Acta Biotechnologica: Volume 6, Number 3 [Reprint 2021 ed.] 9783112533024, 9783112533017

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Atta II

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JL Ä

Journal of microbial, biochemical and bioanalogous technology

Akademie-Verlag Berlin ISSN 0138- 4988 Acta Biotechnol., Berlin 6 (1986) 3, 207 - 310

Volume 6 • 1986 • Number 3

Instructions to Authors

1. Only original papers that have not been published previously will be accepted. Manuscripts may be submitted in English, German or Russian (in dublicate). The name of the institute (with the full address) from which the manuscript originates should be stated below the name(s) of the author(s). The authors are responsible for the content of their contributions. 2. Original papers should not consist of more than 20 typewritten pages (two-lined spacing), including references, tables and figures; short original communications may have a maximum of six typewritten pages. 3. Eaoh paper should be preceded by a summary in English and also by an English translation of the title. 4. Latin names of species as well as passages to be printed in italics for greater emphasis should be denoted by a waving line. Please use only units and symbols of the Si-system. 5. Tables may be used to shorten the text or to make understanding easier. They should be numbered consecutively throughout and supplied with a brief heading. They should not appear in the text, but should be written on separate sheets. 6. The numbers and sizes of illustrations should be limited to the essential minimum, they should be numbered consecutively and be supplied on separate sheets. Line drawings, including graphs and diagrams, should be drawn in black ink. Half-tone illustrations should be supplied as white glossy prints. Figure legends are to be typed in sequence on a separate sheet. The name(s) of the author(s) should be written on the back of each sheet. 7. References listed at the end of the contribution should contain only works quoted in the text. They should be numbered in the order in which they are first mentioned in the text. Please give surnames and initials of all authors, the name of the journal abbreviated according to "Chemical Abstracts — List of Periodicals", volume number, year of publication, issue number or month, first page number. Books are to be cited with full title, edition, volume number, page number, place of publication, publisher and year of publication. 8. Notes to the text may be given as footnotes on the same page. 9. 50 printed copies are free of charge. Additional reprints may be ordered (at full cost). 10. The author will receive two galley proofs for correction. They are to be returned to the managing editor (Permoserstr. 15, Leipzig, 7050 DDR, Dr. L. Dimter) as soon as possible.

Acta BiotBctMlaiica Journal of microbial, biochemical and bioanalogous technology

Edited at the Institute of Biotechnology of the Academy of Sciences of the G.D.R., Leipzig and at the Kombinat of Chemical Plant Construction Leipzig—Grimma by M. Ringpfeil, Leipzig and G. Vetterlein, Leipzig

Editorial Board: P. Moschinski, Lodz A. Moser, Graz M. D. Nicu, Bucharest Chr. Panajotov, Sofia L. D. Phai, Hanoi H. Sahm, Jülich W. Scheler, Berlin R. Schulze, Kothen B. Sikyta, Prague G. K. Skrjabin, Moscow M. A. Urrutia, Habana J . E. Zajic, El Paso

1986

A. A. Bajev, Moscow M. E. Beker, Riga H. W. Blanch, Berkeley S. Fukui, Kyoto H. G. Gyllenberg, Helsinki G. Hamer, Zurich J . Holló, Budapest M. V. Iwanow, Moscow P. Jung, Berlin H. W. D. Katinger, Vienna K. A. Kalunjanz, Moscow J . M. Lebeault, Compiégne D. Meyer, Leipzig

Number 3

Managing Editor:

L. Himter, Leipzig

Volume 6

AKADEM I E-VERLAG

•

B E R L I N

"Acta Biotechnologica" publishes original papers, short communications, reports and reviews from the whole field of biotechnology. The journal is to promote the establishment of biotechnology as a new and integrated scientific field. The field of biotechnology covers microbial technology, biochemical technology and the technology of synthesizing and applying bioanalogous reaction systems. The technological character of the journal is guaranteed by the fact that papers on microbiology, biochemistry, chemistry and physics must clearly have technological relevance. Terms of subscription for the journal "Acta Biotechnologica" Orders can be sent — in the GDR: to Postzeitungsvertrieb or to the Akademie-Verlag Berlin, Leipziger Str. 3—4, P F 1233, Berlin, 1086 D D R ; — in the other socialist countries: to a book-shop for foreign languages literature or to the competent news-distributing agency; — in the FRG and Berlin (West): to a book-shop or to the wholesale distributing agency Kunst und Wissen, Erich Bieber, Wilhelmstr'. 4 - 6 , D-7000 Stuttgart 1; — in the other Western European countries: to Kunst und Wissen, Erich Bieber GmbH, General Wille-Str. 4, CH-8002 Zürich; — in other countries: to the international book- and journal-selling trade, to Buchexport, Volkseigener Außenhandelsbetrieb der DDR, P F 160, Leipzig, 7010 DDR, or to the Akademie-Verlag Berlin, Leipziger Str. 3—4, P F 1233, Berlin, 1086 DDR. Acta Biotechnologica Herausgeber: Institut für Biotechnologie der AdW, Permoserstr. 15, Leipzig, 7050 DDR (Direktor: Prof. Dr. Manfred Ringpfeil) und VEB Chemieanlagenbaukombinat Leipzig—Grimma, Bahnhofstr. 3 - 5 , Grimma, 7240 DDR (Generaldirektor: Obering. G. Wohllebe). Verlag: Akademie-Verlag Berlin, Leipziger Straße 3 - 4 , P F 1233, Berlin, 1086 DDR; Fernruf: 2236201 und 2236229; Telex-Nr.: 114420; Bank: Staatsbank der DDR, Berlin, Konto-Nr.: 6836-26-20712. Redaktion: Dr. Lothar Dimter (Chefredakteur), Käthe Geyler (Redakteur), Permoserstr. 15, Leipzig, 7050 D D R ; Tel.: 2392255. Veröffentlicht unter der Lizenznummer 1671 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: VEB Druckhaus „Maxim Gorki", Altenburg, 7400 DDR. Erscheinungsweise: Die Zeitschrift „Acta Biotechnologica" erscheint jährlich in einem Band mit 4 Heften. Bezugspreis eines Bandes 120,—DM zuzüglich Versandspesen; Preis je Heft 30,—DM. Der gültige Jahresbezugspreis für die DDR ist der Postzeitungsliste zu entnehmen. Bestellnummer dieses Heftes: 1094/6/3. Urheberrecht: Alle Rechte vorbehalten, insbesondere der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translation into foreign languages). No part of this issue may be reproduced in any form, by photoprint, microfilm or any other means, without written permission from the publishers. © 1986 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 42133 03000

Aota Biotechnol. 6 (1986) 3, 209-214

Production of a White Wine and a Protein-Rich Soy Flour by Yeast Fermentation of Soybean Slurry, Soybean Milk and the Whey from Tofu Production Y E E , V . , W E L L I N G T O N , G . H . , O L E K , A . , STEINKBATJS, K . H .

