Journal of Biotechnology, 13 (1990) 257-266 257 Elsevier

BIOTEC 00454

Microbial glycolipid production under nitrogen limitation and resting cell conditions

J in -Seog K i m 1, Michae l Powal la a, S i egmund L a n g a, Fr i tz W a g n e r a, He in r i ch L i in sdor f 2 a n d Vic tor W r a y 2

J Institut )e~r Biochemie und Biotechnologie, Technische Universitgit, and 2 Gesellschaft f~r Biotechnologische Forschun~ D-3300 Braunschweig~ F.R. G.

(Received 6 July 1989; accepted 16 September 1989)

Summary

Rhodococcus erythropolis is able to synthesize an anionic trehalose-2,2',3,4-tetra- ester during cultivation on n-alkanes. Preconditions for an overproduction are nitrogen limitation, temperature- and pH-shift. The opt imum carbon source was technical grade n-C-10, which led to 0.35 g g- i of glycolipid per n-alkane. Electron microscopical observations showed that n-C-14,15 (technical grade) grown cells contained numerous lipid inclusions in contrast to n-C-10 (technical grade) grown cells. Nocardia corynebacteroides synthesizes a novel pentasaccharide lipid and as side products small amounts of trehalose-corynomycolates. Opt imum precursors for overproduction are n-alkanes from n-tetradecane to n-hexadecane with yields in the range of 0.17 g g- i of glycolipid per carbon source.

n-Alkanes utilizing bacteria; Glycolipid; Trehalose-tetraester; Pentasaccharide lipid; Lipid inclusion

Introduction

Some microorganisms are able to overproduce nonionic and ionic surfactants. These substances are of interest as wetting, foaming and emulsifying agents in the

Correspondence to: Dr. Siegmund Lang, lnstitut fiir Biochemie und Biotechnologie, Technische Uni- versit~it, Konstantin-Uhde-Str. 5, D-3300 Braunschweig, F.R.G.

Presented at the 2nd International Symposium on Overproduction of Microbial Products, held in (~eske Bud~jovice, Czechoslovakia, 3-9 July, 1988.

0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

258

field of, for instance, environmental protection of coastal areas and in the cosmetic industry. They are synthesized during the cultivation on various carbon sources, in particular during growth on lipophilic substrates. In some cases they seem to facilitate the uptake of water insoluble n-alkanes. Among the biosurfactants known in the literature, the glycolipids are important compounds as most of them can be overproduced to give a final yield of 0.1 to 0.8 g g-1 of carbon source. Examples are the nonionic and anionic trehalose lipids of Rhodococcus erythropolis (Rapp et al., 1979; Kretschmer et al., 1982; Ristau and Wagner, 1983), the rharrmose lipids of Pseudomonas sp. (Syldatk et al., 1985), the sophorose lipids of the yeast Torulopsis bombicola (Cooper and Paddock, 1984; Asmer et al., 1988; Inoue, 1988) and the anionic cellobiose lipids of Ustilago maydis (Frautz et al., 1986).

Other biosurfactants are lipopolysaccharides, lipids with one or more amino acids (lipopeptides, lipoproteins), and the usual phospholipids and triglycerides of all microorganisms, respectively.

Our intention in this work was to improve the production conditions of known glycolipids as well as to search for novel microbial surfactants.

Materials and Methods

Chemicals

Mihagol L and S were purchased from Wintershall A G (Kassel, F.R.G.). Mihagol L: n-C-10 (86%), n-C-11 (11.5%), n-C-12 (2.4%). Mihagol S: n-C-10 (6%), n-C-14 (76%), n-C-15 (11%), n-C-16 (6%) (values after distillation).

Bacteria

Rhodococcus erythropolis DSM 43215 and Nocardia corynebacteroides SM 1 were isolated from oil-contaminated soil.

Growth conditions

Both strains were maintained on agar slants at 4 ° C and transferred at one-month intervals.

