Yeast flotation viewed as the result of the interplay of supernatant composition and cell-wall hydrophobicity

Yeast flotation viewed as the result of the interplay of supernatant composition and cell-wall hydrophobicity

Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319 www.elsevier.com/locate/colsurfb Yeast flotation viewed as the result of the interplay of ...

238KB Sizes 0 Downloads 4 Views

Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319 www.elsevier.com/locate/colsurfb

Yeast flotation viewed as the result of the interplay of supernatant composition and cell-wall hydrophobicity Sandro R. deSousa a, Karen F. Oliveira a, Crisla S. Souza a, Beatriz V. Kilikian a, Cecilia Laluce b,* a

PPIB-Programa de Po´s-graduac¸a˜o Interunidades em Biotecnologia-USP/IPT/I. BUTANTAN, Instituto de Cieˆncias Biome´dicas IVUSP, Av. Lineu Prestes 1730, 05508-900 Sa˜o Paulo, SP, Brazil b Department of Biochemistry and Technological Chemistry, Instituto de Quı´mica da UNESP, Universidade Estadual Paulista-UNESP, R. Francisco Degni, s/n, P.O. Box 355, CP 355, 14801-970 Araraquara, SP, Brazil Accepted 19 December 2002

Abstract Flotation is a process of cell separation based on the affinity of cells to air bubbles. In the present work, flotability and hydrophobicity were determined using cells from different yeasts (Hansenulla polymorpha , Saccharomyces cerevisiae , Candida albicans ), which were propagated in different media and at different temperatures. Alterations to the supernatant of the cells were also carried out before the flotation assays. The results described here indicate that supernatants of the yeast cells can play a more important role on flotation than cell-wall hydrophobicity. For example, wall-hydrophobicity of strain FLT-01 of S. cerevisiae was high but flotation did not occur when their washed cells were resuspended in water. Additions of neopeptone to cultures of S. cerevisiae and H. polymorpha repressed flotation and increased the volume of foam. An additional task of the present work was to show that the relationship between cellwall hydrophobicity and flotation performance was dependent on the method used for the measurement of hydrophobicity. Based on the assay procedure, two types of hydrophobicity were distinguished: (a) the apparent hydrophobicity for cells suspended in the medium and expressed by the degree of cell affinity to the organic solvent in the two-phase system supernatant/hexane; (b) the standard hydrophobicity, which was determined for cells suspended in a standard solution (acetate buffer, in the present work) within the acetate buffer/hexane system. Flotation of cells of S. cerevisiae and C. albicans were best related to the degree of apparent hydrophobicity (varying with the supernatant composition at the cell/medium interface) rather than to the degree of standard hydrophobicity (varying with the alterations in the wall components, since the liquid phase was constant in the assay). However, depending on the yeast unpredictable results can be obtained. For example, cells of H. polymorpha exhibited good flotation associated to a high degree of standard hydrophobicity while having a lower degree of apparent hydrophobicity. Concerning growth temperature, flotation of cells of C. albicans was strongly repressed when the temperature was raised from 30 to 38 8C while a similar effect was not observed in cultures of S. cerevisiae and H. polymorpha . It is difficult to understand and

* Corresponding author. Tel.: /55-16-201-6673; fax: /55-16-222-7932. E-mail address: [email protected] (C. Laluce). 0927-7765/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0927-7765(03)00019-5

310

S.R. deSousa et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319

predict flotation of yeast cells but simple modifications made to the supernatant of cultures can activate or repress flotation. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Flotation; Hydrophobicity; Saccharomyces cerevisiae ; Hansenula polymorpha ; Candida albicans

1. Introduction Flotation is a very promising tool for the separation of cells and/or products in biotechnological processes [1 /4]. It is based on the affinity of particles to air bubbles that accumulate on the surface of the medium forming a cell-enriched foam. Flocculation is another surface phenomenon based on the cell /cell affinity, which leads to the formation of large cell aggregates. Thus, both processes are related to independent phenomena [4,5]. The methods based the on degree of cell adhesion to hydrocarbons, are ordinarily used for the determination of the cell surface hydrophobicity. However, the role of cell-wall hydrophobicity on flotation phenomenon and the choice of the best method for the measurement of the cellwall hydrophobicity are controversial [6,7]. The degree of hydrophobicity is usually determined by the partitioning of cells in the two-phase system water/organic solvent [8]. It seems that the affinity of the cells to the organic phase is the result of the interplay of hydrophobic and electrostatic interactions at the water/organic solvent interface [9 /11]. Studies based on X-ray photoelectron spectroscopy [12] suggest that the surface hydrophobicity of microorganism depends on the relative amounts of chemical groups present on the cell-wall. In addition, properties such as viscosity, liquid density, and interaction forces (electrostatic forces, dipole /dipole, hydrophobic, and steric interactions) that are usually involved in the frequent interactions occurring among biological molecules of the living world, are also involved in the flotation phenomenon [13]. In order to obtain more general conclusions regarding yeast flotation, yeasts from different strains and genera (Hansenulla polymorpha , Saccharomyces cerevisiae , Candida albicans ), were propagated at 30 and 38 8C in different media.

