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BmR1 antigen production in recombinant E. coli fed-batch fermentation
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Abstracts / Journal of Biotechnology 136S (2008) S460–S495
100 g of CO2 used, 200 bar, 70 ◦ C and 60 min, at which the extraction yields of total chlorophyll and caffeine were 86.39% and 82.41%, respectively. References Park, H.S., Lee, H.J., Shin, M.H., Lee, K.W., Lee, H., Kim, Y.S., Kim, K.O., Kim, K.H., 2007. Effects of cosolvents on the decaffeination of green tea by supercritical carbon dioxide. Food Chemistry 105, 1011–1017. Rasch, V., 2003. Cigarette, alcohol, and caffeine consumption: risk factors for spontaneous abortion. Acta Obstetrics and Gynecology Scandinavia 82, 182–188.
doi:10.1016/j.jbiotec.2008.07.1112 V5-P-044 Inactivation of biofilms formed by Bacillus cereus and Eschericahia coli O157:H7 by using supercritical carbon dioxide Hee Jung Park, Jihyun Bang, Hae Young Kim, Jee-Hoon Ryu, Kyoung Heon Kim ∗ School of Life Sciences and Biotechnology, Korea University, Seoul 136713, Republic of Korea E-mail address: [email protected] (K.H. Kim). The biofilm indicates the gelatinous matrix formed by bacteria, and is generally composed of exopolysaccharides and a small amount of proteins and nucleic acids. When bacterial communities are embedded in biofilms, it is difficult to inactivate the bacteria even with a variety of detergents and antibiotics. It is because its dense extracellular matrix and outer layer of cells protect the bacterial community inside the film. Therefore, we have developed a novel supercritical carbon dioxide (SC-CO2 ) process for inactivating biofilms. For the target microrgoganism, the gram positive Bacillus cereus (B. cereus) and the gram negative Escherichia coli (E. coli) O157:H7, the conditions for SC-CO2 treatment were in the ranges of 35–45 ◦ C and 100–250 bar and 35–70 ◦ C and 80–200 bar, respectively. The vegetative cells of B. cereus in its biofilm showed 0.9–2.6 log reductions of colony forming unit (CFU) per mL at constant pressure, and 3.6–4.6 log reductions of CFU per mL at constant temperature. For the case of E. coli O157:H7 in the biofilm, the SCCO2 treatment was found to completely inactivate the cells in the above-mentioned treatment ranges. For both bacteria, the effectiveness was more efficient than generally used chemical sanitizers. These results are presented as the first report on the inactivation of bacterial biofilms by using SC-CO2 . doi:10.1016/j.jbiotec.2008.07.1113 V5-P-045 BmR1 antigen production in recombinant E. coli fed-batch fermentation Ahmad R.M. Yahya 1,∗ , Norsyahida Arifin 2 , Rahmah Noordin 2 1
School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden Penang, Malaysia 2 Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Minden Penang, Malaysia E-mail address: [email protected] (A.R.M. Yahya). BmR1, presently produced via shaken cultures, is used in Brugia RapidTM kit (MBDr Sdn Bhd), a diagnosis test for the detection of lymphatic filariasis caused by Brugia malayi and Brugia timori (Rahmah et al., 2001). Increasing demand calls for new cultivation methods to boost production, namely fed-batch cultivation. Typi-
cally, controlling the growth at a lower specific growth rate prior to induction resulted in higher product yield. However, applying exponential feeding during the post-induction phase may not result in a constant specific growth rate due to changes in cell physiology and metabolism (Heng et al., 1998). A fed-batch study of the recombinant E. coli expressing BmR1 antigen was performed to investigate the effect of feeding protocols, inducer concentration and time of induction on the antigen production. The experiments were carried out in a Biostat B5 5-L fermenter, equipped with MFCS/Win software (B. Braun, Melsungen, Germany). Cells were grown at controlled specific growth rates prior to induction, namely 0.075, 0.10, and 0.15 h−1 . Upon induction, the culture was fed at a constant feed rate of either 1.9 ml min−1 or 3.9 ml min−1 , keeping the acetic acid concentration below the inhibitory level (1.5 g L−1 ) (Shiloach and Fass, 2005). Comparison of similar post-induction conditions data suggests that pre-induction feeding protocol affects antigen and biomass production, generally inversely related to each other. BmR1 level increased 20% when the culture was induced with 1 mM of IPTG compared to that produced at 5 mM IPTG. Furthermore, applying IPTG induction twice at the late log phase gave a threefold increase in antigen concentration. Overall, fed-batch cultivation and induction protocol has led to a sixfold improvement in BmR1 concentration (11.9 mg L−1 ) compared to that in conventional flask cultivations. Concentration of BmR1 in experiments with the highest biomass and highest BmR1 concentrations are 5.95 mg L−1 and 11.9 mg L−1 , respectively. References Heng, H.W., Yeon, C.K., Sang, Y.L., Ho, N.C., 1998. Effect of post-induction nutrient feeding strategies on the production of bioadhesive protein in Escherichia coli. Biotechnology and Bioengineering 60 (3), 271–276. Rahmah, N., Taniawati, S., Shenoy, R.K., Lim, B.H., Kumaraswami, V., Khairul, A.A., Lokman, H.S., Noor, H.M.I., Chan, B.T., Suharni, M., Ramachandran, C.P., 2001. Specificity and sensitivity of a rapid dipstick test (Brugia Rapid) in the detection of Brugia malayi infection. Transactions of the Royal Society on Tropical Medicine and Hygiene 95 (6), 601–604. Shiloach, J., Fass, R., 2005. Growing E.coli to high cell density—a historical perspective on method development. Biotechnology Advances 23, 345–357.
