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Mixed bacterial culture for remediation of s-triazine contaminated environment and treatment of industrial effluents
S702
Abstracts / Journal of Biotechnology 136S (2008) S678–S707
has the homoplastic bph metabolic pathway as the most aerobic PCBs degradation bacterium (Borja et al., 2005). The plasmid purification and electrophoresis showed that LY402 contains an about 20 kb plasmid, named pLYZ402. The plasmid deletion and transformation indicated the bph genes located in pLYZ402. According to homologs of bph genes, primers were designed and the genes were amplified by PCR. Nearly complete bph gene cluster about 11.2 kb was obtained. Sequence analysis showed that the gene organization and gene sequence were almost completely the same with that of Burkholderia xenovorans LB400. However, bph gene cluster of LB400 is located in a plasmid that is about 1.4 Mb. In conclusion, the advantage of LY402 is that it has strong toxicity resistance and high biodegradability to PCBs. Moreover, the degradative plasmid is very small, so it is easy to gene engineering, for instance, transforming the plasmid to aboriginal bacterium to improve survival ability, or integrating other genes to this plasmid to increase the biodegradability of PCBs or co-metabolic other pollutants. References
that this method will be suitable for bioremediation of atrazinecontaminated soils, even those rich in nitrogen, and for accelerating the process of atrazine mineralization in contaminated natural waters and industrial effluents. Reference Mixed bacterial culture for atrazine degradation. International application published under the Patent Cooperation Treaty WO 2007/0966681 A1.
doi:10.1016/j.jbiotec.2008.07.1629 VII5-P-059 Enhanced metallosorption by engineered Saccharomyces cerevisiae Pavel Kotrba ∗ , Jan Kas, Tomas Ruml Institute of Chemical Technology, Prague, Technicka 3, Prague CZ-166 28, Czech Republic E-mail address: [email protected] (P. Kotrba).
Borja, J., Taleon, D.M., Auresenia, J., Gallardo, S., 2005. Polychlorinated biphenyls and their biodegradation. Process Biochem. 40, 1999–2013. Jia, L.Y., Zheng, A.P., Xu, L., Huang, X.D., Zhang, Q., Yang, F.L., in press. Isolation and characterization of comprehensive polychlorinated biphenyl degrading bacterium, Enterobacter sp. LY402. J. Microbiol. Biotechnol. 18.
doi:10.1016/j.jbiotec.2008.07.1628 VII5-P-058 Mixed bacterial culture for remediation of s-triazine contaminated environment and treatment of industrial effluents ´ Maja Havriluk Dubravka Hrˇsak ∗ , NikolinaUdikovic´ Kolic, Rudjer Boˇskovi´c Institute, Center for Marine and Environmental Research, Zagreb 10 000, Croatia E-mail address: [email protected] (D. Hrˇsak). The present work describes a mixed bacterial culture of natural origin deposited in the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary, as the culture Atz Mix 1 (Anon., 2007). The culture was enriched from an agrochemical factory soil exposed to long-term contamination by s-triazine compounds. Growth kinetics and biodegradation studies have shown that Atz Mix 1 exhibits sustained growth and efficiently degrades atrazine at various temperatures (10–30 ◦ C) in a wide variety of concentrations (several ppb to 10,000 ppm), without formation of toxic metabolites. High mineralization degree of ring-labelled [14 C] atrazine (approximately 80%) further shows that atrazine degradation proceeds to the complete mineralization. PCR assessment of the culture genetic potential reveals the presence of catabolic genes trzN, atzB, atzC coding the enzymes for the degradation of atrazine to cyanuric acid and the gene trzD coding the enzymes for subsequent opening of s-triazine ring. The present work also describes a biodegradation method in which the culture Atz Mix 1 has been used as a biological agent. The method is considered advantageous over the so-far described methods where individual bacterial cultures are employed. This is primarily due to the culture capability of expressing sustained growth and atrazinedegrading efficiency under the conditions that are similar to those occurring in atrazine-contaminated environment (e.g. low/varying temperatures, carbon limitation, low C/N ratio, presence of preferential nitrogen source, etc.) as well as under the conditions that are specific for industrial effluents (high concentration of atrazine and other s-triazine compounds). Accordingly, it can be expected
