Journal of Biotechnology, 12 (1989) 37-44 37 Elsevier

BIOTEC 00420

Biosurfactant production by a chloramphenicol tolerant strain of Pseudomonas aeruginosa *

Catherine N. Mulligan 1, Georges Mahmourides 2 and Bernard F. Gibbs 1

I National Research Council, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada and e University College of Cape Breton, Sydney, Nova Scotia B1P 6L2, Canada

(Received 5 December 1988; accepted 10 May 1989)

Summary

A strain of Pseudomonas aeruginosa ATCC 9027, var. RCII , produced biosurfac- tant in an inorganic phosphate-limited medium supplemented with chloramphenicol (150 /~g ml-1) . This surfactant reduced the surface tension of the culture super- natant to 29 m N m -1 and its concentration was 50 times the critical micelle concentration (CMC).

Several intracellular processes were monitored to correlate biosurfactant produc- tion with metabolic changes. In particular, biosurfactant production was preceded by phosphate depletion, followed by increased secretion of alkaline phosphatase (APase) and glutamate, and induction of transhydrogenase (PATH) and glucose-6- phosphate dehydrogenase (G6PD) activity. In the presence of chloramphenicol, a switch from amino acid catabolism to glucose metabolism (reverse diauxie) corre- lated with the onset of biosurfactant production by P. aeruginosa.

Intracellular enzyme; Biosurfactant production; Pseudomonas; Chloramphenicol; Rhamnolipid

Introduction

Although biosurfactants have many interesting properties (Cooper and Zajic, 1980; Wagner et al., 1981), their industrial importance is dependent upon ease of

Correspondence to: C.N. Mulligan, National Research Council, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada. * NRCC 30548

0168-1656/89/$03.50 © 1989 Elsevier Science Publishers B.V.

38

production (Cooper, 1986). Low yields of biosurfactant are a major factor jeopardiz- ing its popularity. Recently, efforts have been made to increase yields by focussing on nutritional and environmental factors (Cooper et al., 1981; Guerra-Santos et al., 1986).

A rhamnolipid-type biosurfactant produced by Pseudomonas aeruginosa has been reported as a function of growth on hydrocarbons and carbohydrates (Guerra-Santos et al., 1986; Hisatsuka et al., 1971; Itoh et al., 1970). The role of biosurfactants has been determined for production on hydrocarbons but not for carbohydrates. The metabolic processes associated with biosurfactant production need to be better understood.

Earlier studies with P. aeruginosa ATCC 9027 have demonstrated the importance of phosphate metabolism in biosurfactant production. To test further whether the phosphate metabolism aspect was particular to the strain or the genus, subsequent studies have focussed upon a chloramphenicol-tolerant strain of P. aeruginosa ATCC 9027 var. RCII. This paper reports on the shift in metabolic processes of P. aeruginosa ATCC 9027 var. RCII in relation to biosurfactant production in the presence of chloramphenicol.

Materials and Methods

Microorganism The chloramphenicol-tolerant strain used was P. aeruginosa ATCC 9027 var.

RCII. It was maintained at room temperature on Pseudomonas P agar (Difco) slants with 150/~g ml-1 of chloramphenicol and transferred monthly.

Media Two types of media were used: Kay's minimal medium (Warren et al., 1960)

supplemented with 150 btg m1-1 of chloramphenicol for preculture and proteose peptone-glucose-ammonium salts medium (PPGAS) supplemented with the same amount of chloramphenicol (Cheng et al., 1970) for biosurfactant production studies.

Cultivation conditions and sampling P. aeruginosa was inoculated into 100 ml of minimal medium (500 ml flask, 250

rpm shaking and 37 o C). After 24 h, 1 ml of the culture was transferred to PPGAS medium. Duplicate cultures were inoculated - one set as experimental, the other as backup. 13 ml fractions were removed each time for chemical analyses, the same volume being replenished from the backup culture.

Analytical methods Optical density (660 nm) and pH values were measured using standard labora-

tory equipment. Surface tension (Cooper et al., 1979) and critical micelle concentra- tions (CMC 1) (Sheppard and Mulligan, 1987) were determined as previously reported. Glucose (Sheppard and Mulligan, 1987) and inorganic phosphates (Waters,

39

1985) were determined by HPLC methods. Amino acid analyses were performed according to Spackman et al. (1958).

Preparation of cell extracts for enzymatic assays After 10 ml of sample were centrifuged (12 000 x g, 15 min, 4 ° C), the cell pellets

were washed three times (10 mM tricine, pH 7.6) and resuspended in buffer. The debris was removed by centrifugation (27 000 x g, 30 min, 4 ° C) after cell disruption by sonication (60 W, 5 x 1 rain periods) and the supernatant used for enzyme assays.

Chemicals Enzymes and substrates were purchased from Sigma Chemicals. All other re-

agent-grade chemicals were obtained from local supply houses.

