Skip to main content
Log in

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

Antonie van Leeuwenhoek Aims and scope Submit manuscript

Cite this article


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).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others


  • 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 51:595–608

    Google Scholar 

  • 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 5:55–60

    CAS  Google Scholar 

  • Ahmed I, Sin Y, Paek J, Ehsan M, Hayat R, Iqbal M, Chang YH (2014) Description of Lysinibacillus pakistanensis. Int J Agric Biol 16:447–450

    Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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 653–658

  • 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

    Article  CAS  Google Scholar 

  • Castellani A, Chalmers AJ (eds) (1919) Manual of tropical medicine, 3rd edn. Williams Wood and Co., New York

    Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

  • 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

    Article  Google Scholar 

  • Mikami H, Ishida Y (1983) Post-column fluorometric detection of reducing sugars in high performance liquid chromatography using arginine. Bunseki Kagaku 32:E207–E210

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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 16:564–570

    CAS  Google Scholar 

  • Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • Staneck JL, Roberts GD (1974) Simplified approach to identification of aerobic Actinomycetes by thin-layer chromatography. Appl Environ Microbiol 28:226–231

    CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Tindall B (1990b) Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66:199–202. doi:10.1111/j.1574-6968.1990.tb03996.x

    Article  CAS  Google Scholar 

  • 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 5:707–716

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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 8:136–171

    Google Scholar 

  • 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

    Article  Google Scholar 

Download references


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.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Iftikhar Ahmed.

Additional information

The DDBJ/EMBL/GenBank accession numbers for strains NCCP-650T (=LMG 28368T = KCTC 42083T = JCM 30216T), NCCP-667, LMG 22996T, DSM 13975T, JCM 20522T and DSM 16503T are LC001699–LC001704 (gyrB gene) and AB983284–AB983289 (nirK gene), respectively; whereas the 16S rRNA gene accession numbers for strains NCCP-650T and NCCP-667 are AB920828 and AB968096, respectively.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 877 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Abbas, S., Ahmed, I., Iida, T. et al. A heavy-metal tolerant novel bacterium, Alcaligenes pakistanensis sp. nov., isolated from industrial effluent in Pakistan. Antonie van Leeuwenhoek 108, 859–870 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: