Acta Biotechnologica: Volume 8, Number 5 1988 [Reprint 2021 ed.] 9783112581780, 9783112581773

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Acta BiitettnimicB •

^ J.^

Volume 8 • 1988 • Number 5

Journal of microbial, biochemical and bioanalogous technology

Akademie-Verlag Berlin ISSN 0138-4988 Acta Biotechnol., Berlin 8 (1988) 5. 393-472

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1. Only original papers that have not been published previously will be accepted. Manuscripts may be submitted in English, German or Russian (in duplicate). 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 exceed 20 typewritten pages (double spaced), including references, tables and figures; short original communications may contain a maximum of six typewritten pages. 3. Each paper should be preceded by a summary in English and the title in English. 4. Latin names of species as well as passages to be printed in italics for greater emphasis should be marked 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 it more comprehensible. They should be numbered consecutively throughout the text and be 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 a minimum, they should be numbered consecutively and be quoted on separate sheets. Line drawings, including graphs and diagrams, should be drawn in black ink. Half-tone illustrations should be presented as white glossy prints. Figure legends are to be typed in sequence on a separate sheet. The back of each sheet should bear the name(s) of the author(s). 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 presented as footnotes on the same page. 9. 50 offprints are free of charge. Additional ones may be ordered on payment. 10. The author will receive two galley proofs for correction. They are to be returned to the managing editor (Dr. Dimter, Permoserstr. 15, Leipzig, 7050 - DDR) as soon as possible.

Acta Bìatecfemloaica Journal of microbial, biochemical. and bioanalogous technology

Edited by the Institute of Biotechnology of the Academy of Sciences of the G.D.R., Leipzig and by the Kombinat of Chemical Plant Construction Leipzig—Ori ti ì ma by M. Ringpfeil, Berlin and (5. Vetterlein, Leipzig

Editorial Board: L). Meyer, Leipzig P. Moschinski, Lodz A. Moser, Graz M. D. Nicu, Bucharest Chr. Panayotov, Sofia L. D. Phai, Hanoi H. Sahm, Jülich W. Scheler, Berlin R. Schulze, Halle B. Sikyta, Prague G. K . Skrjabin, Moscow M. A. Urrutia, Habana

1988

A. A. Bajew, Moscow M. E. Beker, Riga H. W . Blanch, Berkeley S. Pukui, Kyoto H. G. Gyllenberg, Helsinki G. Hamer, Zurich J. Hollo, Budapest M. V. Iwanow, Moscow L. P. Jones, El Paso F. Jung, Berlin H. W. D. Katinger, Vienna K . A. Kalunyanz, Moscow J. M. Lebeault, Compiegne

Number 5

Managing Editor:

L. Dimter, Leipzig

Volume 8

A K A D E M I

E - V E R L A G

B E R L I N

"Acta Biotechnologica" publishes original papers, short communications, reports and reviews f r o m 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 t h e technology of synthesizing and applying bioanalogous reaction systems. The technological character of the journal is guaranteed b y t h e fact t h a t 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 t h e Akademie-Verlag Berlin, Leipziger Str. 3 - 4 , P F 1233, D D R - 1 0 8 6 Berlin; — în the other socialist countries : to a bookshop for foreign languages literature or to the competent news-distributing agency; — in the FRG and Berlin (West): to a bookshop or to the wholesale distributing agency K u n s t und Wissen, Erich Bieber oHG, Postfach 102844, D-7000 S t u t t g a r t 10; — in the other Western European countries: to K u n s t 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 D D R , P F 160, D D R - 7 0 1 0 Leipzig, or to the Akademie-Verlag Berlin, Leipziger Str. 3 - 4 , P F 1233, D D R - 1 0 8 6 Berlin.

Acta Biotechnologica Herausgeber: I n s t i t u t f ü r Biotechnologie der AdW der D D R Permoserstr. 15, D D R - 7 0 5 0 Leipzig (Prof. Dr. Manfred Ringpfeil) und V E B Chemieanlagenbaukombinat Leipzig—Grimma, Bahnhofstr. 3 - 5 , D D R - 7 2 4 0 Grimma, (Dipl.-Ing. Günter Vetterlein). Verlag: Akademie-Verlag Berlin, Leipziger Straße 3 - 4 , P F 1233, D D R - 1 0 8 6 Berlin; Fernruf: 2236201 und 2 2 3 6 2 2 9 ; Telex-Nr.: 114420; B a n k : Staatsbank der D D R , Berlin, Konto-Nr.: 6836-26-20712. Redaktion: Dr. Lothar Dimter (Chefredakteur), Martina Bechstedt, K ä t h e Geyler, Permoserstr. 15, D D R - 7 0 5 0 Leipzig; Tel.: 2392255. Veröffentlicht unter der Lizenznummer 1671 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: V E B Druckhaus „Maxim Gorki", D D R - 7 4 0 0 Altenburg. Erscheinungsweise: Die Zeitschrift „Acta Biotechnologica" erscheint jährlich in einem Band mit 6 Heften. Bezugspreis eines Bandes 192,— DM zuzüglich Versandspesen; Preis je H e f t 32,— DM. Der gültige Jahresbezugspreis f ü r die D D R ist der Postzeitungsliste zu entnehmen. Bestellnummer dieses Heftes: 1094/8/5. Urheberrecht: Alle Rechte vorbehalten, insbesondere der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner F o r m — 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 p a r t of this issue may be reproduced in any form, by photoprint, microfilm or any other means, without written permission from the publishers. © 1988 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 18520 03000

Acta Biotechnol. 8 (1988) 5, 3 9 5 - 4 0 0

Significance of the Surface of Immobilized Saccharomyces cerevisiae Cells in Ethanolic Fermentation BERGER, R . , RUHLEMANN, I.

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

Summary S. cerevisiae cells immobilized in alginate beads show in many cases an increase of mean single cell volume during long-time fermentations (successive batch cycles). The biomass loading capacity of the gel beads is characterized by a maximum volume but not by a maximum number of cells occupying the gel volume. In our system this loading capacity, i.e. the maximum volume fraction of cells per volume of beads, amounted to about 0.54. As a more important result it must be stated that the specific product formation rate in the case of fermentations negligibly influenced by diffusion hindrance is related to the total surface of the viable cells but not to their total number, total volume or total dry weight.

Introduction We have already shown that the fermentation power of gelentrapped 8. cerevisiae cells in the case of negligible diffusion hindrance is identical with that of free cells, obeying an empiric productivity model for ethanolic fermentation [ 1]. To calculate a specific ethanol formation rate, the dry weight of biomass is usually used as the reference base, reasonable related to the portion of viable cells only. In some cases the ethanol formation rate was related to the number of viable cells, too (e.g. [2]). In this paper we show that at least in the case of S. cerevisiae the specific ethanol formation rate and, therefore, the ethanol productivity, too, depends on the total surface of the viable cells. Experimental Yeast: Five strains of Saccharomyces cerevisiae from a culture collection of our institute were used: Sc 2, 5, 20, 34, and 35. Immobilization: The^ells were entrapped in Ca-alginate beads in the usual manner using a 1% aqueous solution of Na-alginate ( R I E D E L D E H A E N AG) containing 0.6—6 g dry biomass per 100 ml, and a 0.02 M aqueous CaCl2-solution for precipitation, curing, and storage of the beads, respectively. The beads prepared for the different fermentation series had a narrow diameter distribution with mean values ranging from 1.8 to 3.6 mm after preparation. 1*

396

Acta Biotechnol. 8 (1988) 5

Fermentation: A double-walled cylindrical glass vessel with magnetic stirring, pH-control, sampling devices, and C0 2 -outlet has been used. The working volume amounted to 320 ml with a bead to medium volume ratio ranging from 0.15 to 0.40. Four to fifteen cyclic batch fermentations were performed at p H 4 and 32 °C with one and the same bead preparation (one series), each cycle until the C0 2 -evolution diminished. During a whole series all the handlings have been done under an inert atmosphere of C0 2 . The liquid phase consisted of either a complex medium or a sucrose-molasse mixture (weight ratio 1:2) without supplements. The complex medium contained glucose or sucrose, mineral salts, a nitrogen source, 1 or 5 g/1 yeast extract, and in some cases 5 g/1 peptic peptone. The sugar content of the liquid phase ranged from 125—250 g/1.

