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Research article

Environmental Microbiology and Engineering

Detection of Polyhydroxyalkanoate-Accumulating Bacteria from Domestic Wastewater Treatment Plant Using Highly Sensitive PCR Primers

Yu-Tzu Huang 1*, Pi-Ling Chen 1, Galilee Uy Semblante 1 and Sheng-Jie You 1

Department of Bioenvironmental Engineering and R&D Center for Membrane Technology, Chung Yuan Christian University, Chung-Li 32023, Taiwan

Received: November 15, 2011; Accepted: April 16, 2012

J. Microbiol. Biotechnol. 2012; 22(8): 1141-1147

Published August 28, 2012 https://doi.org/10.4014/jmb.1111.11040

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Polyhydroxyalkanoate (PHA) is a class of biodegradable plastics that have great potential applications in the near future. In this study, the micro-biodiversity and productivity of PHA-accumulating bacteria in activated sludge from a domestic wastewater treatment plant were investigated. A previously reported primer set and a selfdesigned primer set (phaCF1BO/phaCR2BO) were both used to amplify the PHA synthase (phaC) gene of isolated colonies. The new primers demonstrated higher sensitivity for phaC, and combining the PCR results of the two primer sets was able to widen the range of detected genera and raise the sensitivity to nearly 90%. Results showed that 85.3% of the identified bacteria were Gram-negative, with Ralstonia as the dominant genus, and 14.7% were Gram-positive. In addition, Zoogloea and Rhizobium contained the highest amounts of intracellular PHA. It is apparent that glucose was a better carbon source than pentone or tryptone for promoting PHA production in Micrococcus. Two different classes, class I and class II, of phaC were detected from alphaproteobacteria, betaproteobacteria, and gammaproteobacteria, indicating the wide diversity of PHA-accumulating bacteria in this particular sampling site. Simultaneous wastewater treatment and PHA production is promising by adopting the high PHAaccumulating bacteria isolated from activated sludge.

