Journal of Biotechnology, 12 (1989) 199-210 199 Elsevier

BIOTEC 00437

The influence of phosphate metabolism on biosurfactant production by Pseudomonas aeruginosa

Catherine N. Mulligan x, Georges Mahmourides 2 and Bernard F. Gibbs 1 1 National Research Council, Biotechnology Research Institute, 6100 Royalrnount Avenue, Montreal, Quebec

H4P 2R2, Canada and 2 University College of Cape Breton, Sydney, Nova Scotia BIP 6L2, Canada

(Received 14 November 1988; accepted 3 July 1989)

Summary

Three types of media were evaluated as substrates for the production of bio- surfactants by Pseudomonas aeruginosa ATCC 9027. An (inorganic) phosphate- limited medium, proteose pep tone /g lucose /ammonium salts, supported the best yield of biosurfactant. Under these conditions of growth, a surface tension of 29 mN m -] and a concentration of surfactant of approximately 30 times the critical micelle concentration were obtained.

In an effort to correlate changes in cell metabolism with the onset of surfactant production, the following enzymes were studied: transhydrogenase (PATH), glu- cose-6-phosphate dehydrogenase (G6PD) and alkaline phosphatase (APase). The results showed that biosurfactant production was induced during the shift in metabolism. In particular, the following events coincided with this process: deple- tion of phosphate, induction of APase activity and a decrease in PATH activity. In summary, it appears that a shift in phosphate metabolism coincided with biosurfac- tant production in P. aeruginosa.

Phosphate metabolism; Biosurfactant production; P s e u d o m o n a s aeruginosa; Rhamnolipid

Correspondence to: B.F. Gibbs, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada.

0168-1656/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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Introduction

Surfactants are potentially useful in virtually every industry dealing with multi- phase systems. They are used as adhesives, flocculating, wetting and foaming agents, de-emulsifiers and penetrants. Other uses include pulp and sludge de-watering and slurry stabilization. Demand is therefore high for surfactants. By 1990, 4.4 billion lb. of surfactants will have been consumed in North America, of which many will be hydrocarbon-based (Layman, 1985).

Biological surfactants, produced as metabolic by-products, are not only poten- tially as effective but offer some distinct advantages over the highly used synthetic surfactants. These include biodegradability and reduced toxicity. In general, they are grouped as glycolipids, phospholipids, fatty acids and neutral lipids, lipo- peptides and polysaccharides (Zajic and Steffens, 1974; Cooper, 1986; Parkinson, 1985).

Most biosurfactant studies have used bacteria grown on hydrocarbon substrates. Few workers (Suzuki et al., 1974; Cooper et al., 1981) have utilized carbohydrates. Production of glycolipids by Pseudomonas aeruginosa is well documented (Hitsatsuka et al., 1971). These surface-active compounds have been identified as rhamnolipids, R1 and R2; they can be produced from hydrocarbons or carbohydrates. The first contains two rhamnoses attached to fl-hydroxydecanoic acid, whereas R2 consists of one rhamnose connected to the identical hydroxyfatty acid. They have been pro- duced in the pilot plant (Reiling et al., 1985). However, the synthesis of these metabolites is not well understood, especially when glucose is the substrate.

Previous authors (Cooper et al., 1981; Guerra-Santos et al., 1986) have focussed upon environmental parameters (pH, temperature, aeration and nutrient require- ments). Unlike these studies, we have examined biosurfactant production in relation to metabolic processes. The secretion of cellular metabolites, enzymatic activities of cell extracts and biosurfactant production were followed during the course of growth of P. aeruginosa.

Materials and Methods

Microorganism The strain used was Pseudomonas aeruginosa ATCC 9027. It was maintained at

room temperature on Pseudomonas agar P (Difco) slants and transferred monthly.

Media Three types of phosphate media were used: (a) phosphate-buffered Kay's minimal

medium (Warren et al., 1960); (b) phosphate-sufficient nutrient broth (Difco); and (c) phosphate-limited proteose peptone/g lucose /ammonium salts medium (PPGAS) (Cheng et al., 1970).

Cultivation conditions All cultures were grown on a New Brunswick Instrument platform shaker, Model

G25, 250 rpm, 37 ° C. P. aeruginosa was inoculated into 100 ml of minimal medium

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(500 ml flask). After 24 h, 1 ml of the culture was transferred to 100 ml of each type of sterile medium.

