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Lichen-Associated Bacterium, a Novel Bioresource of Polyhydroxyalkanoate (PHA) Production and Simultaneous Degradation of Naphthalene and Anthracene

  • Nahar, Shamsun (Department of Environmental Education and Science, Sunchon National University) ;
  • Jeong, Min-Hye (Korean Lichen Research Institute, Sunchon National University) ;
  • Hur, Jae-Seoun (Korean Lichen Research Institute, Sunchon National University)
  • Received : 2018.08.20
  • Accepted : 2018.11.12
  • Published : 2019.01.28

Abstract

Lichens are generally known as self-sufficient, symbiotic life-forms between fungi and algae/cyanobacteria, and they also provide shelter for a wide range of beneficial bacteria. Currently, bacterial-derived biodegradable polyhydroxyalkanoate (PHA) is grabbing the attention of many researchers as a promising alternative to non-degradable plastics. This study was conducted to develop a new method of PHA production using unexplored lichen-associated bacteria, which can simultaneously degrade two ubiquitous industrial toxins, anthracene and naphthalene. Here, 49 lichen-associated bacteria were isolated and tested for PHA synthesis. During the GC-MS analysis, a potential strain of EL19 was found to be a 3-hydroxyhexanoate (3-HHx) accumulator and identified as Pseudomonas sp. based on the 16S rRNA sequencing. GC analysis revealed that EL19 was capable of accumulating 30.62% and 19.63% of 3-HHx from naphthalene and anthracene, respectively, resulting in significant degradation of 98% and 96% of naphthalene and anthracene, respectively, within seven days. Moreover, the highly expressed phaC gene verified the genetic basis of $PHA_{mcl}$ production under nitrogen starvation conditions. Thus, this study strongly supports the hypothesis that lichen-associated bacteria can detoxify naphthalene and anthracene, store energy for extreme conditions, and probably help the associated lichen to live in extreme conditions. So far, this is the first investigation of lichen-associated bacteria that might utilize harmful toxins as feasible supplements and convert anthracene and naphthalene into eco-friendly 3-HHx. Implementation of the developed method would reduce the production cost of $PHA_{mcl}$ while removing harmful waste products from the environment.

