Influence of light wavelengths, light intensity, temperature, and pH on biosynthesis of extracellular and intracellular pigment and biomass of Pseudomonasaeruginosa NR1

2.1 Materials

All the glassware and Petri dishes used in experimentation were purchased from Scott Duran manufacturer (DURAN®, Germany). The glassware consists of good quality glass with clear, colourless properties, having negligibly low absorption spectra in the range about 310 to 2200 nm. Petri dishes used in experiments could transmit light of all wavelengths by more than 90%. Philips 20Watts lamps (Philips India Ltd.) were used as a light source for experimentation. All other chemicals used in experimentation were of analytical grade, and were procured from Merck, India. Light intensity was controlled in both experimental setups by using the same 20 Watt lamps, and by keeping all plates and flasks equidistant (15 cm) from the light source.

2.2 Isolation of pigment producing bacterium

The pigment producing bacterium was isolated from dead Rhinoceros beetle. The insect was collected aseptically and was brought into the laboratory. The feathers of the insect were cut and were washed with sterile distilled water. This water was serially diluted up to a factor of 10^-6 with sterile distilled water. Diluted samples were used for the isolation of pigmented bacteria. A loopful of diluted sample from 10^-6 tube was withdrawn with Nichrome wire loop, and was streaked on sterile nutrient agar (Hi-Media, Mumbai) plates. Plates were incubated at 37 °C in incubator for 48 h. A colony with intense and bright pigment producing ability was selected for the further research studies. The selected bacterium was able to produce dark red intracellular (Fig. 3) and green extracellular pigments (Fig. 4).

Close

Fig. 1

. 16S rRNA of Pseudomonas aeruginosa NR1.

Export to PPT

Close

Fig. 2

. Phylogenetic tree of P. aeruginosa NR1.

Export to PPT

Close

Fig. 3

. Intracellular pigment produced by P. aeruginosa NR1.

Export to PPT

Close

Fig. 4

. Extracellular pigment produced by P. aeruginosa NR1.

Export to PPT

2.3 Identification of isolated pigmented bacterium

The isolated bacterium was identified by morphological, biochemical characteristics, and by the 16S rRNA gene sequencing method. The 16S rRNA gene was amplified by using following 16S universal primers.

•  

  16S Forward primer: 5′ AGA GTT TGA TCC TGG CTC AG 3′

•  

  16S Reverse primer: 5′ AAG GAG GTG ATC CAG CCG CA 3′

2.4 Effect of different light wavelengths on pigment and biomass production

Effect of different light wavelengths on pigment and biomass production was evaluated by using two methods. One was glass paper wrapping method, which has been used by researchers (Soumya et al., 2014; Babitha et al., 2008) to deduce effect of light wavelengths on microbial pigment and biomass production; the second was by using the selective lamp light wavelength method, which is a novel technique and for the first time introduced by our research group. First method was based on the principle that the color glass paper allows only selective wavelength of light to pass through it. If red colored glass paper is used, then only red color light wavelength will pass through the paper. In the second method, only selective colored light wavelength was provided to flasks and plates, which were kept in controlled confined environment. Colored lamps (Philips, 20 W) were used for providing selective wavelengths.

2.5 Experimental set up for method one (glass wrapping method)

For this method, five different color glass papers were used. The papers used in this research study were green, red, yellow, brown, blue in color (Fig. 5). Each glass paper was wrapped around the one flask and a plate and these wrapped plates and flasks were kept around the illumination source, 15 cm away from the light source (Fig. 6). One flask and one plate were directly kept under illumination without wrapping to check effect of direct light on pigment and biomass production. To detect effect of darkness on pigment and biomass production, one flask and one plate were kept in total darkness. For intracellular pigment production, the organism was grown on sterile nutrient agar plates; for extracellular pigment production, as it was diffusible only in liquid medium, the organism was grown in sterile nutrient broth flasks. Flasks and plates were inoculated with the organism with equal volumes (2 mL) of 24 h old bacterial culture and were incubated in a box that had a single illumination source provided centrally.

Close

Fig. 5

. Glass papers wrapped to plates and flasks.

Export to PPT

Close

Fig. 6

Wrapped flasks and plated under light source.

