- Email: [email protected]
Enhancement of zinc and cadmium uptake in Brassicaceae plants by Rhizosphere bacteria
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Abstracts / Journal of Biotechnology 136S (2008) S22–S71
affect the function exertion of microbes in anode chamber due to the different conditions. Reference Liu, H., Stephen, G., Bruce, E.L., 2005. Electrochemically assisted microbial production of hydrogen from acetate. Environ. Sci. Technol. 39, 4317–4320.
doi:10.1016/j.jbiotec.2008.07.055 I1-P-004 Characteristics of 36 kDa ChiA from Bacillus thuringiensis subsp. colmeri and its potential as biocontrol agents Dong Liu ∗ , Jun Cai, Liang Xiao, Yuehua Chen ∗ Department of Microbiology, College of Life Sciences, Nankai University, Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China E-mail address: [email protected] (D. Liu). In this study we investigated characteristics of chitinases from Bacillus thuringiensis and evaluated its biocontrol potentials. One kind of chitinase from Bt15A3, ChiA was expressed in E. coli (Chen et al., 2007). Expression product was purified with ammonium sulfate precipitation, dialysed and Sephadex G200 gel filtration chromatography. The molecular mass of ChiA was estimated by sodium dodecyl sulfate-polyacryamide gel electroresis (SDS-PAGE). We studied some kinds of metal ions’ impacts on the chitinase activity of ChiA. We studied the optimal temperature and pH for ChiA and its stability in different temperatures and pH. We also made researches in the inhibition effects on the sporangia germination of four kinds fungi and if ChiA had synergistic effects on larvicidal activity. The molecular mass of ChiA was estimated 36 kDa by SDSPAGE (de la Vega et al., 2006). Most medal ions had no great effects on the enzyme activity of ChiA. The optimal temperature for ChiA was 50 ◦ C, whereas, the optimal pH was 5.0. The enzyme was quite stable, in the absence of substrate, at temperature less than 60 ◦ C and pH between 4.0 and 8.0. ChiA completely inhibited sporangia germination in four kinds of fungi. Moreover, the bioassays showed that ChiA could reduce the 50% lethal concentration (LC50 ) of the lyophilized crystal protein of Bt15A3 against Spodoptera exigua and Helicoverpa armigera larvae by approximately 26% and 30%, respectively. ChiA not only had stable characteristics, but also had good biocontrol potentials. These results suggested that the prospects of application of ChiA in the future would be more extensive. References Chen, Y.L., Lu, W., Chen, Y.H., Xiao, L., Cai, J., 2007. Cloning, expression and sequence analysis of chiA, chiB in Bacillus thuringiensis subsp. colmeri 15A3. Acta Microbiol. Sin. 47 (5), 169–174. de la Vega, L.M., Barboza-Corona, J.E., Aguilar-Uscanga, M.G., et al., 2006. Purification and characterization of an exochitinase from Bacillus thuringiensis subsp. aizawai and its action against phytopathogenic fungi. Can. J. Microbiol. 52, 651–657.