Enzymatic assays Changes in optical density were monitored at 37°C. Alkaline phosphatase

(APase, EC 3.1.3.1) was assayed by the method of Neu and Heppel (1965). This assay was also performed on supernatants from media. The Ng and Dawes (1973) method was employed for glucose-6-phosphate dehydrogenase estimation (G6PD, EC 1.1.49). P. aeruginosa transhydrogenase (PATH) activities were assayed by the method of Wermuth and Kaplan (1976).

Protein measurement Protein estimation was done by the Lowry method (1951) with bovine serum

albumin as standard. To ensure complete solubilization of protein extracts, 1.0% SDS was added to the 0.2 M NaOH solution.

Results

P. aeruginosa ATCC 9027 var. RCII did not produce surfactant in either minimal or complex media. Both media are phosphate-sufficient. The phosphate-limited medium, PPGAS, induced surfactant production after 6 h of growth (Fig. 1). Initiation was accompanied by exhaustion of inorganic phosphate in the culture medium (Fig. 2), glucose consumption, induction of APase (Fig. 3) and G6PD (Fig. 4) and APase secretion (Fig. 2). These processes were followed by glutamate and glutamine secretion (Fig. 2).

The surface tension decreased until a value of 29 mN m-1 was reached (Fig. 1). A maximum inverse critical micelle concentration (CMC -1) value of 50 was obtained. At this point, glucose was exhausted, pH increased and the optical density of the medium began to decrease slightly.

The secretion of glutamine and glutamate occurred only during surfactant production (Fig. 2). Glutamate secretion was observed upon the initiation of glucose utilization (6 h). Glutamine secretion (10 h) occurred after maximum glutamate

40

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Fig. 1. Growth and surfactant production by P. aeruginosa var. RCI I in P P G A S medium with chloramphenicol. The following parameters were followed: optical density ( 0 ) , pH , (A) surface tension

(m) and C M C - 1 ( + ).

levels were reached (800 nmol ml-1). A maximum glutamine concentration of 400 nmol m1-1 was observed after 15 h of growth.

APase activity was seen in the culture supernatant and within the cells. Maxi- mum intracellular APase (100 nmol min -1 mg -1 protein) occurred after 10 h of growth (Fig. 3), whereas maximum extracellular activity (800 nmol min --1 m1-1) (Fig. 2) was seen at 7 h.

Maximum induction of G6PD, N A D P and N A D specific activities within the chloramphenicol-tolerant strain (Fig. 4) was prior to surfactant production. Maxi- mum levels for both enzymes after 5 -6 h of growth were 700 nmol min -1 mg -~

c

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Fig. 2. Levels of various components in the cell-free medium during growth. These include: inorganic 1)hosphate (m), APase ( + ) , and glutamic acid ( 0 ) and glutamine concentrations (A).

41

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o Bc

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Fig. 3. Intracellular enzymatic activities during growth. The monitored enzymes include: APase ( l ) , PATH 1 (A) and PATH 2 ( x ).

protein. The decrease in activities, beyond 6 h, coincided with decreases in surface tension and glucose concentration.

The overall P. aeruginosa transhydrogenase activity (PATH) in the chlor- amphenicol-tolerant strain was low (Fig. 3). Maximum PATH 1 activity (25 nmol min - ] mg - ] protein) was seen after 5 h of growth. This enzyme catalyses the oxidation of N A D P H and the reduction of NAD. After 6 h, PATH 2 which catalyses the reverse reaction dominated over PATH 1. This phenomenon was also observed in the wild type strain.

80C

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Fig. 4. In t race l lu la r G 6 P D activity during growth, NADP sp. ( , ) and N A D sp. ( + ) .

42

Discussion

Contrary to expectation, the presence of 150 t~g ml 1 of chloramphenicol enhanced biosurfactant production, The CMC 1 of the culture supernatant was increased from 30 for the wild type to 50 despite a 10% lower cell yield. This product yield is in general agreement with other authors (Guerra-Santos et al., 1986).

In addition, the production coincided with the onset of glucose utilization. During growth on PPGAS, P. aeruginosa undergoes two distinct types of metabo- lism: exponential growth linked with amino acid catabolism and stationary growth linked with glucose metabolism. This behaviour is known as reverse diauxic (Ham- ilton and Dawes, 1960). At this transition point, biosurfactant production was initiated.

Co-secretion of APase and biosurfactant occurred to a greater extent in the chloramphenicol-tolerant strain as compared to the wild type. Extracellular APase activity increased 4-fold whilst intracellular decreased 50%. Low intracellular APase levels in the chloramphenicol=tolerant strain resulted from partial repression and higher secretion levels. We observed that cell-free surfactant fractions displayed high APase activity. By affinity interaction, the surfactant may facilitate enzyme secretion through the cytoplasmic membrane.

