Acta Biotechnologica: Volume 8, Number 3 1988 [Reprint 2021 ed.] 9783112581728, 9783112581711

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AGtl Biotechioligica •

^

Volume 8• 1988 • Number3

Journal of microbial, biochemical and bioanalogous technology

Akademie-Verlag Berlin ISSN 0138-4988 Acta Biotechnol., Berlin 8 (1988) 3, 209-304

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Acta BlottdHliiica Journal of microbial, biochemical and bioanalogous technology Volume 8 1988

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—Grimma % M. Ringpfeil, Berlin and jG. Vetterlein, Leipzig

Editorial Board: A. A. Bajew, Moscow M. E. Beker, Riga H. W. Blanch, Berkeley S. Fukui, Kyoto H. G. Gyllenberg, Helsinki G. Hamer, Zurich J . Hollo, Budapest M. V. Iwanow, Moscow P. Jones, El Paso F. Jung, Berlin H. W. D. Katinger, Vienna K. A. Kalunjanz, Moscow J . M. Lebeault, Cotnpiegne

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

Managing Editor:

L. Dimter, Leipzig

Number 3

AKADEMIE-VERLAG

BERLIN

"Acta Biotechnologica" publishes original papers, short communications, reports and reviews from t h e whole field of biotechnology. The journal is to promote t h e 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 t h e journal is guaranteed by t h e fact t h a t papers on microbiology, biochemistry, chemistry and physics must clearly have technological relevance. Terms of subscription for t h e 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, D D R - 1086Berlin; — in the other socialist countries: to a bookshop for foreign languages literature or to t h e competent news-distributing agency; — in the F R 6 and Berlin (West) : to a bookshop or to the wholesale distributing agency K u n s t ' und Wissen, Erich Bieber OHG, Wilhelmstr. 4 - 6 , D-7000 S t u t t g a r t 1; — 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 t h e international book- and journal-selling trade, to Buchexport, Volkseigener Außenhandelsbetrieb der D D R , P F 160, 7 0 1 0 - D D R Leipzig, or to t h e AkademieVerlag Berlin, Leipziger Str. 3 - 4 , P F 1233, D D R -1086 Berlin.

Acta Biotechnologica Herausgeber: I n s t i t u t f ü r Biotechnologie der AdW der DDR, Permoserstr. 15, D D R - 7 0 5 0 Leipzig (Prof. Dr. Manfred Ringpfeil) und V E B Chemieanlagenbaukombinat Leipzig—Grimma, Bahnhofsstr. 3 — 5, D D R - 7 2 4 0 Grimma, (Dipl.-Ing. Günter Vetterlein). Verlag: Akademie-Verlag Berlin, Leipziger Str. 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 (Redaktion), 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: 1094/8/3. 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 p a r t of this issue may be reproduced in any form, by photoprint, microfilm or any other means, without written permission from t h e publishers. © 1988 by Akademie-Verlag Berlin, Printed in t h e German Democratic Republic. AN (EDV) 18520 03000

Acta Biotechnol. 8 (1988) 3, 2 1 1 - 2 2 3

Solid Substrate Growth of White Rot Fungi on Coffee Pulp R o l z , C., d e Leon. R., d e A k b i o l a , M. C. Applied Research Division Central American Research Institute for Industry (ICAITI) P.O. Box 1552 Guatemala City, Guatemale, C.A.

Summary The presence of several antiphysiological factors limit the use of coffee pulp in monogastric and ruminant feeding. Twenty six white rot fungi were grown under solid substrate conditions in previously ensiled and pressed coffee pulp without adding additional sources of nitrogen. All grew and wholly covered the surface of the substrate. Six of them produced fruiting bodies. The weight loss interval ranged between 6.7—28.0% dry matter before fructification and from 17.0 to 48.7% after fructification. Some fungi biodegraded about 70, 55 and 47% of the total polyphenols, caffeine and permanganate lignin present in the original substrate.

Introduction Approximately half of the world coffee harvest is processed by the wet method in which the coffee berry is subjected to mechanical and biological operations in order to separate the bean or seed from the exocarp (skin), mesocarp (mucilagenous pulp) and the endocarp (parchment) [1], The skin and most of the pulp is separated in the pulpers, this fraction represents about 40% in weight of the fresh fruit and presently is underutilized causing serious pollution problems due to natural degradation [2], In Fig. 1 flow diagrams for the two coffee processing methods are presented [3]. Many alternatives have been proposed for its better utilization and these were described in a recent review [4]. Its use in feeding formulas for ruminant or monogastric animals is aa attractive proposition from the technical and economical points of view. There is already an industrial factory operating in San Jose, Costa Rica. On it, coffee pulp is continuously screwpressed, dried and milled. The recommended amounts of pulp in the rations are 12% for dairy cattle, 7.5% for swine and only 5 % for poultry. There are antiphysiological factors present in the material which limit its use beyond the figures given. Present experimental evidence indicate t h a t among these are the relative high caffeine, polyphenolic and potassium contents and the characteristics of its lignocellulose [5], Not much is known about the nature, chemistry and distribution of the pulp polyphenols; only t h a t they are more easily solubilized by diluted ammonium or calcium hydroxide and the condensed tannins seem to represent from 30 to 60% of the total extracted polyphenols depending on the solvent used [6]. I t is also very probable t h a t chlorogenic acid, the main polyphenol present in the coffee bean [7], as such or as closely related derivatives [8] might also be present in coffee pulp; al1*

Acta Biotechnol. 8 (1988) 3, 2 1 1 - 2 2 3

Solid Substrate Growth of White Rot Fungi on Coffee Pulp R o l z , C., d e Leon. R., d e A k b i o l a , M. C. Applied Research Division Central American Research Institute for Industry (ICAITI) P.O. Box 1552 Guatemala City, Guatemale, C.A.

Summary The presence of several antiphysiological factors limit the use of coffee pulp in monogastric and ruminant feeding. Twenty six white rot fungi were grown under solid substrate conditions in previously ensiled and pressed coffee pulp without adding additional sources of nitrogen. All grew and wholly covered the surface of the substrate. Six of them produced fruiting bodies. The weight loss interval ranged between 6.7—28.0% dry matter before fructification and from 17.0 to 48.7% after fructification. Some fungi biodegraded about 70, 55 and 47% of the total polyphenols, caffeine and permanganate lignin present in the original substrate.

Introduction Approximately half of the world coffee harvest is processed by the wet method in which the coffee berry is subjected to mechanical and biological operations in order to separate the bean or seed from the exocarp (skin), mesocarp (mucilagenous pulp) and the endocarp (parchment) [1], The skin and most of the pulp is separated in the pulpers, this fraction represents about 40% in weight of the fresh fruit and presently is underutilized causing serious pollution problems due to natural degradation [2], In Fig. 1 flow diagrams for the two coffee processing methods are presented [3]. Many alternatives have been proposed for its better utilization and these were described in a recent review [4]. Its use in feeding formulas for ruminant or monogastric animals is aa attractive proposition from the technical and economical points of view. There is already an industrial factory operating in San Jose, Costa Rica. On it, coffee pulp is continuously screwpressed, dried and milled. The recommended amounts of pulp in the rations are 12% for dairy cattle, 7.5% for swine and only 5 % for poultry. There are antiphysiological factors present in the material which limit its use beyond the figures given. Present experimental evidence indicate t h a t among these are the relative high caffeine, polyphenolic and potassium contents and the characteristics of its lignocellulose [5], Not much is known about the nature, chemistry and distribution of the pulp polyphenols; only t h a t they are more easily solubilized by diluted ammonium or calcium hydroxide and the condensed tannins seem to represent from 30 to 60% of the total extracted polyphenols depending on the solvent used [6]. I t is also very probable t h a t chlorogenic acid, the main polyphenol present in the coffee bean [7], as such or as closely related derivatives [8] might also be present in coffee pulp; al1*

Acta Bioteclinol. 8 (1988) 3

212 D R Y PROCESS

W E T PROCESS

Berries

Berries I

I

+

Drying

Dehulling

»

Green beans of the trade

i Hulls

1 Beans + mucilage

1 Pulp + Skin

I

Green beans Parchment of the trade (washed coffees) Fig. 1. Processing of coffee berries: unit operations and flow diagram for the wet and dry processes

though its existance has not been reported previously, either in a free form or in complexes with other compounds, i.e. hemicellulose [9 — 11], lignin [12] or with caffeine and potassium [13]. Biological removal of antiphysiological factors is an interesting alternative. A filamentous fungi, Penicillium crustosum, was used to decaffeinate roasted coffee infusions by SCHWIMMER and KURTZMAN [14]; it was also shown capable of using chlorogenic acid as a carbon source and caffeine as a source of nitrogen, when cultured in synthetic media in shake flasks at 30 °C, 200 rpm for 72 h [15]. PENALOZA et al. [16] grew Aspergillus niger strain 10 (ORSTOM, Paris) in fresh, freeze dried coffee pulp for 43 h in solid state culture with practically no reductions in tannins, caffeine or lignin. The substrate was enriched with urea, ammonium salts and phosphate; we believe this to be the cause of such experimental results. MARTINEZ et al. [17] grew two strains of the basidiomycete Pleurotus ostreatus in fresh and previously composted (5 and 10 days) coffee pulp until 3 to 4 flushes of fruiting bodies were obtained. Again no reductions in caffeine were found in the solid residue, but the data is difficult to interpret because no weight losses were reported. With this background we decided to carry out an experimental study in which several white rot fungi were grown on ensiled and pressed coffee pulp in solid state culture

ROLZ,

C.,

DE LEON, R .

et al., Solid Substrate Growth

213

Tab. 1. Basidiomycetes used in the experiments Number

Name

ICAITI collection

Origina

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Agrocybe aegerita Bondarzewia berkeleyi Coriolus versicolor Cyathus africanus Cyathus carina Cyathus pallidus Dichomitus squalens Flammulina velutipes Fomitopsis ulmaria Ganoderma applanatum, Ganoderma applanatum. Ischnoderma resinosum Phlebia radiata Pleurotus flabellatus Pleurotus ostreatus ("Florida") Pleurotus sajor-caju Pycnoporus sanguineus Sporotrichum pulverulentum Phanerochaete chrysosporium Lentinus edodes Phanerochaete chrysosporium Stropharia rugosoannulata Polyporus anceps Trametes versicolor Fomes fomentarius Pleurotus sp.

F-1090 F-1093 F-1095 F-1096 F-1097 F-1098 F-1099 F-1100 F-1101 F-1102 F-1103 F-1105 F-1108 F-1011 F-llll F-1117 F-1112 F-1113 F-1107 F-1115 F-1118 F-1116 F-1119 F-1122 F-1120 F-1133

CBS-388.79 FPL-FP-105839-S FPR-R-105-Sp NRRL-6519 NRRL-6522 NRRL-6529 CBS-432.34 CMI-176670 FPL-L-11682-Sp CMI-157818 FSU-602-7 FPL-L-13682-Sp FPL-FP-101840-sp IARI-1724 NRRL-3526 IBB-FAL CMI-75002 QM-9145 FPL-ME-446 IBB-FAL ETH IBB-FAL NSRF WAG-T15 TUV b

a CBS FPL

= Central bureau voor Schimmelcultures, Baarn, The Netherlands = Forest Products Laboratory, USDA Forest Service, Madison Wisconsin 53705, USA NRRL = Northern Regional Research Center, Peoria, Illinois 61604, USA CMI = Commonwealth Mycological Institute, Kew, UK FSU = Friedrich Schiller University, Biology Section, Weimar, German Democratic Republic IARI = Indian Agricultural Research Institute, New Delhi, India IBB-FAL = Institute of Soil Biology, Federal Research Centre of Agriculture, FAL, Braunschweig, Federal Republic of Germany ETH = Institute of Microbiology, Technological Institute, Zurich, Switzerland NSRF = Nova Scotia Research Foundation, Dartmouth, NSB2Y 3Z7, Canada WAG = University of Wageningen, The Netherlands TUV = Vienna Technical University, Vienna, Austria b = Collected in Izabal, Guatemala, by Dr. Frantisek ZADRAZIL, FAL, Braunschweig, FRG

and the changes on caffeine, polyphenols and lignin were quantified. Such study is justified as an effort to upgrade an agroindustrial waste which is underutilized and available in large quantities. It is estimated that there is a yearly production of 4 million tons of fresh pulp in the Caribbean basin coffee producing countries.

214

Acta Biotechnol. 8 (1988) 3

Materials and Methods Microorganisms I n Table 1 a list of the fungi is given including its name, number in ICAITI's collection and origin. Only one fungi, number 26, was isolated in Guatemala, the rest were from other collections. Preparation of the Substrate Fresh coffee pulp was collected from a local "beneficio". I t was immediately ensiled in plastic bags and left for a four week period at ambient temperature (18— 20°C). Bags were then opened and examined for the present of fungal mycelia. Those positive were discarded. A well ensiled product had the characteristic aroma and its p H had decreased to about 4 from an original value close to 6.5. The pulp was pressed in a pilot plant screw press of local design and construction. The yield of pressed residue was about 72% of the original ensiled pulp wet weight. I t was washed with an equal weight of water and pressed again. Amounts of this material (30 g dry weight) were placed in wide-mouth-glass jars and sterilized for 45 min at 121 °C. Inoculation and Solid State Culture Mycelia from a culture tube of each fungal strain was grown on PDA in petri dishes. After fungal growth covered the whole surface of the dish, approximately two cm 2 were transferred to the test flask. The flasks were then left at ambient temperature until the substrate surface was totally covered by the white mycelium (strain No. 17 produced a red pigment). The date was recorded and the flasks were opened. The contents of one flask were dried employing air at about 60 °C and dry weight recorded. The dried material was analyzed. Periodically water was sprayed to avoid excessive dehydration. The time on which fruiting bodies emerged and fully developed was recorded. The fruiting bodies were removed and the flask contents dried and processed as before. Strain No. 18 was stored at 5—8°C under artificial light for successful fruiting. Those samples which did not produce fruiting bodies, even after a storing period 3-times the initial growth interval, were dried and processed as before. Analytical

Methodology

On the dried material caffeine was determined according to ISHLER et al. [18], total polyphenol using the AOAC methodology [19] and permanganate lignin following t h e V a n SOEST t e c h n i q u e s [20].

Results and Discussion I n Table 2 are shown the average values of at least five different samples done on triplicate each for the chemical compounds of interest present in the ensiled and in the pressed coffee pulp. There is a weight ratio of 0.72:1.00 between pressed and ensiled samples. Hence a straight forward calculation shows t h a t the following amounts of soluble solids, total polyphenols and caffeine were extracted in the coffee pulp juice: 72, 73 and 76% respectively. Lignin as expected stayed in the pressed residue. About 40% of the original ash remained in the pressed residue. These results confirm previous data obtained in pilot trials employing various continuous screw press designs (un-

ROLZ, C., DE LEON, R . e t al., Solid S u b s t r a t e G r o w t h

215

T a b . 2. Chemical analysis of coffee pulp D a t a on d r y basis [ g / 1 0 0 g]

Lignin Soluble solids Ash T o t a l polyphenols Caffeine

E n s i l e d pulp

P r e s s e d pulp

9.59 46.84 7.65 1.94 0.80

13.44

18.01 4.24 0.73

0.26

published data); also are higher than those reported before employing a hydraulic press [21]. From the process point of view continuous screw pressing with an intermediate water wash is a warranted unit operation as it decreases drastically three of the possible antiphysiological factors present in fresh coffee pulp. These reductions are important for the next step: the biological detoxification employing basidiomycetes. I t has been shown in a separate publication [22] that coffee pulp juice inhibits the surface growth of four Pleurotus strains. I n Table 3 the losses of dry weight at two times are presented. Fungi No. 26 was analyzed at three different times. Only seven fungi produced fruiting bodies in that time span. Fruiting was stimulated by opening the flask after the mycelium had covered the substrate surface, which was the time for the first sample for all fungi. F. velutipes was stored at 5—8°C under artificial light as recommended for fruit bodies to develop [23, 24]. The four Pleurotus species produced fruiting bodies as expected [25], so coffee pulp seems a good substrate for the production of this strain [17, 26]. Some fungi were fast growers (under 30 days for the first period), i.e. P. chrysosporium (or S. •pulverulentum, No. 18, 19, 21), T. versicolor (No. 24), F. fomentarius (No. 25), and P. radiata (No. 13). Others were very slow, i.e. the three Gyathus species (No. 4—6). The weight loss interval before the testing for fructification was begun, was from 6 . 7 — 2 8 . 0 % . I n the second period the range increased to 17.0 to 4 8 . 7 % . Those with overall weight losses of more than 4 0 % were S. pulverulentum (or P. chrysosporium, No. 18, 19, 21), C. versicolor (No. 3), and P. ostreatus (No. 15). Experimental data on the weight loss of wheat straw by the growth of S. pulverulentum, in solid state culture has been reported by several authors. ZADRAZIL and BRUNNERT [27] found a range of 38 to 5 8 % for 30 days depending on the cultivation temperature, 2 5 t o 3 5 °C. DUARTE e t al. [ 2 8 ] 3 7 % in l O d a y s , HATTAKA [ 2 9 ] 2 4 % in 2 8 d a y s a t 2 8 °C

and finally AGOSIN and ODIER [30] an interval of 5 — 2 5 % in a period of 5 — 11 days. Practically all of these authors found high substrate degradation by this fungus as a relatively fast grower in such a substrate. I n more lignified material weight losses were lower, i.e. only 1 3 % for aspen after eight weeks at 25°C [31], in cotton straw. 1 4 % after 21 days [32]. C. versicolor caused a 1 3 % weight loss of aspen after eight weeks at 25 °C [31]; another article reported 3 9 % for hardwoods, which increased to 41 for softwoods and 6 4 % for some graminae after 10 weeks at 28°C [33]. The type of lignin present in the substrate seemed a key factor on the weight loss obtained [34], T. versicolor also grew well in coir from coconut [35]. Recently HATTAKKA and PIRHONEN [36] reported losses in dry weight between 7.5 and 12.5% after 14 days for F. fomentarius and P. radiata on untreated wheat straw. The former responded better in coffee pulp, however about the same results were obtained for the latter, which is some what unexpected due to the great differences in chemical composition of the two substrates being compared. Usually the rates of weight loss reported in the

Acta Biotechnol. 8 (1988) 3

216 Tab. 3. Dry weight loss and fructification Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 a

Period 1 Substrate totally covered by mycelium

Period 2 Time left for fruiting bodies to appear

Days

Days

[g]

Loss dry weight [%]

28.0 25.1 20.9 19.9 22.5 26.1 25.1 25.4 20.9 27.1 25.9 26.9 25.9 22.4 26.5 23.8 23.8 24.0 25.0 23.6 25.3 18.9 21.9 nd 23.1 22.7

6.7 16.3 30.3 33.7 25.0 13.0 16.3 15.3 30.3 9.7 13.7 10.3 13.7 25.3 11.7 20.7 20.7 20.0 16.7 21.3 15.7 37.0 27.0 nd 23.0 24.3

85 110 91 nd 141 141 nd 78 97 100 85 100 75 70 85 85 91 101 91 131 89 nd 66 46 46 58

47 56 32 97 87 87 32 32 49 41 41 41 22 49 41 36 29 29 29 36 14 47 32 17 17 46

Dry weight

Dry weight [g]

Loss dry weight [%]

24.9 22.6 15.9 nd 19.0 22.9 nd 20.0 20.7 18.5 19.0 22.6 18.4 19.8 17.5 20.8 19.0 15.4 17.3 23.2 17.0 nd 18.2 19.9 19.3 22.8

17.0 24.7 47.0 nd 36.7 23.7 nd 33.3 31.0 38.3 36.7 24.7 38.7 34.0 41.6 30.8 36.7 48.7 42.3 22.7 43.3 nd 39.3 33.7 35.7 24.0

