Introduction

The genus Alcaligenes was proposed in 1919 (Castellani and Chalmers 1919) and placed in the family Alcaligenaceae (De Ley et al. 1986) with the type species, Alcaligenes faecalis. So far, the genus Alcaligenes contains only two species A. aquatilis and A. faecalis, and the latter has been split into three sub species A. faecalis subsp. faecalis, A. faecalis subsp. parafaecalis and A. faecalis phenolicus (Euzéby 1997). Though the members of this genus were isolated from soil, sediment, bioprocess residues and water (Rehfuss and Urban 2005; Schroll et al. 2001; Van Trappen et al. 2005), some strains have also been reported to be isolated from clinical specimens (Busse and Auling 2005) and thus attract particular interest. This genus comprises of aerobic, motile, Gram-negative non-fermentative coccobacillary rods, which form non-pigmented colonies on nutrient agar. Some organisms were reported to have potential roles in bioremediation due to the presence of genes encoding for copper containing nitrite reductase and phenol hydroxylase (Rehfuss and Urban 2005).

Several microorganisms are reported to tolerate toxic concentrations of heavy metals (Abbas et al. 2014; Affan et al. 2009; Tripathi and Garg 2010; Tripathi et al. 2011; Zahoor and Rehman 2009). These heavy metal tolerant bacteria offer an opportunity to exploit their role in bioremediation of environments contaminated with heavy-metals. During our studies of microbial diversity for heavy-metal tolerant bacteria, strains NCCP-650T and NCCP-667 were isolated on tryptic soy agar (TSA, BD, USA) by a dilution plate method from industrial effluent samples. The aim of this study was to delineate the taxonomic position of the isolated strains by a polyphasic taxonomic characterization. On the basis of results obtained, the strains NCCP-650T and NCCP-667 are considered to represent a novel species of the genus Alcaligenes.

Materials and methods

Isolation and growth of strains

Strain NCCP-650T was isolated from industrial effluent (water and sludge sample) collected from industrial wastewater discharge channel of an industrial area (lat/lon = “33.66N 73.05E”), Islamabad, Pakistan, whereas strain NCCP-667 was isolated from tanneries effluent (water and sludge sample) collected from a leather factory (lat/lon = “31.10N 74.45E”), Kasur, Pakistan. The strains were recovered on TSA supplemented with different concentrations of heavy-metals (Cr+2, As+2, Pb+2 and Cu+2) by a dilution plate method. The purified strains were maintained on agar medium as well as stored in glycerol (35 %, w/v) at −80 °C, and subjected to polyphasic taxonomic characterization experiments. Type strains of closely related taxa, Alcaligenes aquatilis LMG 22996T, A. faecalis subsp. parafaecalis DSM 13975T, A. faecalis subsp. faecalis JCM 20522T and A. faecalis subsp. phenolicus DSM 16503T were used as reference strains in the majority of experiments under the same laboratory conditions. The characterization experiments were performed at 30 °C unless otherwise mentioned.

Heavy-metals tolerance

To demonstrate the tolerance of isolated strains to toxic concentrations of heavy metals, the strains NCCP-650T, NCCP-667 and the reference strains were grown on TSA supplemented separately with different concentrations of heavy-metals (Cr+2, As+2, Pb+2 and Cu+2) for 5–7 days. The concentration of heavy-metals (Cr+2, As+2, Pb+2 and Cu+2) in agar media was in the range of 300–3000 ppm (in an incremental addition of 300 ppm), which were prepared using the salts: K2Cr2O7, Na2HAsO4, Pb(NO3)2 and CuSO4·5H2O, respectively.

