Acta Biotechnologica: Volume 9, Number 6 1989 [Reprint 2021 ed.] 9783112581902, 9783112581896

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Acta Bhtecfeioliglca

Volume 9 • 1989 • Number 6

Journal of microbial, biochemical and bioanalogous technology

Akademie-Verlag Berlin ISSN 0138-4988 Atta Blotechnol., Berlin 9 (1989) 6, 497- 578

Instructions to Authors

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

Acta BtotedMloiia Journal of microbial, biochemical and bioanalogous technology

Volume 9 1989

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 by M. Ringpfeil, Berlin and G. Vetterlein, Leipzig Editorial Board:

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

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

Managing Editor:

L. Dimter, Leipzig

Number 6

AKADEMIE-VERLAG

BERLIN

"Acta Biotechnologica" publishes original papers, short communications, reports and reviews from the whole field of biotechnology. The journal is to promote the establishment of biotechnology as a new and integrated scientific field. The field of biotechnology covers microbial technology, biochemical technology and the technology of synthesizing and applying bioanalogous reaction systems. The technological character of the journal is guaranteed by the fact that papers on microbiology, biochemistry, chemistry and physics must clearly have technological relevance. Terms of subscription for the journal "Acta Biotechnologica" Orders can be sent — in the GDB: to Postzeitungsvertrieb or to the Akademie-Verlag Berlin, Leipziger Str. 3—4, P F 1233, DDR -1086 Berlin; — in the other socialist countries: to a bookshop for foreign languages literature or to the competent news-distributing agency; — in the FRG and Berlin (West): to a bookshop or to the wholesale distributing agency Kunst und Wissen, Erich Bieber oHG, Postfach 102844, D-7000 Stuttgart 10; — in the other Western European countries: to Kunst und Wissen, Erich Bieber GmbH, General Wille-Str. 4, CH-8002 Zürich; — in other countries: to the international book- and journal-selling trade, to Buchexport, Volkseigener Außenhandelsbetrieb der DDR, P F 160, DDR - 7010 Leipzig, or to the AkademieVerlag Berlin, Leipziger Str. 3—4, P F 1233, DDR-1086 Berlin.

Acta Biotechnologica Herausgeber: Institut für Biotechnologie der AdW der DDR, Permoserstr. 15, DDR - 7050 Leipzig (Prof. Dr. Manfred Ringpfeil) und VEB Chemieanlagenbaukombinat Leipzig—Grimma, Bahnhofsstr. 3—5, DDR-7240 Grimma, (Dipl.-Ing. Günter Vetterlein). Verlag: Akademie-Verlag Berlin, Leipziger Str. 3—4, P F 1233, DDR -1086 Berlin; Fernruf: 2236201 und 2236229; Telex-Nr. 114420; Bank: Staatsbank der DDR, Berlin, Konto-Nr.: 6836-26-20712. Redaktion: Dr. Lothar Dimter (Chefredakteur), Martina Bechstedt, Käthe Geyler, Permoserstr. 15, DDR-7050 Leipzig; Tel. 2392255 Veröffentlicht unter der Lizenznummer 1671 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: VEB Druckhaus „Maxim Gorki", DDR-7400 Altenburg. Erscheinungsweise: Die Zeitschrift „Acta Biotechnologica" erscheint jährlich in einem Band mit 6 Heften. Bezugspreis eines Bandes 198,— DM zuzüglich Versandspesen; Preis je Heft 33,— DM. Der gültige Jahresbezugspreis für die DDR ist der Postzeitungsliste zu entnehmen. Bestellnummer dieses Heftes: 1094/9/6. Urheberrecht: Alle Rechte vorbehalten, insbesondere der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translation into foreign languages). No part of this issue may be reproduced in any form, by photoprint, microfilm or any other means, without written permission from the publishers. © 1989 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 18520 03000

Akademie-Verlag Berlin

Acta Biotechnol. 9 (1989) 6, 4 9 9 - 5 0 3

Gerätesystem zur massenspektrometrischen Prozeßüberwachung in der Fermentationstechnik I S K E , U . , SCHÖN, G . , J E C H O B E K , M . , R O S T ,

H.-J.

Akademie der Wissensehaften der D D R Institut für Biotechnologie Permoserstr. 15, Leipzig 7050, D D R Vortrag auf dem 4. Heiligenstädter Kolloquium „Wissenschaftliche Geräte für die Biotechnologie", Heiligenstadt, 2 4 . - 2 7 . 10. 1988, DDR.

Summary A quadrupol mass spectrometer QMS c 300 developed and manufactured from the workshops of the Institute of Nuclear Research of the Hungarian Academy of Sciences, Debrecen, has been cuppled with a 12 1 laboratory steril fermenter. The QMS apparatus is provided with 3 different possibilities for sampling. Via a dynamic air input system the discharged air of the fermenter can be analyzed continuously. The static sampling input allows the off-line measurement of gaseous and volatile compounds. Special membran samplers are used for measurements of volatile and gaseous substrates and microbial metabolites in the fermentation broth. The samplers can be adopted in the reactor vessel directly analogous to the pH and p0 2 -electrodes.

Einleitung Die Entwicklung effektiver biotechnologischer Verfahren und ihre rasche Umsetzung in den industriellen Maßstab erfordert leistungsfähige hochinstrumentierte Fermentationssysteme sowohl im Stadium der Grundlagenforschung als auch in der industriellen Praxis. On-line Messungen ohne oder mit nur geringer Verzögerung in den verschiedenen hierarchischen Ebenen des fermentations- bzw. mikrobiologischen Systems sind die Forderung der Stunde. Neben den traditionellen physikalisch-chemischen und elektrochemischen Meßprinzipien haben in den letzten Jahren optisch-spektroskopische Methoden bereits Eingang in die Analytik von Fermentationsprozessen gefunden [1, 2]. Die Massenspektrometrie als Analysenmethode existiert nahezu 100 Jahre, ihre Nutzung in der Biotechnologie und hier speziell in der Fermentationstechnologie ist jedoch relativ neu [3]. Die Nutzung massenspektrometrischer Meßmethoden besitzt im wesentlichen folgende Vorteile: — schnelle und gut reproduzierbare Einstellung der Meßparameter — Messung in der gasförmigen als auch in der flüssigen Phase möglich — kontinuierliche und gleichzeitige Messung verschiedener Gase und flüchtiger Komponenten möglich * veröffentlicht im Tagungsband des Kolloquiums, AdW der D D R , Zentrum für wissenschaftlichen Gerätebau, Mytron, 1989. 1*

500 — — — — —

Acta Biotechnol. 9 (1989) 6

die Erfassungsgrenzen liegen im Bereich von ppm bis Prozent Möglichkeiten der on- und off-line Messung geringe Totzeiten universelle Nutzbarkeit einfache Eichung.

Auf Grund dieser Vorteile stellt die fermentergekoppelte massenspektrometrische Analyse eine notwendige Ergänzung bzw. Erweiterung der analytischen Möglichkeiten zur Charakterisierung und Steuerung biotechnologischer Prozesse dar. Jedoch erst mit der Erfindung des Quadrupolmeßsystems wurde es möglich, preisgünstige, transportable und leichthandhabbare Spektrometer für die verschiedensten Applikationszwecke zu entwickeln. Aufbau eines Quadrupolmassenspektrometers (QMS) Massenspektrometer können nach 4 funktionellen Gruppen unterteilt werden, Abb. 1. Voraussetzung für die Arbeitsweise von Ionenquelle, Analysator und Detektor ist die Schaffung eines Hochvakuums im Bereich von 10~5—10~9 mbar zur Gewährleistung der erforderlichen Hochspannung für die Ionenquelle und ihres Schutzes vor Verunreinigungen, zur Sicherung der Glühkathode und zur Verhinderung unkontrollierter IonenMolekül-Reaktionen . Massenspektrometer

Abb. 1. Eunktionselemente eines Massenspektrometers

Durch Drehschieberpumpen wird ein Vorvakuum von ca. 10"2 mbar erzeugt, während für das Hochvakuum wahlweise — öldiffusionspumpen — Turbomolekularpumpen oder — Ionengetterpumpen

eingesetzt werden. Durch einen mittels Elektronenstoßionenquelle (Glühkathode) erzeugten Elektronenstrahl von etwa 50—100 eV (bevorzugt 70 eV) werden die zu untersuchenden Gasmoleküle bzw. Atome ionisiert und nach Fokussierung durch eine Ionenoptik in den Analysator ausgestoßen. Hier erfolgt die Trennung der Ionen nach ihrem Massen/Ladungsverhältnis. Im Falle des Quadrupolmeßsystems besteht der Analysator aus 4 Stäben mit hyperbolischen bzw. in der Praxis kreisrunden Stäben (meist Molybdän) gleicher Größe, die mit größter Präzision parallel und in gleicher Entfernung zueinander angebracht sind. Die gegenüberliegenden Stäbe sind dabei elektrisch miteinander verbunden und eine Hochfrequenzspannung, die einer Gleichspannung überlagert ist, wird den 2 Stabpaaren zugeführt. In diesem elektrischen Feld werden die ionisierten Probenmoleküle oder Atome in Schwingungen versetzt. Bei einem definierten Verhältnis von Hochfrequenzspannung zu Gleichspannung können nur Ionen mit einem bestimmten

ISKE, U., SCHÖN, G. U. a., Massenspéktrometrische Prozeßüberwachung lohehquelle

— iE

600

ACCUREL®-MODUL

6

500 V o 2?

400 - 4

/

yT

300 - 3 200

-

2.2 m2

X ......

2

100

50

100

150 Med

[llh]

Abb. 3. Sauerstoffstrom M*, und Konzentrationsdifferenz in Abhängigkeit des Wasserstromes V y e d

Acmiog

Da bereits die Anordnung von 5 Modulen doch aufwendige Rohrleitungsführungen verlangt, da zum Beispiel einzeln sterilisierbare Module gewünscht sind, erscheint die Umsetzung dieses Konzepts in die Praxis als relativ schwierig.