Sampling Duplicate cultures were inoculated; one set would be designated as experimental,

the other as backup. 13-ml samples were removed - 10 ml for the cells, the determination of surface tension activity and critical micelle concentration (CMC) values and 3 ml for both optical density and pH measurement. 13 ml were replenished from the backup culture.

Analytical methods Optical densities were measured at 660 nm with a Bausch & Lomb Spectronic

2000 double beam UV spectrophotometer. Measurements of pH were performed using a Fisher Accumet pH meter. Surface tension of the supernatant was de- termined by the de Nouy method with a Fisher Tensiomat after cell removal by centrifugation (Beckman Centrifuge, Model no. J2-21M; JA-20 rotor, 12100 x g, 15 min, 4 ° C). A 2 ml portion of the supernatant was frozen ( - 7 0 o C) for later analyses for glucose, phosphate and amino acid concentration.

CMC values were determined by measuring the surface tension at various dilutions (Cooper et al., 1981). The logarithm of the dilution was plotted as a function of surface tension. The CMC is the point where surface tension abruptly increases. The reciprocal of the CMC thus increases with surfactant concentration and is an indication of relative concentration.

For emulsification tests, a volume of 4 ml of cell-flee supernatant was vortexed for 2 min with 6 ml of kerosene in a 12 mm diameter test tube and left for 24 h at ambient room temperature.

Glucose and inorganic phosphates were analyzed using a Waters high-perfor- mance liquid chromatograph (HPLC), equipped with a DEC Model 350 Computer. For the glucose analyses, a 250 × 4.6 mm stainless steel column packed with a Spherosorb aminobonded matrix was used. With a mobile phase of acetonitrile and water (85 : 15) and a flow rate of 0.7 ml min -1, glucose was detected by a Waters 401 differential refractometer.

Phosphates were detected by a Waters 430 conductivity detector (sensitivity 0.5 /~S); a mobile phase of 3 mM octane-sulfonic acid and 83.3/~M lithium hydroxide was used at a flow rate of 1.2 ml min -1. The analyses were performed with a Waters ICPAK A 50 × 4.6 mm anion column.

For the amino acid analyses, samples were kept frozen until used to minimize oxidation of glutamine to glutamic acid. A 50 /~1 aliquot was added to an equal volume of 20% trichloroacetic acid, vortexed and centrifuged in a bench top centrifuge for 3 min. The supernatant was diluted (1 : 1, v /v ) with Beckman lithium buffer and injected in a Beckman System 6300 high-performance analyser, equipped with a Beckman Model 7000 data station.

Preparation of cell extracts for enzymatic studies Of the 13 ml taken at each point of sampling, 10 ml were used for enzymatic

assays. After centrifugation of these 10 ml samples, the cell pellets were washed

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three times with 10 mM tricine (pH 7.6) prior to resuspension in 1.5 ml of buffer and immediate storage in a Revco ultra-low freezer at - 7 0 o C. These preparations were thawed on the day of assay and disrupted by sonication.

The sonication conditions were: ultrasonic processor and cell disrupter, Model W-375 (Heat Systems Ultrasonics Inc.), with microtip and maximum output control setting at 5 (60 W). The cell suspensions were subjected to five 1-min sonication periods with intervening 1 min cooling (in ice) periods.

After such disruption, cell debris was removed by centrifugation; Beckman centrifuge with JA-18.1 rotor, 27 000 × g, 30 min, 4 ° C. The supernatants of the cell extracts were used for the enzyme assays.

Chemicals Unless specifically listed, commercially available reagent-grade chemicals were

used. p-Nitrophenylphosphate, glucose-6-phosphate, NAD, NADP, reduced and oxidized forms, reduced thionicotinamide adenine dinucleotide (TNAD) and re- duced thionicotinamide adenine dinucleotide phosphate (TNADP) were purchased from Sigma Chemical Co. Buffers and ninhydrin reagent for amino acid analyses were purchased from Beckman.

Enzymatic assays Alkaline phosphatase (APase), glucose-6-phosphate dehydrogenase (G6PD) and

transhydrogenase activities were assayed using cell extracts. Changes in optical density were monitored at 37 ° C using a Beckman UV/VIS DU-7 scanning spectro- photometer.

APase, orthophosphoric acid monoesterase (EC 3.1.3.1) was assayed by the method of Neu and Heppel (1965) at 420 nm. The millimolar extinction coefficient (EmM) was 13.6. This assay was also performed on culture supernatants.

