1 Introduction

Several structurally diverse surface-active molecules areproducable by a wide spectrum of microorganisms (bacteria,fungi, and yeasts). Compared to naturally occurring lipopep-tides, phospholipids, fatty acids, polysaccharide-proteincomplexes, and lipopolysaccharides, glycolipids belong tothe most important class of biosurfactants [1, 2]. Some examples are the sophorolipids, which can be produced in a commercially usable yield ≥ 300 g l–1 by Candida strains [3–5]. Moreover, trehalose lipids are secreted by Rhodococ-cus, rhamnolipids by Pseudomonas bacteria, and cellobioselipids as well as mannosylerythritol lipids are formed bysome smut fungi (Ustilagospecies) [6–8].

The glycolipids are of increasing interest for commercialuse as natural surfactants with promising characteristics be-cause they offer several advantages such as low toxicity,biodegradability, and ecological acceptance [9, 10]. Biosyn-thesis of these compounds is often associated with growth onhydrocarbons or other lipophilic substrates as for examplevegetable oils and fatty acids. Using these carbon sources,some microorganisms synthesize extracellular or cell-wallassociated glycolipids. Their overproduction is often con-nected with growth-limiting conditions. Tsukamurella spec.

(DSM 44370) produces, as well as Tsukamurella pauro-metabolum (DSM 20162), a mixture of three classes of ex-tracellular glycolipids when grown on sunflower oil (Fig. 1)[11].

The detailed structure elucidation showed that trehalose(GL 1), trisaccharide (GL 2), and tetrasaccharide lipids(GL 3) are formed. While GL 2 and GL 3 contain short-chainfatty acids in the range of 6–8 carbon atoms, GL1 carries al-so longer chain acyl residues with incorporated oleic acid orhexadecenic acid. After initial investigations to optimize me-dia and cultivation conditions in shake flask experiments aswell as in a bioreactor yields up to 5 g l–1 were achieved.

The aim of the following investigations was to increasethe microbial production by using domestic vegetable oils inorder to convert renewable resources into higher value prod-ucts. Moreover, it was focused on the glycolipid composi-tion, and some more interfacial properties were evaluated aswell as biological activities.

2 Materials and Methods

2.1 Microorganisms

Tsukamurella spec. DSM 44370 (isolated from an oil con-taining soil sample) used for all experiments, was main-tained on nutrient broth agar slants (Difco), stored at 4 °C,and transferred at an interval of three months.

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The vegetable oil utilizing bacterial strain Tsukamurella spec. (DSM44370) growing on oleic acid rich sunflower oil was found to produce a mixture of oligosaccharide lipids. In addition to trehalose lipids(GL 1) also trisaccharide (GL 2) and tetrasaccharide lipids (GL 3) weredetected. In submerged culture the tri- and tetrasaccharide lipids wereoverproduced during the stationary phase (nitrogen limitation) and excreted into the supernatant. In contrast, the trehalose lipids were synthesized growth-associated. As a result of bioreactor cultivations ap-proximately 30 g l–1 glycolipids were produced from 110 g l–1 sun-flower oil.Interfacial properties of the crude product and the purified compoundswere investigated. The surface tension of water was reduced to less than30 mN m–1 and the interfacial tension water/n-hexadecane was lowereddown to 5–10 mN m–1. Moreover, the HLB values of the purified compounds were estimated to be between 8 and 10. The glycolipidsshow antimicrobial activities against gram-positive and gram-negativebacteria and one fungal strain.

Microbial conversion of vegetable oils into surface-active di-, tri-, and tetrasaccharide lipids (biosurfactants) by the bacterial strain Tsukamurella spec.

Elke Vollbrecht*, Udo Rau*, and Siegmund Lang*

Mikrobielle Konversion pflanzlicher Öle in oberflächenaktiveOligosaccharidlipide. Der Pflanzenöl-verwertende bakterielleStamm Tsukamurella spec. (DSM 44370) produziert bei Wachstum aufSonnenblumenöl als einziger Kohlenstoffquelle ein Gemisch verschie-dener Oligosaccharidlipide. Neben Trehaloselipiden (GL 1) konntenTrisaccharid- (GL 2) und Tetrasaccharidlipide (GL 3) identifiziert wer-den. In Submerskultur wurden die Tri- und Tetrasaccharidlipide ver-stärkt in der stationären Phase – unter Stickstofflimitierung – gebildetund ins Kulturmedium ausgeschieden. Im Gegensatz dazu scheint dieSynthese der Trehaloselipide wachstumsgekoppelt stattzufinden. ImBioreaktor konnten unter optimierten Bedingungen aus ca. 110 g l–1