Fructification

+ — — — — — —

+ —

-L -

-

+

_L

+ — — — — — — — — —

4-

a

Note: Fungi No. 26 was sampled at 112 days with a dry weight of 19.1 g and a percent of 36.3. nd: Not determined literature for the different Pleurotus strains in other substrates are usually in the same order of magnitude or higher than those shown in Table 3 for coffee pulp which are easily calculated to be 0.49, 0.49 and 0 . 3 6 % weight loss per day for P . flabellatus (No. 14), P. ostreatus (No. 15) and P. sajor-caju (No. 16) at the end of the second period (70—85 days). F o r example with wheat straw the following data have been informed: in 19 days (i) 0.53 and 0 . 3 8 % weight loss per day for P . cornucopiae and P . ostreatus respectively [37]; (ii) 0.36, 0.44 and only 0 . 1 8 % weight loss per day for P . salmoneo, P. florida and P . eryngii respectively in 60 days [38]; (iii) 0.52 to 1 . 0 0 % weight loss per day for a mixed culture of P . ostreatus and Erwinia carotovora in 56 days |.'!9]s (iv) 0 . 8 4 % weight loss per day for P . ostreatus in 28 days [29]; (v) 0 . 4 8 % weight los; per day in 14 days [30]; (vi) 0.41 to 0 . 4 6 % weight loss per day under continuous aereation at 2 5 °C in 14 to 4 0 days [40]; and (vii) 1.43% weight loss per day in 20 days [41] the authors inform that in only 14 days had the fungi covered the straw. Weight loss data for other substrates is given for the rest of the basidiomycetes as follows:

ROLZ,

C.,

DE L E O N , R .

et al., Solid Substrate Growth

217

A. aegerita, sugarcane, lemongrass and citronella bagasse [42,43]; B. berkeleyi aspen [31], sugarcane, lemongrass and citronella bagasse [42,43]; Gyathus sp. kenaf [44]; D. squalens, barley and rape straws [27], wheat straw [ 3 0 , 4 5 ] ; F. velutipes, wheat straw [38], sugarcane, lemongrass and citronella bagasse [ 4 2 , 4 3 ] ; G. applanatum soft and hardwoods [46], wheat straw [38,47], oat straw [48] and hardwoods [49]; I. resinosum, hardwoods [49, 50], sugarcane, lemongrass and citronella bagasse [42, 43]; P. sanguineus, hardwoods [51], sugarcane, lemongrass and citronella bagasse [42, 43]; L. edodes, wheat straw [38], hardwoods [31, 52]; S. rugosoannulata, wheat straw [38, 53], sugarcane, lemongrass and citronella bagasse [42, 43] and P. anceps, oat straw [48], hardwoods [51, 54], sugarcane, lemongrass and citronella bagasse [42, 43]. In Table 4 the changes of polyphenols and caffeine are given in terms of the relative losses respect to the original amount found in coffee pulp. The contents determined analytically for each fungus at the two time periods were transformed and compared with the original amounts as can be illustrated for the strain number 1. Such strain as shown in Table 1 is A. aegerita and at period 1 the solid residue had 0.37 and 0.24g/ 100 g of total polyphenols and caffeine respectively as shown in Table 4. Absolute amount of, polyphenols: 28 g X (0.37/100) = 0.1036 g. Absolute amount of caffeine: 28 g X (0.24/100) = 0.0672 g. Twenty eight grams is the weight of dry matter after period 1 as shown in Table 3. Initial amount of polyphenols: 30 X (0.73/100) = 0.2190 g. Initial amount of caffeine: 30 X (0.26/100) = 0.0780 g. Both figures from Table 2. Relative loss of polyphenols: ((0.2190 - 0.1036)/(0.2190)) X 100 = 52.7. Relative loss of caffeine: ((0.0780 — 0.0672)/(0.0780)) X 100 = 13.9. The rest of the data was calculated on a similar manner. In most samples the relative losses of the two compounds increased from the time period one to two, as expected. This fact implied that both compounds were actively metabolized during active growth and also during fructification. There were eight exceptions for polyphenols (No. 11, G. applanatum,-, No. 13, P. radiata; No. 16. P. sajor-caju-, No. 18, S. pulverulentum; No. 20, L. edodes; No. 21, P. chrysosporium; No. 24, T. versicolor-, and No. 26, Pleurotus sp). and five for caffeine (No. 14, P. flabellatus; No. 19, P. chrysosporium-, No. 25, F. fomentarius; No. 24 and No. 26). In some of the exceptions the differences were small, for example No. 16 P. sajor-caju and No. 20 L. edodes, which gave an average loss for the two time periods of 39.8 and 4 3 . 4 % of total polyphenols, and No. 14 P. flabellatus with an average caffeine loss of 42.2%, hence the polyphenols and caffeine were metabolized by the fungi principally in the first time period during active growth and dry matter weight loss. In some others the differences were higher and in this case we cannot offer a satisfactory explanation why this took place. In terms of polyphenols the following gave the higher losses with a consistent trend: C. carina (No. 5), P. anceps (No. 23), C. pallidus (No. 6), A. aegerita (No. 1), F. ulmaria (No. 9), P. ostreatus (No. 15), P. flabellatus (No. 14), I. resinosum (No. 12), F. fomentarius (No. 25), P. chrysosporium (No. 19), C. versicolor (No. 3) and P. sanguineus (No. 17). With respect to caffeine, the following: S. pulverulentum (No. 18), C. versicolor (No. 13), F. velutipes (No. 8), P. radiata (No. 13), G. canna (No. 5), G. applanatum (No. 11), P. chrysosporium (No. 21), I. resinosum (No. 12), P. sanguineus (No. 17), F. ulmaria (No. 9), G. applanatum, (No. 10), L. edodes (No. 20), P. ostreatus (No. 13), and P. sajor-caju (No. 16). In terms of absolute values not many fungi were able to lower the total polyphenols to less than half of its original value; in other words less than 0.37%. In this case the best were No. 1, A. aegerita, the two Cyathus, canna and pallidus (No. 5 and 6), and P. anceps (No. 23). Those who had 0 . 2 0 % of caffeine or less were C. canna (No. 5), D. squalens (No. 7), F. velutipes (No. 8), G. applanatum (No. 11), I. resinosum (No. 12), P. radiata (No. 13), S. pulverulentum (No. 18), P. chrysosporium (No. 19), and S. rugosoannulata (No. 22).

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Tab. 4. Changes in the contents of polyphenols and caffeine in coffee pulp after fungal growth Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Period

1 2 1 2 1 2 1 1 2 1 2 1 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 1 2 1 2 1 2 1 2 3

a Data on dry basis

Total polyphenolsa [g/100g]

Polyphenols loss [%]

Caffeinea

Caffeine loss

[g/100g]

[%]

0.37 0.34 0.81 0.68 0.66 0.81 0.56 0.27 0.30 0.36 0.37 0.59 0.61 0.56 0.60 0.41 0.81 0.68 0.33 0.52 0.52 0.54 0.28 0.57 0.51 0.50 0.56 0.49 0.54 0.65 0.75 0.71 0.48 0.85 0.66 0.73 0.52 0.54 0.26 0.72 0.59 0.35 0.40 0.60 0.64 0.73 0.63 0.53 0.67 0.87

52.7 61.3 7.2 29.8 37.0 41.2 49.1 72.3 74.0 57.1 61.3 32.3 29.2 48.9 42.7 61.2 0.0 42.6 61.0 54.9 36.1 44.2 66.7 52.1 47.8 54.8 32.2 60.8 41.3 38.3 18.5 38.4 47.4 40.2 31.5 42.3 44.0 42.8 70.0 44.1 49.1 65.0 66.8 58.9 41.8 23.0 44.5 45.1 34.4 24.1

0.24 0.22 0.25 0.26 0.24 0.22 0.25 0.20 0.22 0.29 0.28 0.08 0.23 0.18 0.24 0.23 0.25 0.26 0.24 0.21 0.23 0.19 0.21 0.20 0.20 0.23 0.25 0.29 0.26 0.26 0.22 0.24 0.19 0.22 0.12 0.21 0.27 0.21 0.27 0.24 0.17 0.29 0.32 0.26 0.31 0.22 0.28 0.17 0.24 0.31

13.9 29.8 19.6 24.7 35.7 55.2 36.2 42.3 51.3 3.0 17.8 74.3 25.1 53.8 35.7 39.0 13.1 38.3 20.3 48.8 20.7 45.0 30.3 52.8 42.6 41.7 15.1 34.9 20.7 30.7 32.9 41.5 41.5 56.6 61.5 53.4 18.3 37.5 12.4 47.7 58.8 18.6 25.3 50.0 20.9 40.8 30.1 50.5 30.0 24.1

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Caffeine at certain levels inhibits the synthesis of RNA and protein both for prokaryotic as eukaryotic cells [55] however the effect might be temporal and in certain cases, like the one reported for Neurospora crassa practically reversible [56]. There are in the literature reports of successful and extensive biological decaffeination. both for bacteria, like Pseudomonas aeruginosa [57], Bacillus coagulans [58] and the more extensively studied Pseudomonas putida which has been easily isolated from soil samples [59—61]. as for various filamentous fungi under nitrogen starving conditions, P. crustosum and a Stemphylium species [58, 62]. I t seems that the first enzymatic step in the degrading pathway appears to be a mixed-function oxygenase which demethylates the different methyl-xanthines. Our data seems to be the first reported evidence that under nitrogen limitation basidiomycetes can utilize caffeine. In the technical literature several research groups have reported the transformation of phenolic acids by white rot fungi. Ferulic, cinnamic, p-coumaric and sinapic acids were metabolized to the corresponding alcohols by Trametes sp. under submerged culture conditions for up to 120 h [63], HAARS et al. [64] on the other hand informed that the growth of Pomes annosus in liquid medium was inhibited by various phenols which were oxidized by the fungus. The authors pointed out that the probability that a particular polyphenolic compound inhibits fungal growth is high when it induces laçasse production or when it is bioxidized or both. The laccase induction, as also the increase of lignin degradation rates and hydrogen peroxide produced, by polyphenols has been reported for Polyporus versicolor [65, 66], Agaricus bisporus [67] and P. chrysosporium [68], However the overall picture might be more complicated, for example chlorogenic acid did not stimulate hydrogen peroxide production by P. chrysosporium [68] but it did increase lignin degradation. The same compound inhibited laçasse, protein biosynthesis and carpophores production by A. bisporus [67], We measured the amount of lignin biodegraded by some of the basidiomycetes. The results are shown in Table 5 for eight white rots during their filamentous growth on the pressed pulp. We have also calculated the rate of lignin loss in that period and compared it with the corresponding rate for polyphenols. The first three fungi (C. versicolor (Xo. 3). P. flabellatus (No. 14) and G. applanatum (No. 10)) gave low rates of lignin loss in the growth period, but higher polyphenols losses, with the exception of the last. On the other hand P. sanguineus (No. 17), Pleurotus sp. (No. 26) and S. rugosoannulata (Xo. 22) showed approximately the same biodégradation rates for both compounds. S.pulverulenum (Xo. 18) was also in this group but its rate of polyphenol biodégradation was T a b . 5. Lignin losses for some fungi Number

3 14 10 17 26 18 22 24

First growth period [days]

Permanganate lignin a [g/100 g]

Lignin loss b

32 49 41 29 46 29 47 17

17.70 14.73 13.06 12.94 10.96 12.41 11.29 19.38

8.25 18.17 12.22 23.62 38.30 26.13 47.08 27.90

[%]

a D a t a on dry basis b Expressed as % of original amount in pressed pulp c During days of first growth period

R a t e of lignin 0 loss [%/day]

R a t e of polyphenols loss [%/day]

0.26 0.37 0.30 0.81 0.83 0.90

1.16 0.98 negligible 0.63 0.98 1.63 1.04 3.46

1.00 1.95

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Acta Biotechnol. 8 (1988) 3

almost twice as high. Finally, T. versicolor (No. 24) gave very high rates of biodégradation mainly due to its relatively fast growth rate. The following fungi produced a solid residue with less absolute lignin content than the one found in the original substrate (pressed pulp shown in Table 2) : G. applanatum (No. 10), P. sanguineus (No. 17), Pleurotv.s sp. (No. 26), S. pulverulentum (No. 18) and S. rugosoannulata (No. 22). The lignin losses found for some of the cultures at the end of the second time period are shown in Table 6. Due to the extensive time period and the corresponding dry matter weight loss of the substrate, although the relative amounts of lignin losses were high, the lignin contents of the solid residue were all higher than the amount found in the original substrate. The two P. chrysosporium, strains, Nos. 19 and 21 behaved quite similar. Tab. 6. Lignin losses after the second growth period Number

Time of Growth [days]

Permanganate» lignin [g/100g]

Lignin b loss [g]

3 5 8 13 15 19 21

91 141 78 75 85 91 89

20.17 18.73 14.04 19.67 21.89 18.16 19.21

20.46 11.74 30.36 10.24 4.99 22.08 19.01

a Data on dry basis b Expressed as % of original amount in pressed pulp

P. chrysosporium (or S. pulverulentum) has been shown to biodegrade lignin from angiosperm and gymnosperm woods [69—73] as also residues from annual crops [32, 48, 74] as such or after selective pretreatments [42, 43, 75]. This metabolic activity is produced by multiple extracellular enzymes that oxidize lignins and related compounds [76, 77] and which usually appear only during secondary metabolism under nutrient limitation conditions [78—80]. Several aromatic compounds have been shown to increase initial lignin degradation rates, including veratryl alcohol a natural secondary metabolite of this fungus [68, 80]. G. applanatum is one of the white rots that extensively degrades lignin in the forests [81] producing the so-called '"'palo podrido" [82]. Along with C. versicolor its biodelignification has been shown to be not uniformly distributed in wood, but rather occurring in specific areas [83]; however differences have been observed between angiosperm and gymnosperm woods [84]. P. radiata was shown to be quite effective in degradation of popplar lignin [71] and recently three different ligninases and one oxidase were separated by anion exchange chromatography [85], F. velutipes, an early wood colonizer in nature [86] along with S. rugosoannulata have been shown to degrade lignin in wheat straw [38]. ABBOTT and WICKLOW [87] studies kenaf biodelignification employing twelve Cyathus species and found C. canna the one that showed the highest preference for lignin degradations. P. ostreatus has been shown to degrade lignin in surface culture growth of various lignocellulosic residues [31, 39, 41, 47, 88—93], Conclusions

From our experiments we can conclude that the alternative of biological removing in part some of the antiphysiological factors present in coffee pulp is possible as all of the 26 white rot fungi tested performed well under solid substrate culture. Seven fungi

ROLZ.

221

C., D E L E O N , R . et al., Solid Substrate Growth

produced fruiting bodies including the four Pleurotus species tested. Some were relatively fast growers like 8. pulverulenlum (P. chrysosporium), T. versicolor, F. fomentarius, and P. radiata. However, these were not the ones t h a t biodegraded in larger quantities caffeine and total polyphenols. The slow growers like Cyathus carina were better in this respect. The rate of polyphenol losses were about the same or higher for seven of eight fungi tested. Six fungi gave a solid residue with less lignin content than the one found in the original substrate. Preliminary guidelines can be offered for several possible process alternatives: i) if human food is sought, the various Pleurotus should be cultivated. After fruit body collection the residue could be employed as a "biological compost". Biological tests with animals should also be carried out to find out its suitability as a feed. F. velutipes could also be cultivated however it needs low temperatures for fructification which implies additional energy and it is not suitable for tropical conditions. Further tests employing longer time periods, should also be carried out with L. edodes to check it truiting takes place, ii) The direct use of the product as an animal feed should be established by carrying out biological tests after growing either a relatively fast grower (under 30 days) or a slow grower. T. versicolor or P. chrysosporium are potential examples for the former and 0. applanatum, P. radiata, and C. carina for the latter.

Acknowledgement This work has been done as part of a MIRCEN-UNESCO/UNEP research project under Contract No. UNESCO 2133/SC. Their funding is appreciated as also the support of Dr. Edgar DaSiLVA. We want to thank F. P . LOMBARD, F P L ; L . M. JOSHI, I A R I ; F . ZADRAZIL, IBB-FAL; A. FIBCHTER, E T H A ; A. E. READE, N S R F ; J . W . de B R U I N , WAG and M. ROHR, TUY for sending us fungal strains. The same is extended to scientists a t CBS, Baarn; N R R L , Peoria; CMI, Kew and FSU, Weimar. Received July 6, 1987

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[71] HATTAKA, A. I., UUSI-RAWVA, A. K . : Eur. J . Appi. Microbiol. Biotechnol. 17 (1983), 235. [ 7 2 ] R E A D E , A . E . , M C Q U E E N , R . E . : Can. J . Microbiol. 29 ( 1 9 8 3 ) , 4 5 7 . [73] F A I X , 0 . , M O Z U C H , M . D., K I R K , T. K . : Holzforschung 39 (1985), 203. [74] AGOSIN, E., DAUDIN, J . J., ODIER, E . : Appi. Microbiol. Biotechnol. 22 (1985), 132. [75] BONO, J . J., GAS, G., BOUDET, A. M.: Appi. Microbiol. Biotechnol. 22 (1985), 227. [76] TIEN, M., KIRK, T. K . : Science 2 2 1 (1983), 661. T I E N , M., Technol. S (1986), 27.

[77] KIRK, T. K . ,

CROAN, S . ,

MURTAGH, K .

E.,

FARRELL, R . L .

: Enzyme

Microb.

Biotechnol. 1 ( 1 9 8 4 ) , 1 3 . [79] F A I S O N , B. D . , K I R K , T . K . : Appi. Environ. Microbiol. (1985), 299. [79] F A I S O N , B. D., K I R K , T . K . : Appi. Environ. Microbiol. (1985), 299. [80] FAISON, B. D., KIRK, T. K., FARRELL, R. L.: Appi. Environ. Microbiol. 52 (1986), 251.

[ 7 8 ] ULMER, D . C., LEISOLA, M . S. A . , FIECHTER, A . : J .

[81] BLANCHETTE, R . A.,

OTJEN, L.,

EFFLAND, M. J . ,

ESLYN,

W. E.:

Wood

Sci.

Technol.

19

(1985), 35. [82] ZADRAZIL, F . , GRINBERGS, J . , GONZALEZ, A . : E u r . J . A p p i . M i c r o b i o l . 1 5 (1982), 167.

A.: Appi. Environ. Microbiol. 4 8 ( 1 9 8 4 ) , 6 4 7 . Holzforschung 4 1 (1987), 67. [ 8 5 ] L E E N A , M . , P A A V O L A , N . : Proc. 4 t h I n t . Symp. Wood Pulping Chem. 2 ( 1 9 8 7 ) , 3 0 1 . [ 8 6 ] R A Y N E R , A . S., H E D G E S , M. J . : Trans. Br. Mycol. Soc. 7 8 ( 1 9 8 2 ) , 3 7 0 . [87] ABBOTT, T. P., WICKLOW, D. T.: Appi. Environ. Microbiol. 47 (1984), 585. [ 8 8 ] K A N E S H I R O , T . : Dev. Ind. Microbiol. 1 8 ( 1 9 7 7 ) , 5 9 1 . [89] L I N D E N F E L S E R , L . A., D E T R O Y , R . W . , R A M S T A C K , J . M., W O R D E N , K. A.: Dev. Ind. Micro biol. 20 (1979), 541. [90] J A N S H E K A R , H . , H A L T M A E I R , T., B R O W N , C.: E u r . J . Appi. Microbiol. Biotechnol. 14 (1982), 174. [83] BLANCHETTE, R .

[84]

H I G H L E Y , T . L . , MURMANIS, L . L . :

[91] PLATT, M., CHET, I . , H E N I S , Y . : M u s h r o o m J . 1 2 0 (1982), 4 2 5 .

[92]

B I S A R I A , V. S . , S A X E N A , S . K., M A N I H A R , R . B., G O P A L K R I S H N A N , K . Biotechnol. 9 (1984), 341. [93] COMMANDAY, F., MACY, J . M. : Arch. Microbiol. 142 (1985), 61.