Institute of Food Science Cornell University Geneva, New York 14456, U.S.A.

Summary A dry white wine with an alcoholic content of 10 to 14% v/v was produced by yeast fermentation of slurried ground soybeans, soybean milk and whey from tofu production. Wines from whey and soybean milk were judged by a 20 member taste panel to be acceptable and comparable to a commercial chablis control. Chemical analysis indicated that the high fat and protein contents of soybeans do not cause a problem in the production of wines from soybeans as the lipids and proteins are precipitated by the acid and alcohol formed during the fermentation. The lees recovered following fermentation were dehydrated and ground to a flour having an enriched protein content due to the yeasts and an improved flavor resulting from the yeast fermentation.

Introduction Soybean acceptability as a food is limited by the "beany" flavor and the relatively long cooking times. STEINKRATTS [ 1 ] patented a process in which ethanol was used as a first-stage to improve the flavor of soy flour. Yeast fermentations of either slurried soybeans, soymilk or whey obtained by calcium or acid precipitation of soybean milk (tofu) may be another method of improving the flavor and other organoleptic characteristics of the products (soy flour) recovered from such fermentations. By adding sufficient fermentable sugar to the soybean fractions, it is possible to produce soy wines with ethanol levels of 10 to 15% v/v comparable to many wines. The ethanol and organic acids produced (or added) will precipitate the proteins and it would be expected that the lipids would accompany the protein into the precipitate. The ethanol produced would be expected to modify and hopefully improve the flavor of the soybean flour. The yeast cells accumulating in the lees would result in a higher protein content with possibly improved nutritional value. Potm-EL and R E D D Y [ 2 ] patented a process whereby the whey from an acid process of isolating soy protein was fermented with a species of Saccharomyces. The whey was fortified with fermentable sugar and the final ethanol content ranged from 1 0 to 2 0 % v/v. STTGIMOTO [ 3 ] used a yeast Debaromyces sp. to remove 8 4 % of the biological oxygen demand (BOD) from spent soybean solubles waste. C H I E N [ 4 ] grew a yeast aerobically in a soybean flour slurry to raise the protein content of the flour. Steeping or washing soybeans in ethanol has been shown to remove bitter, beany flavors [ 1 , 5 — 8 ] . MUSTAKAS et al. [ 9 ] used ethanol/isopropanol for the same purpose. B R A D O F [10] produced a bland soybean flour by the use of yeast along with oxidizing and neutra1»

210

Acta Biotechnol. 6 (1986) 3

lizing agents. FTTKUSHIMA [11, 12] reported t h a t the 7S and 11S proteins of soybean

were denatured more rapidly the higher the hydrophobicity of the alcohol at low alcohol concentrations. Under high concentrations of alcohols, the lesser the hydrophobicity the greater the denaturing capacity of the alcohol. This study was undertaken to determine if a satisfactory white wine could be produced by a yeast fermentation of soybeans slurried with water or the filtrate of such a slurry (soymilk) or the whey recovered following calcium sulfate precipitation of the curd (tofu). The residues (lees) of the fermentations were recovered and tested for organoleptic quality and protein enhancement.

Materials and Methods Soybean slurry, soymilk, soymilk residue and soy whey were prepared as follows: soybeans (CORSOY variety) were soaked in 45°C water until they had doubled in weight. The soybeans were then washed, drained, and ground with 90 °C water, using a 1: 9 w/v dry bean to water ratio, in a WAKING blender for 5 min. The resulting slurry was filtered under reduced pressure through a BUCHNEK funnel lined with two layers of Agway Kleen Test 300 milk filters. The soymilk filtrate was then boiled in a steam-jacketed kettle for 5 min. The boiled filtrate was used as a fermentation medium. Soymilk residue used as a fermentation medium was obtained by reconstituting the soymilk spent solids with water to its original volume before the slurry was filtered. The soymilk residue was also cooked and cooled before further use. To prepare soy whey for fermentation, CaS0 4 (0.4% w/v) was stirred into cooked soymilk and the curd allowed to precipitate for 30 min at 75 °C. The resulting precipitate (curd) was cut and transferred to a perforated box lined with 10 layers of cheese cloth. The whey was allowed to drain freely away for a few minutes. While the curd was still warm, it was subjected to compression in a Carver Laboratory Press for 5 min at 45 psi. Defatted soy grits (Central Soya) were also processed in the same way as the whole soybeans to yield the soy slurry and soymilk. A 1: 9 water ratio (based on the protein weight of soy grits per volume of water) was used when soy grits were ground to form the soy slurry.

Fermentation The fermentation media were ameliorated to 21—25° Brix with sucrose; a brix hydrometer was used to measure soluble solids. Citric acid was added to adjust the p H to 3.8. The fermentation was initiated by the addition of 1 g dry yeast (S. cerevisiae var. ellipsoideus) per liter of medium. The media were then placed in bottles corked with waterseal traps, which were thinly layered with paraffin oil. The bottles were swirled to mix the yeast and medium. The fermentation process was allowed to proceed for 2 weeks at 20 °C. Brix and p H were measured periodically during the fermentation. The wines were racked and aged for 9 months at 5°C. Lees from the fermentations were removed by centrifugation, freeze-dried for 3 days and ground, yielding a fine white soy-yeast flour.

Chemical Analysis A SALLERON DUJABDIN ebulliometer was used to determine t h e alcoholic contents of the

wines. Total acidity, volatile acidity and higher alcohols (fusel oils) were measured by t h e m e t h o d s of GUYMON a n d OTTGH [13], GOWANS [14], GUYMON a n d HEITZ [15]. AOAC

[16] methods 31.034—31.036 ai^d 30.081 were used for t h e determination of reducing

sugars and sulfates.