metabolisiertem Sonnenblumenöl 30 g l–1 Glycolipid isoliert werden.Sowohl das Rohprodukt als auch die aufgereinigten Glycolipide zeig-ten interessante oberflächenaktive Eigenschaften. Sie reduzierten dieOberflächenspannung von Wasser auf Werte unter 30 mN m–1. DieGrenzflächenspannung zwischen Wasser und n-Hexadecan wurde auf5–10 mN m–1 erniedrigt. Die HLB-Werte der aufgereinigten Kompo-nenten lagen bei 8 bzw. bei 10. Zusätzlich zeigten die Verbindungen an-timikrobielle Wirkung gegen Gram-positive, Gram-negative Bakterienund den Pilz Ustilago violacea.

* Technical University Braunschweig, Institute for Biochemistry andBiotechnology, Dept. for Biotechnology, Braunschweig, Germany.

Forschungsbeiträge/Research Papers

Antimicrobial activities were tested with following bacte-ria, fungi, and alga, which were purchased from the DSMZ(German strain collection for microorganisms and cellcultures, Braunschweig, Germany): Escherichia coli,Pseudomonas aeruginosa, Staphylococcus aureus, Vibriofischeri, Ustilago violacea, Eurotium repens, Mycothyphamicrospora, Chladosporium cucumerum, Fusarium oxyspo-rum, and Chlorella fusca.

2.2 Microbial production

The experiments were carried out in 500 ml shake flaskswith 100 ml culture broth. The medium contained the fol-lowing mineral salts per liter: MgSO4 × 7 H2O 0.2 g, CaCl2 ×2 H2O 0.2 g, 1 M NaH2PO4/K2HPO4 buffer pH 7.5 100 ml (ifnot specified separately), ZnSO4 × 7 H2O 444µg, CuSO4 × 5H2O 75µg, FeSO4 × 7 H2O 6.3 mg, MnSO4 × 6 H2O 255µg,CoSO4 × 7 H2O 672µg, NiSO4 × 6 H2O 81µg, (NH4)6 MoO24 × 4 H2O 78µg, H3BO3 186µg, KI 30µg, EDTA 0.25 g. As nitrogen source 10 mmol l–1 (NH4)2SO4were used, the carbon source (mainly vegetable oils) wasadded as specified later.

For batch and fed batch cultivations a 10-l-bioreactor (B.Braun Biotech International,Melsungen, Germany) was

used. The culture conditions and the reactor equipment werebasically identical to those described in a previous paper[11].

2.3 Determination of the cultivation parameters

Biomass was measured gravimetrically. 10 ml of culturesuspension were centrifuged for 15 min at 13,000 rpm,washed twice with 5 ml ethanol/butanol (1:1, v/v), and driedat 300 mbar and 75 °C. Ammonia was determined semiquan-titatively with analytical test strips (Merckoquand 10024Ammonium Test, Merck, Darmstadt, Germany).The proteincontent of the culture suspension (cells and aqueous phasetogether) was measured with the method of Lowry [12].

The whole culture suspension was extracted three timeswith the same volume of t-butylmethylether. The organicphase was separated from the aqueous one by centrifugationand decanted carefully. The sunflower oil was detected in thet-butylmethyletherextract by HPLC on a RP-18 column (ET250/3 Nucleosil 120-5C8) with CHCl3/CH3CN (30:70; v:v)as solvent and a flow rate of 0.8 ml min–1, with an evapora-tive light-scattering detector (Alltech, MKII, Deerfield,USA). For TLC of the organic phase, silica plates (Merck 60F254) were developed in CHCl3/MeOH/H2O (65/15/2), thedetecting reagent was α-naphthol-sulphuric acid followedby heating at 180 °C. The content of glycolipid was deter-mined with a densitometer (Desaga CD 60,Heidelberg, Ger-many) by scanning the plates at 580 nm and calculating theresulting peak areas.

GL 1–3 were separated from the culture broth by silica gelcolumn chromatography as reported previously [11].

2.4 Physicochemical properties

Surface tension and interfacial tension of aqueous solu-tions of GL 1–3 were determined with a Tensiomat (MGWLauda, Königshofen, Germany) at 25 °C using the ringmethod.

