A new strategy for the efficient production of pyocyanin, a versatile pigment, in Pseudomonas aeruginosa OG1 via toluene addition

Abstract

Pseudomonas aeruginosa produce pyocyanin, which is an extracellular secondary metabolite and multifunctional pigment. In this study, the effects of several surfactants (Tween 20, Tween 80 and Triton X-100) and organic solvents (toluene and chloroform) on pyocyanin production and cell growth were investigated in submerged culture of P. aeruginosa OG1. Organic solvents were found to be more effective in the production of pyocyanin. The maximum production of pyocyanin (33 mg/L) was achieved when 0.2% toluene was added at the stationary growth phase (30 h), corresponding to significant increase of 312% compared with the control (8 mg/L). With the addition of toluene, pyocyanin production was significantly increased, but bacterial biomass reduced. Production of alkaline protease was also affected by toluene addition. It was found that the ratio of saturated/unsaturated fatty acids in the bacterial biomass significantly increased when toluene addition to the medium. This study revealed that with a novel strategy, the addition of toluene to the fermentation medium significantly increased pyocyanin production. These findings suggest that solvent-assisted fermentation strategy can be used in microbial fermentations to increase the production of biotechnological products such as industrially important pigment and enzyme. This study is a first investigation on the stimulation of pyocyanin release in the medium of P. aeruginosa cultures by the addition of toluene.

Introduction

Some bacteria synthesize either intracellular or extracellular low-molecular-weight natural pigments. The demand for these natural pigments is increasing due to the carcinogenic, allergic, and environmental pollutant properties of the synthetic pigments currently used (Kurbanoglu et al. 2015; Ozdal et al. 2017a).

Pyocyanin is water soluble, nitrogen containing (C₁₃H₁₀N₂O), extracellular phenazine derivative pigment which is produced as a secondary metabolite by Pseudomonas aeruginosa. The pyocyanin has different biological activities such as antimicrobial, anticancer, antimalarial, antiparasitic, immunosuppressive, antioxidant, and antibiofilm. The ability of pyocyanin to support electron transfer has many potential biotechnological applications such as colorimetric redox indicators, sensors in nanotechnology, luminescence-based pH sensor, electron transfer in microbial fuel cells, and organic light emitting devices (Pierson and Pierson 2010; Jayaseelan et al. 2014; Wu et al. 2014). Due to these characteristics, pyocyanin has potential for medical, pharmaceutical, food, textile, bio-control, nanotechnology, and physicochemical applications. For these reasons, pyocyanin is a multifunctional and versatile pigment.

Pyocyanin production by P. aeruginosa is affected by environmental factors including carbon and nitrogen sources, incubation time, concentration of salts, availability of oxygen, temperature, and pH of the medium (Jayaseelan et al. 2014; Patil et al. 2017). It also determined that alkaline protease enhanced pyocyanin production (Liyama et al. 2017).

Many researchers have dealt with to increase the production of microbial products using different stimulants such as fatty acids, organic solvents (toluene, chloroform, acetone, methanol, ethanol), surfactants (Tween 20-60-80, Triton X-100), and vegetable oils (Wang et al. 2013; Xu et al. 2016; He et al. 2016; Lei et al. 2017). It has been explained that these stimulants exchange the composition of the cell membrane and thereby contributing to the improvement of desired products (Wang et al. 2013; He et al. 2016). The use of organic solvents is both economical and easy to separate (Lim and Yun 2006).

Although there are many applications of pyocyanin, it is quite expensive compound in the market (€118/5 mg, Sigma-Aldrich Co. 2019) due to its low yield, high production cost, and lack of large-scale production. In light of the potential applications, a new strategy is required to obtain inexpensive pyocyanin. The aim of this paper was to investigate the effect of toluene on the pyocyanin production of P. aeruginosa OG1. The effect of toluene on P. aeruginosa cell membrane fatty acid profile and alkaline protease production were also determined.