S.:

Appi. Biochem.

Acta Biotechnol. 8 (1988) 3, 224

Book Reviews MUJUMDAR, A . S.

Advances in Drying. Yol. 4 Berlin, Heidelberg, New Y o r k , T o k y o : Springer-Verlag, 1987. 421 pp., 100 fig., 148 DM I n den letzten J a h r e n ist d a s theoretische u n d p r a k t i s c h e Interesse a n P r o b l e m e n der Feststoff t r o c k n u n g sowohl von der Seite der Hersteller v o n T r o c k n u n g s a n l a g e n als a u c h v o n den industriellen A n w e n d e r n beständig gestiegen. Dies f i n d e t u. a. seinen A u s d r u c k in der laufenden Z u n a h m e der Anzahl v o n Veröffentlichungen auf diesem F a c h g e b i e t . Der von A . S. M U J U M D A R herausgegebene 4 . B a n d der Serie „ A d v a n c e s in D r y i n g " f a ß t 8 u m f a n g reichere Originalarbeiten k o m p e t e n t e r Wissenschaftler zu ausgewählten F r a g e n der Feststofft r o c k n u n g z u s a m m e n . I m einzelnen werden Ergebnisse zur Modellierung des Trocknungsprozesses, zur c o m p u t e r g e s t ü t z t e n T r o c k n e r k o n s t r u k t i o n sowie z u m Stoff- u n d W ä r m e ü b e r g a n g bei der F e s t s t o f f t r o c k n u n g mitgeteilt, wobei eine Vielzahl praktischer P r o b l e m e , wie sie z. B. bei der Holz-, Papier- oder Getreidetrocknung a u f t r e t e n , angesprochen werden. D u r c h zahlreiche anschauliche Zeichnungen u n d D i a g r a m m e wird das V e r s t ä n d n i s der A u s f ü h r u n g e n wesentlich erleichtert. D a m i t ist dieser F o r t s c h r i t t s b e r i c h t f ü r d e n Theoretiker wie a u c h f ü r d e n auf d e m F e s t s t o f f t r o c k n u n g s g e b i e t t ä t i g e n P r a k t i k e r gleichermaßen interessant, wobei d u r c h Berücksichtigung der neuesten L i t e r a t u r d e m N u t z e r der Z u g a n g zu a k t u e l l e n w e i t e r f ü h r e n d e n I n f o r m a t i o n e n v e r e i n f a c h t wird. D a s W e r k stellt eine willkommene E r g ä n z u n g zu d e m v o m gleichen H e r a u s g e b e r z u s a m m e n gestellten B a n d „ D r y i n g ' 8 5 " d a r (Review in A c t a Biotechnol. 7 (1987), 6, 514), der eine Vielzahl kürzerer Originalarbeiten zur T r o c k n u n g s p r o b l e m a t i k e n t h ä l t . E s k a n n d e m als Forscher, Entwicklungsingenieur, P r o j e k t a n t oder Betreiber v o n Trocknungsanlagen t ä t i g e n K o n s t r u k t e u r oder Verfahrenstechniker als Nachschlagewerk zur Lösung spezieller Aufgabenstellungen auf d e m Gebiet der F e s t s t o f f t r o c k n u n g empfohlen werden. W.

Thengiz

BIEDERMANN

BEEIDSE

SateUite DNA Berlin, Heidelberg, New Y o r k , T o k y o : Springer-Verlag, 1986. 149 pp., 78 fig., 22 t a b . , 149 DM Dieses B u c h stellt eine ü b e r a r b e i t e t e Version der sowjetischen Ausgabe des J a h r e s 1982 d a r . Der I n h a l t dieses Buches erschließt breit die sehr heterogenen Erscheinungsformen der schwer zu definierenden Satelliten-DNA in d e n bisher u n t e r s u c h t e n Organismen v o n d e n P r o t o z o e n bis z u m Menschen sowie P f l a n z e n . Diese D a t e n w u r d e n in systematischer Weise aneinandergereiht. Z u m besseren Verständnis der beschriebenen B e f u n d e werden in kurzer F o r m methodischexperimentelle Grundlagen wie U l t r a z e n t r i f u g a t i o n im D i c h t e g r a d i e n t e n u n d DNA-Reassoziationskinetik in einfacher F o r m v e r m i t t e l t . I n d e m d a r a n anschließenden kürzeren A b s c h n i t t werden D a t e n verschiedener Organismen, deren C h r o m a t i n s t r u k t u r e n im Z u s a m m e n h a n g m i t der Satelliten-DNA diskutiert. I n d e n drei folgenden letzten A b s c h n i t t e n werden z u s a m m e n f a s s e n d u n d einordnend F r a g e n der universellen S t r u k t u r , der H e r k u n f t u n d der Rolle dieser SatellitenD N A b e t r a c h t e t . Dabei werden bisherige Modelle insbesondere zur E n t s t e h u n g u n d E v o l u t i o n der Satelliten-DNA dargestellt. Die forcierte U n t e r s u c h u n g der Satelliten-DNA als Teil des chromosomalen Genoms e r b r a c h t e in den letzten drei bis vier J a h r e n eine Reihe von Ergebnissen, welche zu einer Vervollständigung beitragen w ü r d e n . Dazu gehören beispielsweise B e f u n d e z u m Y-Chromosom von Drosophila sowie R F L P - U n t e r s u c h u n g e n bei verschiedenen Spezies. Dieses B u c h stellt eine wertvolle D a t e n s a m m l u n g zur Satelliten-DNA d a r u n d ist als einführendes B u c h f ü r Genetiker u n d Molekularbiologen geeignet. H . - P . GÜTTEB

Acta Biotechnol. 8 (1988) 3, 2 2 5 - 2 3 1

The Pretreatment Effect on Wheat Straw Saccharification STOYANOV, I . , ILIEVA, S . , SAVOV, V . , PANAYOTOV, H . , DINEVA, J . , SPASOVA,

D.

K. Ohridski Sofia University Research Laboratory of Biotechnology and Biotechnics 1421 Sofia, 8 Dragan Tsankov, Blvd, Bulgaria

Summary Enzymatic hydrolysis of cellulose is potentially an attractive method for converting cellulose into glucose which can then be used as a chemical feed or as a growth substrate for a number of microorganisms to produce microbial products. An enzymatic hydrolysis of wheat straw with cellulase preparation "Trichocease" was made. The wheat straw used was pretreated mechanically and with NaOH. A procedure of pretreatment was investigated in 26 variants. The dynamics of enzymatic hydrolysis was studied. An assay of this dynamics based on the amount of reducing sugars formed during the cellulase reaction and depending upon enzyme and substrate concentration and time of action was carried out.

The utilization of cellulose-containing raw materials in industry and agriculture results in the accumulation of large amounts of waste products of vegetative origin. Straw, according to 5 0 % of the mass of cereals, is an important raw material for the production of energy, chemicals and single-cell protein [1, 2]. The rational transformation of straw into technologically usuable and economical raw material for the needs of fermentation-type production is associated with the preliminary application of suitable pretreatment (physical, chemical and biological). The data about some pretreatment types indicate an increase of the hydrolysis rate of lignocellular materials to sugars [3, 4]. The lignine elimination from the lignocellulose material considerably increases the substrate sensitivity to acidic and enzymatic hydrolysis. In order to reduce to a minimum the losses during pretreatment at low temperatures and atmospheric pressure F R A N Z , E R K E L et al. [ 5 ] have studied the sodium hydroxide effect [6—8] of sulfuric acid [9] and ethylen-diamine [10] on the saccharification of the lignocellulose material [ I I ] by a multienzymatic system or the merging of the cellulose saccharification and the production of single-cell protein [12] into a unified process. The cellulose acidic hydrolysis used in industry has the widest application of all chemical methods. Recently great interest was aroused by the enzymatic hydrolysis of cellulose-containing raw materials. The last two methods have been outlined as most promising regardless of the number of unsolved problems listed in Tab. 1. Acidic hydrolysis can be used as a preparation for enzymatic hydrolysis or as an efficient method for treating enzymatic hydrolysis residues despite the fact that these combi2

Acta Biotechnol. 8 (1988) 3

Acta Biotechnol. 8 (1988) 3, 2 2 5 - 2 3 1

The Pretreatment Effect on Wheat Straw Saccharification STOYANOV, I . , ILIEVA, S . , SAVOV, V . , PANAYOTOV, H . , DINEVA, J . , SPASOVA,

D.

K. Ohridski Sofia University Research Laboratory of Biotechnology and Biotechnics 1421 Sofia, 8 Dragan Tsankov, Blvd, Bulgaria

Summary Enzymatic hydrolysis of cellulose is potentially an attractive method for converting cellulose into glucose which can then be used as a chemical feed or as a growth substrate for a number of microorganisms to produce microbial products. An enzymatic hydrolysis of wheat straw with cellulase preparation "Trichocease" was made. The wheat straw used was pretreated mechanically and with NaOH. A procedure of pretreatment was investigated in 26 variants. The dynamics of enzymatic hydrolysis was studied. An assay of this dynamics based on the amount of reducing sugars formed during the cellulase reaction and depending upon enzyme and substrate concentration and time of action was carried out.

The utilization of cellulose-containing raw materials in industry and agriculture results in the accumulation of large amounts of waste products of vegetative origin. Straw, according to 5 0 % of the mass of cereals, is an important raw material for the production of energy, chemicals and single-cell protein [1, 2]. The rational transformation of straw into technologically usuable and economical raw material for the needs of fermentation-type production is associated with the preliminary application of suitable pretreatment (physical, chemical and biological). The data about some pretreatment types indicate an increase of the hydrolysis rate of lignocellular materials to sugars [3, 4]. The lignine elimination from the lignocellulose material considerably increases the substrate sensitivity to acidic and enzymatic hydrolysis. In order to reduce to a minimum the losses during pretreatment at low temperatures and atmospheric pressure F R A N Z , E R K E L et al. [ 5 ] have studied the sodium hydroxide effect [6—8] of sulfuric acid [9] and ethylen-diamine [10] on the saccharification of the lignocellulose material [ I I ] by a multienzymatic system or the merging of the cellulose saccharification and the production of single-cell protein [12] into a unified process. The cellulose acidic hydrolysis used in industry has the widest application of all chemical methods. Recently great interest was aroused by the enzymatic hydrolysis of cellulose-containing raw materials. The last two methods have been outlined as most promising regardless of the number of unsolved problems listed in Tab. 1. Acidic hydrolysis can be used as a preparation for enzymatic hydrolysis or as an efficient method for treating enzymatic hydrolysis residues despite the fact that these combi2

Acta Biotechnol. 8 (1988) 3

226

Acta Biotechnol. 8 (1988) 3 Tab. 1. Problems of acidic and enzymatic hydrolysis I. Problems of acidic hydrolysis

— — — — —

cellulose crystallization degree of hydrolysis fast furfural formation hydrooxymethylfurfural formation reaction nonspecificity

II. Problems of enzymatic hydrolysis

— — — — — —

enzyme production substrate concentration lignine-carbohydrate bond kinetics of hydrolysis microbial pollution hazzard enzyme complex inhibition from hydrolytic products

nations also cannot fully solve the problems listed in Tab. 1. Chemical treatment of the lignine-cellulose substrate by NaOH combined with suitable mechanical pretreatment and subsequent enzymatic hydrolysis is presumably a more acceptable alternative. The purpose of this work is to establish the technologically and economically most rational combinations of pretreatment methods (physical, chemical and enzymatic). Materials and Methods Lignocellulosic

Substrate

All experiments described in this paper have been carried out with wheat straw of a specified region as a classical agricultural lignocellulose waste. Wheat straw was ground in a suitable laboratory mill to 3—25 mm particle sizes. The wheat-straw composition was as follows: 40.76% cellulose, 24.00% hemicellulose, 12.50% lignine, 10.60% ashes, 12.10% water and 87.90% total dry substance. Pretreatment The straw pretreatment was carried out in a 3-1 glass jar. The ground straw was suspended in 0.25 N and 0.5 N NaOH solution and 1.0% of H 2 0 2 . The wheat straw percentage content in the samples varied from 5 to 30% dry substance. Then autoclaving at 121 °C for one hour followed. Ionized water as chemical pretreatment agent was used in the experiments. I t was produced by adequate electric current treatment to pH = 11.00. The ionized water prepared in this way was used for suspending the straw in respective percentages which was then incubated for 24 h at room temperature. The material was subjected to correction of pH by concentrated sulphuric acid to pH = 4.8, after enzymatic hydrolysis pretreatment. Enzymatic Production The biosynthesis of the Trichoceasa-SU cellulase preparation was made under the conditions of two-stage fermentation of strain Trichoderma sp. QM from the collection of the Research Laboratory of Biotechnology and Biotechnics of K. Ohridski Uni-

227

STOYANOV, I., ILIEVA, S. et al., W h e a t Straw Sacoharification

versity Sofia. The fungi strain was cultivated in 2 m 3 nutritive fermenter [13]. The fermentation process was 144 hours at 28—30°C, aeration VjV min, agitation 150 rpm, and overpressure 0.03—0.05 MPa. The cellulase preparation activity was determined by the method described in another publication of ours [14]. The Cx activity was found: 5 0 0 U / m l ; Cl — 2 0 0 U / m l ; betaglucosidase — 0.8 U/ml and xylanase — 100 U/ml. The cultural fluid was dried in a pulverizer drier. The preparation produced in this way was used in our hydrolysis experiments. In the dried state the preparation had the following activities in units per gramme: C j : — 5000; Gx — 2000; beta glucosidase — 25 and xylanase — 800. Enzymatic Hydrolysis The enzymatic hydrolysis of the straw was carried out by Trichocease SU preparation in 0.5—1 glass jars at static conditions. The following substrate concentrations were used: 5 % , 10%, 2 0 % and 3 0 % in respect of moisture. The enzyme ratio to the respective substrate ranged from 1:10 to 1:200. The hydrolysis conditions were: pH 4.8. 55°C and 24 h. The enzymatic hydrolysis was observed in its dynamics. Analysis of Sugars and Degree of Hydrolysis The reducing sugar concentrations were determined by the SOMODGY-NEILSON method [8]-

The enzymatic hydrolysis degree was determined as Weight of formed sugars Weight of used substrate

162 180

JQQO/

after 24-hour hydrolysis. Results and Discussion The sequence of the individual stages of the indicated alternative are schematically shown in Fig. 1 for wheat straw substrate ground to particle sizes from 3 to 25 mm. After the pretreatment. the cellulose substrate (wheat straw) decay by the Trichocease technical enzyme preparation is determined by measuring the quantity of reducing sugars freed during incubation. The results of the different combinations of scheme illustrated in this way are given in Tab. 2. I t supplies data which definitely show that the degree of enzymatic hydrolysis of wheat straw depends on the pretreatment alternatives and the substrate concentrations as dry substance as well as on the specified quantity of enzyme involved in the hydrolysis process. Enzymatic production

Pretreatment Fig. 1. Sequence of wheat straw enzymatic sacoharification 2*

Products

228

Acta Biotechnol. 8 (1988) 3

Alternatives 1—5 in Tab. 2 illustrate the results of wheat-straw enzymic hydrolysis without pretreatment. In the investigations the combinations of untreated material were used for reasons of comparison with the remaining alternatives as control. Enzymatic hydrolysis efficiency depends on the enzyme effect on the cellulose functional groups in the lignocellulose complex. As the enzyme cannot penetrate deeply into the fibre, the reaction occurs on its surface on the accessible functional groups and results in incomplete substrate hydrolysis. The enzymatic hydrolysis rate, the cellulose adsorption and the cellobiotic accumulation at the beginning of enzymatic hydrolysis are determined by the pretreatment effect. If the reagent used manages to penetrate into the microfibriles, or in particular into their amorphous zones, then the hydrolysis degree and its rate increase considerably. Maximum hydrolysis can be produced only in those cases when the solvent or reagent used can contribute to more significant surging of the cellulose so that more functional groups can participate in the reaction [15]. Strong mechanical treatment, e.g. dry grinding (crushing) in rollers results in breaking the cellulose molecular bonds. Ground straw in wetted state (about 50% moisture) under the same conditions results in a considerable decrease of particle sizes without really affecting the enzymatic reaction rate. This is obvious from the results after the enzymatic hydrolysis of alternatives Nos. 18, 19 and 21 in comparison with alternatives Nos. 13 and 14. Interesting results were also obtained when ionized water was used: alternatives Nos. 6 and 7. Tab. 2. Content of saccharides and degree of hydrolysis for the different alternatives of wheat straw pretreatment No.

Pretreatment

Substrate [%] (dry)

Enzyme E:S

1. 2. 3. 4. 5. 6.1 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Controls Controls Controls Controls Controls Ionized water Ionized water 0.25 n NaOH 0.25 n NaOH 0.25 n NaOH 0.5 n NaOH 0.5 n NaOH 0.5 n NaOH 0.5 n NaOH 0.5 n NaOH 0.5 n NaOH 0.5 n NaOH Rolled. + 0.5 n NaOH Rolled + 0.5 n NaOH Rolled + 0.5 n NaOH Rolled + 0.5 n NaOH 1% NaOH + 1% H 2 0 2 1% NaOH + 1% H 2 0 2 1% NaOH + 1% H 2 0 2 1% NaOH + 1% H 2 0 2 1% NaOH + 1% H 2 0 2 1% NaOH + 1% H 2 0 2

30 30 30 30 30 20 5 5 5 30 5 5 20 20 30 30 30 20 20 20 20 10 10 10 20 20 20

1 : 12.5 1 : 25 1 :50 1 : 100 1 :200 1:: 50 1 : 50 1::25 1::50 1:: 50 1;: 50 1:: 200 1:: 50 1:: 100 1;:25 1:: 50 1:: 100 1:: 12.5 1::25 1:; 50 1:: 100 1:: 12.5 1: 25 1: 50 1: 12.5 1: 25 1: 50

RS [mg/ml]

Hydrolysis degree [%]

14.0 11.0 11.0 11.0

5.2 3.6 3.6 3.6

14.0 9.3 6.3

6.0 17.0 11.0 5.0 12.0 5.6 8.0 7.0 10.0 18.0 6.0 22.0 13.0 12.0 2.0 11.0 9.0 6.0 9.0 8.0 7.0

6.0 3.2 20.0 18.0 30.0 31.5 23.5 55.0 33.0 30.0 6.0 14.0 12.0 7.7 22.0 19.5 16.5

229

STOYANOV, I., ILIEVA, S. et al., Wheat Straw Saccharification

Cellulose wetted by electrolyte instead of water surges considerably more. This phenomenon is due to hydroxyl adsorption from the hydroxylized ions on the cellulose demanding a greater volume than the water molecule. The larger the hydrolyzed electrolyte and the smaller the above-mentioned anion, the more efficient is surging. This can be seen best from the enzymatic hydrolysis comparison of alternatives Nos. 6 and 7 and alternatives Nos. 1, 23, 4 and 5. An important factor in pretreatment is the delignification of the cellulose-containing materials. Partial delignification is achieved by H 2 0 2 alkaline solution [16]. Alternatives Nos. 22—27 illustrate saccharification and enzymatic hydrolysis degree for straw treated by 1% H 2 0 2 in 1% sodium base solution. Figs. 2 and 3 trace the monosaccharides (monose) yield dynamics in mg/ml per 24 hour incubation.