In addition, alterations were made to the supernatants of the cultures (dilutions, additions of ethanol and neopeptone, and exchanges of supernatants between pairs of different cultures) before flotation to better clarify the roles of both supernatant (liquid phase) and cell-wall hydrophobicity on yeast flotation. Proteins extracted from the cellwall and secreted into the defined medium of strain FLT-01/4b were identified using gel electrophoresis.

2. Experimentals 2.1. Yeast strains The following yeast strains were used: strain FLT-01 of S. cerevisiae exhibiting both high cellwall hydrophobity and flotation capacity [4]; strain FLT-01/4b resulting from sporulation of strain FLT-01; strain CBS 4732 of Hansenula polymorpha showing high flotation ability [2,4]; an isolate (strain LTU) obtained from commercial baker’s yeast from Fleischmann and Royal Ltd., Brazil; an isolate of C. albicans (strain FCFAR250) obtained from an oral lesion of an AIDS patient, kindly donated by Dr Maria J. M. Giannini from the Department of Clinical Analysis, Faculty of Pharmaceutical Sciences (Faculdade de Cieˆncias Farmaceˆuticas de Araraquara), Araraquara, SP, Brazil. 2.2. Culture media and cultivation conditions The defined medium [14] was prepared as follows: (a) in g/l: glucose 20, (NH4)2SO4, 3.12, KH2PO4 1.995, MgSO4 ×/ 7H2O 0.54, CaCl2 0.09, and NaCl 0.11; (b) in mg/l: myo-inositol 30.0, pyridoxine 4.8, thiamine 16.8, calcium pantothenate 4.8, biotin 0.36; (c) in mg/l: ZnSO4 ×/ 7H2O 72.8, CuSO4 ×/ 5H2O 10.4, H3BO3 10.4, FeCl3 ×/

S.R. deSousa et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319

6H2O 52.0, KI 10.4. The pH was adjusted to 4.5 before sterilization. The YPD medium (1% yeast extract, 2% peptone and 2% glucose) and the Sabouraud medium (1% neopeptone and 2% glucose) were also used for propagation. A single colony (grown for 2/3 days on solid YPD medium) was transferred to a 125 ml Erlenmeyer flask containing 25 ml of sterilized medium and the culture was propagated at 30 8C (9/0.5 8C) in a rotary shaker (125 rpm) for 24 h (stationary phase). This pre-culture was used to inoculate a 250 ml Erlenmeyer flask containing 50 ml of medium so that the propagation started with an initial cell density of around 0.25 mg/ml (dry weight). The final culture was propagated for 24 h under agitation before being used in the experiments. Propagation in defined medium was also carried out in a 2 l fermenter from Cole/Parmer (mod. J-1919), operating in a batch process at 30 8C. The pH was maintained at 4.5 during the process. The stirring speeds were 400 rpm for growth and 800 rpm for flotation [4]. The flotation occurred during air injection (5.0 vvm for 20 min) into the 2 l vessel containing 1.8 l medium. The froth started to form, increased rapidly, and the cell-enriched foam overflowed out from the fermenter through a silicone tube connected to the condenser of this apparatus. Stationary phase cells (growth curve) were submitted to flotation in all cases. 2.3. Bench flotation system A flotation column (17 cm high /2 cm i.d.) was used for the flotation assays [14]. The parameters determined were: (a) flotation (FLT, %) /[(Cp/ Cr/Cp)/100] [3]; (b) flotation efficiency [14] or proportion of the total cells transferred to foam from a volume of medium equivalent to the residual medium left in the column after flotation, (FLTeff, %) /[(Cp/Cr)/Cp) /100 /Vr/Vp; (c) Cp, initial cell concentration; (d) Cr, cell concentration in the medium remaining in the column after flotation; (e) Vp, volume of medium added to the flotation column; (f) Vr, volume of medium remaining in the column after flotation. The ratio Vr/Vp increased with the decrease in the volume of foam, which means that this ratio increased when

311

the cell concentration in the foam increased as a consequence of an increase in Vr. Thus, flotation efficiency expresses the capacity of the flotation process to concentrate cells in the foam. 2.4. Biomass determination The cell biomass concentration (expressed as mg/ml) was determined in duplicate assays, by drying the cells of each sample (previously washed in water by filtration using a 0.22 mm Millipore membrane) to a constant weight in an oven at 106 8C for 16 h. 2.5. Standard and apparent hydrophobicity assays [15] Cells (washed twice in water by centrifuging at 4000/g) were resuspended in 0.05 M acetate buffer at pH 4.5 for determination of the standard hydrophobicity. A mixture composed of cell suspension (3.0 ml containing 0.5 mg/ml cells, dry weight) and 1.0 ml n -hexane was vigorously shaken for 50 s and the two-phase system was completely separated after 30 min at room temperature. The portion of cells transferred from the aqueous (medium) to the hexane phase was determined by the absorbance at 570 nm (A570nm) of samples harvested from the aqueous phase (bottom phase) and the hydrophobicity was expressed as the amount (%) of the total cells removed form the aqueous phase (medium) to the solvent organic layer. Thus, hydrophobicity (%) / (A570nm before cell removal/A570nm after cell removal from the medium)/A570nm before removal /100 [16]. The degrees of the apparent hydrophobicity (cells suspended in the culture at pH 2.2) were determined using the same procedure as described above for the determination of the standard hydrophobicity. However, the initial concentration of cells was adjusted to 0.5 mg/ml before the assay of apparent hydrophobicity using the supernatant of the culture (resulting from centrifugation at 4000 /g for 5 min) instead of water. In a few experiments, exchanges of supernatants involving pairs of two different cultures were carried out before the apparent hydrophobicity assay.