doi:10.1016/j.jbiotec.2008.07.056
I1-P-013 Heavy metal uptake potential of various types of white rot fungi: A preliminary study and investigation of metal toxicity Muhammad Asif Hanif 1 , Haq Nawaz Bhatti 1 , Muhammad Asif Ali 2 1
Department of Chemistry, University of Agriculture, Faisalabad 38040, Pakistan 2 Institute of Horticultural Sciences, University of Agriculture, Faisalabad 38040, Pakistan Potentiometric sorption experiments were used for screening of cost-effective 11 strains of white rot fungi native to Pakistan. The heavy metals biosorption capacities of the studied strains of white rot fungi were found to have a significant difference in their uptake capacity. In potentiometric experiment, it was found that the uptake of the metal ions by the different strains was accompanied with proton release, indicating that the metal binding occurs via an ion exchange as well as by electrostatic interaction between carboxylate groups and heavy metal ions. Furthermore, the heavy metals toxicity assays and minimum inhibitory concentration (MIC) values were described in the present study. The obtained results of present study clearly demonstrates that different strains of white rot fungi may have a potential for use as high-value biosorbent of heavy metals and it deserves further investigations into the details of practical application, for example on the development of desorption methods and on sorption process optimization. Keywords: Heavy metals; White rot fungi; Toxicity; MIC; Biosorption doi:10.1016/j.jbiotec.2008.07.057 I1-P-015 Enhancement of zinc and cadmium uptake in Brassicaceae plants by Rhizosphere bacteria
Prayad Pokethitiyook ∗ , Toemthip Poolpak, Phanwimol Tanhan, Sorawit Siangjaeo, Patid Mahakittikun Department of Biology, Mahidol University, Bangkok 10400, Thailand E-mail address: [email protected] (P. Pokethitiyook). The interactions between plant and rhizobacteria on Zn and Cd uptake were investigated. Seed germination of Brassicaceae plants (B. oleracea var. cabitataI, B. campertris var. chinensis, B. oleracea var. alboglabra, and B. juncea) were evaluated for Zn and Cd tolerances. Based on vigor index, B. campertris var. chinensis was selected with the highest germination rate. For heavy metal accumulation study, plants were exposed to 0, 50, 100, and 200 mg/L of Zn and 0, 5, 10, and 20 mg/L of Cd under hydroponic condition. Increasing metal concentration resulted in the increase in metal accumulation by plant. Furthermore, Zn and Cd concentrations accumulated in root were higher than in shoot at all treatments. Screening of heavy metal resistance rhizobacteria was performed to examine their plant growth promotion and heavy metal uptake. Five of Zn and six of Cd tolerance rhizobacteria strains were isolated from Cd and Zn contaminated soil of Padaeng zinc mine, Thailand. The influence of selected bacteria on growth of B. campertris var. chinensis was performed using root elongation assay and the production of indole acetic acid (IAA) (Pedraza et al., 2004). The biosorption capabilities of all bacterial biomass were low meaning that Zn and Cd uptake was caused by plant (Chen et al., 2005; Zouboulis et al., 2004). Based on promotion
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
of plants growth in Zn and Cd stress, Pseudomonas aeruginosa and Serratia marcescens were selected, respectively. Production of IAA expressed only in S. marcescens. Two rhizobacteria enhance plant growth and tend to increase Zn and Cd accumulation capacity of B. campertris var. chinensis. P. aeruginosa promoted zinc accumulation at low concentration (50 mg Zn/L), whereas S. marcescens enhanced cadmium accumulation at high concentration (20 mg Cd/L). This study demonstrates that the two bacteria can increase the growth of B. campertris var. chinensis, a potential plant for phytoremediation, and enhanced their heavy metal accumulation capacity.
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References Alva, S., Anupama, J., Savla, J., Chiu, Y.Y., Vyshali, P., Shruti, M., Yogeetha, B.S., Bhavya, D., Purvi, J., Ruchi, K., Kumudini, B.S., Varalakshmi, K.N., 2007. Production and characterization of fungal amylase enzyme isolated from Aspergillus sp. JGI 12 in solid state culture. Afr. J. Biotechnol. 6 (5), 576–581. Demirel, R., Ilhan, S., Asan, A., Kınacı, E., Oner, S., 2005. Microfungi in cultivated fields in Eskis¸ehir Provience (Turkey). J. Basic Microbiol. 45 (4), 279–293. Kathiresan, K., Manivannan, S., 2006. Alpha-amylase production by Penicillium fellutanum isolated from mangrove rhizosphere soil. Afr. J. Biotechnol. 5, 829–832.
doi:10.1016/j.jbiotec.2008.07.059 I1-P-019
References Chen, X.C., Wang, Y.P., Lin, Q., Shi, J.Y., Wu, W.X., Chen, Y.X., 2005. Biosorption of copper (II) and zinc (II) from aqueous solution by Pseudomonas putida CZ1. Colloid Surf. B 46, 101–107. Pedraza, R.O., Ramirez-Mata, A., Xiqui, M.L., Baca, B.E., 2004. Aromatic amino acid aminotransferase activity and indole-3-acetic acid production by associative nitrogen-fixing bacteria. FEMS Microbiol. Lett. 233, 15–21. Zouboulis, A.I., Loukidou, M.X., Matis, K.A., 2004. Biosorption of toxic metals from aqueous solutions by bacteria strains isolated from metal-polluted soils. Process. Biochem. 39, 909–916.