Time Lh]

Fig. 2. Dynamics of the enzymatic saccharification after pretreatment of the wheat with 0.5 N X a O H in different concentrations of the enzyme O - 1:25; A - 1:50; x - 1:100 30.0r 25.0

-

-20.0

S

I l

i i

5.0

K

10.0 5.0

12 Time Ch]

24

Fig. 3. Effect of the pretreatment variant way on the dynamics of enzymatic saccharification of the wheat straw with 2 0 % dry weight and ration of enzyme and substrate 1 : 5 0 -

0.5 NaOH

-

rolled straw with 0.5 NaOH

-

rolled straw with 1 % NaOH + 1 % H 2 0 2

230

Acta Biotechnol. 8 (1988) 3

The results in Fig. 2 are presented in relation to the amount of the included enzyme. Three enzyme concentrations were used: ( E : S ; Enzyme-substrate ratio) 1:25, 1:50 and 1:100. Fig. 3 illustrates the wheat straw enzyme saccharification dynamics depending on the pretreatment alternatives at 20% substrate dry matter and E : S = 1:50. Three alternatives of pretreatment of 1% sodium base and 1% hydrogen peroxide solution, 0.5 N sodium base and rolled straw with 0.5 N sodium base are illustrated. Conclusion The rate and degree of enzymatic hydrolysis are strictly related to the pretreatment method used for the material in question. The results are interesting from the point of view of the conditions under which the used substrate (wheat straw) saccharification occured. Unlike the classical methods of carrying out enzymatic saccharification, i.e. low percentage content of the substrate and continuous agitation of the hardliquid phase, the experiments described were made with relatively high percentage contents of the substrate up to 30% and at static conditions. The results presented in our paper definitely suggest that the combined material treatment with sodium base (0.5 N) and mechanical force (rolling) has the best effect. After the enzymatic hydrolysis of the material treated in this way we get 22% saccharification or 5.5% sugar syrup, respectively. This alternative most likely offers the most favourable conditions for the enzyme molecules penetrating between the cellulose fibres and their subsequent depolymerization to monosaccharides (monose). Enzymatic hydrolysis dynamics reaffirm the classical dependence of the enzymatic reaction on the concentration of the included enzyme. I n this case the results may serve to characterize the process from the economical point of view, i.e. the relation between the quantity of the enzyme introduced and the yield produced in time. Interesting results were obtained in using ionized water as pretreatment agent. Most likely this method will prove to be very promising and its utilization in practice will demand extra investigations included in the National Programe of the Peoples Republic of Bulgaria for finding new raw material sources and their biological conversion into energy, chemicals or new types of raw materials. Received May 5, 1987

References [1] BIEN-AIME, A.: These senie Cycle Université de Montpellier (1985). [ 2 ] TANAKA,

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57 (1979), 186. [ 7 ] TANIGIJCHI, M . , KOMETANI, Y . , T A N A K A , M . , MATSUNO, R . , K A M I K U B O , T . :

Microbiol. Biotechnol. 14 (1982), 74. [8] SOMODGY, M.: J. Biol. Chem. 193 (1952) 19, 195. [ 9 ] F A N , L . T . , GHARPURAY, M . M . , L E E , Y . H . : Biotechnol. Bioeng. Symp.

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[ 1 0 ] DETROY, R . W . , ILDENFELSER, L . A . , JULIAN, G . S., ORTON, J r . a n d W .

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Bioeng. 10 (1980), 135. [11] ALFANK, F . , CANTARELLA, M., ERTO, L . , SCARDI, V . — I n : E n e r g y f r o m b i o m a s s . 2 n d E . - C .

Conference. London, New York: Applied Science Publishers, 1982, p. 10000. M., T A N A K A , M., M A T S U N O , R., K A M I K U B O , T . : E u r . J . Appi. Microbiol. Biotechnol. 14 (1982), 35. [13] S P A N O , C . A., H A N D E L S , M. J . : Wash. Acad. Sci. 66 (1976), 279. [14] A T E V , A. P . , SPASOV, S . D., P A N A Y O T O V , H . A.: Dokl. Bulg. Akad. Nauki 86 (1983), 533.

[12]

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[ 1 5 ] LADICH, M . R , , LADICH, C. M . , TSAO, G . T . : S c i e n c e 2 0 1 ( 1 9 7 8 ) , 7 4 3 .

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BINDING

Results and Problems in Cell Differentiation A Series of Topical Volumes in Developmental Biology Vol. 12. Differentiation of Protoplasts and of Transformed Plant Cells Berlin. Heidelberg, New York, Tokyo: Springer-Verlag, 1986. 157 pp., 24 fig., 98 DM

Die Techniken der Isolierung, Manipulierung und Regeneration von pflanzlichen Protoplasten sind im letzten J a h r z e h n t bedeutend verbessert worden und haben sich zu einem Arbeitsgebiet entwickelt, das ganz wesentlich zum Fortschritt auf den Gebieten der Pflanzengenetik, Pflanzenzüchtung und Pflanzenphysiologie beiträgt. Der vorliegende Band befaßt sich in den ersten 3 Kapiteln mit der Entwicklung von Protoplasten zu Zellklonen und Regeneratpflanzen, wobei der Protoplasten-Fusion und ihren genetischen Aspekten breiter R a u m gegeben wird. Zunächst werden im

1. K a p i t e l

(S. C. MAHESHWARI, R . GILL, N . MAHESHWARI, P . K . GHARYAL) d i e

all-

gemeinen Grundlagen und Voraussetzungen der Isolierung und Regeneration von Protoplasten höherer Pflanzen besprochen. I m Vordergrund stehen dabei physiologische Probleme, wie die Bedeutung chemischer und physikalischer F a k t o r e n f ü r das Regenerationsvermögen der Protop l a s t e n . D a s 2. K a p i t e l ( H . BINDING, G. KRUMBIEGEL-SCHROEREN, R . NEHLS) b e f a ß t s i c h m i t

der Protoplasten-Fusion und den ersten Entwicklungsschritten der Fusionsprodukte, während i m 3. K a p i t e l ( R . NEIILS, G. KRUMBIEGEL-SCHROEREN, H . BINDING) die w e i t e r e

Entwicklung

der fusionierten Protoplasten behandelt wird. Beide Kapitel zeichnen sich durch eine Fülle von F a k t e n und Problemdiskussionen aus, die in einer sehr klaren und straffen Diktion geboten werden. Insgesamt werden hier über 300 Literaturzitate angeführt. Eine besonders wertvolle Literaturzusammenstellung bietet der Abschnitt „Genetic Traits Utilized in Somatic Hybridization E x p e r i m e n t s " im 3. Kapitel. Das 4. Kapitel (N. S. YADAV) behandelt die Transformation pflanzlicher Zellen mittels Ti- und Ri-Plasmiden sowie Cauliflower Mosaic Virus. Es gibt auf 34 Seiten eine ausgewogene, die Literatur bis zum J a h r e 1984 berücksichtigende Übersicht über diese gegenwärtig wohl aussichtsreichste Forschungsrichtung der Molekulargenetik höherer Pflanzen. Alles in allem bietet dieses Buch eine ausgezeichnete, ungewöhnlich weit in die Details gehende Übersicht über das „genetic engineering" und seine entwicklungsphysiologischen Aspekte bei höheren Pflanzen. Es sollte in keinem auf diesem Gebiet arbeitenden Laboratorium fehlen. P . DÖBEL

Acta Biotechnol. 8 (1988) 3, 232

Book Reviews Angelo Azzi, Lanfranco

MASOTTI,

Arnaldo

VECLI

Membrane Proteins Isolation and Characterization Berlin, Heidelberg, New York, Tokyo, London, Paris, Tokyo: Springer-Verlag, 1986. 181 pp., 58 fig., 38 DM ISBN 3-540-17014-6 Das vorliegende Buch ist das dritte einer Serie von Laborhandbüchern zum Gebiet „Membranproteine". Durch einschlägig tätige Fachexperten wurden Arbeitsvorschriften in Form von Versuchsprotokollen zusammengestellt, die einen schnellen methodischen Zugang zu diesem speziellen biowissenschaftlichen Arbeitsgebiet ermöglichen. Die vorliegenden Arbeitsvorschriften resultieren aus Erfahrungen, die im Verlauf eines durch die F B B S und dem Italienischen Forschungsrat initiierten „International Advanced Course" gesammelt wurden. Die einzelnen Versuche des Versuchsprogramms wurden so ausgearbeitet und dargestellt, daß die Planung und die Ausführung der Experimente so einfach wie möglich ist. Allerdings scheinen jedoch entsprechende Vorkenntnisse eine notwendige Voraussetzung zu sein. Auf der Basis dieser umfassenden und sorgfältig ausgearbeiteten Versuchsanleitungen sollte es in Forschungslaboratorien bei Notwendigkeit recht leicht möglich sein, sich in das Gebiet der „Membranproteine" einzuarbeiten und schnell zu eigenen neuen Experimenten zu kommen. Das Buch dürfte sowohl dem erfahrenen, aber nicht einschlägig orientierten Praktiker den schnellen Zugang zu diesem Arbeitsgebiet ermöglichen als auch zumindest teilweise bei Studentenpraktika Anwendung finden können. Letzteres erfordert jedoch ein sehr hohes Niveau der studentischen Ausbildung sowie die entsprechende materiell-technische Basis. Alles in allem ist dieses auf einem hohen Niveau stehende, kleine H a n d b u c h empfehlenswert und sollte in keiner biowissenschaftlichen Bibliothek fehlen. A.

Rolf

SCHMID,

Saburo

STEUDEL

FUKUI

Dictionary of Biotechnology in English — Japanese — German Berlin, Heidelberg, New York, London, Paris, Tokyo: Springer-Verlag, 1986. 1324 pp., 298 DM, ISBN 3-540-15566 The preface of this "Dictionary of Biotechnology in English-Japanese-German" very aptly describes t h e content of this work. "This "Dictionary of Biotechnology" is the outcome of several years of work with English, German and Japanese texts in this field. It contains nearly 6.000 terms and is divided into 3 main parts: 1. Alphabetically listed technical terms in English accompanied by translations into German and Japanese, 2. Alphabetically listed technical terms in Japanese accompanied by translations into English and German, 3. Alphabetically listed technical terms in German accompanied b y translation into English. Each of the main parts is followed by a list of synonyms in t h e word entry language. An appendix defines over 300 abbreviations using all three languages." This dictionary is of particular value to specialized translaters and biotechnologists who have to deal with Japanese t e x t s in the original. The Japanese terms are easy to find because they are arranged alphabetically according to their roman spelling ("roma-ji"), t h e transcription being based on the Hepburn method. The list of synonyms and the appendix of abbreviations further add t o t h e value of the dictionary. The reviewer gratefully acknowledges t h e relatively large print which makes for easy legibility. All the features mentioned make the dictionary a valuable tool. K.

GEYLER

Acta Biotechnol. 8 (1988) 3, 2 3 3 - 2 4 0

Untersuchungen zur gelpermeationschromatographischen Fraktionierung von Natrium-Ligninsulfonat POLTER, E .

Akademie der Wissenschaften der D D R I n s t i t u t für Biotechnologie, Leipzig Permoserstraße 15, Leipzig, 7050 D D R

Summary The gelpermeation chromatography of sodium ligncsulfonates with bidistilled water as an eluent is influenced b y the volume expansion of the polyelectrolyte, b y the negative changes of the gels, and by the inclusion effect so markedly that true conclusions on the molecular weight distribution of sodium lignosulfonate cannot be drawn. Using electrolytes as an eluent these irregularities can be avoided. Polydispersity of these macromolecules however prevents separation into discrete peaks. As a result of this behaviour changes of molecular weight distribution are only poorly recognized. Therefore, on studying biotransformation of lignosulfonic acids we purpose to use gels by which one part of the lignosulfonates is excluded, and biotransformation m a y be controlled by the ratio of excluded part of the lignosulfonates to that part retarded b y the gels.

Einleitung Lingninsulfonate sind Abprodukte der Sulfitzellstoffherstellung u n d führen zu Umweltbelastungen. Zu ihrer Verwertung sind weltweit umfangreiche Forschungsarbeiten durchgeführt worden, jedoch hat sich bisher nur die Verbrennung zur Gewinnung von Prozeßenergie durchgesetzt. Eine weitere Verwertung stellt möglicherweise die Biotransformation dar. Bei diesem Prozeß treten infolge Polymerisation oder Depolymerisation [1—6] Veränderungen in der Molekulargewichtsverteilung der Ligninsulfonate auf. Zum Nachweis dieser Veränderungen bietet sich die relativ einfache Methode der Gelpermeationschromatographie an. Jedoch ergeben sich hierbei auf Grund des Polyelektrolytcharakters dieser Makromoleküle Besonderheiten, die zu berücksichtigen sind. Bei Polyelektrolyten nimmt der Dissoziationsgrad mit abnehmender Konzentration zu und die Gegenionen ihrer Salze bilden um das Molekül eine Ionosphäre, deren Durchmesser mit abnehmender Konzentration zunimmt und ihr Maximum bei unendlicher Verdünnung in salzfreien Lösungen erreicht. Die im Inneren befindlichen Anionen stoßen sich gegenseitig ab und versteifen das Molekül. Auf Grund dieser elektrostatischen Effekte unterliegt das Polyelektrolytmolekül in verdünnten Lösungen der Volumenexpansion. Trotz Kenntnis dieser Anomalien werden als Elutionsmittel f ü r die Gelpermeationschromatographie von Ligninsulfonaten neben Elektrolytlösungen [3, 7, 8] auch bi-

Acta Biotechnol. 8 (1988) 3, 2 3 3 - 2 4 0

Untersuchungen zur gelpermeationschromatographischen Fraktionierung von Natrium-Ligninsulfonat POLTER, E .

Akademie der Wissenschaften der D D R I n s t i t u t für Biotechnologie, Leipzig Permoserstraße 15, Leipzig, 7050 D D R

Summary The gelpermeation chromatography of sodium ligncsulfonates with bidistilled water as an eluent is influenced b y the volume expansion of the polyelectrolyte, b y the negative changes of the gels, and by the inclusion effect so markedly that true conclusions on the molecular weight distribution of sodium lignosulfonate cannot be drawn. Using electrolytes as an eluent these irregularities can be avoided. Polydispersity of these macromolecules however prevents separation into discrete peaks. As a result of this behaviour changes of molecular weight distribution are only poorly recognized. Therefore, on studying biotransformation of lignosulfonic acids we purpose to use gels by which one part of the lignosulfonates is excluded, and biotransformation m a y be controlled by the ratio of excluded part of the lignosulfonates to that part retarded b y the gels.

Einleitung Lingninsulfonate sind Abprodukte der Sulfitzellstoffherstellung u n d führen zu Umweltbelastungen. Zu ihrer Verwertung sind weltweit umfangreiche Forschungsarbeiten durchgeführt worden, jedoch hat sich bisher nur die Verbrennung zur Gewinnung von Prozeßenergie durchgesetzt. Eine weitere Verwertung stellt möglicherweise die Biotransformation dar. Bei diesem Prozeß treten infolge Polymerisation oder Depolymerisation [1—6] Veränderungen in der Molekulargewichtsverteilung der Ligninsulfonate auf. Zum Nachweis dieser Veränderungen bietet sich die relativ einfache Methode der Gelpermeationschromatographie an. Jedoch ergeben sich hierbei auf Grund des Polyelektrolytcharakters dieser Makromoleküle Besonderheiten, die zu berücksichtigen sind. Bei Polyelektrolyten nimmt der Dissoziationsgrad mit abnehmender Konzentration zu und die Gegenionen ihrer Salze bilden um das Molekül eine Ionosphäre, deren Durchmesser mit abnehmender Konzentration zunimmt und ihr Maximum bei unendlicher Verdünnung in salzfreien Lösungen erreicht. Die im Inneren befindlichen Anionen stoßen sich gegenseitig ab und versteifen das Molekül. Auf Grund dieser elektrostatischen Effekte unterliegt das Polyelektrolytmolekül in verdünnten Lösungen der Volumenexpansion. Trotz Kenntnis dieser Anomalien werden als Elutionsmittel f ü r die Gelpermeationschromatographie von Ligninsulfonaten neben Elektrolytlösungen [3, 7, 8] auch bi-

234

Acta Bioteehnol. 8 (1988) 3

destilliertes Wasser [1, 2, 9—12] verwendet. Die Ergebnisse sind somit untereinander nicht vergleichbar. Für eine Biotransformation ergibt sich somit die Aufgabe, zu überprüfen, welche der Methoden ihren Verlauf richtig widerspiegelt.

Materialien und Methoden Für die Fraktionierung wurde ein Chromatographiesystem der Firma L K B (Bromma, Schweden) eingesetzt. Die Chromatographiesäulen (9 X 500 mm, 9 X 1 0 0 0 mm) wurden vom V E B Kombinat Technisches Gas, Ilmenau, D D R und die Säulenfüllung Sephadex G-75, Sepharose CL-6 B , Sephacryl-S 200 sowie Blue Dextran 2000 von der Firma Pharmacia (Uppsala, Schweden) bezogen. Das Natrium-Ligninsulfonat (Na-LS) wurde von Herrn S T E N L U N D (Finnish Pulp and Paper Research Institute) hergestellt. Die Durchflußgeschwindigkeit wurde mit einer Micro Perpex Pumpe konstant gehalten, die Extinktion mit dem Uvicord S I I bei 278 nm kontinuierlich gemessen und von einem Schreiber registriert. Fraktionen von 1,3—2,0 ml wurde mit einem Fraktionssammler (Multi Rae) aufgenommen. Als Elutionsmittel dienten bidestilliertes Wasser und 0,75 M NaCl-Lösung.

Ergebnisse Gelpermeationschromatographie als Elutionsmittel

von Na-LS

unter Verwendung

von bidestilliertem

Wasser

Die gelchromatographische Fraktionierung erfolgte an Sephadex G-75 und Sepharose CL-6 B (Abb. 1, 2). Wie aus Abb. 1 ersichtlich, wird das Na-LS an Sephadex G-75 bei Verwendung von bidestilliertem Wasser als Elutionsmittel in 4 relativ gut voneinander getrennten

ÜJ 80000 D besitzen. Außerdem ist zu erkennen, daß für die Isolierung der letzten beiden Peaks das Elutionsvolumen das Gelbettvolumen übersteigt, d. h. dieser Teil des Na-LS wird von der Gelmatrix stark retardiert. o o o

N H / + O H + H 2 0 + 3 N A D P Theoretical

Growth

(5a) (5b)

Yields

Ethanol Ethanol is an energy excess substrate with ammonium as the nitrogen source, if, on the basis of Eqs. 2 and 4, a P/O 22 1.85 is realized (Fig. 1). Results with H.polymorpha MH 20 show that, due to the growth yields obtained for different substrates, a P/Oquotient at around 2 is realistic [13, 20], so t h a t at least a balanced carbon/energy ratio can be assumed during growth of this species on ethanol. N A D P H is also in excess, and the possibility of using this coenzyme equivalently as an energy source, as is N A D H [21, 22], is a prerequisite for ethanol being an energy excess substrate at a P/O of 2. Otherwise, the carbon conversion efficiency could not amount to 75%, and any external energy donor like formate should be useful for improving growth yield. With nitrate as the nitrogen source any substrate must in each case be oxidized to generate N A D P H (Tab. 1). Thus, growth yields are clearly below the values for ammonium nitrogen. Their absolute maxima are determined by the sequences or the coenzyme dependencies of the reactions considered for the respective generation or consumption of NADPH. I n yeasts pathways for NADPH-formation are quite limited. I n general, only the hexosemonophosphate pathway (glucose 6-phosphate and 6-phosphogluconate dehydrogenase), the isocitrate dehydrogenase (dual pyridine nucleotide dependency) and the aldehyde dehydrogenase reactions can be employed. This results in different metabolic constellations with regard to NADPH-generation: 1. the sole generation of the required N A D P H via the Ald-DH if a limited function of the TCA cycle or a predominance of NAD-dependency of ICDH are considered (EtOH -»• 2C0 2 + 4 N A D H + 1 N A D P H + 1FADH 2 ),

Acta Biotechnol. 8 (1988) 3

252

Ethanol

Hexadecane

Acetate

Respiratory quotient Fig. 1. Theoretical growth yields of yeasts on C 2 -substrates with ammonium and nitrate as the nitrogen sources Those cases are shown according to the metabolic constellations given in Tab. 1 which have relevance to the experimental growth yield data on the basis of a P / 0 = 2. Plateau regions are indicative of having reached the respective maximum carbon conversion efficiency (this level is shown by the dashed lines in the case of acetate). The following cases are considered with respect to the nitrogen source, the coenzyme dependency of nitrate reduction, the reactions of N A D P H generation and the sequence of oxidation to CO., of substrate: a : NH 4 +; OX. via TCA cycle. b: N 0 3 - ; N a R and NiR (NADPH); ICDH and Ald-DH (NADP); Ox. via TCA cycle. c: N 0 3 ~ ; N a R (NADH), NiR (NADPH); Ald-DH (NADP); Ox. via TCA cycle, d : NH 4 +; Ox. via TCA cycle. e: N 0 3 " ; N a R (NADH), NiR (NADPH); N A D P H via /9-Oxidation and HMP pathway; Ox. via HMP pathway, f: N 0 3 ~ ; N a R and NiR (NADPH); N A D P H via HMP; Ox. via HMP pathway, g : NH 4 +; OX. via TCA cycle. h: N 0 3 ~ ; N a R and NiR (NADPH); N A D P H via HMP pathway; Ox. via TCA cycle. i: N 0 3 " ; N a R (NADH), NiR (NADPH); N A D P H via HMP pathway; Ox. via TCA cycle.