Export to PPT

2.6 Experimental set up for method two (selective wavelength method)

We designed ‘a selective light wavelength’ experiment for evaluating the effect of light on pigment and biomass production. For this experiment, a wooden box was constructed. This box was equally subdivided in nine chambers by partitioning the box with wooden blocks (Fig. 7). The partition was done in such a manner that no light rays from one chamber could pass to another chamber. An electric holder plate was designed with 10 lamp holders (Fig. 8). This plate was designed in such a way that in each chamber a holder socket was present in a central position. At every holder socket, one colored selective wavelength lamp was fixed. The box was closed, so that no external light was able to enter the box. Red, orange, blue, green, yellow, white, pink lamps and one more lamp with multiple light wavelengths that exerts red, blue, green, yellow, white light wavelength one by one repeatedly were used for the study. In one chamber no lamp was fixed, so that total darkness is achieved for incubation in absence of light.

Close

Fig. 7

. Equal chambers made in box.

Export to PPT

Close

Fig. 8

. Electric plate constructed for the experiment.

Export to PPT

2.7 Effect of temperature on pigment and biomass production.

To evaluate effect of temperature on pigment and biomass production, sterile nutrient broth flasks and nutrient agar plates were prepared. For each temperature, one flask and one plate were investigated. All plates and flasks were inoculated with equal amounts of bacterial culture and kept at different temperatures such as 25 °C, 35 °C, 45 °C, 55 °C, 65 °C. After 2 days of incubation, the amount of pigment and biomass was estimated.

2.8 Effect of pH on pigment and biomass production.

To check the effect of pH, one nutrient broth flask and one nutrient agar plate with pH 3, 4, 5, 6, 7, 8, 9, 10 were prepared and inoculated with equal amounts of bacterial culture and were kept for incubation for 2 days at 30 °C. Amount of pigment and biomass was estimated by using spectrophotometric analysis (Elico UV–Visible double beam spectrophotometer).

2.9 Effect of light intensity on pigment production

For detecting the effect of increased light intensity on extracellular and intracellular pigment production, and to get information regarding role of extracellular pigment in photoprotection, this study was undertaken. To study the light intensity effect on pigment production, the plates and flasks producing intracellular and extracellular pigment, respectively, were kept and incubated under increasing light intensity environment by using 15 W, 20 W, 25 W, 30 W, 40 W, 50 W white light lamps.

2.10 Inoculation

2 mL of inoculum medium was used for all flasks. Twenty-four hours old broth of P. aeruginosa NR1 was used for inoculation. Plates and flasks were inoculated with desired bacterial culture and all were kept for incubation at their places for 72 h at 35 °C. The room temperature was maintained at 35 °C with the help of room heater.

2.11 Intracellular and extracellular pigment estimation

Extracellular pigment concentration was estimated by measuring absorption of pigmented solution at its λ_max, i.e., 382 nm. Broth was centrifuged with relative centrifugal force of 3600 × g for 20 min by using REMI research centrifuge (REMI, India). The supernatant was filtered and cleared, colored filtrate was obtained. Equal amount of Chloroform was used for the complete extraction of extracellular pigment from the aqueous broth. The colored chloroform was used for spectrophotometric analysis (Elico UV–Visible double beam spectrophotometer). For estimating intracellular pigment concentration, the intracellular pigment was extracted by using acetone. 10 mL of acetone was added in a Petri plate with well grown pigmented colonies. The pigment slowly started to dissolve in solvent. When colonies turned colorless, the acetone was removed and collected in a test tube. 5 mL of acetone was again added to extract the remaining traces of pigment completely from colonies. The red colored acetone was filtered with Whatmann filter paper GF/C disc (47 mm). The absorption maxima were determined for the red pigment, and the concentration of pigment in each plate was estimated by measuring the absorption in spectrophotometer (Elico UV–Visible double beam spectrophotometer). Acetone was used as blank in the analysis.

