Degradation and detoxification of phenanthrene by actinobacterium Zhihengliuella sp. ISTPL4

  • Arti MishraEmail author
  • Rashmi Rathour
  • Rashmi Singh
  • Taruna Kumari
  • Indu Shekhar ThakurEmail author
Sustainable Industrial and Environmental Bioprocesses


Polycyclic aromatic hydrocarbons (PAHs) are universal environmental contaminants of great concern with regard to their potential exposure and deleterious effect on human health. The current study is the first report of phenanthrene degradation by a psychrotolerant (15 °C), halophilic (5% NaCl), and alkalophilic (pH 8) bacterial strain Zhihengliuella sp. ISTPL4, isolated from the sediment sample of the Pangong Lake, Ladakh, Jammu and Kashmir, India. Degradation studies revealed that the optimum specific growth rate was observed at 250 ppm of phenanthrene with 81% and 87% removal of phenanthrene in 72 h and 168 h, respectively. During the degradation of phenanthrene; 9,10-dihydrophenanthrene; 1-phenanthrenecarboxylic acid; and phthalic acid were detected as intermediates. Whole-genome sequencing of strain ISTPL4 has predicted phenanthrene; 9,10-monooxygense; and epoxide hydrolase B that are involved in the phenanthrene metabolism. Phenanthrene cytotoxicity was evaluated with human hepatic carcinoma cell line (HepG2) and it was observed that the cytotoxicity decreased with increased duration of bacterial incubation and maximum cell viability was observed at 168 h (89.92%). Our results suggest, Zhihengliuella sp. ISTPL4 may promise a great potential for environmental remediation applications.


Polycyclic aromatic hydrocarbons Phenanthrene Biodegradation Zhihengliuella sp. ISTPL4 MTT assay Whole genome sequencing 



The authors are thankful to Dr. Kristina Medhi (SES, JNU, New Delhi) for helping in editing the manuscript. The authors are also thankful to AIRF and JNU for analytical analysis.


The authors would like to express their sincere thanks to the University Grant Commission (UGC), Government of India, UPoE-II, for financial support and also for providing the Dr. D.S. Kothari Fellowship to Arti Mishra and Rashmi Singh. Rashmi Rathour is grateful to the UGC: NFSC, New Delhi, for the Junior Research Fellowship.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

11356_2019_5478_MOESM1_ESM.docx (346 kb)
ESM 1 (DOCX 346 kb)


  1. Alharbi SA, Zayed ME, Chinnathambi A, Alharbi NS, Wainwright M (2014) Isolation and characterization of PAH-biodegrading marine bacteria. J Food Agric Environ 12:793–796Google Scholar
  2. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F (2008) The RAST server: rapid annotations using subsystems technology. BMC Genomics 9:75CrossRefGoogle Scholar
  3. Baik KS, Lim CH, Park SC, Choe HN, Kim HJ, Kim D, Lee KH, Seong CN (2011) Zhihengliuella aestuarii sp. nov., isolated from tidal flat sediment. Int J Syst Evol Microbiol 61:1671–1676CrossRefGoogle Scholar
  4. Bhagat J, Sarkar A, Ingole BS (2016) DNA damage and oxidative stress in marine gastropod Morula granulata exposed to phenanthrene. Water Air Soil Pollut 227:114CrossRefGoogle Scholar
  5. Bücker M, Glatt HR, Platt KL, Avnir D, Ittah Y, Blum J, Oesch F (1979) Mutagenicity of phenanthrene and phenanthrene K-region derivatives. Mutat Res 66:337–348CrossRefGoogle Scholar
  6. Camenzuli D, Freidman BL (2015) On-site and in situ remediation technologies applicable to petroleum hydrocarbon contaminated sites in the Antarctic and Arctic. Polar Res 34:24492CrossRefGoogle Scholar
