848 R.Liu,F.Shen Bioresource Technology 99 (2008)847-854 Cell immobilization can be more effective because cell Table 1 washout in continuous operation is prevented,and,hence. The composition of culture media cell separation and/or recycle are not required for main- Composition of Slant culture Liquid medium Fermentation taining high cell density in the bioreactor;thus,the biopro- culture media medium (g(100mL)- medium cesses can be operated more efficiently (Tzeng et al.,1991). (g(100mL)- (g(100mL)-) Many researches concerned with immobilized cells have Glucose or sucrose 5.0 5.0 10.0 been carried out throughout the world.Particularly,there is Yeast extract 0.5 0.5 0.5 Peptone 0.5 0.5 0.5 an increasing interest in the practical applications of immo- K,HPO 0.1 0.1 0.1 bilized cells in ethanol production(Kobayashi and Nakam- MgSO4·7H,O 0.1 0.1 0.1 ura,2004),and considerable researches have been Agar 2.0 performed over the last 20 years into the use of immobi- lized cell systems for the production of fuel and potable- grade ethanol (Bardi et al..1996). the yeast cells were up to 108 cellsmL-observed by micro- There are many factors that have influence upon the eth- scope,the yeast cells in liquid culture medium were mature; anol yield and fermentation rate in fermentation process, Fermentation culture was carried in a 500mL flask with a such as fermentation temperature,agitation rate,pH and working volume of 200-250 mL.The flask was inoculated particles stuffing rate that is defined as a ratio of immobi- with 20-25 mL mature yeast cells of liquid culture medium lized yeast particles weight to fermentation solution weight. and incubated at 30C and 150 rpm for about 48h.When The immobilization process changes the environmental, the yeast cells were up to 10 cellsmL-,the yeast cells in physiological and morphological characteristics of cells, fermentation culture medium were mature,and could be along with the catalytic activity (Prasad and Ishra,1995). used for the yeast cells immobilization. As a result,the fermentation conditions are different between free yeast and immobilized yeast fermentation. 2.3.Yeast cells immobilization and proliferation The aim of the current work was to investigate the effect of main variables (fermentation temperature,agitation rate, Na-alginate (A.R)powder was dissolved in distilled particles stuffing rate and pH)on ethanol yield and CO2 water in a 75-80C water bath for 30min at the ratio of weight loss rate and to determine optimal condition for eth- 2.5-3.0:100.The mature yeast cells of fermentation culture anol fermentation by immobilized yeast from stalk juice of medium were mixed with the Na-alginate solution at the sweet sorghum. ratio of 1:10.The mixture was injected into 0.05 M calcium chloride(A.R)solution to form Ca-alginate particles by the 2.Methods equipment that was designed by us.The produced Ca-algi- nate particles were of near-spherical shape about 3 mm in 2.1.Sweet sorghum and organisms diameter.After 12h gelation,the immobilized yeast parti- cles were stored at 4C. Sweet sorghum cultivar SN Tianza No.2 was cultivated Immobilized yeast particles were added into the fermen- in the farm of Shanghai Jiao Tong University.The fresh tation culture medium at the ratio of 1:10(W/W)in 500mL crops were harvested and leaves were stripped from the flasks.The flasks were incubated at 30C and 150rpm for fresh stalks by hand before the stalks were squeezed by a 48 h.To determine the quantity of yeast in particles,the three-roller mill to obtain fresh juice.The fresh juice was immobilized yeast particles were dissolved in 0.1 M PBS sterilized in autoclave sterilizer at 0.15 MPa and 121C for solution and then observed by microscope.When the quan- 15min and stored at 4C in a refrigerator before it was tity of yeast cell in immobilized yeast particles was up to used for fermentation. 108 cellsmL-1,the immobilized yeast could be used for fer- Laboratory strain of Saccharomyces cerevisiae CICC mentation. 1308 (obtained from Centre of Industrial Culture Collec- tion of China)was used.The strain was stored on slant cul- 2.4.Equipment ture medium at 4C in a refrigerator. The equipment used in this study was a 5L bioreactor 2.2.Culture media and microorganism culture (GUJS-5C,East Biotech.Co.Zhenjiang China).The sche- matic diagram of the 5L bioreactor is shown in Fig.1. The composition of culture media is shown in Table 1. The activated Saccharomyces cerevisiae CICC 1308 was 2.5.Experiment design inoculated into flesh slant culture medium in a test tube and cultivated in a thermotank at 30C for 1-3d until the white The L16(45)orthogonal table was designed to investigate lawn appeared on the slant culture and distributed evenly; the influence of main parameters,namely the fermentation Precultures were incubated for 20-24h at 30+0.5C with temperature,agitation rate,particles stuffing rate and pH orbital shaking at 150 rpm in a 25 mL-test tube containing a on bioethanol fermentation.According to the literature loopful of yeast cells in 10mL liquid culture medium.When (Zheng and Jiang,2004)and our practical experience of848 R. Liu, F. Shen / Bioresource Technology 