Materials and methods

Microorganism and preparation of inoculum

Pseudomonas aeruginosa strain OG1 used in this study possesses the ability to biodegrade endosulfan (Ozdal et al. 2016) and produce rhamnolipid (Ozdal et al. 2017b). Cultures were grown on Nutrient Agar (Oxoid, USA) at 30 °C 20 h and then transferred to a 250-mL flask that contained 50 mL of the Nutrient Broth and subsequently incubated at 30 °C for 24 h with shaking at 150 rpm. One milliliter (OD₆₀₀:1) of bacterial suspension was used as inoculum. All experiments were performed in shake flask cultures of 250-mL flasks with 50 mL liquid. For pyocyanin production, the inoculated flasks were incubated for 96 h at 30 °C and 180 rpm to determine optimum fermentation time using production media (Nutrient Broth containing 1% glycerol).

Effect of different surfactants and organic solvents on pyocyanin production

To evaluate the effect of different surfactants and organic solvents on pyocyanin production, three surfactants (Tween 20, Tween 80, and Triton X-100) and two organic solvents (toluene and chloroform) at three different concentrations (0.1, 0.2, and 0.3%, v/v) were incorporated into the production media after 24 h of cultivation. The control flask was devoid of any stimulant. The appropriate concentration (0.1–0.3%, v/v) and additional time (0–48 h) of the best stimulating agent in production media (Nutrient Broth containing 1% glycerol) were determined. The fermentation was carried out for 72 h at 30 °C, initial medium pH 7.2. The flasks were harvested at selected time intervals for measurement of the biomass and pyocyanin contents. Stimulants were aseptically added to the production medium after filtration with a 0.22-µm membrane.

Measurement of biomass and pyocyanin

At regular intervals of fermentation, the microbial biomass and pyocyanin content were determined. Bacterial biomass was harvested by centrifuging the fermented broth at 5000×g for 10 min, washed twice with sterile distilled water, and dried in an oven at 80 °C for 24 h. To determine prodigiosin, a 2.5-mL cell-free supernatant was extracted with 1.5 mL of chloroform and 0.5 mL of 0.2-N HCl. The pyocyanin content of the cell-free supernatant in the acidic form was measured based on the absorbance (A) at 520 nm (Essar et al. 1990). The amount of pyocyanin was estimated by the following formula:

$$\text{Pyocyanin (mg}/\text{L)}=A_{520}\times 17.072.$$

Purification of pyocyanin

After incubation, the culture broth was centrifuged at 5000×g for 10 min (4 °C). Two volumes (100 mL) of chloroform were added to one volume (50 mL) of cell-free supernatant. The solution was mixed well using shaker for 2 min, and again centrifugation at 10,000×g for 15 min. Two distinct layers separated out, in which one was the pigment and the other remaining material of culture. The pyocyanin was purified by TLC (Silica gel 60 F254, Merck) on silica gel using chloroform:methanol (90:10) as an eluent. The obtained pyocyanin was dissolved in methanol. HPLC analysis (LC20AD, Shimadzu), chromatographic separation was carried out using a C18 column for reverse-phase chromatography (PDA C18, 259 nm), with a flow rate of 0.8 mL/min, and the column oven temperature was 24 °C. The mobile phase was acetonitrile:water (pH 2.5 HCl) in the ratio 85:15.

Assessment of alkaline protease activity

To investigate the effect of toluene on protease production from P. aeruginosa OG1, the bacterium was grown in protease production media in the presence of different concentrations of toluene (0.1–0.3%, v/v). Toluene was added after 30 h of culture. For the production of alkaline protease, bacteria were inoculated in production medium consisted of 7 g/L K₂HPO₄, 2 g/L KH₂PO₄, 0.2 g/L MgSO₄, 4 g/L casein, 6 g/L yeast extract, 0.5 g/L NaCl, 7 mL/L glycerol, 0.1 g/L CaCl₂, and pH 7.0, and kept in a shaker incubator at 30 °C and 180 rpm for 60 h. One unit alkaline protease activity was defined as the amount of enzyme required to produced peptides equivalent to 1.0 µg of tyrosine per minute at pH 8.0 and 30 °C as described by Gupta and Khare (2006).