Analytics and Calculations The evolved C0 2 -volume was determined every 10 minutes collecting the gas over satured NaCl solution at 20 °C and correcting the values to normal pressure. The dry weight of immobilized biomass at the end of a series was gravimetrically measured after dissolving a representative lot of rinsed beads in phosphate buffer. The total wet gel volume has been measured by means of water displacement. The number, viability and linear dimensions of the yeast cells inside the beads have been microscopically estimated after dissolving a small but representative lot of rinsed beads. The cell number was estimated using a THOMA counting chamber, the viability after staining the cells with methylene blue. Each population of immobilized cells (series) has additionally been characterized at different fermentation times (at the end of different batch cycles) by a mean value of the single cell volume and single cell surface. For this purpose we have measured with a calibrated ocular micrometer the length and breadth of non-budding and budding cells, determining in the latter case these linear dimensions for both the mother cells and buds [3]. These values have been used for the calculation of the mean volume of non-budding and budding cells, respectively, assuming a prolate spheroid for the cell shape. Using the resulting values with different weight corresponding to the statistical distribution of budding and non-budding cells in the examined population we have calculated the mean volume of a single cell for characterizing the size of the cells in the population at the corresponding time of fermentation. From this mean cell volume we have calculated the mean surface of a single cell in the corresponding population, but with the assumption of a sphere for the cell shape, because the mean surface of the budding cells would be overestimated assuming a prolate spheroid and using the mean length and mean breadth of both the mother cells and buds, which would yield a noncorrect averaging on separate large and small cells. By multiplication of the estimated total number of all the cells or of the viable cells inside the gel with the calculated mean volume and mean surface of a single cell, respectively, one gets the total volume and total surface of all the cells or of the viable cells inside the gel, respectively. (It should be mentioned that the total volume of immobilized yeast cells estimated with a simplified method, taking into consideration the linear dimensions of non-budding cells only, did not very differ from the value estimated as described [3]). Similarly to other authors [4—6], we have used the Conformation rate as a criterion of ethanol formation rate, because the gas volume can be measured with the highest accuracy and with nearly any abundance. I n our case, the gas volume-time-plots have always shown a straight-line part [cf. 6], from which we have calculated the C0 2 -formation rate. This procedure has been verified by numerious GC-measurements of the ethanol concentration in the liquid phase (ranging from 5—35 g/1). The product formation

BERGER,

R.,

RÜHLEMANN,

I., Immobilized Ceils in Ethanolic Fermentation

397

rates estimated in this manner were only unsignificantly influenced by diffusion hindrance at the fermentation conditions used (high agitation speed, porous gel matrix, high driving force of diffusion on account of the sugar concentration existing during the fermentation time at which the amount of evolved C0 2 linearly depends on time). For the calculation of the different specific C0 2 -formation rates ("specific productivities") we used as reference base the gravimetrically measured HTS as well as the arithmetical averages of Zh Zi • Vz, and Zx • Oz, respectively, calculated from the corresponding values measured at the start and the end of each respective batch cycle. Results and Discussion The number of cells inside the gel beads increased during the batch cycles at the outset of a series, thereafter it nearly remained constant. The extent of this increase depends on the amount and viability of the immobilized cells at the start of a series as well as 011 the fermentation conditions [cf. 7]. The cells were rather homogeneously distributed inside the gel beads, i.e. the cell concentration was not much higher near the bead surface [cf. 8,9,18]. At the last batch cycles of almost each series so-called "free cells" occured in the liquid phase, but only during the second half of these batch cycles. The ratio of free cells to free plus immobilized cells in the reactor amounted to 0.005—0.05 in such cases. The contribution of these free cells to the C0 2 -formation has been allowed for by substraction in the calculation of the specific C0 2 -formation rates of the immobilized cells. The bead volume also increased during each series until a steady state was reached, on the triple in the extreme [cf. 1], This increase in the bead volume results from the increase of biomass inside the beads as well as from internal gas bubble formation by the evolving C0 2 [11]. In some series, especially in such with the larger beads, one could observe after several batch cycles a distinct cavity in the inside part of the majority of the beads after cutting them. Of course, the expansion of the gel matrix also promotes the cell leakage [cf. 7]. The gel beads posess a maximum biomass loading capability, as has been found by other authors, too [7, 9, 12], This loading capacity could not exactly be characterized neither by a maximum number of cells per ml gel, as will be discussed later, nor by a maximum amount of dry biomass per ml gel, as has been done among ¡others by LEE et al. [10]. The dry biomass is an improper reference base because the content of dry biomass per ml wet cells may vary in dependence upon the fermentation conditions, fermentation time, the yeast strain used, and the state of cells (immobilized or free cells) [3]. However, in each case the maximum loading of the gel with cells could well be characterized by a maximum volume of viable plus non-viable cells occupying the gel volume. In our system this maximum volume fraction amounts to 0.54 (with a standard deviation s = 8%) [13]. If present, the cavities inside of the beads, mentioned above, were allowed for in the calculation of this maximum loading capacity of the gel by substraction of the microscopically appraised cavity volume from the measured total volume of the wet gel. The maximum loading of the gel with biomass cannot be reached using a high initial cell concentration at the immobilization but only after some time of fermentation [cf. 9, 12], As already shown [13], the mean single cell volume has increased during the fermentation in many cases (Tab. 1). This increase depends on the cell strain and in particular on the fermentation conditions. Other authors have also discussed a variation and/or increase of the mean single cell volume of aerobically growing Saccharomyces cerevisiae cells, being depended on the fermentation time (generation) and/or fermentation conditions [ 1 4 - 1 6 ] , Because of changes of the mean single cell volume during the fermentations, the biomass loading capacity of the gel could not be well characterized by a maximum

398

Acta Biotechnol. 8 (1988) 5

number of cells. The maximum loading of the gel beads can be reached with a high number of small cells as well as with a lower number of large cells. Therefore, on the contrary to the mode of other authors [9, 12, 17], the number of cells may not be a proper reference base for describing the maximum loading capacity of a gel. The number of cells should only be used if one can rule out a change of the cell volume during fermentation. Tab. 1. Mean volume of the single cells and specific product formation rates related to the total dry weight, to the total volume, to the total number, and to the total surface of viable cells, respectively, measured at different fermentation series with the immobilized yeast strain Sc 5 Series and cycle

1. 1. 2. 2. O. 3. 4. 4. 5. 5. 6. 7. 8. 8. 9. 10. 10. 10.

2 8 3 11 3 6 1 9 3 5 4 12 2 12 15 2 4 9

Total time of fermentation [h]

Zl • 10"10 per batch cycle

18 53 14 91 17 26 4 25 19 28 16 64 13 53 73 10 18 42

5.8 6.5 9.5 5.2 7.5 9.4 8.7 20.5 8.7 10.6 8.9 7.9 5.2 10.8 8.1 6.9 11.6 8.7

Vz

C0 2 /(HTS),

COJZvVz

C02/Zl

[¡jun3]

[1/h-g]

[1/h • ml]

[1/h • 1010 cells]

0.120 0.110 0.117 0.100 0.128 0.126 0.124 0.119 0.130 0.113 0.104 0.119 0.125 0.102 0.109 0.124 0.105 0.124

0.197 0.315 0.227 0.355 0.199 0.262 0.193 0.240 0.240 0.243 0.302 0.311 0.259 0.267 0.288 0.278 0.244 0.272

0.400

0.117

0.261

0.145

0.043 (10.7%)

0.010 (8.5%)

0.044 (16.9%)

0.008 (5.5%)

164 288 194 355 157 208 156 201 185 215 291 262 208 260 264 224 232 220 mean value:

±

0.323 0.382 0.439 0.468 0.389

0.403 0.389

0.411

COJZvOz

[1/h-m2] 0.136 0.150 0.140 0.147 0.141 0.154 0.138 0.145 0.153 0.140 0.142 0.157 0.152 0.135 0.145 0.156 0.133 0.154

Concerning the main intention of our investigations, i.e. to find the proper reference base for describing the specific ethanol productivity, it must be emphasized that the only proper reference base is the total surface of immobilized viable cells in case of a varying cell volume. As can be seen in Tab. 1, the surface-related specific productivity shows under conditions of optimum fermentation for many different batch cycles of different series with the strain Sc 5 the lowest variation around a mean value, amounting in this case to 0.145 1 C02/h • m2 (s = 5.5%) and 0.28 g EtOH/h • m2, respectively. These values have not been influenced by the different medium compositions used. Such a nearly linear dependence of the C02-formation rate on a reference base could by far not be reached, neither with the total number of viable cells as reference base (s = 16.9%), nor with the total weight of dry biomass of the viable cells as a reference base (s = 10.7%). The specific productivity related to the total volume of immobilized viable cells also shows a somewhat greater variation around the apparent mean value (s = 8.5%).

BERGER, R . , RUHLEMANN,

399

I., Immobilized Cells in Ethanolic Fermentation

T h e checkup of generalization of this pronounced significance of t h e t o t a l cell surface a t other S. cerevisiae strains has shown, as can be seen in T a b . 2, t h a t these strains generate t h e p r o d u c t s with t h e same cell surface-related specific formation r a t e (0.145 1 C 0 2 / h • m 2 ; s = 6.2%). Moreover, f r o m T a b . 2 one can see t h a t for distinct different cell sizes ( V x ) t h e a p p a r e n t m e a n value of t h e specific p r o d u c t i v i t y related to t h e t o t a l n u m b e r of viable cells is meaningless (s = 32.0%). The same holds for t h e specific p r o d u c t i v i t y related t o t h e t o t a l volume of immobilized viable cells (Zt • Vz), not only by reason of t h e higher s t a n d a r d deviation a r o u n d an a p p a r e n t m e a n value (s = 16.4%), b u t more pronounced because of its distinct dependence upon t h e cell size (Y z ), which also exist with t h e specific p r o d u c t i v i t y related to t h e cell n u m b e r . I n contrast, such a dependence of t h e surface-related specific p r o d u c t formation rate is not obvious, neither upon t h e cell n u m b e r , nor upon t h e cell size. Tab. 2. Mean volume of single cells and specific product formation rates related to the total volume, to the total number, and to the total surface of viable cells, respectively, measured at different batch cycles of different fermentation series with the immobilized yeast strains Sc 2, Sc 5, Sc 20, Sc 34, and Sc 35 Strain

Series and cycle

• io- 1 0 per batch cycle

[(im3]

CO JZ, • F* [1/h • ml]

CO JZ, [1/h • 1010 cells]

CO J Z r O . [1/h • m 2 ]

Sc 34

11. 11. 11. 12. 12. 13. 13. 14. 14. 14. 1. 6. 2.