312

S.R. deSousa et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319

2.6. Extraction of cell-wall proteins Before extraction, cells were washed twice in 0.1 M Tris /HCl buffer solution (containing 10% glycerol and 0.15 M NaCl at pH 7.5) by centrifugation for 5 min at 4000 /g . The crude cell-wall extract was obtained from a wet cell biomass (300 mg washed cells), which was resuspended in 1.0 ml of 0.2 M Tris /HCl buffer at pH 9.0 (containing 2% nonidet P-40, 1.2% NaCl and 0.9% DTT) and incubated for 20 min at 4 8C in a 40 kHz ultrasonic cleaning bath from Thornton T-14 (Thornton Inpec Electronic Ltd., Vinhedo, SP, Brazil). Nonidet P-40 (IGEPAL CA-250) was purchased from LKB, BROMMA, Sweden. The cell extract was separated by centrifuging at 8000 /g for 5 min. 2.7. Preparation of protein concentrates from supernatants of cultures Samples of supernatant (50 ml) were dialyzed for 24 h at 4 8C against 500 ml water, which was exchanged for distilled, water every 4 h. The dialyzed supernatant was lyophilized and the resulting powder was dissolved in 1 ml water. A sample of the concentrated protein solution was then immediately used. 2.8. SDS /acrylamide electrophoresis Samples were prepared for electrophoresis as follows: a 10 ml sample containing protease inhibitors (1 mM EDTA, 1 mM phenylmethylsufonyl fluoride, 1 mM EGTA, 1 mM benzamidine, 5 mg/ml pepstatin A, 5 mg/ml leupeptin, 5 mg/ml aprotinin, 20 mg/ml soybean trypsin inhibitor) as described in literature [17], 17 ml of 5.0 mM Tris / HCl buffer at pH 6.8 (containing 30% urea, 1.0% SDS, 50% glycerol and 0.5% bromophenol blue) plus 5 ml b-mercaptoethanol. After boiling for 3 min, samples of wall extract (4 ml), standard protein mixtures (5 ml) and concentrated supernatant samples (23 ml) were applied to the gel. The gel (12.5% acrylamide gels) was prepared following the procedure described by Hames [18]. Standard protein mixtures of high molecular sizes (HMW standard from Amersham Pharmacia, cat.

no.17-0445-01) and low molecular sizes (LMW standard from Amersham Pharmacia, cat. no. 170446-01) were used. Electrophoresis was run at 4 8C in a 25 m M Tris /HCl running buffer at pH 8.3 (containing 0.192 M glycine and 0.1% SDS) for 20 min at 17.5 mA per gel (constant current) followed by an additional 80 min running period at 35 mA per gel. A Mini-Protean II electrophoresis apparatus (supplied by BioRad Laboratories, Hercules, CA, USA) was used. The gel was silver stained following the procedure as described by Nielsen and Brown [19]. 2.9. Total protein assay The total protein was assayed following the procedure based on the reaction of proteins with Coomassie brilliant blue R-250 [20] using HCl to acidify the reaction mixture and soroalbumin as standard.

3. Results The degrees of flotation and hydrophobicity (apparent and standard hydrophobicity) were determined for cells of three different yeast genera grown on three different media (YPD, defined, and Sabouraud media) at 30 and 38 8C. In addition, exchanges between pairs of supernatants from different cultures were carried out to study the effects of the supernatant on both flotation and hydrophobicity. 3.1. Flotation and cell-wall hydrophobicity of strains of Saccharomyces cerevisiae In the present work, the flotation capacity of the hydrophobic strain FLT-01 of S. cerevisiae was not dependent on the growth phase (data not shown). 3.1.1. Effect of media composition and temperatures on the growth of strain FLT-01 The effects of both media composition and the growth temperatures on flotation and hydrophobicity (apparent or standard values) of the cells of strain FLT-01 of S. cerevisiae are described in

S.R. deSousa et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319

313

Table 1 Variations in degrees of flotation and hydrophobicity after growth of cells of strain FLT-01 of S. cerevisiae in both defined and Sabouraud media at 30 and 38 8C Strains and growth conditions

Flotation (%, pH 2.2)

Hydrophobicity (%)

Flotation

Efficiency

Apparent (pH 2.2)a

Standard (pH 4.5)b

Defined medium 30 8C 38 8C

95.09/2.1 78.79/7.0

77.99/2.6 75.89/3.6

80.69/3.3 90.69/2.1

97.69/2.6 94.99/1.2

Sabouraud medium 30 8C 38 8C

37.29/3.6 46.69/1.8

13.69/1.9 17.69/1.2

4.89/2.7 13.79/2.8

82.59/2.1 87.09/6.5

YPD medium 30 8C

43.39/7.8

19.89/3.3

25.79/7.0

96.49/0.9

a b

Apparent hydrophobicity for cells suspended in the indicated media (0.5 mg/mlcells) at pH 2.2. Standard hydrophobicity for washed cells resuspended in 0.05 M sodium acetate buffer solution at pH 4.5.