Carboxyl-terminus of alpha-agglutinin (AGalpha1Cp) was employed to anchor CP2 S(GCGCPCGC)2 G or HP3 peptides S(GHHPH)3 G, promoting metallosorption by E. coli (Ruml and Kotrba, 2003), and/or the metal fixation motif MDCPTEEALIR (NP peptide) of P1-ATPases on the surface of the yeast biosorbent. The AGalpha1Cp variants were found covalently attached to cell wall glucan and glycosylated (Vinopal et al., 2007). Display of the mere anchoring domain multiplied the metallosorption capability of S. cerevisiae from 100 M metal solutions twofold for Cd2+ (to 19.5 ± 2.3 nmol Cd2+ mg−1 dry cell weight [dwt]) and Zn2+ (to 14.3 ± 1.6 nmol mg−1 dwt) and fourfold for Cu2+ (to 25.2 ± 1.8 nmol mg−1 dwt). Display of CP2 additionally increased Cd2+ sorption by 30%, HP3 display improved Zn2+ sorption by 20% and NP display increased Pb2+ sorption by 22% (to 61.5 ± 1.0 mg−1 Pb2+ dwt. We showed in vitro that HP3 forms high-affinity 3 Zn2+ binding sites of Kd of 1.2 × 10−7 M. Accordingly, the increase in amount of Zn2+ sorbed by yeasts due to HP3 display was equivalent to 3.7 Zn2+ per displayed HP3 peptide. Our data indicate that overproduction of cell wall mannoproteins is a promising approach to improve the metallosorption by a yeast biosorbent. Displayed specific and high-affinity peptides do not favour metal displacement over other cell wall components, observed with modified gram-negative bacterial cell walls (Ruml and Kotrba, 2003), and their contribution to the overall yeast metallosorption capacity was thus only moderate. Acknowledgement Funded by Czech Ministry of Education, grants no. 1M6837805002 and MSM 6046137305. References Ruml, T., Kotrba, P., 2003. Microbial control of metal pollution: an overview. In: Fingerman, M., Nagabhushanam, R. (Eds.), Recent Advances in Marine Biotechnology. Science Publishers, Inc., Enfield, NH, pp. 81–153. Vinopal, S., Ruml, T., Kotrba, P., 2007. Biosorption of Cd2+ and Zn2+ by cell surfaceengineered Saccharomyces cerevisiae. Int. Biodeter. Biodegr. 60, 96–102.
doi:10.1016/j.jbiotec.2008.07.1630
Abstracts / Journal of Biotechnology 136S (2008) S678–S707
has the homoplastic bph metabolic pathway as the most aerobic PCBs degradation bacterium (Borja et al., 2005). The plasmid purification and electrophoresis showed that LY402 contains an about 20 kb plasmid, named pLYZ402. The plasmid deletion and transformation indicated the bph genes located in pLYZ402. According to homologs of bph genes, primers were designed and the genes were amplified by PCR. Nearly complete bph gene cluster about 11.2 kb was obtained. Sequence analysis showed that the gene organization and gene sequence were almost completely the same with that of Burkholderia xenovorans LB400. However, bph gene cluster of LB400 is located in a plasmid that is about 1.4 Mb. In conclusion, the advantage of LY402 is that it has strong toxicity resistance and high biodegradability to PCBs. Moreover, the degradative plasmid is very small, so it is easy to gene engineering, for instance, transforming the plasmid to aboriginal bacterium to improve survival ability, or integrating other genes to this plasmid to increase the biodegradability of PCBs or co-metabolic other pollutants. References
that this method will be suitable for bioremediation of atrazinecontaminated soils, even those rich in nitrogen, and for accelerating the process of atrazine mineralization in contaminated natural waters and industrial effluents. Reference Mixed bacterial culture for atrazine degradation. International application published under the Patent Cooperation Treaty WO 2007/0966681 A1.
doi:10.1016/j.jbiotec.2008.07.1629 VII5-P-059 Enhanced metallosorption by engineered Saccharomyces cerevisiae Pavel Kotrba ∗ , Jan Kas, Tomas Ruml Institute of Chemical Technology, Prague, Technicka 3, Prague CZ-166 28, Czech Republic E-mail address: [email protected] (P. Kotrba).
