Isolation and Screening of Bacteria with Ability to Decolorize Selected Synthetic Dyes – Preliminary Results

For the screening four synthetic dyes belonging to three chemical groups were used:

All dyes were produced by Sigma Aldrich.

The spectral properties of dyes, including the wavelength corresponding to the maximum absorbance, were determined experimentally using the Hitachi 1900 UV-VIS spectrophotometer. The characteristics of all used dyes are shown in Table 1.

Aqueous solutions of dyes (BG, CV, EB, Er), prepared in sterile distilled water were mechanically sterilized (cellulose syringe filters with the pore diameter 0.2 µm) before usage. After sterilization the real concentration of dyes in aqueous solutions was estimated (spectrophotometer UV-VIS Hitachi 1900). The solutions of dyes were used for supplementation of solid and liquid bacteria growth media. The initial concentration of dyes in samples was at the level 0.1 gL^-1.

The culture of bacteria was carried out on the solid and liquid growth media. Two solid growth media, different in compositions were used for isolation, purification of strains and confirmation of their decolorization capabilities. Nutrient agar (BTL) and minimal mineral medium MM were used (composition gL^-1: 1.75 g K₂HPO₄; 0.75 g KH₂PO₄; 0.025 g MgSO₄x7H₂O; 0.25 g (NH₄)2SO₄; 20 g agar-agar). Before using the growth media were autoclaved (121°C, 15 min.). The sterile media were supplemented with the solutions of BG or EB (dye concentration in medium 0.1 gL^-1). At the second stage of the experiment the sterile (autoclaved at 121°C for 15 min.) liquid nutrient broth (BTL) was used.

Dilution of source materials and preparation of bacteria suspensions were done in the sterile physiological salt (0.85% water solution of NaCl). For storage and multiplication of bacteria the nutrient agar slants (BTL) were used.

The main criterion for the choose in materials for isolation of bacteria strains was expected presence of microorganisms active at decomposition of the complex organic matter. The used source materials were:

The compost and the rotten wood were pretreated (after grinding they were sieved through 2 mm mesh sieves).

The solutions of raw materials (sewages, compost, rotten wood) were prepared in physiological salt (ranged: sewages and compost 10^-1–10^-8; rotten wood 10^-1–10^-5). Diluted samples were sieved by the spread plate method on solid media at two series:

Bacteria were cultured at temperature 26°C for 96 h. For further experiment bacteria strains were chosen grown as colonies with the decolorized area around them and/or having strongly dyed biomass what pointed on the dye sorption process. Chosen strains were purified and checked in terms of their decolorization abilities (on growth media supplemented with BG or EB) and then they were multiplied on agar slants.

Obtained strains were signed with codes, which among others define the source of obtaining, kind of medium and the strain number:

Abilities of decolorization of three synthetic dyes (two triphenylmethane dyes: brilliant green (BG), crystal violet (CV) and fluorone erythrosine (Er)) by isolated bacteria were estimated. Decolorization experiment was conducted on liquid medium (10 ml of medium in each tube). Each strain was tested in three series (BG, CV, and Er) in four replications. For each series the control series without biomass were performed. Samples were inoculated with the bacteria suspensions prepared in physiological salt from 48 hours aged biomass. The optical density of all biomass suspensions was estimated by the McFarland scale and was set at the level of 15x10⁸ cfu/ml. Tubes with the sterile liquid media were inoculated with 0.1 ml of given bacteria suspension. Samples were incubated at temperature 26°C for 48 h in the aim of multiplication of biomass. The next step was the introduction into tubes the sterile water solutions of dyes (BG, CV or Er)) in volumes which allowed to obtain the initial dyes concentration in samples on level 0.1 g/l. The samples with dyes were incubated at temperature 26°C for 96 h. After every 24 h the visual estimation of changes were done and in decolorized samples the percentage removal of dyes were spectrophotometrically estimated. The rest of the samples were incubated maximum up to 96 h and then measured.

In order to determine the dyes removal, the samples were centrifuged (5000 rpm/10 min). The dyes concentrations were estimated in obtained supernatants spectrophotometrically (spectrophotometer UV-Vis Hitachi U-1900) at a proper wavelength (BG – 624 nm; CV – 590 nm; Er – 527 nm). The dyes concentrations in samples were calculated based on performed calibration curves. The percentage removal of dyes was calculated by the formula (1): (1) R% = ( K-B/K ) x 100 %

where:

R% – percentage removal of dye [%];

K – concentration of dye in control sample (sample without biomass) [mg/l];

B – concentration of dye in sample with bacteria biomass [mg/l].