Begasung mit Silicon-Schläuchen Einige Fermentorhersteller bieten seit geraumer Zeit im Fermentor eingebaute Begasungsschläuche zur blasenfreien Begasung an. Meistens wird Silicon als Membranmaterial verwendet und Sauerstoff mittels Druckerhöhung im Begasungsschlauch ins Medium transportiert. Der Transportvorgang setzt sich aus einer Diffusion durch den Siliconschlauch und einem konvektiven Transport von der Schlauchoberfläche ins Medium zusammen. Der Gesamttransport ist von verschiedenen Einflußgrößen abhängig : — Material des Begasungsschlauches — Wanddicke des Begasungsschlauches (d. h. Länge des Diffusionsweges) und Durchmesser des Schlauches — Druckdifferenz zwischen Schlauchinnerem und dem Medium (d. h. treibendes Druckgefälle über dem Diffusionsweg) — Strömungsgeschwindigkeit des Mediums über die Schlauchoberfläche (d. h. Grenzschichtdicke und damit Widerstand in der laminaren Unterschicht) — Konzentration des Sauerstoffs im Schlauchinnern und Volumenstrom des Feedgases im Schlauch (wegen der Entmischung) — Lähge des Schlauches und Unterteilung in parallele Pässe

Die Abhängigkeiten sind also beeinflußt vom Material, von der konstruktiven Gestaltung, von Betriebsbedingungen und von Herstell- und Betriebskosten. Zur Auslegung eines Reaktors mit Schlauchbegasung müssen deshalb folgende Schritte unternommen werden:

KOPP,

— — — —

TH., Blasenfreie Begasung

509

Festlegung des Schlauchmaterials und -dimensionen Festlegung der Betriebsbedingungen (Gaskonzentration, -fluß) Berechnen der notwendigen Schlauchlänge Berechnung des Druckabfalles und eventuell Aufteilen des Gasflusses in mehrere parallele Pässe

In einem 7 1 Fermentor mit eingebautem Korb und aufgewickeltem Begasungsschlauch wurde die sogenannte dynamische Methode angewendet, das heißt, die mit N2 ausgegaste Flüssigkeit (meist Wasser) wird ab der Zeit t = 0 mit Sauerstoff oder mit Luft begast. Aus dem Anstieg des p 0 2 = /(Zeit) können OTR-Werte leicht berechnet werden. Abb. 4 zeigt die Abhängigkeit des p 0 2 -Anstiegs von der Rührerdrehzahl. Werden solche Experimente nun mit verschiedenen Schläuchen durchgeführt, interessiert natürlich besonders die Aufschlüsselung der Abhängigkeiten von den einzelnen Effekten. Vor allem die Abhängigkeit vom Material und von der Dimension des Schlauches ist für einen Scale-up von größter Wichtigkeit. Mittels eines vereinfachten Modellls können die Abhängigkeiten teilweise entkoppelt werden:

Zeit [min] Abb. 4. Sättigungsexperiment p 0 2 (i) = f(t) bei Schlauchbegasung mit Rührerdrehzahl als Parameter • n = 600 rpm • n = 300 rpm • n = 100 rpm O n = 50 rpm

Es wird ein Schlauch von definierter Geometrie (Schlauchlänge Ls und Schlauchmitteldurchmesser d s ) in einer Flüssigphase mit dem Volumen FL angenommen. Beim Einbringen eines Gases mit dem Druck px entsteht ein Sauerstofftransport N*(t) durch den Schlauch der zur Gelöstsauerstoffkonzentration c(t) führt. Der Druck soll im folgenden konstant gehalten werden, ebenfalls die Gaskonzentration, das heißt Entmischungseffekte werden vernachlässigt. N*

mmol

rmmol] [ m2 • s J

-fr]

• As-

de

[

mmol

± V]L

'

dc(t) dt

510

Acta Biotechnol. 9 (1989) 6

Der Sauerstoffstrom N*(t) ist gemäß der Theorie vom Stoffübergang proportional zur Austauschfläche .4 S und der treibenden Konzentrationsdifferenz Ac. Die transportierte Menge an Sauerstoff ist gleich der im geschlossenen Flüssigkeitssystem aufgenommenen Menge. Umgeformt erhalten wir die Ausgangsgleichung:

Wieder nach der Theorie des Stoffüberganges ist die Stoffdurchgangskonstante k abhängig vom inneren Stoffübergangskoeffizienten ßi, vom äußeren Stoffübergangskoeffizienten ßa, von der Diffusionskonstante D und von der Wanddicke d. Da der Bewegungswiderstand eines Sauerstoffmoleküls in der Gasphase viel kleiner ist als in der Flüssigphase gilt die Vereinfachung, das nur ßa zu berücksichtigen bleibt. i--J_4.A4._L

A

J_

Da der Wandwiderstand und der Widerstand in der äußeren Grenzschicht schwierig zu trennen sind, gilt folgende Vereinfachung: 6 _L M k D*

Dies ist für die spätere Interpretation natürlich nur dann zulässig, wenn wir für die Vergleiche der verschiedenen Schlauchtypen nur Versuche mit gleicher Überströmgeschwindigkeit und damit gleichem Grenzschichtverhalten verwenden. Aus unserer Gleichung kann n u n abgeleitet werden: dg)

=

^

X s ^ J s

, (c+ _

c(0 2 -Wert

krit.pO2 p02-Min. \ (Startdrehzahl) \ reduz. Drehzahl Sättigung Die Funktion der Vorrichtung ist vom Grundsatz her dadurch charakterisiert, daß der Übertrager dem aktuell in der Kulturlösung gemessenen p 0 2 -Wert nach Absinken desselben unter den am jp0 2 -Regler vorgewählten SchwellwertS i einen aktuellen pH-Sollwert zuordnet und diesen dem pH-Regler in Form des elektrischen Eingangssignals in der Betriebsart externe Sollwertvorgabe vorschreibt. Der pH-Regler bewirkt mittels Ansteuerung der Pumpen f ü r Säure bzw. Lauge die Anpassung des aktuellen pH-Werts der Kulturlösung im Fermentor an den vom p 0 2 -Wert und die Übertragungscharakteristik vorgegebenen aktuellen pH-Sollwert.

526

Acta Biotechnol. 9 (1989) 6

Somit verursacht der Abfall des p 0 2 -Wertes unterhalb des Schwellwertes S 1 infolge wachstumsbedingter Zunahme des 0 2 -Verbrauches der Fermentationskultur eine dieselbe vermindernde definierte Absenkung (oder entsprechend G B O S S E et al. [8] auch Anhebung) des pH-Wertes der Kulturlösung (Selbstkopplung) im Anschluß an die Phase des pH-Freilaufs, die durch p 0 2 -Werte größer als S 1 gekennzeichnet ist. Der Koppler bewirkt also mittels kulturspezifisch vorgewählter selbstgekoppelter pH-Verschiebung, daß der p 0 2 -Wert vorübergehend ein stationäres p 0 2 -Minimum als Ausdruck der mit dem erreichten pH-Minimum verbundenen Balance zwischen dem festen 0 2 -Eintrag des Fermentors und dem 0 2 -Verbrauch der wachsenden Kultur annimmt. Die Übertragungscharakteristik wird so vorgegeben, daß das p 0 2 -Minimum größer als i>0 2 k r l t ist. Während der pH-Absenkung wächst die Kultur trotz überschüssig vorliegender C-, N- und PSubstrate mit einer suboptimalen maximalen spezifischen Wachstumsrate; die Stationarität des Wachstums, verbunden mit der Verminderung des 0 2 -Verbrauchs, tritt erst mit der Limitation eines der essentiellen Substrate ein. Die Erfassungseinheit Ap02/At gibt nach Erreichen des Wertes ApOJAt = 0 (stationäres p 0 2 -Minimum) die in der Koppler-Untereinheit pH-Festsollwertgeber vorgewählte und gespeicherte pH-Schwelle frei. Diese Schwelle wird als fester Sollwert der weiteren pH-Regelung aktiviert, wenn der p 0 2 -abhängige aktuelle pH-Wert durch den Übertrager infolge des durch eine Substratlimitation verursachten p 0 2 -Anstiegs dieselbe oder das zusammen mit ihr vorgewählte Toleranzintervall erreicht. Damit endet die Punktion des Übertragers. Mit der Aktivierung des festen pH-Sollwertes kann die Betriebsart externe Sollwertvorgabe des Drehzahlreglers gestartet werden. Die Koppler-Untereinheit Drehzahlgeber übernimmt die Sollwertvorgabe, beginnend mit der bislang schon realisierten Drehzahl und endend bei der Minimaldrehzahl, die der p 0 2 -Sollwertregelung an der vorgewählten Schwelle S 2 des p 0 2 -Reglers entspricht. Prinzipiell steht mit der Einbeziehung des Drehzahlreglers natürlich auch in der Phase der p 0 2 -Regelung über die pH-Absenkung die Drehzahlerhöhung zur OTR-Steigerung zur Verfügung. Diese Wirkungsvariante der Vorrichtung ist dann von Bedeutung, wenn trotz Absenkung des pH-Wertes an den Rand des physiologischen Toleranzintervalls keine Stationarität des p 0 2 Wertes oberhalb p 0 2 k r i t eintritt. Die direkte Charakteristik des Übertragers laut Abb. 2 entsteht im einfachsten Fall durch Zuleitung des Ausgangssignals des p O a -Meßverstärkers zum Eingang für die externe Sollwertvorgabe des pH-Reglers (Pegel z. B. 0 — 10 V für beide Signale). Als Modifikation tritt die Addition oder Subtraktion einer konstanten oder variablen Spannung zum bzw. vom Ausgangssignal des p 0 2 Verstärkers hinzu. Diese Maßnahme legt den Wert des stationären p 0 2 -Minimums fest.

Anwendungsbeispiel Die fermentative Herstellung des Enzyms Glucoseisomerase wurde im Laborfermentor LF 2 (zur Charakterisierung vgl. [9, 10]) nach den Regimen I bzw. II durchgeführt, die in Abb. 3 erläutert bzw. bezüglich des sich jeweils ergebenden prozeßkinetischen Verlaufs dargestellt sind. Als einheitliche Grundbedingungen wurden verwendet: — Mikroorganismus: Streptomyces chrysomallus, Selektante X K 10—4 [11]; — Vorkultur: 300 ml einer 48 h alten Schüttelkultur, angezogen aus einer Mycelkonserve im VKMedium der Zusammensetzung 15 g Soja-Extraktionsschrot, 15 g Glucose, S g NaCl, 1 g CaC0 3 , 0,3 g K H 2 P 0 4 je Liter Wasser; — Hauptkulturmedium: 2,7 1 der Zusammensetzung 40 g Maisquellwasser, 30 g Maisstärke, 2 g MgS0 4 , 1,4 g (NH 4 ) 2 S0 4 , 1,4 g K H 2 P 0 4 , 10 g Xylose je Liter Wasser, Autoklavensterilisation 30 Minuten bei 121 °C ohne die Anteile Xylose und K H 2 P 0 4 (jeweils getrennt sterilisiert); — Permentationsparameter: Belüftung mit 150 1 Luft/h, Temperatur 28 °C, 3etagiges Rührwerk und Strombrecheranordnung entsprechend [9], 31 Arbeitsvolumen nach Zugabe des Inokulums.

Die Regime I und II unterscheiden sich dadurch, daß im Regime II die Vermeidung der 0 2 -Limitation auf herkömmliche Weise mittels kontinuierlicher automatischer Anhebung der Rührerdrehzahl gesichert und der pH-Wert lediglich zum Prozeßende durch automatische NH4-OH-Titration bei 6,7 geregelt wurde. Der benötigten Drehzahl-

GBOSSE, H . H . , HILLIGER, M .