Morphology and phenotypic characterization

Colony morphology of the isolated strains was observed on isolated colonies grown on TSA for 2 days. A phase-contrast microscope (Nikon Optiphot-2, Japan) was used to examine cells of the isolated strains grown on TSA for 24–48 h and further detailed by scanning electron microscope. For the electron microscopic analysis, cells were fixed in a 2.5 % (v/v) paraformaldehyde–glutaraldehyde mixture buffered with 0.1 M phosphate (pH 7.2) for 2 h, dehydrated in graded ethanol, substituted by isoamyl acetate and after drying at critical point sputter-coated with gold (SC502, Polaron) and observed using a scanning electron microscope (FEI Quanta 250 FEG). Gram staining was performed using commercial color (Gram-staining) kit (Cat. 55542, bio Mérieux, France) according to manufacturer’s instructions. The motility of strains was determined with M medium (bio Mérieux, France) in addition to microscopy. Relation to oxygen was determined on TSA by incubation in an anaerobic chamber (Mitsubishi Gas Chemicals Co., Inc.) for 10 days.

The optimum and range of pH for growth was determined in tryptic soy broth (TSB; BD, USA) by adjusting to a range of pH 4.0–10.5 (at increment of 0.5 pH unit) and OD600 was monitored using a spectrophotometer (Beckman Coulter Model DU730, USA). The pH values adjusted by using buffers, HCl or Na2CO3 (Sorokin 2005) and were verified after autoclaving. The temperature range for growth was determined on TSA (pH 7.0) by incubating at different temperatures (3, 5, 10, 15, 20, 25, 30, 33, 37, 40, 45, 50 °C) for 6 days. Growth at various NaCl concentrations was investigated in TGE (pH 7.0), which contains (per litre): beef extract (6 g), tryptone (10 g), dextrose (2 g); agar (15 g) and supplemented with various concentration of NaCl (0–10 %; w/v), at adjusted pH 7.0, and incubated for 6 days.

Physiological and biochemical characteristics were determined using API 20E (Cat. 20100), API 20NE (Cat. 20050) and API 50CH (Cat. 50300) galleries (bio Mérieux, France). Since the strains showed mainly negative reactions for utilization of various carbon sources with API 20E, API 50CH and API 20NE, thus an extended array of metabolic features of the strains was analyzed using the Biolog GN2 characterization system (Biolog Inc. USA). Biolog test was performed by growing the strain NCCP-650T along with reference strains on Biolog Universal Growth (BUG) agar medium according to the instructions of the manufacturer. Catalase and oxidase activities were determined by using API Color Catalase (Cat. 55561) and API Oxidase (Cat. 55635) reagents (bio Mérieux, France). Resistance to antibiotics was assessed with an ATB-VET (Cat. 14289) strip (bio Mérieux, France) and enzyme activities were determined with an API ZYM (Cat. 25200) strip (bio Mérieux, France). API suspension medium was used to inoculate the strips. All commercial kits were used according to the manufacturers’ protocols.

Amplification and sequencing of 16S rRNA, gyrase subunit B (gyrB) and nitrite reductase (nirK) genes and the phylogenetic analysis

Nearly complete 16S rRNA gene sequence of the isolated strains was amplified and sequenced as previously described (Roohi et al. 2014); whereas, gyrB gene was amplified using the primers and PCR conditions described earlier by Brady et al. (2008). To demonstrate the presence of genes coding for copper containing nitrite reductase (NirK) and the phenol hydroxylase in the isolated strains in comparison with the reference species, PCR was performed using the primers and PCR conditions as described previously by Rehfuss and Urban (2005). All the purified PCR products were sequenced using the same primers on an ABI DNA analyzer. The sequences obtained were assembled using BioEdit software to get consensus sequence of the genes and submitted to DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/). The DDBJ/EMBL/GenBank accession numbers for strains NCCP-650T (=LMG 28368T = KCTC42083T = 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.