25.9 30.4 37.3 8.2 13.4 17.6 20.0 8.5 10.2 11.3 6.5 8.9 5.2

105 105 105 110 110 133 148 235 234 226 288 291 355

0.162 0.154 0.132 0.156 0.137 0.126 0.139 0.110 0.109 0.122 0.110 0.104 0.100

0.170 0.162 0.139 0.172 0.151 0.168 0.206 0.259 0.255 0.276 0.315 0.302 0.355

0.158 0.150 0.129 0.155 0.136 0.133 0.153 0.141 0.139 0.154 0.150 0.142 0.147

Sc 20 Sc 35 Sc 2 Sc 5

6 7 8 2 3 3 11 3 6 7 8 4 11

V,

mean value :

0.128

0.225

±

«:• 0.021 (16.4%)

0.072 (32.0%)

0.145 0.009 (6.2%)

Therefore, t h e surface of t h e viable cells is t h e significant criterion of specific ethanol f o r m a t i o n r a t e of t h e yeast cells, presumably n o t only with 8. cerevisiae. This indicates t h a t a mass t r a n s f e r into or o u t of t h e cells through t h e cell m e m b r a n e is t h e r a t e limiting s t e p of ethanol formation a t o p t i m u m s u p p l y of cells. Of course, t h e shown regularity f o u n d with immobilized cells has t o be hold with free cells, too. Consequently, for kinetic investigations it does clearly n o t suffice t o look a t the actual mass or n u m b e r of viable cells only, b u t additional informations concerning a possible variation of t h e cell size during a f e r m e n t a t i o n are needed. Abbreviations HTS = total weight of dry biomass inside the gel O z = mean surface of a single cell V1 — mean volume of a single cell r A = total number of cells inside the gel

400

Acta Biotechnol. 8 (1988) 5

Subscript I = viable

Acknowledgement T h e a u t h o r s wish to t h a n k D r . sc. K . R I C H T E R for helpful discussion, D r . V. T E N C K H O F F for his aid in determination of t h e cell dimensions, a n d Mrs. E . N A U M A N N , Mrs. M. S U C K E R a n d Mrs. A. BUHE for their conscientious technical assistance. Received December 1, 1987

References K Ü H L E M A N N , I . , B E C K E R , U . , B E R G E R , R . : Acta Biotechnologica, s u b m i t t e d [2] WADA, M., KATO, J . , CHIBATA, 1.: E u r . J . Appi. Microbiol. Biotechnol. 10 (1980), 275. [3] RÜHLEMANN; I., TENCKHOFF, V.: Poster a t t h e 4. Symposium of Socialist Countries on Biotechnology. W a r n a , May 1986. [4] CHOTANI, G. K., CONSTANTINIDES, A.: Biotechnol. Bioeng. 26 (1984), 217. [5] AMIN, G., STANDAERT, P., VERACHTERT, H . : Appi. Microbiol. Biotechnol. li) (1984), 91. [ 6 ] J O H A N S E N , A . , F L I N K , J . M.: E n z y m e Microb. Technol. 8 ( 1 9 8 6 ) , 1 4 5 . [7] SHIOTANI, T., YAMANE, T . : E u r . J . Appi. Microbiol. Biotechnol. 13 (1981), 96. [8] WANG, H . Y., HETTWEIÌ, D. J . : Biotechnol. Bioeng. 24 (1982), 1827. [9] G O D I A , F.. CASAS, C . , SOLA, C . : J . Chem. Technol. Biotechnol. 35 B (1985), 139. [10] LEE, T. H., AHN, J . C., RYU, D. D. Y . : E n z y m e Microb. Technol. 5 (1983), 41. [11] KROUWEL, P. G., KOSSEN, N. W. F . : Biotechnol. Bioeng. 23 (1981), 651. [12] SIMON, J . P., MESSENGUY, F., DUBOIS, E . : Proc. 4. E u r o p . Congr. Biotechnol., A m s t e r d a m 1987, Vol. 3 (1987), 445. [13] B E R G E R , R . , R Ü H L E M A N N , I., R I C H T E R , K . : Poster on t h e 16. FEBS-Meeting, J u n e 1984, Moscow [ 1 4 ] A L B E R G H J N A , L . , M A R I A N I , L., M A R T E G A N I , E . , V A N O N I , M . : Biotechnol. Bioeng. 2 5 ( 1 9 8 3 ) , [1] RICHTER, K . ,

1295.

[15]

RANZI,

[16] RANZI,

B. B.

M . , COMPAGNO, C . , M A R T E G A N I , M., PORRO, D . , MARTEGANI,

E.,

E . : Biotechnol. Bioeng. 28 (1986), 185. : Proc. 4 . E u r o p . Congr. Biotech-

ALBERGIUNA, L .

n o l . , A m s t e r d a m 1987, V o l . 3 (1987), 3 3 1 .

[17] WADA, M., KATO, J . , CHIBATA, I . : E u r . J . Appi. Microbiol. Biotechnol. 10 (1980), 275. £18] Kim, H. S., Ryli, D. D. Y . : Biotechnol. Bioeng. 24 (1982), 2167.

Acta Biotechnol. 8 (1988) 5, 4 0 1 - 4 0 5

Stable Ionotropic Gel for Cell Immobilization Using High Molecular Weight Pectic Acid B E R G E R , R . , RTTHLEMANN, I .

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

Summary It is shown that Ca- and Al-pectate gel beads prepared by use of high molecular weight poly, galacturonic acid (viz. polygalacturonic acid having a high STAUDINGER index) are well suited for cell immobilization. The pectate beads are much more insensitive to those ions and chemical agents which destructively act on alginate beads (such as phosphate, citrate, gluconate, lactate as well as a high excess of sodium, potassium, and/or ammonium ions), even without addition of gelling cations to the liquid phase.

Introduction As can be seen in the literature [e.g. 1—4], biotechnological product syntheses with the help of microbial cells, especially with viable cells, which are immobilized by entrapment in a gel-like polymeric lattice, find continuously increasing interest. Originally the mainly used matrix material was polyacrylamide [4], but at present one prefers the entrapment in ionotropic gels or other gel-like matrices which can be produced under mild conditions as well as the entrapment in polyurethane foam (polyurethane gel) or in photo-cross-linked resins using the suitable prepolymers [5]. For preparing immobilized cells being entrapped in ionotropic gels Na-alginate or Na-carrageenan are most widely applied as the basic material [1, 6—9], In both cases the resulting gels possess the disadvantage that they rapidly lose their mechanical stability or even dissolve under certain medium-conditions. This may be prevented by a suitable aftertreatment of the gel, but such procedure is an expensive and time-consuming one, and cell damage cannot be ruled out in many cases (toxic hardening or cross-linking agents). For example, potassium-, ammonium- or calcium carrageenan gel dissolves in physiological saline, at least at a temperature S; 37—40 °C [10], The stability of Ca-alginate gel is greatly diminished upon contact with media containing a ten- to twentyfold molar excess of sodium, potassium, ammonium, and/or magnesium ions over calcium ions [11]. Furthermore, an instability of Ca-alginate even resulting in dissolution of the gel occurs upon contact with media containing a noticeable amount of yeast extract [12] and/or such calcium complexing agents as phosphate, citrate, lactate etc., especially in the absence of additional calcium ions in the medium [13]. (Carrageenan gelled by a diamine or polyamine dissolves after some time in a medium containing citrate, too [14]). Therefore, product syntheses necessitating phosphate

Acta Biotechnol. 8 (1988) 5

402

ions as well as the biotechnological manufacture of citric acid, gluconic acid or lactic acid with microbial cells immobilized in Ca-alginate cannot be conducted in a continuous manner over a long time without copious addition of calcium ions, which on their part may interfere with other process requirements. Only a few references concerning with the successful pratical application of pectinate gel for cell immobilization and with use of pectinate entrapped cells for biotechnological substrate conversions are known [ 1 1 , 1 5 — 1 9 ] , NAVARRO et al. [ 1 5 , 1 6 ] employed a citrus pectin (from Sigma Chemical Co.) for alcoholic fermentation with cells entrapped in Ca-pectinate and a complex medium containing calcium ions for gel stabilization. VORLOP [ 1 1 ] has tested a low esterified Na-pectinate from Denmark ( 4 . 3 % methoxy groups) as basic material for preparation of Ca- and Mg-pectinate beads. Both types of gel beads possessed a mechanical stability similar to Ca-alginate beads provided that the loading of the pectinate gel with cells does not exceed a value of about 0.2 g wet cells per gram gel. VORLOP has not communicated fermentation tests using these pectinate beads, but swelling experiments showed that both the Ca- and Mg-pectinate beads after drying in an air stream exhibit a greater tolerance for ions (e.g. N a + , phosphate) destroying the mechanical stability of air-dried Ca-alginate beads in a medium also containing some calcium of magnesium ions for gel stabilization, viz. the pectinate beads show a much lower swelling than Ca-alginate beads in such cases [11]. In this paper it is shown that Ca- or Al-pectate beads prepared by use of high molecular weight pectic acid ( S T A U D I N G E R index 200 ml/g) containing a very low methoxy group content (sS 3 % ) are well suited for cell immobilization, and that these pectate gels show a much higher tolerance for ions or chemical agents fast destroying the stability of Ca-alginate beads, even without addition of gel-stabilizing ions (such as C a + + , A l + + + ) to the liquid surrounding the pectate beads.