Emulsifying ability was tested by the addition of 2µg gly-colipid to test emulsions, that contain 4 ml water and 100µlhydrophobic phase. The tubes were vortexed for exactly1 min. Subsequently, the stability of the emulsion was ob-served 3 h by measurement of the optical density at 623 nm.

For determination of the HLB value a mixture of cyclo-hexane and soybean oil was used as hydrophobic phase. TheHLB value needed for the optimum emulsion of the hy-drophobic phase was calculated as [13, 14]:

A =HLBneeded–HLBB)

HLBA–HLBB

A % cyclohexaneHLBA HLB value of cyclohexane (= 15)HLBB HLB value of soybean oil (= 6)

The hydrophobic mixture, which leads to the most stableemulsion, gives the HLB-value of the used surfactant.

2.5 Antimicrobial activities

The antimicrobial activities were followed qualitativelyby a paper disc/agar diffusion assay. For determination of theIC50 value in general 2 ml cultures were supplemented with40µl glycolipid solutions (dimethylsulfoxide solutions) andcultivated using conventional growth conditions (dependingon the used microorganism) at 30 °C or 27 °C and 240 rpm.The nematicidic effect on Caenorhabditis elegans was mea-sured at the Institute for Pharmaceutical Biology, TechnicalUniversity Braunschweig.

390 Fett/Lipid 101 (1999), Nr. 10, S. 389–394

Fig. 1.Structures of the three classes of glycolipids produced by Tsuka-murella spec.

3 Results and Discussion3.1 Substrate screening

Initial studies indicated that Tsukamurella spec. is able togrow on water-soluble substrates as well as on hydrophobiccarbon sources [11]. Improving the glycolipid productionduring substrate screening the aim was to use renewableresources for product formation. As shown in Tab. 1, the in-fluence of various carbon substrates on growth and glyco-lipid formation of Tsukamurella spec. was tested. Complexmedia containing yeast extract, malt extract, and peptone aswell as mineral salt media with hydrophilic substrates as car-bohydrates and hydrophobic carbon sources as n-alkanes,fatty alcohols, fatty acids, and vegetable oils were used. Oncomplex media excellent growth occurs, however, glyco-lipid could not be detected. GL 1, GL 2, and GL 3 containseveral glucose molecules, which form a trehalose-unit, andin GL 3 additionally a galactose unit is incorporated. There-fore, the use of these sugars as precursors of glycolipid for-mation was tested. With glucose only traces of glycolipidswere formed, while sucrose, trehalose, and galactose did notinduce any product synthesis. Lactose was not degraded byTsukamurella spec. Using n-alkanes and fatty alcohols weobserved only slow growth and in comparison longer chainlength substrates led to better results. Best growth and gly-colipid production was achieved by using natural vegetableoils. Glycerol enabled neither growth nor glycolipid produc-tion.

Fig. 2 presents a quantitative comparison of several veg-etable oils after shake flask experiments. Among theselipophilic substrates the oleic acid rich oils and rapeseed oil(C 22:1) show the best results. Sunflower oil (C 18:1) leadsto the highest product yield of 5 g l–1 glycolipid resulting in abiomass based yield coefficient YP/X of 1.10. Similar data ofapproximately 4 g l–1 can be reached with olive oil and rape-seed oil (C 22:1). For the biosynthesis of glycolipids fromtriglycerides some possible pathways are reported, however,for other microorganisms [7, 15]. The first step is the hydro-lysis into fatty acids and glycerol, that is not used by Tsuka-murella spec. Thus, the glycolipids are formed from the fat-

ty acids, which undergo β-oxidation ultimately to acetate.Gluconeogenesis leads to the carbohydrate backbone. Thefatty acids can be incorporated directly, after elongation,modification or they are synthesized de novo.Both, carbohy-drates and fatty acids, are combined to form the glycolipids.

Although the extracellular lipase activity in the culturebroth is low, Tsukamurella spec. grows on oils rapidly com-pared to free fatty acids. Due to their action against cellmembranes, fatty acids themselves are toxic and only toler-ated beyond a low concentration within and outside the cell[16].