U. a., Prozeßsteuerung des O a -Partialdruckes

Regime I Festdrehzahl 800min' 1 18.-22.h: pH=pH(p02) .

100 |

Q

NFYOH

Fest-SW

N

a:

\

\

20

60

CL

4-0 \

\

pOfMin.15%

10

6,7.

\

\

a)

527

30

-

20

t[h]

b) Abb. 3. a. Glucoseisomerase-Fermentation mit der Kopplung des pH-Wertes an den p 0 2 - W e r t bei fester Rührerdrehzahl Abb. 3. b. Glucoseisomerase-Fermentation mit kontinuierlicher Drehzahlsteigerung zur Vermeidung der O a -Limitation

Steigerung von 700 min - 1 auf 1050 min - 1 entsprechen OTR-Werte der Fermentorkonfiguration im Bereich von 3 g 0 2 / l • h bis 5 g 0 2 /l • h (bezogen auf die stationäre Methode der Na-Sulfit-Oxidation in Wasser mittels Messung der Abluft-0 2 -Konzentration). Dagegen entspricht Regime I mit einer Festdrehzahl von 800 min - 1 (OTR = 3,8 g 0 2 / l • h) und Benutzung der Kopplung des pH-Wertes an den p 0 2 -Wert der Fermentorkultur zwischen der 18. und 22. Stunde dem dargestellten neuen Konzept der Vermeidung der 0 2 -Limitation. Die Minimalwerte pH = 5,2 bzw. p02 = 15% resultieren aus der gewählten Übertragungscharakteristik pH-Pegel (V) = p0 2 -Pegel (V) + 1,7V,

528

Acta Biotechnol. 9 (1989) 6

wobei dem Pegelbereich 0—10 V die Bereiche 0—100% (p0 2 ) bzw. 2—12 (pH) in der Fermentor-Steuerelektronik zugeordnet sind. Zur Absenkung des pH-Wertes bzw. zu seiner Anhebung, gekoppelt an den ^0 2 -Wert, wurden automatisch 10%ige H 2 S0 4 -Lösung bzw. 25%ige NH4OH-Lösung zugepumpt. Die pH-Festsollwertregelung erfolgte an der Schwelle 6,7 mittels Zugabe von NH4OHLösung. Im Regime I begann die pH-Festsollwertregelung zur 22. Stunde, nachdem die Funktion des Übertragers mit dem Erreichen dieser Schwelle während der pH-Anhebung beendet war. Abb. 4 weist aus, daß die im Regime I erreichte Glucoseisomerase-Ausbeute deutlich über der des Regimes I I liegt. Die Maximalwerte zum Abbauzeitpunkt (32. Stunde) betragen 12500 bzw. 8500 Units pro Liter Kulturlösung. Dieses Ergebnis deutet darauf hin, daß die Steigerung der mechanischen Scherbelastung des Mycels im Regime I I ausbeutebegrenzend wirkt und der Variante mit Absenkung des pH-Wertes der Vorzug zu geben ist. Dementsprechend liegen die Anwendungsvorteile vorzugsweise bei Hochleistungsfermentationen mit filamentösen Mikroorganismen (Streptomyceten, Pilze). 75 0 00 -

= 10000

. ]

Abb. 3. Reduzierende Zucker, die aus verschiedenen Substraten: Lösliche Stärke ( —O —); unlösiche Stärke (—•—); Amylose (—o—) und Amylopectin (—o—) entstehen

Weiterhin wurde die Temperaturabhängigkeit der Hydrolysegeschwindigkeit von löslicher Stärke (2% w/v) mittels thermostabiler «-Amylase („Amylotherm") untersucht. Zu diesem Zweck wird die Enzym Wirkung bei Temperaturen von 30 °C und 90 °C verfolgt. Dabei werden die Angaben für die Veränderung der reduzierenden Produkte im Laufe der Zeit in Abb. 4 vorgestellt. Sie bestätigen die Hypothese, daß die Substrat-

6

3N 0» £t 20

40

60 tfminj

Abb. 4. Reduzierende Zucker, die aus Stärke mittels thermostabilen a-Amylasepräparates „Amylotherm" bei 30°C (—•—) und 90 °C (—•—) hergestellt werden

DOBREVA, E . , IVANOVA, V., a-Amylase und Stärkehydrolysate

553

Spaltung bei 30 °C mit niedrigerer Geschwindigkeit als bei 90 °C verläuft. Aus der dargestellten chromatographischen Analyse (Abb. 5) ist ersichtlich, daß die Temperatur nicht die Art der entstehenden Maltozucker, sondern nur ihre Quantität beeinflußt. Bei 90 °C ist, im Vergleich zu 30 °C, eine Zunahme der Menge von Glucose, Maltose und Maltotriose und eine Abnahme von Maltopentose zu beobachten.

Abb. 5. Dünnschichtchromatographie der Hydrolysate, die aus löslicher Stärke (1) und unlöslicher Stärke (2) bei 30 °C (A) und 90 °C (B) gewonnen wurden

Schlußfolgerung Die aus den Untersuchungen der Hydrolyse der Stärkesubstrate mittels ¡%-Amylase aus Bacillus licheniformis 44MB82 erhaltenen Ergebnisse widerspiegeln seine Endoenzymwirkung. Das Präparat führt zur schnellen Stärkehydrolyse, indem das Substrat während der ersten 10—15 min. gründlich zu Dextrinen abgebaut wird. Die Verlängerung der Enzymeinwirkung ruft eine Zunahme der Maltozucker mit Polymerisationsgraden von 1 bis 5 hervor. Diese Endprodukte werden auch nach 6stündiger Hydrolyse beobachtet. Die Geschwindigkeit der Amylosespaltung, die durch die Veränderung der Menge der reduzierenden Stoffe im Verhältnis zur Zeit dargestellt wird, ist niedriger, als bei den anderen untersuchten Substraten. Die Erhöhung der Temperatur von 30 °C auf 90 °C führt zur Veränderung des Verhältnisses der entstehenden Maltozucker, d. h. zur Zunahme der Maltose und Maltotriose und Abnehme der Maltopentaose. Die vorliegende Arbeit wurde vom Komitee der Wissenschaften der Volksrepublik Bulgarien finanziert (Vertrag Nr. 289). Eingegangen: 30. 6. 1988

Acta Biotechnol. 8 (1989) 6

554

Literatur [ 1 ] MADSEN, G . B . , NORMAN, B . E . , SLOTT, S . : D i e S t ä r k e 2 5 ( 1 9 7 3 ) , 3 0 4 .

[2] SAITO, N.: Arch. Biochem. Biophys. 155 (1973), 290. [ 3 ] CHIANG, J . P . , A L T E R , J . E . , S T E R N B E R G , M . : Die Stärke

3 1 (1979), 86. [ 4 ] MORGAN, P . J . , P R I E S T , F . G . : J . A p p l . B a c t e r i o l o g y 5 0 ( 1 9 8 1 ) , 1 0 7 . [ 5 ] J O H N , M . , SCHMIDT, J . , W A N D R E Y , C h . , SALM, H . : J . C h r o m . 2 4 7 ( 1 9 8 2 ) , 2 8 1 . [ 6 ] ROLLINGS, J . E., OKOS, M . R . , TSAO, G . T . : Found. Biochem. Eng. Kinet. Thermodyn.

Biol. Syst., Winter Symp. ACS Div. Ind. Eng. Chem., Boulder Colo, 17—20. 01.1982, Washington DC, Abstracts (1983), 443.

[ 7 ] EAJibCHC A . E . , EAXMATOBA, H . B . , HIOPJIHC, T . K . , TJIEMHTA, A . A . , CHHHI^HH, A . TAJIEBOPOBCKAH, H . K . : EHOXHMHH 5 1 , ( 1 9 8 6 ) 3 , 3 7 8 . [ 8 ] SCHIMIZU, M . , K A N N O , M . , TAMURA, M . , S U E K A N E , M . : B i o l . C h e m . 4 2 ( 1 9 7 8 ) , 1 6 8 1 .

II.,

[9] UCHINO, F . : Agr. Biol. Chem. 46 (1982), 7. [ 1 0 ] BESCHKOV, M . , EMANTJILOVA, E . , GRIGOROVA, R . , KOSTURKOVA, P . , DOBREVA, E . , TONKOVA, A . , SCHIVAROVA, N . , KAMENOV, K . , KOMINKOVA, V . : Bulg. Urh.-schein Nr. 3 7 7 6 5 , ( 1 9 8 3 ) . [ 1 1 ] PANTSCHEV, C h . , K L E N Z , G . , H Ä F N E R , B . : L e b e n s m i t t e l i n d . 2 8 ( 1 9 8 1 ) , 7 1 .

[12] SOMOGYI, M.: J. Biol. Chem. 195 (1952), 19. [13] DAviDEK, J., Laboratorni priruöka analyzy potravin (pod ved. J . Davidka) SNTL — Praha (1981), 255. [14] LINKO, Y . Y., SAARINEN, P . , LINKO, M . : Biotechnol. Bioeng. 17 (1975), 153.

Book Review M. A.

BOROWITZKA,

L. J.

BOROWTTZKA

(Eds.)

Micro-algal Biotechnology Cambridge, New York, New Rochelle, Melbourne, Sydney: Cambridge University Press, 1988, 477 S., 86 Tab., 37 Abb., $ 79.50, ISBN 0521-32349-5

Ein hervorragendes Werk, geschrieben von 19 prominenten Vertretern der Biotechnologie der Mikroalgen. In 16 Kapiteln und einem Anhang, der die Zusammensetzung häufig gebrauchter Nährmedien sowie eine Aufzählung wichtiger Algen-Stammsammlungen enthält, findet der Leser nahezu alles Wissenswerte über biotechnologisch relevante Algenspezies, über Produkte und Anwendungen der Mikroalgen sowie über technologische Aspekte der Algen-Massenkultivierung. Die gesamte Darstellung berücksichtigt modernste Erkenntnisse und läßt die gewaltigen Fortschritte erkennen, die in den vergangenen Jahren mit dem Ziel der biotechnischen Nutzung der Potenzen photosynthisierender Mikroorganismen erzielt wurden. Besonders wertvoll sind die allgemeingültigen Kapitel zum Generalthema. Die Gliederung des Buches bringt es mit sich, daß Aussagen, die für alle Mikroalgen gelten, teilweise lediglich am Beispiel einer Gattung getroffen werden. Vielleicht hätte die zusammengefaßte Behandlung von Spezies und zugehörigem Produkt bzw. Anwendungsgebiet mehr Raum für Ausführungen über die spezifischen Eigenschaften phototropher Mikroorganismen geschaffen. Das Buch orientiert sich überwiegend an den Problemen der Freilandkultivierung in klimatisch bevorzugten Regionen und setzt sich nur selten mit den Potenzen mikrobieller Photosynthese in gemäßigten Klimazonen und unter Nutzung von Kunstlicht auseinander. Sowohl Ausstattung als auch die bis ins Detail sorgfältige Behandlung des Stoffes gewähren eine ausgezeichnete Lesbarkeit des Buches, dessen Lektüre für alle Biotechnologen und an biotechnologischen Fragestellungen Interessierte ein großer Gewinn ist. P . ROTH

Acta Biotechnol. 8 (1989) 6

554

Literatur [ 1 ] MADSEN, G . B . , NORMAN, B . E . , SLOTT, S . : D i e S t ä r k e 2 5 ( 1 9 7 3 ) , 3 0 4 .