The strain was identified using the sequence of 16S rRNA gene on Ez-Taxon Server (http://eztaxon-e.ezbiocloud.net) and BLAST search at the DDBJ/NCBI servers. The 16S rRNA gene sequences of closely related validly named type strains were retrieved from the database of the EzTaxon Server and phylogenetic trees were constructed as described previously (Ahmed et al. 2014) using three algorithms: neighbor joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods. The stability of the relationship was assessed with bootstrap analysis, by performing 1000 re-sampling for the tree topology. The sequence similarities of gyrB and nirK genes of isolated strains were compared with the sequences of reference strains using the Kimura 2-parameter model contained in MEGA 6 software package.

DNA base composition and DNA–DNA hybridization

For DNA G+C content analysis and DNA–DNA hybridization, genomic DNA of strains NCCP-650T and NCCP-667 and the reference strains were isolated using Qiagen Genomic-tip 500/G following the manufacturer’s protocol, with a minor modification in which RNase T1 was used in addition to RNase A. To confirm that strains NCCP-650T and NCCP-667 belong to the same species, DNA–DNA hybridization was performed at 45 °C with photobiotin-labelled DNA and microplates as described by Ezaki et al. (1989), using a Fluoroskan Ascent Plate Reader (Thermo Lab Systems, USA) for fluorescence measurements. To establish a separate identity of the isolated strains NCCP-650T and NCCP-667 as a new species, DNA–DNA hybridization between strain NCCP-650T and with the reference strains, Alcaligenes aquatilis LMG 22996T, A. faecalis subsp. parafaecalis DSM 13975T, A. faecalis subsp. faecalis JCM 20522T and A. faecalis subsp. phenolicus DSM 16503T, were performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values were converted to percentage DNA–DNA relatedness values.

To determine DNA G+C contents, the genomic DNA was digested with P1 nuclease and alkaline phosphatase. The DNA G+C contents were analyzed by HPLC (model UFLC, Shimadzu, Japan) at 270 nm using solvent NH4H2PO4 (0.02 M) –CH3CN (v/v 20:1) with Cosmosil 5C18 column (4.6 by 150 mm; Nacalai Tesque, Japan; reversed phase silica gel; C18).

Chemotaxonomic analyses

For cellular fatty acids analysis, the isolates and the reference strains were grown on TSA for 24 h. The cellular fatty acid methyl esters were prepared (Sasser 1990) and were analyzed by GC (6890; Hewlett Packard) according to the standard protocol of the Sherlock Microbial Identification System (MIDI Sherlock version 4.5, MIDI database TSBA40 4.10). Respiratory quinone and polar lipids of strain NCCP-650T and the closely related reference strains were extracted and analyzed from 100 mg lyophilized cells grown in PYE (0.3 % peptone from casein, 0.3 % yeast extract, pH 7.2) for 24 h as described by Tindall (1990a, b) and Altenburger et al. (1996). Polyamines were extracted and analyzed from biomass grown in PYE medium as described by Busse and Auling (1988). For HPLC analysis, conditions were applied reported by Busse et al. (1997). HPLC equipment applied for analyses of quinones and polyamines was reported by Stolz et al. (2007). Strain NCCP-650T along with the reference species were analysed for whole cell sugars using lyophilized cells as described by Staneck and Roberts (1974) with the modification that sugars were identified on HPLC instead of TLC (Mikami and Ishida 1983).

Results and discussion

The isolated strains, NCCP-650T and NCCP-667, formed off-white colonies, which were circular, low-convex with smooth surface and older colonies spread with irregular margins on TSA medium. Cells of the strains were Gram-stain negative, aerobic, motile short rods, mostly occurring in pairs, sometimes in single and rarely in quadrant form (Fig. 1). Cells grew at pH range of 5.5–10.0 (optimal at pH 6.5–7.5) and showed no growth at pH 5.0 or 10.5. The strains tolerated 0–7 % NaCl (w/v) (optimum 0–1 %) in TSB medium. The growth of the strains was observed at temperature range of 10–37 °C (optimal growth at 25–33 °C); no growth was observed at 40 °C after 6 days, which differentiated strain NCCP-650T from all the reference species that exhibited growth at 40–42 °C with optimum growth at 33–37 °C (Rehfuss and Urban 2005). Characterization by API 50CHB, API 20E and API 20NE kits produced mostly negative results (except positive results for acetoin production, citrate utilization and assimilation of capric acid, malate, trisodium citrate and phenyl acetic acid) and were similar to those of the closely related reference species; however, several physiological (mainly obtained in Biolog GN2 characterization) and some biochemical characteristics also differentiated the novel strains from the reference species (detailed results are provided in Table 1 and in the species description). Both their isolates were overall similar in the phenotypic features but some variable results were also obtained in Biolog tests and API ATB-Vet for analysis of resistance to antibiotics (Table 1).