Bioplastics from activated sludge treating pulp and paper wastewater S. Yan 1 , S. Bala Subramanian 1 , R.D. Tyagi 1,∗ , R.Y. Surampalli 2 1
INRS Eau, Terre et Environnement, Université du Québec, 490 rue de la Couronne, Québec, G1K 9A9 Canada 2 U.S. Environmental Protection Agency, P.O. Box 17-2141, Kansas City, KS 66117, United States E-mail address: [email protected] (R.D. Tyagi).
doi:10.1016/j.jbiotec.2008.07.058 I1-P-016 Production of fungal amylase enzyme from Aspergillus species in solid-state culture R. Demirel 1,∗ , N. Yılmaz Sarıözlü 1 , S. I˙ lhan 2 1
Department of Biology, Faculty of Science, University of Anadolu, 26470 Eskis¸ehir, Turkey 2 Department of Biology, Faculty of Science and Art, University of Eskis¸ehir Osmangazi, 26480 Eskis¸ehir, Turkey E-mail address: [email protected] (R. Demirel). Amylases are important enzymes employed in the starch processing industries for the hydrolysis of polysaccharides (Alva et al., 2007). Microbial amylases meet industrial demands; a large number of them are available commercially (Kathiresan and Manivannan, 2006). Several microorganisms can be obtained to amylases enzyme (Alva et al., 2007). However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors. Fungal sources are confined to terrestrial isolates, mostly to Aspergillus and Penicillium (Kathiresan and Manivannan, 2006). We used Aspergillus species for production of amylase. Aspergillus species isolated from agricultural soil from Eskis¸ehir, Turkey. Obtained isolates were classified based on morphological features. Thirteen different Aspergillus species were determined (Demirel et al., 2005). All of the species were screened for their ability to produce amylase. An Aspergillus species that showed the highest amylase activity were selected and used for solid-state fermentation. Different substrates and their combinations were screened for enzyme production. After the determination of suitable substrate, optimum conditions for production of amylase enzyme were designated (Demirel et al., 2005). As a result of this study, we were determined suitable cultural condition and substrate combination. For determination of industrially availability, advanced experimental properties such as characterization and enzyme kinetics will be investigated.
∗ Corresponding author. Fax: +90 222 320 49 10.
Due to the problem and harmful effects of conventional plastics on the environment, there has been a considerable interest in the development of biodegradable plastics since last few decades (Dias et al., 2006; Kumar et al., 2004; Yan et al., 2006) [4]. In this study, activated sludge from full-scale pulp-paper industry wastewater treatment plant was used as a source of microorganisms to produce biodegradable plastics (polyhydroxyalkanotes—PHA). The experiments were conducted in shake flask as well as in computercontrolled fermentors. Acetate and wastewater was used as carbon source, respectively. Effect of different activated sludge suspended solids concentrations was studied on PHA production with different initial acetate concentration and employing different operating system (batch, fed-batch and continuous feeding strategy). The results showed that copolymers (polyhydroxybutyrates—PHB and polyhydroxyvalerates—PHV) were produced by the sludge whose molar composition varied with the process time. The results also showed that activated sludge of 15 g/l was the optimum concentration for PHA production. The continuous feeding of acetic acid further enhanced the PHA content and productivity. The maximum PHA content reached up to 60% (w/w), a substantial amount of sludge could be reduced and therefore less sludge was left for final disposal. By this approach, it could significantly reduce the cost of PHA production and at the same time, reduce the quantity of excess sludge from the wastewater treatment process that required further treatment, and a new way for carbon recycle by the conversion of organic pollutants in waste into valuable products. Furthermore, PHA accumulating bacteria were isolated and identified from the pulp and paper activated sludge. Initially Nile blue A staining was performed to detect lipid cellular inclusions [5]. Lipid-positive isolates were then grown in a synthetic medium containing acetate as sole carbon source to promote accumulation of PHA. The PHA was analyzed by gas chromatography linked to mass spectroscopy (GCMS) to further confirm the presence and the concentration of PHA. It was found that six bacterial strains isolated from pulp and paper activated sludge can produce PHA (PHA content obtained from 5.22 to 35.5%), These sludge microorganisms were identified based on their 16S rDNA sequences and these
∗ Corresponding author. Tel.: +1 418 654 2617; fax: +1 418 654 2600.
