Expression and characterization of a potential exopolysaccharide from a newly isolated halophilic thermotolerant bacteria Halomonas nitroreducens strain WB1

Sampling

Most hot spring tanks showed a thin layered microbial mat at the photic zone; however, to avoid the algal deposition, we selected the hot spring named Agnikunda. Six sampling stations were identified surrounding Agnikunda, among the seven springs of Bakreshwar in December 2015. Each of these tanks was sampled thrice. We collected 3–5 g of surface soil sample submerged at a depth of 0.5 m below the water table and a water sample from the same depth. Water samples were collected before soil samples in order to reduce any particle dispersion. Physicochemical properties of the soil and water such as pH, Eh, temperature, and dissolved oxygen, were measured in situ and the samples were stored in sterile containers and kept at 4 °C until carried to the laboratory. The soil pH was measured by making 2:5 ratio of soil: water suspensions, using a glass electrode (Digital pH meter; Systronics, Ahmedabad, India). The electrical conductance of soil-saturation extract was also measured (Sartorius PY-Y12, Göttingen, Germany).

Isolation of EPS producing bacteria

The collected soil samples were used as primary inoculums to isolate the bacterial strains. 1 g of the soil was dissolved in 10 ml of 5% NaCl solution and was further diluted upto 10⁷. The suspension was spread on saline Malt extract/yeast extract (MY) agar plates (1% dextrose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, 5% NaCl, and 1.5% agar; Moraine & Rogovin, 1966). The plates were prepared in triplicates for each sample and were then incubated in inverted condition for 48 h at 60 °C. High incubation temperature was selected based on the water temperature of the spring. This also allowed only indigenous thermophilic bacterial strains from the hot spring to grow, inhibited the growth of any mesophilic contaminant; expelling the chances of handling error. Of the MY agar plates prepared from the six station, the sample from the first station didn’t show any growth. In the other 15 plates, very small colonies were observed. Of which only five different bacterial strains showed a slimy surface (an indication of EPS production) and were isolated and sub-cultured in the MY agar plates, and re-incubated at 60 °C for one week. One of the isolates showed a high production rate of a thick gel-like substance which got deposited on the lid of the Petri plate as incubated inversely. This strain named as WB-1 was then further characterized by the following downstream analyses.

Taxonomic characterization of EPS producing bacteria

Polyphasic taxonomic approaches were taken to identify the bacterial strain. The oxidase activity of WB-1 oxidizing bacterial isolates was detected using 1% p-aminodimethylaniline oxalate and the catalase activity was detected using 3% (v/v) H₂O₂ solution. Utilization of 30 different carbon sources was tested using KB009 HiCarbo™ Kit (HiMedia, Mumbai, India) following the manufacturer’s instructions.

For molecular characterization of WB-1, its genomic DNA was extracted (Sambrook & Russel, 2001) and the polymerase chain reaction (PCR) amplification of its partial 16S rDNA was targeted using previously published eubacterial primers Fc27 (5′-AGAGTTTGATCCTGGCTCAG-3′) and Rc1492 (5′-TACGGCTACCTTGTTACGACTT-3′; Lane, 1991). Each of these PCR reactions consisted of 0.5 μl DNA Dream Taq polymerase (5 U/μl) (Fermentas), 5.0 μl 10× Dream Taq buffer, 5.0 μl dNTPs (final concentration 0.2 mM), 5.0 μl MgCl₂ (final concentration 2.0 mM), 0.5 μl of each primers (final concentration 5 μM), 0.5 μl (∼20 ng) DNA template, 0.5 μl BSA (1 mg/ml), and nuclease-free water to make a final volume of 50 μl. The reaction conditions are as follows: initial denaturation at 95 °C for 10 min, 36 cycles of 95 °C for 1 min, 55 °C for 1 min, 72 °C for 3 min 30 sec, and a final extension at 72 °C for 10 min. The PCR reactions were performed in triplicates pooled down and purified using the Gel Purification Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions. The purified product was then sequenced in both directions using the same primer in an ABI Prism 3730 Genetic Analyzer based on Big Dye Terminator chemistry (Applied Biosystems, California, United States). The sequence was checked in Bellerophon (Huber, Faulkner & Hugenholtz, 2004) for chimera and had been submitted to GenBank database accession number MG923810. It was then compared against nucleotide databases (GenBank/EMBL/DDBJ) using the blastn tool (Camacho et al., 2008). The top five 16S rDNA sequences in the match result were used for phylogenetic representation (Fig. 2). The phylogenetic representation of the identified strain was done using Maximum likelihood (ML; Saitou & Nei, 1987) method based on Kimura 2 parameter model (Kimura, 1980) for constructing the tree. The phylogenetic tree was rooted using the 16S rDNA sequence of Methanobrevibacter smithii strain B181 (accession number U55235) as outgroup.