[2] SAITO, N.: Arch. Biochem. Biophys. 155 (1973), 290. [ 3 ] CHIANG, J . P . , A L T E R , J . E . , S T E R N B E R G , M . : Die Stärke

3 1 (1979), 86. [ 4 ] MORGAN, P . J . , P R I E S T , F . G . : J . A p p l . B a c t e r i o l o g y 5 0 ( 1 9 8 1 ) , 1 0 7 . [ 5 ] J O H N , M . , SCHMIDT, J . , W A N D R E Y , C h . , SALM, H . : J . C h r o m . 2 4 7 ( 1 9 8 2 ) , 2 8 1 . [ 6 ] ROLLINGS, J . E., OKOS, M . R . , TSAO, G . T . : Found. Biochem. Eng. Kinet. Thermodyn.

Biol. Syst., Winter Symp. ACS Div. Ind. Eng. Chem., Boulder Colo, 17—20. 01.1982, Washington DC, Abstracts (1983), 443.

[ 7 ] EAJibCHC A . E . , EAXMATOBA, H . B . , HIOPJIHC, T . K . , TJIEMHTA, A . A . , CHHHI^HH, A . TAJIEBOPOBCKAH, H . K . : EHOXHMHH 5 1 , ( 1 9 8 6 ) 3 , 3 7 8 . [ 8 ] SCHIMIZU, M . , K A N N O , M . , TAMURA, M . , S U E K A N E , M . : B i o l . C h e m . 4 2 ( 1 9 7 8 ) , 1 6 8 1 .

II.,

[9] UCHINO, F . : Agr. Biol. Chem. 46 (1982), 7. [ 1 0 ] BESCHKOV, M . , EMANTJILOVA, E . , GRIGOROVA, R . , KOSTURKOVA, P . , DOBREVA, E . , TONKOVA, A . , SCHIVAROVA, N . , KAMENOV, K . , KOMINKOVA, V . : Bulg. Urh.-schein Nr. 3 7 7 6 5 , ( 1 9 8 3 ) . [ 1 1 ] PANTSCHEV, C h . , K L E N Z , G . , H Ä F N E R , B . : L e b e n s m i t t e l i n d . 2 8 ( 1 9 8 1 ) , 7 1 .

[12] SOMOGYI, M.: J. Biol. Chem. 195 (1952), 19. [13] DAviDEK, J., Laboratorni priruöka analyzy potravin (pod ved. J . Davidka) SNTL — Praha (1981), 255. [14] LINKO, Y . Y., SAARINEN, P . , LINKO, M . : Biotechnol. Bioeng. 17 (1975), 153.

Book Review M. A.

BOROWITZKA,

L. J.

BOROWTTZKA

(Eds.)

Micro-algal Biotechnology Cambridge, New York, New Rochelle, Melbourne, Sydney: Cambridge University Press, 1988, 477 S., 86 Tab., 37 Abb., $ 79.50, ISBN 0521-32349-5

Ein hervorragendes Werk, geschrieben von 19 prominenten Vertretern der Biotechnologie der Mikroalgen. In 16 Kapiteln und einem Anhang, der die Zusammensetzung häufig gebrauchter Nährmedien sowie eine Aufzählung wichtiger Algen-Stammsammlungen enthält, findet der Leser nahezu alles Wissenswerte über biotechnologisch relevante Algenspezies, über Produkte und Anwendungen der Mikroalgen sowie über technologische Aspekte der Algen-Massenkultivierung. Die gesamte Darstellung berücksichtigt modernste Erkenntnisse und läßt die gewaltigen Fortschritte erkennen, die in den vergangenen Jahren mit dem Ziel der biotechnischen Nutzung der Potenzen photosynthisierender Mikroorganismen erzielt wurden. Besonders wertvoll sind die allgemeingültigen Kapitel zum Generalthema. Die Gliederung des Buches bringt es mit sich, daß Aussagen, die für alle Mikroalgen gelten, teilweise lediglich am Beispiel einer Gattung getroffen werden. Vielleicht hätte die zusammengefaßte Behandlung von Spezies und zugehörigem Produkt bzw. Anwendungsgebiet mehr Raum für Ausführungen über die spezifischen Eigenschaften phototropher Mikroorganismen geschaffen. Das Buch orientiert sich überwiegend an den Problemen der Freilandkultivierung in klimatisch bevorzugten Regionen und setzt sich nur selten mit den Potenzen mikrobieller Photosynthese in gemäßigten Klimazonen und unter Nutzung von Kunstlicht auseinander. Sowohl Ausstattung als auch die bis ins Detail sorgfältige Behandlung des Stoffes gewähren eine ausgezeichnete Lesbarkeit des Buches, dessen Lektüre für alle Biotechnologen und an biotechnologischen Fragestellungen Interessierte ein großer Gewinn ist. P . ROTH

Acta Biotechnol. 9 (1989) 6, 555—564

Akademie-Verlag Berlin

Simultaneous Saccharification and Fermentation of Cellulose: Effect of Ethanol and Cellulases on Particular Stages SZCZODBAK, J . 1 , TABGONSKI, Z. 2 1

2

Maria Curie-Sklodowska University Department of Applied Microbiology 19 Akademicka Street, 20-033 Lublin, Poland Agricultural University Department of Food Technology 13 Akademicka Street, 20-934 Lublin, Poland

Summary The effects of ethanol and Trichoderma reesei cellulase on the saccharification and fermentation processes as well as the tolerance of the cellulase complex for ethanol have been investigated. The studies were conducted with respect to their usefulness in the process of simultaneous saccharification and fermentation of cellulose to ethanol. The following results were obtained. 1) Fermentative activity of Kluyveromyces fragilis yeasts was gradually depressed with increasing initial ethanol concentrations and temperature of fermentation between 35—46 °C. 2) Crude cellulase preparation introduced to the culture broth to a final enzyme activity of 0.5 to 2.0 FPU/ml had not distinct effect on the biomass production, ethanol yield, and glucose uptake by yeasts in 48 h fermentation at 43 °C. On the other hand, only a negligible decrease in the cellulase complex activity was observed during fermentation process. 3) Saccharification of wheat straw was inhibited by at least 1% w/v ethanol. 4) The enzymes of the cellulase system showed a high stability to exposure to ethanol for 48 h at 43 °C.

Introduction The production of ethanol from renewable cellulosic materials is attractive and seems promising [1, 2]. This process is composed of two different steps: saccharification of cellulose to glucose b y microbial cellulases and yeast or bacterial fermentation of glucose. A one-stage system coupling the enzymatic conversion of cellulose with ethanol fermentation, known as simultaneous saccharification and fermentation (SSF), has been reported by T A K A G I et al. [3]. A number of studies of SSF have been conducted employing different cellulases and ethanol-producing organisms [4—8]. The economics of the process based on Trichoderma reesei cellulase and Saccharomyces cerevisiae have been evaluated by the Gulf Oil Company after pilot-scale studies [9, 10]. SSF process offers a t least four potential benefits compared to the conventional two-step approach: one reactor and associated equipment are eliminated, the reaction time is considerably shortened, the presence of ethanol in the fermentation broth reduces the possibility of infection, and end product inhibition of cellulase by the product sugars is substantially diminished results in faster saccharification rates and higher yields of ethanol. Restrictions associated with the process include different temperature optima for the action of enzymes and ethanol-producing cultures, problem of the compatibility between fermenting cells and enzyme, and the possible inhibitory effect of ethanol on the cellulase system and fermentation activity.

556

Acta Biotechnol. 9 (1989) 6

Due to complexity of the S S F process, the investigation of the e x t e n t and degree of the mutual influences between cellulase used, fermenting organism and ethanol produced is impossible in one-stage system. To fully understand the process of S S F , however, it is important to determine and optimize these effects in order to make the simultaneous system efficient. A few studies on this theme have been reported but the presented results are inconsistent [11 — 14], Recently [15], we have selected the thermotolerant yeast strain Kluyveromyces fragilis F T 2 3 able to produce high ethanol yield a t temperatures above 4 0 °C. I t was then successfully utilized together with T. reesei cellulase for ethanol production from straw in an optimized S S F t y p e system [16]. I n this paper, the effects of ethanol and cellulases on saccharification and fermentation processes as well as the tolerance of cellulase preparation for ethanol were evaluated in cellulolysis conditions.

Materials and Methods Microorganisms The thermotolerant yeast strain Kluyveromyces fragilis FT23 selected earlier [15] was employed in ethanolic fermentations. The composition of basal medium for yeast maintenance was given previously [15], The precultures (100 ml in 500-ml conical flasks) were inoculated from malt agar slants and incubated with shaking for 24 h at 28 °C in the basal medium with 20 g/1 glucose. This seed was then used to inoculate the fermentation tests. The mould Trichoderma reesei F-522 (QM9414) was taken from the Czechoslovak Collection of Microorganisms in Brno. Conidia for the inoculations were produced on potato dextrose agar slants for 7 days at 28 °C. Cellulases Cellulases were obtained from 5 d shake flask cultures of T. reesei at 28 °C on M A N D E L S and W E B E R [17] medium (pH 5.5) using 0 . 7 % cellulose powder (CP-123, S C H L E I C H E R and S C H U L L , GmbH, F.R.G.) as inducing carbon source. The medium was seeded with 10% (v/v) of the 48 h mycelium, previously incubated with shaking in the same medium but with 20 g/1 glucose. Crude cellulase preparation (also including hemicellulases) was obtained from the culture supernate by about threefold concentration in a rotary vacuum evaporator [18]. I t contained the following enzyme activities (IU/ml): cellulase (FPase), 2.61; endoglucanase, 5.9; /3-glucosidase, 0.097; and xylanase, 43.78. Effect of Ethanol and Cellulases on Alcoholic

Fermentation

Glucose fermentations by Kl. fragilis were conducted in 300-ml conical flasks, each containing 200 ml basal medium (pH 5.1) with initial glucose concentration of 140 g/1. Different initial concentration of ethanol ranging from 0 to 80 g/1 were added to the fermentation medium before inoculation. The flasks were inoculated with a 1 0 % v/v seed culture of yeast. Fermentations were carried out for 48 h in the temperature range 35—46 °C on a water bath rotary shaker at 150 rpm. Ethanol evaporation was prevented by rubber stoppers with fermentative tubes filled with 5 0 % H 2 S 0 4 . The effect of cellulases on ethanolic fermentation was studied in 48 h at 43 °C in shake flask cultures with 90 g glucose/1. Crude preparation was introduced to the culture medium to a final cellulases activity of 0.5, 1.0, and 2.0 F P U (Filter paper unit) per ml. The tests with thermally inactivated enzymes served as a control. Fermentations were run in 100-ml conical flasks with a 80-ml working volume. All other conditions were as described. Samples were harvested aseptically and assayed for ethanol yield, glucose uptake, and production of yeast biomass. In the case of the cellulase effect, they were additionally analyzed after fermentation for the activities of the enzymes. To eliminate the influences of ethanol during determination of enzyme activity, the samples were first vacuum-distilled at 39 °C and then filled with buffer to make up for lost volume.