Fig. 1
figure 1

Scanning electron micrograph of cells of Alcaligenes pakistanensis NCCP-650T grown on tryptic soy agar medium at 30 °C for 48 h

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Table 1 Characteristics that differentiate novel strain NCCP-650T from the type strains of closely related species of the genus Alcaligenes
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The two isolates, NCCP-650T and NCCP-667 showed tolerance to toxic concentrations of heavy metals, including Cr+2, As+2, Pb+2 and Cu+2. They grew well on TSA medium containing Cr 1500 ppm, As 3000 ppm, Pb 2100 ppm and Cu 1800 ppm. In comparison, the closely related reference strains were also tested for tolerance to heavy-metals under similar conditions. It was found that the isolated strains differed for tolerance to Cr+2 and Pb+2 from the closely related reference species (Table 1), however, no difference was observed for tolerance to Cu, As and boron when compared with the reference species, suggesting that the members of genus Alcaligenes are mostly tolerant to heavy metals. Compared with the previously reported heavy-metal tolerant bacteria (Abbas et al. 2014; Affan et al. 2009; Tripathi and Garg 2010; Tripathi et al. 2011; Zahoor and Rehman 2009), the strains NCCP-650T and NCCP-667 can be considered as highly tolerant to toxic concentrations of heavy-metals.

Phylogenetic analysis, DNA–DNA hybridization and DNA base composition

Comparative sequence analyses of the 16S rRNA, gyrB and nirK genes were used to confirm the inter species relatedness of isolated strains NCCP-650T and NCCP-667 with type strains of closely related reference species. Sequence comparison of the 16S rRNA genes of strains NCCP-650T and NCCP-667 were carried out using Ez-Taxon Server database (http://www.ezbiocloud.net/). The 16S rRNA gene sequence of strain NCCP-650T showed the highest similarity (98.79 %) with A. aquatilis LMG 22996T (GenBank accession no. JX986976); the similarity values with other established species of the genus Alcaligenes were 98.76–98.22 %. The 16S rRNA gene sequence similarity between the isolated strains was 99.22 %. A neighbor-joining phylogenetic tree constructed based on a comparison of 1321 aligned nucleotides (without gaps and ambiguous nucleotides) showed that both isolates formed a coherent unit at a high bootstrap value (95 %) within the clade comprising species of genus Alcaligenes (Fig. 2). The nodes of this clade also appeared with the same species at high bootstrap values, when phylogenetic trees were constructed using maximum-likelihood and maximum parsimony algorithms (Supplementary Fig 2 & 3), suggesting a strong affiliation of the isolated strains NCCP-650T and NCCP-667 with the established species of the genus Alcaligenes. The sequence similarity values of housekeeping genes gyrB and nirK of the isolates NCCP-650T and NCCP-667 were 100 % with each other, respectively; however, there were notably low similarity values (89.1–85.1 and 92.4–89.7 %, respectively) with the species of genus Alcaligenes. The analysis based on deduced amino acid sequences of the gyrB gene of the isolates showed 95.2–89.7 % similarity with the reference species (the highest similarity with A. faecalis subsp. parafaecalis DSM 13975T).