MÜLLER, R. H., BABEL, W., C 2 -Compounds for Microbial Growth

253

2. the inclusion of the TCA cycle and assuming the predominance of the NADPdependent ICDH activity (EtOH ^ 2C0 2 + 3NADH + 2NADPH + 1FADH 2 ), 3. the functioning of the HMP pathway (EtOH

2C0 2 + 1.5NADH + 4NADPH + 0.5FADH 2 - 1.5 ATP).

From these equations it becomes evident that, besides NADPH, inevitably a distinct amount of energy equivalents is synthesized. For this reason, the final growth yields, 1.e. the metabolism-determined limits of carbon conversion, are reached at comparatively lower P/O-quotients than with ammonium nitrogen. As expected, the smallest part of substrate must be used for generating a given amount of NADPH when the HMP pathway is employed (presuming that the fructose bisphosphatase operates). But this is the only case in which too low an amount of reduction equivalents remain available for energy generation so that a further part of substrate must be oxidized to satisfy this requirement. Consequently, this is also the only case in which formate could excert a stimulating effect on growth yield. n-Alkanes Similar statements as for ethanol were hold for n-alkanes. These substrates generate an excess in energy and reduction equivalents (if the /¡¡-oxidation results in the generation of NADPH) with ammonium as the nitrogen source and comparable P/O-quotients (Fig. 1, Tab. 1). The differences in the final growth yield are quite pronounced, because the metabolic conditions considered result in distinctly different quantities of substrate which must be oxidized to meet biosynthetic requirements. In Fig. 1 the two extreme situations are shown for nitrate as the nitrogen source. Acetate In the case of acetate no excesses in energy or reduction equivalents can be reached and only if a P/O = 3 is taken into account, a balanced carbon/energy ratio is approximated for both nitrogen sources (Fig. 1). As sequences for NADPH-generation only the TCA cycle (ICDH) or the HMP pathway are available and, depending on which pathway is used, growth yields will either be reduction equivalent-limited (with the TCA cycle) or energy-limited (with the HMP pathway). In the first case a great amount of energy equivalents is generated if the substrate used for NADPH-synthesis is oxidized completely to C0 2 and, consequently, a balanced carbon/energy ratio is obtained with ammonium and a large excess in energy with nitrate as the nitrogen source (Tab. 1). With the HMP pathway the limited amount of substrate needed for NADPH-generation again gives rise to the necessity to further oxidize substrate merely for energy generation, thus enabling an external energy source to improve growth yield. 2. Experimental Growth Yields Ethanol

These experiments were performed with H. polymorpha MH 20. Growth on ammonium as the nitrogen source results in growth yields between 0.75 and 0.77 g/g (Tab. 1). Accordingly, the maximum carbon conversion efficiency was obtained with this yeast on this substrate.

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(14)

mit Ms =

eo

• X0 • Y0 • Z0l(m0 + 1)

(15)

Die Temperaturabhängigkeit der Verdampfungsenthalpie des Wassers kann Tabellenbüchern entnommen werden. Der Wärmeübergang einschließlich Gutsschrumpfung ist experimentell zu ermitteln. Mit der Energiebilanz (Gl. 13) wird für festgelegte Zeitschritte At die Temperaturänderung zl#s = möglich.

— #S,n-l

(16)

264

Acta Biotechnol. 8 (1988) 3

Simulationsmodell für die Trocknung thermolabiler biologischer Produkte Die Qualitätsänderungen bei der Trocknung können in Abhängigkeit von den Prozeßbedingungen und den Produkteigenschaften durch Computersimulation rechnerisch verfolgt werden. Ein dazu erforderliches Simulationsprogramm soll nachfolgend kurz skizziert werden. 1. Aus experimentell bestimmten Trocknungsverläufen oder Literaturdaten werden die konstanten Parameter m k , m 0 i l , m 0i2 , #s,a und &L in den Rechner eingegeben. 2. E s wird die Trocknungszeit für die erste fallende Periode für den Feuchtegehalt von m A bis m 0 i l mit Gl. 10 berechnet. Für die zweite fallende Periode für den Feuchtegehalt von m 0 i l bis m 0 i 2 wird anstelle des Diffusionskoeffizienten D der Diffusionskoeffizient D x in der A R R H E N i u s - F o r m der Gl. 12 verwendet. 3. Die nach 2 berechnete Trocknungszeit wird in die Zeitinkremente At = tn — tn_x aufgeteilt. Die Anfangsbedingung für die erste fallende Periode ist gekennzeichnet durch die Anfangsbedingung m = m A , bei t = 0 für alle x/X. Es existiert am Anfang kein Feuchteprofil. 4. Mit der Energiebilanz (Gl. 13) wird = #S.n 5. 6. 7. 8. 9.

fls.n-1

berechnet. Das Trocknungsgut mit der halben Dicke X wird in die Inkremente Ax aufgeteilt. Mit Gl. 11 in Verbindung mit einer Gl. 10 analogen Beziehung wird für jedes Inkrement der Feuchtegehalt berechnet. I n einem ersten Unterprogramm wird mit dem über den Querschnitt gemittelten Feuchtegehalt wt_n = m t>n _j + Arn weitergerechnet. In einem zweiten Unterprogramm wird mit den in Punkt 5 berechneten Feuchteprofilen weitergerechnet. E s werden mit Gl. 5 die Qualitätsänderungen nach dem ersten Unterprogramm für mittlere Feuchtegehalte oder nach dem zweiten Unterprogramm für Feuchteprofile berechnet. Die Berechnungen werden für die gesamte erste fallende Periode durchgeführt. Das Feuchteprofil am Ende der ersten fallenden Periode stellt die Anfangsbedingung für die zweite fallende Periode dar. Analog zu den Punkten 6, 7 und 8 wird die Rechnung zu Ende geführt.

Variiert werden die Temperatur der Trocknungsluft, Form, Größe und physikalische Eigenschaften des Gutes und die qualitätsbestimmenden Parameter. Für die Durchführung von Optimierungsrechnungen werden Trocknungszeit, Endfeuchtegehalt, maximale und minimale Lufttemperatur und maximale Qualitätsänderung vorgegeben. Erhalten werden Lufttemperatur-Trocknungszeit-Profile, die die Grundlage für Mikrorechnersteuerungen von Trocknern sind. Das Optimierungsmodell ist einer weiteren Publikation vorbehalten. Computersimulation der Trocknung eines stückigen Gutes Als Trocknungsmodell diente ein dreidimensionaler plattenförmiger Körper der Abmessungen 0,05 m X 0,05 m X 0,005 m. Mit diesem Körper wurde die Heißlufttrocknung eines stückigen biologischen Produktes simuliert. Leitsubstanzen für die Qualität waren der Ascorbinsäureverlust und die nichtenzymatische Bräunung. Untersucht wurden der Einfluß konstanter Lufttemperatur und die Wirkung von Temperaturprofilen auf die Qualitätserhaltung. Die gesamte Trocknungszeit betrug 360 min und die Zeitschritte 10 min.

M o h r , K.-H., V o n d r a n , J., Trocknung biologischer Produkte

265

Abb. 2. Prozentuale Änderung des Peuchtegehaltes ( — ) , des Ascorbinsäuregehaltes ( ) und der nichtenzymatischen Bräunung ( ) mit der Trocknungszeit t in min bei Trocknungstemperaturen von 40°C, 50°C, 60°C, 70°C, 80°C und 90°C

Abb. 3. Prozentuale Änderung des Feuchtegehaltes ( — ) , des Ascorbinsäuregehaltes ( ) und der nichtenzymatischen Bräunung ( ) mit der Trocknungszeit t in min bei stündlich veränderter Trocknungslufttemperatur von 60/60/70/75/85/90°C (1), 85/80/65/65/75/85°C (2) und 90/90/85/70/63/65°C (3)

266

Acta Biotechnol. 8 (1988) 3

Der Einfluß konstanter Lufttemperatur # L = 40...90°C auf Trocknungsverlauf, nichtenzymatische Bräunung und Ascorbinsäureabbau ist in Abb. 2 grafisch dargestellt. Diesem Bild kann entnommen werden, daß mit steigender Temperatur der Trocknungsluft die Trocknungszeit sinkt, der Ascorbinsäureabbau und die nichtenzymatische Bräunung aber steigen. Der Einfluß verschiedener Trocknungsprofile, simuliert durch eine stündliche Änderung der Temperatur der Trocknungsluft auf den Trocknungsverlauf, den Ascorbinsäureabbau und die nichtenzymatische Bräunung ist in der Abb. 3 grafisch dargestellt. Diesem Bild ist zu entnehmen, daß das Trocknungsprofil einen entscheidenden Einfluß auf die Qualitätserhaltung bei der Trocknung hat. Günstig wirken sich Temperaturprofile mit hoher Anfangs- und niedriger Endtemperatur aus. Dagegen bewirken hohe Endtemperaturen der Trocknungsluft unabhängig von der Anfangstemperatur hohe Qualitätseinbußen. Charakteristisch ist der Trocknungsschwanz. Er kann minimiert werden durch eine starke Temperaturabsenkung am Ende der Trocknung oder eine Trocknungsteilung mit Nachtrocknung und Kühlung bei tiefen Temperaturen. Symbole a Bi c C Cp D D*

d, b

Ea

e Fi H k Le M m n ß T t # X Y x, y, z Z A Ci ß e

Temperaturleitfähigkeitskoeffizient Biot-Zahl Konzentration normierte Konzentration spezifische Wärmekapazität Diffusionskoeffizient Diffusionskoeffizient für die zweite fallende Periode Koeffizienten der Gl. 3, 4 und 5 Aktivierungsenergie Koeffizient für den Ascorbinsäureabbau Fick-Zahl Verdampfungsenthalpie Reaktionsgeschwindigkeitskcnstante Lewis-Zahl Masse Wassergehalt Laufzahl der Gl. 7 Universelle Gaskonstante Absolute Temperatur Zeit Temperatur Halbe Plattenlänge Halbe Plattenbreite Ortskoordinaten Halbe Plattenhöhe Fläche Wärmeübergangskoeffizient Stoffübergangskoeffizient Dichte

Indices A B E

Anfang Nichtenzymatische Bräunung Ende

m 2 /s —

kg/m 3 —

J/kg • K m 2 /s m 2 /s J/mol — —

J/kg s- 1 —

kg kg/kg —

J/mol • K K s °C m m m m m2 J/m 2 • s • K m/s kg/m 3

MOHR,

K.-H.,

L m n 0

VONDBAN,

J., Trocknung biologischer Produkte

267

Luft mittlere Größe Laufzahl Bezugszustand Feststoff Zeit Wasser

S

t

w

Eingegangen: 8. 5. 1987

Literatur [1] KABEL, M . : Z.

Lebensmitteltechnol. Verfahrenstechnik

3 5 (1984), 6.

Food Technol. 2 ( 1 9 5 5 ) , 4 3 3 . M I S H K I N , K . , K A B E L , M . , S A G U Y , J . : Food Technol. 7 (1982), 101. M O H E , K.-H.: Acta Biotechnol. 6 (1986), 189. S Y K E S , S. M., K E L L Y , F. H. C.: J . Sei. Food Agric. 20 (1969), 654. A G U I L E R A , J. M., C H I B I F E , J., F I N K , J . M., K A B E L , M.: Lebensmittel Wiss. Technol. 8 (1975),

[2] H E N D E L , C. E . , SILVEIEA, V . G . , HARRINGTON, W . O . :

[3] [4] [5] [6]

128.

[7] MOHE, K.-H.: Lebensmittelindustrie 32 (1985) 10, 110; 32 (1986) 253; 34 (1987), in Druck. [8] I G L E S I A S , H. A., C H I B I F E , J . : Handbook of Food Isotherms. New York: Academic Press, 1982.

Book Review H . - J . REHM, G. R E E D

W . SCHÖNBOBN

Vol. 8. Microbial Degradation Weinheim: VCH Verlagsgesellschaft, 1986. 725 pp. 217 fig., 104 tab., 495 DM Mit dem Band 8 der Biotechnologie-Monographie, herausgegeben von S C H Ö N B O R N , liegt ein Buch vor, das den lohnenswerten und nach Ansicht des Rezensenten auch gelungenen Versuch unternommen hat, Prozesse der mikrobiologischen Degradation, beginnend von Abwasserbiologien für die Gewinnung von Trinkwasser bis zum Abbau von Xenobiotika nicht nur phänomenologisch darzustellen, sondern in einem erforderlichen Maße auch den naturwissenschaftlichen Hintergrund der Prozesse aufzuzeigen. Besonders erwähnenswert ist die zusammenfassende Darstellung aktueller Entwicklungen auf dem Gebiet des Abbaues von halogenierten aromatischen Verbindungen, eine Entwicklung, die international im wesentlichen noch Forschungsgegenstand ist und gerade industrielles Neuland wird. Unter gleichen Aspekten sei die zusammenfassende Darstellung der mikrobiellen Korrosion von Werkstoffen genannt, deren Bekämpfung in den hochentwickelten Industriestaaten gleichrangig neben die klassischen Prozesse der Korrosionsinhibierung bei Eisen und Stahl tritt. Der vorliegende Band kann den Anspruch erheben, ein Kompendium mikrobiologischer Degradationsprozesse zu sein und erlaubt dem Nutzer in Forschung und Praxis durch die am Ende der jeweiligen Kapitel angefügten Literaturzitate eine eingehendere Beschäftigung mit den dargelegten Problemen. Das Buch ist darum über den Biotechnologen hinaus vielen industriellen Praktikern zur Nutzung zu empfehlen. D . MEYER

Acta Biotechnol. 8 (1988) 3, 268

Book Review A n t h o n y P . F . TURNER, I s a o KARUBE, G e o r g e S. WILSON

Biosensors — Fundamentals and Applications Oxford, New York, T o k y o : Oxford University Press, 1987. 720 pp., 60 £

Das Buch stellt einen ersten Versuch dar, das sich in den beiden letzten J a h r z e h n t e n stürmisch entwickelnde Gebiet der Biosensoren umfassend zu beschreiben. B e k a n n t e Autoren u n d Gruppen aus Großbritannien, USA, Schweiz, Schweden, J a p a n , F r a n k r e i c h u n d der D D R lieferten Beit r ä g e f ü r die insgesamt 37 Kapitel. Der I n h a l t gliedert sich in die größeren Gruppen Biokomponente, Bioelektrochemie, m i t den U n t e r g r u p p e n potentiometrische Sensoren, amperometrische Sensoren u n d Analyse der elektrischen I m p e d a n z , mechanische u n d akustische I m p e d a n z , Kalorimetrie, Fotometrie, A n w e n d u n g von Mikroprozessoren sowie einen Ausblick auf kommerzielle Anwendungen u n d zukünftige Entwicklungsmöglichkeiten. E s ist klar, d a ß durch eine solche Aneinanderreihung individueller Beiträge Überschneidungen nicht i m m e r zu vermeiden sind u n d die Geschlossenheit der Darstellung m i t u n t e r leidet. E s wird d a v o n ausgegangen, d a ß bei den in vollem F l u ß befindlichen Gebieten in den meisten Fällen die Schlußfolgerungen noch offen bleiben müssen u n d somit dem Leser überlassen bleiben. T r o t z d e m ist das Buch f ü r alle auf diesem Gebiet arbeitenden Wissenschaftler sowie an der Applikation interessierten Techniker von u n s c h ä t z b a r e m W e r t , da es f ü r den Einzelnen k a u m mehr möglich ist sich, in der Fülle der Veröffentlichungen zurechtzufinden, u n d er n u n m i t dem Buch eine Quelle erhalten h a t , sich an Übersichtsartikeln, denen ausführlichere L i t e r a t u r a n g a b e n angegliedert sind, über den jetzigen S t a n d zu informieren. E s ist an dieser Stelle natürlich weder möglich, den I n h a l t auch n u r einigermaßen vollständig zu beschreiben, noch alle Konsequenzen der möglichen A n w e n d u n g von Biosensoren zu berücksichtigen. Deshalb sollen n u r einige Hinweise auf besonders f ü r die Biotechnologie bedeutende Darstellungen gegeben werden. Dabei darf m a n nicht übersehen, d a ß die überwiegende Zahl der Entwicklungen von medizinischen Belangen ausgegangen ist. D o r t wird o f t eine schnelle und spezifische Diagnose durch Anwendung analytischer Hilfsmittel erforderlich, wobei Biosensoren den meist aufwendigen u n d teueren A n a l y s e n a u t o m a t e n vorgezogen werden k ö n n t e n . Elektroden m i t biologischen Sensoren wie E n z y m e n , Mikroorganismen, Gewebezellen, Antikörpern u n d dergleichen eignen sich sehr g u t f ü r diese Zwecke, einschließlich der Miniaturisierung u n d I m plantierbarkeit. Dabei wird auch auf die Möglichkeit der gezielten Modifizierung der biologischen K o m p o n e n t e durch Gentechnik u n d spezifische Proteinsynthese eingegangen. Besonders amperometrische Sensoren lassen sich auch zur L e b e n s m i t t e l ü b e r p r ü f u n g auf Frischheit einsetzen, weiterhin zur Kontrolle von Fermentationsprozessen. F ü r in-vivo-Experimente wurden besonders kleine Dünnfilm- oder Nadelelektroden entwickelt. Auch andere Sensortypen werden behandelt, wie Biomassebestimmung durch A n w e n d u n g der Dispersion der elektrischen Leitfähigkeit, akustische Spektroskopie, Kalorimetrie u n d F o t o m e t r i e mittels Faseroptik. Weitere Kapitel beschäftigen sich m i t der Anwendung von Silizium-Chips als chemisch sensitive Elemente u n d mit der K o m b i n a t i o n der Sensoren m i t Mikroprozessoren u n d Mikrorechentechnik. F ü r alle an den g e n a n n t e n Methoden Interessierten wird das B u c h einen seit langem gehegten W u n s c h erfüllen. E s ist in seiner Anlage geeignet, in späteren Auflagen die zusammenfassende I n f o r m a t i o n über den Wissensstand auf dem Gebiet der Biosensoren stets auf dem laufenden zu halten. ,

G. GEPPERT

Acta Biotechnol. 8 (1988) 3, 2 6 9 - 2 7 4

Rapid Determination of L-Lysine with an Enzyme Electrode by Steady State and Kinetic Measurement W E I S S B A C H , F . 1 , K R E I B I C H , G . 1 , BARTELS, K . 2 , SCHÜLKE, W . 2

1

2

VEB Pharmazeutisches Kombinat GERMED Dresden VE Forschungszentrum Biotechnologie Berlin Alt-Stralau 62, Berlin 1017, DDR Akademie der Wissenschaften der DDR Zentrum für wissenschaftlichen Gerätebau Rudower Chaussee 6, Berlin 1199, DDR

Summary Fast and simple methods of determination of L-lysine by a potentiometric enzyme electrode based on a C0 2 electrode and L-lysine decarboxylase are described. Measuring devices for manual and automated operation for steady state response measurement as well as kinetic measurement are compared. Sample frequency may be increased by decreasing the time of a measuring cycle.