SZCZODRAK, J., TARGOI^SKI, Z., Saccharification and Fermentation of Cellulose

557

Effect of Ethanol on Saccharification Wheat straw was utilized as waste cellulose in the saccharification experiments. It was milled and the median fraction (0.2 to 0.5 mm) was either exposed for 10 min to autohydrolysis at 200 °C [19], or chemical pulping by a mixture of equal volumes of 96% ethanol and 8% NaOH (30 min at 170°C) [20]. After the pretreatment the samples were thoroughly water washed, pressed out to obtain straw of about 26 wt.% dry matter and stored at — 18°C until use. Hydrothermally and chemically transformed straw contained 63.6% and 85.5% carbohydrates (hexosans plus pentosans), respectively. The neutral straw material (wet) was hydrolyzed with cellulase. The reaction mixture (50 ml) in 0.05 M citrate buffer pH 4.5 contained: 2.5% (dry basis) of the pretreated straw, 20 FPU/g straw of T. reesei cellulase, and ethanol ranging from 0 to 10% (w/v). The hydrolysis was carried out in plugged conical flasks (100 ml) in the presence of 0.01% sodium azide. The flasks were incubated for 48 h at 43 °C on a water bath shaker, agitated at 150 rpm. Samples were removed periodically, centrifuged, and the supernatants were assayed for tota' reducing sugars. The percent of saccharification was calculated using the equation: „ , .„,. reducing sugars formed X 0.9 Sacharification (%) = —2 X 100 carbohydrates in straw Effect of Ethanol on Stability and Activity of Cellulases The stability of the cellulase preparation for ethanol was evaluated by incubating it for 48 h at 43 °C in the presence of alcohol but in the absence of substrate. The reaction mixture (80 ml) in 0.05 M citrate buffer pH 4.5 contained: 0.5 FPU/ml of T. reesei cellulase, 0 to 10% w/v of ethanol, and 0.01% of sodium azide. All tests were conducted in stopped conical flasks (100 ml) on a water bath shaker at 150 rpm. Samples were withdrawn at a regular interval, and after vacuum distillation analyzed for enzyme activities. The effect of ethanol on cellulase and xylanase activities was tested in the presence of substrate. Ethanol was added to the incubating mixtures for determinations of the activities of particular enzymes in final concentration of 0 to 10% w/v. The reaction time was 30 min for /9-glucosidase and xylanase, 10 min for endoglucanase, and 60 min for cellulase (FPase). Temperature was 50°C and pH 4.8. The activities were expressed as a percentage of the values that were obtained when the enzymes were assayed in the absence of ethanol. Assays Cellulase (FPase) and /3-1,4-glucosidase (/?GDase) activities were assayed by the methods recommended by IUPAC [21], using WHATMAN NO 1 filter paper and cellobiose as substrates. Endoglucanase (CMCase) activity was estimated viscometrically with carboxymethylcellulose (KochLight Laboratories Ltd) as a substrate [22]. Xylanase activity was determined by the release of xylose from a xylan solution [23]. The activities of the enzymes were expressed in International Units (|xmol product/min/ml enzyme). In the case of CMCase, unit represents ¡xmol of /3-1,4-glucosidic bonds broken per min per ml enzyme. Total reducing sugars and glucose were measured by the dinitrosalicylic acid [24] and glucose oxidase-peroxidase [25] methods, respectively. Ethanol was analyzed according to KELLERMANN [26]. The growth of yeast cells (biomass) was monitored by correlating cell dry weight with optical density at 650 nm [27]. The content of hexosans and pentosans in straw was determined with o-toluidine reagent [28].

Results and Discussion Effect of Ethanol and CeUulases on Fermentation Activity of Yeasts An attempt was made to evaluate the tolerance of selected yeast strain Kluyveromyces fragilis F T 2 3 to initial ethanol concentration ranging from 0 to 80 g/1 at temperatures 35, 40, 43 and 46 °C. It is noteworthy that the total ethanol concentration to which the

558

Acta Biotechnol. 9 (1989) 6

yeast cells were exposed during the course of fermentation was the initial amount added to the broth plus the additional ethanol produced. The results (Fig. 1) indicate that supplemental ethanol had a strongly inhibitory effect on cell growth, glucose uptake rate, and ethanol efficiency which were gradually decreased with increasing alcohol concentration and temperature of fermentation. The most inhibition of fermentation activity was observed at 46 °C. For example, at initial ethanol concentration of 0 and 40 g/1 the net ethanol concentration produced by yeasts at 35 °C was found to decrease 70 60 50

oc a -c Uj

40

30

30

140

0

120

20

50

30, %O

~\o

360

20

100 80 60

20

30

60,80

0

20

40

40

20 10

< b < oo0 s

10

20

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30 40

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20

40 50

20

30

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30

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8 6

20 30

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4

50

60 "ISO

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D £ Temperature

20 30. mp 43

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Fig. 1. Effect of different initial ethanol concentrations (g/1, see numbers on the diagrams) on biomass formation, glucose utilization, and net ethanol production by Kl. fragilis at various temperatures after 48 h shake flask cultures with 140 g/1 glucose. Initial biomass concentration was about 1.8 g/1 and medium pH 5.3. After fermentation, p H of the culture was near 4.4

from 59 to 18 g/1 whereas at 46 °C it reached values of 35 and 0, respectively. I t should be stressed that the yeasts did not grow or produce ethanol at 35 and 40 °C as the initial alcohol concentration in the liquid medium increased over 60 g/1 or 40 g/1 at 43 and 46 °C. Fig. 1 illustrates also the effect of temperature on ethanolic fermentation (see control tests containing no ethanol). The optimal temperature was 40°C at which the maximal ethanol yield (64 g/1) and total sugar utilization were recorded after 48 h fermentation. I t should be noted that at 43 °C the overall ethanol efficiency was only 12.5% lower as compared with optimal conditions. Therefore, this temperature (nearly optimal for the enzymatic hydrolysis of cellulose) was applied in further studies. In order to increase the ethanol yield attemps were made to acclimatize the yeast by keeping the inoculum at 43 °C in medium with increasing ethanol concentrations or by

SZCZODRAK, J . , T A B G O N S K I , Z . ,

Saccharification and Fermentation of Cellulose

559

successsive subculturing it in anaerobic conditions. In both cases, however, the negative results were obtained. In further studies, the Kl. fragilis yeasts were exposed at 43°C to the action of crude T. reesei cellulase preparation in a final enzyme activity of 0.5, 1.0, and 2.0 FPU per ml of medium. Fig. 2 shows the effect of cellulases on the time course of biomass produc-

Fig. 2. Effect of added cellulases on the ethanolic fermentation of glucose (90 g/1) by K. fragilis in 48 h at 43 °C Cellulase concentration (FPU/ml) : o , 0 (control with thermally inactivated enzymes); o , 0.5; o , 1.0; o , 2.0

tion, glucose consumption, and ethanol formation by fermenting organism. The results indicate that the added enzymes at concentration of 0.5 and 1.0 FPU/ml did not have a significant effect on the fermentation activity of yeasts. At cellulase concentration of 2.0 FPU/ml only small decrease (particularly in the first 24 h fermentation) in biomass formation, glucose utilization, and ethanol yield was noted. The changes in the activities of individual enzymes of the crude cellulase preparation after 48 h fermentation are shown in Tab. 1. It was found that only a negligible loss (maximum 2.5% for filter

560

Acta Biotechnol. 9 (1989) 6 Tab. 1. Activity of T. reesei cellulase preparation after 48 h glucose fermentation by Kl. fragilis at 43 °C Time

Enzyme activity* [IU/ml]

[h]

FPase

CMCase

/ÎGDase

Xylanase

0

0.50 1.00 2.00

1.40 2.80 5.60

0.019 0.037 0.074

8.39 16.77 33.56

48

0.46 0.91 1.75

1.36 2.72 5.50

0.017 0.032 0.070

6.61 11.10 22.20

* Abbreviations: FPase = cellulase, CMCase = endo-l,4-/S-D-glucanase. (SGDaae = l,4-|8-D-glucosidase

paper activity, 2.9% for endoglucanase, and 13.5% for /?-glucosidase) in the cellulase complex activity took place during fermentation process. Xylanase appeared to be the most labile enzyme in fermentation conditions. After 48 h cultivation, the activity of this enzyme decreased from 21% to about 34% compared with its initial activity level. Effect of Ethanol on

Saccharification

The cellulase of T. reesei is a system of synergistic enzymes, and for this reason it is important to measure the effect of ethanol not only on the components of the system, but also on the system as a whole. To accomplish this, saccharifications of hydrothermally and chemically treated wheat straw were conducted to which ethanol was added initially. Saccharification-time profiles of the two treated straw kinds in the presence of ethanol are shown in Fig. 3. The production of reducing sugars decreased by at least 1% (w/v) ethanol and the decreament becomes large with an increase in the concentration of ethanol. A concentration of 10% w/v ethanol decreased after 48 h the saccharification efficiency from 26.5 (for autohydrolysis-treated straw) to 37% (for ethanol-alkali pulped straw) as compared to a control containing no ethanol. In SSF, there remains a problem as to whether the ethanol produced inhibits the enzymatic hydrolysis. Published data in this area are conflicting. BLOTKAMP et al. [11] have reported no inhibitory effect on T. reesei cellulase up to the concentration of 5% w/v ethanol in the reaction mixture. On the other hand, TAKAGI et al. [3] have shown that the saccharification of 10% cellulose by the same enzyme as that of BLOTKAMP is depressed to 70% at ethanol concentrations up to 6% w/v. GHOSH et al. [12] have reported that T. reesei QM9414 cellulase is inhibited uncompetitively by at least 0.75% ethanol and a concentration of 7.5% w/v ethanol was needed to halve the saccharification rate. Similar results for Trichoderma viride cellulase were obtained by OOSHEVLA et al. [14]. Our findings confirmed an inhibitory effect of ethanol on the enzymatic hydrolysis of cellulose but the extent of alcohol inhibition was less than that given by other authors. The mechanism of the inhibitory effect of ethanol on saccharification process is not exactly known. OOSHIMA et al. [14] suggested that ethanol disturbs the adsorption of exoglucanase on the surface of cellulose, resulting in the depression of the saccharification rate.