Analysis of fatty acid methyl esters by GC/MS

Bacteria were cultivated in production media (Nutrient Broth containing 1% glycerol) with or without toluene on shaker at 30 °C for 72 h. Toluene was added directly to the culture medium after 30 h of cultivation. About 40 mL of fermentation broth was centrifuged at 5000×g for 20 min at 4 °C and washed twice with sterile distilled water. Bacterial cellular lipids were extracted and converted to fatty acid methyl ethers (FAME) as previously described by Sasser (1990). The composition of FAME was analyzed using a chromatography–mass spectrometry system (Agilent 7820A/5977) on an HP-5 MS column (30 m × 0.25 mm I.D., USA). The temperatures of the inlet, transfer line and detector were 260, 290n and 240 °C, respectively. Identification of the components was carried out using a standard mixture of methyl ester FAs.

Results

Growth curve and production of pyocyanin in production medium of P. aeruginosa

The time-course studies on the production of pyocyanin by P. aeruginosa were carried out for a period of 144 h in the production media, as shown in Fig. 1. After 24 h of growth phase, cells entered the stationary growth phase and reached the maximum concentration of 1.86 g/L at 24 h. The maximum of 8 mg/L pyocyanin production was obtained at 72 h. After this, optimum fermentation time did not show any increase in pyocyanin production.

Fig. 1.

Effect of time on pyocyanin production in production medium. An asterisk denotes a value significantly greater than other pyocyanin values (P < 0.05)

Effects of surfactants and organic solvents on pyocyanin production

The influences of two different organic solvents (e.g., toluene and chloroform) and three surfactants (e.g., Tween 20, Tween 80, and Triton X-100) on the pyocyanin production in P. aeruginosa OG1 were studied by supplementing 0.1–0.3% (v/v) of each into production media on the 24 h of fermentation (Fig. 2). Bacterial biomass of cultures containing Tween 80 and Triton X-100 remained fairly close to the control, but chloroform and toluene significantly reduced the bacterial biomass (Fig. 2a). As shown in Fig. 2b, all surfactants had an increasing effect on the production of pyocyanin, to different extents, except Tween-80. However, the pyocyanin production was improved by 125.4 and 275% when chloroform (0.3%, v/v) and toluene (0.2%, v/v) were used, respectively. Maximum pyocyanin production with chloroform was 28 mg/L at a concentration of 0.5% (data not shown). The maximum pigment yield (30 mg/L) was obtained with the addition of toluene (0.2%, v/v). Based on these results, the addition of toluene resulted in the highest pyocyanin production and chosen for further use in optimization.

Fig. 2.

Effects of various stimulants on the bacterial biomass (a) and pyocyanin (b) production in cultures of P. aeruginosa. Each fermentation was carried out for 72 h at 30 °C. The data are expressed as mean ± S.E. The surfactants and organic solvents were added to the production medium at a volume fraction of 0.1–0.3% after 24 h of cultivation

Effect of toluene addition time on bacterial biomass and pyocyanin production

To better understand the effect of toluene on bacterial biomass and the production of pyocyanin in submerged fermentation of P. aeruginosa OG1, 0.2% toluene was added at 0, 12, 18, 24, 30, 36, and 42 h of fermentation (Fig. 3). When toluene was added at the beginning of fermentation (0 h), cell growth was completely inhibited. As is well known, secondary metabolite (such as pyocyanin) production begins after cells reach the stationary growth phase. As shown in Fig. 1, after 24 h of fermentation, the cells were in the stationary growth phase. The biomass was lower than the control when toluene was added at anytime. This indicating that there was important effect of toluene on bacterial growth. However, addition of toluene at 24 h or later resulted in a clear increase in pyocyanin production yield compared with earlier addition times (12 and 18 h). The maximum production of pyocyanin (33 mg/L) yield was achieved when toluene was added at 30 h of fermentation. Equally importantly, when toluene was added at 12 h of fermentation, the concentration of pyocyanin was 10.8 mg/L, which was 25% higher than that of the control. Based on the above results, the addition time of toluene had a significant influence on the pyocyanin production (P < 0.05). Pyocyanin yield improved among all toluene concentration additions at stationary phase (Fig. 3). Therefore, toluene should be added after cells are in the stationary growth phase. The results suggested that pyocyanin yield could be improved if a suitable concentration of toluene was added at cells stationary phase.

Fig. 3.

