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Anaerobic degradation of polychlorinated biphenyls
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Abstracts / Journal of Biotechnology 131S (2007) S211–S241
Further, using biomass support particles (BSPs) as a cell immobilized matrix, immobilized recombinant Streptomyces lividans continuously produced phospholipase D (PLD) in a yield of about 1.5 × 104 U/l in each of eight batches. In contrast to the original strain Streptoverticillium cinnamoneum, this heterologous expression system with an immobilization method is capable of producing secretory PLD with an eightfold greater efficiency. The presence of both glucose and tryptone in the initial culture medium also promoted secretory production, and PLD activity around 3.0 × 104 U/l were achieved. In addition, the promoter region of PLD ORF was deduced, and three types of plasmid having different lengths of promoter sequence were constructed. The deduced sequence had same effect on either of PLD production or mycelium immobilization, and the transformants harboring each of three plasmids showed the similar cultivation profiles (3.0 × 104 U/l). A combination of the immobilization method with BSPs and S. lividans transformant harboring the deduced plasmid has the potential for producing secretory PLD in the culture supernatant continuously. doi:10.1016/j.jbiotec.2007.07.433 57. Anaerobic degradation of polychlorinated biphenyls Vlasta Dudkova ∗ , Katerina Demnerova Institute of Chemical Technology in Prague, Technicka 5, 18000, Praha 6, Czech Republic Polychlorinated biphenyls (PCB) are organic xenobiotics contaminating environment for at least 50 years. The extensive application of the PCBs resulted in their wide distribution in the environment. PCBs are actually a large family of 209 congeners that all share the biphenyl backbone but differ by the number and the position of the chlorine atoms on the biphenyl ring. The degree of chlorine substitution influences their biodegradability which decreases with increasing chlorination. PCBs should be eliminated from environment by the activity of various organisms under different condition. Our work is focused on the PCBs biodegradation under anaerobic conditions. Suitable microbial consortium was isolated from sediment of Str´azˇ sk´y canal (located near by factory producing PCBs in past). This consorcium was able to dechlorinate polychlorinated biphenyls under anoxic condition. The effectiveness of this process was tested under different cultivation temperature (4 and 28 ◦ C) and by addition of dechlorination inducers: 2,6- or 4-4dibrombiphenyls. Bacterial medium with 4-4-dibrombiphenyl was amended by Aroclor 1248 or Aroclor 1260. For further dechlorination improvement pyruvate or lactare or acetate as an electrone donors were added. In all cases dechlorination proceeds at para position. The best results were obtained with pyruvate.
Acknowledgements This work was sponsored by Centrum 1M06011, FRVSˇ ˇ 04/03/H066. 1632/G4/2007, NPV II 2B06151 and GACR doi:10.1016/j.jbiotec.2007.07.434 58. Lipase production by a newly isolated Bacillus thermoamylovorans strain Francisco J. Deive ∗ , M. Angeles Sanroman, Maria A. Longo Institute of Chemical Technology in Prague, Technicka 5, 18000, Praha 6, Czech Republic Lipases and esterases are versatile enzymes, with a wide range of potential applications (Pandey et al., 1999). Some thermophilic microorganisms have been shown to produce this kind of activity, although the enzyme expression level is usually low, and the protein is seldom extracellular (Dominguez et al., 2004, 2007). In recent years, thermophilic enzymes have received considerable attention, mainly due to their stability in severe reaction conditions (i.e. high temperature, extreme pH, chemical reagents). However, the number of well-known enzyme-producing thermophiles is still rather limited (Hough and Danson, 1999). In this work, several hot springs in Northwest Spain were investigated as potential sources of lipase-producing thermophilic microorganisms. Nine strains were isolated, by means of tributyrin-agar and Rhodamine B-olive oil-agar plates, and their ability to produce lipolytic enzymes at 55 ◦ C on a complex liquid medium was ascertained, in flask cultures. Especial attention was paid to the location of the lipolytic enzymes (intracellular, extracellular or membrane-bound). Also, some relevant properties of the produced enzymes (i.e. optimum pH and temperature, thermostability) were assessed. Most of them showed maximum activity at alkaline pH and temperatures ranging from 55 to 95 ◦ C. Thermal stability was also very satisfactory, with high residual activities after incubation for at least 1 h at 65 or 95 ◦ C, depending on the enzyme. The most promising strains, in terms of production capability and enzyme advantageous properties, were genetically identified by 16S rRNA sequencing, and found to belong to the genus Bacillus and Anoxybacillus. One of them, tentatively denominated Bacillus thermoamylovorans CHB6, was chosen for further study of the culture conditions for lipase production. It is noteworthy that most of the lipolytic enzyme produced by this microorganism was extracellular. This is very interesting, since it would mean a significant reduction in downstream processing costs, should large-scale production be attempted. First, the most adequate pH and temperature for the production process were investigated in shake flask cultures, and found to be 7.0 and 55 ◦ C, respectively. Then, the culture was carried out in a stirred tank bioreactor, using the optimized conditions. A preliminary study of the influence of agitation and aeration rates on enzyme pro-