2.12 Biomass estimation

The bacterial biomass was estimated as dry weight by the conventional method described by Li and Mira (2010) with some minor modification. Broth was centrifuged with relative centrifugal force of 3600 × g for 20 min by using REMI research centrifuge (REMI, Centrifuge, India) for 20 min. The pellet was washed with distilled water and was mixed in 5 mL distilled water. The empty weight (in gram) of the evaporating dish was measured and recorded (initial weight, IW). The bacterial mass was kept in evaporating dish. The dish was kept in oven at 100 °C until all water was evaporated, and only bacterial mass remained in dish. After complete drying, the final weight (in gram) of the dish with the dried bacterial mass was recorded (Final weight, FW). The actual weight of bacterial cells was determined by subtracting the initial weight from the final weight. $$\text{Dry weight}=\text{FW}-\text{IW}$$ where,

•  

  FW – final dish weight (with dried bacterial mass)

•  

  IW – initial dish (empty dish weight)

2.13 Statistical analysis

Each experiment was repeated at least three times. Numerical data are presented as mean ± SD. All the results were analyzed by means of one-way ANOVA. All statistical analyses were performed by using SPSS 11.5 software. P < 0.05, P < 0.01 was considered statistically significant.

3.1 Isolation and identification of pigmented bacterium-

The red colored bacterium was identified as P. aeruginosa NR1 by 16srRNA gene sequencing and considering it’s morphological and biochemical characteristics. The bacterium was able to catabolize many sugars including glucose, galactose, sucrose, maltose, fructose, mannose, dextrose, xylose, but was found unable to catabolize rhamnose, lactose, arabinose, and mannitol (Table 1). Moreover, the bacterium was oxidase and catalase positive. The 16S rRNA gene sequence and phylogenic tree is shown in Figs. 1 and 2 respectively.

3.2 Effect of light wavelengths, light intensity, temperature and pH on pigment and biomass production.

Propst and Lubin (1979) have also reported the light mediated changes in P. aeruginosa cultures. In their research study, they found that cultures of Pseudomonas aeruginosa PA0 grown under uninterrupted broad-spectrum light showed pigmentation different from dark-grown cultures; dark-grown bacteria produced pigments, which resulted in blue-purple colored agar, while light-grown organisms produced red colored plates. We didn’t observe complete change in pigment color, but a slight change in color shade was observed when our bacterium was kept under light and total darkness. In our experiment, higher concentration of pigment was obtained for the flasks and plates of ‘selective wavelengths lamp light method’ than for the ‘glass paper wrapped’ flasks and plates (Table 2). This higher concentration of pigment in selective wavelengths method shows its superiority and efficacy over the glass paper wrapping method. For intracellular pigment production, yellow light wavelength was found most suitable in our experiment. Under yellow light wavelength, maximum intracellular pigment production was recorded (SLP- 4.672 ± 0.1 OD/ml; GPP- 4.395 ± 0.03 OD/ml), followed by green (SLP – 3.816 ± 0.02 OD/ml; GPP –3.611 ± 0.07 OD/ml), orange (SLP – 3.500 ± 0.03 OD/ml), and pink (SLP – 2.913 ± 0.04 OD/ml). Minimum intracellular pigment production was observed under red (GPP – 2.124 ± 0.01 OD/ml; SLP- 2.252 ± 0.02 OD/ml), blue (GPP- 2.308 ± 0.01 OD/ml; SLP – 2.412 ± 0.02 OD/ml) and white light wavelengths (SLP – 2.890 ± 0.03 OD/ml). Our light experiments revealed that under blue and white light, intracellular pigment production decreases, which is in accordance with the findings by Nobutaka et al. (2004) and Chen et al. (2016). As a difference between our result and those by Nobutaka et al. (2004) results, red light negatively influenced the pigment formation in our experiments, while in Nobutaka et al. (2004) red light didn’t affect the pigment and biomass concentration. Our result that under blue light the intracellular pigment production decreases, was similar with the result by De Lucca et al., (2012), as they reported that the blue light effectively inhibits bacterial and fungal growth, whereas Miyake et al. (2005) in a research study reported opposite observation that under blue light pigment production was maximum by Monascus ruber N. Darkness was not that much effective for the organism for intracellular pigment production. On the other hand, extracellular pigment was produced maximum under darkness (4.042 ± 0.13 OD/mL). This result is in accordance with Babitha et al., (2008), Soumya et al., (2014), and Velmurugan et al., 2010, who also reported that darkness was beneficial for the pigment production by their organism. Our this result is significant as this observation refutes the photo protective role of our pigment as proposed by Dieringer et al. (1977), Yong and Lee (1991), Salih et al., (2000), and Seagle et al., (2005). Using red light wavelength (Selective lamp flask 3.714 ± 0.2 OD/mL; Glass paper flask, 3.317 ± 0.07 OD/mL) and blue wavelength (Selective lamp flask 3.638 ± 0.19; Glass paper flask, 3.590 ± 0.02), extracellular pigment production was also pronounced. The lowest extracellular pigment concentration was obtained under green light wavelength (GPP-2.151 OD/mL; SLP- 2.232 OD/mL). The biomass was produced maximum under yellow (Selective lamp flask 830 ± 5.7 µg/0.01 L; Glass paper flask, 810 ± 2.88 µg/0.01 L) and green light (Selective lamp flask 731 ± 4.4 µg/0.01 L; Glass paper flask, 708 ± 4.4 µg/0.01 L) wavelengths. This means that the biomass and intracellular pigment was produced at the maximum concentration under yellow and green light. This result was expected since the concentration of intracellular pigment should be directly proportional to the concentration of biomass; however, in a research published by Alejandro et al. (2011), the opposite observation was reported namely no direct relationship between biomass concentration and pigment production. Pigment production by the organism was not completely inhibited under any particular light wavelength.