YEE, V., WELLINGTON, G. H. et al., Production of a White Wine Crude protein content of the lees from the fermentation process was determined by the KJELDAHL method [16]. Lipids were determined by ether extraction using method 7.062 [16]. Following extraction with diethyl ether for 72 h in a soxhlet extractor, the samples were dried in a vacuum oven for 24 h before weighing. Ash was determined by heating the lees at 550 °C for 24 h. A BECKMAN-SPINCO Model 120C Amino Acid Analyser was used for amino acid analysis. Protein samples of 16 mg each were prepared for analysis by digestion in an ampoule with 5 ml conc HC1, 4 ml water, and 1 ml norleucine solution (internal standard). After freezing the contents of the ampoule, it was evacuated to less than 50 u pressure and heated to 110°C for 22 h. The hydrolysate was then filtered, and 2 ml of the filtrate was dried under NaOH in a vacuum desiccator for about 18 h. The dried sample was dissolved in 4 ml p H 2.2 sodium citrate buffer. Peak areas from the chromatograms were determined by triangulation, and the amount of amino acid was reported as amount per 16 g nitrogen. Protein quality [17] was calculated as proposed by USDA- APHIS (100 X (Lys + Thr + Val + Met + lie + Leu + Phe + Trp)/total amino acid). Organoleptic Evaluation Randomized samples were served to 20 semi-trained panelists. New York Chablis wine: (Great Western) was used for comparison. The panelists were asked to evaluate characteristics such as clarity, color, aroma, bouquet, total acidity, tannin, body, sugar, general flavor and overall acceptability on a score sheet from the American Wine Society. Results and Discussion Soy Wines The taste panel found all the soy wines to be acceptable. Wines made from whey and from acidified soy milk and soybean slurry received scores comparable to panel scores for New York State chablis wine (Great Western) used as a control. Wines made from defatted soy grits had a slight off flavor. The chemical evaluation of the wines is presented in Table 1. Although there were no significant compositional differences (P < 0.01) between wines made from different media (10—14% alcohol by vol., 0.20—0.35 g/1 fusel oil, 0 . 3 - 5 . 0 g/100 ml reducing sugar, 3.1—6.8 g/1 total solids except alcohols), their fermentation processes proceeded by different modes. While the p H of the acidified media remained constant at about p H 3.8 throughout the fermentation process, the p H of the unacidified media decreased from 6.6 to 4.5 during the early stages of the fermentation. Wines made from whey and acidified media had lower levels of volatile acidity than wine made from unacidified soybean slurry or unacidified soy milk (P < 0.01). This may be due to the lower p H of whey and acidified soymilks which may inhibit microbial degradation. The concentration of sulfate in soy wines made from soymilks was 10—180 ppm, significantly below the restricted maximum level of 2000 ppm sulfate established for commercial wines. The wines that were produced from whey had a sulfate range of 3050 to 3150 ppm. Sulfate levels were very high in these wines since CaS0 4 was used to precipitate the soy curd, of which whey is a by-product. The use of a non-sulfate coagulant would reduce the sulfate level of wines made from whey to conform within the legal limits. The yield of soy wines from the fermentation process increased in the order of soybean slurry, milk residue, soymilk and soy whey. The yield of dried lees increased in the reverse

212

A c t a Biotechnol. 6 (1986) 3

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213

Table 2. Gross Composition of Soy-Yeast Flour Media Type

°Brix

Protein [%]

Fat[%]

21 21 21 25 21 21 25

31.45 27.37 41.96 42.58 34.44 21.27 15.86

± ± ± ± ± ± ±

0.61 2.53 0.30 0.51 0.73 0.05 0.66

28.71 27.79 36.15 36.39 33.17 19.10 18.56

± ± ± ± ± ± ±

0.21 0.25 0.28 0.42 0.34 2.01 0.61

2.77 2.42 2.40 2.24 2.44 2.28 1.63

21 23 21

64.30 68.25 65.64 35.04 41.17 16.96 69.71 27.51

± 2.01 ± 0.21 ± 0.01

3.17 1.10 1.33 25.32 24.78 6.42 2.14 7.22

± 0.35 ± 0.62 ± 0.08

2.96 2.70 2.38 4.34 5.76 18.01 6.80 2.18

Ash[%]

Whole Soybean Slurry Acidified slurry Milk Milk Acidified milk Milk residue Milk residue

± ± ± ± ± ± ±

0.05 0.10 0.14 0.01 0.06 0.17 0.26

Defatted soy grits Slurry Slurry Acidified slurry Whole soybean Milk Whey Defatted soy slurry Defatted soy milk

± 0.12 ± 0.20

± 0.13

± 0.15 ± 0.13 ± 0.04

± 0.18

order. Whey produced negligible amounts of lees. High f a t and protein contents of soybeans did not hinder the use of soy products in the production of soy wine. Most of the f a t was precipitated with the protein by acid/alcohol denaturation during fermentation. The use of defatted soybean material as a medium did not affect the fermentation. Soy Yeast Flour Grinding the dried lees produced a fine white soy-yeast flour which did not have the typical n u t t y flavor of soy flour. However, the flour did have a slight yeasty flavor. Table 2 shows the gross composition of the soy-yeast flour. The protein content of the lees obtained from milk and slurry media were quite similar to the protein content of the raw products from which they were made. The f a t and ash contents of the lees differ from t h a t of the dried media (P < 0.01). The reduction of ash in the lees relative to the medium was probably caused by the consumption of the inorganic compounds needed for growth by the yeast. Table 3. Protein qualities of soy-yeast flour amino acids [%]

soy slurry

soymilk

milk residue

Lys Thr Val Met He Leu Phe Protein quality [%]

5.9 4.1 4.0 1.1 3.9 7.4 4.4 33.2

5.8 4.5 4.6 1.5 4.3 8.5 5.1 32.9

5.6 4.1 3.9 1.0 3.5 6.9 4.1 34.4

Acta Biotechnol. 6 (1986) 3

214

There were no significant differences in the amino acid composition of soy-yeast flour made from the lees of soy slurry, soymilk and milk residue (Table 3). The protein qualities of the soy-yeast flour meet the minimum legal protein quality for meat products and m a y be used as a meat extender. Received July 29, 1985

References [1] STEINKRATJS, K. H.: Method for defatting soybean meal. U.S. Patent 3,721,569. March 20 (1973). [2] POTJR-EL, A., REDDY, G. V.: Preparation of alcoholic beverages from oil seed whey. U.P. P a t e n t 3,769,437. O c t . 3 0 (1973).

[3] SUGIMOTO, H.: J . Food Sci. 39 (1974), 934. [4] CHIEN, H. C.: Treatment of soybeans with yeast. U.S. Patent 3,810,997. May 14 (1974). [5] TEETER,'H. M., GAST, L . E . , GEU, E . W., SCHNEIDER, W . J . : J . A m . Oil Chem. Soc. 32 (1955),

390. [6] WANG,.L. C.: J . Agric. Food Chem. 17 (1969), 335. [7] STEINKRAUS, K. H.: Process for producing defatted and debittered soybean meal. U.S. Patent 4,496,599. J a n . 29 (1985). [8] ELDRIDOE, A. C.: Chem. E n g . N e w s 5 3 (1975), 20.

[9] MUSTAKAS, G., GRIFFIN, E., KIRK, L.: Production of undenatured debittered soybean product. U . S . P a t e n t 3,023,107. F e b . 27 (1962).

[10] BRADOF, R. W.: Process for improving the flavor of soy flour. U.S. Patent 2,930,700. March 29 (1960).

