R.Liu.F.Shen Bioresource Technology 99 (2008)847-854 851 was 5.0,which indicates that the pores on the matrix of Ca- the order of influence is temperature>agitation rate>pH> alginate are large enough to keep the nutrition materials particles stuffing rate.The optimal condition for accelerat- and inorganic salt ions go through from the outside of ing fermentation rate was determined as A B:CD, immobilized yeast particles to its inside freely;The results namely,temperature,agitation rate,particles stuffing rate showed that as the particles stuffing rate was increased the and pH were 37C,200rpm,25%and 3.5,respectively. ethanol yield was increasing up to 25%.After this particles The results of analysis of variance (ANOVA)showed stuffing rate the ethanol yield was decreasing.A particles that F values of four factors were as follows:Ftemperature= stuffing rate of 25%resulted the maximum ethanol yield of 7100.67,Fagitation rate=2542.64,Fparticles stuling rate920.53, 86.18%.The growth and metabolism of yeast cells would be FpH=1510.77,which were all greater than the critical F restricted when too high immobilized yeast particles value=4.40 (p<0.01).It could be concluded that the four stuffing rate is offered in the ethanol fermentation because factors were all high significant in influencing fermentation the nutrition material is not infinite.In addition.when the rate. particles stuffing rate is in a lower value,the quantity of The CO2 weight loss rates at different levels and factors immobilized yeast used for fermentation would be in fall. are displayed in Fig.3.The CO2 weight loss rate increased Hence,fermentation efficiency would be decreased at a with the increased temperature from 28C to 37C.The lower particles stuffing rate.The highest ethanol yield of highest CO,weight loss rate was 0.801 gh-at a fermenta- 85.73%was obtained at an agitation rate of 200 rpm.Both tion temperature of 37C.This could be attributed to many the permeation intensity of nutrition materials from the fer- kinds of enzymes in the yeast cells which work for the etha- mentation solution to the inside of yeast cells and that of nol formation in different biochemistry processes.In fact, ethanol from the inside of yeast cells to the fermentation the ethanol fermentation is the enzyme catalysis processes solution could be improved with the increased agitation Therefore,the fermentation rate would increase with the rate.These processes would enhance the sugar utilization increased fermentation temperature (Qi and Zhang,1999). and weaken the inhibition of ethanol to the yeast cells. The highest CO,weight loss rate of 0.706gh-was However,too high agitation rate would not work for obtained at agitation rate of 200rpm and kept almost improvement of ethanol yield any more because of the limi- invariable when the agitation rate was increased from tation of metabolism ability of the yeast cells.Therefore, 200rpm to 250rpm.This is probably due to the fact that the ethanol yield was not enhanced when the agitation rate the fluidity of substrate in fermentation has direct influence exceeded 200 rpm on the mass transfer between the substrate and the immobi- lized yeast during a certain range in the cells immobiliza- 3.3.Impacts of multi-factors on CO2 weight loss rate of tion system(Cheng,2000).When the fluidity of substrate ethanol fermentation solution exceeds a certain value.it is the membrane thick- ness of immobilized yeast particles to control the mass CO,is the other product of ethanol fermentation from transfer.As a result,fermentation rate would be enhanced sugar by S.cerevisiae.When batch fermentation rate was with the increased agitation rate from 100rpm to 200rpm, considered,the CO2 weight loss rate could represent fer- and kept almost invariable from 200rpm to 250 rpm. mentation rate instead of ethanol production rate.Table 5 shows the range analysis of L(4)orthogonal experiments 0.85 of CO2 weight loss rate.Range (R)of factor A was 0.353, -Temperature which ranked the first.Factor B was 0.194,which ranked 0.80- 一o一A接i准tion ra1e Partides stufling rate second.Factor D was 0.161,which ranked third.The factor C was 0.120,which ranked the last.According to the range, 0.75 0.70 Table 5 The range analysis of L(4)orthogonal experiments of CO,weight loss rate 0.60 Temperature Agitation Blank Particles pH rate stuffing rate 0.55 A B C 0.50- K1 5.381 6.136 7.605 7.397 8.509 7.346 7.656 7.120 7.993 7.958 0.45- K 8.208 8.468 7.648 8.296 6.579 9.613 8.288 8.175 6.862 7.502 0.40 30 40 0.448 0.511 0.634 0.616 0.709 pH 4.5 0.612 0.638 0.539 0.666 0.663 Temperature (C) k3 0.684 0.706 0.637 0.691 0.548 100 10 700 20 Agitation rate(rpwm） ka 0.801 0.691 0.681 0.571 0.625 15 R 0.353 0.194 0.142 0.120 0.161 Particles stuming rate ( Q As B3 C3 Fig.3.The