Growth of the strain and EPS production

The isolated bacterial strain was maintained in saline MY media (mentioned above). In the same medium, various growth conditions were tested, such as, NaCl concentration (2.5%, 5%, 7.5%, and 10% wt/vol), growth temperature (25, 35, 40, 50, and 60 °C) and pH (5.5, 6, 6.5, 7, 7.5, and 8). The production of EPS was carried out in a 1 l flask containing 300 ml of saline (5% NaCl) MY media (pH 7.5). The flasks were incubated on a rotary shaker incubator at speed 110 rpm and temp 60 °C for one week.

Isolation and extraction of EPS

The bacterial cells were harvested from 2 l of saline MY medium, using extended high-speed centrifugation, 8,000 rpm for 30 mins to the supernatant, 2 vol of chilled isopropanol (99.5%; Sigma-Aldrich, Bengaluru, India) was added and kept at 4 °C for 24 h for precipitation. It was then centrifuged at 10,000 rpm for 30 min in a cold centrifuge and the precipitated white material was slowly dried at 50 °C in a hot air oven.

Characterization of physical properties of EPS

The dried EPS was dissolved in sterile mili Q water (0.5% w/v) and the rheological property was measured at a shear rate 0–100 s⁻¹ at 25 °C in a controlled-stress Rheolyst AR1000N (TA Instruments, Bengaluru, India). To measure the emulsifying property of the EPS, a previously described method was used (Cooper & Goldenberg, 1987; Llamas et al., 2006). Triton X-100 and Tween 80 (Sigma-Aldrich, Bengaluru, India) were used as control surfactants. Equal volume of each of these solutions in mili Q water (0.5% w/v) were mixed with hydrophobic substances: sunflower oil, mustard oil, olive oil (commercial products), octane, Vaseline oil, hexadecane, and toluene (Sigma-Aldrich, Bengaluru, India), mixed vigorously and kept at 4 °C undisturbed for 24 h. The emulsifying property is described as the percentage (%) of the volume of the emulsion.

Metal-binding property of the EPS was characterized by equilibrium dialysis experiments as described previously (Geddie & Sutherland, 1993; Morillo et al., 2006). For the dialysis, 200 ml 0.1 mM solution of the cations (Cadmium, Cobalt, Copper, Lead, Nickel, and Zinc) was prepared in mili Q water. A mixed solution (0.1 mM) of all the six selected cations was also used to study the selective adsorption of metal to the polymer. Dialysis tubing (Sigma D9777, 12,000 Da and above) was pre-treated by washing under running distilled water for 3–4 h and then treating it with a mixture of 0.3% (w/v) sodium sulphide and 1 mM EDTA at 80 °C for 60 sec followed by mili Q water at 60 °C for 2 min and acidification with 0.2% H₂SO₄. The acid was then removed by washing the tube with hot mili Q water. Five ml of EPS solution in mili Q water (0.5% w/v) was placed into the dialysis tube and was released into the flask containing 200 ml of the respective cation solution. The flask was then gently shaken at 100 rpm for 24 h at room temperature. The cation solution was sampled at 0 h and 24 h and immediately acidified with 1% nitric acid solution. The cations were quantified using an atomic absorption spectrophotometer (PerkinElmer 5100, Waltham, MA, United States).

The metal uptake Q in the solution was determined as follows: $$Q=\frac{V(C_i-C_f)}{m}$$ Where, C_i is the metal concentration in solution of volume (V) at 0 h, C_f is the equilibrium concentration of the metal in solution (at 24 h), and m is the mass of the EPS (Morillo et al., 2006).

Characterization of chemical properties of the EPS

The total spectrophotometric quantification of the following was done: total carbohydrate (Dubois et al., 1956), total protein content (Bradford, 1976), acetyl residues (McComb & McCready, 1957), pyruvate (Sloneker & Orentas, 1962), and hexosamines (Johnson, 1971). Ash content was determined gravimetrically after baking the EPS at 550 °C for 6 h. To analyze the net ionic charge, the purified EPS was loaded on an anion exchange cartridge: quaternary methyl ammonium column (1.5 m × 20 cm; Waters, Millipore). The flow rate in the column of 0.05 M NH₄HCO₃ (pH 8.0) was 2 ml/min followed by a linear gradient of 0.05–2 M NaCl in 0.05 M NH₄HCO₃ (pH 8.0). The monitoring of EPS was done at 210 nm (UV). Fractions of 5 ml were collected to determine their total saccharine composition and molecular mass as described earlier Mata et al. (2006).