[11] FUKTTSHIMA, D.: Cereal Chem. 45 (1968), 203. [12] FUKUSHIMA, D . : Cereal Chem. 46 (1969), 156. [ 1 3 ] GUYMON, J . F „ OUGH, C. S . : A m . J . E n o l . V i t i c u l t . 1 3 (1962), 4 0 .

[14] GOWANS, W. J . : J . Assoc. Off. Agric. Chem. 47 (1964), 722. [15] GUYMON, J . F., HEITZ, J . E.: Food Technol. 6 (1952), 359. [16] Association of Official Agricultural Chemists: Official methods of analysis of the AO AC, 14th ed. (Ed.: S. WILLIAMS) AOAC, Washington, D.C., 1984. [17] F e d e r a l register: 41(82): 17535. P a r t 319.3, 1976.

Acta Biotechnol. 6 (1986) 3, 215—219

Some Theoretical Considerations on Overflow Production of Metabolites* BABEL,

W.

Academy of Sciences of the G.D.R. Institute of Biotechnology, Leipzig PermoserstraBe 15, Leipzig, 7050k G.D.R.

Summary The overflow production of metabolites appears to be an energy spilling process in terms of life because part of the energy of the primary substrate remains in the metabolite produced. The other part of energy, which is liberated as reducing equivalents and/or ATP along the way to the product, must be wasted. This part is discussed to be responsible for the discrepancies between the theoretically possible and experimentally obtained product yields, because for the wasting process substrate or product are consumed. By reducing the amount of this superfluous energy the product yield should be increased. The auxiliary substrate concept occurs to be an appropriate method.

The objective of microbial life is to maintain the individual and the species. Growth and multiplication serve this objective. Multiplication represents the offensive strategy of living systems for survival. With balanced nutrition media — under the condition of chemostatic cultivation — in addition to cell substance as the proper result of growth and multiplication only carbon dioxide, water and heat are produced. Product formation, especially overflow production, is the result of an imbalance, which leads to disregulation and slips in terms of reproduction, or of already existing genetic defects. Overflow production is an energy-spilling process in terms of life in so far as part of the biologically utilizable energy of the primary substrate remains in the metabolite produced (see the following metabolic equations, Table 1) and the other part of energy generated, which is in itself biologically useful, might not be used for growth and reproduction under the conditions of overflow production, e.g. because there is no nitrogen [1]. Nevertheless, overflow production is to be considered as positive in terms of life since it is a physiological mode for getting rid of energy (e.g. of NADH) which cannot be transferred into A T P as is the case, for instance, with anaerobiosis. Since the reducing equivalents cannot be regenerated by aerobic oxidation, they are removed by internal hydrogen compensation, i.e. the energy is deposited in carbon skeletons (fermentation). * Shortened version of the lecture given at the UNESCO course "Biotechnology", September 23-October 13, 1985, Leipzig.

Acta Biotechnol. 6 (1986) 3

216

Table 1. Metabolic equations for the overflow production of some metabolites and of their complete oxidation 1 Glucose 1 Glucose

EMP ED

2 Ethanol

+

2 ATP

2 Ethanol

+

1 ATP

+ 2 C0 2 + 2 C0 2

1 Ethanol 1 Glucose

-V

2 Lactate

+

1 Lactate 1 Glucose

+

1 Glucose 1 CiAc 1 Glucose + NHS 1 Glu

-NH,

3 Ethanol

+

1 CiAc

+

4 NAD(P)H

+

1 FADH 2 + 3 C0 2 +

3 NAD(P)H

1 ATP

+ + +

2 ATP

+

7 NAD(P)H

+

2 FADHJ

3 ATP

+

8 NAD(P)H

+

1 FADH 2

2 ATP

1 Glu

1 FADHA + 2 COJ

1 ATP 1 ATP

1 CiAc

+

2 ATP 2 ATP

1 Ethanol 1 Lactate

5 NAD(P)H

7 NAD(P)H

+

2 FADH 2

3 NAD(P)H

1C02

+ 6 C0 2 +

1C02

+ 5 C0 2

Glucose Hexadecane

GlcA Ethanol Ethanol

1.02 0.47 0.34

CiAc

0.8

CiAc

1.6

-^theor 1.09 0.51 0.311 0.511 0.61 1 1.072 0.713 2.26

-1

product yields [g • g ] 1 2 3

depending on metabolism oxalacetate synthesis via heterotrophic C0 2 fixation oxalacetate synthesis via glyoxylate cycle

BABEL,

W., Overflow Production of Metabolites

217

given substrate/product couple. These reducing equivalents and ATP must be annihilated. I t can take place by oxidation or by hydrolysis, respectively, which is not coupled with an endergonic reaction. Since carbon is supplied in any amount (in batch process) in form of the primary substrate this energy may be utilized for reductive syntheses (e.g. for polyols, lipids) or for the, synthesis of polysaccharides. While nitrogen is still present (NH 3 as a conservative substrate, pool amino acids) even growth and multiplication can occur. This can be quite desirable because in this way the catalyst can be kept active due to its renewal. If the discharge of ATP (and reducing equivalents) does not take place by means of ATP-ases or via futile cycles, but it happens by means of syntheses, this discharge is accompanied by diminishing product yields, in which case either more substrate is used for product formation than needed according to the balance equation, or the product already built is, so to speak, consumed as the substrate for other synthesis. This also applies to NAD(P)H if it is used for reductive syntheses. With respiring cells NAD(P)H is mainly converted by oxidation whereby product formers are doubtless able to throttle the ATP yield down to zero by forming inefficient respiratory chains; hence the energy form of ATP needed for real syntheses is not generated at all. The product yield of approximately 100% in so-called incomplete oxidations very well supports the arguments given here to explain the discrepancies between theoretical and experimental product yields. For products representing a metabolic "dead end" the yield is almost 100%, too. Gluconic acid is a metabolic "dead end" because it cannot be taken up by cells under the condition of high glucose concentration and because gluconokinase is repressed [2]. L-glutamate is a metabolic "dead end", too, for it cannot be oxidized completely because the «-ketoglutarate dehydrogenase is absent or does not operate under the condition of overflow production. Therefore glutamate can at most be transaminated. Ethanol is both a metabolic and,energetic "dead end" in so far as it is produced anaerobically. In the absence of oxygen the TCA cycle necessary for energy generation does not operate since the TCA cycle for the dehydrogenation of acetate to C0 2 is endergonic. If the moles of ATP are made available enroute to ethanol used for cell substance synthesis, then the theoretically possible yield of 0.51 g • g _ 1 is reduced to about 0.42 g • g _ 1 . This value is practically attained, but seldom exceeded (Table 2). In the case of overflow production of citric acid only 70 to 75% of metabolism-determined possible yield are reached. This is not surprising, but rather to be expected against the background of the above consideration. By using reducing equivalents plus ATP made available from glucose on the way to citric acid a maximum of 0.78 moles of glucose could be consumed for growth; thus, the carbon conversion efficiency of citric acid would be reduced to about 56%, i.e. the product yield would amount to 0.60 g • g' 1 . The experimentally obtained yield on glucose closely approximates the value calculated in this way. But the fact was not taken into consideration that growth is restricted by NH 3 limitation. Therefore, glucose must simply be oxidized to C0 2 , for the dissimilatory capacity is always higher, and apparently glucose uptake is hardly controlled or not at all. While a t the beginning of citric-acid overproduction other synthesis processes (e.g. polyole) might be more responsible for the reduction of product yield, complete oxidation is later supposed to be more and more important because the energy charge might decrease during the process and, therefore, citric acid and isocitric acid, respectively, can flow off in increasing amounts. The overflow production of both ethanol and citric acid can be considered to be a growthassociated process. "Growth-associated" does not mean that overflow production is obligately coupled with growth, it rather means that growth might take place. Growth