A single PCR product of the nirK gene (expected size of ~470 bp) was amplified, but no amplicon (expected size ~700 bp) of phenol hydroxylase (LmPH) gene was visualized on an ethidium bromide gel for the isolated strains. The similarities of deduced amino acids for nirK gene of the isolates were 99.10–97.26 % with A. faecalis, but low similarity (95.39 %) was observed with A. aquatilis LMG 22995T. Both the isolates showed 100 % similarity of deduced amino acids sequences of nirK and gyrB genes, suggesting that these belong to the same species.

Fig. 2
figure 2

Neighbour-joining phylogenetic tree inferred from 16S rRNA gene sequence showing inter-relationship of strain NCCP-650T with the closely related species of the genus Alcaligenes and other related genera. Data with gaps and ambiguous nucleotides were removed during alignment for the construction of tree, which was generated using the MEGA 6.0 software package (Tamura et al. 2013) based on a comparison of 1321 nucleotides and was rooted by using Kerstersia gyiorum LMG 5906T (AY131213) as an out-group. Bootstrap values (only >50 % are shown), expressed as a percentage of 1000 replications, are given at the branching points. Bar 0.5 % sequence divergence. The accession number of each type strain is shown in parentheses

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In summary, the analyses of 16S rRNA, gyrB and nirK gene sequences showed that the strains NCCP-650T and NCCP-667 are closely related to the members of genus Alcaligenes; however, based on physiological and biochemical features (Table 1), they are also distinct from all the recognized species of this genus. Although 16S rRNA gene sequences of the isolates exhibited greater than 97 % similarity with the members of genus Alcaligenes, the findings of the low sequence similarity of housekeeping gene sequences for gyrB and nirK supported the hypothesis that these strains belong to a novel species. To confirm this hypothesis, DNA–DNA hybridization analysis was also performed. It was found that the DNA–DNA relatedness between both the isolated strains NCCP-650T and NCCP-667 was 93.6 (±3.1) %, confirming that these strains belong to the same species. However, DNA–DNA hybridization values between NCCP-650T and the reference strains, Alcaligenes faecalis subsp. parafaecalis DSM 13975T, A. faecalis subsp. phenolicus DSM 16503T, A. faecalis subsp. faecalis JCM 20522T and A. aquatilis LMG 22996T were determined to be 39.8 (±4.1), 34.7 (±4.7), 31.6 (±5.5) and 10.8 (±3.2) %, respectively. These values are clearly below the threshold value of 70 %, demonstrating that the two isolates are representatives of a novel species (Wayne et al. 1987). The DNA G+C content of strain NCCP-650T was 55.5 mol% as determined by HPLC. These results are consistent with members of the genus as previously reported (Rehfuss and Urban 2005; Van Trappen et al. 2005), which support affiliation of the isolates to the genus Alcaligenes.

Chemotaxonomic analysis

The cellular fatty acid profile of the isolated strains consisted predominantly of summed features 3 (C16 :1 ω7c/iso-C15 :0 2-OH as defined by MIDI) and C16:0 followed by Summed features 2 (iso-C16 :1 I/C14 :0 3-OH), C17:0 Cyclo, C18:1 ω7c, C12:0 2-OH, C14:0 and C10:0 and other minor components (Table 2). Major components of this profile are similar to those found in other members of the genus, albeit some variation in values of these components clearly differentiated our strains from the closely related reference species of the genus. The presence of summed features 3, C16:0, summed features 2 and C17:0 Cyclo as major components has been observed in members of the Alcaligenaceae (Coenye et al. 2003; Vandamme et al. 1995, 1996).

