APPLIED AND ENVIRONMENTAL MICROBIOLOGY, NOV. 1989, p. 3016-3019
`0099-2240/89/113016-04$02.00/0
`Copyright © 1989, American Society for Microbiology
`
`Vol. 55, No. 11
`
`Correlation of Nitrogen Metabolism with Biosurfactant Production
`by Pseudomonas aeruginosaf
`
`CATHERINE N. MULLIGAN1 AND BERNARD F. GIBBS2*
`Biochemical Engineering1 and Protein Engineering2 Sections, Biotechnology Research Institute, National
`Research Council, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2
`
`Received 10 July 1989/Accepted 3 August 1989
`
`A direct relationship between increased glutamine synthetase activity and enhanced biosurfactant production
`was found in Pseudomonas aeruginosa grown in nitrate and Proteose Peptone media. A chloramphenicol-
`tolerant strain showed a twofold increase in biosurfactant production and glutamine synthetase activity.
`Increased ammonium and glutamine concentrations repressed both phenomena.
`
`Pseudomonas aeruginosa produces two types of rhamno-
`lipid-type biosurfactants, R1 and R2, on either hydrocarbon
`or carbohydrate media (6). R1 contains two rhamnoses
`attached to p-hydroxydecanoic acid, whereas R2 consists of
`one rhamnose unit connected to the same hydroxy-fatty
`acid. Higher yields of these rhamnolipids must be realized to
`stimulate commercial interest. Although previous authors (5)
`have examined the effects of ammonium and nitrate as
`sources of nitrogen, the role of amino acids has not been
`investigated. Pseudomonads utilize nitrates, ammonia, and
`amino acids as nitrogen sources. Nitrates must be reduced to
`nitrite and then ammonia (9). Ammonia can then be assimi­
`lated either by glutamate dehydrogenase (GDH; EC 1.4.1.4)
`to form glutamate (2) or, with glutamate, by glutamine
`synthetase (GS; EC 6.3.1.2) to form glutamine. Glutamine
`and a-ketoglutarate are then converted to glutamate by
`L-glutamine 2-oxoglutarate aminotransferase (GOGAT; EC
`1.4.1.13) (2, 7).
`This paper describes the relationship of the activities of
`these enzymes to biosurfactant production with various
`nitrogen sources. Two strains of P. aeruginosa, a wild type,
`ATCC 9027, and a chloramphenicol-tolerant strain, ATCC
`9027 var. RCII, are compared. The latter strain, derived
`from the wild type, was chosen for its ability to tolerate large
`doses of the antibiotic chloramphenicol (8b). The strains
`were maintained on Pseudomonas agar P (Difco Laborato­
`ries). The chloramphenicol-tolerant strain was isolated by
`repeated subculturing of the wild type in the presence of
`increasing amounts of chloramphenicol until a concentration
`of 150 |Ag/ml was achieved.
`Kay minimal medium (10) was used for preculture, and
`Proteose Peptone-glucose salts (PPGS) medium (4) was used
`for biosurfactant studies. Nitrogen sources were varied for
`each experiment. Chloramphenicol (150 |xg/ml) was added
`for experiments with the tolerant strain to maintain toler­
`ance. All batch cultures were grown in a stirred-tank CHE-
`MAP 3.7-liter fermentor under the following conditions: 30 h
`of incubation, agitation rate of 100 rpm, 15 ml of inoculum,
`aeration at 3.5 liters/min, 370C, 1.5-liter working volume,
`and pH of 7.0. A CHEMAP Fundafom mechanical foam
`breaker was used. Growth was monitored turbidimetrically
`at 660 nm. One optical density unit is equivalent to 0.185 g
`
`Corresponding author.
`t NRCC paper no. 30698.
`
`(dry weight) of cells per liter. The surface tension, critical
`micelle concentration (CMC), and glucose, nitrate, and
`ammonium analyses were determined as described by de-
`Roubin et al. (4a ). P; and amino acid analyses and prepara­
`tion of cell extracts by sonication for enzymatic assays were
`performed by the method of Mulligan et al. (8b).
`Commercially available reagent-grade chemicals were ob­
`tained from local supply houses. All enzymes and substrates
`were purchased from Sigma Chemical Co. (St. Louis, Mo.).
`All enzyme assays were carried out at 370C. GS activity is
`expressed in terms of nanomoles of 7-glutamyl hydroxamate
`formed per minute per milligram of protein (7). GDH and
`GOGAT were assayed spectrophotometrically by measuring
`NAD(P)H oxidation (7). Protein estimation was done by the
`Lowry method (8), with bovine serum albumin as standard.