The screening studies showed that compost, rotten wood as well as municipal sewages may be a proper sources for obtaining the bacteria strains able to decolorize the synthetic dyes. All used materials had the common feature – the complex composition and the ongoing biological decomposition processes.

The highest efficiency of removal of the used synthetic dyes was reached in the case of triphenylmethane brilliant green (BG) (Fig. 1–3). The most numerous group of bacteria with abilities to decolorize BG was obtained from sewage - 18 strains (Fig. 1), then from compost – 9 strains (Fig. 2), and from rotten wood – 6 strains (Fig. 3). The BG removal efficiency higher than 50% after 96 h of incubation was observed in the case of 83% of strains from sewage, 78% of strains from compost and 100% of strains from rotten wood. The removal of BG higher than 80% was obtained in the case of 50%, 56% and 66% of strains respectively and respectively 39%, 11% and 33% of them reach this level already after 48 h of culture. The average removal of BG by the bacteria strains isolated from the sewage was on the level of 76.9%, from the compost 71.6%, from the rotten wood 83.2%.

Figure 1.

Figure 2.

Figure 3.

It is noteworthy that the highest efficiency of decolorization of BG was observed in the case of the strains isolated from rotten wood. It may be connected with the complexity of this substrate (polymers, mainly cellulose and lignin) what requires the production of special groups of enzymes necessary for utilization of such complex substrates, what probably allowed to decompose also the complex aromatic structure of BG.

The second studied triphenylmethane dye was crystal violet (CV). The much worse results of the bacterial decolorization of this dye than BG (Fig. 1–3) were probably connected with the differences in chemical structure between them. More structurally complex crystal violet has in molecule six methyl groups bonded with the three phenyl rings, while the brilliant green has four methyl groups bonded with the two phenyl rings (Tab. 1).

It was proved by the other authors, that the dyes’ biodegradability decreases with the increasing complexity of their chemical structure, which is connected among others with the presence of a higher number of functional groups in the molecule [17, 25, 28, 31, 32].

The best results of decolorization of crystal violet were achieved by the strains isolated from sewages (Fig. 1) then from rotten wood (Fig. 3) and compost (Fig. 2). After 96 h of incubation the average decolorization of CV was 29.9%, 25.9%, 12.0% respectively. Only 22% of strains isolated from sewages were able to remove more than 50% of CV. It is noteworthy, that all of these strains showed also the highest efficiency of decolorization of BG and additionally they were obtained from cultures on MM medium where the dye was the only source of carbon and energy. The removal of CV higher than 80% was observed only in the case of one strain (Sz3m) isolated on MM medium, which removed 92% of this dye already after 48 h of incubation (similarly in the case of BG – the removal of 98% after 48 h). None of the strains isolated from the compost and rotten wood removed 50% of CV after 96 h.

The fluorone erythrosine (Er), as well as CV, was more difficult for removal than BG. After 96 h of incubation, the average results of decolorization of Er by the strains isolated from sewage, compost and rotten wood were comparable and were on the level of 24.1%, 24.3%, 23.2% respectively. None of strains removed 50% of this dye from solutions.

One of the selective factors applied at screening studies was the composition of the growth media used for isolation of the bacteria strains. Both of the used growth media were supplemented with synthetic dyes (BG or EB). One of them was a nutrient agar – rich in easily bioavailable organic ingredients, the other was a mineral medium (MM), free from organic substrates (after supplementation, the only source of carbon and energy was synthetic dye introduced as supplement).

The results of conducted screening depended on the composition of the growth medium used for isolation of the decolorizing bacteria strains (Fig. 4–6). It was observed that from used materials (sewage, compost and rotten wood) much more bacteria strains with the ability for dyes removal were isolated on the nutrient agar (24 strains) then on MM medium (9 strains). But it should be pointed out that the lower number of microorganisms isolated on MM medium (supplemented with the given dye) reached the higher average of decolorization results than a higher number of strains isolated on nutrient agar.

Figure 4.

Figure 5.

Figure 6.