SZCZODRAK, J . , TAKGO:6SKI, Z., Saccharification and Fermentation of Cellulose

561

Fig. 3. Effect of various ethanol concentrations (% w/v, see numbers on the curves) on saccharification of hydrothermally (A) and ethanol-alkali (B) treated wheat straw Substrate, 2 . 5 % ; T. reesei cellulase, 20 F P U / g straw; 43°C; pH 4.5

The effect of ethanol on the and determination cellulase activities is presented in Tab. 2. Effect of Ethanol on Activity Stability of ofCelluloses Ethanol was added to culture filtrates of T. reesei in final ethanol concentrations of 0 to 10%, and enzymes were assayed according to standard methods. Both the FPase and xylanase were significantly inhibited at ethanol concentration above 2%. A concentration of 10% w/v ethanol was needed to halve the enzyme effectiveness as compared with a control containing no ethanol. The endoglucanase and /S-glucosidase activities were not affected by the supplemental ethanol. Other results in this area were obtained by HOGAN et al. [13] for the cellulase and xylanase systems of Trichoderma harzianum E58. They have shown that the introduced ethanol did not affect the cellulase activities while the xylanase activity was slightly enhanced at higher ethanol concentration. The stability of the cellulase preparation in exposure to ethanol was tested by incubating it for 48 h at 43 °C in the presence of a range of ethanol concentrations (0 to 10% w/v) but in the absence of cellulosic substrate. Following exposure to ethanol the samples were vacuum-distilled and their effectiveness determined through measuring the enzymatic activity for a given substrate. No effect of exposure to ethanol on the activity of the cellulase complex was observed (Tab. 3). Only xylanase activity was depressed by ethanol. For example, after 48 h incubation in 10% ethanol its activity was decreased 5 Acta. Biotechnol. 9 (1989) 6

562

Acta Biotechnol. 9 (1989) 6 Tab. 2. Effect of ethanol on the determination of T. reesei cellulase and xylanase activities* Ethanol added [ % w/v]

% Effectiveness FPase

CMCase

/SGDase

Xylanase

0 2 4 6 8 10

100 81 72 70 61 50

100 100 100 100 102 100

100 101 100 95 93 93

100 92 82 77 71 58

* Abbreviations as in Tab. 1 The detailed experimental conditions are described in the Materials and Methods section

Tab. 3. Effect of ethanol on the stability of T. reesei cellulase preparation in 48 h incubation at 43 °C and pH 4.5 Ethanol added [ % w/v]

Time

Enzyme activity* [IU/ml]

[h]

FPase

CMCase

ßGBaae

Xylanase

0.0 1.0 2.5 4.0 6.0 8.0 10.0 0.0 1.0 2.5 4.0 6.0 8.0 10.0 0.0 1.0 2.5 4.0 6.0 8.0 10.0 0.0 1.0 2.5 4.0 6.0 8.0 10.0

0

0.69 0.68 0.70 0.71 0.68 0.58 0.59 0.65 0.62 0.66 0.65 0.64 0.57 0.56 0.74 0.86 0.81 0.91 0.80 0.71 0.70 0.78 0.85 0.84 0.82 0.78 0.75 0.72

1.96 1.90 1.96 1.96 1.93 1.90 1.90 1.96 1.92 1.93 1.92 1.96 1.92 1.90 1.96 1.92 1.92 1.92 1.90 1.90 1.90 1.90 1.88 1.88 1.88 1.88 1.90 1.88

0.026 0.026 0.026 0.026 0.025 0.023 0.021 0.027 0.025 0.026 0.027 0.025 0.026 0.025 0.028 0.034 0.033 0.033 0.028 0.027 0.027 0.030 0.031 0.030 0.031 0.027 0.028 0.027

16.39 17.01 16.55 16.09 13.96 12.54 10.23 15.17 13.82 13.96 14.57 13.35 12.00 8.28 13.64 15.01 13.51 14.58 9.25 7.75 6.65 13.35 12.72 11.66 11.22 6.36 6.43 5.21

4

24

48

•Abbreviations as in Tab. 1

SZCZODRAK, J . , TAEGOÄSKI, Z., Saccharification and Fermentation of Cellulose

563

by 61% compared with the unexposed control. These results are comparable with data compiled by G H O S H et al. [ 1 2 ] , who have demonstrated the high stability of T. reesei cellulase in 24 h incubation at 35—50 °C in the presence of ethanol concentration between 0 to 7 . 5 % w/v. Also OOSHIMA et al. [ 1 4 ] have found endoglucanase from T. viride not to be affected in 2 4 h at 4 0 ° C by less than 1 8 . 4 wt% ethanol. B y contrast, H O G A N et al. [13] reported a reduction in effective cellulase and xylanase activities at ethanol concentrations up to 10% w/v after 24 h incubation at 45°C. I t should be noted that the authors have not eliminated (by dilution or distillation) the inhibitory effect of ethanol during determination of enzyme activities. The presented results show the good compatibility of K. fragilis cells with T. reesei cellulases. The fermenting yeasts at 43 °C tolerated the enzyme concentration of 2 F P U per ml medium and had a considerable tolerance to ethanol under such conditions. On the other hand, the small decrease in the cellulase complex activity during fermentation process indicate that yeast cells and/or their metabolic products were to a certain degree neutral in relation to enzymatic preparation. These facts together with a high stability of T. reesei cellulase for ethanol seem to lend hope to wider utilization of these two biosystems for the simultaneous saccharification and fermentation of cellulose. Received October 27, 1988

Acknowledgements This work was supported by the Polish Scientific Project No 04.11./2.27 and by the Maria CurieSklodowska University ( B W / 2 ) .

References P. J . , E M E R T , G. H . — In: Fuels from Biomass and Wastes. Eds.: D . L. KLASS and G. H. EMERT, Ann Arbor Science Publication , Ann Arbor, Michigan, 1981, p. 395. [2] WOODWARD, J . : Spec. Publ. Soc. Gen. Microbiol. 21 (Carbon Substrates Biotechnol.) (1987), 45. [ 3 ] T A K A G I , M . , A B E , S., S U Z U K I , S., E M E R T , G . H . , Y A T A , N. — In: Proceedings of Byconversion Symposium. E d . : T. K . GHOSE, IIT, New Delhi, 1977, p. 551. [ 4 ] D E S H P A N D E , V . , SIVARAMAN, H . , R A O , M . : Biotechnol. Bioeng. 2 5 ( 1 9 8 3 ) , 1 6 7 9 .

[ 1 ] B E C K E R , D . K . , BLOTKAMP,

[5] GHOSE, T . K . , ROYCHOTJDHURY, P . K . , GHOSH, P . : Biotechnol. Bioeng. 2 6 (1984), 3 7 7 .

S.: Hakkokogaku 6 5 ( 1 9 8 5 ) , 4 2 7 . G. H . : Biotechnol. Bioeng. 2 8 ( 1 9 8 6 ) , 1 1 5 . [8] W Y M A K , C . E . , S P I N D L E R , D . D . , GROHMANN, K . , LASTIO, S. M . : Biotechnol. Bioeng. Symp. 17 (1986), 221. [ 9 ] E M E R T , G . H . , K A T Z E N , R . : Chemtech. 1 0 ( 1 9 8 0 ) , 6 1 0 . [ 1 0 ] E M E R T , G . H . , K A T Z E N , R . , F R E D R I C K S O N , R . E . , KAITPISCH, K . F . : Chem. Eng. Prog. 7 6 [ 6 ] A R A I , M . , OOI, T . , H A Y A S H I , H . , M U R A O , [ 7 ] SPANGLER, D . J . , E M E R T ,

(1980), 47.

[ 1 1 ] BLOTKAMP, P . J . , TAKAGI, M., PEMBERTON, M. S., EMERT, G. H . : A m . I n s t . Chem. E n g . S y m p .

Ser. No. 181, 74 (1978), 85. N. B . , M A R T I N , W . R . B . : Enzyme Microb. Technol. 4 ( 1 9 8 2 ) , 4 2 5 .

[ 1 2 ] GHOSH, P . , PAMMENT,

[ 1 3 ] HOGAN, C. M., MES-HARTREE, M., SADDLER, J . N. — I n : Proceedings of Third E u r o p e a n Con-

gress on Biotechnology. Munich, West Germany, Verlag Chemie, Weinheim, 2 ( 1 9 8 4 ) , 3 9 5 . Biotechnol. Bioeng. 2 7 ( 1 9 8 5 ) , 3 8 9 . [15] SZCZODRAK, J . , TARGONSKI, Z.: Biotechnol. Bioeng. 31 (1988), 300. [16] SZCZODRAK, J . : Biotechnol. Bioeng. 82 (1988), 77.1.

[ 1 4 ] OOSHIMA, H . , I S H I T A N I , Y . , H A R A N O , Y . :

5*

Acta Biotechnol. 9 (1989) 6

564

[ 1 7 ] MANDELS, M . , W E B E B , J . : A d v . C h e m . S e r . 9 5 ( 1 9 6 9 ) , 3 9 1 . [ 1 8 ] ROGALSKI, J . , SZCZODRAK, J . , ILCZUK, Z . : A c t a M i c r o b i o l . P o l o n . 3 2 , ( 1 9 8 3 ) , 3 6 3 .

[19] TARGONSKI, Z.: Enzyme Microb. Technol. 7 (1985), 126. [ 2 0 ] MARTON, R . , GRANZOW, S . : T a p p i 6 5 ( 1 9 8 2 ) , 1 0 3 .

[21] GHOSE, T. K.: Pure Appi. Chem. 59 (1987), 257. [ 2 2 ] RABINOVICH, M . L . , KLYOSOV, A . A . , BIEBIEZIN, J . V . : B i o o r g a n i c h e s k a y a k h i m i y a 8 ( 1 9 7 7 ) ,

405. [23] DESCHAMPS, F . , HUET, M. C.: Appi. Microbiol. Biotechnol. 22 (1985), 177. [24] MILLER, G. L . : A n a l . Chem. 3 1 (1959), 426. [ 2 5 ] LLOYD, J . B . , WHELAN, W . J . : A n a l . B i o c h e m . 3 0 ( 1 9 6 9 ) , 4 6 7 .