The metabolic shift, described earlier, must result in enzyme activities coinciding with the induction of new enzymes. In this experiment, secretion of glutamate, glutamine and APase occurred simultaneously. Glutamine and glutamate secretion levels for the chloramphenicol-tolerant strain were 6-fold and 4-fold higher, respec- tively, than the wild type. Q6PD and PATH induction also occurred simultaneously. Biosurfactant production coincided with these processes. Such co-regulation has been previously described in the case of the synthesis by E. coli of an outer membrane protein 'e' (Tommassen and Lugtenberg, 1980) and an sn-glycerol-3- phosphate transport system (Argast and Boos, 1980) with APase secretion.

In P. aeruginosa, wild type and chloramphenicol-tolerant strains, the metabolic shift and biosurfactant production was under the influence of phosphate. Neither APase activity nor surfactant production occurred in phosphate-sufficient media. Phosphate metabolism plays an important role in surfactant production, APase induction and glucose metabolism.

References

Argast, M. and Boos, W. (1980) Co-regulation in Escherichia coli of a novel transport system for sn-glycerol-3-phosphate and outer membrane protein Ic (e,E) with alkaline phosphatase and phos- phate binding protein. J. Bacteriol. 143, 142-150.

Cheng, K.J., Ingram, J.M. and Costerton, J.W. (1970) Release of alkaline phosphatase of Pseudomonas aeruginosa by manipulation of cation concentration and pH. J. Bacteriol. 104, 748 753.

Cooper, D.G. (1986) Biosurfactants. Microbiol. Sci. 3, 145 149. Cooper, D.G., MacDonald, C.R., Duff, S.J.B. and Kosaric, N. (1981) Enhanced production of surfactin

from Bacillus subtilis by continuous product removal and metal cation additions. Appl. Environ. Microbiol. 42, 408-412.

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Cooper, D.G. and Zajic, J.E. (1980) Surface active compounds from microorganisms. Adv. Appl. Microbiol. 26, 229-256.

Cooper, D.G., Zajic, J.E. and Gerson, D.F. (1979) Production of surface active lipids by Corynebacterium lepus. Appl. Environ. Microbiol. 37, 4-10.

Guerra-Santos, L., K~ippeli, O. and Fiechter, A. (1986) Dependence of Pseudomonas aeruginosa continu- ous culture biosurfactant production on nutritional and environmental factors. Appl. Microbiol. Biotechnol. 24, 443-448.

Hamilton, W.A. and Dawes, E.A. (1960) The nature of the diauxic effect with glucose and organic acids in Pseudomonas aeruginosa. Proc. Biochem. Soc. Biochem. J. 76, 70.

Hisatsuka, K., Nakahara, T., Sano, N. and Yamada, K. (1971) Formation of rhamnolipid by Pseudo- monas aeruginosa and its function in hydrocarbon fermentation. Agric. Biol. Chem. 35, 686-693.

Itoh, S., Honda, H., Tomita, F. and Suzuki, T. (1970) Rhamnolipid produced by Pseudomonas aeruginosa grown on n-paraffin. J. Antibiot. 24, 855-859.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.G. (1951) Protein measurement with the Folin reagent. J. Biol. Chem. 193, 265-275.

Neu, H.C. and Heppel, L.A. (1965) The release of enzymes from Escherichia coli by osmotic shock and during formation of spheroplasts. J. Biol. Chem. 240, 3685-3692.

Ng, F.M.-W. and Dawes, E.A. (1973) Chemostat studies on the regulation of glucose metabolism in Pseudomonas aeruginosa by citrate. Biochem. J. 132, 129-140.

Sheppard, J.D. and Mulligan, C.N. (1987) The production of surfactin by Bacillus subtilis grown on peat hydrolysate. Appl. Microbiol. Biotechnol. 27, 110-116.

Spackman, D.H., Stein, W.H. and Moore, S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206.

Tommassen, J. and Lugtenberg, B. (1980) Outer membrane protein 'e' of Escherichia coli K-12 as co-regulated with alkaline phosphatase. J. Bacteriol. 143, 151-157.

Wagner, F., Bock, H. and Kretscher, A. (1981) In: Lafferty, R.M. (Ed.), Fermentation, Springer Verlag, Wien, pp. 181-192.

Waters Scientific Ltd. (1985) Waters Sourcebook for Chromatography Columns and Supplies, Cat. No. 07335, p. 38.

Warren, A.J., Ellis, A.F. and Campbell, J.J.R. (1960) Endogenous respiration of Pseudomonas aeruginosa. J. Bacteriol. 79, 875-880.

Wermuth, B. and Kaplan, W.O. (1976) Pyridine nucleotide transhydrogenase from Pseudomonas aeru- ginosa. Purification by affinity chromatography and physico-chemical properties. Arch. Biochem. Biophys. 176, 13-19.