The Ng and Dawes method (1973) for G6PD (EC 1.1.49) was used. Both NAD and NADP specific activities were assayed (EmM = 6.22).

Pseudomonas aeruginosa transhydrogenase (PATH) activities were assayed by the method of Wermuth and Kaplan (1976) which is based on the absorption of the reduced TNADP and TNAD derivatives at 400 n m ( E m M = 11.3). For each 1 ml assay, the addition of 0.1 ml of 0.1 M Tris-HC1 (pH 7.4) and 0.1 ml of cell extract was required with either 0.3 ml of 0.1 mM NADPH 2 and 0.5 ml of 0.1 mM TNAD for PATH 1 or 0.3 ml of 0.1 mM NADH: and 0.5 ml of 0.1 mM TNADP for PATH 2.

Protein measurement The protein estimation method of Lowry et al. (1951) with bovine serum albumin

as protein standard was used. The 0.2 N NaOH solution contained 1.0% SDS to ensure complete solubilization of protein extracts.

Results

Three types of phosphate media were evaluated as possible substrates for the production of biosurfactants by P. aeruginosa ATCC 9027. Optical density (Fig.

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Fig. 1. The effect of three types of phosphate media on the growth of P. aeruginosa. The types of media included: minimal medium (11), nutrient broth (+) and PPGAS (0). (A) Optical density was monitored.

(B) pH w a s m o n i t o r e d .

1A), pH (Fig. 1B), surface tension (Fig. 2A) and C M C - 1 (Fig. 2B) were fol lowed for 24 h after inoculation. It was found that the ability of P. aeruginosa to produce biosurfactant and growth behavior were dependent on the type of phosphate medium used.

204

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Fig. 2. The effect of three types of phosphate media on biosurfactant production by P . a e r u g i n o s a . The types of media included: minimal medium (111), nutrient broth (+) and PPGAS (0) . (A) Surface tension

was followed. (B) CMC ~1 was followed.

205

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Fig. 3. Levels of various components in the cell-free broth during growth of P. aeruginosa . These include: inorganic phosphate (I), APase (+), glutarnic acid (0 ) and glutamine concentrations (A).

In phosphate-buffered minimal medium (37 000 nmol ml-1 Pi), the growth of P. aeruginosa was very slow; no significant change in pH was seen. A small temporary decrease (18 h) in surface tension (40 mN m -1) was seen during stationary growth phase.

In nutrient broth (Difco; 310 nmol m1-1 Pi), a phosphate-sufficient medium, a significantly higher growth rate and yield of P. aeruginosa did not support surfac- tant production. The pH of the medium increased throughout growth.

Using a phosphate-limited complex medium (65 nmol m1-1 Pi), PPGAS, ex- ponential growth was accompanied by a slight increase in surface tension (6 h). At 8 h of growth, surface tension was significantly reduced to 33 mN m-1 (Fig. 2A), indicating surfactant production. This event also coincided with the appearance of a blue pigment and acidification of the medium. With PPGAS, the curve of CMC- (Fig. 2B) showed significant biosurfactant accumulation above the CMC between 10 and 20 h (maximum CMC-1 = 30). The surface tension was 29 mN m-1.

Emulsification ability and interfacial tension determinations were undertaken to characterize the surface activity of the supernatants taken from the PPGAS medium after growth. Emulsification tests showed no significant ability of the medium to emulsify kerosene and water. However, determinations of the interfacial tensions of the supernatants against hexadecane were very low, 1.3 mN m -1.

Characterization of metabolite secretion and nutrient utilization during growth was carried out using the PPGAS medium (Fig. 3). As predicted, phosphate was indeed limiting. Complete depletion occurred at 7 h. Glucose, not shown on the graph, was not used until 6 h and was exhausted by 16 h. Glutamic acid (Fig. 3) was taken up during exponential growth. Such uptake was temporarily arrested as the cells entered late exponential growth phase (6 h). Thereafter, the excretion of

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Fig. 4. Intracellular enzymatic events during growth. The monitored enzymes included: (A) APase (I), G6PD (NADP sp.) (+) and (NAD sp.) (~); (B) PATH 1 (*) and PATH 2 ( x ).

207

glutamic acid coincided with the secretion of the intracellular enzyme, APase, into the medium. Glutamine, an intracellular metabolite, was subsequently secreted at 10 h. All these processes occurred simultaneously with the initiation of biosurfactant production (6-8 h).