3.2 Bioreactor cultivations

Initial studies [11] showed that nitrogen limited condi-tions favored the overproduction of glycolipids by Tsuka-murella spec. Yields of 5 g l–1 glycolipids were obtained in a10 l bioreactor. Product analysis revealed different concen-trations of the individual components. GL 3 was the mainproduct after 65 h with an amount of 55% of the mixture.During the course of the cultivation the production of GL 1starts in the growth phase and continues in a constant rate

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Tab. 1. Influence of different carbon sources (10 g l–1) on growth and glycolipid formation of Tsukamurella sp.; CP: yeast extract 10 g l–1, glucose10 g l–1, pH 6.2, MPY: malt extract 20 g l–1, peptone 2,5 g l–1, yeast extract 2.5 g l–1.

C-source Growth Glycolipid formation C-source Growth Glycolipid formation

complex media fatty alcohols

nutrient broth (Difco) ++ – 1-dodecanol – –

CP ++ – 1-tetradecanol + +

MPY ++ – 1-octadecanol + +

sugars fatty acids

glucose + + lauric acid – –

galactose + – myristic acid + –

sucrose + – palmitic acid ++ +

trehalose + – stearic acid ++ +

lactose – – oleic acid – –

n-alkanes vegetable oils

n-decane – – coconut oil ++ ++

n-dodecane + + corn oil ++ ++

n-tetradecane + + rapeseed oil (C 22:1) ++ ++

rapeseed oil (C 18:1) ++ ++

glycerol – – sunflower oil (C 18:1) ++ +++

olive oil ++ ++

Fig. 2. Glycolipid formation by Tsukamurella spec. on vegetable oils(20 g l–1 carbon source). YP/X = glycolipid yield based on biomass.

during growth as well as stationary phase. The synthesis ofGL 2 and GL 3 starts after nitrogen-limitation is reached.

As in general secondary metabolite production is influen-cable by phosphate limitation[17], now the correspondingcultivation was carried out (Fig. 3). In this case, sunfloweroil and aqueous media together formed a stable emulsion, sothat it was not possible to remove the biomass by centrifuga-tion. Instead of biomass the protein content of the whole cul-ture broth was measured for growth. Protein and glycolipidcontents increased in parallel. After 25 h growth stopped dueto phosphate limitation (initial phosphate concentration0.35 mmol l–1). 22 g l–1 sunflower oil were consumed to form5 g l–1 glycolipids. The product composition is quite differentas in the case of nitrogen limitation. The trehalose lipid GL 1produced during growth phase was the main product (59%).GL 2 had an amount of 27%. GL 3 (14%) was synthesizednot before phosphate limitation. Therefore, it is possible todirect the product composition by choosing different cultureconditions.

Mycolic acids, which are associated with carbohydratesas trehalose or cell wall arabinogalactan, are characteristiccompounds occurring in the cell wall of Actinomycetes [18].Tomiyasu et al. (1986) [19] isolated trehalose and mycolicacid containing glycolipids from the cell wall of Rhodo-coccus aurantiacus (actual classification Tsukamurellapaurometabolum). An asymmetric substituted trehalose-2, 3,6’-trimycolate with chain lengths of 64 to 72 carbon atoms,was identified. The positions of the acyl residues are identi-

cal with those we found in GL 1 and GL 1B. This indicatesthat the synthesis of GL 1 and GL 1B is related to cell wallsynthesis. This may explain the formation of GL 1 during ex-ponential growth phase.

Recent experiments (not shown here) indicated that theglycolipid production of Tsukamurellaspec. is inhibited byhigher phosphate concentrations. Therefore, a phosphateconcentration of 0.2 mol l–1 led to only 0.7 g l–1 glycolipid in-stead of 5 g l–1 glycolipid when 0.1 mol l–1 phosphate wasused. With a modified medium containing only 2.5 mmol l–1

K2HPO4 in a nitrogen-limited, but phosphate excess (provenby an addition of phosphate without resulting in furthergrowth) fed batch cultivation, up to 22 g l–1 biomass andmore than 30 g l–1 glycolipid were achieved after consump-tion of 107 g l–1 sunflower oil (Fig. 4). The resulting yield co-efficients were Yglycolipid/biomass= 1.4 [w/w] and Yglycolipid/oil =0.29 [w/w]. The volumetric productivity increased up to0.19 g l–1 h–1. Following the product composition during thecultivation, Fig. 4 shows that the three glycolipids are pro-duced approximately in the same amount. Similar effects ofphosphate limitation on the formation or distribution of gly-colipids are known from Ustilago maydis [20]. The produc-tion of glycoglycerolipids from Microbacterium spec. is in-hibited by phosphate, too [21].

