Antonie van Leeuwenhoek

, Volume 108, Issue 4, pp 859–870 | Cite as

A heavy-metal tolerant novel bacterium, Alcaligenes pakistanensis sp. nov., isolated from industrial effluent in Pakistan

  • Saira Abbas
  • Iftikhar Ahmed
  • Toshiya Iida
  • Yong-Jae Lee
  • Hans-Jürgen Busse
  • Toru Fujiwara
  • Moriya Ohkuma
Original Paper


Two strains, NCCP-650T and NCCP-667, were isolated from industrial effluent and their taxonomic positions were investigated using a polyphasic taxonomic approach. The strains were found to be Gram-stain negative, strictly aerobic, motile short rods, which are tolerant to heavy-metals (Cr+2, As+2, Pb+2 and Cu+2). Cells were observed to grow at a temperature range of 10–37 °C (optimal 25–33 °C), pH range of 5.5–10.0 (optimal 6.5–7.5) and can tolerate 0–7 % NaCl (w/v) (optimum 0–1 %) in tryptic soya agar medium. Sequencing of the 16S rRNA gene and two housekeeping genes, gyrB and nirK, of the isolated strains revealed that both strains belong to the Betaproteobacteria showing highest sequence similarities with members of the genus Alcaligenes. The chemotaxonomic data [major quinones as Q-8; predominant cellular fatty acids as summed features 3 (C16 :1 ω7c/iso-C15 :0 2OH) and C16:0 followed by Summed features 2 (iso-C16 :1 I/C14 :0 3OH), C17:0 Cyclo and C18:1 ω7c; major polar lipids as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminolipid] also supported the affiliation of the isolated strains with the genus Alcaligenes. DNA–DNA hybridizations between the two strains and with closely related type strains of species of the genus Alcaligenes confirmed that both isolates belong to a single novel species within the genus Alcaligenes. On the basis of phylogenetic analyses, physiological, biochemical characteristics and DNA–DNA hybridization, the isolated strains can be differentiated from established Alcaligenes species and thus represent a novel species, for which the name Alcaligenes pakistanensis sp. nov. is proposed with the type strain NCCP-650T (=LMG 28368T = KCTC42083T = JCM 30216T).


Heavy metals tolerance Industrial effluent Alcaligenes pakistanensis nirK gene gyrB gene 



The financial support from Higher Education Commission of Pakistan to S.A. under International Research Support Initiative Program (IRSIP) is gratefully acknowledged. This work was also supported partly by financial assistance from PSDP funded Project Research for Agricultural Development Project (RADP) under a sub-project (Grant No. CS-55/RADP/PARC) entitled “Establishment of Microbial Bio-Resource Laboratories: National Culture Collection of Pakistan (NCCP)” from Pakistan Agricultural Research Council (PARC), Islamabad, Pakistan and partially from Japan Society for Promotion of Science (JSPS) under fellowship program to I.A.

Supplementary material

10482_2015_540_MOESM1_ESM.docx (877 kb)
Supplementary material 1 (DOCX 877 kb)