Keywords

polyhydroxyalkanoate, wastewater, PHA synthase gene, PCR, activated sludge

References

  1. Amann, R. I., W. Ludwig, and K. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59: 143-169.
    Pubmed PMC
  2. Bengtsson, S., A. Werker, M. Christensson, and T. Welander. 2008. Production of polyhydroxyalkanoates by activated sludge treating a paper mill wastewater. Bioresour. Technol. 99: 509-516.
    Pubmed CrossRef
  3. Cerrone, F., M. Sánchez-Peinado, B. Juárez-Jiménez, J. GonzálezLópez, and C. Pozo. 2010. Biological treatment of two-phase olive mill wastewater (TPOMW, alpeorujo): Polyhydroxyalkanoates (PHAs) production by Azotobacter strains. J. Microbiol. Biotechnol. 20: 594-601.
    Pubmed
  4. Chakravarty, P., V. Mhaisalkar, and T. Chakrabarti. 2010. Study on poly-hydroxyalkanoate (PHA) production in pilot scale continuous mode wastewater treatment system. Bioresour. Technol. 101: 2896-2899.
    Pubmed CrossRef
  5. Chanprateep, S. 2010. Current trends in biodegradable polyhydroxyalkanoates. J. Biosci. Bioeng. 110: 621-632.
    Pubmed CrossRef
  6. Chua, A. S. M., H. Takabatake, H. Satoh, and T. Mino. 2003. Production of polyhydroxyalkanoates (PHA) by activated sludge treating municipal wastewater: Effect of pH, sludge retention time (SRT), and acetate concentration in influent. Water Res. 37: 3602-3611.
    CrossRef
  7. Ciesielski, S., A. Cydzik-Kwiatkowska, T. Pokoj, and E. Klimiuk. 2006. Molecular detection and diversity of mediumchainlength polyhydroxyalkanoates-producing bacteria enriched from activated sludge. J. Appl. Microbiol. 101: 190-199.
    Pubmed CrossRef
  8. Ciesielski, S., P. Tomasz, and E. Klimiuk. 2010. Cultivationdependent and –independent characterization of microbial community producing polyhydroxyalkanoates from rawglycerol. J. Microbiol. Biotechnol. 20: 853-861.
    Pubmed CrossRef
  9. Ferris, M. J., G. Muyzer, and D. M. Ward. 1996. Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl. Environ. Microbiol. 62: 340-346.
    Pubmed PMC
  10. Gomez, J. G. C., M. F. A. Rodrigues, R. C. P. Alli, B. B. Torres, C. L. B. Netto, M. S. Oliveira, and L. F. da Silva. 1996. Evaluation of soil Gram-negative bacteria yielding polyhydroxyalkanoic acids from carbohydrates and propionic acid. Appl. Microbiol. Biotechnol. 45: 785-791.
    CrossRef
  11. Heuer, H., M. Krsek, P. Baker, K. Smalla, and E. Wellington. 1997. Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl. Environ. Microbiol. 63:3233-3241.
    Pubmed PMC
  12. Khardenavis, A. A., M. S. Kumar, S. N. Mudliar, and T. Chakrabarti. 2007. Biotechnological conversion of agro-industrial wastewaters into biodegradable plastic, poly beta-hydroxybutyrate. Bioresour. Technol. 98: 3579-3584.
    Pubmed CrossRef
  13. Kim, Y. and R. Lenz. 2001. Polyesters from microorganisms. Adv. Biochem. Eng. Biotechnol. 71: 51-79.
    CrossRef
  14. Liu, W. T., T. L. Marsh, H. Cheng, and L. J. Forney. 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63: 4516-4522.
    Pubmed PMC
  15. Mengmeng, C., C. Hong, Z. Qingliang, S. N. Shirley, and R. Jie. 2009. Optimal production of polyhydroxyalkanoates (PHA) in activated sludge fed by volatile fatty acids (VFAs) generated from alkaline excess sludge fermentation. Bioresour. Technol. 100: 1399-1405.
  16. Reddy, C. S. K., R. Ghai, Rashmi, and V. C. Kalia. 2003. Polyhydroxyalkanoates: An overview. Bioresour. Technol. 87:137-146.
    CrossRef
  17. Shah, A. A., F. Hasan, and A. Hameed. 2010. Degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by a newly isolated Actinomadura sp. AF-555, from soil. Int. Biodeteriol. Biodegrad. 64: 281-285.
    CrossRef
  18. Shamala, T. R., A. Chandrashekar, S. V. N. Vijayendra, and L. Kshama. 2003. Identification of polyhydroxyalkanoate (PHA)producing Bacillus spp. using the polymerase chain reaction (PCR). J. Appl. Microbiol. 94: 369-374.
    Pubmed CrossRef
  19. Sheu, D. S., Y. T. Wang, and C. Y. Lee. 2000. Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR. Microbiology 146: 20192025.
  20. Solaiman, D. K. Y., R. D. Ashby, and T. A. Foglia. 2000. Rapid and specific identification of medium-chain-length polyhydroxyalkanoate synthase gene by polymerase chain reaction. Appl. Microbiol. Biotechnol. 53: 690-694.
    Pubmed CrossRef
  21. Solaiman, D. K. Y., R. D. Ashby, and T. A. Foglia. 2002. Synthesis of poly(hydroxyalkanoates) by Escherichia coli expressing mutated and chimeric PHA synthase genes. Biotechnol. Lett. 24: 1011-1016.
    CrossRef
  22. Steinbüchel, A. and T. Lütke-Eversloh. 2003. Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms. Biochem. Eng. J. 16: 81-96.
    CrossRef
  23. Suriyamongkol, P., R. Weselake, S. Narine, M. Moloney, and S. Shah. 2007. Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants - A review. Biotechnol. Adv. 25: 148-175.
    Pubmed CrossRef
  24. Taguchi, S., H. Nakamura, T. Kichise, T. Tsuge, I. Yamato, and Y. Doi. 2003. Production of polyhydroxyalkanoate (PHA) from renewable carbon sources in recombinant Ralstonia eutropha using mutants of original PHA synthase. Biochem. Eng. J. 16:107-113.
    CrossRef
  25. Tsuge, T. 2002. Metabolic improvements and use of inexpensive carbon sources in microbial production of polyhydroxyalkanoates. J. Biosci. Bioeng. 94: 579-584.
    Pubmed

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Article

Research article

J. Microbiol. Biotechnol. 2012; 22(8): 1141-1147

Published online August 28, 2012 https://doi.org/10.4014/jmb.1111.11040

Copyright © The Korean Society for Microbiology and Biotechnology.