3.3 Physicochemical properties

The presence of both a hydrophobic and a lipophilic areawithin one molecule creates the typical surfactant propertiesof these glycolipids. The effects of the biosurfactants wereestimated by measuring the minimum surface tension valuereached and the critical micelle concentration. Different sur-face active properties were observed. The experimental data

392 Fett/Lipid 101 (1999), Nr. 10, S. 389–394

Fig. 3. Growth, glycolipid production (top), and product composition(bottom) of Tsukamurellaspec. in a fed batch cultivation on sunfloweroil (30 g l–1) with phosphate limitation. Conditions: 10-l-bioreactor,mineral salt medium, 30 °C, 800 rpm, 0.5 v/vm, pH adjusted at 7.5.

Fig. 4. Growth, glycolipid production (top), and product composition(bottom) of Tsukamurella spec. in a fed batch cultivation on sunfloweroil (in total 107g l–1) with nitrogen limitation. Conditions: 10-l-bioreac-tor, mineral salt medium, 20 g l–1 sunflower oil at the beginning, 0.44 g/lK2HPO4, 30 °C, 800 rpm, 0.4 v/vm, pH adjusted at 7.5.

given in Fig. 5 show that the trehalose lipid GL 1 reduces thesurface tension of water from 72 mN m–1 down to 35 mN m–1

with a cmc value of 10 mg l–1 or 0.15 × 10–4mol l–1, respec-tively. The highest reduction in surface tension was detectedwith the more polar glycolipids GL 2 and GL 3 resulting insurface tension values of 23 and 24 mN m–1. The cmc dataare around 100 mg l–1 (GL 2 1.05 × 10–4mol l–1, GL 3 0.91 ×10–4mol l–1).

Moreover, all glycolipids investigated lower the interfa-cial tension between water and n-hexadecane at 25 °C to lessthan 10 mN m–1.

The surface properties of the crude glycolipid mixturewere compared to some commercially available carbohy-drate surfactants (Tab. 2) as alkylpolyglycoside APG 1200®(Henkel), β-octylglucoside, and β-dodecylmaltoside (Sigma).The Tsukamurella glycolipids were found to be as effectiveas the commercial surfactants. They attain lower surface ten-sion values accompanied by a lower cmc, so that in compar-ison reduced surfactant concentrations are needed to obtainthe same surface tension.

The HLB value conception [14] (hydrophilic-liphophilic-balance) is an often used tool for the emulgator choice. TheHLB is calculated as:

HLB = 20 × MH × M–1

MH = molecular weight of the hydrophilic moietyM = molecular weight of the whole molecule.

In the case of ionic surfactants or if hydrophilic and hydrophobic moieties are not arranged equally within themolecule the formula gives no proper results. Therefore, theHLB value was measured experimentally, too.

The calculated HLB values for the three classes of glyco-lipids are 10.3 for GL 1, 10.5 for GL 2, and 11.9 for GL 3.

The experimental data were HLB 8 for GL 1 and GL 3,while GL 2 yielded a value of 10 (Fig. 6). The substrate sun-flower oil has an HLB value of 8. Therefore, the producedglycolipid mixture possesses the ability to cause a stableemulsion, that facilitates the supply of the microorganismswith carbon source and mineral salts.

3.4 Biological activities

The pure microbial glycolipids were tested against gram-positive and gram-negative bacteria, some fungi, and an alga in a qualitative paper disc/agar assay. The glycolipidsshowed some activity against gram-positive and gram-nega-tive bacteria. The pathogenic strains Pseudomonas aerugi-nosa and Staphylococcus aureus were not inhibited. Amongthe fungi growth of Ustilago violacea was strongly inhibited(Tab. 3). Some serial dilution tests were performed withBacillus megaterium, Escherichia coliand Ustilago violaceato quantify the antimicrobial activities. GL 2 showed onlysmall activity against E. coli, while GL 1 and GL 3 had no effect. With B. megaterium the IC50 data were between50 mg ml–1 for GL 3 and 150 mg ml–1 for GL 1. The corre-sponding data for Ustilago violacea indicate that GL 1 andGL 2 are more efficient against this fungus than GL 3. Addi-tionally GL 2 showed antimicrobial activity against Vibriofischeri and GL 3 revealed a small nematicidic effect onCaenorhabditis elegans.