Table 1. The highest degrees of flotation were obtained in defined medium. That is, cells of strain FLT-01 grown in defined medium showed higher degrees of flotation and flotation efficiency than cells grown in Sabouraud and YPD media containing proteinaceous materials (neoptone and peptone, respectively). In addition, the degrees of apparent hydrophobicity were more closely related to the degrees of flotation than the degrees of standard hydrophobicity. The decreases in flotation capacity were followed by decreases in apparent hydrophobicity either in Sabouraud or YPD media. The values of standard hydrophobicity (cells suspended in acetate buffer solution at pH 4.5) were high in the three media (82.5 /98.4%) and not affected by the growth temperature.

3.1.2. Alterations in supernatants of strain FLT-01 before flotation Table 2 describes the alterations in flotation and apparent hydrophobicity of cells of strains FLT-01 resulting from modifications in the supernatants (addition of NaCl to washed cells resuspended in water, additions of neopeptone and ethanol to the culture propagated in defined medium and dilutions of cultures with water). The additions of neopeptone to the cultures (before flotation) reduced the degree of both flotation and apparent hydrophobicity while the addition of ethanol reduced flotation without affecting the apparent

hydrophobicity. Nevertheless, decreases in flotation were observed with the addition of ethanol at a concentration ]/10% (v/v). The flotation degree was extremely reduced in pure water but the flotation was fully restored with the addition of sodium chloride (5%, w/v) to the washed cells resuspended in water. In addition, dilutions of the culture with water led to small effects on apparent hydrophobicity but the flotation degree decreased.

3.1.3. Exchanging supernatants between pairs of different cultures before flotation In order to establish correlation between the supernatants of cells and both the flotation capacity and apparent hydrophobicity, exchanges between pairs of supernatants from two different cultures were carried out before flotation (Table 3). The differences in supernatants (concerning their components and physico-chemical properties before flotation) were more related to flotation degree than the degrees of apparent hydrophobicity of cells. For instance, the degrees of both flotation and apparent hydrophobicity obtained for cells of strain FLT-01 grown in Sabouraud medium were low but both flotation and apparent hydrophobicity were raised when these cells were re-suspended in the supernatant resulting from growth in defined medium. Cells of this yeast lost their flotation capacity when resuspended in pure water. In water, the apparent hydrophobicity was

S.R. deSousa et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319

314

Table 2 Variations in flotation and apparent hydrophobicity resulting from alterations (before assays) made to the supernatants of cultures of strain FLT-01 of S. cerevisiae propagated in defined medium at 30 8C Supernatants

Flotation (%, pH 2.2) Flotation

Efficiency

Apparent hydrophobicity (%, pH 2.2)a

Control experiment (w/o alterations)

95.09/2.1

77.99/2.6

80.69/3.3

Dilutions of supernatants with water (1:1, v/v) (1:3, v/v) NaCl (5%, w/v) added to washed cells resuspended in water Neopeptone (0.5%, w/v) added to the supernatant

81.59/4.0 64.99/3.5 94.49/2.8 66.79/3.2

80.89/3.5 64.39/3.4 52.59/2.3 20.19/0.5

92.29/2.5 93.89/3.2 94.29/3.3 13.09/6.0

Ethanol added to the supernatant (10%, v/v) (20%, v/v)

74.79/1.7 4.69/4.0

66.99/1.6 4.69/4.0

86.89/5.2 89.89/2.1

a

Apparent hydrophobicity for cells suspended in the indicated (0.5 mg/mlcells) supernatants at pH 2.2.

high, but flotation did not occur because foam was not formed. Cells of baker’s yeast, strain LTU, showed very low degrees of both flotation and hydrophobicity after growth on defined medium (Table 3). However, flotation did not occur when these cells (strain LTU) were resuspended in the supernatant resulting from growth of strain FLT-01 (cells showing both elevated hydrophobicity and flotation capacity) in defined medium. On the other

hand, cells of strain FLT-01 grown on defined medium did not loose their high flotation capacity when resuspended in the supernatant resulting from growth of strain LTU, but the flotation efficiency declined. 3.1.4. Changes in protein distribution during flotation Flotation was carried out with cells of strain FLT-01/4b of S. cerevisiae grown up to the

Table 3 Variations in flotation and apparent hydrophobicity resulting from alterations (before assays) made to supernatants of cultures of S. cerevisiae (strains FLT-01 and LTU) propagated at 30 8C in Sabouraud and defined media Growth media (strains)