Introduction The specific determination of the amino acid L-lysine is of major importance for food science, plant breeding, nutrition and biotechnology. A high specific and accurate determination method is the chromatographic one with amino acid analyzers. Disadvantages of this method are expensive equipment and maintenance costs which do not allow routine use. Over recent years enzyme electrodes for L-lysine have been developed. The basis of such electrodes are gas-sensing electrodes combined with L-lysine decarboxylase [1—3] or L-lysine «-amino oxidase [4]. Their advantages are high specificity, low costs for apparatus and maintenance. A disadvantage of this technique in routine use are the relatively long response times from 5 to 10 minutes and times for a measuring cycle from 10 to 20 minutes in the case of potentiometric probes. I n a previous paper [5] we described an enzyme probe for determination of L-lysine based on L-lysine decarboxylase (E.C. 4.1.1.18) from Klebsiella pneumoniae [6] and a SERViNGHAUs-type gas-sensing potentiometric electrode for C0 2 . In this contribution we present the results of our work directed towards the developement of fast and specific L-lysine determination methods. Experimental Section Enzyme Electrode and Working Conditions The enzyme electrode was prepared by a combination of the Model EMC0 2 N (Forschungsinstitut „ K U R T S C H W A B E " Meinsberg) C0 2 gas-sensing electrode with an enzyme membrane as described previously [5]. The enzyme (1 mg, 4 U) was fixed by an adhesive

Acta Biotechnol. 8 (1988) 3, 2 6 9 - 2 7 4

Rapid Determination of L-Lysine with an Enzyme Electrode by Steady State and Kinetic Measurement W E I S S B A C H , F . 1 , K R E I B I C H , G . 1 , BARTELS, K . 2 , SCHÜLKE, W . 2

1

2

VEB Pharmazeutisches Kombinat GERMED Dresden VE Forschungszentrum Biotechnologie Berlin Alt-Stralau 62, Berlin 1017, DDR Akademie der Wissenschaften der DDR Zentrum für wissenschaftlichen Gerätebau Rudower Chaussee 6, Berlin 1199, DDR

Summary Fast and simple methods of determination of L-lysine by a potentiometric enzyme electrode based on a C0 2 electrode and L-lysine decarboxylase are described. Measuring devices for manual and automated operation for steady state response measurement as well as kinetic measurement are compared. Sample frequency may be increased by decreasing the time of a measuring cycle.

Introduction The specific determination of the amino acid L-lysine is of major importance for food science, plant breeding, nutrition and biotechnology. A high specific and accurate determination method is the chromatographic one with amino acid analyzers. Disadvantages of this method are expensive equipment and maintenance costs which do not allow routine use. Over recent years enzyme electrodes for L-lysine have been developed. The basis of such electrodes are gas-sensing electrodes combined with L-lysine decarboxylase [1—3] or L-lysine «-amino oxidase [4]. Their advantages are high specificity, low costs for apparatus and maintenance. A disadvantage of this technique in routine use are the relatively long response times from 5 to 10 minutes and times for a measuring cycle from 10 to 20 minutes in the case of potentiometric probes. I n a previous paper [5] we described an enzyme probe for determination of L-lysine based on L-lysine decarboxylase (E.C. 4.1.1.18) from Klebsiella pneumoniae [6] and a SERViNGHAUs-type gas-sensing potentiometric electrode for C0 2 . In this contribution we present the results of our work directed towards the developement of fast and specific L-lysine determination methods. Experimental Section Enzyme Electrode and Working Conditions The enzyme electrode was prepared by a combination of the Model EMC0 2 N (Forschungsinstitut „ K U R T S C H W A B E " Meinsberg) C0 2 gas-sensing electrode with an enzyme membrane as described previously [5]. The enzyme (1 mg, 4 U) was fixed by an adhesive

270

Acta Biotechnol. 8 (1988) 3

between a dialysis membrane and the gas-selective plastic membrane (Teflon 0.012 mm and Silicon film Type D 606 Radiometer Copenhaven, resp.). The internal electrolyte was 5 • 10" 3 M NaHC0 3 in 0.1 M KCl. A pH/mV meter was used for response. Calibration and sample measurement were made in C0 2 -free phosphate buffer (pH6.0; 0.5 M) with 10" 5 M pyridoxal-5-phosphate as coenzyme. The temperature was 37 °C. Measuring

Devices

All measurements were made with a Praecitronic Model MV 88 pH/mV meter in conjunction with an Endim 621.02 Potentiometrie recorder. An automated slope analyzer for measurement of the first derivative of the response curve was developed according to [7], Kinetic measurements were made by the automated slope analyzer combined with the pH meter. Automated handling of the samples was made with an MLW Model APS 4 sampler. Three methods were applied: 1) A simple manual method for response measurement in steady state and recording the full measuring cycle with the steps: filling of the measuring cell, insertion of electrode, measurement, discharging, rinsing, filling etc.; 2) An automated method for response measurement in steady state followed by a short time for rinsing with flow cell, peristaltic pump, sampler and strip-chart recorder; 3) An automated method for kinetic measurement? with an additional slope analyzer for determination of maximum slope of response of the enzyme probe.

Results and Discussion

The use of an enzyme electrode based on a gas-sensing electrode in routine measurement is determined by several properties such as sensitivity, measuring range, response time to reach steady state and time for return to base line, the sum of which is the time for one measuring cycle, long time stability as well as repeatability of the determination of sample concentration. The properties depend strongly upon the kind of the base sensor, upon the enzyme activity and stability during electrode preparation and at working conditions, upon the material and dimensions of the gas-permeable membrane and upon all the facts which influence the mass transfer between the bulk solution and the electrochemical active surface of the electrode. In literature practical results and models to describe the response behaviour of protentiometric gas-sensing electrodes [8. 9] and enzyme electrodes based on it [10] are discussed widely. Enzyme electrodes working well in routine measurement are always a compromise in relation to the above-named properties and working conditions. Measuring

Cycle

For a given enzyme electrode based on a potentiometric gas sensing electrode and the working conditions such as temperature, pH, buffer capacity and stirring, the time for a full measuring cycle only depends on sample concentration. The L-lysine electrode showes such behaviour (Fig. 1 a). This is in agreement with the behaviour of a C0 2 electrode of the SERVINGHAUS type [9]. The time for a measuring cycle is 10 to 15 minutes and the response time is 3 to 5 minutes. In the response curves the response time is defined as time t (At, AE) at which the potential change with time is 0.1 mV per 1 minute according to [11]. The time for an

WEISSBACH,

F.,

KEEIBICH,

G. et al. Determination of L-Lysine

271

Fig. 1. Response curves of the L-lysine electrode a) Quasi-steady state response measurement, full measuring cycle b) Automated method with steady state response measurement and with short rinsing time c) Automated method with kinetic measurement of response by a slope analyzer and with full rinsing time equilibrium can not be determined. In the enzyme layer of the L-lysine electrode L-lysine is decarboxylized according to the equation L-Lysine

de^rboxyiLe

C02

+ Cadaverine

The mass transfer and the enzyme reaction leads only to a quasi-steady state. The electrode response is not constant over a long measurement period especially in the case of measurement in a small-volume sample. However measurement in flow systems leads to a constant electrode response over a long time period since the sample is continuously replaced at the electrode. Deviations from this behaviour are often caused by instrumental drift. For the developement of a simple and fast method for determination of L-lysine the measuring time can be decreased by two kinds of measuring principles. Firstly, quasisteady state measurement with a short rinsing time and, secondly, kinetic measurement with monitoring the slope of response. Both lead to a decrease in time of a measuring

272

Acta Biotechnol. 8 (1988) 3

cycle by one half. The response curves (Fig. l b and lc) show that this results in the saving of time for returning to the base line and in saving response time, respectively, compared with the complete measuring cycle for the two measuring principles. In the case of short rinsing time the washing out of the measured sample is done by a small volume of buffer solution followed by the next sample until the response of this sample is reached. The kinetic measurement starts at a fixed level of electrode response which must not be identical with the base line under the measuring conditions [7]. The slope of-response is monitored by an automated slope analyzer which differentiates the response curve. The maximum of slope of the electrode response is proportional to the concentration of L-lysine in the sample according to the N E R N S T equation. This maximum value is reached 15 to 30 seconds after the start of the reaction. After reaching the slope maximum the sample is replaced by buffer solution, and electrode rinsing follows until the base line is reached. The results and characteristics of the two measuring principles are summarized in Tab. 1. I t is shown that sample frequences may be increased from 5 to 10 and more samples per hour by application of the above described measuring principles and devices in comparison to the manual method with monitoring the complete measuring cycle. Sample handling and timing by a sampler is a pre-requisite for the repeatability of the measurements. Tab. 1. Properties of the L-lysine electrode and measuring devices Properties

Simple method

Practical measuring range M Slope of response per decade mV min Measuring cycle Response time to ziE = 0.1 mV/min min s Time for max. slope of response Time for return to base line min Time for rinsing s Stability weeks Repeatability of lysine determination /o Samples per hour Flow speed ml/min Measuring

5 • 10-4-10"2 53-59 10-15 3-5

Automated methods Steady state

Kinetic

5 • 10~4 —10~2 53-59 fixed to 6 3-5

10~4 —2 • 1 0 50-55 2-6

—

7

11)

—

3: 3 ±(2-4) 4-6 stirring

—

fixed to 15 3-6 ±(2-4) 10 0.5

—

15-30 1.5-5.5 —

3-6 ±5 10-20 1

Range

Determination of L-lysine by the enzyme probe gave the best results in accuracy and repeatability when the measurements were done in a range of concentration which is represented by the first half of a concentration decade in the linear range of the calibration curve. The slope of the electrode response per decade of L-lysine concentration was 53 to 59 mV for steady state measurements (Tab. 1). The limit of L-lysine detection was found from 5 • 10"5 to 1 0 4 M for steady state measurement. In the case of kinetic measurement the detection limit of L-lysine depends on the time period At for the differentiation and was found from 5 • 10~5 to 10"4 M for At = 0.5 seconds and 10" 3 M for At = 0.15 seconds. The upper limit of the linear measuring range depends on the concentration of the internal NaHC0 3 solution in the electrolyte film of the C0 2 electrode. The L-lysine electrode with 5 • 10" 3 M NaHC0 3 as internal solution showed the limits 1 0 2 and 2 • 1 0 2 M for steady state and kinetic measurement, respectively.

WEISSBACH, F . , K E B I B I C H , G . e t a l . , D e t e r m i n a t i o n o f L - L y s i n e

273

Stability The stability of the enzyme electrode during a long time period depends above all on the stability of the enzyme membrane which is sensitive to mechanical and chemical stresses. Therefore all handling of the enzyme probe must be done carefully. The influence of pH, temperature, ionic strength, coenzyme and inhibitors must be noticed carefully during the development of the working conditions and during the preparation of samples for the L-lysine determination. During the development of the described enzyme electrode and measuring devices for determination of L-lysine in microbial production of L-lysine the manual method had some disadvantages in routine measurement. The mechanical stability was lower than in automated systems with flow cell and sampler. Especially, rupture of the dialysis membrane was the main factor of instability. A long time stability of more than three weeks is usual for the flow systems (Tab. 1). For the conservation of the activity of the enzyme L-lysine decarboxylase the coenzyme pyridoxal-5-phosphate must be present at a concentration of 10~5 M in the buffer solution for rinsing and in the samples. When not in use the enzyme electrode was stored in the rinsing buffer with the coenzyme at room temperature.

Conclusions The described methods for determination of L-lysine by the enzyme electrode combined with the measuring devices for a short measuring cycle monitoring have some advantages in comparison with the manual method with the complete measuring cycle. They make possible the determination of L-lysine with a sample frequency of 10 per hour in the case of steady state measurement and of more than 10 per hour by the kinetic one. The ranges of concentration for L-lysine determination are similar for the described methods. For low concentrations the steady state method is preferred. High concentrations of L-lysine are better determined by the kinetic method with automated slope analyzer. The short contact time from 15 to 30 seconds of electrode and sample reduces the time for a measuring cycle, the stress for the enzyme by the sample is reduced, and the capacity of the NaHC0 3 solution in the electrolyte film of the C 0 2 electrode is not exhausted as in the case of steady state measurement at high concentrations of L-lysine. The slopes of the calibration curves and the sensitivities of the methods are in the range of the N E R N S T factor and thus optimal conditions for the potentiometric measuring principle are granted. The repeatabilities of the determination of L-lysine by the enzyme electrode combined with the described measuring devices are in the normal range of potentiometric methods with ± ( 0 - 5 to 1.0) mV for voltage measurement corresponding to ± ( 2 to 4 ) % in the concentration determinations. The accuracy of the methods was established by comparison with amino acid analyzer determinations for L-lysine in samples from L-lysine fermentations. A pre-requisite for the repeatability and accuracy of L-lysine determination by the described methods is the stability of all the outside conditions, such as temperature, pH, ionic strength, speed of flow or stirring, concentration of coenzyme and the specificity of the enzyme, as well as the stability of the pH electrode. Concentration of interfering C0 2 in the sample must be very low compared with the concentration of L-lysine. 5

Acta Biotechnol. 8 (1988) 3

Acta Biotechnol. 8 (1988) 3

274

The long time stability of the L-lysine electrode depends on the mechanical stability of the enzyme membrane at working conditions. Stability in flow systems is higher than in the manual operation method. The values of the measuring cycle time, response time to steady state and time for return are characteristic for an enzyme electrode and are important for routine application. Usually, most time consuming and labour intensive is not the measurement of response but the whole sample preparation beginning with hydrolysis of protein in the case of grain or with separation of biomass in the case of samples from fermentation. Enzyme electrodes and measuring devices of the described types have been used by us successfully for process control of L-lysine fermentations for several years. Received June 29, 1987

References [1] WHITE, G. C., GOTLBAULT, G. G.: Anal. Chem. 50 (1978), 1481. [ 2 ] SKOGBERG, D . , RICHARDSON, F . : Cereal C h e m . 5 6 (1979), 147.

[3] TRAN, N. D., ROMETTE, J. L., THOMAS, D.: Biotechnol. Bioeng. 25 (1983), 329. [ 4 ] ROMETTE, J . L . , YANG, J . S., KUSAKABE, H . , THOMAS, D . : B i o t e c h n o l . B i o e n g . 2 5 ( 1 9 8 3 ) , 377. [5] WEISSBACH, P . , SCHINDLER, W . , HUBER, J . , KREIBICH, G . : A c t a B i o t e c h n o l . 7 ( 1 9 8 7 ) ,

in press. [6] HUBER, J., WEISSBACH, F.: Acta Biotechnol. 6 (1986), 273. [7] SKOGBERG, D . , RICHARDSON, F . , BLASCZYK, T . : A n a l . C h e m . 5 1 ( 1 9 7 9 ) , 2 0 5 4 .

[8] Ross, J. W., RISEMAN, J. H., KRFEGER, J. A.: Pure Appl. Chem. 36 (1973), 473. [9] JENSEN, M. A . , RECHNITZ, G. A . : A n a l . C h e m . 5 1 (1979), 1 9 7 2 .

[10] CARR, P. W., BOWERS, L. D.: Immobilized enzymes in analytical and clinical chemistry. V o l . 5 6 of C h e m i c a l A n a l y s i s . E d s . ELVING, P . J . , WINEFORDNER, J . D . , KOLTHOFT, I . M . ;

New York: J. Wiley & Sons, N. Y„ 1980, Capt. 5. [11] UEMASU, I., UWEZAWA, Y.: Anal. Chem. 54 (1982), 1200.

Acta Biotechnol. 8 (1988) 3, 2 7 5 - 2 8 3

Hopf Bifurcation for a Family of Two-State Microbial Growth Models BLEY, Th.1, WEGNER, B . 2

1

2

Academy of Science of the G.D.R. Institute of Biotechnology PermoserstraBe 15, Leipzig 7010, G.D.R. Academy of Sciences of the G.D.R. Karl-WeierstraB-Institute of Mathematics Mohrenstrafie 39, PF 1304, Berlin 1086, G.D.R.

Summary We describe a family of two-state microbial growth models, in which growth and maintenance are assigned to two different cell states. The way of splitting periodic solutions for low dilution rates of a continuous fermentation is shown. The existence of these periodic solutions is mainly influenced by the properties of substrate consumption, which the maintenance rate in the second cell state amounts to.

Introduction I n dependence on the process control conditions we find oscillatory behaviour of microbial growth in continuous fermentation processes. Frequently described, and by the use of models theoretically founded, the inhibition of the growth rate by metabolic products is one reason for this [1, 2], However, in using this inhibition kinetics, oscillations of another type cannot be explained. These oscillations are characterized by periodic changes in the state distribution of the microbial population [3]. By states we will understand different successive phases in the proliferation cycle of microorganisms. In addition to morphological and biochemical features these states are characterized by different kinetics of carbon substrate consumption [4], If these states are used for modelling the growth of a population, it should not be understood as the result of biochemical reactions, but as a motion in that space which is spanned by different cell states (cf. [5, 6]). The practical suitability of two-state models can be shown for the description of the growth of yeasts, which are especially important in biotechnological processes [7]. In this case one state corresponds to the single cell phase and the other to the budding cell phase of the yeast cell cycle. The different features of these states will be explained by the following model. By means of computer simulations autonomous oscillations of the state distribution depending on the model parameters were found for a special two-state model [8, 5]. We derived the hypothesis that, if we consider different substrate consumption kinetics in the two states and a transition rate from the single to the double cell state depending on the milieu conditions, which are both supported by biological investigations, periodic 5*

Acta Biotechnol. 8 (1988) 3, 2 7 5 - 2 8 3

Hopf Bifurcation for a Family of Two-State Microbial Growth Models BLEY, Th.1, WEGNER, B . 2

1

2

Academy of Science of the G.D.R. Institute of Biotechnology PermoserstraBe 15, Leipzig 7010, G.D.R. Academy of Sciences of the G.D.R. Karl-WeierstraB-Institute of Mathematics Mohrenstrafie 39, PF 1304, Berlin 1086, G.D.R.

Summary We describe a family of two-state microbial growth models, in which growth and maintenance are assigned to two different cell states. The way of splitting periodic solutions for low dilution rates of a continuous fermentation is shown. The existence of these periodic solutions is mainly influenced by the properties of substrate consumption, which the maintenance rate in the second cell state amounts to.