218

Acta Biotechnol. 6 (1986) 3

can even be desired since it can stabilize the production phase because the producer is renewed again and again. Furthermore, it opens up a chance of making the process continuous. Such a procedure has been developed with the production of gluconic acid from glucose by means of Acinetdbacter calcoaceticus. Generally, gluconic acid is produced microbially batchwise [3]. After a certain time the production activity decreases. This decrease can be reduced or even prevented by using a second heterotrophic substrate which is assimilated while the producer oxidizes glucose. Acinetdbacter calcoaceticus is able to oxidize glucose and assimilate acetate simultaneously. Ì h e product yield is about 95% and productivity amounts to about 3 g • 1 _1 • h - 1 .

Mixed Substrate Utilization and its Consequences Mixed substrate utilization is, according to the auxiliary substrate concept, an approach to improving the growth yield (and growth rate) in SCP production [4—7]. In the abovementioned product synthesis it is evident that overflow production can be made continuous by using a mixture of heterotrophic substrates. Is this method also appropriate for improving the product yield and formation rate? The auxiliary-substrate effect in SCP production is based on balancing the carbon/energy ratio. By mixing substrates with different carbon/energy ratios the energy generated enroute from the substrates to the central precursor (PGA) must be so high that the substrates need not be oxidized to C0 2 merely for producing ATP and/or reducing equivalents. Then the carbon conversion efficiency approximates the carbon-metabolism conditioned limit, which is determined by unavoidable oxidative decarboxylations during the synthesis of cell constituents. These limits are 85% for glycolytic substrates and 75% for gluconeogenetic or non-glycolytic substrates [8]. Such an energetic aspect cannot readily be perceived at first sight for the overflow production of metabolites. Hence the question arises as to the strategy of chosing substrate couples and the expectations to be put on using substrate mixtures. If it is possible to shorten and linearize the pathway from the substrate to the product by skilfully mixing substrates the specific product formation rate should become higher [1] since there is a proportionality between this product formation rate and the number of enzymatic steps required [9], As in this manner the number of possible competitive reactions (branching points) and the outflow of intermediates are reduced an improvement in yield might result [1]. In the case of citric-acid overflow production, glucose and a non-glycolytic substrate offer themselves as a substrate couple. Acetyl coenzyme A is supplied by the gluconeogenetic substrate, and oxalacetate comes from glucose via the heterotrophic carboxylation of pyruvate or phosphoenolpyruvate. In fact, by using a mixture of glucose and hexadecane the production rate is higher than on glucose or hexadecans alone (Table 3). Table 3. Specific product formation rate with Yarrowia lipolytica E H 59 on glucose, paraffine, and a mixture

n [h- 1 ]

Glucose

Paraffine

Glucose plus Paraffine

0.17

0.18

0.24

WEISSBRODT, E . and BEHRENS, U., personal communication

219

BABEL, W., Overflow Production of Metabolites

Furthermore, and this is in close agreement with biochemically based predictions, the citric acid/isocitric acid ratio becomes more favourable than that obtained if hexadecane is used alone and approaches that of glucose (Table 4). Taking into account that the energy provided simultaneously during product accumulation is essentially responsible for the discrepancies between the theoretically possible and experimentally obtained carbon conversion efficiencies attention must be paid to keeping this energy low, which might be attained by combining suitable substrates. Table 4. Ratio of citric acid to isocitric acid in dependence on substrates used with Yarrowia lipolytica EH 59 [10, 12, 13] Glucose

Paraffine

(CiAc) (CiAc) + IsCiAc)

Glucose plus Paraffine 0.59(1:1)

0.94 [11]

0.56 [11]

0.71(2:1)

Figures in parenthesis indicate the mixing proportion in gram per gram

The choice of substrates for overflow production of metabolites should take place from such an energetic angle. Since overproduction of metabolites is released by imbalances in nutrition media, e.g. by lacking oxygen or nitrogen, overflow production of certain metabolites, which does not take place under such conditions using a single heterotrophic substrate, might be caused by using a mixture of heterotrophic substrates. Received July 9, 1985

References [1] BABEL, W.: Proc. National Summer School, Bulg. Acad. Sei., Primorsko, September 29 to October 3, 1984. [2] OLIJVE, W.: Ph. Thesis Groningen, 1978. [3] REHM, H. J.: Industrielle Mikrobiologie. Berlin, Heidelberg, New York Tokyo: SpringerVerlag, 1980. [ 4 ] B A B E L , W . : Z . Allg. Mikrobiol. 1 9 (1979), 671. [ 5 ] B A B E L , W . : Abhandl. Akad. Wiss. D D R , N 2 „Biotechnologie", (Hrsg. M . R I N G P F E I L ) . Berlin: Akademie-Verlag, 1982, 183 — 188. [6] BABEL, W.: Proc. 3rd Symp. Soc. Countries Biotechnol. Bratislava, April 25—29, 1983, 169-176. [7] B A B E L , W . , MÜLLER, R . H.: J. Gen. Microbiol. 1 3 1 ( 1 9 8 5 ) , 3 9 . [ 8 ] B A B E L , W., MÜLLER, R . H.: Appl. Microbiol. Biotechnol. 2 2 ( 1 9 8 5 ) , 2 0 1 . [9] CHRISTNER, A.: Z. Allg. Mikrobiol. 16 ( 1 0 7 6 ) , 1 5 7 . [ 1 0 ] B E H R E N S , U . , W E I S S B R O D T , E . , L E H M A N N , W . : Z . Allg. Mikrobiol. 18 ( 1 9 7 8 ) , 5 4 9 . [11] TABUCHI, T., HARA, S.: Nippon Nogei Kagaku Kaishi 48 (1974), 417. [ 1 2 ] STOTTMEISTER, U . , BEHRENS, U . , GÖHLER, W . : Z. A l l g . Mikrobiol. 2 1 (1981), 677. [ 1 3 ] STOTTMEISTER, U . , B E H R E N S , U . , W E I S S B R O D T , E . , B A R T H , G . , F R A N K E - R I N K E R , D . , SCHULZE, E.:

Z. Allg. Mikrobiol. 22

(1982), 399.