For Rhodococcus erythropolis the slants were incubated for 2 d at 30 ° C. After inoculation 100 ml of shake flask cultures were shaken at 100 rpm and 30 ° C for 2 d (Mihagol S) or 5 d (Mihagol L), respectively; 25 ml of this preculture I were added to 250 ml of fresh medium and this (preculture II) was incubated for 3 d (Mihagol S) or 7 d (Mihagol L), respectively. The p H was adjusted semi-continuously.

The reaction mixtures of eight 250 ml-cultures were combined and transferred to 18 1 of the main culture (N limitation) in a 20-1 bioreactor (b 20, Giovanola Fr6res SA, Monthey, Switzerland), equipped with an intensor system. Conditions: 1000- 1500 rpm; aeration rate 0.5 to 1.0 ( v / v / m ) ; pH 7, T = 30 °C; after ammonium consumption: shifts to p H 6 to 6.5, T = 22 ° C.

259

Media (g l- 1) (a) Agar slant: as described by Rapp et al. (1979). (b) Preculture I: similar to (a) but Mihagol L or S as carbon sources (1-2%). (c) Preculture II: KH2PO4, 0.5; K 2 H P O 4 • 3 H20, 1.0; N a 2 H P O 4 • 2 H20, 0.5;

MgSO 4 • 7 H20, 0.1; KC1, 0.1; trace element solution, 0.5 ml 1-1 (composition: as described by Rapp et al., 1979); Mihagol L, 30.0, or Mihagol S, 60.0; yeast extract, 1.0 or 5.0; p H 7.

(d) Main culture: yeast extract, 1.0; citric acid. H20, 1.24; (NH4) 2 SOn, 4.71; K H 2 P O 4, 0.5; N a 2 H P O 4 • 2 H20, 0.5; H3PO 4 (85%), 1.0 ml 1-1; CaC12 • 2 H 2 0 , 0.05; FeC13 • 6 H20, 0.02; MgSO 4 • 7 H 2 0 , 1.1; FeSO 4 • 7 H20, 0.2; Mihagol L or S, 100.0; pH 7.

Resting cell conditions At first Rhodococcus erythropolis was cultivated on a nitro- gen-saturated medium similar to the above main culture, with the following excep- tions: yeast extract, 10.0; (NH4)2SO 4, 15.0; Mihagol L or S, 20.0; incubation time 72 h.

After continuous centrifugation at 17,000 rpm (Cepa Z 41, Padberg, Lahr, F.R.G.) 800 g of wet biomass (corresponding to 160 g dry weight) were transferred to 16.4 1 of a reaction medium consisting of 0.1 M phosphate buffer, pH 6.5, and 80 g 1-1 of Mihagol L or S. Conditions: 20-1 bioreactor (see above); 1000-1500 rpm; aeration rate 0.5 to 1.0 ( v / v / m ) ; pH 6 to 6.5; T = 22°C.

For Nocardia corynebacteroides the slants were incubated for 3 d at 27 o C and the 100 ml precultures for 48 h at 100 rpm and 22 ° C. Then 15 ml were added to 500 ml of the main culture in 2-1 shake flasks, which were shaken for a period of 144 h. The p H was adjusted at 24-h intervals.

Media (g l- 1) (a) Agar slant: Standard I-nutrient broth (E. Merck, Darmstadt , F.R.G.). (b) Pre- and main culture: N a N O 3, 2.0; KH2PO 4, 1.0; N a 2 H P O 4 • 2 H20, 2.5;

FeC13 • 6 H20, 0.13; MgSO 4 • 7 H20, 0.75; CaCI 2 • 2 H20, 0.75; MnSO 4 • H20, 0.2; ZnSO 4 • 7 H20, 0.02; yeast extract, 3.0; n-alkanes C-9 to C-18, 20.0; pH 6.6.

Analytical methods

Biomass, n-alkane, glycolipid content and composition (TLC) and structure (1H and 13C NMR, G C coupled with MS) (Asmer et aL, 1988), ammonium ions (Fawcett and Scott, 1960), nitrate ions (enzymatic kit from Boehringer, Mannheim, F.R.G.) were determined by methods described previously.