  7. Chen YG, Tang SK, Zhang YQ, Liu ZX, Chen QH, He JW, Cui XL, Li WJ (2010) Zhihengliuella salsuginis sp. nov., a moderately halophilic actinobacterium from a subterranean brine. Extremophiles 14:397–402CrossRefGoogle Scholar
  8. Dandie CE, Thomas SM, Bentham RH, McClure NC (2004) Physiological characterization of Mycobacterium sp. strain 1B isolated from a bacterial culture able to degrade high-molecular-weight polycyclic aromatic hydrocarbons. J Appl Microbiol 97:246–255CrossRefGoogle Scholar
  9. Dias RL, Ruberto L, Calabró A, Balbo A, Lo D, Panno MT, Mac Cormack WP (2015) Hydrocarbon removal and bacterial community structure in on-site biostimulated biopile systems designed for bioremediation of diesel-contaminated Antarctic soil. Polar Biol 38:677–687CrossRefGoogle Scholar
  10. Evans WC, Fernley HN, Griffiths E (1965) Oxidative metabolism of phenanthrene and anthracene by soil pseudomonads. Biochem J 95:819–831CrossRefGoogle Scholar
  11. Gao S, Seo JS, Wang J, Keum YS, Li J, Li QX (2013) Multiple degradation pathways of phenanthrene by Stenotrophomonas maltophilia C6. Int Biodeterior Biodegradation 79:98–104CrossRefGoogle Scholar
  12. Gran-Scheuch A, Fuentes E, Bravo DM, Jiménez JC, Pérez-Donoso JM (2017) Isolation and characterization of phenanthrene degrading bacteria from diesel fuel-contaminated Antarctic soils. Front Microbiol 8:1634CrossRefGoogle Scholar
  13. Grifoll M, Selifonov SA, Gatlin CV, Chapman PJ (1995) Actions of a versatile fluorine-degrading bacterial isolate on polycyclic aromatic compounds. Appl Environ Microbiol 61:3711–3723Google Scholar
  14. Guo C, Dang Z, Wong Y, Tam NF (2010) Biodegradation ability and dioxgenase genes of PAH-degrading Sphingomonas and Mycobacterium strains isolated from mangrove sediments. Int Biodeterior Biodegradation 64:419–426CrossRefGoogle Scholar
  15. Hamada M, Shibata C, Tamura T, Suzuki K (2013) Zhihengliuella flava sp. nov., an actinobacterium isolated from sea sediment and emended description of the genus Zhihengliuella. Int J Syst Evol Microbiol 63:4760–4764CrossRefGoogle Scholar
  16. Hilyard EJ, Jones-Meehan JM, Spargo BJ, Hill RT (2008) Enrichment, isolation, and phylogenetic identification of polycyclic aromatic hydrocarbon-degrading bacteria from Elizabeth River sediments. Appl Environ Microbiol 74:1176–1182CrossRefGoogle Scholar
  17. Ifegwu OC, Anyakora C (2015) Polycyclic aromatic hydrocarbons: part I. Expo In Adv Clin Chem 72:277–304CrossRefGoogle Scholar
  18. Jha B, Singh VK, Weiss A, Hartmann A, Schmid M (2015) Zhihengliuella somnathii sp. nov., a halotolerant actinobacterium from the rhizosphere of a halophyte Salicornia brachiata. Int J Syst Evol Microbiol 65:3137–3142CrossRefGoogle Scholar
  19. Kanehisa M, Goto S (2000) KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28:27–30CrossRefGoogle Scholar
  20. Khosla K, Rathour R, Maurya R, Maheshwari N, Gnansounou E, Larroche C, Thakur IS (2017) Biodiesel production from lipid of carbon dioxide sequestrating bacterium and lipase of psychrotolerant Pseudomonas sp. ISTPL3 immobilized on biochar. Bioresour Technol 245:743–750CrossRefGoogle Scholar
  21. Kim YH, Freeman JP, Moody JD, Engesser KH, Cerniglia CE (2005) Effects of pH on the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii PYR-1. Appl Microbiol Biotechnol 67:275–285CrossRefGoogle Scholar