Keywords

Acknowledgement

Supported by : National Research Foundation

References

  1. Grube M, Cardinale M, Jr de Castro JV, Muller H, Berg G. 2009. Species-specific structural and functional diversity of bacterial communities in lichen symbioses. ISME J. 3: 1105-1115. https://doi.org/10.1038/ismej.2009.63
  2. Bates ST, Cropsey GWG, Caporaso JG, Knight R, Fierer N. 2011. Bacterial communities associated with the lichen symbiosis. Appl. Environ. Microbiol. 77: 1309-1314. https://doi.org/10.1128/AEM.02257-10
  3. Iskina RY. 1938. On nitrogen fixing bacteria in lichens. Isv. Biol. Inst. Permsk. 11: 133-139.
  4. Panosyan AK, Nikogosyan VG. 1966. The presence of Azotobacter in lichens. Akad. Nauk. Armian. SSR Biol. Zhurn. Armen. 19: 3-11.
  5. Henkel PA, Plotnikova TT. 1973. Nitrogen-fixing bacteria in lichens. Izv. Akad. Nauk SSR Ser. Biol. 1973: 807-813.
  6. Gonzalez I, Ayuso-Sacido A, Anderson A, Genilloud O. 2005. Actinomycetes isolated from lichens: evaluation of their diversity and detection of biosynthetic gene sequences. FEMS Microbiol. Ecol. 54: 401-415. https://doi.org/10.1016/j.femsec.2005.05.004
  7. Aschenbrenner IA, Cernava T, Berg G, Grube M. 2016. Understanding microbial multi-species symbioses. Front. Microbiol. 7: 180.
  8. Grube M, Cernava T, Soh J, Fuchs S, Aschenbrenner I, Lassek C, et al. 2015. Exploring functional contexts of symbiotic sustain within lichen-associated bacteria by comparative omics. ISME J. 9: 412-424. https://doi.org/10.1038/ismej.2014.138
  9. Nampoothiri KM, Nair NR, John RP. 2010. An overview of the recent developments in polylactide (PLA) research. Bioresour. Technol. 101: 8493-8501. https://doi.org/10.1016/j.biortech.2010.05.092
  10. Chen G-Q. 2010. Plastics Completely Synthesized by Bacteria: Polyhydroxyalkanoates, pp. 17-37. In Chen G-Q (ed.), Plastics from Bacteria: Natural functions and Applications, vol. 14. Microbiology Monographs, Springer Berlin Heidelberg, Germany.
  11. Suriyamongkol P, Weselake R, Narine S, Moloney M, Shah S. 2007. Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants - a review. Biotechnol. Adv. 25: 148-175. https://doi.org/10.1016/j.biotechadv.2006.11.007
  12. Doi Y, Kitamura S, Abe H. 1995. Microbial synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 28: 4822-4828. https://doi.org/10.1021/ma00118a007
  13. Shahid S, Mosrati R, Ledauphin J, Amiel C, Fontaine P, Gaillard J-L, et al. 2013. Impact of carbon source and variable nitrogen conditions on bacterial biosynthesis of polyhydroxyalkanoates: Evidence of an atypical metabolism in Bacillus megaterium DSM 509. J. Biosci. Bioeng. 116: 302-308. https://doi.org/10.1016/j.jbiosc.2013.02.017
  14. Verlinden R, Hill D, Kenward M, Williams C, Radecka I. 2007 Bacterial synthesis of biodegradable polyhydroxylalkanoates. J. Appl. Microbiol. 102: 1437-1449. https://doi.org/10.1111/j.1365-2672.2007.03335.x
  15. Gao X, Yuan XX, Shi ZY, Guo YY, Shen XW, Chen JC, et al. 2012. Production of copolyesters of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by E. coli containing an optimized PHA synthase gene. Microb. Cell Fact. 11: 130. https://doi.org/10.1186/1475-2859-11-130
  16. Matias F, Brandt CA, da Silva ES, de Andrade Rodrigues MF. 2017. Polyhydroxybutyrate and polyhydroxydodecanoate produced by Burkholderia contaminans IPT 553. J. Appl. Microbiol. 123: 124-133. https://doi.org/10.1111/jam.13469
  17. Mohan SV, Reddy MV. 2013. Optimization of critical factors to enhance polyhydroxyalkanoates (PHA) synthesis by mixed culture using Taguchi design of experimental methodology. Bioresour. Technol. 128: 409-416. https://doi.org/10.1016/j.biortech.2012.10.037
  18. Nikodinovic J, Kenny ST, Babu RP, Woods T, Blau WJ, O'Connor KE. 2008. The conversion of BTEX compounds by single and defined mixed cultures to medium-chain-length polyhydroxyalkanoate. Appl. Microbiol. Biotechnol. 80: 665-673. https://doi.org/10.1007/s00253-008-1593-0