Isolation and purification of glycolipids At the end of the cultivation the reaction mixture of the bioreactor or of a shake

flask was extracted twice with d ichloromethane/methanol (2/1, v / v ) or ethyl acetate. After evaporation of the solvent the crude products were separated by the method of Asmer et al. (1988), initially using medium pressure liquid chromatogra- phy with ch lo ro form/methano l mixtures as developing system (100/0, v /v , to 60/40; 5% steps) and then thick layer chromatography.

260

Alkaline hydrolysis of the glycolipids for removal and determination of fatty acids was carried out by refluxing with 1 N ethanolic N a O H solution.

Acidic hydrolysis of the oligosaccharide (after alkaline desacylation of the native glycolipid of Nocardia corynebacteroides) was performed by stirring in 0.5 N H2SO 4 at 70 °C for 4 h under a nitrogen atmosphere.

Electron microscopy After conventional fixation with aqueous 1% OsO 4 solution (w/v) , removal of

water, embedding in epoxy resin (Spurr, 1969) and staining by means of uranyl acetate and lead citrate (Reynolds, 1963), ultra-thin sections (120 to 150 nm) of cells of Rhodococcus erythropolis were studied with a transmission electron microscope 10-B (Zeiss, Oberkochen, F.R.G.).

For freeze fracture studies the cells were frozen in Freon 22 at -165 ° C, fractured at - 1 0 3 ° C and sublimated at 1 × 10 -7 mbar (Biotech 2000, Leybold Hereaus, K~51n, F.R.G.). P t / C replicas were purified in hypochloric acid.

Surface tension was measured using the ring method in a Lauda Tensiomat (Lauda-Wobser, K~Snigshofen, F.R.G.) after previous emulsification of the glyco- lipid by ultrasonic treatment in water. Interracial tension was measured in a similar way against n-hexadecane.

Results

Trehalose-tetraester production by Rhodococcus erythropolis

Ten years ago the bacterium Rhodococcus erythropolis was found to synthesize certain lipids with surfactant properties when grown on n-alkanes. Under nitrogen saturation conditions t~, a-trehalose-6, 6 '-dicorynomycolates and a, a-trehalose-6- monocorynomycolates were the major components (Rapp et al., 1979; Kretschmer et al., 1982). In later experiments both nitrogen deficiency and a temperature shift from 30°C to 22°C at the beginning of N limitation caused the formation of a novel anionic a, t~-trehalose-2, 2' , 3, 4-tetraester (Ristau and Wagner, 1983). Using technical grade n-C-14,15 mixtures as carbon source two decanoyl, one octanoyl and one succinoyl residues were detected as dominant acyl donors for the disaccharide. In 50-1 bioreactor cultivations 8 to 9 g 1-1 of trehalose lipids (trehalose-tetraester: 90%, trehalose-corynomycolates: 10%) were produced (Ristau, 1983).

Continuing these studies we found initially that a p H shift from 7 to about 6 to 6.5 improved the synthesis. Then we examined the substrate specificity with regard to a higher overproduction of the biosurfactant. On water soluble substances such as glucose or ethanol although microbial growth was observed, production was zero or low. Vegetable oils were also unsuitable. On testing pure n-alkanes we observed that a chain length of 10 to 14 carbon atoms affected an overproduction in the range of more than 2 g 1-1; n-decane led to the highest value of 4 g 1-1. As there was a possible technical application of our microbial process the influence of two techni- cal grade hydrocarbons was also investigated. The result of these shake flask

261

30

5 q 25 ~ Shift FI ~ r ~ J _.__.__D

2

o:

F--

0 40 8'0 150 160

INCUBATION TIME (h)

100

i

60 ..~ IM

40 2: <

20 ~ <

0

6"7

4 ~ <

2 E

0 m 2OO

Fig. 1. Growth and glycolipid formation of Rhodococcus erythropolis on 10% technical grade n-C-10 (Mihagol L). Conditions: 20-1 bioreactor; mineral salts medium; T = 30 o C and pH 7 at the beginning,

later T = 22°C, pH 6.5.

experiments indicated that technical grade Mihagol L (mainly n-C-10) was superior to technical grade Mihagol S (mainly n-C-14,15).