CO,weight loss rate at different levels and factorsR. Liu, F. Shen / Bioresource Technology 99 (2008) 847–854 851 was 5.0, which indicates that the pores on the matrix of Ca￾alginate are large enough to keep the nutrition materials and inorganic salt ions go through from the outside of immobilized yeast particles to its inside freely; The results showed that as the particles stuYng rate was increased the ethanol yield was increasing up to 25%. After this particles stuYng rate the ethanol yield was decreasing. A particles stuYng rate of 25% resulted the maximum ethanol yield of 86.18%. The growth and metabolism of yeast cells would be restricted when too high immobilized yeast particles stuYng rate is oVered in the ethanol fermentation because the nutrition material is not inWnite. In addition, when the particles stuYng rate is in a lower value, the quantity of immobilized yeast used for fermentation would be in fall. Hence, fermentation eYciency would be decreased at a lower particles stuYng rate. The highest ethanol yield of 85.73% was obtained at an agitation rate of 200 rpm. Both the permeation intensity of nutrition materials from the fer￾mentation solution to the inside of yeast cells and that of ethanol from the inside of yeast cells to the fermentation solution could be improved with the increased agitation rate. These processes would enhance the sugar utilization and weaken the inhibition of ethanol to the yeast cells. However, too high agitation rate would not work for improvement of ethanol yield any more because of the limi￾tation of metabolism ability of the yeast cells. Therefore, the ethanol yield was not enhanced when the agitation rate exceeded 200 rpm. 3.3. Impacts of multi-factors on CO2 weight loss rate of ethanol fermentation CO2 is the other product of ethanol fermentation from sugar by S. cerevisiae. When batch fermentation rate was considered, the CO2 weight loss rate could represent fer￾mentation rate instead of ethanol production rate. Table 5 shows the range analysis of L16(45 ) orthogonal experiments of CO2 weight loss rate. Range (R) of factor A was 0.353, which ranked the Wrst. Factor B was 0.194, which ranked second. Factor D was 0.161, which ranked third. The factor C was 0.120, which ranked the last. According to the range, the order of inXuence is temperature > agitation rate > pH > particles stuYng rate. The optimal condition for accelerat￾ing fermentation rate was determined as A4B3C3D1, namely, temperature, agitation rate, particles stuYng rate and pH were 37 °C, 200 rpm, 25% and 3.5, respectively. The results of analysis of variance (ANOVA) showed that F values of four factors were as follows: FtemperatureD 7100.67, Fagitation rateD2542.64, Fparticles stuYng rateD 920.53, FpH D 1510.77, which were all greater than the critical F valueD 4.40 (p< 0.01). It could be concluded that the four factors were all high signiWcant in inXuencing fermentation rate. The CO2 weight loss rates at diVerent levels and factors are displayed in Fig. 3. The CO2 weight loss rate increased with the increased temperature from 28 °C to 37 °C. The highest CO2 weight loss rate was 0.801 g h¡1 at a fermenta￾tion temperature of 37 °C. This could be attributed to many kinds of enzymes in the yeast cells which work for the etha￾nol formation in diVerent biochemistry processes. In fact, the ethanol fermentation is the enzyme catalysis processes. Therefore, the fermentation rate would increase with the increased fermentation temperature (Qi and Zhang, 1999). The highest CO2 weight loss rate of 0.706 g h¡1 was obtained at agitation rate of 200 rpm and kept almost invariable when the agitation rate was increased from 200 rpm to 250 rpm. This is probably due to the fact that the Xuidity of substrate in fermentation has direct inXuence on the mass transfer between the substrate and the immobi￾lized yeast during a certain range in the cells immobiliza￾tion system (Cheng, 2000). When the Xuidity of substrate solution exceeds a certain value, it is the membrane thick￾ness of immobilized yeast particles to control the mass transfer. As a result, fermentation rate would be enhanced with the increased agitation rate from 100 rpm to 200 rpm, and kept almost invariable from 200 rpm to 250 rpm. Table 5 The range analysis of L16(45 ) orthogonal experiments of CO2 weight loss rate Temperature Agitation rate Blank Particles stuYng rate pH A B CD K1 5.381 6.136 7.605 7.397 8.509 K2 7.346 7.656 7.120 7.993 7.958 K3 8.208 8.468 7.648 8.296 6.579 K4 9.613 8.288 8.175 6.862 7.502 k1 0.448 0.511 0.634 0.616 0.709 k2 0.612 0.638 0.539 0.666 0.663 k3 0.684 0.706 0.637 0.691 0.548 k4 0.801 0.691 0.681 0.571 0.625 R 0.353 0.194 0.142 0.120 0.161 Q A4 B3 C3 D1 Fig. 3. The CO2 weight loss rate at diVerent levels and factors