transmembrane properties of bioflocculant-producing bacterium Agrobacterium tumefaciens F2, build the prediction model of bioflocculant yield produced from mixed carbo n sources of glucose, galactose and mannose and develop a new technology of fermentation using mycelial pellet as a biomass carrier.Glucose, galactose and mannose are chosen as sole substrate with different initial concentration from 25 mmol/L to 150 mmol/L. The carbohydrates concentration was tested by HPLC every other 6 hours in 36 hours. According to cell kinetics and Michaelis-Menten equation, this dissertation is to calculate the intracellular glycolysis rate, transmembrane rate and permeability coeff icient. The results revealed that glycolysis rate of glucose was higher than the counterpart of mannose, while the rate of galactose was the lowest among the three carbon sources. Eventually, initial concentration of 10 mmol/L was employed for the next calculation. Transmembrane rate of the three carbohydrates increased first and then decreased while permeability coefficient kept declining when the initial concentration of extracellular carbohydrates are in the range of 25-150 mmol/L. The peak value of transport rate for each carbohydrates was achieved under the concentration of 100 mmol/L after 18 hours cultured. During the culture time of 18-30 hours, cells’ permeability was in high level and the figures of galactose were less than those of glucose and mannose.In order to realize the high concentration of organic wastewater with glucose, galactose and mannose for bioflocculant production, growth kinetics of strain F2 in each monose as sole carbon source was examined. Besides the cost and maximum growth rate were taken into consideration, it was reasonnable to choose 9 g/L as the initial concentration of carbon sources. The results showed that the maximum biomass was obtained with the dosage proportion of three carbohydrates in 5:1:3. Neural networks was adopted for prediction model construction of bioflocculant yield, with glucose, galactose and mannose as input layers meanwhile flocculation efficiency and bioflocculant yield as output layers. The optimal results of carbon substrates concentration were 6.59 g/Lglucose, 1.32 g/L galactose and 3.57 g/L mannose. Under this conditions, the peak of flocculation efficiency and bioflocculant yield were 95.09% and 2.91 g/L respectively, which were comparable to the prediction values, proving the good fitting ability of the model with only 4% errors in the range. Metabolism of mixed carbon sources were measured by HPLC after pre-column derivatization. According to the bacterial biomass and product synthesis, strain F2 produced bioflocculant mainly during the latter period of logarithmic phase(14-18 h) and the former time of steady phase(18-26 h). The maximum yield(2.92 g/L) was achieved after the culture of 24 hours.On the basis of the material balance in chemometrics, bacterial growth, product synthesis, substrates consumption and other carbon conversation were measured and calculated to investigate different fermentation modes including sole carbohydrates(glucose, galactose, mannose), mixed carbon sources in free-cell and immobilization fermentation. It was showed that the sole carbon source was converted more into other pathway instead of bacterial biomass, EPS or protein accumulation. However, the conversation of mixed carbon sources substrates was mainly in biomass, EPS production and protein. This contributed to the mixed substrates of glucose, galactose and mannose by reducing the energy consumption during glycosyl changes one another. Obviously, glucose, galactose and mannose were supplied directly for bioflocculant production which was beneficial to condensation reaction and could enhance the conversation efficiency of substrates.Immobilization method and carrier performance were investigated by taking Aspergillus nige Y3 as biomass carrier. Adsorption equilibrium was attained after 48 h contact by adsorption method, bacterial quantities absorbed by mycelial pellet could reach 3.6 × 1012 cfu/g(mycelium dried weight). Response surface methodology was adopted for optimization of immobilized fermentation. Under the optimal predicted inoculum dose(8.85%) and cultured time(27 h), the highest yield could be 3.37 g/L, which was better than that of free-cell fermentation(only 2.93 g/L). Based on the above results, bioflocculant powder lyophilized from immobilization fermentation was employed for the optimization of f locculation conditions. The best prediction value of flocculation efficiency could be reached to 97.89% under the conditions of 0.03 g/L bioflocculant powder lyophilized, 1.09 m L/L Ca Cl2 dose and p H=8.29. After repeated experiments, the tow response surface models were proved in high accuracy. Bioflocculant production efficiency in a single circle and semi-continuous circles were compared and the growth phases kept the same but the high yield of bioflocculant(3.35 g/L) in immobilization production delayed 2 hours compared with free-cell culture. 30 Mycelial pellets carrier could be transferred more than 30 cycles to realize the semi-continuous fermentation, and meanwhile the bacterial biomass and yield could be kept stable due to the increasing absorbed bacterial cells.In sum, in order to accelerate the industrialization of bioflocculant and reduce the cost of large scale production, the study builded a prediction mode of bioflocculant yield cultred from mixed carbon sources including glucose, galactose and mannose and explored semi-continuous fermentation of bioflocculant by immobilization technology using mycelial pellet as a biomass carrier. The significance of this dissertation is to bring down the input cost during bioflocculant fermentation through using diversity wasterwater comprising of glucose, galactose and mannose. In addition, the exploitation of a new fermenting technology is benefical to guarantee bacterial biomass in system effectively, enhance bacterial resistance for enverimental change, skip the preparation of seed liquid diminish the microbial contamination as a result of withour inoculation, contribute to solid-liquid separation and dispose of carrier. All the results inidict that immobilizing semi-continuous fermentation could cut down the input cost as well as boost yield, making a promotion for easy and continuous fermentation of bioflocculant. |