Borja, J., Taleon, D.M., Auresenia, J., Gallardo, S., 2005. Polychlorinated biphenyls and their biodegradation. Process Biochem. 40, 1999–2013. Jia, L.Y., Zheng, A.P., Xu, L., Huang, X.D., Zhang, Q., Yang, F.L., in press. Isolation and characterization of comprehensive polychlorinated biphenyl degrading bacterium, Enterobacter sp. LY402. J. Microbiol. Biotechnol. 18.
doi:10.1016/j.jbiotec.2008.07.1628 VII5-P-058 Mixed bacterial culture for remediation of s-triazine contaminated environment and treatment of industrial effluents ´ Maja Havriluk Dubravka Hrˇsak ∗ , NikolinaUdikovic´ Kolic, Rudjer Boˇskovi´c Institute, Center for Marine and Environmental Research, Zagreb 10 000, Croatia E-mail address: [email protected] (D. Hrˇsak). The present work describes a mixed bacterial culture of natural origin deposited in the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary, as the culture Atz Mix 1 (Anon., 2007). The culture was enriched from an agrochemical factory soil exposed to long-term contamination by s-triazine compounds. Growth kinetics and biodegradation studies have shown that Atz Mix 1 exhibits sustained growth and efficiently degrades atrazine at various temperatures (10–30 ◦ C) in a wide variety of concentrations (several ppb to 10,000 ppm), without formation of toxic metabolites. High mineralization degree of ring-labelled [14 C] atrazine (approximately 80%) further shows that atrazine degradation proceeds to the complete mineralization. PCR assessment of the culture genetic potential reveals the presence of catabolic genes trzN, atzB, atzC coding the enzymes for the degradation of atrazine to cyanuric acid and the gene trzD coding the enzymes for subsequent opening of s-triazine ring. The present work also describes a biodegradation method in which the culture Atz Mix 1 has been used as a biological agent. The method is considered advantageous over the so-far described methods where individual bacterial cultures are employed. This is primarily due to the culture capability of expressing sustained growth and atrazinedegrading efficiency under the conditions that are similar to those occurring in atrazine-contaminated environment (e.g. low/varying temperatures, carbon limitation, low C/N ratio, presence of preferential nitrogen source, etc.) as well as under the conditions that are specific for industrial effluents (high concentration of atrazine and other s-triazine compounds). Accordingly, it can be expected
Carboxyl-terminus of alpha-agglutinin (AGalpha1Cp) was employed to anchor CP2 S(GCGCPCGC)2 G or HP3 peptides S(GHHPH)3 G, promoting metallosorption by E. coli (Ruml and Kotrba, 2003), and/or the metal fixation motif MDCPTEEALIR (NP peptide) of P1-ATPases on the surface of the yeast biosorbent. The AGalpha1Cp variants were found covalently attached to cell wall glucan and glycosylated (Vinopal et al., 2007). Display of the mere anchoring domain multiplied the metallosorption capability of S. cerevisiae from 100 M metal solutions twofold for Cd2+ (to 19.5 ± 2.3 nmol Cd2+ mg−1 dry cell weight [dwt]) and Zn2+ (to 14.3 ± 1.6 nmol mg−1 dwt) and fourfold for Cu2+ (to 25.2 ± 1.8 nmol mg−1 dwt). Display of CP2 additionally increased Cd2+ sorption by 30%, HP3 display improved Zn2+ sorption by 20% and NP display increased Pb2+ sorption by 22% (to 61.5 ± 1.0 mg−1 Pb2+ dwt. We showed in vitro that HP3 forms high-affinity 3 Zn2+ binding sites of Kd of 1.2 × 10−7 M. Accordingly, the increase in amount of Zn2+ sorbed by yeasts due to HP3 display was equivalent to 3.7 Zn2+ per displayed HP3 peptide. Our data indicate that overproduction of cell wall mannoproteins is a promising approach to improve the metallosorption by a yeast biosorbent. Displayed specific and high-affinity peptides do not favour metal displacement over other cell wall components, observed with modified gram-negative bacterial cell walls (Ruml and Kotrba, 2003), and their contribution to the overall yeast metallosorption capacity was thus only moderate. Acknowledgement Funded by Czech Ministry of Education, grants no. 1M6837805002 and MSM 6046137305. References Ruml, T., Kotrba, P., 2003. Microbial control of metal pollution: an overview. In: Fingerman, M., Nagabhushanam, R. (Eds.), Recent Advances in Marine Biotechnology. Science Publishers, Inc., Enfield, NH, pp. 81–153. Vinopal, S., Ruml, T., Kotrba, P., 2007. Biosorption of Cd2+ and Zn2+ by cell surfaceengineered Saccharomyces cerevisiae. Int. Biodeter. Biodegr. 60, 96–102.
doi:10.1016/j.jbiotec.2008.07.1630