Table 2 Cellular fatty acid profiles (%) of strain NCCP-650T and type strains of reference species of the genus Alcaligenes
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Ubiquinone Q-8 was observed in strain NCCP-650T as the major component of respiratory lipoquinone. Ubiquinone Q-8 was also detected in all the reference species. Previously, the type strain of the reference species, A. faecalis subsp. parafaecalis DSM 13975T was reported to contain ubiquinone Q-8 system (Schroll et al. 2001). Our results are in agreement with those reported in A. faecalis subsp. parafaecalis DSM 13975T and also conforms the reports that the predominant presence of Q-8 is a common trait of members of Betaproteobacteria including members of the Alcaligenaceae such as A. faecalis subsp. faecalis IAM 12586T and A. faecalis subsp. parafaecalis DSM 13975T (Schroll et al. 2001; Yokota et al. 1992).

The polar lipid profile of strain NCCP-650T showed high similarity with those of the reference species (Supplementary Fig. 1). The polar lipids of strain NCCP-650T were predominantly identified to be diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and unidentified aminolipid (AL1). Furthermore, moderate to minor amounts of phosphatidylserine (PS), another unidentified aminolipid (AL2) and six unidentified polar lipids (L1–6) without an amino residue, a phosphate residue or sugar moiety were also observed. Though the presence of polar lipid L5 distinguished NCCP-650T from all reference species and the presence of aminolipid AL2 from all reference species except A. faecalis subsp. parafaecalis DSM 13975T (Supplementary Fig. 1) not too much significance should be given to this observation because the amounts detected of these two lipids were rather low. Also the presence of several unidentified polar lipids in the reference species but absent in NCCP-650T were too low to be considered as a robust distinguishing trait.

The polyamines pattern was composed of putrescine [45.9 µmol (g dry weight)−1], spermidine [2.2 µmol (g dry weight)−1] and traces of cadaverine and spermine [<0.1 µmol (g dry weight)−1]. The absence of any detectable 2-hydroxyputresence is very rarely observed among Betaproteobacteria but the close relative of NCCP-650T, A. faecalis subsp. parafaecalis DSM 13975T was also reported to lack 2-hydroxyputresence (Schroll et al. 2001), whereas the type species of A. faecalis subsp. faecalis was shown to contain this polyamine though in relatively low amounts (Busse and Auling 1988). However, the absence of this polyamine in both NCCP-650T and A. faecalis subsp. parafaecalis reflects the close relatedness between the two (Fig. 2).

Ribose was found to be the major whole cell sugar in strain NCCP-650T and the reference species, whereas mannose (15 %), and glucose (5 %) were detected as minor components in strain NCCP-650T. The comparison of molar ratio of sugars in all the strains is presented in Table 1. Mannose was absent in A. aquatilis LMG 22996T, A. faecalis subsp. parafaecalis DSM 13975T and A. faecalis subsp. faecalis JCM 20522T, whereas a significant amount (13 %) of galactose was detected in A. faecalis subsp. phenolicus DSM 16503T (Table 1). Our data demonstrate that the genus Alcaligenes is heterogeneous in terms of minor whole cell sugars.

On the basis of phenotypic, genotypic, chemotaxonomic data and phylogenetic analyses, both the isolated strains NCCP-650T and NCCP-667 belong to a single novel species of the genus Alcaligenes, for which the name Alcaligenes pakistanensis sp. nov., is proposed with the type strain NCCP-650T and its description is given below:

Description of Alcaligenes pakistanensis sp. nov.

Alcaligenes pakistanensis (pa.kis.tan.en’sis. N.L. masc. adj. pakistanensis from Pakistan, where the organism was isolated).