Materials and Methods Polymers Na-alginate was obtained from Riedel de H A E N (STAUDINGER index = 7 1 1 ml/g), pectic acid (MW ca. 30000; S T A U D I N G E R index = 85 ml/g), and citrus pectin (methoxy group content > 6.7%) from S E R V A . The high molecular weight pectic acid (polygalacturonic acid) samples are kindly provided by the Central Institute of Nutrition of the Academy of Sciences of the G . D . R . ; PGA-1 with a STAUDINGER index = 210 ml/g and a degree of esterification = 3.0%, PGA-2 with a STAUDINGER index = 373 ml/g and a methoxy group content = 0. The STAUDINGER indices have been extrapolated using viscosity measurements on dilute solutions (c ^ 0.003 g/ml) in 0.05 M aqueous NaCl at pH 6.0 and 25°C. Microorgan

isms

Saccharomyces cerevisiae S c 5 and Streptococcus collection of our institute. Preparation

of the Gel Beads I Cell

thermophilus

are strains from a culture

Immobilization

2 g Na-alginate were dissolved in 100 ml distilled water at room temperature with stirring and this solution was added dropwise to 400 ml gentle stirred 0.1 M CaCl2 solution or 0 . 1 M Al-sulfate solution using a device similar to that described by V O R L O P [11]. After that the beads formed have been stirred another two hours in the same but renewed hardening solution.

BERGER, R . , RÜHLEMANN, I . , Cell I m m o b i l i z a t i o n

403

2 or 4 g citrus pectin or 3 or 4 g pectic acid PGA-1 and PGA-2, respectively, suspended in 60—70 ml distilled water, were dissolved at room temperature by means of cautious, dropwise addition of 2 N NaOH with stirring until a p H 7 — 8 had been reached. Thereafter this solution was filled up to 100 ml with distilled water and used for the bead preparation in the same manner as described for alginate beads, applying a 0.1—0.4 M CaCl2 solution or 0.2 M Al-sulfate solution for precipitation and hardening, respectively. In each case the beads prepared had a narrow diameter distribution with mean values ranging from 0.8 to 1.5 mm. For cell immobilization the Streptococcus cells were entrapped in Ca pectate beads ( 0 = 0 . 8 mm) using the 4% PGA-2 solution (3.7 g dry biomass/100 ml) and 0.4 M CaCl2 solution. The yeast cells were entrapped in Ca-pectate as well as Ca-alginate using the 3% PGA-1 solution (2.5 g dry biomass/100 ml) and 0.1 M CaCl2-solution or the 2% alginate solution (2.5 g dry biomass/100 ml) and 0.05 M CaCl 2 -solution; in both cases the mean diameter of the beads formed amounted to 1.5 mm. Stability

Tests

The Ca- and Al-alginate beads as well as the Ca- and Al-pectate beads prepared by use of the 3% PGA-2 solution and 0.1 M CaCl2 solution or 0.2 M Al-sulfate solution were shaken 42 h at 80 °C on a vibrating table-water bath at ca. 300 rpm in the following solutions: 0.5 M phosphate buffer p H 8; 20% aqueous gluconic acid adjusted to p H 3.5 with NaOH; 10% aqueous citric acid adjusted to p H 4.8 with NaOH; 10% aqueous lactic acid adjusted to pH 6.0 with NaOH. These p H values were chosen to simulate fermentation conditions which may occur at production of the corresponding acids. The citrus pectin beads (0.2 M CaCl2-solution) were shaken in 0.5 M phosphate buffer pH 8.0 and in 0.1 M phosphate buffer pH 7.0. Additionally, the Ca-alginate beads and Ca-pectate beads prepared by use of the 4% PGA-1 solution and 0.2 M CaCl2 solution as well as the 3% PGA-2 solution and 0.1 M CaCl2 solution have been shaken 48 h at 80 °C in 1 M and 2 M NaCl solution. For each test 5 beads per 10 ml of the corresponding aqueous solution in a 25 ml volumetric flask have been used. In a further test 20 g of the Ca-pectate beads loaded with the Streptococcus cells were continuously stirred for twelve days at 50 °C in 30 ml of the 10% lactic acid solution (pH 6.0) with a stiff-blade agitator (two 10 X 15 mm blades) in a 100 ml round bottom boiling flask at ca. 400 rpm, viz. without addition of calcium ions, too. Alcoholic

Fermentation

Continuous ethanol fermentations have been accomplished using a special horizontal column reactor [20] filled with the Ca-pectate and Ca-alginate beads containing the yeast cells, respectively. The inflowing unbuffered complex medium adjusted to p H 6.0 contained glucose, yeast extract, and mineral salts but no additional calcium ions. The molar sum of sodium, potassium, and ammonium cations in the medium exceeded that of bivalent cations by at least the hundredfold. Results The stability test demonstrated that Ca-pectate (as well as Al-pectate) is much more insensitive to those ions and calcium (aluminium) complexing agents which diminish the stability of Ca- and Al-alginate beads, respectively, even without addition of gelstabilizing cations (such as Ca + + , Al + + + ) to the liquid environment of the pectate beads, but on condition that the pectate gel beads are prepared by use of a high molecular

404

Acta Biotechnol. 8 (1988) 5

weight pectic acid ( S T A U D I N G E R index Si 200 ml/g, methoxy group content 3%). With the pectic acid from SERVA (4% solution) no beads were formed in the stirred precipitating liquid (0.2 M CaCl2 solution). Whereas the Ca- and Al-alginate beads dissolved in the 10% citric acid solution already after 3 h and in the 0.5 M phosphate buffer, 10% lactic acid, and 20% gluconic acid solution after 24 h at the latest, the Ca- and Al-pectate beads did not dissolve, they only swelled up some after 42 h at 80°C, maximally to the 1.1-fold of the original diameter. The Ca-alginate beads also dissolved in 0.1 or 0.2 M NaCl-solution after 20 h whereas the pectate beads prepared by use of PGA-1 and PGA-2, respectively, showed no change of diameter after 48 h at 80°C. The citrus pectin beads dissolved in the phosphate buffers after 2 h at the latest. Likewise, the diameter (if the Ca-pectate beads with entrapped Streptococcus cells did not change after 12 days continuous agitation in the 10% lactic acid solution at 50°C. Despite of the vigorous stirring only 10% of the applied beads were mechanically injured. The applicability of Ca-pectate gel as an immobilization matrix at continuous ethanol fermentation with entrapped cells has been examined using immobilized yeast cells and compared with the behaviour of Ca-alginate under the same conditions. The ethanol fermentation in the horizontal column reactor using the yeast-containing alginate beads must be stopped after 30 h because of high swelling of the alginate beads. This bead swelling caused plugging of the packed bed by which steady flow of the liquid medium was no longer assured. To the contrary there was only a low swelling of the pectate beads and therefore no plugging of the packed bed. The liquid medium steadily flowed through the column from the outset up to the seventh day of fermentation, after w hich the fermentation was interrupted, because of plugging of the bed caused by free cells. Acknowledgement The authors wish to thank Dr. R. MAUNE (Central Institute of Nutrition of the Academy of Sciences of the G.D.R.) for supply of the pectic acid samples, Dr. sc. K. RICHTER for helpful suggestions as well as Mrs. E. NAUMANN, Mrs. A. BUHE, and Mrs. M. HENNIG for their conscientious technical assistance. Received January 19, 1988

References [1] CHIBATA, I., TOSA, T., FUJIMURA, M. : Annu. Rep. Ferment. Processes 6 (1983), 1. [ 2 ] K E N N E D Y , J . F . , CABRAL, M . S . J . : A p p l . B i o c h e m . B i o e n g . 4 ( 1 9 8 3 ) , 1 8 9 .

[3] MESSING, R. A. : Annu. Rep. Ferment. Processes 4 (1980), 105. [4] [5] [6] |7] [8]

BERGER, R . : A c t a B i o t e c h n o l . 1 ( 1 9 8 1 ) , 7 3 ; 2 ( 1 9 8 2 ) , 3 4 3 . FUKUI, S . , TANAKA, A . : A n n u . R e v . M i c r o b i o l . 3 6 ( 1 9 8 2 ) , 1 4 5 , TAKATA, I . , TOSA, T . , CHIBATA, I . : J . S o l i d - P h a s e B i o c h e m . 2 ( 1 9 7 7 ) , 2 2 5 . HACKEL, U . , KLEIN, J . , MEGNET, R . , WAGNER, R . : E u r . J . A p p l . M i c r o b i o l . 1 ( 1 9 7 5 ) , 2 9 1 . KLEIN, J . , WAGNER, F . : D e c h e m a M o n o g r . 8 2 ( 1 9 7 8 ) , 1 4 2 .

[9] KLEIN, J., WAGNER, F.: DE-OS 2835875 (1980). [ 1 0 ] TOSA, T . , SATO, T . , MORI, T . , YAMAMOTO, K . , TAKATA, I . , NISHIDA, Y . , CHIBATA, I . :

technol. Bioeng. 21 (1979), 1697. [11] VORLOP, K.-D.: Dissertation, TU Braunschweig, 1984. [12] LUONG, J. H. T.: Biotechnol. Bioeng. 27 (1985), 1652.