In our research study, we used two methods to detect the effect of light wavelengths on pigment production. Both methods, i.e., glass paper wrapping method and selective wavelength lamp method, gave good results, but more satisfactory and more efficient results were obtained in method two, i.e., selective lamp light wavelength method; as the pigment concentrations were higher under selective light lamps than the glass paper wrapped flasks and plates. In different light wavelengths, we observed some differences in color shade of extracellular pigment. This might be due to the variation in composition of pigment as suggested by Velmurugan et al. (2010). Our result and conclusion is also in agreement with Miyake et al. (2005), who stated that pigment concentration changes with the variation in light wavelength. When bacterial plates and flasks were incubated under increasing light intensity, no considerable changes in pigment concentration were observed (Table 5). These results also refute the role of our pigment in photoprotection. The effect of temperature on extracellular and intracellular pigment and biomass production is shown in Table 3. The maximum extracellular (3.924 ± 0.04 OD/mL) and intracellular pigment concentration (3.698 ± 0.01 OD/mL) was recorded when temperature was 35 °C and 30 °C, respectively. Maximum biomass concentration (656 ± 3.05 µg/0.01 L) was obtained when temperature was 30 °C. Above 45 °C, neither intra- nor extracellular pigment production was observed. Growth was observed up to 55 °C, but not at higher temperature. This shows that this bacterium is a moderate thermopile. Maximum intracellular pigment (3.856 ± 0.02 OD/mL), extracellular pigment (3.548 ± 2.2 OD/ml) and biomass concentration (800 µg/0.01 L) was recorded at pH 7. Above pH 10 and below pH 5, no pigment production was recorded. The bacterial growth was observed up to pH 10, which means the isolated bacterium is an alkalophilic strain. Table 4 shows the effect of pH on extracellular, intracellular pigment and biomass production by the bacterium. It is apparent from this research study that the pigment of isolated bacterium doesn’t have any role in photoprotection. This fact raises many questions in mind like why does the bacterium produce the pigment? Many bacterial and fungal pigments have been well recognized for their virulent activities (Liu and Nizet, 2009; Hall et al., 2016; Ortiz-Castro et al., 2014; Carbonell et al., 2000). As we had isolated the pigment producing bacterium from dead insect, we may speculate that the pigment of the bacterium might have acted as virulent factor for the insect and it might have served a reason for insect’s death. Another role of the pigment (considering its source of isolation) might be in degradation of cellular components (like chitin) of insect for making energy for bacterial cellular processes. This presumption seems more convincing after considering the ability of the bacterium to produce chitinase enzyme (the isolated bacterium was able to produce chitin in colossal amount). The pigment might be having roles other than these two hypothesized roles. The pigment may help the bacterium in competition with other microbes for getting needed nutrients for its growth and survival or it could have a role in bacterial resistance to naturally produced antimicrobial compounds by other microbes or plants. The pigment might be helpful for the bacterium to cope up with harsh and extreme conditions like high temperature, high pressure, high salinity and cold. Many researchers have reported the role of pigments in salt tolerance by haloarchae (Rebecca et al., 2016; Oren, 2010).