Scaling up to a 20-1 bioreactor and improvement of the cultivation conditions such as oxygen supply and maintenance of pH value, the yield of trehalose lipids reached 32 g 1 -~ after 160 h when 100 g 1-1 of Mihagol L were used as carbon source (see Fig. 1). This cultivation also demonstrates that the overproduction of the trehalose-tetraester begins after the exponential phase of growth following the nearly total consumption of ammonium ions and after the temperature and pH shift. In Fig. 2 the influence of both technical grade n-alkanes on the glycolipid formation is compared. As shown Mihagol S finally led to only about 8 g 1-1. Furthermore, the values of the specific glycolipid production related to cell dry

r-',

d h i t/} o < T

I . - -

35

30-

25-

20-

15-

10-

5-

0 O

w ~

40 e o 15o 16o 200 T I M E ( h )

Fig. 2. Trehalose-2,2',3,4-tetraester production by Rhodococcus erythropofis on technical grade n-alkanes under N limitation. Conditions: 20-1 bioreactor; mineral salts medium; T = 3 0 ° C and pH 7 at the

beginning, later T = 22 ° C, pH 6.5. A,B: Mihagol S-cells; C,D: Mihagol L-cells.

262

30

25 ̧

~-' 2 0 ̧

o_ 15

o

~o.

5-

0 0 5 0 100 150

T i m e (h)

2 0 0

Fig. 3. Production of trehalose-2,2',3,4-tetraester with resting cells of Rhodococcus erythropolis in a 20-1 bioreactor. Conditions: 0.1 M phosphate buffer, pH 6.5, T = 2 2 ° C . ( o ) Mihagol S-grown cells+8%

Mihagol S (e) Mihagol L-grown cells + 8% Mihagol L.

weight, were higher in the case of the shorter chain substrate: 4 and 1 g g-~ per biomass, respectively. Related to Mihagol L consumption, the specific glycolipid production was 0.35 g g-1.

As the overproduction began at a low ammonium concentration the complete separation of growth and production phase was attempted. In resting cell experi- ments using a phosphate buffer solution and 80 g 1-1 of hydrocarbons, Mihagol S (n-C-14,15) grown cells converted Mihagol S to 7 g 1-1 trehalose-tetraester whereas Mihagol L (n-C-10) grown cells transformed Mihagol L to more than 20 g 1-1 biosurfactant (see Fig. 3). Crossover experiments with Mihagol L grown cells and Mihagol S as substrate for conversion and vice versa led to lower amounts.

By means of electron microscopy the effects of both hydrocarbon mixtures on the cell morphology were studied. As shown in Fig. 4, the thin section of Rhodococcus erythropolis has numerous lipid inclusions in the case of the technical grade n-C-14,15. Also in freeze-fractured samples (Fig. 4, right column), these inclusions can be observed as smooth areas beside the amorphous cytoplasm. In contrast these peculiarities were not detected in n-C-10 grown cells.

Pentasaccharide lipid production by Nocardia corynebacteroides

During a recent screening on n-alkane utilizing microorganisms we isolated a bacterium from oil contaminated soil. The taxonomical investigations led to the species Nocardia corynebacteroides. After cultivation on technical grade n-C-14,15 and extraction of the reaction mixture with ethyl acetate three glycolipids were detected by thin-layer chromatography (Rf values 0.36, 0.48 and 0.78, respectively). After isolation and purification the molecular structures were elucidated by chem- ical methods, NMR, GC-MS coupling as well as elemental analysis. As a result a

263

F - l / , l q - r ~ l l ¢

a l l s - - - -

500 nm 500 nrn Fi~. 4. Electron micrographs: thin sections (left) and freeze-fractures (right) of Rhodococcus erythropolis during glycolipid production phase (136 h) on different technical grade n-alkanes. L = lipid inclusion; H = membrane monolayer; G = amorphorous phase; P = polyphosphate granules. * = glycogen granule.

novel nonionic pentasaccharide lipid (Fig. 5) was characterized together with the known trehalose-mono- and -dicorynomycolates. The percentage content of the three compounds was estimated to be 92%/2%/6%. The details of downstream processing as well as the structural characterization of this pentaglucose lipid are

described elsewere (Powalla et al., 1989). We initially tested the effects of temperature, pH and various nitrogen sources on

growth and glycolipid synthesis using Mihagol S in shake flask experiments. The best results were obtained at T = 22°C and pH 6 to 7.5, with 0.3% of yeast extract and 0.2% of NaNO 3. TLC control of glycolipid formation versus growth and time