Effect of toluene addition time on biomass yield (a) and pyocyanin production (b) by P. aeruginosa. Toluene (0.2%, v/v) was added to the broth at different time points (after 0, 12, 18, 24, 30, and 42 h of culture). Values with the same letter are not significant (P < 0.05)

As shown in graphical abstract, the color of the culture broth containing toluene became dark blue–green due to pyocyanin content. It was clearly observed that toluene had a stimulatory effect on the pyocyanin biosynthesis (Fig. 4).

Fig. 4.

Effect of toluene on pyocyanin production. Toluene (0.2%, v/v) was added after 30 h of incubation

Characterization of pyocyanin

The chromatogram of blue color spot on TLC plate indicated an Rf value of 0.7 related to pyocyanin pigment. The spot showed a similarity between the standard and the used molecule of pyocyanin with the same RF (Fig. 5a). Figure 5b indicated that pyocyanin was separated by HPLC, with 94.2% purity and a single peak at retention time of 2.445 min. The TLC chromatogram and HPLC analysis confirmed the pyocyanin produced by P. aeruginosa OG-1 in the presence of toluene.

Fig. 5.

TLC profiles of pure (1) and produced pyocyanin (2) (a), and HPLC chromatogram of pure and produced pyocyanin (b)

Effect of toluene on alkaline protease production

The effects of toluene on bacterial biomass and alkaline protease activity were investigated. Alkaline protease production was increased in the presence of toluene (0.1–0.3%, v/v), while the biomass decreased after adding toluene due to the direct effect of toluene on P. aeruginosa OG1 (Fig. 6). With 0.2% toluene, alkaline protease activity reached the maximum value of 1540 U/mL, which increased by 40% compared to control (1100 U/mL).

Fig. 6.

Effect of toluene on protease production from P. aeruginosa. Different toluene concentrations (0.1–0.3%, v/v) were added after 30 h of culture. An asterisk denotes a value significantly greater than other alkaline protease values (P < 0.05)

Effect of toluene on membrane fatty acids

As seen in Table 1, different fatty acid profiles were determined when the bacteria were grown in the presence of the toluene (0–0.3%, v/v). With increasing concentrations of toluene, the composition of palmitic acid (C16:0) was significantly increased from 34.01% to 67.2%, whereas the composition of 3-hydroxydecanoic acid (10:0 3OH) and octadecanoic acid (18:0) was decreased. Palmitic acid (16:0) and cis-vaccenic acid (18:1 w7c) had the highest percentage in all treatments. Octanoic acid (8:0) was acquired after toluene (0.2–0.3%) addition. At highest concentration of toluene (0.3%, v/v), several fatty acids (12:0, 12:0 2OH, 12:0 3OH, 14:0, 16:1 w7c, 17:0 cyclo, and 19:0 cyclo w8c) were lost. It was found that the ratio of saturated/unsaturated fatty acids was significantly increased from 0.91 to 2.11.

Table 1.

Fatty acid composition of P. aeruginosa OG1 grown with and without toluene

Fatty acids (%)	Control	Toluene 0.1%	Toluene 0.2%	Toluene 0.3%
8:0	ND	ND	0.31	0.21
10:0 3OH	3.91	1.34	0.47	0.29
12:0	2.12	1.82	4.6	ND
12:0 2OH	2.32	2.65	5.01	ND
12:0 3OH	2.43	2.7	4.8	ND
14:0	0.22	0.27	4.21	ND
16:1 w7c	4.88	4.1	3.6	ND
16:0	34.01	36.15	47.84	67.2
17:0 cyclo	3.70	4.29	1.87	ND
18:1 w7c	36.1	33.5	24.7	31.9
18:0	0.93	0.68	0.52	0.35
19:0 cyclo w8c	8.84	12.35	2.29	ND
S/Uns	0.91	1.03	2.02	2.11

ND not determined, S saturated, Uns unsaturated

Discussion

The maximum pyocyanin production (8 mg/L) was measured after 72 h and further increase in incubation time did not enhance pyocyanin production (Fig. 1). It might be due to depletion of nutrients in fermentation medium. Pyocyanin is a bacterial secondary metabolite. It is well known that secondary metabolites produce during the end or near the stationary phase of growth (Kurbanoglu et al. 2015). The different optimal fermentation times reported in the literature might be due to the differences in the types of strain, composition of fermentation medium, and culture conditions (Lundgren et al. 2013). Özcan and Kahraman (2015) reported that 72 h was the optimal period for pyocyanin production (6.35 mg/L) by P. aeruginosa NRRL B-771.