[26] KELLERMANN, E.: Kvasny prumysl 6 (1960), 11. [27] HUGHES, D. B., TUDROSZEN, N. J., MOYE, C. J . : Biotechnol. Lett. 6 (1984), 1. [ 2 8 ] KOBNEICHIK, T . V . , BOBOVSKAYA, L . A . , ZIL'BEBGLEIT, M . A . : K h i m i y a d r e v e s i n y 5 ( 1 9 8 6 ) , 42.

Book Review F . B . ALTHATTS, C H . R I C H T E R

ADP-Ribosylation of Proteins Enzymology and Biological Significance Berlin, Heidelberg, New York, London, Paris, Tokyo: Springer-Verlag, 1987. 237 S., 16 Abb., 10 Tab., DM 1 9 8 , - , ISBN 3-540-17734-5

Die Autoren haben sich der Aufgabe unterzogen, die in ca. 20 Jahren Forschung erzielten Ergebnisse in einer Monographie zu ordnen. Sie haben den gesamten Stoff zweigeteilt. Im Teil 1 (Verfasser F. R. ALTHAUS) werden die Poly-ADP-Ribosylierungsreaktionen (122 Seiten) und im 2. Teil (Verfasser C. RICHTEB, 100 Seiten) die Mono-ADP-Ribosylierungsreaktionen behandelt. Die Autoren haben sich zu dieser Aufteilung eher aus Zweckmäßigkeitsgründen entschlossen; sachlichinhaltlich gibt es keinen zwingenden Grund, denn es besteht — wie die Autoren selbst feststellen — wahrscheinlich ein enger funktioneller Zusammenhang zwischen beiden Klassen, wofür ihre Koexistenz in Eukaryoten durchaus spricht. Teil 1 umfaßt 9 Kapitel. Sie beinhalten die Struktur, Eigenschaften und quantitative Bestimmung, die Biosynthese und den Katabolismus von Poly(ADP-Ribose), die Kernakzeptorproteine und die funktionellen Konsequenzen der Poly-ADP-Ribosylierung für die Akzeptorspezies. Dargestellt werden Poly-ADP-Ribosylierungsreaktionen am Chromatin, bei der Erholung von Säugerzellen nach DNA-Schädigung, ihre Rolle bei der DNA-Synthese, im Zellzyklus, für die Zelldifferenzierung und Genexpression sowie Erbkrankheiten. Der 2. Teil enthält die Kapitel Signalwandlung, Cholera-, Pertussis- und Diphtherie-Texin, zelluläre Transferasen und Mitochondrien. Auf den letzten 10 Seiten des Kapitels "The Bond" werden dieBindungs- und „Verbindungs"möglichkeiten mit den verschiedensten „Akzeptoren" diskutiert; die ester-, diphthamid-, arginin- und cystein-spezifischen ADP-Ribosylierungen werden ebenso behandelt wie Ribosylierungen an unbekannten Akzeptorstellen, nichtenzymatisch katalysierte Ribosylierungen (z. B. kovalente Addukte mit Poly-L-Lysin) und die Freisetzung von Akzeptorsteilen. Jedes Kapitel schließt mit einem Literaturverzeichnis, das hilft, weiter in die doch sehr komprimiert dargestellte Problematik vorzudringen. Es handelt sich um ein Buch, das nach Inhalt und Form hohen Ansprüchen gerecht wird. Es wendet sich vor allem an in Lehre und Forschung stehende Biochemiker, Molekularbiologen und Mediziner, nicht so sehr an den praktizierenden Arzt, und ist mit großem Gewinn zu lesen. W . BABEL

Acta Biotechnol. 9 (1989) 6

564

[ 1 7 ] MANDELS, M . , W E B E B , J . : A d v . C h e m . S e r . 9 5 ( 1 9 6 9 ) , 3 9 1 . [ 1 8 ] ROGALSKI, J . , SZCZODRAK, J . , ILCZUK, Z . : A c t a M i c r o b i o l . P o l o n . 3 2 , ( 1 9 8 3 ) , 3 6 3 .

[19] TARGONSKI, Z.: Enzyme Microb. Technol. 7 (1985), 126. [ 2 0 ] MARTON, R . , GRANZOW, S . : T a p p i 6 5 ( 1 9 8 2 ) , 1 0 3 .

[21] GHOSE, T. K.: Pure Appi. Chem. 59 (1987), 257. [ 2 2 ] RABINOVICH, M . L . , KLYOSOV, A . A . , BIEBIEZIN, J . V . : B i o o r g a n i c h e s k a y a k h i m i y a 8 ( 1 9 7 7 ) ,

405. [23] DESCHAMPS, F . , HUET, M. C.: Appi. Microbiol. Biotechnol. 22 (1985), 177. [24] MILLER, G. L . : A n a l . Chem. 3 1 (1959), 426. [ 2 5 ] LLOYD, J . B . , WHELAN, W . J . : A n a l . B i o c h e m . 3 0 ( 1 9 6 9 ) , 4 6 7 .

[26] KELLERMANN, E.: Kvasny prumysl 6 (1960), 11. [27] HUGHES, D. B., TUDROSZEN, N. J., MOYE, C. J . : Biotechnol. Lett. 6 (1984), 1. [ 2 8 ] KOBNEICHIK, T . V . , BOBOVSKAYA, L . A . , ZIL'BEBGLEIT, M . A . : K h i m i y a d r e v e s i n y 5 ( 1 9 8 6 ) , 42.

Book Review F . B . ALTHATTS, C H . R I C H T E R

ADP-Ribosylation of Proteins Enzymology and Biological Significance Berlin, Heidelberg, New York, London, Paris, Tokyo: Springer-Verlag, 1987. 237 S., 16 Abb., 10 Tab., DM 1 9 8 , - , ISBN 3-540-17734-5

Die Autoren haben sich der Aufgabe unterzogen, die in ca. 20 Jahren Forschung erzielten Ergebnisse in einer Monographie zu ordnen. Sie haben den gesamten Stoff zweigeteilt. Im Teil 1 (Verfasser F. R. ALTHAUS) werden die Poly-ADP-Ribosylierungsreaktionen (122 Seiten) und im 2. Teil (Verfasser C. RICHTEB, 100 Seiten) die Mono-ADP-Ribosylierungsreaktionen behandelt. Die Autoren haben sich zu dieser Aufteilung eher aus Zweckmäßigkeitsgründen entschlossen; sachlichinhaltlich gibt es keinen zwingenden Grund, denn es besteht — wie die Autoren selbst feststellen — wahrscheinlich ein enger funktioneller Zusammenhang zwischen beiden Klassen, wofür ihre Koexistenz in Eukaryoten durchaus spricht. Teil 1 umfaßt 9 Kapitel. Sie beinhalten die Struktur, Eigenschaften und quantitative Bestimmung, die Biosynthese und den Katabolismus von Poly(ADP-Ribose), die Kernakzeptorproteine und die funktionellen Konsequenzen der Poly-ADP-Ribosylierung für die Akzeptorspezies. Dargestellt werden Poly-ADP-Ribosylierungsreaktionen am Chromatin, bei der Erholung von Säugerzellen nach DNA-Schädigung, ihre Rolle bei der DNA-Synthese, im Zellzyklus, für die Zelldifferenzierung und Genexpression sowie Erbkrankheiten. Der 2. Teil enthält die Kapitel Signalwandlung, Cholera-, Pertussis- und Diphtherie-Texin, zelluläre Transferasen und Mitochondrien. Auf den letzten 10 Seiten des Kapitels "The Bond" werden dieBindungs- und „Verbindungs"möglichkeiten mit den verschiedensten „Akzeptoren" diskutiert; die ester-, diphthamid-, arginin- und cystein-spezifischen ADP-Ribosylierungen werden ebenso behandelt wie Ribosylierungen an unbekannten Akzeptorstellen, nichtenzymatisch katalysierte Ribosylierungen (z. B. kovalente Addukte mit Poly-L-Lysin) und die Freisetzung von Akzeptorsteilen. Jedes Kapitel schließt mit einem Literaturverzeichnis, das hilft, weiter in die doch sehr komprimiert dargestellte Problematik vorzudringen. Es handelt sich um ein Buch, das nach Inhalt und Form hohen Ansprüchen gerecht wird. Es wendet sich vor allem an in Lehre und Forschung stehende Biochemiker, Molekularbiologen und Mediziner, nicht so sehr an den praktizierenden Arzt, und ist mit großem Gewinn zu lesen. W . BABEL

Acta Biotechnol. 9 (1989) 6, 5 6 5 - 5 7 5

Akademie-Verlag Berlin

Biochemical Reactions of Brevibacterium flavum Depending on Medium Stirring Intensity and Flow Structure RUKLISHA, M. P. 1 , VAIVAGS, J . J . 1 , RIKMANIS, M. A . 1 , TOMA, M. K . 1 , VIESTURS, U . E . 2

1 2

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

Summary Authors have studied the dependence of the physiological and biochemical characteristics of lysine producers Brevibacterium flavum on medium stirring intensity upon constant p02 during fermentation processes. Various factors affecting the rate of biochemical reactions in cells upon a changeable intensity of medium stirring in apparata with turbine stirrers; oxygen mass transfer intensity in the system; stirring characteristics in the fermenter; biochemical changes in the cells due to gradients of energy introduced by the stirrer in the fermenter, etc. have been studied. A comparison of the experimentally established values, characterizing macrostirring (e) and microstirring (KR), with the physiological and chemical characteristics of bacteria during fermentation made us suppose t h a t the character of the kinetic energy distribution in the volume of fermenter is one of the factors regulating bacterial metabolism. Besides, there exists the value e c r l t , upon which there can be observed essential changes of the physiological and biochemical properties of the cells. The effect of medium flows upon the cells is characterized by index jP 3tress , which takes into account both, the possible presence of the cells in the zone with e 2: e c r i t , and the degree of medium flow and cell interaction, numerically equal to value (e — e crit ) 0 - 6 .

Introduction The most often used aeration intensity characteristics in the processes of microbiological synthesis is partial pressure of oxygen (p0 2 ). During microorganism cultivation the optimum value of the latter is ensured by control over oxygen (air) feed, or by medium stirring rate. Experimentators use controllable (N2, 0 2 ) gas mixtures. We have previously demonstrated that upon constant cultivation conditions (p02 included) changes in medium stirring intensity lead to changes in the growth rate and lysine synthesis in Brevibacterium flavum [1,2]. The regulatory activity of medium stirring rate is expressed by the changes in the activity of central carbon metabolism pathway enzymes in Brevibacterium flavum cells and depends also on glucose or sucrose concentration in the medium, which, in its turn, changes the rate of their transport into the cell. I t can be suggested, that beside substrate concentrations, stirring intensity, and 0 2 and C0 2 dissolved in the medium, the hydrodynamic factors of fermentation liquid flow, leading to structural and functional changes of the cells, can also be metabolism regulating factors. And more than that, it has been earlier demonstrated, that within a certain range, the limit concentration of 0 2 and stirring intensity are interchangeable values [3].