doi:10.1016/j.jbiotec.2008.07.1114 V5-P-046 Recovery of acetone–butanol–ethanol from fermentation broth by liquid–liquid extraction Mohd Sobri Takriff ∗ , Siti Jamilah Hanim Mohd Yusof, Abdul Amir Hassan Kadhum, Jamaliah Jahim, Abdul Wahab Mohammad Chemical and Process Engineering Department, Faculty of Engineering, Universiti Kebangsaan Malaysia, Bangi, Malaysia E-mail address: [email protected] (M.S. Takriff). Conventional distillation process for the recovery of acetone–butanol–ethanol (ABE) fermentation product is unfavorable due to its high energy consumption for product purification, thus effecting the economically viability of ABE fermentation. A more energy efficient recovery processes is desired and liquid–liquid extraction appears to be particularly promising (Boudreau and Gordon, 2006; Dadgar and Foutch, 1988; Qureshi and Maddox, 1995). The objectives of this study are to identify potential solvents for the acetone–butanol–ethanol (ABE) recovery and to investigate the performance of separation process through liquid–liquid extraction. In the previous studies, model solutions consist of water–ABE and POME-ABE solutions were prepared according to the common ABE ratio (A:B:E = 3:6:1). A volume of solvent is added to the model solution in a separating funnel with
Abstracts / Journal of Biotechnology 136S (2008) S460–S495
100 g of CO2 used, 200 bar, 70 ◦ C and 60 min, at which the extraction yields of total chlorophyll and caffeine were 86.39% and 82.41%, respectively. References Park, H.S., Lee, H.J., Shin, M.H., Lee, K.W., Lee, H., Kim, Y.S., Kim, K.O., Kim, K.H., 2007. Effects of cosolvents on the decaffeination of green tea by supercritical carbon dioxide. Food Chemistry 105, 1011–1017. Rasch, V., 2003. Cigarette, alcohol, and caffeine consumption: risk factors for spontaneous abortion. Acta Obstetrics and Gynecology Scandinavia 82, 182–188.
doi:10.1016/j.jbiotec.2008.07.1112 V5-P-044 Inactivation of biofilms formed by Bacillus cereus and Eschericahia coli O157:H7 by using supercritical carbon dioxide Hee Jung Park, Jihyun Bang, Hae Young Kim, Jee-Hoon Ryu, Kyoung Heon Kim ∗ School of Life Sciences and Biotechnology, Korea University, Seoul 136713, Republic of Korea E-mail address: [email protected] (K.H. Kim). The biofilm indicates the gelatinous matrix formed by bacteria, and is generally composed of exopolysaccharides and a small amount of proteins and nucleic acids. When bacterial communities are embedded in biofilms, it is difficult to inactivate the bacteria even with a variety of detergents and antibiotics. It is because its dense extracellular matrix and outer layer of cells protect the bacterial community inside the film. Therefore, we have developed a novel supercritical carbon dioxide (SC-CO2 ) process for inactivating biofilms. For the target microrgoganism, the gram positive Bacillus cereus (B. cereus) and the gram negative Escherichia coli (E. coli) O157:H7, the conditions for SC-CO2 treatment were in the ranges of 35–45 ◦ C and 100–250 bar and 35–70 ◦ C and 80–200 bar, respectively. The vegetative cells of B. cereus in its biofilm showed 0.9–2.6 log reductions of colony forming unit (CFU) per mL at constant pressure, and 3.6–4.6 log reductions of CFU per mL at constant temperature. For the case of E. coli O157:H7 in the biofilm, the SCCO2 treatment was found to completely inactivate the cells in the above-mentioned treatment ranges. For both bacteria, the effectiveness was more efficient than generally used chemical sanitizers. These results are presented as the first report on the inactivation of bacterial biofilms by using SC-CO2 . doi:10.1016/j.jbiotec.2008.07.1113 V5-P-045 BmR1 antigen production in recombinant E. coli fed-batch fermentation Ahmad R.M. Yahya 1,∗ , Norsyahida Arifin 2 , Rahmah Noordin 2 1