Acta Biotechnol. 6 (1986) 3, 220

Book Review A . E I N S E L E , W . SAMHABBB, R . K . F I N N

Mikrobiologische und biochemische Verfahrenstechnik Weinheim: VCH Verlagsgesellschaft, 1985. 248 S., 195 Abb., 40 Tab., 88 DM. Die Fortschritte, die in den letzten Jahren in der technischen Nutzung von Enzymen und Zellkulturen erzielt wurden, sind enorm. Die Biotechnologie gilt heute als ein Industriebereich mit großen Wachstumschancen. Die Wirtschaftlichkeit biotechnischer Verfahren hängt aber entscheidend, davon ab, daß es den Ingenieurwissenschaften gelingt, das von Chemikern, Biologen und Genetikern geschaffene Potential der Stoffumwandlung verfahrenstechnisch handhabbar zu machen. Voraussetzung dafür ist eine Ausbildung, die das gesamte Spektrum der biochemischen Verfahrenstechnik berücksichtigt. Dieses Buch bietet eine Einführung in die mikrobiologische und biochemische Verfahrenstechnik. Die langjährige Erfahrung der Autoren in Hochschule und Industrie gewährleistet eine ausgewogene und an den Erfordernissen der Praxis orientierte Darstellung. Anhand von Beispielen und Berechnungen lernt der Leser Lösungen technischer Probleme kennen, die bei der mikrobiellen und biochemischen Stoffwandlung auftreten. Meß- und regeltechnische Aspekte, die häufig für das Gelingen eines Bioprozesses ausschlaggebend sind, werden besonders ausführlich behandelt. Verfahren zur Produktisolation und Produktreinigung sowie neue Entwicklungen in der Membrantechnik und der Chromatographie sind weitere Themen dieser Einführung. Sie ist für Biologen, Biochemiker und Ingenieure gleichermaßen verständlich geschrieben und wendet sich nicht nur an Studenten, sondern auch an Praktiker in Forschungsinstituten und in der Industrie.

Acta Biotechnol. 6 (1986) 3, 2 2 1 - 2 3 1

Partikelelektrophoretische Charakterisierung der Oberflächeneigenschaften von alkanutilisierenden Hefezellen: chemische Zusammensetzung und Tensidadsorption L E R C H E , K . - H . , KBETZSCHMAB, G .

Akademie der Wissenschaften der DDR Zentralinstitut für Organische Chemie Rudower Chaussee 5, Berlin, 1199 DDR

Summary By using particle electrophoresis and quantitative analysis the interaction of anionic surfactants (sodium dodeeylculfate, sodium decylbenzene sulfonate) with a hydrocarbon-grown yeast and a commercial baker's yeast has been investigated to obtain further informations about the chemical composition of the surface region (3—6 nm) of the cell wall of this yeasts. A correlation is found between the chemical composition and the different adsorption behaviour of various batches of the same yeasts. I t was found that surface-localized hydrophobic glycoproteins, probably proteophosphomannan are responsible for the strong p H dependent adsorption behaviour of a typical Candida species. In contrast the cell surface of a typical Saccharomyces species was strongly hydrophilic and showed no surfactant adsorption. This can be explained by the presence of a polysaccharide probably phosphomannan in the surface region only. The implication of proteins in the adsorption process is confirmed by model experiments too. The models for the yeast cell wall proposed by LAMPEN and KIDBY et al. were critically discussed in terms of our results.

Einleitung Bei einer Reihe derzeitiger Verfahren zur Futtereiweißgewinnung durch Konvertierung flüssiger Kohlenwasserstoffe mit Hilfe von Mikroorganismen (in der Hauptsache Hefen) spielen grenzflächenaktive Stoffe (Tenside) eine erhebliche Rolle [1, 2]. Tenside werden im Fermentationsprozeß zur Emulgierung des wasserunlöslichen Substrats, zur Erhöhung der Koaleszenzstabilität des Systems und für die Intensivierung des Stoffübergangs gasförmig/flüssig eingesetzt. , In dem der Fermentation nachgeschalteten Trennprozeß dienen Tenside zur Phasentrennung. Anionische (Alkylsulfate, Alkansulfonate) und nichtionogene Tenside (EthylenoxidPropylenoxid-Copolymere, Sorbitanfettsäureester, Alkylphenolpolyethoxylate u. a.) zählen dabei zu den bevorzugten grenzflächenaktiven Verbindungen in diesen Prozeßstufen [1]. Trotz ihrer vielfachen Anwendung ist die Wirkungsweise der Tenside an der biologischen Zellbegrenzung weitgehend unbekannt [1, 3, 4].

222

Acta Biotechnol. 6 (1986) 3

Dies hängt damit zusammen, daß die Zellbegrenzung selbst in chemischer, physikalischer und kolloidchemischer Hinsicht unzureichend charakterisiert ist [5—8], Obwohl bekannt ist, daß am Aufbau der etwa 100 nm dicken Hefezellwand hauptsächlich Polysaccharide, Proteine, Lipide und Phosphopolymere beteiligt sind [9—12], liegen Informationen über die Lokalisation dieser Komponenten in der äußersten Region der Zellwand (bis zu einer Dicke von ca. 6 nm) nur in begrenztem Umfang vor. Selbst diese Informationen müssen kritisch bewertet werden, da sie überwiegend mit Methoden gewonnen wurden, die außer der Oberfläche auch tiefere Bereiche der Zellwand miterfassen. Dazu gehören die immunchemischen [13—15], elektronenoptischen [16, 17], potentiometrischen [18] und enzymatischen Verfahren [19, 20]. Im Gegensatz dazu stellen die Partikelelektrophorese [21—23] und die ESCA-Spektroskopie [7, 24] echte Oberflächenanalysenmethoden dar, mit denen direkt die äußerste Zone der Zellwand (1—6 nm) erfaßt und die in ihr vorkommenden makromolekularen Verbindungen und funktionellen Gruppen identifiziert werden können. Unter Verwendung der Partikelelektrophorese wurden von uns in einer früheren Arbeit [23] vergleichende Untersuchungen zur Oberflächenchemie von auf Kohlenwasserstoffen und Kohlenhydraten gewachsenen Hefen (Candida guilliermondii und Saccharomyces cerevisiae) durchgeführt. Zur weiteren physiko-chemischen und kolloidchemischen Charakterisierung der Zelloberflächeneigenschaften dieser Hefen wird in der vorliegenden Arbeit ihre Wechselwirkung mit anionischen Tensiden untersucht. Durch kombinierte Anwendung von Partikelelektrophorese, quantitativen Messungen und Modellexperimenten sollen dabei weitergehende Aussagen zur stofflichen Zusammensetzung der Zelloberfläche, zum Adsorptionsverhalten der Hefezellen unter verschiedenen experimentellen Bedingungen (pH-Wert, Tensidkonzentration) und zum Wechselwirkungsmechanismus Hefezelle/Aniontensid getroffen werden. Material und Methoden Für die Untersuchungen wurden die alkanutilisierende Hefe Candida guilliermondii vom Institut für Biotechnologie der Akademie der Wissenschaften der D D R , Leipzig sowie kommerzielle Backhefe (Saccharomyces cerevisiae) verwendet. Das biologische Material, die eingesetzte Partikelelektrophoreseapparatur sowie die Ausführung der Messungen sind an anderer Stelle [23] ausführlich beschrieben. Als anionische Tenside wurden Na-Dodecylsulfat und Na-Decylbenzensulfonat verwendet. Die Tenside wurden durch Oberflächenspannungs-Konzentrations-Isothermen (Ringmethode) charakterisiert. Als Reinheitskriterium für die Tenside wurde das Fehlen von Minima in der Nähe der CMC herangezogen. Zur quantitativen Erfassung der Adsorption von Na-Decylbenzensulfonat an den Hefezellen wurde die UV-Spektroskopie eingesetzt. Sämtliche dazu notwendigen Arbeiten (Erstellen einer Eichkurve, Bestimmung der Resttensidkonzentration im Zentrifugat nach Abtrennung der Hefezellen) wurden am VSU 2-P der Firma Carl Zeiss Jena ausgeführt.