Bio-

BERGER,

R.,

RUHLEMANN,

I., Cell Immobilization

405

[13] BIRNBAUM, S., PENDLETON, T., LARSSON, P.-O., MOSBACH, K . : Biotcchnol. L e t t . 3 (1981),

393. [14] BORGLUM, G. B., MARSHALL, J . J . : Appi. Biochem. Biotechnol. 9 (1984), 117. [15] NAVARRO, A. R . , RUBIO, M. C., CALLIERI, D . A. S.: E u r . J . Appi. Microbiol. Biotechnol. 17

(1983), 148. [16] NAVARRO, A. R . ,

MARANGONI, H . ,

PLAZA, I . M.,

CALLIERI, D . A. S. : B i o t e c h n o l .

(1984), 465. [17] NOGUCHI, S., NAGASHIMA, M., AZUMA, A., FURUKAWA, S . : CA 1 1 9 1 0 9 8 (1985).

[18] Anon. J P 8313387 (1983); Chem. Abstr. 98: 196384. [19] Anon. J P 8313391 (1983); Chem. Abstr. 98: 214167. [20] BERGER, R . , RUHLEMANN, I . : D D 221470 (1985).

Lett.

6

Acta Biotechnol. 8 (1988) 5, 406

Book Review Morton H.

FRIEDMAN

Principles and Models of Biological Transports Berlin, Heidelberg, N e w York, T o k y o : Springer-Verlag, 1986. 260 pp., 105 fig., DM 1 6 5 . -

I n t h e preface this book is characterized as a t e x t b o o k for advanced students, a n d in f a c t one feels t h a t it is t h e result of a successful teaching practice. The logical s t r u c t u r e , m a n y clear, v e r b a l explanations of physical facts a n d m a t h e m a t i c a l derivations, interspersed conclusions a n d a n a logies as well as references t o their biological significance m a k e this book suitable for self-instruction. Also t h e reader, n o t very familiar with biological t r a n s p o r t , will be easily enabled t o i n t r u d e into t h e m a t t e r . The m a t h e m a t i c a l a p p a r a t u s avoids all unnecessary details a n d special cases. T h e a r r a n g e m e n t of chapters begins with physical-chemical f u n d a m e n t a l s (chemical, electrochemical potential, equilibria across membranes, free a n d electrodiffusion). The following p a r a g r a p h s t r e a t with facilitated diffusion a n d active t r a n s p o r t , essentially in t h e usual m a n n e r . The references t o biological structures a n d t h e avoidance of pure, a b s t r a c t model conceptions m u s t be emphasized. One t h i r d of t h e book is devoted t o t r a n s p o r t p h e n o m e n a in characteristic biological m e m b r a n e systems (e. g. ion- a n d monosachharide t r a n s p o r t across red cell membrane, excitation a n d propagation of actions potentials in nerves, synaptic transmission, coupled transcellular t r a n s p o r t s in small intestine a n d kidney, oxygen diffusion in capillaries a n d tissue). The index of recommended literatur is restricted to f u n d a m e n t a l papers a n d reviews. T h e book can be recommend w i t h o u t a n y limitation as i n t r o d u c t i o n a r y literature for s t u d e n t s of biology a n d related disciplines (medicine, biophysics, biochemistry) a n d for all scientists whose research topics touch t r a n s p o r t phenomena. F . Miri,i,ER

Acta Biotechnol. 8 (1988) 5, 4 0 7 - 4 1 3

Biosynthesis of Protein by Microscopic Fungi in Solid State Fermentation. I. Selection of Aspergillus Strain for Enrichment of Starchy Raw Materials in Protein CZAJKOWSKA, D . , ILNICKA-OLEJNICZAK, 0 .

Institute of Fermentation Industry Department of Technical Microbiology and Biochemistry Rakowiecka 36, 02532 Warszawa, Poland

Summary A fungal strain suitable for protein enrichment of starchy raw materials was selected by evaluation of the growth rate, results of protein biosynthesis in solid state fermentation (SSP) and assessment of fungal biomass. The strain Aspergillus oryzae A.or. 11 selected for further studies was characterized by the radial growth rate Kr of about 300 (J.m/h and by the specific growth rate ¡i of about 0.100 h - 1 . Fungal biomass contained about 3 2 % of crude protein in dry matter. The digestibility of this protein in vitro was close to 75%. Protein analysis for amino acids showed that the content of exogenous amino acids approached that in protein of the FAO standard. As a result of 34 — h culture of this strain in solid medium, net protein increased by about 5.0 g/100 g of starting medium d.m. at the cost of decomposition of about 20 g carbohydrates.

Introduction On account of the steadily increasing protein deficiency on a world scale, it is indispensable to search for new sources and new procedures for protein obtainment. In recent years attention has been directed to utilization of microbiological biosynthesis of protein by microscopic (filamentous) fungi exhibiting — similarly as yeasts — a high growth rate, big protein content in biomass, high protein digestibility and an interesting amino acid composition. According to ¡LOBANOK [1], in the major part of filamentous fungi the specific growth rate fi is between 0.100—0.300 h" 1 ; however, in some species these values are much higher, amounting for e.g. Neurospora sitophila to 0.400 h - 1 , and for Oeotrichum candidum to 0.610 h _ 1 . For fodder yeast, the values of fi (determined at the

Institute of Fermentation Industry in Warsaw [2]) have been found to be between 0.065—0.500 h _ 1 . Protein content in the biomass of microscopic fungi and yeasts depends on the species and kind of medium. GREGORY [3] has cultured 130 fungal species in a

medium with manioc, and found protein contents of 36.7—49.5% of biomass dry matter. CHRISTIAS [4] h a s grown Aspergillus

niger,

Fusarium

oxysporum

a n d Fusarium

monili-

forme in a synthetic medium and obtained protein contents of 39.1 — 58.2% of biomass dry matter. In the biomass of strain Trichoderma album applied for protein biosynthesis

on lignocellulosic raw materials, protein content was 5 3 — 6 3 % of d.m. [5]. The biomass

of yeasts is generally assumed to contain about 50% of protein in d.m. The literature concerning the amino acid composition of fungal proteins testifies to great differences in contents of amino acids within each group of microorganisms (yeasts

408

Acta Bioteohnol. 8 (1988) 5

and filamentous fungi). However, in moat cases the content of amino acids (with the exception of methionine and cystine) fluctuates near the values reported for the FAO standard [5, 6]. Authors taking up utilization of microscopic fungi for protein biosynthesis stress — moreover — other favourable properties of these organisms, namely their ability to form an enzymic complex permitting transformation — into protein — of various raw materials as well as of different agricultural and industrial by-products; the low content of nucleic acids in fungal biomass is also emphasized. In the mid — seventies, in several countries (France, U S S R , Mexico, Poland) research work has been started on fungal protein biosynthesis in solid state fermentation. Most of these studies have been performed on a laboratory scale [7—12]; at present these investigations are carried out also on a pilot plant scale [13—15], Fungal culture in solid medium affords products with protein content more than 100% higher than that in the raw material; furthermore these products contain all residual medium components which have not been transformed into fungal biomass. Studies of this kind, initiated at the Institute of Fermentation Industry in Warsaw in the beginning of the eighties, were aimed at the development of a technology for obtainment of high-protein feeds from raw materials or from agricultural and food industry by-products. It was intended to introduce this kind of culture and immediate utilization of the resulting feeds at state, co-operative or private farms.

Materials and Methods Microorganisms Use was made of 10 Aspergillus strains maintained in our collection. They included five Aspergillus oryzae strains ( A.or. 6/2, A.or. 9, A.or. 10, A .or. 11, A.or. 13), four Aspergillus niger strains (A.n. 39, A.n. 40, A.n. 41, A.n. 50) and one Aspergillus awamori strain (A.a. 2). The inoculum consisted of spores from a 7 — day fungal culture on wort-agar slants. To 100 g of solid medium, 10 7 —10 8 conidia were added. Media Fungal culture was carried out in solid medium (60% of moisture, pH about 6.5) containing about 3 2 % of coarse rye meal and 8 % of dry beet pulp. In the experiments concerning protein biosynthesis addition was made — per 100 g of a mixture of 80 g coarse rye meal and 20 g dry beet pulp — of 2.1 g nitrogen in the form of (NH 4 ) 2 S0 4 and of 0.7 g phosphorus in the form of K H 2 P 0 4 . For evaluation of the strain growth rate (/% and Kr) the C Z A P E K - D O X medium with soluble starch was applied. For multiplication of the biomass of the selected strains, the C Z A P E K - D O X medium with sucrose was used. Fungal

Culture

Fungal culture in solid medium was performed in a laboratory bioreactor [ 16] at 30 °C ± 1 °C, the thickness of medium layer being about 12 cm (this corresponded to 25 g of medium per culture tube). Tubes were aerated with 160 dm 3 of water-saturated air per h and per 1000 g medium. The duration of culture depended on the kind of experiment. In the preliminary experiments dealing with the strain's ability of amylolytic enzymes biosynthesis, the strain growth rate and sporulation rate, this duration was 48 h.