Detection of Polyhydroxyalkanoate-Accumulating Bacteria from Domestic Wastewater Treatment Plant Using Highly Sensitive PCR Primers

Yu-Tzu Huang 1*, Pi-Ling Chen 1, Galilee Uy Semblante 1 and Sheng-Jie You 1

Department of Bioenvironmental Engineering and R&D Center for Membrane Technology, Chung Yuan Christian University, Chung-Li 32023, Taiwan

Received: November 15, 2011; Accepted: April 16, 2012

Abstract

Polyhydroxyalkanoate (PHA) is a class of biodegradable
plastics that have great potential applications in the
near future. In this study, the micro-biodiversity and
productivity of PHA-accumulating bacteria in activated
sludge from a domestic wastewater treatment plant were
investigated. A previously reported primer set and a selfdesigned
primer set (phaCF1BO/phaCR2BO) were both
used to amplify the PHA synthase (phaC) gene of isolated
colonies. The new primers demonstrated higher sensitivity
for phaC, and combining the PCR results of the two
primer sets was able to widen the range of detected genera
and raise the sensitivity to nearly 90%. Results showed
that 85.3% of the identified bacteria were Gram-negative,
with Ralstonia as the dominant genus, and 14.7% were
Gram-positive. In addition, Zoogloea and Rhizobium
contained the highest amounts of intracellular PHA. It is
apparent that glucose was a better carbon source than
pentone or tryptone for promoting PHA production in
Micrococcus. Two different classes, class I and class II, of phaC
were detected from alphaproteobacteria, betaproteobacteria,
and gammaproteobacteria, indicating the wide diversity
of PHA-accumulating bacteria in this particular sampling
site. Simultaneous wastewater treatment and PHA
production is promising by adopting the high PHAaccumulating
bacteria isolated from activated sludge.

Keywords: polyhydroxyalkanoate, wastewater, PHA synthase gene, PCR, activated sludge