Abstracts / Journal of Biotechnology 131S (2007) S211–S241
Further, using biomass support particles (BSPs) as a cell immobilized matrix, immobilized recombinant Streptomyces lividans continuously produced phospholipase D (PLD) in a yield of about 1.5 × 104 U/l in each of eight batches. In contrast to the original strain Streptoverticillium cinnamoneum, this heterologous expression system with an immobilization method is capable of producing secretory PLD with an eightfold greater efficiency. The presence of both glucose and tryptone in the initial culture medium also promoted secretory production, and PLD activity around 3.0 × 104 U/l were achieved. In addition, the promoter region of PLD ORF was deduced, and three types of plasmid having different lengths of promoter sequence were constructed. The deduced sequence had same effect on either of PLD production or mycelium immobilization, and the transformants harboring each of three plasmids showed the similar cultivation profiles (3.0 × 104 U/l). A combination of the immobilization method with BSPs and S. lividans transformant harboring the deduced plasmid has the potential for producing secretory PLD in the culture supernatant continuously. doi:10.1016/j.jbiotec.2007.07.433 57. Anaerobic degradation of polychlorinated biphenyls Vlasta Dudkova ∗ , Katerina Demnerova Institute of Chemical Technology in Prague, Technicka 5, 18000, Praha 6, Czech Republic Polychlorinated biphenyls (PCB) are organic xenobiotics contaminating environment for at least 50 years. The extensive application of the PCBs resulted in their wide distribution in the environment. PCBs are actually a large family of 209 congeners that all share the biphenyl backbone but differ by the number and the position of the chlorine atoms on the biphenyl ring. The degree of chlorine substitution influences their biodegradability which decreases with increasing chlorination. PCBs should be eliminated from environment by the activity of various organisms under different condition. Our work is focused on the PCBs biodegradation under anaerobic conditions. Suitable microbial consortium was isolated from sediment of Str´azˇ sk´y canal (located near by factory producing PCBs in past). This consorcium was able to dechlorinate polychlorinated biphenyls under anoxic condition. The effectiveness of this process was tested under different cultivation temperature (4 and 28 ◦ C) and by addition of dechlorination inducers: 2,6- or 4-4dibrombiphenyls. Bacterial medium with 4-4-dibrombiphenyl was amended by Aroclor 1248 or Aroclor 1260. For further dechlorination improvement pyruvate or lactare or acetate as an electrone donors were added. In all cases dechlorination proceeds at para position. The best results were obtained with pyruvate.
Acknowledgements This work was sponsored by Centrum 1M06011, FRVSˇ ˇ 04/03/H066. 1632/G4/2007, NPV II 2B06151 and GACR doi:10.1016/j.jbiotec.2007.07.434 58. Lipase production by a newly isolated Bacillus thermoamylovorans strain Francisco J. Deive ∗ , M. Angeles Sanroman, Maria A. Longo Institute of Chemical Technology in Prague, Technicka 5, 18000, Praha 6, Czech Republic Lipases and esterases are versatile enzymes, with a wide range of potential applications (Pandey et al., 1999). Some thermophilic microorganisms have been shown to produce this kind of activity, although the enzyme expression level is usually low, and the protein is seldom extracellular (Dominguez et al., 2004, 2007). In recent years, thermophilic enzymes have received considerable attention, mainly due to their stability in severe reaction conditions (i.e. high temperature, extreme pH, chemical reagents). However, the number of well-known enzyme-producing thermophiles is still rather limited (Hough and Danson, 1999). In this work, several hot springs in Northwest Spain were investigated as potential sources of lipase-producing thermophilic microorganisms. Nine strains were isolated, by means of tributyrin-agar and Rhodamine B-olive oil-agar plates, and their ability to produce lipolytic enzymes at 55 ◦ C on a complex liquid medium was ascertained, in flask cultures. Especial attention was paid to the location of the lipolytic enzymes (intracellular, extracellular or membrane-bound). Also, some relevant properties of the produced enzymes (i.e. optimum pH and temperature, thermostability) were assessed. Most of them showed maximum activity at alkaline pH and temperatures ranging from 55 to 95 ◦ C. Thermal stability was also very satisfactory, with high residual activities after incubation for at least 1 h at 65 or 95 ◦ C, depending on the enzyme. The most promising strains, in terms of production capability and enzyme advantageous properties, were genetically identified by 16S rRNA sequencing, and found to belong to the genus Bacillus and Anoxybacillus. One of them, tentatively denominated Bacillus thermoamylovorans CHB6, was chosen for further study of the culture conditions for lipase production. It is noteworthy that most of the lipolytic enzyme produced by this microorganism was extracellular. This is very interesting, since it would mean a significant reduction in downstream processing costs, should large-scale production be attempted. First, the most adequate pH and temperature for the production process were investigated in shake flask cultures, and found to be 7.0 and 55 ◦ C, respectively. Then, the culture was carried out in a stirred tank bioreactor, using the optimized conditions. A preliminary study of the influence of agitation and aeration rates on enzyme pro-