School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden Penang, Malaysia 2 Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Minden Penang, Malaysia E-mail address: [email protected] (A.R.M. Yahya). BmR1, presently produced via shaken cultures, is used in Brugia RapidTM kit (MBDr Sdn Bhd), a diagnosis test for the detection of lymphatic filariasis caused by Brugia malayi and Brugia timori (Rahmah et al., 2001). Increasing demand calls for new cultivation methods to boost production, namely fed-batch cultivation. Typi-
cally, controlling the growth at a lower specific growth rate prior to induction resulted in higher product yield. However, applying exponential feeding during the post-induction phase may not result in a constant specific growth rate due to changes in cell physiology and metabolism (Heng et al., 1998). A fed-batch study of the recombinant E. coli expressing BmR1 antigen was performed to investigate the effect of feeding protocols, inducer concentration and time of induction on the antigen production. The experiments were carried out in a Biostat B5 5-L fermenter, equipped with MFCS/Win software (B. Braun, Melsungen, Germany). Cells were grown at controlled specific growth rates prior to induction, namely 0.075, 0.10, and 0.15 h−1 . Upon induction, the culture was fed at a constant feed rate of either 1.9 ml min−1 or 3.9 ml min−1 , keeping the acetic acid concentration below the inhibitory level (1.5 g L−1 ) (Shiloach and Fass, 2005). Comparison of similar post-induction conditions data suggests that pre-induction feeding protocol affects antigen and biomass production, generally inversely related to each other. BmR1 level increased 20% when the culture was induced with 1 mM of IPTG compared to that produced at 5 mM IPTG. Furthermore, applying IPTG induction twice at the late log phase gave a threefold increase in antigen concentration. Overall, fed-batch cultivation and induction protocol has led to a sixfold improvement in BmR1 concentration (11.9 mg L−1 ) compared to that in conventional flask cultivations. Concentration of BmR1 in experiments with the highest biomass and highest BmR1 concentrations are 5.95 mg L−1 and 11.9 mg L−1 , respectively. References Heng, H.W., Yeon, C.K., Sang, Y.L., Ho, N.C., 1998. Effect of post-induction nutrient feeding strategies on the production of bioadhesive protein in Escherichia coli. Biotechnology and Bioengineering 60 (3), 271–276. Rahmah, N., Taniawati, S., Shenoy, R.K., Lim, B.H., Kumaraswami, V., Khairul, A.A., Lokman, H.S., Noor, H.M.I., Chan, B.T., Suharni, M., Ramachandran, C.P., 2001. Specificity and sensitivity of a rapid dipstick test (Brugia Rapid) in the detection of Brugia malayi infection. Transactions of the Royal Society on Tropical Medicine and Hygiene 95 (6), 601–604. Shiloach, J., Fass, R., 2005. Growing E.coli to high cell density—a historical perspective on method development. Biotechnology Advances 23, 345–357.
doi:10.1016/j.jbiotec.2008.07.1114 V5-P-046 Recovery of acetone–butanol–ethanol from fermentation broth by liquid–liquid extraction Mohd Sobri Takriff ∗ , Siti Jamilah Hanim Mohd Yusof, Abdul Amir Hassan Kadhum, Jamaliah Jahim, Abdul Wahab Mohammad Chemical and Process Engineering Department, Faculty of Engineering, Universiti Kebangsaan Malaysia, Bangi, Malaysia E-mail address: [email protected] (M.S. Takriff). Conventional distillation process for the recovery of acetone–butanol–ethanol (ABE) fermentation product is unfavorable due to its high energy consumption for product purification, thus effecting the economically viability of ABE fermentation. A more energy efficient recovery processes is desired and liquid–liquid extraction appears to be particularly promising (Boudreau and Gordon, 2006; Dadgar and Foutch, 1988; Qureshi and Maddox, 1995). The objectives of this study are to identify potential solvents for the acetone–butanol–ethanol (ABE) recovery and to investigate the performance of separation process through liquid–liquid extraction. In the previous studies, model solutions consist of water–ABE and POME-ABE solutions were prepared according to the common ABE ratio (A:B:E = 3:6:1). A volume of solvent is added to the model solution in a separating funnel with