`The effect of a nitrogen source on surfactant production by
`P. aeruginosa was investigated in PPGS medium for both
`
`TABLE 1. Effect of nitrogen source on enzyme activities
`and biosurfactant production by P. aeruginosa
`
`Strain and
`medium
`
`ATCC 9027
`20 mM NH4+ +
`PPGS
`20 mM NCV +
`PPGS
`PPGS
`
`ATCC 9027 var.
`RCII
`20 mM NH4+ +
`PPGS
`20 mM N(V +
`PPGS
`PPGS
`6 mM glutamine
`+ PPGS
`
`Enzyme activity
`(nmol/min per mg of protein)
`
`GS"
`
`GDH
`(NADP)
`
`GDH
`(NAD)
`
`GOGAT
`
`CMC-10
`
`' 272
`
`362
`
`1,199
`
`705
`
`859
`
`2,468
`139
`
`61
`
`16
`
`8
`
`34
`
`61
`
`32
`9
`
`15
`
`25
`
`30
`
`6
`
`5
`
`8
`9
`
`16
`
`33
`
`13
`
`18
`
`38
`
`39
`37
`
`8
`
`10
`
`13
`
`15
`
`17
`
`25
`2
`
`" The CMC is determined by measuring the surface tension of the medium
`at various dilutions, and the logarithm of the dilution is plotted as a function
`of surface tension. The CMC is the point of abrupt surface tension increase,
`and CMC-1 increases with surfactant concentration and is an indication of
`relative concentration.
`b Total GS activity.
`
`3016
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`EXHIBIT NO. 1027 Page 1 of 4
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`VOL. 55, 1989
`
`NOTES
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`FIG. 1. Growth and biosurfactant production by P. aeruginosa var. RCII. Symbols: •, optical density; •, surface tension; +, CMC ';
`• , GS activity.
`
`strains (Table 1). The production of biosurfactant was high­
`est when Proteose Peptone was the sole nitrogen source. In
`the supplemented media, ammonium ions and nitrates were
`used at slightly higher rates by the chloramphenicol-tolerant
`strain than by the wild type (22 and 40%, respectively,
`compared with 7 and 10% for the wild type). The preferred
`nitrogen source was glutamic acid from
`the peptone (80%
`utilization). Growth without Proteose Peptone was very
`poor (optical density of <0.3).
`
`The activities of enzymes involved in nitrogen metabolism
`were examined to deduce a regulatory role during rhamno-
`lipid production (Table 1). All results shown are for single
`experiments. All assays were performed in duplicate and
`were reproducible to ±5%. For both strains, total GS
`activity was significantly higher in PPGS medium. In the
`nitrate medium, there was more GS synthesis than in the
`ammonium medium. GDH (NADP) repression was also
`evident. GOGAT activity did not differ significantly in the
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`FIG. 2. Concentration of medium components during the growth of P. aeruginosa var. RCII. They include phosphate (+), glutamic acid
`(A), glucose (•), and ammonium (•).
`
`PETITIONERS
`
`EXHIBIT NO. 1027 Page 2 of 4
`
`

`

`3018
`
`NOTES
`
`APPL. ENVIRON. MICROBIOL.
`
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`FIG. 3. Intracellular enzyme activities of P. aeruginosa var. RCII during growth. Enzymes include GDH (NADP) ( •). GDH (NAD) (+),
`and GOG AT (•).
`
`various media. GDH activity (NAD specific) was evident in
`all types of media but was not as affected by the medium as
`was the NADP-dependent form. The addition of glutamine,
`an end product in the GS reaction, inhibited both GS activity
`and biosurfactant production by the tolerant strain. Glu­
`tamine was taken up quickly (4 h) by the bacteria. It has been
`reported that an elevated amount of glutamine has a detri­
`mental effect On GS activity in Klebsiella aero genes (1).
`In PPGS medium, as the growth of P. aeruginosa var.
`RCII slowed (Fig. 1), surface tension started to decrease (at
`7 h) until a value of 28 mN/m was reached. Subsequently, the
`CMC-1 and GS activity increased significantly. Similar
`trends were observed for the wild type, with a reduction of
`50% for both phenomena. Ammonium and phosphate levels
`(Fig. 2) were depleted at 6 h. After an initial increase in free
`glutamic acid concentration, significant amounts were uti­
`lized as the growth rate increased (4 h). As the growth rate
`slowed, however, excess glutamic acid was secreted into the
`medium (9 h) and there was simultaneous glucose consump­
`tion. Carbon was not exhausted until 15 h.
