Introduction

The increased demand of biopolymers for various scientific and industrial applications in recent years has led to a renewed interest in the production of exopolysaccharides (EPS) from biological origins (Shao et al. 2014; Trabelsi et al. 2015). EPS are linear and/or branched sugar polymers composed of heterogeneous monomers which are linked together by glycosidic bonds. Among the EPS, microbial EPS have received much attention due to their simple, biodegradable and non-toxic properties (Bafana 2013; Lordan et al. 2011). Microbial EPS is one of the major groups of industrially important polymeric substances because of their wide range of applications in food and pharmaceutical industries (Raza et al. 2012). They are also used as thickening agents, emulsifiers and adhesives in detergent, textile, paper and paint industries. In addition, EPS are used as metal removers and bioadsobers in oil recovery, mining and petroleum industries (Biswas and Paul 2014; Janaki et al. 2012; Mittelman and Geesey 1985). Among the microbial EPS, the use of bacteria has been widely investigated because the large-scale production of bacterial by-products is simpler than other microorganisms. Several bacterial isolates belonging to the genus Pseudomonas, Bacillus, Streptococcus and Serratia have been reported as efficient EPS producers (Bezawada et al. 2013; Celik et al. 2008; Wu et al. 2014).

Although in recent years polysaccharides of bacterial derivation have been reported, there is a demand for novel EPS with improved physical and/or chemical characteristics (Luo et al. 2009). Heavy metal-resistant bacteria have developed unique metabolic and physiological capabilities to thrive in extreme habitats and produce metabolic products which are not habitually present in other terrestrial origin of bacterial species. Thus, the present study is focused on the isolation of EPS-producing heavy metal-resistant bacteria from ore-contaminated soil from Tamil Nadu Magnesite ore (TANMAG, Salem), located in the state of Tamil Nadu, India, and evaluation of their EPS production efficiency under invitro conditions.

Materials and methods

Sampling and microbial strain isolation

Soil samples (0–15 cm depth) were collected from ore-contaminated site located in Salem of Tamil Nadu, India (Tamil Nadu Magnesite Limited, Salem). The collected soil sample was immediately transferred to laboratory and stored at 4 °C before analysis. The serially diluted ore suspension (0.1 mL) was plated using the spread plate technique onto nutrient agar plates. The inoculated plates were incubated at 37 °C for 2 days and observed for the bacterial growth. Morphologically different colonies were subjected to the identification and purification and stored at 4 °C for further study. Isolation and purification of the isolates were carried out at the Department of Biotechnology, Mahendra Arts and Science College (Autonomous), Kalippatti, Tamil Nadu, India.

Minimum inhibitory concentration (MIC) analysis

Minimum inhibitory concentration (MIC) has been defined as the lowest concentration of the heavy metals added that completely inhibits the bacterial growth in the medium. The MIC of the heavy metals was determined by agar dilution method (Kamala-Kannan and Krishnamoorthy 2006). Stock solutions of the metal salts were added to the Luria–Bertani (LB) agar plates (1/4 strength). Mid-log-phase culture of the isolates was aseptically inoculated onto LB agar plates. The plates were incubated at 37 °C for 24 h and observed for bacterial growth. Experiments were carried out in duplicate. Based on the screening results of MIC and EPS production efficiency, the isolate MG was chosen for the current study and confirmed by molecular characterization.

16S rDNA sequencing

The isolate was grown in LB broth, and cell lysate was made for DNA isolation and polymerase chain reaction (PCR). Genomic DNA of the isolate was extracted using DNA extraction kit (Gene All, South Korea). 16S rDNA fragment was amplified with the forward primer 27f (5′ AGA GTT TGA TCC TGG CTC AG 3′) and reverse primer 907r (5′ CCC CGT CAA TTC ATT TGA GTT T 3′) by using genomic DNA as a template. Cycling parameters included initial denaturing for 1 min at 98 °C, 35 cycles of 45 s at 96 °C, 1 min at 55 °C and 3 min at 72 °C and final elongation for 10 min at 72 °C. The PCR product was purified (Qiagen, CA, USA) and sequenced using an automated sequencer ABI PRISM (Model 3700, CA, USA). The sequences were compared using BLAST program for the identification of isolates.

Production of EPS

To prepare the inoculation for EPS production, the isolate Halomonas sp. MG culture was inoculated with 50 mL of LB broth. The inoculated flask was then incubated at room temperature of 30 °C and 200 rpm in a shaker incubator for 24 h. The actively grown cells were used as inoculum for EPS production in the shake-flask experiment. Strain Halomonas sp. MG was inoculated [108 cells/mL (0.8 OD) at 600 nm] in 2000-mL Erlenmeyer flasks containing 1000 mL of LB broth, and the flasks were maintained at 30 °C and 200 rpm in a shaker incubator for 48 h. The bacterial growth was determined by measuring samples turbidity at 660 nm on a UV-1800 UV–Vis spectrophotometer (Shimadzu, Japan). The EPS in the supernatant were recovered by centrifuging the cells in a stationary phase of growth (at 10,000× g for 15 min). The resulting supernatants were precipitated overnight at 4 °C with six volumes of 95 % ethanol (Celik et al. 2008). Precipitated EPS were recovered by centrifugation, and the ethanol precipitation step was repeated once again. After centrifugation (10,000 g for 15 min at 4 °C), pellets were dissolved in nanopure purified water (Barnstead NANOpure, Waltham, MA, USA). Then, the washed pellets were freeze-dried and used for characterization studies.