  22. Kumar M, Singhal A, Thakur IS (2016a) Comparison of submerged and solid state pretreatment of sugarcane bagasse by Pandoraea sp. ISTKB: enzymatic and structural analysis. Bioresour Technol 203:18–25CrossRefGoogle Scholar
  23. Kumar S, Stecher G, Tamura K (2016b) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  24. Kumari M, Ghosh P, Thakur IS (2016) Landfill leachate treatment using bacto-algal co-culture: an integrated approach using chemical analyses and toxicological assessment. Ecotoxicol Environ Saf 28:44–51CrossRefGoogle Scholar
  25. Lawal AT (2017) Polycyclic aromatic hydrocarbons. A review. Cogent Environ Sci 3:1339841CrossRefGoogle Scholar
  26. Luan TG, Yu KS, Zhong Y, Zhou HW, Lan CY, Tam NF (2006) Study of metabolites from the degradation of polycyclic aromatic hydrocarbons (PAHs) by bacterial consortium enriched from mangrove sediments. Chemosphere 65:2289–2296CrossRefGoogle Scholar
  27. Luo Y, Dong D, Su Y, Wang X, Peng Y, Peng J, Zhou C (2018) Transcriptome analysis of Brassica juncea var. tumida Tsen responses to Plasmodiophora brassicae primed by the biocontrol strain Zhihengliuella aestuarii. Funct Integr Genom:1–4Google Scholar
  28. Medhi K, Singhal A, Chauhan DK, Thakur IS (2017) Investigating the nitrification and denitrification kinetics under aerobic and anaerobic conditions by Paracoccus denitrificans ISTOD1. Bioresour Technol 242:334–343CrossRefGoogle Scholar
  29. Mei X, Wu YY, Mao X, Tu YY (2011) Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols. Environ Pollut 159:164–168CrossRefGoogle Scholar
  30. Mishra M, Das MT, Thakur IS (2014) Mammalian cell-line based toxicological evaluation of paper mill black liquor treated in soil microcosm by indigenous alkalotolerant Bacillus sp. Environ Sci Pollut Res 21:2966–2976CrossRefGoogle Scholar
  31. Mishra A, Jha G, Thakur IS (2018) Draft genome sequence of Zhihengliuella sp. strain ISTPL4, a psychrotolerant and halotolerant bacterium isolated from Pangong Lake, India. Genome Announc 6:1533–1517Google Scholar
  32. Mohan SV, Kisa T, Ohkuma T, Kanaly RA, Shimizu Y (2006) Bioremediation technologies for treatment of PAH-contaminated soil and strategies to enhance process efficiency. Rev Environ Sci Biotechnol 5:347–374CrossRefGoogle Scholar
  33. Moody JD, Freeman JP, Doerge DR, Cerniglia CE (2001) Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. Strain PYR-1. Appl Environ Microbiol 67:1476–1483CrossRefGoogle Scholar
  34. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefGoogle Scholar
  35. Park JY, Lee HH, Kim SJ, Lee YJ, Yang JW (2007) Surfactant-enhanced electrokinetic removal of phenanthrene from kaolinite. J Hazard Mater 140:230–236CrossRefGoogle Scholar
  36. Prabhu Y, Phale PS (2003) Biodegradation of phenanthrene by Pseudomonas sp. stain PP2: novel metabolic pathway, role of biosulfactant and cell surface hydrophobicity in hydrocarbon assiminlation. Appl Environ Microbiol 61:342–351Google Scholar
  37. Rathour R, Gupta J, Tyagi B, Kumari T, Thakur IS (2018) Biodegradation of pyrene in soil microcosm by Shewanella sp. ISTPL2, a psychrophilic, alkalophilic and halophilic bacterium. Bioresour Technol Reports 4:129–136CrossRefGoogle Scholar
  38. Rehmann K, Steinberg C, Kettrup A (1996) Branched metabolic pathway for phenanthrene degradation in a pyrene-degrading bacterium. Polycycl Aromat Compd 11:125–130CrossRefGoogle Scholar