The activities of intracellular enzymes were determined. The G6PD reaction initially favored a specific preference for NADP cofactor for the first 4 h (Fig. 4A). Thereafter, the enzymatic activity was greatest with NAD cofactor. Intracellular APase activity reached significant levels after 5 h of growth. From Fig. 3, we have already observed the initiation of the secretion of this enzyme into the medium at 6 h. In Fig. 4B, a dominance of PATH 1 over PATH 2 for the first 5 h of growth was shown. After 8 h, PATH 2 dominated. A distinct shift in metabolic behavior (4 to 8 h) was indicated by the components in the supernatant and the intracellular enzyme activities.

Discussion

Pseudomonas aeruginosa ATCC 9027 produced the highest yield of biological surfactant in the phosphate-limited PPGAS medium. Proteose peptone was the sole source of phosphate. Both nutrient broth and the minimal medium which contained substantially higher levels of inorganic phosphate were very poor media for bio- surfactant production. This evidence suggested that phosphate metabolism played an important role in biosurfactant production.

Hitsatsuka et al. (1971) showed that if hydrocarbons were used for the growth of P. aeruginosa, emulsifier was produced. They concluded that the rhamnolipid biosurfactant was the emulsifier. Later experiments (Hitsatsuka et al., 1972) re- vealed that a 'protein-like activator' mixed with the rhamnolipid enhanced emulsifi- cation. In the PPGAS medium, P. aeruginosa produced biosurfactant but not emulsifier. This was shown by Hitsatsuka et al. (1971) when a glucose substrate was used. It is evident that biosurfactants must be utilized for other purposes than to emulsify the carbon source.

To determine what physiological events coincide with biosurfactant production, the metabolic processes of the cells were monitored during growth in the PPGAS medium. Such information would be necessary for the optimization of the surfac- tant formation process. This approach differs markedly from other biosurfactant studies where only external changes were monitored. Here, metabolite secretion and intracellular enzyme activities were related to nutritional conditions and biosurfac- tant secretion.

From the analyses of extracellular components, an indication of cell processes was gained. The secretion of APase (4-6 h) and a characteristic blue pigment, reduced uptake of glutamic acid (4 h), increased uptake of glucose (6 h) and the delayed excretion of glutamine coincided with the initiation of surfactant produc- tion (6 h). A common link to all these metabolic processes was the availability of Pi- APase activity was induced upon phosphate limitation in the cell and was subse- quently secreted into the medium (Cheng et al., 1970). Phosphate depletion initiated

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the formation of glutamine from ammonium and glutamate so that Pi could be obtained from ATP.

A few key enzymes involved in phosphate metabolism were assayed to correlate the aforementioned extracellular changes in the medium with intracellular processes. Transhydrogenase (PATH) studies monitored the shifting cofactor preferences of P. aeruginosa during growth. PATH 1 catalyzed the oxidation of N A D P H and the reduction of NAD. The PATH 2 reaction proceeded in the opposite direction (Widmer and Kaplan, 1977). Glucose metabolism was followed by G6PD which catalyses the phosphorylation of glucose to glucose,6-phosphate before oxidation. Glucose phosphorylation reactions are known to be responsible for phosphate depletion (Ng and Dawes, 1973). Intracellular APase was also followed.

The following observations of the metabolic behavior of P. aeruginosa were made. Transhydrogenase activity with the active form (PATH 1) predominated in the first hours of growth. This reaction was necessary to provide G6PD activity with a sufficient supply of NADP. G6PD activity demonstrated an initial preference for the N A D P cofactor. After 12 h, the preference was for the N A D cofactor. Accordingly, transhydrogenase activity was reversed; PATH 2 now produced N A D and NADPH. Surfactant production was induced at this point of transition. The transition from PATH 1 to PATH 2 was influenced by the availability of inorganic phosphate (Widmer and Kaplan, 1977) brought on by the glucose phosphorylat ion step.

Biosurfactant production, in this case unrelated to substrate emulsification, could serve another purpose. It can function as a solubilizer for hydrophobic proteins (such as APase), facilitating their movement across the hydrophobic cell membrane into the medium.

In conclusion, this study related cell metabolic processes with biosurfactant production. Phosphate metabolism played an important role. Other limitations in the medium could be equally important and are under current investigation.

Acknowledgement

The authors wish to thank Bivan Consultants Inc. (Montreal, Quebec) for their continuing support throughout the course of these studies.

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