264

H H t CH2e I C,H2OH

H ~= i N • UH

H H H O H H O ~ 7 ~ • H O - ~ ' ~ O H

O . ~ Eel H H

H H

O = R

CH3COO (2 ~ ) CH3CH2COO and CH3(CH2)2COO (3 ~') CH3(CH2)6COO (2 K- ) HOOC(CH2)2COO ( 1 * ) . . . . . . . . . . . . . . . . . . . . . . . .

Fig. 5. Structure of pentasaccharide lipid from Nocardia corynebacteroides after growth on Mihagol S.

showed that the overproduction occurred after consumption of the nitrogen sources. The study of the influence of the chain length of pure n-alkanes from C-9 to C-18 on the pentasaccharide lipid production of Nocardia corynebacteroides showed that n-C-14, n-C-15 and n-C-16 were the most suitable substrates; glycolipid yield: about 2.75 g 1-1 (see Fig. 6). Related to the consumption of about 16 g 1-1 n-alkane (not shown here), the specific production was 0.17 g g-1. Other cultivations in the presence of water soluble compounds such as carbohydrates or ethanol not shown here, did not indicate unusual amounts of oligosaccharide lipid.

As to preliminary resting cell experiments using a phosphate buffer or the main culture medium without nitrogen sources, they did not lead to a remarkable overproduction of the pentasaccharide lipid.

,4

:t 1

0 9 10 11 12 13 14 15 16 17 18

Chain Length ( C - a t o m s )

Fig. 6. Influence of the chain length of pure n-alkanes on the pentasaccharide lipid production of Nocardia corynebacteroides after 144 h. Conditions: 1130 ml cultures (shake flasks); mineral salts medium;

20 g 1-1 of n-alkanes; pH 6.6; T = 22 ° C.

265

TABLE 1

SURFACTANT PROPERTIES OF AMPHIPHILIC COMPOUNDS FROM RHODOCOCCUS ERYTHROPOLIS AND NOCARDIA CORYNEBACTEROIDES IN WATER AT 40 °C

Surfactant Minimum Minimum Critical surface interfacial micelle tension tension * concentr. (mN m -l ) (mN m -1 ) (mg 1-1 )

Trehalose-dicorynomycolates 36 Trehalose-monocorynomycolates 32 Trehalose-2,2',3,4-tetraester 26 Pentasaccharide lipid 26

17 4 14 4

<1 15 < i 30

* Against n-hexadecane.

The studies of the surfactant properties of all the glycolipids from Rhodococcus erythropolis and Nocardia corynebacteroides are presented in Table 1. As can be seen 4 mg 1-1 each of both trehalose-corynomycolates reduced the surface tension of water from 72 mN m -1 down to 36 and 32 mN m -1, respectively, and the interfacial tension against n-hexadecane from 43 mN m -1 to 17 and 14 mN m -1, respectively. Higher values of 26 mN m -1 and lower than 1 mN m -~, respectively, were reached with trehalose-2, 2', 3, 4-tetraester and the pentasaccharide lipid.

Discussion

Our present studies on the formation of biosurfactants show that Rhodococcus erythropolis and Nocardia corynebacteroides need lipophilic carbon sources such as n-alkanes for the induction of glycolipid synthesis and their overproduction. Water soluble substrates were unsuitable.