Ergebnisse und Diskussion Wegen der Heterogenität in der chemischen Natur der funktionellen Oberflächengruppen verschiedener Hefechargen (Hefen aus zeitlich auseinanderliegenden Fermentationsansätzen) erfolgte in unserer vorangegangenen Arbeit eine Einteilung der Hefen in die Gruppen I, I I und III. Danach besitzen die Hefen der Gruppe I nur eine Art von funktionellen Gruppen an der Zelloberfläche.

LEBOHE,

K.-H.,

RRETZSCHMAR,

G., Partikelelektrophoretische Charakterisierung

223

Die Gruppe I I faßt Hefen zusammen, die Säuregruppen unterschiedlicher Azidität aufweisen und die Gruppe I I I charakterisiert Heferi mit amphoterem Oberflächenverhalten (Vorhandensein von sauren und basischen Gruppen an der Zelloberfläche). In der vorliegenden Arbeit wird an dieser Einteilung festgehalten. Für die Untersuchungen der Wechselwirkung Hefezelle/Aniontensid werden bevorzugt die Hefen der Gruppe I (C. guilliermondii, 8. cerevisiae), die Säccharomyceshefe'der Gruppe I I und die Candidahefe der Gruppe I I I ausgewählt, da über sie die meisten Kenntnisse zum elektrostatischen und chemischen Oberflächenverhalten der Zellen aus unseren vorangegangenen Untersuchungen [23] vorliegen. ' Die wichtigsten Oberflächeneigenschaften der verwendeten Hefen sind zusammenfassend in Tab. 1 dargestellt. Tabelle 1. Für die Tensidwechselwirkung verwendete Hefen und ihre wichtigsten Oberflächeneigenschaften [23]. Hefen

Hefen der Gruppe I

:

u/pH-Charakteristik

der Hefen

10'sm!v'1s'1

^

U

C.guilliermondii [1J

J

-

(C.guilliermondiil

-PO^H IS. cerevisiae) \

/ 2 U

i i

i

i

i

6

8

10

pH

10'8m2v'1s'1

S. cerevisiae

Hefen der Gruppe E:

-C00H

-12)

5. cerevisiae 121

Hefen der Gruppe E:

funktionelle Gruppen an der Zelloberfläche

- C00H, -PO^H

U

i

i

2

i

i

i

i

6

8

10 pH

ftT'/nVi"* -C00H,

Cguilliermondii

-PO^H

-NH2

i /i

0 + 2 / i

T 6-8

i

i 10

pH

Charakterisierung der Wechselwirkung von Hefen der Gruppe I mit anionischen Tensiden — C. guilliermondii, S. cerevisiae Zeitabhängige Untersuchungen der Adsorption von Aniontensiden an Hefen ergaben, daß sich nach ca. 60 min das Adsorptionsgleichgewicht eingestellt hat [2]. Bei allen nachfolgend aufgeführten Meßreihen wurde diese Adsorptionszeit eingehalten. Zunächst wurde der Einfluß steigender Mengen Na-Dodecylsulfat auf die elektrophoretische Beweglichkeit von C. guilliermondii und S. cerevisiae in 0,02 mol/dm 3 NaCl untersucht. Das Ergebnis dieser Messungen ist in Abb. 1 dargestellt. 2

Acta Biotechnol. 6 (1986) 3

224

Acta Biotechnol. 6 (1986) 3

Man erkennt, daß nur die alkanpositive Hefe eine Zunahme der anodischen Beweglichkeit mit steigender Tensidkonzentration erfährt. Die elektrophoretische Beweglichkeit der Backhefe hingegen bleibt von der Tensidkonzentration unbeeinflußt. Dies läßt den Schluß zu, daß nur an der Hefe C. guilliermondii Tensidmoleküle an die Zelloberfläche gebunden wurden. Aus Beweglichkeitsmessungen der beiden Hefen in Abhängigkeit vom pH-Wert konnte gezeigt werden [23], daß sie über vergleichbare Oberflächeneigenschaften (negative Oberflächenladung, Anwesenheit von Polysacchariden im peripheren Bereich der Zellbegrenzung) verfügen. Der hier festgestellte Unterschied in der Affinität der Hefen gegenüber dem anionischen Tensid legt die Vermutung nahe, daß an der Zelloberfläche der Candidahefe eine Zweitkomponente mit hydrophoben Eigenschaften wie z. B. an Polysaccharid gebundenes Lipid oder Protein vorkommt, das diese Tensidadsorption hervorruft.

I s

^•—-05

' 10-*

1 • • •• i 1 L S-10-'' 10 Cna-Dodec/Isulfa! tmol dm'3]

i

i

Abb. 1. Einfluß der Konzentration von Na-Dodecylsulfat auf die elektrophoretische Beweglichkeit von S. cerevisiae ( • ) und C. guilliermondii (x —) bei konstanter Ionenstärke I = 0,02 mol/dm 3 .