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
affect the function exertion of microbes in anode chamber due to the different conditions. Reference Liu, H., Stephen, G., Bruce, E.L., 2005. Electrochemically assisted microbial production of hydrogen from acetate. Environ. Sci. Technol. 39, 4317–4320.
doi:10.1016/j.jbiotec.2008.07.055 I1-P-004 Characteristics of 36 kDa ChiA from Bacillus thuringiensis subsp. colmeri and its potential as biocontrol agents Dong Liu ∗ , Jun Cai, Liang Xiao, Yuehua Chen ∗ Department of Microbiology, College of Life Sciences, Nankai University, Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China E-mail address: [email protected] (D. Liu). In this study we investigated characteristics of chitinases from Bacillus thuringiensis and evaluated its biocontrol potentials. One kind of chitinase from Bt15A3, ChiA was expressed in E. coli (Chen et al., 2007). Expression product was purified with ammonium sulfate precipitation, dialysed and Sephadex G200 gel filtration chromatography. The molecular mass of ChiA was estimated by sodium dodecyl sulfate-polyacryamide gel electroresis (SDS-PAGE). We studied some kinds of metal ions’ impacts on the chitinase activity of ChiA. We studied the optimal temperature and pH for ChiA and its stability in different temperatures and pH. We also made researches in the inhibition effects on the sporangia germination of four kinds fungi and if ChiA had synergistic effects on larvicidal activity. The molecular mass of ChiA was estimated 36 kDa by SDSPAGE (de la Vega et al., 2006). Most medal ions had no great effects on the enzyme activity of ChiA. The optimal temperature for ChiA was 50 ◦ C, whereas, the optimal pH was 5.0. The enzyme was quite stable, in the absence of substrate, at temperature less than 60 ◦ C and pH between 4.0 and 8.0. ChiA completely inhibited sporangia germination in four kinds of fungi. Moreover, the bioassays showed that ChiA could reduce the 50% lethal concentration (LC50 ) of the lyophilized crystal protein of Bt15A3 against Spodoptera exigua and Helicoverpa armigera larvae by approximately 26% and 30%, respectively. ChiA not only had stable characteristics, but also had good biocontrol potentials. These results suggested that the prospects of application of ChiA in the future would be more extensive. References Chen, Y.L., Lu, W., Chen, Y.H., Xiao, L., Cai, J., 2007. Cloning, expression and sequence analysis of chiA, chiB in Bacillus thuringiensis subsp. colmeri 15A3. Acta Microbiol. Sin. 47 (5), 169–174. de la Vega, L.M., Barboza-Corona, J.E., Aguilar-Uscanga, M.G., et al., 2006. Purification and characterization of an exochitinase from Bacillus thuringiensis subsp. aizawai and its action against phytopathogenic fungi. Can. J. Microbiol. 52, 651–657.
doi:10.1016/j.jbiotec.2008.07.056
I1-P-013 Heavy metal uptake potential of various types of white rot fungi: A preliminary study and investigation of metal toxicity Muhammad Asif Hanif 1 , Haq Nawaz Bhatti 1 , Muhammad Asif Ali 2 1
Department of Chemistry, University of Agriculture, Faisalabad 38040, Pakistan 2 Institute of Horticultural Sciences, University of Agriculture, Faisalabad 38040, Pakistan Potentiometric sorption experiments were used for screening of cost-effective 11 strains of white rot fungi native to Pakistan. The heavy metals biosorption capacities of the studied strains of white rot fungi were found to have a significant difference in their uptake capacity. In potentiometric experiment, it was found that the uptake of the metal ions by the different strains was accompanied with proton release, indicating that the metal binding occurs via an ion exchange as well as by electrostatic interaction between carboxylate groups and heavy metal ions. Furthermore, the heavy metals toxicity assays and minimum inhibitory concentration (MIC) values were described in the present study. The obtained results of present study clearly demonstrates that different strains of white rot fungi may have a potential for use as high-value biosorbent of heavy metals and it deserves further investigations into the details of practical application, for example on the development of desorption methods and on sorption process optimization. Keywords: Heavy metals; White rot fungi; Toxicity; MIC; Biosorption doi:10.1016/j.jbiotec.2008.07.057 I1-P-015 Enhancement of zinc and cadmium uptake in Brassicaceae plants by Rhizosphere bacteria