409

CZAJKOWSKA, I ) . , ILNICKA-OLEJNICZAK, O . , B i o s y n t h e s i s of P r o t e i n

Culture in liquid medium was carried out in E R L E N M A Y E R flasks ( 5 0 0 cm 3 ) containing 100 cm 3 of medium: a laboratory shaker (200 rev./min) was employed. Culture was carried out for 48 h at 30 °C. For evaluation of protein biosynthesis, before and after the culture determination was made of dry matter, content of total carbohydrates and protein content. This permitted calculation of: — the increase in net protein in g/100 g of starting medium d.m. — taking into account the decrease in dry matter at a given time of culture, — the degree of utilization of carbohydrates in g carbohydrates/100 g of starting medium d.m., — the protein biosynthesis yield calculated either per carbohydrates utilized. Analytical Methods — Crude protein was determined by K J E L D A H L ' S method. — True protein was assayed by L O W R Y ' S method after treatment of the sample with 1 N NaOH at 100°C for 10 min. [8]. — Protein digestibility in vitro was estimated by the pepsin method. — Total carbohydrates calculated per glucose were determined colorimetrically by the method with DNS after 3 h acid hydrolysis of the sample at 100 °C [17]. — Glucoamylase activity (in GA units) was determined by the NOVO method modified b y KUJAWSKI

[18].

— The activity of alpha-amylase (in AS units) was assayed by the method of a n d RODZEWICZ

KLIMOVVSKI

[19].

— Determination of amino acids in hydrolyzates resulting from acid hydrolysis of fungal proteins was performed by gas chromatography according to G E H R K E , in a modification by K U B A C K A [ 2 0 , 2 1 ] . Results and Discussion At the first stage of studies on selection of a strain for protein enrichment of starchy raw materials, determination was made of the ability of some selected fungal strains to biosynthesize amylolytic enzymes in S S F . At the same time, under the same conditions fungal growth and sporulation intensity were evaluated macroscopically (Tab. 1). Among 10 investigated Aspergillus strains, only four: A .or. 9 A.or. 10, A.or. 11 and A.or. 13, were characterized by rapid growth ( + + + + or + + + ), with evident limitation of sporulation. These strains exhibited alpha-amylase and glucoamylase activities. The remaining strains mainly biosynthesized glucoamylase. At the next stage, for four selected strains the growth rate was determined by measurements of KT and p, and the yield of protein biosynthesis was evaluated in S S F (Fig. 1 A, B , C). The values of (i were between 0.050—0.100 h" 1 and those of K r were between 230—300 [xm/h. There was a significant correlation between the specific growth rate fi and radial growth rate Kr; it was expressed by the equation: KT = 1511.144/1 + 128.870

r = 0.968

« = 0.05

Moreover, there was a significant correlation between Kr and protein yield, expressed by the equation: Y = 0.033K r -

3.780

r = 0.951

« = 0.05

Y — increase in yield of net protein in g. 2

Acta Biotcchnol. 8 (1988) 5

410

Acta Biotechnol. 8 (1988) 5 Tab. 1. Growth and sporulation of Aspergillus strains, and amylolytic enzyme activities in SSF Growth

Fungal strain

Aspergillus

niger A.n. A.n. A.n. A.n.

Aspergillus

oryzae A.or. A.or. A.or. A.or. A.or.

Aspergillus

39 40 41 50

6 9 10 11 13

Sporulation

Enzyme activities [U/g d.m. of product] 1 U AS

U AG

791 850 1100 540

++ + ++ + ++ +

intense intense beginning absent

1.5 2.0 3.0 1.0

+ +++ + ++ + ++++ +++ +

absent absent absent beginning beginning

13.0 21.0

+ +

intense

31.0' 19.0

11.0

89.0 107.0 330.0 404.0 294.0

1.8

1800.0

awamori A.a. 2

Enzyme activities were determined in extracts of products (2 g of product extracted with 48 cm 3 of dist. water for 1 h at room temperature) Growth evaluation: 1

+ + + -f + + + + + +

slight medium good very good

The yields of net protein were highest (4.4—5.2 g/100 g of starting medium d.m.) for three most rapidly growing strains A.or. 9, A .or. 11 and A .or 13, whereas they were lowest for slowly growing strain A.or. 10. This was paralleled by differences in utilization of total carbohydrates, fluctuating between 10.0—19.2 g per 100 g of starting medium d.m. Protein yield calculated per carbohydrates utilized was closely similar for all investigated strains, amounting to 23.9—27.6%. Final selection of strain was based on evaluation of fungal biomass of four strains: A.or. 9, A.or. 10, A.or. 11 and A.or. 13 (Tab.2). Crude protein content in the biomass was similar for all four strains (33.1 — 34.8%). Likewise, protein digestibility was closely similar (72.8—78.8%). There were no evident differences between strains in the amino acid composition of protein. Only in case of strain A.or. 10, the threonine content was lowest and the lysine content — highest. However, the strain A.or. 10 was not utilized for further technological studies, because of its poor protein biosynthesis. Finally, for technological studies we selected the strain A.or. 11 which — similarly as strain A .or. 13 — afforded a high yield of net protein (about 5.0 g/100 g of starting medium d.m.), and exhibited a high protein yield calculated per carbohydrates utilized (about 27%, Fig. 1 C). This strain, as compared with strains A.or. 9 and A.or. 13, was characterized by the highest lysine content in biomass protein.

411

Czajkowska, D., Ilnicka-Olejniczak, O., Biosynthesis of Protein 0.100

0.080

-

0.060

0.040

-

0.020

-

Time

[hi

34 h

20

culture 27.6

23.9%

26.0

210

6

"b

6 =»_

5.2

kM £

24 Time

34 [hi

4

A. or.

2.7

/ 9

/10

/11

/13 Strain

Fig. 1. A, B — Growth rate of Aspergillus oryzae strains (/i and Kr) A.or. 9, o o A.or. 10, A.or. 11, A.or. 13; C — Protein biosynthesis and carbohydrate utilization in solid state fermentation ggjgjl net protein biosynthesized, | | total carbohydrates utilized, % protein yield calculated per carbohydrates utilized

Conclusions The strain Aspergillus oryzae A.or. 11 was selected for further studies on protein enrichment of starchy raw materials, because of a high yield of protein biosynthesis in SSF, and on the grounds of evaluation of the biomass. This strain afforded during 34 h culture in SSF an increase in net protein by about 5.0 g/100 g of starting medium d.m., 2*

Acta Biotechnol. 8 (1988) 5

412

Tab. 2. Characterization of biomass of the selected Aspergillus oryzae strains Strain

A.or. 9

A.or. 10

A.or. 11

A.or. 13

Crude protein Ntotal X 6,25 [ % of biomass d.m.]

34.8

33.6

32.4

31.1

Digestibility of protein

72.8

78.8

76.2

75.0

Amino acid composition [g/16 g N] Ala Val Gly lieu Leu Pro Thr Ser Met Phe Asp Glu Lys

4.80 4.48 3.30 3.59 4.60 2.57 3.33 3.59 0.52 2.64 6.49 10.69 5.69

5.24 4.28 3.42 3.21 4.67 2.50 2.65 3.39 0.48 2.86 6.22 9.82 6.61

4.82 4.47 3.63 3.45 4.61 2.64 3.27 3.13 0.64 2.72 6.23 9.72 6.08

4.98 4.35 3.41 3.35 4.92 2.65 3.08 3.41 0.63 2.84 6.19 9.78 5.62

2J Amino acids

56.29

55.35

55.41

55.21

24.85

24.76

25.20

24.79

[%]

Exogenous amino acids

a t the cost of utilization of 20 g of total carbohydrates. The biomass of this fungus contained 32 % of crude protein (digestibility about 75 % ). The composition of exogenous amino acids of this protein approached t h a t of the F A O standard. Received December 3, 1987 References [1]

T O B A Í T O K , A. G., B A B I C K A J A , W. G. : Miceljalnyje griby kak producenty belkovych vesóestv. Nauka i Technika, Minsk, 1981. [ 2 ] I L N I C K A - O L E J N I C Z A K , O . , L I P I E C , M. : Prace Instytutów i Laboratoriów Badawczych Przemyslu Spozywczego 33 (1980), 43. [3] G R E G O R Y , K. F . : Anim. Feed. Sci. Technol. 2 (1977), 1, 7. [ 4 ] C H R I S T I A S , C . , C O U V A R A K I , C . , G E O R Q O P O U L O S , S. G . , M A O R I S , B . , V O N V O Y A N N I , V . : Appi. Microbiol. 29 (1975), 2, 250. [5] S T A R O N , T . : Patent Great Britain, No 1604781, C 12, n 1/04, 1981. [6] Y A Z I C I O G L U , T. : Production and Feeding of Single Cell Protein Proceedings of a COST Workshop Zurich, Appi. Sc. Publish., London, 1983, 159. [ 7 ] B E D N A R S K I , W., J A K U B O W S K I , J . , P O Z X A Ñ S K I , S. : Przemysl Fermcntacyjny i Rolny 1 4 (1970), 10,

21.

Eur. J . Appi. Microbiol. Biotechnol. 9 (1980), 3 , 199. A., D E LA T O R R E , M . , C A S A S - C A M P I L L O , C . : Production and Feeding of Single Cell Protein Proceedings of a COST Workshop Zurich, Appi. Sc. Publish., London,

[ 8 ] RAIMBAULT, M . , ALAZARD, D . : [9]

RAMOS-VALDIVIA,

1983,

104.