  19. Narancic T, Kenny ST, Djokic L, Vasiljevic B, O'Connor KE, Nikodinovic-Runic J. 2012. Medium-chain-length polyhydroxyalkanoate production by newly isolated Pseudomonas sp. TN301 from a wide range of polyaromatic and monoaromatic hydrocarbons. J. Appl. Microbiol. 113: 508-520. https://doi.org/10.1111/j.1365-2672.2012.05353.x
  20. Povolo S, Basaglia M, Fontana F, Morelli A, Casella S. 2015. Poly(hydroxyalkanoate) production by Cupriavidus necator from fatty waste can be enhanced by phaZ1 inactivation. Chem. Biochem. Eng. Q 29: 67-74. https://doi.org/10.15255/CABEQ.2014.2248
  21. Filonov AE, Puntus IF, Karpov AV, Kosheleva IA, Kashparov KI, Slepenkin AV, et al. 2004. Efficiency of naphthalene biodegradation by Pseudomonas putida G7 in soil. J. Chem. Technol. Biotechnol. 79: 562-569. https://doi.org/10.1002/jctb.998
  22. Reddy MV, Mawatari Y, Yajima Y, Seki C, Hoshino T, Chang Y-C. 2015. Poly-3-hydroxybutyrate (PHB) production from alkylphenols, mono and poly-aromatic hydrocarbons using Bacillus sp. CYR1: a new strategy for wealth from waste. Bioresour. Technol. 192: 711-717. https://doi.org/10.1016/j.biortech.2015.06.043
  23. Cernava T, Muller H, Aschenbrenner IA, Grube M, Berg G. 2015a. Analysing the antagonistic potential of the lichen microbiome against pathogens by bridging metagenomic with culture studies. Front. Microbiol. 6: 620.
  24. Lee YM, Kim EH, Lee HK, Hong SG. 2014. Biodiversity and physiological characteristics of Antarctic and Arctic lichens-associated bacteria. World J. Microbiol. Biotechnol. 30: 2711-2721. https://doi.org/10.1007/s11274-014-1695-z
  25. Cernava, T. 2015. Exploring the Substantial Contributions and the Global Interactions of the Microbiome in an Ancient Symbiosis. Doctoral thesis, Graz University of Technology, Graz. 15-16.
  26. Eymann C, Lassek C, Wegner U, Bernhardt J, Fritsch OA, Fuchs S, et al. 2017. Symbiotic interplay of fungi, algae, and bacteria within the lung lichen Lobaria pulmonaria L. Hoffm. as assessed by state-of-the-art metaproteomics. J. Proteome Res. 16: 2160-2173. https://doi.org/10.1021/acs.jproteome.6b00974
  27. Castro-Sowinski S, Burdman S, Matan O, Okon Y. 2010. Natural functions of bacterial polyhydroxyalkanoates, pp. 39-61. In Chen G-Q (ed.), Plastics from Bacteria: Natural functions and Applications, Microbiology Monographs, Springer, Berlin, Heidelberg.
  28. Pham TH, Webb JS, Rehm BH. 2004. The role of polyhydroxyalkanoate biosynthesis by Pseudomonas aeruginosa in rhamnolipid and alginate production as well as stress tolerance and biofilm formation. Microbiology 150: 3405-3413. https://doi.org/10.1099/mic.0.27357-0
  29. Goh YS, Tan IKP. 2012. Polyhydroxyalkanoate production by Antarctic soil bacteria isolated from Casey Station and Signy Island. Microbiol. Res. 167: 211-219. https://doi.org/10.1016/j.micres.2011.08.002
  30. Wang Q, Nomura CT. 2010. Monitoring differences in gene expression levels and polyhydroxyalkanoate (PHA) production in Pseudomonas putida KT2440 grown on different carbon sources. J. Biosci. Bioeng. 110: 653-659. https://doi.org/10.1016/j.jbiosc.2010.08.001
  31. Borrero-de Acuna JM, Bielecka A, Haussler S, Schobert M, Jahn M, Wittmann C, et al. 2014. Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida. Microb. Cell Fact. 13: 88. https://doi.org/10.1186/1475-2859-13-88
  32. Ostle AG, Holt JG. 1987. Nile blue A as a fluorescent stain for poly-${\beta}$-hydroxybutyrate. Appl. Environ. Microbiol. 44: 238-241. https://doi.org/10.1128/aem.44.1.238-241.1982
  33. Oehmen A, Keller-Lehmann B, Zeng RJ, Yuan Z, Keller J. 2005. Optimisation of poly-${\beta}$-hydroxyalkanoate analysis using gas chromatography for enhanced biological phosphorus removal systems. J. Chromatogr. A 1070: 131-136. https://doi.org/10.1016/j.chroma.2005.02.020
  34. Dib MA, Bendahou M, Bandiabdellah A, Djabou N, Allali A, Tabti B, et al. 2010. Partial chemical composition and antimicrobial activity of Daucus crinitus Desf. extracts. Grasas Y Aceites 61: 271-278. https://doi.org/10.3989/gya.122609