The experiments involving cultivations under growth-limiting and resting cell conditions, respectively, showed that the energy gain by n-alkane degradation was high enough to permit the biosynthesis of new metabolites over a long period. Only for Rhodococcus erythropolis was the shorter chain substrate (n-C-10) a better precursor than n-C-14,15 for glycolipid production. A possible explanation could be derived from electron microscopy studies. From the literature it is known that the growth of microorganisms on hydrocarbons usually results in distinct alterations compared to cultivation on water soluble substrates. Intracytoplasmic inclusions are typical for hydrocarbon cultivations (Atlas and Heinz, 1973; Scott and Finnerty, 1976; Hiray et al., 1972; Cooney et al., 1980; Smucker and Cooney, 1981). In this sense the morphology of n-C-14,15 grown cells corresponds to the literature data whereas that of n-C-10 grown cells was not in accordance with these observations. The absence of lipid granules points to other utilization of the shorter hydrocarbon chain with possibly more glycolipid biosynthesis than intracytoplasmic lipid storage. Additional information about these differences should be derived from future studies on the biosynthetic enzymes after cell rupture.

266

References

Asmer, H.J., Lang, S., Wagner, F. and Wray, V. (1988) Microbial production, structure elucidation and bioconversion of sophorose lipids. JAOCS 65, 1460-1466.

Atlas, R.M. and Heinz, C,E. (1973) Ultrastructure of two species of oil degrading marine bacteria. Can. J. Microbiol. 19, 43-45.

Cooney, J.J., Siporin, C. and Smucker, R.A. (1980) Physiological and cytological responses to hydro- carbons by the hydrocarbon using fungus Cladosporium resinae. Bot. Mar. 23, 227-232.

Cooper, D.G. and Paddock, D.A. (1984) Production of a biosurfactant from Torulopsis bombicola. Appl. Environ. Microbiol. 47, 173-176.

Fawcett, J.K. and Scott, J.E. (1960) A rapid and precise method for the determination of urea. J. Clin. Pathol. 13, 156-159.

Frautz, B., Lang, S. and Wagner, F. (1986) Formation of cellobiose lipids by growing and resting cells of Ustilago maydis. Biotechnol. Lett. 8 (11), 757-762.

Hiray, M., Shimizu, S., Teranishi, Y., Tanaka, A. and Fukui, S. (1972) Effects of hydrocarbons on the morphology of Candida tropicalis pK 233. Agr. Biol. Chem. 36, 2335-2343.

Inoue, S. (1988) Biosurfactants in cosmetic applications. In: Applewhite, T.H. (ed.), Proceedings of World Conference on Biotechnology for the Fats and Oils Industry, pp. 206-210.

Kretschmer, A., Bock, H. and Wagner, F. (1982) Chemical and physical characterization of interfacial active lipids from Rhodococcus erythropolis grown on n-alkanes. Appl. Environ. MicrobioL 44, 864-870.

Powalla, M., Lang, S. and Wray, V. (1989) Penta- and disaccharide lipid formation by Nocardia corynebacteroides grown on n-alkanes. AppL Microbiol. Biotechnol., 31,473-479.

Rapp, P., Bock, H., Wray, V. and Wagner, F. (1979) Formation, isolation and characterization of trehalose dimycolates from Rhodococcus erythropolis grown on n-alkanes. J. Gen, Microbiol. 115, 491-503.

Reynolds, E.S. (1963) The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell. Biol. 17, 208-212.

Ristau, E. (1983) Mikrobielle Produktion von grenzfl~ichenaktiven Trehaloselipiden aus n-Alkanen; Strukturaufkl~irung eines anionischen Trehalosetetraesters. Ph. D. thesis, Technical University Braunschweig, F.R.G.

Ristau, E. and Wagner, F. (1983) Formation of novel anionic trehalose-tetraesters from Rhodococcus erythropolis under growth limiting condition. Biotechnol. Lett. 5, 95-100.

Scott, C.C.L. and Finnerty, W.R. (1976) A comparative analysis of the ultrastructure of hydrocarbon oxidizing microorganisms. J. Gen. Microbiol. 94, 342-350.

Smucker, R,A. and Cooney, J.J. (1981) Cytological responses of Cladosporiurn resinae when shifted from glucose to hydrocarbon medium. Can. J. Microbiol. 27, 1209-1218.

Spurr, A.R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultra- struct. Res. 26, 31-43.

Syldatk, C., Lang, S., Matulovic, U. and Wagner, F. (1985) Production of four interfacial active rhanmolipids from n-alkanes or glycerol by resting cells of Pseudomonas spec. DSM 2874. Z. Naturforsch. 40c, 61-67.