Prayad Pokethitiyook ∗ , Toemthip Poolpak, Phanwimol Tanhan, Sorawit Siangjaeo, Patid Mahakittikun Department of Biology, Mahidol University, Bangkok 10400, Thailand E-mail address: [email protected] (P. Pokethitiyook). The interactions between plant and rhizobacteria on Zn and Cd uptake were investigated. Seed germination of Brassicaceae plants (B. oleracea var. cabitataI, B. campertris var. chinensis, B. oleracea var. alboglabra, and B. juncea) were evaluated for Zn and Cd tolerances. Based on vigor index, B. campertris var. chinensis was selected with the highest germination rate. For heavy metal accumulation study, plants were exposed to 0, 50, 100, and 200 mg/L of Zn and 0, 5, 10, and 20 mg/L of Cd under hydroponic condition. Increasing metal concentration resulted in the increase in metal accumulation by plant. Furthermore, Zn and Cd concentrations accumulated in root were higher than in shoot at all treatments. Screening of heavy metal resistance rhizobacteria was performed to examine their plant growth promotion and heavy metal uptake. Five of Zn and six of Cd tolerance rhizobacteria strains were isolated from Cd and Zn contaminated soil of Padaeng zinc mine, Thailand. The influence of selected bacteria on growth of B. campertris var. chinensis was performed using root elongation assay and the production of indole acetic acid (IAA) (Pedraza et al., 2004). The biosorption capabilities of all bacterial biomass were low meaning that Zn and Cd uptake was caused by plant (Chen et al., 2005; Zouboulis et al., 2004). Based on promotion
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
of plants growth in Zn and Cd stress, Pseudomonas aeruginosa and Serratia marcescens were selected, respectively. Production of IAA expressed only in S. marcescens. Two rhizobacteria enhance plant growth and tend to increase Zn and Cd accumulation capacity of B. campertris var. chinensis. P. aeruginosa promoted zinc accumulation at low concentration (50 mg Zn/L), whereas S. marcescens enhanced cadmium accumulation at high concentration (20 mg Cd/L). This study demonstrates that the two bacteria can increase the growth of B. campertris var. chinensis, a potential plant for phytoremediation, and enhanced their heavy metal accumulation capacity.
S31
References Alva, S., Anupama, J., Savla, J., Chiu, Y.Y., Vyshali, P., Shruti, M., Yogeetha, B.S., Bhavya, D., Purvi, J., Ruchi, K., Kumudini, B.S., Varalakshmi, K.N., 2007. Production and characterization of fungal amylase enzyme isolated from Aspergillus sp. JGI 12 in solid state culture. Afr. J. Biotechnol. 6 (5), 576–581. Demirel, R., Ilhan, S., Asan, A., Kınacı, E., Oner, S., 2005. Microfungi in cultivated fields in Eskis¸ehir Provience (Turkey). J. Basic Microbiol. 45 (4), 279–293. Kathiresan, K., Manivannan, S., 2006. Alpha-amylase production by Penicillium fellutanum isolated from mangrove rhizosphere soil. Afr. J. Biotechnol. 5, 829–832.
doi:10.1016/j.jbiotec.2008.07.059 I1-P-019
References Chen, X.C., Wang, Y.P., Lin, Q., Shi, J.Y., Wu, W.X., Chen, Y.X., 2005. Biosorption of copper (II) and zinc (II) from aqueous solution by Pseudomonas putida CZ1. Colloid Surf. B 46, 101–107. Pedraza, R.O., Ramirez-Mata, A., Xiqui, M.L., Baca, B.E., 2004. Aromatic amino acid aminotransferase activity and indole-3-acetic acid production by associative nitrogen-fixing bacteria. FEMS Microbiol. Lett. 233, 15–21. Zouboulis, A.I., Loukidou, M.X., Matis, K.A., 2004. Biosorption of toxic metals from aqueous solutions by bacteria strains isolated from metal-polluted soils. Process. Biochem. 39, 909–916.