Supernatants

Flotation (%, pH 2.2)

Apparent hydrophobicity (%, pH 2.2)a

Flotation Efficiency Sabouraud medium (FLT-01)

Defined medium (LTU) Defined medium (FLT-01)

a

Without alteration Resulting from growth of strain FLT-01 medium Washed cells re- suspended in water Without alteration Resulting from growth of strain FLT-01 medium Without alteration Washed cells resuspended in water Resulting from growth of strain FLT-01 aud medium Resulting from growth of strain LTU in medium

in defined

37.29/3.6 13.69/1.9 4.89/2.7 78.99/2.5 58.99/1.9 31.19/1.0

in defined

2.49/1.4 2.49/1.4 88.19/5.8 31.69/0.8 20.89/1.6 4.99/2.3 24.69/2.6 21.49/2.1 21.69/4.1

95.09/2.1 77.99/2.6 80.69/3.3 2.79/2.2 2.79/2.2 89.39/4.2 in Sabour- 42.79/1.0 21.79/1.6 18.79/6.0 defined

91.79/1.1 48.09/3.5 86.89/1.3

Apparent hydrophobicity for cells suspended in the indicated media (0.5 mg/mlcells) at pH 2.2.

S.R. deSousa et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319

stationary phase in 2 l fermenter to obtain enough volumes of cell-wall extracts and supernatants to be used in gel electophoresis. The values of flotation and hydrophobicity resulting from the growth in the fermenters were as follows (not described in tables): 85.69/0.1% flotation, 17.09/ 8.0% apparent hydrophobicity, and 72.49/2.4% standard hydrophobicity. Cell-wall extracts obtained from cells present in the medium (before and after flotation) and collected from the foam exhibited similar protein profiles, which showed a large number of bands (Fig. 1). However, protein bands obtained for the cells harvested from the foam were slightly more intense. On the other hand, the supernatants of the cells, harvested from the liquid foam and from the medium (before and after flotation), showed a small number of proteins bands in their electrophoretic profiles (Fig. 1) as follows: a protein band of high molecular size (330 kDa), two narrow bands (molecular sizes between 107 and 120 kDa) and two very intense bands

315

(molecular sizes of around 60/67 kDa) were identified among the proteins, which were present in the medium and accumulated in the foam during flotation. 3.2. Flotation and cell-wall hydrophobicity of Candida albicans Cells of C. albicans were grown in Sabouraud and defined media at both 30 and 38 8C (Table 4). These yeast cells showed high degrees of both flotation and hydrophobicity after growth in defined medium at 30 8C. After growth at 38 8C, cells of C. albicans exhibited remarkable lower degrees of hydrophobicity (both apparent and standard hydrophobicity) and lower degrees of adhesion to air bubbles. However, a partial recovery in flotation capacity was obtained when cells grown in defined medium at 38 8C were resuspended in the supernatant resulting from the growth of the same yeast in defined medium at 30 8C. In addition, cells grown at 30 8C lost their flotation capacity when resuspended in the supernatant resulting from growth at 38 8C. 3.3. Flotation and cell-wall hydrophobicity of Hansenulla polymorpha

Fig. 1. Silver stained polyacrylamide gel (a 12.5% SDS-PAGE) showing protein bands for samples (supernatants and cell extracts) taken from a 2 l fermenter: Line 1a /5 ml standard solution of denatured proteins (LMW standard); Line 2a /4 ml wall-extract of cells collected from the culture before flotation; Line 3a/4 ml wall extract of cells collected from the foam; Line 4a/4 ml wall-extract of cells collected from the residual medium left in the column after flotation; Line 1b/23 ml concentrated supernatant obtained from the culture left in the column after flotation; Line 2b/23 ml concentrated supernatant obtained from the culture before flotation; Line 3b /23 ml concentrated supernatant obtained from the supernatant of the liquid foam resulting from the foam collapse; Line 4b/5 ml standard solution of denatured proteins (HMW standard).

Cells of H. polymorpha were also grown in Sabouraud and defined media at 30 and 38 8C (Table 5). The degrees of flotation were higher in the defined medium than in Sabouraud medium as also observed for strain FLT-01 of S. cerevisiae (Table 1). The addition of neopeptone to the defined medium did not repressed flotation of H. polymorpha (Table 5) while flotation of strain FLT-01 of S. cerevisiae was repressed in the presence of neopeptone (Table 2). However, partial recoveries in both flotation and flotation efficiency were obtained, when cells of H. polymorpha (propagated in Sabouraud medium at 30 8C) were re-suspended in a supernatant resulting from growth of the cells in defined medium at 30 8C. In Sabouraud medium, the values of standard hydrophobicity were high after growth at both 30 and 38 8C while the values of apparent hydrophobicity were much lower. Thus, the growth temperature did not exert any significant

S.R. deSousa et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319

316

Table 4 Variations in degrees of flotation and hydrophobicity of cells of strain FCFAR-250 of C. albicans , resulting from alterations in growth conditions (media and temperatures) and from exchanges of supernatants between pairs of two different cultures carried out before assays Growth conditions Supernatants