Da Proteine von uns nicht in der Oberflächenschicht dieser Hefe nachgewiesen werden konnten [23], ist mit hoher Wahrscheinlichkeit anzunehmen, daß die Tensidadsorption durch Oberflächenlipide vermittelt wird. Die mangelnde Fähigkeit der Saccharomyceshefe der Gruppe I, anionische Tenside zu adsorbieren, läßt sich damit erklären, daß ihre Oberfläche aus einem reinen Polysaccharid besteht. Diese Schlußfolgerung wird gestützt durch eine Reihe von Arbeiten [25—28], wonach zwischen Polysacchariden und anionischen Tensiden keine Wechselwirkungen auftreten. Faßt man diese und die Ergebnisse unserer vorangegangenen Arbeit zusammen, so ergibt sich für die untersuchte alkanutilisierende Hefe der Gruppe I das folgende Bild: die Oberfläche dieser Hefe ist durch Dissoziation von Carboxylgruppen mit einem pkWert von ca. 3 unter physiologischen pH-Bedingungen negativ geladen. Aufgrund der Anwesenheit von Lipiden in der äußersten Zone der Zellwand erlangt die Zelloberfläche hydrophobe Eigfenschaften und damit die Fähigkeit, anionische Tenside durch hydrophobe Wechselwirkung zu binden. Für die betrachtete Saccharomyceshefe (Hefe der Gruppe I) ergibt sich demgegenüber das folgende Bild: die Oberfläche dieser Hefe besteht aus einem stark sauren und äußerst hydrophilen Polysaccharid, das als Phosphomannan identifiziert wurde. Dieses Polysaccharid weist diestergebundene Phosphatgruppen mit einem pK-Wert von ca. 1 auf, die für die negative und vom pH-Wert des Suspensionsmediums weitgehend

LERCHE,

K.-H.,

KRETZSCHMAR,

G., Partikelelektrophoretische Charakterisierung

225

unabhängige Oberflächenladung der Zellen verantwortlich sind. Das alleinige Vorkommen dieses Polysaccharids an der Zelloberfläche der Saccharomyceshefe erklärt, weshalb im Vergleich zur Candidahefe keine anionischen Tenside adsorbiert werden. Charakterisierung — S. cerevisiae

der Wechselwirkung von Hefen der Gruppe II mit anionischen

Tensiden

Für die Klärung der Frage nach dem Einfluß der Oberflächenladung auf die Tensidadsorption wurden die Hefen der Gruppe I I und I I I herangezogen, da, sich ihre Oberflächenladung stark mit dem pH-Wert ändert (vgl. Tab. 1). Die Adsorptionsexperimente wurden zu diesem Zweck bei drei ausgewählten pH-Werten (3; 5,5 und 11,5) im Konzentrationsbereich von 10~6 — 10~3 mol/dm3 Na-Dodecylsulfat durchgeführt und anschließend die Beweglichkeit der Hefezellen vermessen. Das Ergebnis dieser Untersuchungen zeigt Abb. 2.

C "0,41

I

10'S

i

i i i i-1 i i I

510-5 W" Na-Dcdecrlsulfot

C

i Imol

i i ' i i 111

HO'4 10-3 dm'3]

Abb. 2. Einfluß dei Konzentration von Na-Dodecylsulfat auf die elektrophoretische Beweglichkeit von S. cerevisiae bei verschiedenen pH-Werten (I = 0,02 mol/dm3). • pH = 11,5; X pH = 5,6; A pH = 3,0

Zum besseren Verständnis wurde die von uns früher erhaltene u/pH-Charakteristik der Saccharomyceshefe in das Diagramm mit aufgenommen. Bemerkenswert am Verhalten der hier untersuchten Saccharomyceshefe ist, daß trotz der über den gesamten pH-Bereich negativen Ladung der Hefezellen bei allen pHWerten (3; 5,5 und 11,5) eine deutliche Zunahme der Beweglichkeit mit steigender Dodecylsulfatkonzentration zu beobachten ist. Dies deutet im Gegensatz zu der vorstehend beschriebenen Saccharomyceshefe auf eine hydrophobere Oberfläche hin. Es kann angenommen werden, daß eine ähnliche Zellwandkomponente an der Oberfläche lokalisiert ist wie bei der diskutierten Candidahefe, d. h. ein Lipopolysaccharid, das durch hydrophobe Wechselwirkung mit den apolaren Kohlenwasserstoffketten der Tensidmoleküle eine Adsorption und damit eine Erhöhung der anodischen Mobilität verursacht. Das Zustandekommen dieser Wechselwirkung wird im wesentlichen von der Oberflächenladungsdichte der Hefen bei dem entsprechenden pH-Wert abhängen. 2*

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Acta Biotechnol. 6 (1986) 3

In Verbindung mit der u/pH-Kürve der Hefe kann man erwarten, daß diese Bindung des anionischen Tensids bei pH 3 wegen der hier geringeren Anzahl negativer Gruppen wesentlich stärker sein wird als bei pH 5,5 und 11,5, wo vergleichbare Tensidmengen adsorbiert werden sollten. Dies wird auch tatsächlich gefunden, wie quantitative Messungen zeigen. Auf diese Ergebnisse wird weiter unten eingegangen. Charakterisierung der Wechselwirkung von Hefen der Gruppe III mit anionischen Tensiden — ein weiterer Befund für die Anwesenheit einer Proteinkomponente an der Oberfläche von C. guilliermondii durch Vergleichstnessungen an einem Modellprotein In viel ausgeprägterem Maße als bei der vorangehend diskutierten Saccharomyceshefe sollte sich bei der hier verwendeten Candidahefe der Einfluß der Oberflächenladung auf die Tensidadsorption bemerkbar machen. Nach [23] gibt es drei ausgewählte pH-Werte (3; 5,5 und 11,5), bei denen die Zelloberfläche ein unterschiedliches Ladungsmuster aufweist: bei pH 3 ist die Hefezelle positiv geladen, bei pH 5,5 dominiert eine Zwitterionenstruktur und bei pH 11,5 liegt eine hochgeladene negative Zelloberfläche vor. Wie sich diese unterschiedlichen Ladungszustände auf die Adsorption von Na-Dodecylsulfat auswirken, zeigt Abb. 3. Auch hier wurde die Beweglichkeitsänderung der Hefezellen in Abhängigkeit von der Tensidkonzentration (10 - 6 — 10 - 3 mol/dms Na-Dodecylsulfat) zusammen mit der von uns früher erhaltenen u/pH-Charakteristik der Hefezellen dargestellt. Daraus geht hervor, daß eine Tensidadsorption nur bei pH 3 und pH 5,5, nicht aber bei pH 11,5 erfolgt. Die hier festgestellte ungeänderte Beweglichkeit selbst in Gegenwart hoher Tensidkonzentrationen spricht gegen eine Tensidadsorption an der biologischen Phasengrenze. Diese Abhängigkeiten werden, verständlich, wenn man die Chemie der Zelloberfläche berücksichtigt. Nach unseren Untersuchungen [23] weist die Candidahefe der Gruppe I I I

CO^

1

¿if

I I I I I

b