Bioplastics from activated sludge treating pulp and paper wastewater S. Yan 1 , S. Bala Subramanian 1 , R.D. Tyagi 1,∗ , R.Y. Surampalli 2 1
INRS Eau, Terre et Environnement, Université du Québec, 490 rue de la Couronne, Québec, G1K 9A9 Canada 2 U.S. Environmental Protection Agency, P.O. Box 17-2141, Kansas City, KS 66117, United States E-mail address: [email protected] (R.D. Tyagi).
doi:10.1016/j.jbiotec.2008.07.058 I1-P-016 Production of fungal amylase enzyme from Aspergillus species in solid-state culture R. Demirel 1,∗ , N. Yılmaz Sarıözlü 1 , S. I˙ lhan 2 1
Department of Biology, Faculty of Science, University of Anadolu, 26470 Eskis¸ehir, Turkey 2 Department of Biology, Faculty of Science and Art, University of Eskis¸ehir Osmangazi, 26480 Eskis¸ehir, Turkey E-mail address: [email protected] (R. Demirel). Amylases are important enzymes employed in the starch processing industries for the hydrolysis of polysaccharides (Alva et al., 2007). Microbial amylases meet industrial demands; a large number of them are available commercially (Kathiresan and Manivannan, 2006). Several microorganisms can be obtained to amylases enzyme (Alva et al., 2007). However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors. Fungal sources are confined to terrestrial isolates, mostly to Aspergillus and Penicillium (Kathiresan and Manivannan, 2006). We used Aspergillus species for production of amylase. Aspergillus species isolated from agricultural soil from Eskis¸ehir, Turkey. Obtained isolates were classified based on morphological features. Thirteen different Aspergillus species were determined (Demirel et al., 2005). All of the species were screened for their ability to produce amylase. An Aspergillus species that showed the highest amylase activity were selected and used for solid-state fermentation. Different substrates and their combinations were screened for enzyme production. After the determination of suitable substrate, optimum conditions for production of amylase enzyme were designated (Demirel et al., 2005). As a result of this study, we were determined suitable cultural condition and substrate combination. For determination of industrially availability, advanced experimental properties such as characterization and enzyme kinetics will be investigated.
∗ Corresponding author. Fax: +90 222 320 49 10.
Due to the problem and harmful effects of conventional plastics on the environment, there has been a considerable interest in the development of biodegradable plastics since last few decades (Dias et al., 2006; Kumar et al., 2004; Yan et al., 2006) [4]. In this study, activated sludge from full-scale pulp-paper industry wastewater treatment plant was used as a source of microorganisms to produce biodegradable plastics (polyhydroxyalkanotes—PHA). The experiments were conducted in shake flask as well as in computercontrolled fermentors. Acetate and wastewater was used as carbon source, respectively. Effect of different activated sludge suspended solids concentrations was studied on PHA production with different initial acetate concentration and employing different operating system (batch, fed-batch and continuous feeding strategy). The results showed that copolymers (polyhydroxybutyrates—PHB and polyhydroxyvalerates—PHV) were produced by the sludge whose molar composition varied with the process time. The results also showed that activated sludge of 15 g/l was the optimum concentration for PHA production. The continuous feeding of acetic acid further enhanced the PHA content and productivity. The maximum PHA content reached up to 60% (w/w), a substantial amount of sludge could be reduced and therefore less sludge was left for final disposal. By this approach, it could significantly reduce the cost of PHA production and at the same time, reduce the quantity of excess sludge from the wastewater treatment process that required further treatment, and a new way for carbon recycle by the conversion of organic pollutants in waste into valuable products. Furthermore, PHA accumulating bacteria were isolated and identified from the pulp and paper activated sludge. Initially Nile blue A staining was performed to detect lipid cellular inclusions [5]. Lipid-positive isolates were then grown in a synthetic medium containing acetate as sole carbon source to promote accumulation of PHA. The PHA was analyzed by gas chromatography linked to mass spectroscopy (GCMS) to further confirm the presence and the concentration of PHA. It was found that six bacterial strains isolated from pulp and paper activated sludge can produce PHA (PHA content obtained from 5.22 to 35.5%), These sludge microorganisms were identified based on their 16S rDNA sequences and these
∗ Corresponding author. Tel.: +1 418 654 2617; fax: +1 418 654 2600.