Defined medium 30 8C 30 8C 38 8C 38 8C

Flotation

Efficiency

Apparent (pH 2.2)a Standard (pH 4.5)b

Without alteration 74.49/1.1 Supernatant resulting from growth at 38 8C 6.09/1.5 Without alteration 6.09/1.4 Supernatant resulting from growth at 30 8C 50.39/2.5

39.19/1.8 5.79/1.3 6.09/1.4 47.79/3.3

78.49/1.5 76.69/4.2 6.39/1.2 11.59/4.5

80.09/4.2 79.49/3.2 12.69/3.0 42.59/1.5

2.79/1.5 2.69/1.8

2.99/1.5 4.89/3.2

14.69/1.9 11.19/6.7

Sabouraud medium 30 8C Without alteration 38 8C Without alteration a b

Flotation (%, pH 2.2) Hydrophobicity (%)

5.39/1.5 5.09/3.0

Apparent hydrophobicity for cells suspended in the indicated media (0.5 mg/mlcells) at pH 2.2. Standard hydrophobicity for washed cells re-suspended in 0.05 M sodium acetate buffer solution (0.5 mg/mlcells) at pH 4.5.

effect on flotation and wall hydrophobicity in Sabouraud medium.

4. Discussion Flotation and formation of cell aggregates (cell / cell interactions) can be independent phenomena as shown by strains FLT-01 [4] and DMS 2155 [2] of S. cerevisiae. The hydrophobic cells of this strain did not form cell /cell aggregates but showed great affinity to air bubbles [4]. In addition, foam formation and cell affinity to air

bubbles can also occur as independent phenomena (each phenomenon can occur without the other occurring) as shown in the present work. In this work, the adherence of yeast cells to hexane occurred in the assay of hydrophobicity indicating that the global charge of these cells possibly supplanted hydrophobicity allowing their attachment to the hexane. Hexane was described in literature as having a negative charge when its droplets (resulting from agitation) come into contact with a phosphate buffer solution at pH 7.0 [21]. In addition, the flotation capacity of the hydrophobic strain FLT-01 of S. cerevisiae was

Table 5 Degrees of flotation and hydrophobicity resulting from alterations in growth conditions (media and temperatures) and in supernatants (addition of neopeptone and exchanges of supernatants involving pairs of two different cultures before the assays) of strain CBS 4732 of H. polymorpha Growth conditions

Supernatants

Flotation (%)

Hydrophobicity (%)

Flotation Efficiency Apparent (pH 2.2)a

Standard (pH 4.5)b

Defined medium 30 8C 30 8C 38 8C

Without alteration Addition of 0.5%, (w/v) neopeptone Without alteration

85.69/2.3 67.39/4.6 78.29/3.4 41.29/5.3 78.99/1.3 69.39/2.6

24.39/2.8 34.79/6.8 23.8 9/.3.0

96.29/1.4 87.59/2.4 84.19/1.6

Sabouraud medium 30 8C 30 8C 38 8C

Without alteration Resulting from growth of H. polymorpha Without alteration

47.09/5.8 25.79/1.1 61.49/8.5 52.29/9.1 53.09/7.6 22.79/3.9

14.89/5.2 15.29/2.5 12.59/1.9

64.79/1.7 67.39/2.2 73.59/7.1

a b

Apparent hydrophobicity for cells suspended in the indicated media (0.5 mg/mlcells) at pH 2.2. Standard hydrophobicity for washed cells re-suspended in 0.05 M sodium acetate buffer solution (0.5 mg/mlcells) at pH 4.5.

S.R. deSousa et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319

not dependent on the growth phase. This is in agreement with the behavior of a strain of sake yeast capable of flotation during growth [22]. It seemed that the lower the values of apparent hydrophobicity of strain FLT-01 of S. cerevisiae, the lower the values obtained for flotation. Values of apparent hydrophobicity (cells suspended in the growth medium) were closer to the degree of flotation than the values of standard hydrophobicity (obtained for cells suspended in buffer solution, [15]), indicating dependence of flotation on the supernatant. In a previous work, variations in the concentration of each component of defined medium (e.g. carbon and nitrogen sources) did not lead to variations in degrees of flotation capacity of strain FLT-01 of S. cerevisiae [23]. This indicated that flotation is more dependent on the interactions that occurred between the cell surface and the liquid phase at the cell/medium interface rather than on individual components of the medium. Ethanol reduced flotation (including the flotation efficiency) but not the apparent hydrophobicity of cells of strain FLT-01, when added to the cultures resulting from growth in defined medium at concentrations ]/10% (v/v) before flotation. However, the degrees of standard hydrophobicity were kept at a high value. It is known that alcohol acts as a frother at low concentrations but induces foam collapse at high concentrations [13]. The ethanol addition decreased flotation efficiency as a consequence of increases in the foam volume. In fact, the degrees of flotation efficiency (expressing the capacity of the process to concentrate cells in the foam) declined whenever the foam volume increased. In water, the apparent hydrophobicity of cells of strain FLT-01 was high but foam was not formed and flotation did not occur. This indicates that a high degree of standard hydrophobicity cannot be used as the sole parameter to predict flotation. Simple experiments based on the exchange of supernatant between pairs of the two different cultures of strains of S. cerevisiae (such as strains FLT-01 and baker’s yeast LTU grown in defined medium) did not lead to modifications in their flotation capacity. This indicated that the supernatants of strain LTU propagated in defined-