99 (2008) 847–854 Cell immobilization can be more eVective because cell washout in continuous operation is prevented, and, hence, cell separation and/or recycle are not required for main￾taining high cell density in the bioreactor; thus, the biopro￾cesses can be operated more eYciently (Tzeng et al., 1991). Many researches concerned with immobilized cells have been carried out throughout the world. Particularly, there is an increasing interest in the practical applications of immo￾bilized cells in ethanol production (Kobayashi and Nakam￾ura, 2004), and considerable researches have been performed over the last 20 years into the use of immobi￾lized cell systems for the production of fuel and potable￾grade ethanol (Bardi et al., 1996). There are many factors that have inXuence upon the eth￾anol yield and fermentation rate in fermentation process, such as fermentation temperature, agitation rate, pH and particles stuYng rate that is deWned as a ratio of immobi￾lized yeast particles weight to fermentation solution weight. The immobilization process changes the environmental, physiological and morphological characteristics of cells, along with the catalytic activity (Prasad and Ishra, 1995). As a result, the fermentation conditions are diVerent between free yeast and immobilized yeast fermentation. The aim of the current work was to investigate the eVect of main variables (fermentation temperature, agitation rate, particles stuYng rate and pH) on ethanol yield and CO2 weight loss rate and to determine optimal condition for eth￾anol fermentation by immobilized yeast from stalk juice of sweet sorghum. 2. Methods 2.1. Sweet sorghum and organisms Sweet sorghum cultivar SN Tianza No. 2 was cultivated in the farm of Shanghai Jiao Tong University. The fresh crops were harvested and leaves were stripped from the fresh stalks by hand before the stalks were squeezed by a three-roller mill to obtain fresh juice. The fresh juice was sterilized in autoclave sterilizer at 0.15 MPa and 121 °C for 15 min and stored at 4 °C in a refrigerator before it was used for fermentation. Laboratory strain of Saccharomyces cerevisiae CICC 1308 (obtained from Centre of Industrial Culture Collec￾tion of China) was used. The strain was stored on slant cul￾ture medium at 4 °C in a refrigerator. 2.2. Culture media and microorganism culture The composition of culture media is shown in Table 1. The activated Saccharomyces cerevisiae CICC 1308 was inoculated into Xesh slant culture medium in a test tube and cultivated in a thermotank at 30 °C for 1–3 d until the white lawn appeared on the slant culture and distributed evenly; Precultures were incubated for 20–24 h at 30§ 0.5 °C with orbital shaking at 150 rpm in a 25 mL-test tube containing a loopful of yeast cells in 10 mL liquid culture medium. When the yeast cells were up to 108 cells mL¡1 observed by micro￾scope, the yeast cells in liquid culture medium were mature; Fermentation culture was carried in a 500 mL Xask with a working volume of 200–250 mL. The Xask was inoculated with 20–25 mL mature yeast cells of liquid culture medium and incubated at 30 °C and 150 rpm for about 48 h. When the yeast cells were up to 108 cells mL¡1 , the yeast cells in fermentation culture medium were mature, and could be used for the yeast cells immobilization. 2.3. Yeast cells immobilization and proliferation Na-alginate (A.R) powder was dissolved in distilled water in a 75–80 °C water bath for 30 min at the ratio of 2.5–3.0:100. The mature yeast cells of fermentation culture medium were mixed with the Na-alginate solution at the ratio of 1:10. The mixture was injected into 0.05 M calcium chloride (A.R) solution to form Ca-alginate particles by the equipment that was designed by us. The produced Ca-algi￾nate particles were of near-spherical shape about 3 mm in diameter. After 12 h gelation, the immobilized yeast parti￾cles were stored at 4 °C. Immobilized yeast particles were added into the fermen￾tation culture medium at the ratio of 1:10 (W/W) in 500 mL Xasks. The Xasks were incubated at 30 °C and 150 rpm for 48 h. To determine the quantity of yeast in particles, the immobilized yeast particles were dissolved in 0.1 M PBS solution and then observed by microscope. When the quan￾tity of yeast cell in immobilized yeast particles was up to 108 cells mL¡1 , the immobilized yeast could be used for fer￾mentation. 2.4. Equipment The equipment used in this study was a 5 L bioreactor (GUJS-5C, East Biotech. Co. Zhenjiang China). The sche￾matic diagram of the 5 L bioreactor is shown in Fig. 1. 2.5. Experiment design The L16(45 ) orthogonal table was designed to investigate the inXuence of main parameters, namely the fermentation temperature, agitation rate, particles stuYng rate and pH on bioethanol fermentation. According to the literature (Zheng and Jiang, 2004) and our practical experience of Table 1 The composition of culture media Composition of culture media Slant culture medium (g (100 mL)¡1 ) Liquid medium (g (100 mL)¡1 ) Fermentation medium (g (100 mL)¡1 ) Glucose or sucrose 5.0 5.0 10.0 Yeast extract 0.5 0.5 0.5 Peptone 0.5 0.5 0.5 K2HPO4 0.1 0.1 0.1 MgSO4 · 7H2O 0.1 0.1 0.1 Agar 2.0 – –