In conclusion, gram-positive bacteria were more sensitiveagainst GL 1–3 than gram-negative, which were slightly ornot inhibited. In comparison, sophorolipids and rhamno-lipids are known to inhibit growth of gram-positive bacteriain concentrations of 10–100µg ml–1 [22]. The mannosylery-thritollipids MEL A and MEL B from Candida antarcticashow high activity against B. subtilis and S. aureus [23].Similar resulting MIC (lowest concentration of glycolipidenabling any growth) values in a range of 20–200µg ml–1

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Fig. 5. Influence of the purified glycolipids from Tsukamurella spec. onthe surface tension of water and the interfacial tension between waterand n-hexadecane at 25 °C.

Tab. 2. Surface activity characteristics of the glycolipid mixture fromTsukamurellaspec. compared to different commercially available carbohydrate surfactants (APG 1200 Plantaren®, Henkel; β-octylgluco-side, Sigma; β-dodecylmaltoside, Sigma).

Properties Tsukamurella APG 1200 β-Octyl- β-Dodecyl-glycolipids Plantaren® glucoside maltoside

cmc [mg l–1] 20 20 3000 100

σmin [mN m–1] 25 27 30 33

Fig. 6. Experimental HLB data of the three glycolipids produced byTsukamurella spec.

have been reported by Deml et al. [24] using Schizonellin,isolated form a culture of the smut fungus Schizonellamelanogramma.

Acknowledgements We would like to thank V. Wray and M. Nimtz (GBF, Braunschweig)

for the structure elucidation and the German Ministry for Nutrition,Agriculture and Forestry for funds (grant 97NR172-F). The vegetableoils and fatty acids used were kindly donated by Henkel KGaA, Düs-seldorf, Germany.

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[12] O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, Pro-tein measurement with the Folin phenol reagent, J. Biol. Chem.193 (1951) 265–275.

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[15] D. Kitamoto, H. Yanagishita, K. Haraya, and H. K. Kitamoto, Con-tribution of a chain-shortening pathway to the biosynthesis of thefatty acids of mannosylerythritol lipid (biosurfactant) in the yeastCandida antarctica: effect of β-oxidation inhibitors on biosurfac-tant synthesis, Biotechnol. Letters 20 (1998), 813–818.

[16] R. K. Hommelund C. Ratledge: Biosynthetic mechanisms of lowmolecular weight surfactants and their precursor molecules, in:Biosurfactants, production, properties, applications, Kosaric(Ed.), Marcel Dekker Inc., New York-Basel-Hong Kong 1993,pp. 3–63.

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[18] M. Goodfellow: The family norcardiaceae, in: Prokaryotes, Ahandbook on the biology of bacteria, ecophysiology, isolation,identification, application, A. Balous, H. G. Trüper, M. Dworkin,W.H. Tno, and K. H. Schleifer(Eds), second edition vol. III,Springer-Verlag, Berlin 1992, 1188–1207.

[19] I. Tomiyasu, J. Yoshinaga, F. Kurano, Y. Kato, K. Kaneda, S.Imaizumi, and I. Yano, Occurrence of a novel glycolipid, trehalose2, 3, 6’-trimycolate in a psychrophilic acid-fast bacterium,Rodococcus aurantiacus (Gordona aurantiaca), FEBS Lett. 203(1986) 239–242.

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Address of the authors: Dipl.-Chem. Elke Vollbrecht,PD Dr. UdoRau, and PD Dr. Siegmund Lang (author to whom correspondenceshould be addressed), Technical University Braunschweig, Institute forBiochemistry and Biotechnology, Dept. for Biotechnology, Spiel-mannstraße 7, 38106 Braunschweig, Germany.

[Received: May 11, 1999; accepted: June 25, 1999].

394 Fett/Lipid 101 (1999), Nr. 10, S. 389–394

Tab. 3. Antimicrobial activities of glycolipids from Tsukamurella spec.against several different microorganisms, – no inhibition, + inhibition,++ strong inhibition, nt not tested so far.

Qualitative measurementMicroorganism Crude product GL 3 Growing cells of

Tsukamurellaspec.

B. megaterium + ++ +

E. coli + + +

P. aeroginosa – – nt

S. aureus – – nt

U. violacea ++ + ++

E. repens – – +

M. microspora – – +

C. cucumerum – – nt

F. oxysporum – – –

Chl. fusca – + +

Quantitative measurement/IC 50 value*Microorganism GL 1 GL-2 GL 3

[µg ml–1] [µl ml–1] [µg ml–1]

B. megaterium < 150 < 100 < 50

E. coli > 500 < 500 > 500

U. violacea < 100 < 100 < 200

* concentration leading to 50 % of growth inhibition.