317

medium did not contain any flotation depressant, while strain FLT-01 did not produce any unspecific flotation agent (independent of strain used) able to induce flotation in strain LTU. Thus, cells of hydrophobic strain FLT-01 simply exhibited less affinity to the defined medium (liquid phase) than to the more hydrophilic cells of strain LTU. The simple dilution of the culture of strain FLT-01 was able to reduce flotation without affecting the standard hydrophobicity, which is more related to changes in cell-wall than in supernatant composition. This indicates that the global ionic strength of the liquid phase plays an important role on flotation. Another possibility is that a flotation agent (activator) present in the culture might have a critical concentration below which, a decline in the flotation capacity occurs. In the present work, the highest values of both flotation and hydrophobicity occurred for yeast cells of Saccharomyces , Candida , and Hansenula , grown in defined medium deprived of proteinaceous materials (proteins or peptides). In addition, the experiments based on the exchanges between pairs of supernatant from two different cultures indicated that the supernatant composition was the major determinant of yeast flotation. For example, when the cells of strain FLT-01 propagated in Sabouraud medium (containing neopeptone) were re-suspended in the supernatant resulting from propagation in defined medium, the flotation capacity was recovered. This indicates that neopeptone (used in the preparation of the Sabouraud medium) exerted an inhibitory effect of on flotation. The addition of neopeptone to a supernatant of the cells of strain FLT-01 of S. cerevisiae (also propagated in defined medium) led to significant decreases in flotation and apparent hydrophobicity. The decreases in apparent hydrophobicity of cells of Saccharomyces and Candida in the presence of neopeptone indicated that the wall properties of the cells changed possibly as a consequence of the attachment of the proteinaceous material to the cell surface. Dramatic increases in the volume of the foam were observed in the presence of neopeptone. Froth-forming sake yeast cells showed low flotability when casamino acids were added in excess to the medium [24].

318

S.R. deSousa et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319

Regarding apparent hydrophobicity, cells of S. cerevisiae and C. albicans , grown in defined medium at 30 8C, showed an opposite behavior when compared with cells of H. polymorpha . That is, values of apparent hydrophobicity shown by cells of H. polymorpha propagated in defined medium were lower than the values obtained for cells of the two other yeasts. This indicates that the cell surface of H. polymorpha became more hydrophilic than the cell surfaces of Saccharomyces and Candida . An explanation for this fact found in literature [25] is that exoproteins (glycoproteins) secreted by the cells H. polymorpha possibly acted as collectors and/or foamers (as defined by the flotation technique) allowing flotation. In the present work, strain FLT-01/4b propagated in a fermenter showed a degree of flotation of around 85.69/0.1% but its apparent hydrophobicity was only 17.09/8.0%. This is in agreement with the thought that hydrophobicity cannot be used as the sole parameter for prediction of the flotation capacity. For example, in the culture of strain FLT-01/4b both the flotation and the formation (see Fig. 1) of a proteinenriched foam occurred despite the low value of cell-wall apparent hydrophobicity. It is known that substances produced by cells can act as flothing agents, (increasing the affinity of the cells to the air bubbles) or foamers (inducing foam formation). Flothers and collectors are names used in flotation processes [13]. Although controversial results are described in literature [26 /29], both the growth temperature and medium composition showed a strong influence on the cell surface hydrophobicity of most strains of C. albicans . A strong adhesion to buccal epithelial cells was observed when cells of C. albicans were grown at 25 8C rather than at 37 8C [28], suggesting that an extracellular factor synthesized at 25 8C would play an important role in the buccal infections. In the present work, a pathogenic strain of C. albicans lost both its flotation capacity and hydrophobicity after growth in and defined medium at 38 8C. However, flotation of cells of C. albicans grown at 38 8C in defined medium was partially recovered when cells were resuspended in the supernatant of the culture propagated at 30 8C in the same medium. This

reinforced the thought that cells of C. albicans grown at 30 8C possibly synthesized a flotation activator during growth at 30 8C but not at 38 8C. In defined medium, the surface properties of C. albicans (flotation and hydrophobicity) were more affected by the growth temperature than the surface properties of cells of S. cerevisiae and Hansenula polymorpha propagated in the same medium.