CZAJKOWSKA, D . , ILNICKA-OLEJNICZAK, 0 . , B i o s y n t h e s i s o f

Protein

413

[ 1 0 ] SANTARELLI, S . R . : P a t e n t F r a n c e , N o 2 3 0 0 8 0 6 , C 1 2 , d 1 3 / 0 6 , 1 9 7 6 . [ 1 1 ] SZABTTNINA, T . , B I L A J , T . I . , SLIUSARENKO, T . P . : M i k r o b i o i . Z u r n a l 4 7 ( 1 9 8 5 ) , 2 , 6 3 . [ 1 2 ] VEZINHET, P . , ROGER, M . , O T E N O - G Y A N G , K . , GALZY, P . : R e v . F e r m e n t . I n d . A l i m e n t .

32

(1977), 3, 65. [ 1 3 ] D U R A N D , A . , A R N O U X , P . , TEILHARD DE CHARDIN,

O.: Production

a n d F e e d i n g of S i n g l e

Cell Protein Proceedings of a COST Workshop Zurich, Appi. Sc. Publish., London, 1983, 120. [14] SENEZ, J . C. : Production and Feeding of Single Cell Protein Proceedings of a COST Workshop Zurich, Appi. Sc. Publish., London, 1983, 101. [15] SENEZ, J . C. : Solid state fermentation of starchy substrates. Symp. „Bioconversion of organic residues for rural communities" Guatemala, 1978. [ 1 6 ] CZAJKOWSKA, D . , MYSZKA, L . , ZAKRZEWSKI, A . : B i o t e c h n o l o g i j a i B i o t e c h n i k a 5 ( 1 9 8 7 ) , 3 7 .

[17] MILLER, G. L.: Anal. Chem. 31 (1959), 3, 426. [ 1 8 ] K U J A W S K I , M . , ZAJAC, A . : S t ä r k e 2 6 ( 1 9 7 4 ) , 3 , 9 3 .

[19] PKONTN, C. J . : Amyloliticeskie fermenty i ich roi v piscevoj promyslennosti. Piscepromizdat, Moskwa, 1953. [20] GEHRE, C. W . , ROACH, G., ZUMWALT, W . : Q u a n t i t a t i v e g a s l i q u i d c h r o m a t o g r a p h y of a m i n o

acids in proteins. Monograph, 1968. [ 2 1 ] K U B A C K A , W . , KTJBACKI, J . S . : P r z e m . S p o z . 2 8 ( 1 9 7 4 ) , 2 4 6 .

A c t a Biotechnol. 8 (1988) 5, 414

Book Review J . LASCH

Enzymkinetik

Heidelberg, Berlin, N e w York, L o n d o n , Paris, T o k y o : Springer-Verlag, 1987. 148 S., 62 Abb., 18 Tab., DM 7 9 , - I S B N 17041

D a s Voranschreiten der Biotechnologie in volkswirtschaftliche Dimensionen b r i n g t a u c h ein zun e h m e n d e s Interesse a n der N u t z u n g enzymatischer Prozesse m i t sich. I n diesem Z u s a m m e n h a n g ist es n u r selbstverständlich, d a ß auch eine breitere B e s c h ä f t i g u n g interessierter Kreise m i t e n z y m kinetischen Fragestellungen die Folge ist. Diesem B e d ü r f n i s t r ä g t das vorliegende L e h r b u c h Rechnung. D e m A u t o r ist es gelungen, abgeleitet aus seinen Vorlesungserfahrungen, eine gründliche a b e r a u c h anschauliche E i n f ü h r u n g in die E n z y m k i n e t i k zu geben. D a s L e h r b u c h ist insgesamt didaktisch g u t a u f g e b a u t , wobei der A u t o r nicht n u r die E r f a h r u n g e n a u s bereits vorliegenden P u b l i k a t i o n e n zur gleichen T h e m a t i k n u t z t e , s o n d e r n sie m i t eigenen E r f a h r u n g e n bereicherte. Der I n h a l t v e r m i t t e l t alle n o t w e n d i g e n G r u n d k e n n t n i s s e , die z u r Bea r b e i t u n g enzymkinetischer Aufgabenstellungen benötigt w e r d e n . D u r c h die E i n f ü g u n g zahlreicher Beispiele u n d experimenteller Hinweise wird d e m Leser das E i n d r i n g e n in die relativ komplexe P r o b l e m a t i k weiter erleichtert. D e r kleine E x k u r s a m E n d e des Buches zu F r a g e n d e r R e a k tionskinetik immobilisierter E n z y m e ist f ü r d e n k ü n f t i g e n E n z y m t e c h n o l o g e n v o n b e s o n d e r e m Interesse, d a diese F o r m der E n z y m a n w e n d u n g z u n e h m e n d im technischen M a ß s t a b p r a k t i z i e r t wird. Die deutschsprachige Abfassung des Lehrbuches d ü r f t e vor allem d e n potentiellen I n t e r e s s e n t e n in diesem S p r a c h r a u m e n t g e g e n k o m m e n u n d die Erschließung dieses gewiß nicht leicht v e r s t ä n d lichen Gebietes erheblich schneller ermöglichen. Alles in allem ist das vorliegende L e h r b u c h so konzipiert, d a ß selbst der interessierte N i c h t f a c h m a n n sich relativ schnell m i t diesem Arbeitsgebiet v e r t r a u t m a c h e n k a n n . H a u p t s ä c h l i c h ist dieses B u c h jedoch a n Biowissenschaftler u n d Chemiker gerichtet, die ihre K e n n t n i s s e auf diesem Gebiet auffrischen bzw. vertiefen wollen. D a r ü b e r hianus ist es als L e h r b u c h f ü r S t u d e n t e n d e r Biochemie n u r zu empfehlen. A.

STEUDEL

Acta Biotechnol. 8 (1988) 5, 415-426

Acid-Mechanical, Alkaline and Enzymatic Treatment of Agricultural Wastes to obtain Substrate for Microbiological Conversion KATKEVICH, J . J . 1 , KATKEVICH, R . H . 1 , VIESTUBS, U . E . 1 , SAKSE, A . K . 2 ,

LEITE, M. P . 2

1

2

Institute of Wood Chemistry, Latvian SSR Academy of Sciences, 226006 Riga, 27, Akademijas Street, Latvian SSR, USSR August Kirchenstein Institute of Microbiology, Latvian SSR Academy of Sciences, 226067 Riga, 1, August Kirchenstein Street, Latvian SSR, USSR

Summary The present paper deals with almost wasteless technologies of pretreatment and obtaining of sugars and biomass from straw and potato tops. Several variants ensure a 1 t of straw per hour productivity using original auger reactors 290 and 150 mm in diameter. The authors have studied the submerged and solid state cultivation of various microorganisms applying the obtained substrates.

Introduction The aim of the present studies was to compare the variants of lignified material pretreatment with alkali or acids in order to obtain substrates fit for a subsequent biochemical or microbiological treatment, as well as to use the product as an improved rough feed. In the given studies wheat straw and potato tops were the initial raw material. For the obtaining of biomass the authors used Polyporus squamosus, a Trichoderma sp. fungus, while for the obtaining of sugars and substrate evaluation — the enzyme complex of Oeolrichum candida — cellokandin.

Materials and 3Iethods Wheat straw, gathered after thrashing on the collective farm "Uzvara", Bauska Region of the Latvian SSR, was ground in a DKU mill to 0.6—10 mm particle size. The chemical composition of straw from various portions (% dry matter -DM-): ash 6.6—7.3; water-soluble substances (WSS) — 7.8—9.8, reducing substances (RS) included — 2 . 3 - 2 . 7 ; easily hydrolysible substances (EHS) - 21.1-29.1, including RS 24.5-30.9; poorly hydrolysible substances (PHS) - 42.2—44.4, including RS - 43.2 to 44.9; acid-nonhydrolysible residue — 16.9—20.2. Sugar content in hyd roly sates: glucose — 38%, xylose — 23%, arabinose — 2.4%, galactose 1%. The tops of early potatoes were gathered in July on the Experimental Station of Selection at Priekuli, Latvian SSR. We used the stems, leaves removed, after extraction with hot water, then dried at 20°C and ground to 0.2—1.6 mm particle size. The stems constitute 33% of all the dry mass of the tops. The chemical composition (in % of absolute DM;