  39. Roy M, Khara P, Dutta TK (2012) Meta-Cleavage of hydroxynaphthoic acids in the degradation of phenanthrene by Sphingobium sp. strain PNB. Microbiology 158:685–695CrossRefGoogle Scholar
  40. Saichek RE, Reddy KR (2003) Effect of pH control at the anode for the electrokinetic removal of phenanthrene from kaolin soil. Chemosphere 51:273–287CrossRefGoogle Scholar
  41. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425Google Scholar
  42. Satyavani K, Gurudeeban S, Ramanathan T, Balasubramanian T (2012) Toxicity study of silver nanoparticles synthesized from Suaeda monoica on Hep-2 cell line. Avicenna J Med Biochem 4:35–39Google Scholar
  43. Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069CrossRefGoogle Scholar
  44. Seo JS, Keum YS, Hu Y, Lee SE, Li QX (2006) Phenanthrene degradation in Arthrobacter sp. P1-1: Initial 1,2-, 3,4- and 9,10-dioxygenation, and meta- and ortho-cleavages of naphthalene-1,2-diol after its formation from naphthalene-1,2-dicarboxylic acid and hydroxyl naphthoic acids. Chemosphere 65:2388–2394CrossRefGoogle Scholar
  45. Seo JS, Keum YS, Hu Y, Lee SE, Li QX (2007) Degradation of phenanthrene by Burkholderia sp. C3: Initial 1,2- and 3,4-dioxygenation and meta- and ortho-cleavages of naphthalene-1,2-diol. Biodegradation 18:123–131CrossRefGoogle Scholar
  46. Seo JS, Keum YS, Li Q (2009) Bacterial degradation of aromatic compounds. Int J Environ Res Public Health 6:278–309CrossRefGoogle Scholar
  47. Seo JS, Keum YS, Li QX (2012) Mycobacterium aromativorans JS19b1T degrades phenanthrene through C-1, 2, C-3, 4 and C-9, 10 dioxygenation pathways. Int Biodeterior Biodegradation 70:96–103CrossRefGoogle Scholar
  48. Shetty AR, de Gannes V, Obi CC, Lucas S, Lapidus A, Cheng JF, Goodwin LA, Pitluck S, Peters L, Mikhailova N, Teshima H (2015) Complete genome sequence of the phenanthrene-degrading soil bacterium Delftia acidovorans Cs1-4. Stand Genomic Sci 10:55CrossRefGoogle Scholar
  49. Siddikee MA, Chauhan PS, Anandham R, Han GH, Sa T (2010) Isolation, characterization, and use for plant growth promotion under salt stress, of ACC deaminase-producing halotolerant bacteria derived from coastal soil. J Microbiol Biotechnol 20:1577–1584CrossRefGoogle Scholar
  50. Song X, Xu Y, Li G, Zhang Y, Huang T, Hu Z (2011) Isolation, characterization of Rhodococcus sp. P14 capable of degrading high-molecular-weight polycyclic aromatic hydrocarbons and aliphatic hydrocarbons. Mar Pollut Bull 62:2122–2128CrossRefGoogle Scholar
  51. Song MK, Song M, Choi HS, Kim YJ, Park YK, Ryu JC (2012) Identification of molecular signatures predicting the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs). Toxicol Lett 212:18–28CrossRefGoogle Scholar
  52. Subashchandrabose SR, Venkateswarlu K, Naidu R, Megharaj M (2019) Biodegradation of high-molecular weight PAHs by Rhodococcus wratislaviensis strain 9: overexpression of amidohydrolase induced by pyrene and BaP. Sci Total Environ 651:813–821CrossRefGoogle Scholar
  53. Sukhdhane KS, Pandey PK, Ajima MN, Jayakumar T, Vennila A, Raut SM (2019) Isolation and characterization of phenanthrene-degrading bacteria from PAHs contaminated mangrove sediment of Thane Creek in Mumbai, India. Polycycl Aromat Compd 39:73–83CrossRefGoogle Scholar