5. Conclusions Yeast flotation depends on foam formation and increasing affinity of hydrophobic cells to air bubbles at the cell/air interfaces. This is then followed by a decreasing affinity of cells to the medium at the medium/cell interface. In addition, cell surface heterogeneity and complexity of the surface forces are greater in the microbial cells than in mineral particles. As shown here, a simple dilution of the culture with water can affect flotation. Trials for improving flotation based on simple modifications to the supernatant of the cultures can be carried out using a simple device such as that described by deSousa and Laluce [14]. Variations in ionic strength, surface tension, charge and hydrophobic interactions at both the cell/supernatant and cell/air bubble interfaces can improve the flotation capacity. Recovery of mica (mineral particle) by flotation was achieved by adding an organic surfactant to the mineral suspension flowed by an appropriated adjustment of the pH [30]. However, the organic surfactants commonly used in particle flotation can cause an unwanted damage to the cell-wall.

Acknowledgements This research was supported by a grant from FAPESP (Proc. no. 1998/4299-9), and the authors also wish to thank FAPESP for the fellowship awarded to Sandro R. de Sousa (Proc. no.98/ 16224-3). The authors are also grateful to Fernando Delfino for his valuable collaboration in coordinating all the technical procedures under way in our laboratory during the present research.

S.R. deSousa et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 309 /319

References [1] J.K. Edzwald, Water Sci. Technol. 31 (1995) 1. [2] R. Gehle, T. Sie, T. Kramer, K. Schugerl, J. Biotechnol. 17 (1991) 147. [3] K.H. Bahr, K. Schugerl, Chem. Eng. Sci. 47 (1992) 11. [4] M.C. Palmieri, W. Greenhalf, C. Laluce, Biotechnol. Bioeng. 50 (1996) 248. [5] P. Romano, G. Suzzi, L. Vannin, Colloids Surf. B: Biointerf. 2 (1994) 511. [6] D.S. Jones, C.G. Adair, W.M. Mawhinney, S.P. Gorman, Int. J. Pharm. 131 (1996) 83. [7] F. Ahimou, M. Paquot, P. Jacques, P. Thonart, P.G. Rouxhet, J. Microbiol. Methods 45 (2001) 119. [8] M. Rosenberg, Crit. Rev. Microbiol. 18 (1991) 159. [9] G.I. Geertsema-Doornbusch, H.C. van der Mei, H.J. Busscher, J. Microbiol. Methods 18 (1993) 61. [10] H.J. Busscher, B. van de Belt-Gritter, H.C. van der Mei, Colloids Surf. B: Biointerf. 5 (1995) 111. [11] H.C. van der Mei, B. van der Belt-Gritter, H.J. Busscher, Colloids Surf. B: Biointerf. 5 (1995) 117. [12] P.G. Rouxhet, N. Mozes, P.B. Dengis, Y.F. Dufreˆne, P.A. Gerin, M.J. Genet, Colloids Surf. B: Biointerf. 2 (1994) 369. [13] W. Stumm, Chemistry of the Solid /Water Interface. Processes at the Mineral /Water and Particle /Water Interface in Natural Systems (Chapter 7), Wiley, New York, 1992. [14] S.R. deSousa, C. Laluce, Biotechnol. Lett. 22 (2000) 753. [15] M. Rosenberg, D. Gutnick, E. Rosenberg, FEMS Microbiol. Lett. 9 (1980) 29. [16] G.A. Farris, M. Sinigaglia, M. Budroni, M.E. Guerzoni, Lett. Appl. Microbiol. 17 (1993) 215.

319

[17] S.M. Jazwinski, in: M.P. Deutscher (Ed.), Methods in Enzymology, vol. 182, Academic Press, New York, 1990, p. 154. [18] N. Rawat, in: D. Hames, D. Hames Rickwood (Eds.), Gel Electrophoresis of Proteins. A Practical Approach, IRL Press, Oxford, 1990, p. 30. [19] B.L. Nielsen, L.R. Brown, Anal. Biochem. 141 (1984) 311. [20] C.M. Stoscheck, in: M.P. Deutscher (Ed.), Methods in Enzymology, vol. 182, Academic Press, New York, 1990, p. 50. [21] Boss, H.C. van der Mei, H.J. Busscher, FEMS Microbiol. Rev. 23 (1999) 179. [22] Y. Nunokawa, H. Toba, K. Ouchi, J. Ferment. Technol. 49 (1971) 959. [23] C. Duarte, Flotac¸a˜o espontaˆnea de leveduras, Ph.D. thesis, Faculdade de Filosofia Cieˆncias e Letras da USP de Ribeira˜o Preto, Brazil, 2001. [24] Y. Nunokawa, Y. Sato, K. Ouchi, J. Ferment. Technol. 51 (1973) 551. [25] H. Bahr, H. Weisser, K. Schugerl, Enzyme Microbiol. Technol. 13 (1991) 747. [26] K.C. Hazen, B.W. Hazen, J. Microbiol. Methods 6 (1987) 289. [27] B.W. Hazen, K.C. Hazen, Infect. Immunol. 56 (1988) 2521. [28] J.H. Samaranayake, P.C. Wu, L.P. Samaranayake, M. So, APMIS 103 (1995) 707. [29] M. Kennedy, R.L. Sandin, J. Med. Vet. Mycol. 26 (1988) 79. [30] R.J. Pugh, M.W. Rutland, E. Manev, P.M. Claesson, Int. J. Miner. Proc. 46 (1996) 245.