Introduction I n dependence on the process control conditions we find oscillatory behaviour of microbial growth in continuous fermentation processes. Frequently described, and by the use of models theoretically founded, the inhibition of the growth rate by metabolic products is one reason for this [1, 2], However, in using this inhibition kinetics, oscillations of another type cannot be explained. These oscillations are characterized by periodic changes in the state distribution of the microbial population [3]. By states we will understand different successive phases in the proliferation cycle of microorganisms. In addition to morphological and biochemical features these states are characterized by different kinetics of carbon substrate consumption [4], If these states are used for modelling the growth of a population, it should not be understood as the result of biochemical reactions, but as a motion in that space which is spanned by different cell states (cf. [5, 6]). The practical suitability of two-state models can be shown for the description of the growth of yeasts, which are especially important in biotechnological processes [7]. In this case one state corresponds to the single cell phase and the other to the budding cell phase of the yeast cell cycle. The different features of these states will be explained by the following model. By means of computer simulations autonomous oscillations of the state distribution depending on the model parameters were found for a special two-state model [8, 5]. We derived the hypothesis that, if we consider different substrate consumption kinetics in the two states and a transition rate from the single to the double cell state depending on the milieu conditions, which are both supported by biological investigations, periodic 5*

276

Acta Biotechnol. 8 (1988) 3

solutions appear for low dilution rates, independent of the nature of the growth kinetics in the single cell state. The intention of our paper is to prove this hypothesis for a family of models which fulfils the above-mentioned conditions. We consider the system of ordinary autonomous differential equations: [f*(S)

=

— s

=

~

—

X

(Z) -¡-

Jc^x^ —a/u(s)

xj —

fc^s) fix2

KSX2

\ +

j

(1)

j

— (s0

— s)

D.

ki (i = 1, 2), 0, consequently, D(s) > 0 for these s and dD _ k2(/i — D) + fi'(D ds

+

M

(14)

2D + fcj + k2s — fi

The equation g(D(s), s) = 0 means D(a) + k2s + k, =

D(S)

^

D(s) + k2s

=

D(S)

, +

D(s) J)W

k,

+ k,s-

(15)

From this we conclude D(s) > 0 for all ,s gS s for which D(s) exists, since /i(s) > 0 for s € (0, a]. Further, for s > 0 from (15) we obtain D(s) + k,>

(16)

fi(s) > D(s)

and (D, s) =

-(D

+ k

1

- p ) - ( D + M

< 0,

(17)

i.e., D(s) exists for all s € [0, §]. Because of (16) we find from (14) that ^ ds

> 0 for s € (0, «]. Then the map s -> D(s),

s € [0,a], is monotonously increasing and continuously differentiable. Therefore, on the interval [0, s] the variable s can be assumed as a function of the parameter D: D s(D), where D € [0, D : = D(S)], = « for s € [0, 5], the map a is continuous on [0, D ] and differentiable in (0, Z)]. As we pursue the idea that small periodic solutions originate from low dilution rates D, we restrict our investigation to the interval [0, D']. Therefore, with (7) and (8) we

Two-State Microbial Growth Models

BLEY, TH., WEGNER, B.,

279

find the nontrivial steady states xx{D) =

2>|>o - «(X>)] [D + k2s(D)] «fi(a(D)) [D + k2s(D)] + fa '

(D) =

x

"

(18)

Dh(s0-S(D)) x/t(a{D)) [D + k2s(D)] + fa ' 0, D],

s(D),De[

with ^(0) = £¡¡(0) = s(0) = 0 .

Nonvanishing of the Eigenvalues of fx(x(D),

D) for D 6 (0, D)

We use the Lemma 1. Let (x, D) be a steady state of (1) with x2 =)= 0. D > 0, 5 > 0. Then 0 6 a (fx(x, D)j if and only if

(s, D) = 0.

Proof. For x2 =)= 0, D > 0 we have f(x, Z>) = 0 if and only if f(x, D) := ((/, + U) (x, D), f2(x, D), /,(*, D))T = 0. Because of (8) and (10), (/a + f2) (x, D) =

gr(s, D). This implies

det fx(x, D) = 0 if and only if det /x(a;, D) = det I \ —Oifl = % (M + Ms)

(D +

From k^ + 0_the assumption follows, q.e.d. Our statement holds for D € (0, D] because of (11) and dg/ds > 0.

Conditions for the Existence of a Pair of Purely Imaginary Eigenvalues of fx(x(D), D) with Transversal Transition

The basis for this is Lemma 2. A polynomial of degree 3, P(Q) = Qs

+

+

«HE +

«O.

with real coefficients a-t, i = 0, 1, 2, has a pair of purely imaginary zeros ±ico, o> if and only if a, > 0,

and

axa2 — aa = 0 .

(19)

0, (20)

280

Acta Biotechnol. 7 (1988) 3

Let the coefficients A, be dependent on a parameter: A, = A,\{D), *1 ) +—AÏ227(D*)) DD

has the zeros

=)= 0, and

ZSZIOJ, W2

-F(e) = (E — EO) (E +

3

(21)

if and only if

2

i.e. A2 = —QO, «i = w2 > 0, a 0 = a 2 ap From we obtain the equation

P(V(D)

+ vw v +

+

{D * ] •

) = E — EO° + w2G — G0CO2,

2

,3 + aa(jD)

G0

- ^

+ IW(D)}

= 0, for the real part

A M « A D ) - A M

=0

{22)

Differentiation of (22) with respect to D at D = D* gives the relation (21) for the imaginary root V(DIF) = 0, a)(D*) = co* =|= 0, since A^D*) > 0, q.e.d. We look at the characteristic polynomial of the matrix JJX{D), Z>), D £ [0, D}. and from linear algebra we take the formula det (GID —

M

)=

— tr

G3

— det

M G2 + M G

(23)

M ,

where M is a 3 X 3-matrix, tr M = : trace of M, M : = the sum of the adjoints of M of second order. The linear map FX(X, D) has the form FX(X, D) = : A — D id with

(

[¿(S) — KX

K2S

/N'(S) ^

+

K2X2\

FCJ

— K2S

—K2X2

J.

—# by higher dilution rates D, i.e., to a destabilization of the system. This shall be demonstrated by numerical calculations. This numerical estimation was carried out by using Eq. (1) with an inhibition growth s Jci kinetics fi(s) = /(max ^ ^ ([11]). The used parameters were: =

1, k, = 0.1, ki = 5, kt = 1/0.1/0.02, k2 = 1, « = 2, s0 = 5

The results are shown in Fig. 2. The period length of oscillating states is given by T

a-y and demonstrated in the lower part of Fig. 2.

Conclusions The result shows that a microbial population which grows in a continuous fermentation process, according to a kinetics that is characterized by a reversal transition between two kinetically different states, changes from a steady state to stable oscillations depending on the dilution rate if appropriate kinetic parameters are given. The result is of interest firstly for the interpretation of fermentation processes in which an oscillatory behaviour occurs, and secondly for the control of fermentation processes at relatively low dilution rates which are chosen, for instance, for the continuous synthesis of metabolic products. W e have shown that there needs not be an inhibition

282

Acta Biotechnol. 8 (1988) 3

Fig. 2. Plot of bifurcation behaviour and period length for different parameters in dependence of

kinetics by metabolic products for generating oscillations in continuous fermentation processes. I t should be remembered that the equation investigated is a model for other processes as well. I t is, for instance, a special case of modelling mutation and reversal mutation of microbial strains having kinetically different properties. I t was stated (cf. [16]) that oscillations cannot be expected for this type of model. This contradicts our result and therefore the possibility of periodic fluctuations of different mutants in a continuous process should be considered in further investigations. Acknowledgement We should like to thank Dr. habil. Received July 2, 1987

K . R . SCHNEIDER

for stimulating discussions.

BLEY, TH., WEGNER, B., Two-State Microbial Growth Models

283

References [ 1 ] DEGN, H . , HARRISON, D . E . F . : J . T h e o r . B i o l . 2 2 ( 1 9 6 9 ) , 2 3 8 .

[2] KNORRE, W. A., GUTHKE, R. : Oszillationen in metabolischen und epigenetischen Systemen: Biophysikalische Modelle und Computersimulation. Abh. AdW der DDR 5N (1977), 393. [3] MEYENBURG, H. K. : Stable synchrony oscillations in continuous cultures of Saccharomyces eerevisiae under glucose limitations. — I n : Biol, and biochem. Oscillators. New York, London: Academic Press, 1973. [4] BLEY, Th. et al.: Z. Allg. Mikrobiol. 20 (1980), 275. [5] BLEY, Th. : Strukturierte Wachstumsmodelle — vergleichende Untersuchungen und Anwendung auf Probleme der technischen Mikrobiologie. Diss. A. Leipzig, 1980. [6] LEWIS, E. R.: Network models in population biology. Heidelberg: Springer-Verlag. 1977. [7] BLEY, Th., HEINRITZ, B. : Modelling and control of yeast growth in biotechnical systems. — I n : I n t . Workshop on Dyn. Syst. and Env. Mod., 16.—21. 3. 1986. Wartburg, Eisenach (to appear). [ 8 ] BLEY, T h . , SCHMIDT, A . : S t u d , b i o p h y s . 7 8 ( 1 9 8 0 ) , 1 1 - 1 2 + M i c r o f i c h e 1 / 1 - 1 1 . [9] BLEY, T h . , HEINRITZ, B . , SCHMIDT, A . : S t u d , b i o p h y s . 9 8 ( 1 9 8 4 ) , 1 1 9 .

[10] MONOD, J . : Ann. Rev. Microbiol. 3 (1949), 371. [ 1 1 ] ANDREWS, J . F . : B i o t e c h n o l . B i o e n g . 1 0 ( 1 9 6 8 ) , 7 0 7 . [ 1 2 ] TAKAMATSU, T . , SHIOYA, S . , OKUDA, K . : F e r m e n t . T e c h n o l . 5 9 ( 1 9 8 1 ) , 1 3 1 .

[13] FREDRICKSON, A. G., TSUCHIYA, H. M.: Microbial kinetics and dynamics. — I n : Chem. Reactor Theory. Englewood Cliffs, 1977. pp. 405. [14] CRANDALL, M. G., RABINOWITZ, P. H.: The Hopf bifurcation theorem. Madison: Univ. of Wisconsin, Math. Res. Center, 1976. (Technical summary report; 1604). [15] MARSDEN, J . E., MCCRACKEN, M.: The Hopf bifurcation and its application. New York: Springer-Verlag, 1976. [16] NOACK, D.: Biophysikalische Prinzipien der Populationsdynamik in der Mikrobiologie. Leipzig: VEB Georg Thieme, 1968.

Book Review I . F . K E N N E D Y , G . 0 . P H I L L I P S , P . A . WILLIAMS

Wood and Cellulosics Chichester: Ellis Horwood Limited Publishers, 1987. 664 pp., 69.50 £ Der vorliegende Band gibt wesentliche Ergebnisse der Cellucon-Tagung des Jahres 1986 speziell zu den Gebieten der Cellulose-Chemie und ausgewählter Applikationsgebiete von Cellulose speziell unter den Belangen der Zellstoffindustrie wieder. Neben ausgesprochen chemischen Problemstellungen bzw. solchen zu Zellstoffgewinnungstechnologien war ein großes Gebiet der Biotechnologie auf diesem speziellen Feld gewidmet. Sowohl der Abbau von cellulosehaltigen Abprodukten via Glukose zu Ethanol und Biomassen als auch ausgewählten Fragen des Ligninabbaues mit genetisch veränderten Mikroorganismen waren Gegenstand der Vorträge und der Diskussion in dieser Sektion. Sie wurden ergänzt durch analytische Problemstellungen und die Nutzung von Cellulosederivaten als Träger f ü r Immobilisierungssysteme. Das vorliegende Buch hat durch die Anfügung weiterführender Literatur an die jeweiligen Kapitel einen partiell monographischen Charakter und ist darum für den auf dem Arbeitsgebiet der Cellulosechemie und Lignocellulosebiotechnologie Tätigen ein empfehlenswertes Nachschlagewerk über ausgewählte aktuelle Entwicklungstendenzen. D . MEYER

A c t a Biotechnol. 8 (1988) 3, 284

Book Review Boyd HARDESTY, Gisela

KRAMER

Structure, Function, and Genetics of Ribosomes Springer Series in Molecular Biology (Series Editor: Alexander

RICH)

Berlin, Heidelberg, New Y o r k , L o n d o n , Paris, T o k y o : Springer-Verlag, 1986. 810 pp., 294 fig., 420 DM, I S B N 0-387-96233-6

D a s neue „ R i b o s o m e n - B u c h " erschien im R a h m e n der „Springer-Series in Molecular Biology". Der Springer-Verlag v e r s u c h t m i t dieser Serie d e m interessierten Leser den neuesten Wissenss t a n d , aber a u c h B e k a n n t e s , auf dem Gebiet der Molekularbiologie zu v e r m i t t e l n . Somit t r ä g t diese Serie d e m r a p i d e n Anwachsen der E r k e n n t n i s s e auf d e m Gebiet der Molekularbiologie in den l e t z t e n J a h r z e h n t e n R e c h n u n g . Die F l u t der E r k e n n t n i s z u n a h m e , allein auf d e m Gebiet der R i b o s o m e n f o r s c h u n g , wird in 45 K a p i t e l n (810 Seiten) dieses W e r k e s übersichtlich dargestellt. D a s „ R i b o s o m e n - B u c h " ist d a s E r g e b n i s der , , R i b o s o m e n k o n f e r e n z " , die im April 1985 in P o r t A r a n s a s (Texas) s t a t t f a n d . Die R i b o s o m e n sind wegen ihrer zentralen Rolle in der P r o t e i n b i o s y n t h e s e intensiv erforscht worden. Allein die T a t s a c h e , d a ß ein so u m f a n g r e i c h e s B u c h n u r einer Organelle g e w i d m e t ist, zeigt das Interesse, das viele Wissenschaftler den Ribosomen entgegenbringen. An dieser Organelle f i n d e t die eigentliche U m s e t z u n g der genetischen I n f o r m a t i o n s t a t t , die in der D N A verschlüsselt ist. Hier erfolgt die T r a n s l a t i o n der m - R N A in die Aminosäuresequenz eines Proteins. Schon beim D u r c h a r b e i t e n der ersten K a p i t e l e r k e n n t der Leser, d a ß f ü r das V e r s t ä n d n i s der F u n k t i o n der R i b o s o m e n solide K e n n t n i s s e über deren S t r u k t u r n o t w e n d i g sind. Aus diesem G r u n d e w u r d e ein breites S p e k t r u m chemischer, physikalischer, immunologischer u n d genetischer M e t h o d e n a n g e w e n d e t , u m die S t r u k t u r der Protein- u n d R N A - K o m p o n e n t e n der Ribosomen a u f z u k l ä r e n . D u r c h A n w e n d u n g dieser M e t h o d e n k o n n t e z. B. die P r i m ä r s t r u k t u r aller Proteinu n d RNA-Moleküle der Escherichia coK-Ribosomen b e s t i m m t werden. Dies k a n n a u c h in naher Z u k u n f t f ü r Bacillus stearothermophilus e r w a r t e t werden. W ä h r e n d der erste Teil des Buches h a u p t s ä c h l i c h s t r u k t u r e l l e n F r a g e n gewidmet ist, b e s c h ä f t i g t sich der zweite Teil vorwiegend m i t f u n k t i o n e l l e n Fragestellungen. E s werden neue Ergebnisse bezüglich der I n t e r a k t i o n von m - R N A u n d t - R N A m i t d e n Ribosomen vorgestellt. Der letzte Teil b e s c h ä f t i g t sich m i t der G e n a n o r d n u n g , Genexpression u n d m i t der R e g u l a t i o n der S y n t h e s e der ribosomalen K o m ponenten. H e r v o r z u h e b e n ist, d a ß jeder B e i t r a g eine u m f a n g r e i c h e L i t e r a t u r s a m m l u n g e n t h ä l t , die ein wertvolles A r b e i t s m i t t e l f ü r den Wissenschaftler darstellt. Alle K a p i t e l sind d u r c h Schemen, Abbildungen u n d graphische Darstellungen i n f o r m a t i v g e s t a l t e t . Dieses B u c h ist allen zu e m p fehlen, die sich etwas intensiver m i t den Ribosomen b e s c h ä f t i g e n wollen bzw. denen, die detailliertes Wissen über die S t r u k t u r , F u n k t i o n oder die Genetik der R i b o s o m e n benötigen. R.SCHRÖDER

Acta Biotechnol. 8 (1988) 3, 2 8 5 - 2 8 9

Short Communications Oszillierende-fed-batch-Technik bei der Cellulasegewinnung KERNS, G., KUDE, J . , MEYER,

D.

Akademie der Wissenschaften der D D R I n s t i t u t f ü r Biotechnologie Permoserstraße 15, Leipzig 7050, D D R

Summary F r o m different fungal genera some m u t a n t strains were selected which in cellulase formation exhibit reduced catabolite-repression related to several soluble carbon sources. The cellulase production by these m u t a n t strains on t h e basis of soluble carbon sources is possible if a fedbatch-technique is used for feeding t h e substrate. The optimum procedure is an oscillating-fed-batch-technique (OFB) with feed-back-controlled intermittent addition of t h e substrate. The substrate concentration in the medium is a d j u s t e d in such a manner to realize oscillations between a limitation for mycelium growth and non-limiting conditions under a substrate concentration which is yet below the repressing concentration for cellulase formation. A suitable feed-back-parameter for the control of substrate addition is t h e specific C o n f o r m a t i o n of the mycelium. Using OFB the growth in cellulase activity is nearly proportional to t h e feeded substrate whereas the growth in mycelium is reduced. The productivity of cellulase formation is enhanced in comparison to continuous substrate addition as well as under limiting and non-limiting conditions for growth of mycelium.

Einführung Die Cellulasefermentation mit Cellulose als Kohlenstoffquelle weist eine Reihe prozeßtechnischer Nachteile auf, insbesondere da die drei Teilprozesse Myzelwachstum, Cellulasebildung/-ausscheidung und die enzymatische Cellulosespaltung miteinander verknüpft sind und deren Optima im Fermentationsprozeß nicht zusammenfallen. Der geschwindigkeitsbestimmende Schritt liegt beim enzymatischen Celluloseabbau. Die Möglichkeiten bei Wildstämmen, mittels Zuführung löslicher Substrate zu einer Erhöhung der Cellulasebildung zu gelangen, sind begrenzt, da bereits bei niedrigen Konzentrationen dieser Substrate trotz Aufrechterhaltung einer Substratlimitation die Cellulasebildung zumindest partiell reprimiert ist. Für eine Erhöhung der Cellulasebildung auf Basis löslicher Substrate ist neben geeigneten Mutanten auch eine geeignete, den jeweils eingesetzten Mutanten „angepaßte" Prozeßführung erforderlich. Ziel der vorliegenden Untersuchungen war es, Cellulase-Mutanten mit reduzierter Katabolitrepression herzustellen und eine geeignete Prozeßführung für die Cellulasegewinnung mit löslichen Substraten zu erarbeiten.

Acta Biotechnol. 8 (1988) 3, 2 8 5 - 2 8 9

Short Communications Oszillierende-fed-batch-Technik bei der Cellulasegewinnung KERNS, G., KUDE, J . , MEYER,

D.

Akademie der Wissenschaften der D D R I n s t i t u t f ü r Biotechnologie Permoserstraße 15, Leipzig 7050, D D R

Summary F r o m different fungal genera some m u t a n t strains were selected which in cellulase formation exhibit reduced catabolite-repression related to several soluble carbon sources. The cellulase production by these m u t a n t strains on t h e basis of soluble carbon sources is possible if a fedbatch-technique is used for feeding t h e substrate. The optimum procedure is an oscillating-fed-batch-technique (OFB) with feed-back-controlled intermittent addition of t h e substrate. The substrate concentration in the medium is a d j u s t e d in such a manner to realize oscillations between a limitation for mycelium growth and non-limiting conditions under a substrate concentration which is yet below the repressing concentration for cellulase formation. A suitable feed-back-parameter for the control of substrate addition is t h e specific C o n f o r m a t i o n of the mycelium. Using OFB the growth in cellulase activity is nearly proportional to t h e feeded substrate whereas the growth in mycelium is reduced. The productivity of cellulase formation is enhanced in comparison to continuous substrate addition as well as under limiting and non-limiting conditions for growth of mycelium.