Antioxidant properties of the EPS

The hydroxyl radical scavenging property of EPS was detected using Fenton reaction in comparison to ascorbic acid (Zhang et al., 2013). Each reaction mixture consisted of EPS sample of various concentrations (0.1, 0.25, 0.5, 1, 2, 3, 4, 5 mg/ml) in mili Q water, 2.0 ml of FeSO₄ (0.5 mM), 1.0 ml of brilliant green (0.435 mM) and 1.5 ml of H₂O₂ (3% w/v). The absorbance was measured spectrophotometrically at 624 nm. The scavenging activity (SA) is reported as: $$\text{SA}\%=\left[\left(\text{AS}-\text{A0}\right)/\left(\text{A}-\text{A0}\right)\right]\times 100$$ Where, AS is the absorbance of the sample, A0 is the absorbance of the control and A is the absorbance of the Fenton reaction mixture at 624 nm.

The scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals by the EPS was quantified using the previously published method (Kao & Chen, 2006) with some modifications (Zhang et al., 2013). The SA% is reported as mentioned above.

Biochemical and molecular identification

The isolated EPS producing bacteria was identified by various biochemical and molecular characterizations. The biochemical tests including C-source utilization for 30 different C-sources has been detailed in Table 1. The 16S rDNA of the strain WB1, showed 99% identity with Halomonas nitroreducens strain 11S (Accession No. NR_044317), at nucleotide level, and inferred as H. nitroreducens strain WB1. The phylogenetic identification of the strain showed that it belongs to the bacterial phylum Proteobacteria, class Gammaproteobacteria, order Oceanospirillales, family Halomonadaceae, genus Halomonas (Fig. 2).

Bacterial growth kinetics vs EPS production

EPS production by the bacterial strain in the saline MF media was monitored for seven days at 60 °C at 110 rpm. The 1% dextrose present in the media sustained and increased the biomass production and EPS secretion. The EPS secretion reaches a peak during the late log phase and continues in the stationary phase (Fig. 3). During the death phase the residual glucose levels increased from 10.5% to 14%.

Physical properties of the EPS

The rheological property of the EPS studied has been plotted in Fig. 4. It shows that the EPS is highly viscous and the viscosity decreased with increase in shear rate, indicating a pseudo-plastic nature of the polymer.

The emulsifying activity of the EPS secreted by the studied bacterial strain was measured with respect to two other surfactants Tween X-100 and Tween 80, and detailed in Table 2. The EPS stabilized the oil and water mixture with greater efficiency than Tween 80 and Tween 100. The highest emulsifying activity was detected with sunflower oil.

Among the six metals used to test the metal binding efficiency of the EPS produced by the studied bacterial strain, the affinity for Pb was the highest (Fig. 5). In the mixed metal solution, the partial affinity of EPS for Pb was 182 mg/g, however in uni-metal solution it was 263 mg/g. The binding affinity followed the order Pb>Zn>Cd>Cu>Co>Ni.

Chemical properties of the EPS

Under optimum conditions the EPS produced by the strain WB1, composed of 68.9% (w/w) carbohydrates, 2.2% (w/w) proteins, 0.7% (w/w) acetyls, 7.2% (w/w) pyruvic acid, and 1.9% (w/w) hexosamines. All the values represent the average of triplicates of the quantification.

Three chromatographic peaks were detected when loaded on an anion-exchange cartridge, inferring that the polysaccharide from H. nitroreducens strain WB1 contains at least three species (Fig. 6). The size distribution of these peaks showed peak A of 5.2 × 10⁶ Da, peak B 3.0 × 10⁴ Da and peak C of 1.3 × 10³ Da. The monosaccharide composition of each peak of the studied EPS is detailed in Table 3. The primary components of high molecular weight fraction (peak A) were glucose (28%) and mannose (64%) with small quantities of galactose (6%). The main components of peak B were glucose (18.5%), mannose (44%) and low quantity of rhamnose and arabinose. The low molecular weight fraction (peak C) consisted of glucose (19%), mannose (56.5%), galactose (14.2%) and low quantities of galactouronic acid. It showed no peaks in a cation-exchange CM Sep-Pak cartridge.

Antioxidant properties of the EPS

Hydroxyl radical scavenging activity

Hydroxyl radicals are the most reactive species causing severe damage to the living cells and biomolecules. The ability of the studied EPS to scavenge these radicals is detected using the Fenton reaction with respect to the scavenging property of ascorbic acid (Fig. 7). The scavenging activity had a linear relation with that of concentration. The EPS of the organism in this study can scavenge from 0.1 to 4.8 mg/l. The scavenging activity was higher at higher concentrations (Fig. 7) than ascorbic acid.

DPPH radical scavenging activity

The scavenging activity of the studied EPS was measured spectrophotometrically, with respect to ascorbic acid. Alike hydroxyl ion, the DPPH ion scavenging also takes place higher at higher concentrations. However, after 1 mg/l concentration of EPS, the rate of increase in scavenging activity reduced down (Fig. 8). At the concentration of 5.0 mg/ml, the EPS and ascorbic acid showed 83.3% and 90.2% of DPPH radical scavenging activity, respectively.