  1. Abbas S, Ahmed I, Kudo T, Iida T, Ali GM, Fujiwara T, Ohkuma M (2014) Heavy metal-tolerant and psychrotolerant bacterium Acinetobacter pakistanensis sp. nov., isolated from a textile dyeing wastewater treatment pond. Pak J Agri Sci 51:595–608Google Scholar
  2. Affan Q-u-A, Shoeb E, Badar U, Akhtar J (2009) Isolation and characterization of bacterial isolates having heavy metal tolerance. J Basic Appl Sci 5:55–60Google Scholar
  3. Ahmed I, Sin Y, Paek J, Ehsan M, Hayat R, Iqbal M, Chang YH (2014) Description of Lysinibacillus pakistanensis. Int J Agric Biol 16:447–450Google Scholar
  4. Altenburger P, Kämpferb P, Makristathisc A, Lubitza W, Bussea H-J (1996) Classification of bacteria isolated from a medieval wall painting. J Biotechnol 47:39–52. doi: 10.1016/0168-1656(96)01376-4 CrossRefGoogle Scholar
  5. Brady C, Cleenwerck I, Venter S, Vancanneyt M, Swings J, Coutinho T (2008) Phylogeny and identification of Pantoea species associated with plants, humans and the natural environment based on multilocus sequence analysis (MLSA). Syst Appl Microbiol 31:447–460. doi: 10.1016/j.syapm.2008.09.004 CrossRefPubMedGoogle Scholar
  6. Busse J, Auling G (1988) Polyamine pattern as a chemotaxonomic marker within the Proteobacteria. Syst Appl Microbiol 11:1–8. doi: 10.1016/S0723-2020(88)80040-7 CrossRefGoogle Scholar
  7. Busse H-J, Auling G (2005) Genus I. Alcaligenes. Castellani and Chalmers 1919, 936AL. In: Brenner DJ, Krieg NR, Staley JT (eds) Bergey’s manual of systematic bacteriology, vol 2, 2nd edn. Springer, USA, pp 653–658Google Scholar
  8. Busse H-J, Bunka S, Hensel A, Lubitz W (1997) Discrimination of members of the family Pasteurellaceae based on polyamine patterns. Int J Syst Bacteriol 47:698–708. doi: 10.1099/00207713-47-3-698 CrossRefGoogle Scholar
  9. Castellani A, Chalmers AJ (eds) (1919) Manual of tropical medicine, 3rd edn. Williams Wood and Co., New YorkGoogle Scholar
  10. Coenye T, Vancanneyt M, Cnockaert MC, Falsen E, Swings J, Vandamme P (2003) Kerstersia gyiorum gen. nov., sp. nov., a novel Alcaligenes faecalis-like organism isolated from human clinical samples, and reclassification of Alcaligenes denitrificans Ruger and Tan 1983 as Achromobacter denitrificans comb. nov. Int J Syst Evol Microbiol 53:1825–1831. doi: 10.1099/ijs.0.02609-0 CrossRefPubMedGoogle Scholar
  11. De Ley J, Segers P, Kersters K, Mannheim W, Lievens A (1986) Intra-and intergeneric similarities of the Bordetella ribosomal ribonucleic acid cistrons: proposal for a new family, Alcaligenaceae. Int J Syst Bacteriol 36:405–414. doi: 10.1099/00207713-36-3-405 CrossRefGoogle Scholar
  12. Euzéby JP (1997) List of bacterial names with standing in nomenclature: a folder available on the Internet ( Int J Syst Bacteriol 47:590–592 doi: 10.1099/00207713-47-2-590
  13. Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39:224–229. doi: 10.1099/00207713-39-3-224 CrossRefGoogle Scholar
  14. Mikami H, Ishida Y (1983) Post-column fluorometric detection of reducing sugars in high performance liquid chromatography using arginine. Bunseki Kagaku 32:E207–E210CrossRefGoogle Scholar
  15. Rehfuss M, Urban J (2005) Alcaligenes faecalis subsp. phenolicus subsp. nov. a phenol-degrading, denitrifying bacterium isolated from a graywater bioprocessor. Syst Appl Microbiol 28:421–429. doi: 10.1016/j.syapm.2005.03.003 CrossRefPubMedGoogle Scholar
  16. Roohi A, Ahmed I, Khalid N, Iqbal M, Jamil M (2014) Isolation and phylogenetic identification of halotolerant/halophilic bacteria from the salt mines of Karak, Pakistan. Int J Agric Biol 16:564–570Google Scholar
  17. Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101Google Scholar
  18. Schroll G, Busse HJ, Parrer G, Rolleke S, Lubitz W, Denner EB (2001) Alcaligenes faecalis subsp. parafaecalis subsp. nov., a bacterium accumulating poly-beta-hydroxybutyrate from acetone-butanol bioprocess residues. Syst Appl Microbiol 24:37–43. doi: 10.1078/0723-2020-00001 CrossRefPubMedGoogle Scholar