Many organic solvents such as benzene, xylene, and toluene are highly toxic when added directly to the growth medium and kills most organisms at low concentrations (Li et al. 1995; Pacífico et al. 2018). OprF is the main porin-forming protein in the outer membrane of P. aeruginosa and provides the passage of hydrophilic solutions to the periplasm. Toluene passes through the OprF porin channel in the outer membrane to periplasm and kills the toluene-sensitive cells. When OprF is lost in the toluene resistance mutants, toluene cannot pass through the outer membrane (Li et al. 1995). Pyocyanin production may be reduced due to very low bacterial biomass at high toluene concentrations. These results indicate that P. aeruginosa OG1 is sensitive to toluene (Fig. 2a). It is also seen in Fig. 2 that the increase in the concentration of pyocyanin does not depend on the amount of biomass.

Interestingly, although surfactants are commonly known as potential cell-permeating agents (Wang et al. 2013; Byreddy et al. 2017), they have contributed to the production of less pyocyanin than organic solvents (Fig. 2b). Similar results were observed by Lim and Yun (2006), who found that toluene (0.3%, v/v) had a remarkable promoting effect on exopolysaccharide secretion by Collybia maculata TG-1, increasing the exopolysaccharide yields over twofold. To enhance synthesis of secondary metabolites, addition of surfactants or organic solvents to the fermentation production medium may be a potential strategy, since this strategy has proved to be feasible in many applications. The increase in yield may vary depending on the interaction between the added chemical and the microorganism. Many researchers have reported remarkable results about increased production of microbial metabolites such as enzyme, organic acid, pigment, and polysaccharide, using different types of stimulating agents (Table 2). The addition of 1.0% (v/v) Tween 80 to a liquid fermentation of Schizochytrium S31 increased the lipase activity (Byreddy et al. 2017). The maximum production of pigment by Monascus purpureus was increased significantly by 88.4% after the addition of Triton X-100 at 1.5% (Wang et al. 2013). He et al. (2016) found that Tween 80, acetone, and chloroform at 0.2% (v/v) concentration displayed a stimulatory effect on exopolysaccharide production by Lentinus tigrinus. Triton X-100 at 2.5% (w/v) exhibited a remarkable promoting effect on pigment (hypocrellin A) production of Shiraia bambusicola (Lei et al. 2017).

Table 2.

Effects of surfactants and organic solvents on microbial productions

Microorganism	Product	Stimulant—concentration (%)	Addition time (h)	Increased yield (%)	References
Collybia maculata	Exopolysaccharide	Toluene—0.3	108	86	Lim and Yun (2006)
Monascus purpureus	Pigment	Triton X-100—1.5	24	240	Wang et al. (2013)
Lentinus tigrinus	Exopolysaccharide	Acetone—0.2	196	107.2	He et al. (2016)
Yarrowia lipolytica	Erythritol	Span 20—0.025	0	15	Rakicka et al. (2016)
Aureobasidium pullulans	Pulluan	Tween 80—0.5	0	41	Sheng et al. (2016)
Rhodosporidium toruloides	Microbial lipid	Sodium lignosulfonate—0.2	0	15.7	Xu et al. (2016)
Shiraia bambusicola	Hypocrellin A	Triton X-100—2.5	36	55.9	Lei et al. (2017)
Schizochytrium S31	Lipase	Tween 80—1.0	0	ND	Byreddy et al. (2017)
P. aeruginosa OG1	Pyocyanin	Toluene—0.2	30	312	This study

ND not determined

Type, addition time, and concentration of stimulants have to be chosen carefully, as some of them are toxic to the microorganisms and affect production of metabolites (Table 2). Figure 3 results suggested that the optimal addition time of toluene was during the early and middle stages of the stationary phase. Lim and Yun (2006) reported that exopolysaccharide production in Collybia maculata TG-1 was enhanced by nearly 86% when 0.3% (v/v) toluene was added to the production medium at 108 h of fermentation (i.e., at the late growth phase). The optimum acetone (0.2%, v/v) addition time for the production of exopolysaccharide by Lentinus tigrinus was determined to be 196 h when exopolysaccharide production was measured after 288 h of growth (He et al. 2016).