References

  1. Amann, R. I., W. Ludwig, and K. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59: 143-169.
    Pubmed KoreaMed
  2. Bengtsson, S., A. Werker, M. Christensson, and T. Welander. 2008. Production of polyhydroxyalkanoates by activated sludge treating a paper mill wastewater. Bioresour. Technol. 99: 509-516.
    Pubmed CrossRef
  3. Cerrone, F., M. Sánchez-Peinado, B. Juárez-Jiménez, J. GonzálezLópez, and C. Pozo. 2010. Biological treatment of two-phase olive mill wastewater (TPOMW, alpeorujo): Polyhydroxyalkanoates (PHAs) production by Azotobacter strains. J. Microbiol. Biotechnol. 20: 594-601.
    Pubmed
  4. Chakravarty, P., V. Mhaisalkar, and T. Chakrabarti. 2010. Study on poly-hydroxyalkanoate (PHA) production in pilot scale continuous mode wastewater treatment system. Bioresour. Technol. 101: 2896-2899.
    Pubmed CrossRef
  5. Chanprateep, S. 2010. Current trends in biodegradable polyhydroxyalkanoates. J. Biosci. Bioeng. 110: 621-632.
    Pubmed CrossRef
  6. Chua, A. S. M., H. Takabatake, H. Satoh, and T. Mino. 2003. Production of polyhydroxyalkanoates (PHA) by activated sludge treating municipal wastewater: Effect of pH, sludge retention time (SRT), and acetate concentration in influent. Water Res. 37: 3602-3611.
    CrossRef
  7. Ciesielski, S., A. Cydzik-Kwiatkowska, T. Pokoj, and E. Klimiuk. 2006. Molecular detection and diversity of mediumchainlength polyhydroxyalkanoates-producing bacteria enriched from activated sludge. J. Appl. Microbiol. 101: 190-199.
    Pubmed CrossRef
  8. Ciesielski, S., P. Tomasz, and E. Klimiuk. 2010. Cultivationdependent and –independent characterization of microbial community producing polyhydroxyalkanoates from rawglycerol. J. Microbiol. Biotechnol. 20: 853-861.
    Pubmed CrossRef
  9. Ferris, M. J., G. Muyzer, and D. M. Ward. 1996. Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl. Environ. Microbiol. 62: 340-346.
    Pubmed KoreaMed
  10. Gomez, J. G. C., M. F. A. Rodrigues, R. C. P. Alli, B. B. Torres, C. L. B. Netto, M. S. Oliveira, and L. F. da Silva. 1996. Evaluation of soil Gram-negative bacteria yielding polyhydroxyalkanoic acids from carbohydrates and propionic acid. Appl. Microbiol. Biotechnol. 45: 785-791.
    CrossRef
  11. Heuer, H., M. Krsek, P. Baker, K. Smalla, and E. Wellington. 1997. Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl. Environ. Microbiol. 63:3233-3241.
    Pubmed KoreaMed
  12. Khardenavis, A. A., M. S. Kumar, S. N. Mudliar, and T. Chakrabarti. 2007. Biotechnological conversion of agro-industrial wastewaters into biodegradable plastic, poly beta-hydroxybutyrate. Bioresour. Technol. 98: 3579-3584.
    Pubmed CrossRef
  13. Kim, Y. and R. Lenz. 2001. Polyesters from microorganisms. Adv. Biochem. Eng. Biotechnol. 71: 51-79.
    CrossRef
  14. Liu, W. T., T. L. Marsh, H. Cheng, and L. J. Forney. 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63: 4516-4522.
    Pubmed KoreaMed
  15. Mengmeng, C., C. Hong, Z. Qingliang, S. N. Shirley, and R. Jie. 2009. Optimal production of polyhydroxyalkanoates (PHA) in activated sludge fed by volatile fatty acids (VFAs) generated from alkaline excess sludge fermentation. Bioresour. Technol. 100: 1399-1405.
  16. Reddy, C. S. K., R. Ghai, Rashmi, and V. C. Kalia. 2003. Polyhydroxyalkanoates: An overview. Bioresour. Technol. 87:137-146.
    CrossRef
  17. Shah, A. A., F. Hasan, and A. Hameed. 2010. Degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by a newly isolated Actinomadura sp. AF-555, from soil. Int. Biodeteriol. Biodegrad. 64: 281-285.
    CrossRef
  18. Shamala, T. R., A. Chandrashekar, S. V. N. Vijayendra, and L. Kshama. 2003. Identification of polyhydroxyalkanoate (PHA)producing Bacillus spp. using the polymerase chain reaction (PCR). J. Appl. Microbiol. 94: 369-374.
    Pubmed CrossRef
  19. Sheu, D. S., Y. T. Wang, and C. Y. Lee. 2000. Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR. Microbiology 146: 20192025.
  20. Solaiman, D. K. Y., R. D. Ashby, and T. A. Foglia. 2000. Rapid and specific identification of medium-chain-length polyhydroxyalkanoate synthase gene by polymerase chain reaction. Appl. Microbiol. Biotechnol. 53: 690-694.
    Pubmed CrossRef
  21. Solaiman, D. K. Y., R. D. Ashby, and T. A. Foglia. 2002. Synthesis of poly(hydroxyalkanoates) by Escherichia coli expressing mutated and chimeric PHA synthase genes. Biotechnol. Lett. 24: 1011-1016.
    CrossRef
  22. Steinbüchel, A. and T. Lütke-Eversloh. 2003. Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms. Biochem. Eng. J. 16: 81-96.
    CrossRef
  23. Suriyamongkol, P., R. Weselake, S. Narine, M. Moloney, and S. Shah. 2007. Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants - A review. Biotechnol. Adv. 25: 148-175.
    Pubmed CrossRef
  24. Taguchi, S., H. Nakamura, T. Kichise, T. Tsuge, I. Yamato, and Y. Doi. 2003. Production of polyhydroxyalkanoate (PHA) from renewable carbon sources in recombinant Ralstonia eutropha using mutants of original PHA synthase. Biochem. Eng. J. 16:107-113.
    CrossRef
  25. Tsuge, T. 2002. Metabolic improvements and use of inexpensive carbon sources in microbial production of polyhydroxyalkanoates. J. Biosci. Bioeng. 94: 579-584.
    Pubmed