  19. Sorokin DY (2005) Is there a limit for high-pH life? Int J Syst Evol Microbiol 55:1405–1406. doi: 10.1099/ijs.0.63737-0 CrossRefPubMedGoogle Scholar
  20. Staneck JL, Roberts GD (1974) Simplified approach to identification of aerobic Actinomycetes by thin-layer chromatography. Appl Environ Microbiol 28:226–231Google Scholar
  21. Stolz A, Busse HJ, Kampfer P (2007) Pseudomonas knackmussii sp. nov. Int J Syst Evol Microbiol 57:572–576. doi: 10.1099/ijs.0.64761-0 CrossRefPubMedGoogle Scholar
  22. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. doi: 10.1093/molbev/mst197 PubMedCentralCrossRefPubMedGoogle Scholar
  23. Tindall B (1990a) A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst Appl Microbiol 13:128–130. doi: 10.1016/S0723-2020(11)80158-X CrossRefGoogle Scholar
  24. Tindall B (1990b) Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66:199–202. doi: 10.1111/j.1574-6968.1990.tb03996.x CrossRefGoogle Scholar
  25. Tripathi M, Garg S (2010) Studies on selection of efficient bacterial strains simultaneously tolerant to hexavalent chromium and penta chlorophenol isolated from treated tannery effluent. Res J Microbiol 5:707–716CrossRefGoogle Scholar
  26. Tripathi M, Vikram S, Jain RK, Garg S (2011) Isolation and growth characteristics of chromium(VI) and pentachlorophenol tolerant bacterial Isolate from treated tannery effluent for its possible use in simultaneous bioremediation. Ind J Microbiol 51:61–69. doi: 10.1007/s12088-011-0089-2 CrossRefGoogle Scholar
  27. Van Trappen S, Tan TL, Samyn E, Vandamme P (2005) Alcaligenes aquatilis sp. nov., a novel bacterium from sediments of the Weser Estuary, Germany, and a salt marsh on Shem Creek in Charleston Harbor, USA. Int J Syst Evol Microbiol 55:2571–2575. doi: 10.1099/ijs.0.63849-0 CrossRefPubMedGoogle Scholar
  28. Vandamme P et al (1995) Bordetella hinzii sp. nov., isolated from poultry and humans. Int J Syst Bacteriol 45:37–45. doi: 10.1099/00207713-45-1-37 CrossRefPubMedGoogle Scholar
  29. Vandamme P et al (1996) Bordetella trematum sp. nov., isolated from wounds and ear infections in humans, and reassessment of Alcaligenes denitrificans Rüger and Tan 1983. Int J Syst Bacteriol 46:849–858. doi: 10.1099/00207713-46-4-849 CrossRefPubMedGoogle Scholar
  30. Wayne LG et al (1987) Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37:463–464. doi: 10.1099/00207713-37-4-463 CrossRefGoogle Scholar
  31. Yokota A, Akagawa-Matsushita M, Hiraishi A, Kataya-Ma Y, Urakami T, Yamasato K (1992) Distribution of quinone systems in microorganisms: gram-negative eubacteria. Bull JFCC 8:136–171Google Scholar
  32. Zahoor A, Rehman A (2009) Isolation of Cr(VI) reducing bacteria from industrial effluents and their potential use in bioremediation of chromium containing wastewater. J Environ Sci 21:814–820. doi: 10.1016/s1001-0742(08)62346-3 CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Saira Abbas
    • 1
    • 2
    • 3
    • 4
  • Iftikhar Ahmed
    • 1
    • 2
    • 3
    • 4
  • Toshiya Iida
    • 3
  • Yong-Jae Lee
    • 5
  • Hans-Jürgen Busse
    • 6
  • Toru Fujiwara
    • 4
  • Moriya Ohkuma
    • 3
  1. 1.National Culture Collection of PakistanNational Institute for Genomics and Advanced Biotechnology (NIGAB), National Agricultural Research Centre (NARC)IslamabadPakistan
  2. 2.Department of Plant Genomics and BiotechnologyPARC Institute of Advanced Studies in Agriculture, National Agricultural Research Centre (NARC)IslamabadPakistan
  3. 3.Japan Collection of Microorganisms, RIKEN BioResource CenterTsukubaJapan
  4. 4.Laboratory of Plant Nutrition and Fertilizers, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life SciencesThe University of TokyoTokyoJapan
  5. 5.Korean Collection for Type Cultures, Biological Resources Centre, KRIBBDaejeonRepublic of Korea
  6. 6.Institut für Mikrobiologie, Veterinärmedizinische Universität WienViennaAustria

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