`Both GDH (NADP) and GOGAT reached maxima at 7 h
`and then declined (Fig. 3). Increased GS levels (Fig. 1)
`coincided with decreased GDH (NADP) activity, indicating
`a preference for the GS-GOGAT pathway over the GDH
`pathway during biosurfactant production. This condition can
`be obtained in an organic nitrogen medium with a small
`amount of ammonium ions and by the specific selection of
`strains with elevated GS activity. NAD-dependent GDH
`increased as the glutamic acid levels decreased. During
`rhamnolipid biosynthesis, lipid, not sugar, formation is the
`rate-determining factor (3). Nutrient limitation (via nitrogen)
`may promote lipid accumulation.
`
`In this paper, we have shown with both strains that GS
`activity and biosurfactant production are regulated by gluta-
`mate, glutamine, and ammonia levels. As growth slows,
`glutamate and ammonia are utilized while GS activity in­
`creases. Glutamine inhibits this process. Cell metabolism is
`then switched from nitrogen as it becomes limiting (glutamic
`acid) to glucose, resulting in rhamnolipid production.
`To produce biosurfactants economically, increased yields
`are necessary. We have recently shown that a mutant of
`Bacillus subtilis could produce significantly higher amounts
`of biosurfactant than the wild-type strain (8a). In the present
`case, since a quick method for obtaining rhamnolipid pro­
`ducers is not known, screening for mutants of P. aeruginosa
`which have increased levels of GS activity could lead to
`enhanced production of biosurfactants and promote com­
`mercial interest.
`We thank Bivan Consultants, Inc., Montreal, Quebec, for its
`continuing support throughout the course of these studies (grant
`6238).
`
`LITERATURE CITED
`1. Bender, R. A., and B. Magasanik. 1977. Regulatory mutations in
`the Klebsiella aerogenes structural gene for glutamine syn­
`thetase. J. Bacteriol. 132:100-105.
`2. Brown, C. M., D. S. Macdonald, and S. O. Stanley. 1972.
`Inorganic nitrogen metabolism in marine bacteria: nitrogen
`assimilation in some marine pseudomonads. J. Mar. Biol. As­
`soc. U.K. 52:793-804.
`3. Boulton, C. A., and C. Ratledge. 1987. Biosynthesis of lipid
`precursors to surfactant production, p. 47-87. In N. Kosaric,
`W. L. Cairns, and N. C. C. Gray (ed.), Biosurfactants and
`biotechnology. Marcel Dekker, Inc., New York.
`4. Cheng, K.-J., J. M. Ingram, and J. W. Costerton. 1970. Release
`
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`EXHIBIT NO. 1027 Page 3 of 4
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`VOL. 55, 1989
`
`NOTES
`
`3019
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`of alkaline phosphatase from cells of Pseudomonas aeruginosa
`by manipulation of cation concentration and of pH. J. Bacteriol.
`104:748-753.
`4a.de Roubin, M. R., C. N. Mulligan, and B. F. Gibbs. 1989.
`Correlation of enhanced surfactin production with decreased
`isocitrate dehydrogenase activity. Can. J. Microbiol. 35:854-
`859.
`5. Guerra-Santos, L. H., O. Kappeli, and A. Fiechter. 1986. De­
`pendence of Pseudomonas aeruginosa continuous culture bio-
`surfactant production on nutritional and environmental factors.
`Appl. Microbiol. Biotechnol. 24:443^148.
`6. Hitsatsuka, K., T. Nakahara, N. Sano, and K. Yamada. 1971.
`Formation of rhamnolipid by Pseudomonas aeruginosa and its
`function in hydrocarbon fermentation. Agric. Biol. Chem. 35:
`686-692.
`7. Janssen, D. B., H. J. M. Op Den Camp, P. J. M. Laenen, and C.
`
`Van Der Drift. 1980. Enzymes of the ammonia assimilation of
`Pseudomonas aeruginosa. Arch. Microbiol. 124:197-203.
`8. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.
`1951. Protein measurement with the Folin phenol reagent. J.
`Biol. Chem. 193:265-275.
`Sa.Mulligan, C. N., T. Y.-K. Chow, and B. F. Gibbs. 1989.
`Enhanced surfactin production by a Bacillus subtilis mutant.
`Appl. Microbiol. Biotechnol. 31:486-489.
`8b.Mulligan, C. N., G. Mahmourides, and B. F. Gibbs. 1989.
`Biosurfactant production by a chloramphenicol-tolerant strain
`of Pseudomonas aeruginosa. J. Biotechnol. 12:37-44.
`9. Painter, H. A. 1970. A review of the literature on inorganic
`nitrogen metabolism in microorganisms. Water Res. 4:392-450.
`10. Warren, R. A. J., A. F. Ells, and J. J. R. Campbell. 1960.
`Endogenous respiration of Pseudomonas aeruginosa. J. Bacte-
`riol. 79:875-879.
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