The strains isolated from sewage on the nutrient agar (13 strains) removed dyes with various efficiency (Fig. 4). Only in the case of BG some of them were able to remove more than 50% of this dye. After 96 h of incubation such a result was obtained in the case of 10 strains (77%), amongst them 4 strains (31%) removed more than 80% of BG and 2 of them removed more than 90% already after 48 h. The average efficiency of decolorization of dyes by bacteria isolated from sewage on the nutrient agar was: 68.1% BG, 15.9% CV, 22.3% Er. Whereas the strains isolated from sewage on the MM (5 strains) removed dyes more efficiently (Fig. 4). All of them removed more than 90% of BG after 48 h (about 100% removal after 96 h). The decolorization of CV higher than 70% was reached by 80% of strains isolated on MM, and one of them (Sz3m) removed more than 90% of CV already after 48 h. The worst results (removal has not exceeded 50%) were obtained in the case of Er. The average decolorization efficiency of used dyes by bacteria isolated from sewage on the MM was: 99.7% BG, 66.6% CV, 28.8% Er.

Majority of strains isolated from compost were obtained on nutrient agar (8 from 9 strains) (Fig. 5). The best removal results were reached for BG (average removal efficiency 68%), then for Er (average removal 22.3%) and CV (average removal 8.8%). Only in the case of BG the removal efficiency exceeded the level of 50% (75% of strains), and in the case of 50% of strains the results were higher than 80%. From compost on MM medium was obtained only one strain (KZ4m) which was able to decolorize efficiently BG.

Amongst six strains isolated from the rotten wood three of them were obtained on nutrient agar (Fig. 6). The average removal results obtained by these strains depended on the dye and was 78.9% for BG, 20.7% for CV and 26.8% for Er. Strains isolated from rotten wood on MM medium removed BG and CV with higher efficiency (average removal 87.4% and 31% respectively) than these isolated on nutrient agar (average removal 78.9% and 20.7% respectively).

Two synthetic dyes (triphenylmethane brilliant green (BG) and disazo Evans blue (EB)) were used as selective factors at screening studies. Dyes were used for growth media supplementation.

The influence of used dye on decolorization abilities of the obtained strains was observed (Fig. 7–9).

Figure 7.

Figure 8.

Figure 9.

The presence of the BG in the growth medium allowed to obtain from the sewage the strains with the higher abilities of decolorization of this dye than in the case of using the azo EB for isolation (average removal of BG 82.6% and 71.2% respectively) (Fig. 7). After 96 h of incubation 100% of strains isolated on medium with the BG removed more than 50 % of this dye, whereas in the case of strains isolated on medium supplemented with EB the removal of BG exceeded 50% in case of 71% of strains. Surprisingly after 48 h of incubation, this result was reached by 25% and 36% of strains respectively, which pointed on higher efficiency of removal of BG by strains isolated on medium with EB. It should be emphasized that the majority of these strains were isolated on MM medium, where the only source of carbon and energy was used a given dye.

Similar results were obtained in case of isolation of bacteria strains from compost (Fig. 8). The average removal of BG by strains isolated on medium with BG was 80.1% and 64.4% for strains isolated on medium with EB. All strains isolated from rotten wood independently of the dye used in isolation medium had the high abilities of decolorization of BG (average removal after 96 h of incubation was over 80%) (Fig. 9).

The efficiency of removal of CV by strains isolated from sewage and the compost on medium with BG had higher decolorization abilities than in case of strains isolated on medium with EB, but in both cases the average results of CV removal did not exceed 50 % (it was 34.6%, 17.1% and 25.3%, 79% respectively) (Fig. 7–8). In case of strains isolated from rotten wood the higher decolorization potential of CV had strains isolated on medium with azo EB than with BG (41.8% and 10.0% respectively) (Fig. 9).

The abilities of removal of fluorone erythrosine were low and in none case exceeded 50% after 96 h of incubation (Fig. 7–9). However, the strains isolated from sewage, compost and rotten wood on medium with BG reached higher average results of removal of Er than these isolated on medium with azo EB (28.2%, 30.6%, 25.3% and 20.0%, 19.3%, 21.2% respectively).

In spite of isolation of the largest group of decolorizing bacteria strains from sewage, the compost and rotten wood may also be considered as potential sources of obtaining such microorganisms. The usage of the mineral medium at the screening procedure allowed to obtain the lower number but much more efficient bacteria strains. Usage of the certain dye during the isolation process had a lower impact on decolorization abilities of isolated strains, however the usage of the BG allowed to obtain the strains with higher efficiency of removal of studied dyes than in case of using the EB.

Isolated bacteria strains with the highest decolorization potential should be tested on other dyes belonging to various chemical groups and also their mixtures. The selection carried out in this way will allow to obtain of strains with high decolorization potential and create the basis for the creation of biopreparations supporting the removal of dyes from colored wastewater.