When toluene was added to the fermentation medium, the production of pyocyanin significantly enhanced as it increased the activity of some enzymes (Fig. 4). Several researchers have succeeded in increasing enzyme production using organic solvents. Sumarsih et al. (2017) reported that addition of hydrocarbons (such as toluene and hexadecane) increased the oxygenase activity of bacteria. Gaur and Khare (2009) demonstrated that tetradecane, dodecane, isooctane, and heptane increased lipase production in Pseudomonas aeruginosa PseA. Thumar and Singh (2009) informed that the production of alkaline protease in Streptomyces clavuligerus was significantly increased by the addition of xylene benzene, butanol (0.1%, v/v), and acetone (0.3%, v/v). Alkaline protease is known to increase pyocyanin production (Liyama et al. 2017). In this study, it was found that addition of toluene to fermentation medium increased alkaline protease production (Fig. 6). Protease degrades protein to amino acids which are precursor (alanine, glutamic acid, proline, leucine, and isoleucine) of pyocyanin (Frank and DeMoss 1959).

Moreover, toluene has been reported to stimulate oxidative stress (Svenningsen et al. 2015). Jeong et al. (1999) reported that β-carotene synthesis was increased by oxidative stress in Blakeslea trispora. It is known that pyocyanin has antioxidant activity (Vinckx et al. 2010; Laxmi and Bhat 2016). In general, given the fact that pyocyanin serves as antioxidant protective against cellular damage by scavenging active oxygen species, it seems fairly possible that pyocyanin production would be enhanced by oxidative stresses.

Toluene could increase the contents of saturated fatty acids in bacterial membrane to alter membrane composition (Table 1). Santos et al. (2019) have explained an increase in saturated fatty acids of many Gram-positive and Gram-negative bacteria in the presence of organic solvents (ethanol, propanol, benzene, and toluene). This mechanism increase membrane rigidity to prevent the entry of toxic compounds into the cell (Torres et al. 2011). Some surfactants such as Tween 20, Tween 80, and Triton X‐100 could notably affect the fatty acid composition of microorganisms by remarkably increasing the ratio of unsaturation of the membrane lipids (Zhang and Cheung 2011; Lei et al. 2017). As understood from the above results, the increased membrane stiffness of P. aeruginosa did not reduce pyocyanin production. This can be explained by the fact that pyocyanin can easily cross biological membranes, since it has a low molecular weight and a zwitterion structure (Hall et al. 2016).

P. aeruginosa can produce different pigments such as pyocyanin, pyoverdine, pyomelanin, and pyorubin (Orlandi et al. 2015). Among them, pyocyanin is a blue color (Cheluvappa 2014). The retention time of pyocyanin extracted from supernatant was similar to the standard pyocyanin. Therefore, this study demonstrated that the extracted pyocyanin of bacteria is a pyocyanin pigment (Fig. 5).

Pyocyanin has a significant role as an electron shuttle in microbial fuel cells (MFC). It was observed that the addition of pyocyanin improved performance of MFCs (Wu et al. 2014). For this reason, the cost of adding pyocyanin can potentially be decreased by inoculating the pyocyanin producing P. aeruginosa as co-culture in the generation of electricity by toluene biodegradation in MFC.

Up till now, literature lacks reports on the production of pyocyanin by P. aeruginosa in the medium with the presence of surfactants and organic solvents. The results suggested that pyocyanin yield could be improved if a suitable concentration of toluene was added at cells stationary phase. Addition of toluene supported protease production to a greater extent, although the bacterial biomass was decreased in comparison to control media. Toluene can be used to damage the cytoplasmic membrane of microorganisms or to limit the growth of cells. This strategy can be used to increase the efficiency of many components (pigment, biosurfactant, enzymes, etc.) produced via fermentation.

Author contributions

All the experiments were designed and executed by MO.

Compliance with ethical standards

Conflict of interest

The corresponding author states that there is no conflict of interest.