Einführung Die Cellulasefermentation mit Cellulose als Kohlenstoffquelle weist eine Reihe prozeßtechnischer Nachteile auf, insbesondere da die drei Teilprozesse Myzelwachstum, Cellulasebildung/-ausscheidung und die enzymatische Cellulosespaltung miteinander verknüpft sind und deren Optima im Fermentationsprozeß nicht zusammenfallen. Der geschwindigkeitsbestimmende Schritt liegt beim enzymatischen Celluloseabbau. Die Möglichkeiten bei Wildstämmen, mittels Zuführung löslicher Substrate zu einer Erhöhung der Cellulasebildung zu gelangen, sind begrenzt, da bereits bei niedrigen Konzentrationen dieser Substrate trotz Aufrechterhaltung einer Substratlimitation die Cellulasebildung zumindest partiell reprimiert ist. Für eine Erhöhung der Cellulasebildung auf Basis löslicher Substrate ist neben geeigneten Mutanten auch eine geeignete, den jeweils eingesetzten Mutanten „angepaßte" Prozeßführung erforderlich. Ziel der vorliegenden Untersuchungen war es, Cellulase-Mutanten mit reduzierter Katabolitrepression herzustellen und eine geeignete Prozeßführung für die Cellulasegewinnung mit löslichen Substraten zu erarbeiten.

286

Acta Biotechnol. 8 (1988) 3

Ergebnisse Mittels Mutation und geeignetem Screening wurden — in Anlehnung an die Methode von L A B U D O V A [ 1 ] — Trichoderma- und Aspergillus-Mutanten isoliert, welche bezüglich verschiedener C-Quellen (Glucose, Fructose, Lactose, Galactose, Xylose, Glycerol) eine reduzierte Katabolitrepression aufweisen. Gegenüber den Wildstämmen setzt die Repression der Cellulasebildung erst bei höheren Konzentrationen dieser C-Quellen ein. Das katabolitdereprimierte Cellulasebildungsvermögen wurde ermittelt, indem der Repressor in Kombination mit mikrokristalliner Cellulose (MKC) in das Nährmedium gegeben und diejenige Konzentration bestimmt wurde, bei welcher die Cellulasebildung einsetzt. Die so ermittelten Repressorkonzentrationen waren auch vom Alter des Myzels abhängig. Die stärkste Reduzierung der Katabolitrepression wurde bei einer T. virideMutante gefunden (Abb. 1). 40

30 = z> 20 3

10

100 Zeit Chi

Abb. 1. Diskontinuierliche Cellulasefermentation mit T. viride 9k im 301-Fermentor C-Quelle: 2% Glucose + 1% Cellulose (PP- und CMC-Aktivität gemäß IUPAC-Empfehlung [10])

Mittels dieser partiell katabolitdereprimierten Mutanten ist die Möglichkeit gegeben, lösliche C-Quellen als Substrat für die Cellulasefermentation einzusetzen, vorausgesetzt, die aktuelle Substratkonzentration im Fermentationsmedium wird unterhalb der reprimierenden gehalten. Für eine Optimierung des fed-batch-Regimes ist von Bedeutung, inwieweit das Optimum der Cellulasebildung bei einer C-Limitation oder aber bei einer nichtlimitierenden (noch nicht reprimierenden) Konzentration der CQuelle im Medium liegt. Es wurden deshalb fed-batch-Versuche mit unterschiedlichen Regimes der Substratzugabe durchgeführt. Die oszillierende-fed-batch-Technik (OFB) erwies sich als geeignetste Prozeßführungsvariante für die Cellulasegewinnung auf Basis löslicher Substrate (Tab. 1). In der fedbatch Periode wird die diskontinuierliche Substratzugabe in bezug auf die Menge sowie die zeitlichen Abstände der jeweiligen Zugaben so bemessen, daß die Substratkonzentrationen zwischen Limitation und einer solchen nicht-limitierenden Konzentration oszilliert, welche noch nicht reprimierend auf die Enzymbildung wirkt. Die Steuerung der Substratzugabe erfolgt zweckmäßig in Form einer indirekten feed-backKontrolle über die spezifische C0 2 -Bildung des Myzels. Beispielsweise ist es möglich, die

K e r n s , G., K t j d e , J . u . a., C e l l u l a s e g e w i n n u n g

287

Tab. 1. Einfluß der fed-batch-Technik auf die Cellulasebildung bei T. reesei M 18 Substrat

Fed-batch-Technik

Aktivität [FPU/ml]

Produktivität [FPU/1 • h]

5% Lactose 3,4% Lactose

OFB substratlimitierte Zuführung (konstant. Dosierung) fed-back-kontrollierte Zuführung (nicht-substratlimitierte stetige Zuführung)

10,2 4,8

62 29

5,6

48

5% Lactose

Ansteuerung der Substratpumpe mittels der C0 2 -Konzentration des Fermentationsabgases in Verbindung mit der Konzentration des Myzelproteins vorzunehmen. Bei den T. reesei-Mutanten, welche auch bezüglich Glucose eine reduzierte Katabolit repression aufweisen, konnte mittels OFB die Cellulasegewinnung auf Basis von Glucose durchgeführt werden, falls das Grundnährmedium induzierende Substanzen (wie z. B. Weizenkleie) enthielt. Lösliche induzierende Substrate sind als einzige C-Quelle für die Cellulasegewinnung geeignet (Abb. 2). Batch

F e d - b a t c h (OFB)

25

10 I

1/1

1n

-10° -

ol5

e

5

£

100 Zeit [h]

Abb. 2. Cellulasebildung bei T. reesei M 18 mit Lactose in OFB-ProzeBfiihrung (301-Fermentor) 2,5% Lactose im Grundnâhrmedium ; 4% Lactose in der fed-batch-Periode zudosiert; Begasungsintensitât: 45 N1 Luft/h • kg

Mittels OFB wird bei der Cellulasegewinnung die Produktivität der extrazellulären Komponenten des Cellulasekomplexes erhöht, nicht jedoch derjenigen Komponenten, welche erst durch partielle Lyseprozesse freigesetzt werden, wie beispielsweise ßGlucosidase. Die Ursache dafür liegt offenbar bei den verschiedenen Optima für die Cellulasebildung einerseits und die /?-Glucosidasebildung andererseits. Die Cellulasebildung in der fed-batch-Periode ist weitgehend proportional der Substratzuführung (Abb. 3). Die Zusammensetzung der Cellulasekomplexe hinsichtlich deren Einzelkomponenten ist abhängig vom induzierenden Substrat. Verglichen mit MKC als Substrat bildet die Mutante T. reesei M 18 auf Lactose mehr Endoglucanase und Xylanase, während die Cellobiohydrolase I um ca. 30% verringert ist (Tab. 2).

288 F e d - b a t c h (OFB) 150

rrn

Acta Biotechnol. 8 (1988) 3

30 r E 3 S

20 u .

; 100

ID

to s

50

0

50

100

150 200 Zeit [h]

250

300

Abb. 3. Cellulasebildung bei T. reeaei ZIMET 43803 in OFB-Prozeßführung mit Lactose (301-Fermentor) • — FPA, • — Lactosezuführung (in 20%iger Lösung); | Verringerung des Arbeitsvolumens um 8%, 3% Holzzellstoff plus 1% Weizenkleie im Grundnährmedium

Tab. 2. Einfluß v o n Substrat und Prozeßführung auf die Zusammensetzung des Cellulasekomplexes von T. reesei M 18 Substrat

MKC Lactose Lactose + MKC

Prozeßführung

batch OFB OFB (Lactose)

Enzymaktivitäten [U/mg Protein] Endoglucanase [8]

Cellobiohydrolase [9]

Xylanase [7]

2,83 4,53 5,88

0,19 0,13 0,14

1,97 2,28

Diskussion und Schlußfolgerungen Von ALLEN [2] wurde bereits für eine T. reesei-Mutante die Cellulasegewinnung auf Basis von Lactose mittels einer feed-back-kontrollierten Substratzuführung beschrieben. Die Lactosezuführung erfolgt dabei in einer solchen Weise, daß das Profil der spezifischen C0 2 -Bildung einer Fermentation unter sonst gleichen Bedingungen, jedoch mit MKC als Substrat, realisiert wird. Bei der vorliegenden OFB-Technik wird in der diskontinuierlichen Phase das Myzel angezogen und in der anschließenden fed-batch-Periode unter „induzierenden" Bedingungen mit Substrat soweit versorgt, daß keine Lyse auftritt und andererseits das Myzelwachstum eingeschränkt ist. Die OFB-Technik steht im Einklang mit Untersuchungen zur Korrelation von ATP-Konzentration und Cellulasebildung [3,4]. Maximale Cellulasebildung wird festgestellt, wenn bei starker Verringerung des Myzelwachstums noch hohe ATP-Konzentrationen gemessen werden. Analog hierzu haben Messungen der Dehydrogenaseaktivität [5] gezeigt, daß bei Substratlimitation verbunden mit einem Abfall der Dehydrogenaseaktivität auch ein Rückgang der Cellulasebildung auftritt. Die Anwendung einer fed-batch-Technik mit periodisch intermittieren-

K E R N S , G . , K U D E , J . U. a . ,

Cellulasegewinnung

289

der Glucosezuführung bei der Gewinnung von /S-Glucosidase führt zu einem Anstieg der intrazellulären /S-Glucosidase, nicht jedoch zur erhöhten Ausscheidung [6], Die OFB-Technik ist für die Gewinnung induzierbarer extrazellulärer Enzyme vorteilhaft und sollte somit auch für die Enzymgewinnung mit trägerfixiertem Myzel bzw. mit Myzelrückhaltung geeignet sein. E i n g e g a n g e n : 2. 6. 1987

Literatur [ 1 ] LABUDOVA, I . , FARKAS, V . : F E M S M i c r o b i o l . L e t t . 2 0 ( 1 9 8 3 ) 2 , 2 1 1 . [ 2 ] ALLEN, A . L . , MORTENSEN, R . E . : B i o t e c h n o l . B i o e n g . 2 3 ( 1 9 8 1 ) , 2 6 4 1 . [ 3 ] COCHBT, N . , T Y A G I , R . D . , G H O S E , T . K . , L E B E A U L T , J . M . : B i o t e c h n o l . L e t t . 6 ( 1 9 8 4 ) ,

155.

[ 4 ] F A R K A S , V . , K E R N S , G . , LISKOVA, M . , B A U E R , S . : F o l i a M i c r o b i o l . 8 1 ( 1 9 8 6 ) , 2 7 7 . [ 5 ] G R U N D I G , B . , B E H R E N S , U . , K E R N S , G . , T H I E R S C H , A . : A c t a B i o t e c h n o l . 8 ( 1 9 8 8 ) 1, 7 1 . [ 6 ] K E R N S , G . , O K U N E V , O . N . , A N A N I N , V . M . , GOLOVLEV, E . L . : A c t a B i o t e c h n o l . 7 ( 1 9 8 7 ) 6 , 535.

[7] GHOSE, T. K., BISARIA, V. S.: Measurement of Hemicellulase Activities. P a r t 1. Xylanases. Commission on Biotechnology, I n t e r n a t i o n a l Union of P u r e a n d Applied Chemistry, 1984. [ 8 ] RABINOVITSCH, M . L . , K L Y O S O V , A . A . , B E R E Z I N , I . V . : B i o o r g . K h i m . 3 ( 1 9 7 7 ) , 4 0 5 . [ 9 ] RABINOVITSCH, M . L . ,

MELNICK, M . S . ,

NOVIKOVA, T . V . ,

TICHOMIROV, D . F . ,

TALEBA-

K . , SHCHEGOLEV, A . A . , K L Y O S O V , A . A . : Bioorg. K h i m . 1 2 ( 1 9 8 6 ) , 1 5 4 9 . [10] GHOSE, T . : M e a s u r e m e n t of Cellulase Activities. Commission on Biotechnology, I n t e r n a t i o n a l Union of P u r e a n d Applied Chemistry, 1984. ROVSKAYA, I .

Book Review H.

G . SCHLEGEL

(unter Mitarbeit von K.

SCHMIDT)

Allgemeine Mikrobiologie 6. überarbeitete Auflage S t u t t g a r t , New Y o r k : Georg T h i e m e Verlag, 1985. 571 S „ 240 Abb., 39 Tab., 185 Lit., 3 4 , - DM N a c h d e m f ü r die 5. Auflage zahlreiche K a p i t e l neu geschrieben u n d erweitert worden waren, w u r d e n f ü r die 6. Auflage n u r wenige Kapitel neu gegliedert. Den F o r t s c h r i t t e n der vergleichenden Biochemie, Physiologie u n d Ökologie der Mikroorganismen wurde in allen K a p i t e l n R e c h n u n g getragen. Die meisten Ä n d e r u n g e n b e t r e f f e n die a n a e r o b e n B a k t e r i e n , d a r u n t e r Archaebakterien. Die großen F o r t s c h r i t t e der Physiologie, vergleichenden Biochemie u n d Ökologie der Mikroorganismen schlagen sich nicht i m m e r in grundlegenden Ä n d e r u n g e n der L e h r m e i n u n g nieder, sie m a c h e n aber K o r r e k t u r e n , Weglassungen, E r g ä n z u n g e n u n d Verlagerungen der Akzente notwendig. Bei den meisten K a p i t e l n h a t sich die Ü b e r a r b e i t u n g auf derartige geringfügige Veränderungen b e s c h r ä n k t . N a c h d e m in der v o r a n g e h e n d e n Auflage die K a p i t e l über Viren, spezielle G ä r u n g e n , Bakteriengenetik u n d Regulation des Stoffwechsels u m g e s t a l t e t u n d die Grundkonzepte der Ökologie, Geomikrobiologie u n d Symbioseforschung einbezogen worden waren, h a t j e t z t n u r das K a p i t e l über die S y s t e m a t i k der B a k t e r i e n eine tiefgründige Überarbeit u n g erfahren. Die Zahl der Lehrbeispiele u n d S c h e m a t a w u r d e n i c h t e r h ö h t . I n der L i t e r a t u r ü b e r s i c h t wurde d u r c h Weglassen der experimentellen Originalarbeiten P l a t z f ü r Hinweise auf Übersichtsartikel, Monographien u n d relevante L e h r b ü c h e r der Grenzgebiete geschaffen. D a d u r c h wird die B e n u t z u n g der Bibliotheken zur Vertiefung der S t o f f k e n n t n i s erleichtert. 6

Acta Biotechnol. 8 (1988) 3

Acta Biotechnol. 8 (1988) 3, 290

Book Review K A L U N J A N Z , K . A . , L . I . G O L G E R , W . E . BALASCHOW

Ausrüstungen für mikrobiologische Produktionen Moskau: Verlag Agropromisdat, 1987. 398 S., 176 Abb., 54 Tab. (in Russisch) Das als Hochschullehrbuch in der UdSSR eingeführte Buch für Studenten der Fachrichtung „Technologie der mikrobiologischen Produktionen" schließt an das von den gleichen Verfassern (KALUNJANZ/GOLGER) 1979 herausgegebene Werk „Mikrobielle Enzympräparate" (Rezension siehe Acta Biotechnol. 0 (1980), 68) an, das inzwischen auch in deutscher Sprache vorliegt (Leipzig: Fachbuchverlag, 1984). In diesem Lehrbuch werden ausführlich moderne Ausrüstungen für die verschiedenen mikrobiologischen Technologien (nicht nur auf die Enzymherstellung beschränkt) vorgestellt. Orientiert am studentischen Benutzerkreis wurden für die wichtigsten davon auch die erforderlichen Berechnungsunterlagen aufgenommen. In den einzelnen Kapiteln werden folgende Schwerpunkte behandelt, nachdem einleitend einige allgemeine Ausführungen zur Klassifizierung der Ausrüstungen und der Optimierung der Anlagenstruktur gemacht sowie Beispiele der wichtigsten Technologien zur Herstellung biotechnologischer Produkte anhand von Apparateschemata erläutert werden: — Transportausrüstungen (einschließlich wichtiger physikalischer Parameter der meisten in der Biotechnologie zu transportierenden Stoffe), — Hilfsausrüstungen (wie Behälter, Mischer, Pumpen, Dosiereinrichtungen, Reinigungsapparaturen), — Sterilisatoren für die Nährmedien einschließlich Zubehör (insbes. Wärmeübertrager) und Hydrolyseapparate, — Ausrüstungen zur Luftsterilisation, Verdichter und Gebläse, — Extraktoren, Pressen, Filter- und Flotationsapparate, — Ausrüstungen zur Mikroorganismenkultivierung auf festen Substraten, — Fermentoren für die Submerskultivierung der Mikroorganismen (Apparate für sterile und unsterile Prozesse, Berechnung von Belüftungseinrichtungen), — Ausrüstungen zur Trennung der flüssigen von der festen Phase (Zentrifugen, Separatoren, Baktofugen), — Apparate zur Aufkonzentrierung und Reinigung von Lösungen biologisch aktiver Stoffe (u. a. Eindampfapparate, Kondensatoren, Vakuumpumpen), — Membrantrennanlagen für solche Lösungen, — Trockner unterschiedlichster Bauart, — Mühlen, Granulatoren sowie Ausrüstungen zur Standardisierung und Mikrokapsulierung. Abschließend werden einige Ausführungen zum spezifischen Arbeits- und Umweltschutz sowie zur Sicherheitstechnik in biotechnologisch arbeitenden Betrieben gemacht. Der vorliegende Titel zeichnet sich durch eine klare, systematische Behandlung der Teilgebiete aus und geht auf eine große Zahl von technischen Lösungen besonders aus der Produktion der UdSSR, jedoch auch des Auslandes sehr ausführlich ein. Zu kurz kommen bzw. fehlen solche wichtigen Teilgebiete der Bioprozeßtechnik wie die Geräte- und Automatisierungstechnik, moderne Bioreaktorsysteme (für Zellkultivierung, anaerobe Prozesse, immobilisierte Enzyme bzw. ganze Zellen) und wichtige Details steriler Prozesse (Armaturen, Dichtungen, Werkstoffauswahl). Insgesamt jedoch stellt das Buch eine wichtige Bereicherung der noch immer in geringem Umfang verfügbaren technischen Literatur für die Biotechnologie dar. Es kann nicht nur Studenten, sondern auch Entwicklern, Projektanten und Betriebspraktikern eine Vielzahl von nützlichen Anregungen, Hinweisen und theoretischen Grundlagen vermitteln. KAUP.UFF

Acta Biotechnol. 8 (1988) 3, 2 9 1 - 2 9 3

Transformation of 7,8-dihydrocodeinone to 7,8-dihydroisocodeine by Aureobasidium pullulans FUSKA, J . 1 , PROKSA, B . 2 , FUSKOVA, A . 3 , KHANDLOVA, A . 1

1

2

3

Department of Biochemical Technology Faculty of Chemistry Slovak Technical University CS-81237 Bratislava, Czechoslovakia Institute of Chemistry Slovak Academy of Sciences CS-84238 Bratislava, Czechoslovakia State Forest Products Research Institute CS-82619 Bratislava, Czechoslovakia

Summary. Aureobasidium pullulans reduced stereospecifically 7,8-dihydrocodeinone to 7,8-dihydroisocodeine. Activity of the culture was inhibited by some substances present in the malt extract.

Introduction Natural codeine and thebaine are also important as the basic material for the synthesis of the new analgetics. By microbial transformation of codeine by a culture Cunning hamella bainieri in addition to the codeine N-oxide was also obtained 17-norcodeine [1]. Demethylation of the codeine was carried out by Streptomyces paucisporogenes and Streptomyces lincolnensis but with relatively low yields [2], Codeine N-oxide was not demethylated by microorganisms. Streptomyces griseus (ATCC 10137) transformed codeine to a mixture of 17-norcodeine and 14/Miydroxycodeine (in the ratio 4:1), in the yields 7% to the used substance [3], A culture Trametes sanguined reduced nonspecifically the 6-oxoalkaloids to a mixture of alcohols. This culture reduced 7,8dihydrocodeinone to a mixture of 7,8-dihydrocodeine (28.6%) and 7,8-dihydroisocodeine (36.4%) [4, 5]. The transformation of morphinanes and their derivatives remains still attractive, therefore we tried to transform codeine as well as 7,8-dihydrocodeinone. But, only one of the screened cultures i.e. Aureobasidium pullulans gave positive results.

Materials and Methods From the collection of 28 cultures of microorganisms only A. pullulans transformed specifically 7,8-dihydroisocodeinone (1) [(5a, 6