416

Acta Biotechnol. 8 (1988) 5

nitrogen - 1.9; ash - 6.6; E H S - 43.0, including RS - 22.9; P H S - 46.7, including R S — 44.2; lignin —10.4. The hydrolysate contained 40.6% of glucose, 8.3% of xylose, 2.7% of arabinose, 3.4% of galactose. WSS were determined as to the mass variance of the sample prior to and after storage at 40 °C in 0.05 acetate buffer (1:50) for 2 days in the presence of toluene. E H S — I N H 2 S 0 4 (1:50) for 5 h at 98 °C, P H S — after separation of E H S by treating the sample i n a 7 2 % H 2 S 0 4 ( 1 : 8 ) f o r 2 4 h a t 2 0 ° C w i t h a s u b s e q u e n t i n v e r s i o n of 1 n H 2 S 0 4 f o r 5 h

at 98°C. Insoluble, non-hydrolysible residue consists mainly of lignin. Lignin, soluble in cadoxene was determined upon a 280 nm length of light wave, extinction coefficient 18.0 was used to determine lignin. Ash was determined by combustion of the sample at 5 0 0 ° C , nitrogen — by a DUMAS micromethod, R S content — by SOMOGYI method and calculated as glucose. The number of separate monosaccharides in acid or fermentative hydrolysates was determined as to R S after the separation of their mixtures by paper chromatography method and a subsequent eluation of separate sugars from the chromatograms by water. To estimate the level of enzymatic hydrolysibility [1, 2] (EH) we used cellokandin — enzymatic complex produced by "Biolar" (Olaine, Latvian SSR) — in 0.05 M acetate or benzoate [3] buffer a t 40°C. Concentration of the substrate (S), enzyme (E), duration of hydrolysis and other conditions were set for each separate case. The level of E H was determined as to RS yield. In order to estimate the level of E H with a removal of reaction products we used 2—15 ml fermenters, mounted with a dialysing membrane, ensuring a continuous separation of macromolecular products and R S formed during hydrolysis [1]. Sugars for microbiological treatment were obtained in fermenters (1 — 3 1) equipped with a dialysing membrane. Polyporus squamosus was chosen as a producer of biomass on solutions and sugar suspensions. A mineral nitrogen and phosphorus source — ammonium nitrate and potassium biphosphate, while corn steep liquor was used as a biofactor. Treatment of nutrient medium for the growth of the fungus was carried out in 750 ml flasks with a 100 ml filling on a shaker with 220 r p m at 29 i 1 °C. The balance of the process was established on a laboratory equipment FU-6 [4] batchwise, 2.0 1 of nutrient medium. A 16-hour culture Polyporus squamosus was used as seeds, the culture was grown on a nutrient medium of the following composition: molasses — 2.2% (as t o RS), N H 4 N 0 3 — 0.3%, K H 2 P 0 4 — 0.12%. The amount of the seeds in all the experiments — 10 vol.%. Fermentation process was carried out at 29 ± 1°C, medium p H 5.5—7.1 and p02 — 15—20% of the saturation. In the latter case R S were determined by t h e ebuliostatic method. The obtained biomass was estimated as to the conditional protein content (N x 6.25), true protein, nucleic acid, lipid, vitamin B 1 ; B 2 , P P content, and also as to the quantitative and qualitative composition of amino acids. Fungus Trichoderma viride L-333 was used to obtain biomass on solid substrates. The cultivation of the fungus was carried out in 5 and 30 1 fermenters in pilot fermentation equipment on a medium of the following composition (g/1): K H 2 P 0 4 — 2.0; (NH 4 ) 2 0 4 - 3.0 ; MgS0 4 • 7 H 2 0 - 0.3; CaCl 2 -2H 2 0 - 0.3; straw - 20; alfalfa juice 100 ml; solution of microelements — 1 ml. Solution of microelements: F e S 0 4 - 7 H 2 0 — 5 m g / l ; M n S 0 4 - H 2 0 — 1.56 mg/1; ZnS0 4 • 7 H 2 0 - 1.4 mg/1; CaCl 2 -6H 2 0 - 3.7 mg/1; medium p H - 4.5. During the processes of submerged fermentation (SF) there were used special stirring systems ensuring a sparing regime for the mycelium [5]. Cultivation conditions: t° = 30°C; p H = 4.0—5,0; p 0 2 = 10—30% of saturation; air expenditure — 1 vol/vol to 1.5 vol/vol of culture medium per minute; N = 150 r p m . Seeds were multiplied by seeding connidia on fresh wort agar media, grown for 4 days in thermal chamber a t f = 30 ± 1 °C and stored for 2 weeks at room temperature. 2 ml of connidia suspension were sown into shaker flasks with the above medium, connidia

KATKEVICH,

J. J.,

KATKEVICH,

R.

H.

et al., Treatment of Agricultural Wastes

417

were rinsed off the wort agar surface by 10 ml of sterile nutrient medium. Cultivation of Tr. viride L - 3 3 3 was carried out in shaker flasks at 1 8 0 - 2 0 0 rpm, t° = 30 ± 1°C for 36 hours. Seeds with an increased cellulase activity were obtained as follows: a 36 hour filtered mycelium of Tr. viride L - 3 3 3 was added with a filtrate of the culture liquid of the same fungus with the maximum necessary activity. Growth of Trichoderma viride L - 3 3 3 under solid substrate fermentation was performed in the following way. Cultivation process was carried out as follows: 400 g (DM) of the substrate containing 9 0 % of milled and pretreated straw and 1 0 % of wheat bran were added with 300 ml of nutrient medium of the following composition : ( N H 4 ) 2 S 0 4 — 6 0 g ; K H 2 P 0 4 - 1.75 g; K 2 H P 0 4 - 1 . 7 5 g ; proteinless alfalfa juice - 2 0 0 m l ; t a p water — upto 11. T h e substrate was stirred for 15 min. After that there were added previously grown seeds: Tr. viride L - 3 3 3 — 700 ml or Tr. viride L - 3 3 3 — 500 ml, yeasts — 200 ml. The substrate was mixed with the seeds for 2 min. Subsequently, the substrate with 7 0 % moisture was used in variants of solid substrate fermentation ( S S F ) . In apparatus with stirring the regime of the stirrer was as follows: 1 sec of stirring with 60 sec interval, etc. Sterile compressed air was used for aeration. After S S F the samples were dried on cuvettes in a drying chamber with air circulation a t t° = 8 0 — 8 5 ° C , to reach dry weight 9 0 - 9 5 % . Methods of the evaluation and analysis of cultivation processes have been published [6, 7], Treatment variant I [8]: Straw was ground in a D K U mill, mixed with alkaline solution to 3 6 . 5 % moisture in a doubleshaft mixing device, equipped with a sprayer, and was fed to mechanical treatment division. T h e main equipment for the latter (auger) consists of a disperger-mixer of a cyllindrical form with a cigar-shaped rotor of the " s c u t t l e " type, equipped with blades. The original auger reactor are 290 or 150 mm in diametre. I n a separate part of the apparatus there is ensured a 110— 130°C temperature and the mixture of straw and alkali is subjected to an additional abrasion. Variant IA uses N a O H solution — 5.5 weight units per 100 weight units of dry straw. Variant I B uses a mixture of alkali NaOH, N H 3 and Ca(OH) 2 , respective 5.5, 0.4 and 1.35 weight units per 100 weight units of dry straw. Treatment variant I I [9]: Treatment was analogous to t h a t of variant I yet the chemical reagent was a mixture of H 2 S 0 4 , H N 0 3 and H 3 P 0 4 , respective 7.8, 3.9 and 0.8 weight units per 100 weight units of dry straw. Moisture upon treatment 2 6 . 5 % . Treatment variant I I I [10]: Treatment of straw or stems of potato tops with a NaOH solution (1 — 1 . 5 % ) was carried out under laboratory conditions in reactors with external heating. T h e t y p e and volume of laboratory reactor are indicated in each separate case, as well as the liquid module, i.e., a ratio of the dry material mass to the volume of alkaline solution. T h e temperature of solution boiling — 9 8 — 1 0 0 ° C is maintained in the reaction medium during the treatment. After the treatment the insoluble residue is separated by way of squeezing on filter or by way of centrifugation. After rinsing or right after the separation of lye from the insoluble product — substrate, it is mixed with a limited amount of a solution containing mineral acids for the neutralization of residual lye in the product. Upon a multiple use of lye, rinse water or a new portion of water and N a O H are added, let lye reach its initial volume, and the amount of NaOH is the difference between N a O H amount in the medium necessary for treatment, and NaOH amount in the used lye.

418

Acta Biotechnol. 8 (1988) 5

Results Samples, obtained in variants IA, Hi and I I were analyzed, the results are demonstrated in Tab. 1. Tab. 2 and 3 contain data on straw treatment by several variations of variant I I I . Experiments 1 — 6 were carried out to specify several conditions necessary to elaborate the technology: how do the degree of rinsing, a decrease in the liquid module, NaOH concentration, time of treatment affect the degree of E H . The use of alkali solution in the above experiments was a single one. In experiment 5 the reaction mass after the treatment was at once neutralized by an acid, then it was rinsed. In experiment 7 the lye was used 9 times. Up to the 5th use of the lye the dry matter content and potential RS increase in it, then stabilize on a definite level —10.6 and 2.0%, respectively. In experiments 1 — 7 there was used as 5-litre reactor with double shaft stirrer, which is efficient in stirring the quite thick mass with DM content of about 10%. Experiment 8 used a 50-litre reactor with a propeller-shaft. The lye was used 7 times. There was no rinsing of the substrate. The latter modification of variant I I I was used to obtain substrate used in the production of microbial biomass. Tab. 1. Samples of straw treated as to variants I and II Paramétrés % of abs. dry sample

Treatment variant IA

IB

II

Water-soluble substances potential RS included Easily-hydrolysible substances RS included Poorly-hydrolysible substances RS included Lignin Level of E H as to RS yield from initial sample from water-insoluble part of sample

17.4 3.9 24.4 20.3 44.8 43.2 15.7 30.2

20.6 4.6 24.2 19.8 40.8 40.7 14.4 33.2

39.2 25.8 8.1 5.6 37.6 38.7 15.2 34.4

29.6

34.2

19.4

Experiment 9 was carried in a 11 flask equipped with a stirrer. In this case the substrate was rinsed and the rinse water was further used to prepare a new dose of alkali. After neutralization and water extraction to a 65—80% moisture all the samples were dried at 20 °C. The stems of potato tops after water extraction were treated as to variant I I I in a 3 1 laboratory reactor. Treatment conditions: a 1.5% NaOH solution, module 1:20, oo®t^coao©q

t> o a o c3

io CO CO OS o to td -H ©