Cells are Gram-stain negative, strictly aerobic, motile and short rods, mostly occur in pairs, sometimes in single and rarely occur in quadrant form. Colonies are off-white in color, circular, low-convex with smooth surface; older colonies spread with irregular margins on TSA medium. Cells grow at temperature range of 10–37 °C (optimal growth at 25–33 °C), pH range of 5.5–10.0 (optimal at pH 6.5–7.5) and in 0–7 % NaCl (w/v) (optimum 0–1 %) in TSB medium. Tolerant to heavy-metals (i.e. Cr+2, As+2, Pb+2 and Cu+2). Possesses the copper containing nitrite reductase nirK gene. Catalase and oxidase activities are positive. Positive for Voges–Proskauer reaction and citrate utilization but negative for nitrate reduction to N2, indole production, lysine & ornithine decarboxylases, arginine dihydrolase, tryptophane deaminase, β-galactosidase (2-nitrophenyl-β-d-galactopyranoside) and H2S production. Gelatin, urea and esculin are not hydrolyzed. Can assimilate capric acid, trisodium citrate, phenyl acetic acid and malate but not glucose, mannose, mannitol, arabinose, potassium gluconate, adipic acid and maltose. No oxidation/fermentation of d-glucose, d-sorbitol, amygdalin, l-arabinose, inositol, l-rhamnose, d-sucrose, d-melibiose, and d-mannitol. No acid is produced from substrates in the API-50CH system (bio Mérieux, France). Strong enzyme activity observed for acid phosphatase, leucine arylamidase, alkaline phosphatase, esterase (C 4), esterase lipase (C 8), valine arylamidase, naphthol-As-BI-phosphohydrolase, but weak enzyme activity for lipase (C 14), cystine arylamidase, trypsin, α-chymotrypsin, whereas no enzyme activity for α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase (API-Zym, bio Mérieux, France). The following compounds are used as sole carbon sources as determined by Biolog GN plates: methyl pyruvate, cis-aconitic acid, citric acid, formic acid, α-hydroxy butyric acid, β-hydroxy butyric acid, γ-hydroxy butyric acid, p-hydroxy phenylacetic acid, d,l-lactic acid, malonic acid, propionic acid, succinic acid, bromo succinic acid, succinamic acid, l-alaninamide, d-alanine, l-alanine, l-asparagine, l-glutamic acid, l-histidine, l-leucine, l-ornithine, l-phenylalanine, l-proline, d-serine, l-serine, l-threonine, urocanic acid, phenylethylamine, mono-methyl-succinate (weak), acetic acid (weak), whereas variable results for Tween 40, Tween 80, l-arabinose, d-arabitol, d-fructose, l-fucose, d-galactose, maltose, d-mannitol, d-mannose, l-alanyl-glycine, l-aspartic acid, but the following substrates are not used as carbon source: dextrin, glycogen, d-cellobiose, l-erythritol, α-d-lactose, lactulose, α-keto butyric acid, α-keto glutaric acid, α-keto valeric acid, quinic acid, d-saccharic acid, sebacic acid, glycyl-l-aspartic acid, glycyl-l-glutamic acid, hydroxy-l-proline, l-pyroglutamic acid, d,l-carnitine, γ-amino butyric acid, inosine, putrescine, d,l-α-glycerol phosphate, glucose-1-phosphate, glucose-6-phosphate. Major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unidentified aminolipid (AL1). Moderate to minor amounts of phosphatidylserine, one unidentified aminolipid (AL2) and six unidentified polar lipids (L1–6). The polyamine pattern contains the major compound putrescine and moderate amounts of spermidine. 2-hydroxyputrescine is absent. Predominant cellular fatty acids are summed features 3 (C16 :1 ω7c/iso-C15 :0 2OH as defined by MIDI) and C16:0 followed by Summed features 2 (iso-C16 :1 I/C14 :0 3OH), C17:0 Cyclo, C18:1 ω7c, C12:0 2-OH, C14:0 and C10:0. The major quinone is ubiquinone Q-8. The DNA G+C content of the type strain is 55.5 mol%.

Strain NCCP-650T (=LMG 28368T = KCTC42083T = JCM 30216T) is the type strain, isolated from an industrial effluent (water and sludge) sample collected from Industrial waste water discharge channel of Sector I-9 Industrial area, Islamabad, Pakistan.

The DDBJ/EMBL/GenBank accession numbers for the type strain NCCP-650T are AB920828 (16S rRNA gene), LC001699 (gyrB gene) and AB983284 (nirK gene).