  35. Lin W, Wang Y, Gorby Y, Nealson K, Pan Y. 2013. Integrating niche-based process and spatial process in biogeography of magnetotactic bacteria. Sci. Rep. 3: 1643. https://doi.org/10.1038/srep01643
  36. Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, et al. 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 62: 716-721. https://doi.org/10.1099/ijs.0.038075-0
  37. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic. Acids Res. 25: 4876-4882. https://doi.org/10.1093/nar/25.24.4876
  38. Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.
  39. Kumar S, Stecher G, Tamura K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33: 1870-1874. https://doi.org/10.1093/molbev/msw054
  40. Solaiman DKY, Ashby RD, Foglia TA. 2000. Rapid and specific identification of medium-chain-length polyhydroxyalkanoate synthase gene by polymerase chain reaction. Appl. Microbiol. Biotechnol. 53: 690-694. https://doi.org/10.1007/s002530000332
  41. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2- ${\Delta}{\Delta}CT$ method. Methods 25: 402-408. https://doi.org/10.1006/meth.2001.1262
  42. Chaudhry WN, Jamil N, Ali I, Ayaz MH, Hasnain S. 2011. Screening for polyhydroxyalkanoate (PHA)-producing bacterial strains and comparison of PHA production from various inexpensive carbon sources. Ann. Microbiol. 61: 623-629. https://doi.org/10.1007/s13213-010-0181-6
  43. Selbmann L, Zucconi L, Ruisi S, Grube M, Cardinale M, Onofri S. 2010. Culturable bacteria associated with Antarctic lichens: affiliation and psychrotolerance. Polar Biol. 33: 71-83. https://doi.org/10.1007/s00300-009-0686-2
  44. Cernava T, Aschenbrenner IA, Grube M, Liebminger S, Berg G. 2015. A novel assay for the detection of bioactive volatiles evaluated by screening of lichen-associated bacteria. Front. Microbiol. 6: 398. https://doi.org/10.3389/fmicb.2015.00398
  45. Honegger R, Edwards D, Axe L. 2013. The earliest records of internally stratified cyanobacterial and algal lichens from the Lower Devonian of the Welsh Borderland. New Phytol. 197: 264-275. https://doi.org/10.1111/nph.12009
  46. Ramsay BA, Saracovan I, Ramsay JA, Marchessault RH. 1992. Effect of nitrogen limitation on long-side-chain poly-beta-hydroxyalkanoate synthesis by Pseudomonas resinovorans. Appl. Environ. Microbiol. 58: 744-746. https://doi.org/10.1128/AEM.58.2.744-746.1992
  47. Hoffmann N, Rehm BHA. 2005. Nitrogen-dependent regulation of medium-chain length polyhydroxyalkanoate biosynthesis genes in pseudomonads. Biotechnol. Lett. 27: 279-282. https://doi.org/10.1007/s10529-004-8353-8
  48. Thakor NS, Patel MA, Trivedi UB, Patel KC. 2003. Production of poly(${\beta}$-hydroxybutyrate) by Comamonas testosteroni during growth on naphthalene. World J. Microbiol. Biotechnol. 19: 185-189. https://doi.org/10.1023/A:1023295009846
  49. Leneva NA, Kolomytseva MP, Baskunov BP, Golovleva LA. 2010. Enzymes of Naphthalene Metabolism by Pseudomonas fluorescens 26K Strain. Biochemistry (Moscow) 75: 562-570. https://doi.org/10.1134/S0006297910050044
  50. Panda B, Jain P, Sharma L, Mallick N. 2006. Optimization of cultural and nutritional conditions for accumulation of poly-${\beta}$-hydroxybutyrate in Synechocystis sp. PCC 6803. Bioresour. Technol. 97: 1296-1301. https://doi.org/10.1016/j.biortech.2005.05.013
  51. Ward PG, Goff M, Donner M, Kaminsky W, O'Connor K. 2006. A two step chemo-biotechnological conversion of polystyrene to a biodegradable plastic. Environ. Sci. Technol. 40: 2433-2437. https://doi.org/10.1021/es0517668
  52. Tan GYA, Chen CL, Ge L, Li L, Tan SN, Wang JY. 2015. Bioconversion of styrene to poly(hydroxyalkanoate) (PHA) by the new bacterial strain Pseudomonas putida NBUS12. Microbes Environ. 30: 76-85. https://doi.org/10.1264/jsme2.ME14138
  53. Chung AL, Jin HL, Huang LJ, Ye HM, Chen JC, Wu Q, et al. 2011. Biosynthesis and characterization of poly (3-hydroxydodecanoate) by ${\beta}$-oxidation inhibited mutant of Pseudomonas entomophila L48. Biomacromolecules 12: 3559-3566. https://doi.org/10.1021/bm200770m