  54. Swati GP, Thakur IS (2017) An integrated approach to study the risk from landfill soil of Delhi: Chemical analyses, in vitro assays and human risk assessment. Ecotoxicol Environ Saf 143:120–128CrossRefGoogle Scholar
  55. Swati GP, Das MT, Thakur IS (2014) In vitro toxicity evaluation of organic extract of landfill soil and its detoxification by indigenous pyrene-degrading Bacillus sp. ISTPY1. Int Biodeterior Biodegrad 90:145–151CrossRefGoogle Scholar
  56. Takarada H, Sekine M, Kosugi H, Matsuo Y, Fujisawa T, Omata S, Kishi E, Shimizu A, Tsukatani N, Tanikawa S, Fujita N (2008) Complete genome sequence of the soil actinomycete Kocuria rhizophila. J Bacteriol 190:4139–4146CrossRefGoogle Scholar
  57. Tang SK, Wang Y, Chen Y, Lou K, Cao LL, Xu LH, Li WJ (2009) Zhihengliuella alba sp. nov., and emended description of the genus Zhihengliuella. Int J Syst Evol Microbiol 59:2025–2032CrossRefGoogle Scholar
  58. Tausson WO (1927) Naphthalin als Kohlenstoffquelle für Bakterien. Planta 4:214–256CrossRefGoogle Scholar
  59. Thakur IS (1995) Structural and functional characterization of 4-chlorosalicylic acid degrading stable bacterial community in a chemostat. World J Microbiol Biotechnol 11:643–645CrossRefGoogle Scholar
  60. Treccani V (1965) Microbial degradation of aliphatic and aromatic hydrocarbons. Z Allg Mikrobiol 5:332–341CrossRefGoogle Scholar
  61. Van Loo B, Kingma J, Arand M, Wubbolts MG, Janssen DB (2006) Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis. Appl Environ Microbiol 72:2905–2917CrossRefGoogle Scholar
  62. Waigi MG, Kang F, Goikavi C, Ling W, Gao Y (2015) Phenanthrene biodegradation by sphingomonads and its application in the contaminated soils and sediments: a review. Int Biodeterior Biodegradation 104:333–349CrossRefGoogle Scholar
  63. Wang H, Lou J, Gu H, Luo X, Yang L, Wu L, Liu Y, Wu J, Xu J (2016) Efficient biodegradation of phenanthrene by a novel strain Massilia sp. WF1 isolated from a PAH-contaminated soil. Environ Sci Pollut Res 23:13378–13388CrossRefGoogle Scholar
  64. Weis LM, Rummel AM, Masten SJ, Trosko JE, Upham BL (1998) Bay and baylike regions of polycyclic aromatic hydrocarbons were potent inhibitors of gap junctional intercellular communication. Environ Health Perspect 106:17–22CrossRefGoogle Scholar
  65. World Health Organization (1983) Evaluation of the carcinogenic risk of chemicals to humans-polycyclic aromatic compounds. Part I: Chemical, environmental, and experimental data, p 32Google Scholar
  66. Yang HY, Jia RB, Chen B, Li L (2014) Degradation of recalcitrant aliphatic and aromatic hydrocarbons by a dioxin-degrader Rhodococcus sp. strain p52. Environ Sci Pollut Res 21:11086–11093CrossRefGoogle Scholar
  67. Zeinali M, Vossoughi M, Ardestani SK (2008) Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium. J Appl Microbiol 105:398–406CrossRefGoogle Scholar
  68. Zhang YQ, Schumann P, Yu LY, Liu HY, Zhang YQ, Xu LH, Stackebrandt E, Jiang CL, Li WJ (2007) Zhihengliuella halotolerans gen. nov., sp. nov., a novel member of the family Micrococcaceae. Int J Syst Evol Microbiol 57:1018–1023CrossRefGoogle Scholar
  69. Zwietering MH, Jongenburger I, Rombouts FM, Van't Riet K (1990) Modeling of the bacterial growth curve. Appl Environ Microbiol 56:1875–1881Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Environmental SciencesJawaharlal Nehru UniversityNew DelhiIndia
  2. 2.Department of StatisticsUniversity of DelhiNew DelhiIndia

Personalised recommendations