Characterization of EPS

Surface morphology of the produced EPS was studied under field emission scanning electron microscope (FESEM, AURIGA, Carl Zeiss AG, Jena, Germany) after gold coating with an accelerated voltage of 10–20 kV. The major functional groups of the EPS were detected by Fourier transform infrared (FTIR) spectroscopy, and the spectrum of the EPS was obtained using KBr method on a Perkin-Elmer FTIR spectrophotometer (Norwalk, USA) in the region of 4000–400 cm−1. The infrared spectral resolution was 4 cm−1. X-ray diffractometer (XRD) was used to determine the crystalline nature of the EPS pellicle using a Rigaku X-ray diffractometer (Rigaku, Japan), operated at 2θ from 30° to 80° at 0.041°/min with a time constant of 2 s. MALDI-TOF analysis was performed on a Voyager-DE STR BioSpectrometry Workstation (Allied Biosystems, Foster City, CA, USA) in a linear mode. The MALDI matrix used for the analysis was performed according to Oh et al. (2011).

Results and discussion

The present study represents an attempt to evaluate the potential of heavy metal-resistant bacteria from an ore-contaminated soil for the production of industrially important EPS. Three morphologically different heavy metal-resistant bacterial colonies were isolated from the ore soil at Tamil Nadu Magnesite Limited (Salem, Tamil Nadu, India). Among the isolates, MG exhibited maximum resistance to arsenic, copper and zinc (data not shown). Hence, the isolate MG was selected for EPS production. Priester et al. (2006) reported that heavy metal-resistant bacteria produced more EPS to protect themselves from the heavy metal-contaminated harsh environment. The 16S rDNA nucleotide sequence was subjected to BLAST search. The homology of the sequence search results of the isolate MG resembles 99 % with available DNA sequence in NCBI database. Based on 16S rDNA sequence homology, the isolate MG was identified as Halomonas sp. The partial 16S rDNA of the isolate MG was deposited in GenBank (accession number: KR262893). A phylogenetic tree was derived from the partial 16S rDNA sequences on the isolate Halomonas sp. MG with existing sequences in the NCBI database.

The microstructures of the EPS from Halomonas sp. MG are represented by FESEM (Fig. 1). The micrographs of the EPS showed relatively stable three-dimensional structural porous webs. The results are consisting with previous studies reporting the porous in nature of the EPS (Iqbal et al. 2002; Prasanna et al. 2012; Wang et al. 2015). FTIR spectroscopy is an important tool for the identification of functional groups and interactions between molecules. The FTIR spectrum of EPS, obtained from Halomonas sp. MG, reveals characteristic functional groups (Fig. 2). Some pronounced peaks were observed at 3300, 1660, 1400, 1070 and 542 cm−1 region. The medium stretch of frequency range at 3500–3300 cm−1 is assigned to N–H stretching. The peaks observed at 1650 and 1400 cm−1 are assigned to N–H and C–C stretching, respectively. The spectral band at wave number 1070 cm−1 was based on the C–O–C vibrations, exhibited the character of polysaccharides or polysaccharides-like substances (Song et al. 2004). The peak appearing in the range of 542 cm−1 is mainly attributed to the stretching vibration of primary amines. X-ray diffractograms of the EPS exhibited the non-crystalline nature of the material with a broad peak at around 15°–45° (Fig. 3). The XRD results are in an agreement with previous studies reporting a non-crystalline nature of EPS (Xu et al. 2007). MALDI-TOF mass spectrometry is a convenient tool for the structural analysis of polysaccharides. The MALDI-TOF mass spectrum of partially acid digested EPS represented a series of masses (Fig. 4). Based on the results, we have identified O-linked and N-linked polysaccharide moieties. The peaks observed at m/z 173.71, 213.80, 235.84, 381.28. 447.34, 658.84 and 674.84 were O-linked polysaccharide moieties. The N-linked polysaccharide moieties were observed at m/z 1016.18, 1864.85 and 2252.32. The results revealed that the peaks obtained are mostly from polysaccharides in the EPS, although negligible peaks could be from peptides (Hasan et al. 2011).

Fig. 1
figure 1

FESEM micrographs of the EPS from isolate Halomonas sp. MG

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Fig. 2
figure 2

FTIR spectra of EPS from isolate Halomonas sp. MG

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Fig. 3
figure 3

X-ray diffractogram of the EPS isolate Halomonas sp. MG

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Fig. 4
figure 4

MALDI-TOF MS spectra of EPS from Halomonas sp. MG

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Conclusion

In this study, an EPS-producing isolate MG was isolated from ore-contaminated soil and was identified as Halomonas sp. The production of functional EPS from Halomonas sp. MG is a promising alternative. The FESEM images demonstrated porous in nature of the EPS. The EPS produced by heavy metal-resistant Halomonas sp. MG may find possible application as a polymer for environmental bioremediation and biotechnological processes. Further work will address the chemical and rheological properties of EPS and possible applications in bioremediation of heavy metals.