  54. Elbahloul Y, Steinbuchel A. 2009. Large-scale production of poly(3-hydroxyoctanoic acid) by Pseudomonas putida GPo1 and a simplified downstream process. Appl. Environ. Microbiol. 75: 643-651. https://doi.org/10.1128/AEM.01869-08
  55. Gao J, Ramsay JA, Ramsay BA. 2016. Fed-batch production of poly-3- hydroxydecanoate from decanoic acid. J. Biotechnol. 218: 102-107. https://doi.org/10.1016/j.jbiotec.2015.12.012
  56. Reddy MV, Yajima Y, Mawatari Y, Hoshino T, Chang YC. 2015b. Degradation and conversion of toxic compounds into useful bioplastics by Cupriavidus sp. CY-1: relative expression of the PhaC gene under phenol and nitrogen stress. Green Chem. 17: 4560-4569. https://doi.org/10.1039/C5GC01156F
  57. Follonier S, Henes B, Panke S, Zinn M. 2012. Putting cells under pressure: a simple and efficient way to enhance the productivity of medium-chain-length polyhydroxyalkanoate in processes with Pseudomonas putida KT2440. Biotechnol. Bioeng. 109: 451-461. https://doi.org/10.1002/bit.23312
  58. Le Meur S, Zinn M, Egli T, Thony-Meyer L, Ren Q. 2012. Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440. BMC Biotechnol. 12: 53. https://doi.org/10.1186/1472-6750-12-53
  59. Kaur G, Roy I. 2015. Strategies for large-scale production of polyhydroxyalkanoates. Chem. Biochem. Eng. Q. 29: 157-172. https://doi.org/10.15255/CABEQ.2014.2255
  60. Oksanen I. 2006. Ecological and biotechnological aspects of lichens. Appl. Microbiol. Biotechnol. 73: 723-734. https://doi.org/10.1007/s00253-006-0611-3
  61. Hoffmann N, Steinbuchel A, Rehm BHA. 2000. Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylasemediated polyhydroxyalkanoate biosynthetic pathway. Appl. Microbiol. Biotechnol. 54: 665-670. https://doi.org/10.1007/s002530000441
  62. Popp N, Schlomann M, Mau M. 2006. Bacterial diversity in the active stage of a bioremediation system for mineral oil hydrocarbon-contaminated soils. Microbiology 152: 3291-3304. https://doi.org/10.1099/mic.0.29054-0
  63. Kenny ST, Nikodinovic-Runic J, Kaminsky W, Woods T, Babu RP, Keely CM, et al. 2008. Up-cycling of PET (polyethylene terephthalate) to the biodegradable plastic PHA (polyhydroxyalkanoate). Environ. Sci. Technol. 42: 7696-7701. https://doi.org/10.1021/es801010e
  64. Feijoo-Siota L, Rosa-Dos-Santos F, de Miguel T, Villa TG. 2008. Biodegradation of naphthalene by Pseudomonas stutzeri in marine environments: Testing cells entrapment in calcium alginate for use in water detoxification. Bioremediat. J. 12: 185-192. https://doi.org/10.1080/10889860802477168
  65. Karimi B, Habibi M, Esvand M. 2015. Biodegradation of naphthalene using Pseudomonas aeruginosa by up flow anoxic-aerobic continuous flow combined bioreactor. J. Environ. Health. Sci. Eng. 13: 26. https://doi.org/10.1186/s40201-015-0175-1
  66. Jacques RJS, Santos EC, Bento FM, Peralba MCR, Selbach PA, Sa ELS, et al. 2005. Anthracene biodegradation by Pseudomonas sp. isolated from a petrochemical sludge land farming site. Int. Biodeter. Biodegr. 56: 143-150. https://doi.org/10.1016/j.ibiod.2005.06.005
  67. Huisman GW, Wonink E, Meima R, Kazemier B, Terpstra P, Witholt B. 1991. Metabolism of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans. Identification and sequences of genes and function of the encoded proteins in the synthesis and degradation of PHA. J. Biol. Chem. 5: 2191-2198.
  68. Ciesielski S, Cydzik-Kwiatkowska A, Pokoj T, Klimiuk E. 2006. Molecular detection and diversity of medium-chain-length polyhydroxyalkanoates-producing bacteria enriched from activated sludge. J. Appl. Microbiol. 101: 190-199. https://doi.org/10.1111/j.1365-2672.2006.02973.x
  69. McCool GJ, Cannon MC. 2001. PhaC and PhaR are required for polyhydroxyalkanoic acid synthase activity in Bacillus megaterium. J. Bacteriol.183: 4235-4243. https://doi.org/10.1128/JB.183.14.4235-4243.2001
  70. Catone MV, Ruiz JA, Castellanos M, Segura D, Espin G, Lopez NI. 2014. High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes. PLoS One 9: e98873. https://doi.org/10.1371/journal.pone.0098873

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