566

Acta Biotechnol. 8 (1989) 6

The article presents experimental data, demonstrating the regulatory role of medium stirring intensity in Brevibacterium flavum, depending on sugar concentration upon constant p02 values. The authors have compared the hydrodynamical characteristics of the medium flow with the rate of biochemical reactions and metabolic flows in the cells. Materials and Methods The objects of the study were lysine producers Brevibacterium flavum 22LD [4] and Brevibacterium flavum RC-115 [5]. Bacteria were cultivated in fermenters FU-6 and FU-8 [3, 6] with double-tier turbine stirrers and 3 1 working volume. Cultivation media contained: sucrose or molasses within 1—15% (RS); corn steep liquor —2% (DM); (NH 4 ) 2 S0 4 - 2 % ; K H 2 P 0 4 - 0.05%; K 2 H P 0 4 - 0.05%. Cultivation temperature was 30 ° zt 1 °C; aeration intensity, registered as to the partial pressure of oxygen (p02) — 30% of saturation, under various cultivation conditions it was reached by changing the air and nitrogen ratio in the aerating air. Value p02 = 30% falls into the zone of p02, in which there can be observed neither limiting nor inhibition of Brevibacterium flavum growth by oxygen. Velocity of stirrer revolution — from 300 to 1000 rpm. Methods for the determination of the main physiological activity parameters of lysine producers, cell disintegration and metabolism enzyme activity have been described by us earlier [7]. The total reducing activity (TRA) of cells was determined as to the rate of 2,3,5-triphenyltetrazoliumchloride (TTC) upon cell incubation in 1/15 M K/Na phosphate buffer (pH 7.2) in the absence of substrate at 30 °C. We modified the method of cell TRA determination on the basis of the method for the determination of succinate dehydrogenase activity [8]. Cell TRA was expressed in ¡xg of TTC-formasan, formed upon incubation of 100 mg of cells. RNA, DNA and protein content in the cells was determined by methods we have described earlier [1]. Parameters (e — kinetic energy of medium flow fluctuations, KR — an indication of microstirring, that provide a quantitative characteristics of the hydrodynamic state in the fermenter,) were determined as to a method, elaborated at our institute [9, 10]. The method is based on the transformation of medium flow interactions into a proportional electric signal by piezoelectric transducers, placed at various points of the apparatus. Medium viscosity (rj) was determined by viscosimeter REOTEST-2. Medium stirring characteristics were determined in the horizontal plane of fermentation medium, the height of which corresponds to the middle of the height of the upper stirrer. The choice is determined by the fact, that the highest values of dissipated energies and their gradients are observed in this zone. Using a mathematical model of regression [11] there were obtained data used to determine the dependence of kinetic energy e on the following variable parameters: n — velocity of stirrer shaft revolution, r p m ; I — distance of the fermenter point under study from the tip of stirrer blade, mm. 6 = - 3 4 8 3 - 2738 x A - 504.8/B + 1252/A + 704.7 X A/B - 494.4.4.A 3 (1) 3

where A = 0.5 + 0.729 X 10 x n B = 0.5 + 0.024 x I The given dependence was obtained in 5% sucrose media. Information on the distribution of kinetic energy e in fermenter volume is a prerequisite

RTTKLISHA,

M. P.,

VANAGS,

J. J. et al., Medium Stirring Intensity

567

for the determination of values t h a t could characterize the effect of medium flow structure on cell metabolism. To establish the threshold of specific changes in the metabolic pathway of microbial synthesis we determined the maximum energy e raax dissipated in the fermenter. If we assume t h a t in turbine reactors the maximum energy is dissipated in close proximity to the stirrer and the value of I in formula (1) extrapolate to 0, we obtain e max for various stirring regimes : e crit ), determined as follows: W = TkjT

(3)

where T — time of cell circulation (or time of passage through all the zones) [s] Tk — time of cell presence in the critical zone [s] T = V/Q [s]

(4) 3

where V — volume of stirred medium [m ] Q — stirrer productivity [13] [m 3 • s - 1 ] Q = 1c • dz • n

(5)

where k — proportionality coefficient with a turbine stirrer of Standard dimensions ; h = 0.6-1.0 d — stirrer diameter [m] n — velocity of stirrer revolution [rpm]. A model of outlet jet was used to calculate Tk [12, 13] : ik ik Tk = / dljv{l) = 4jrIQ / (dj2 + l)(h + 2lx tg ocj2) dl o o

(6)

Zk — distance of the critical point from the stirrer [m] h — height of stirrer blade [m] d — stirrer diameter [m] ot — angle of outlet jet, grad; it may be within 5° and 30° depending on stirring regime. Since the given case deals with n > 700 rpm and a constant Q, it can be assumed t h a t ex = const. Calculations show t h a t changes of e cr i t produces a mechanical effect on the cell. I n order to establish the e-dependent effect of medium flow upon the cell we have used the criterion Fatre8S : (k tk F*u = f (e(l) - ecrhy dt(l) = / (e(Z) - e c r i t )>(*) dl

(7)

568

Acta Biotechnol. 9 (1989) 6

Besides, it is assumed that the degree of the effect of medium flow on the cell is proportional to product dt(l) X (e(d) — eCrit)a> where dt(l) — duration of cell presence in the zone dl in the distance I from the stirrer. Degree 6 is determined experimentally, comparing to enzyme activity. In the case when < 5 ^ 0 , Fstr - > W. The parameters W and Fatr are aimed at an unambiguous characterization of the hydrodynamic situation in the fermenter. In this case all the parameters determining stirring regime (velocity of stirrer revolution, aerating gas uptake Q, physical and chemical properties of cultivation medium, fermenter construction etc.) are replaced by value W or FstI. Results and Discussion Regulatory activity of medium stirring rate was studied on Br. flavum cultivation media with sucrose concentration within 1—15% or on molasses media of analogous concentrations. Fig. 1 shows the main indications of cell physiological activity (ju, q3, qp) and Fig. 2 — central metabolic pathway enzyme activities at 300 rpm and 1000 rpm of the stirrer. The data indicate to the fact that upon low medium stirring rates an increase of sucrose concentration in Brevibacterium flavum causes as certain activation of TCA-cycle enzymes, decreases the activity of glycolysis enzymes, accelerates growth rate and lysine synthesis by the cells. LDH activity equals nil, at that.

0.3

1.0 0.3 0.8

. 0.2

• 0.2

!0.6

OA 0.1

0.1

-•3

*0.2

4

8

12

S [%]

16

Fig. 1. Changes of physiological activity parameters in Brevibacterium flavum 22LD upon changes in medium stirring rate (I — 300 rpm, II — 1000 rpm) (Conditions of the experiments: 8 — 1 —16%; j»Oa = 30% of saturation) 1 2 - fi; 3 - q3

An increase of sucrose concentration from 2.5 to 10% upon high velocity of stirrer revolution (1000 rpm) produces in the cells of Brevibacterium flavum signs typical of the Crabtree effect (Fig. 2): the activity of TCA-cycle enzymes (AC, SDH) decreases, while that of glycolysis enzymes (F-l, 6-DPA), MDH, GLDH, as well as LDH increases. Under these conditions there can be observed a drastic increase of the endogenous respiration of cells, registered as to cell TRA (Fig. 3), and a drastic decrease of the enzyme DAP-DC activity, which takes part in lysine biosynthesis (Fig. 4)'. If sucrose concentration reaches 10% and stirrer revolution velocities are high, the RNA and protein content, as well as cell growth rate are observed to grow (See Fig. 1,5).

RTJKLISHA, M. P., VANAGS, J . J . et al., Medium Stirring Intensity

5 [%1

St'/, J

Fig. 2. Changes in metabolic enzyme activity in Brevibacterium flavum 22LD upon changes in medium stirring rate (I — 300 rpm, I I — 1000 rpm) (Conditions of the experiments: S — 1 —16%; p02 = 30% of saturation) 1 - P F K ; 2 - L D H ; 3 - AC; 4 - GLDH; 5 - SDH; 6 - MDH

-

^

—%2

^

8

S t%]

12

16

Fig. 3. Changes of cell TRA in Brevibacterium flavum 22LD upon acceleration of stirrer revolution from 300 ( ) to 1000 ( ) rpm Conditions of the experiments see in Fig. 1

5. 3[ Ol s

Fig. 4. Changes in DAP-DC activity in Brevibacterium flavum 22LD upon acceleration of stirrer revolution from 300 ( ) to 1000 ( ) rpm Conditions of the experiments see in Fig. 1

2

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8

12

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570

Acta Biotechnol. 9 (1989) 6

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_

.C £ O

5 % ; n = 1000 rpm) causes a drastic acceleration of oxygen uptake rate by the cells (Qo,). Analogous changes of metabolic enzyme activity in Brevibacterium flavum are observed upon an intensive aeration in the fermenter (Fig. 6), [14], with some changes in the conditions of insufficient aeration intensity [15], as well as upon a direct inhibition of electron transport along the respiratory chain of bacteria [16, 17]. The activity of TCAcycle enzymes decreases, while t h a t of glycolysis enzymes increases and, if there is no limitation of medium ingredients, the sucrose uptake rate grows to a level ensuring a constant rate of A T P synthesis in the cells [16].

p02l% 1

Fig. 6. Changes in PFK (1), AK (2) and SDH (3) activity in Brevibacterium flavum, 22LD upon an increase of pO% from 5 to 60% Conditions of the experiment: 8 = 3.5%, n = 500 rpm

The appearance of the Crabtree effect upon high medium stirring intensity cannot be explained by the regulatory activity of increased sucrose concentrations, since upon low medium stirring intensity there cannot be observed any analogous effect. There are several reasons for the appearance of the effect in Brevibacterium flavum upon an intensive stirring of the medium: 1. The latter may cause an acceleration of oxygen transfer in the system liquid-cell, j.e. the regulatory effect of medium stirring and t h a t of p02 increase are analogous. 2. Cell membrane properties may change, including also damages of the oxidative phosphorylation chain. Recently the Crabtree effect in bacteria has been explained b y a repression of phosphorylation site I of the electron transport chain, namely, NADH 2 dehydrogenase and a repression of succinate dehydrogenase [18]. I t is possible t h a t inactivation of such functions in the respiratory chain of bacteria may be caused b y mechanical membrane damages. An indirect indication of the phenomenon is a drastic decceleration of sucrose uptake rate by the cells upon high medium stirring rate (Fig. 1). We have earlier demonstrated t h a t the motive force of sugar transport in Brevibacterium flavum is the electrochemical membrane potential of the cells [19].

572

Acta Biotechnol. 9 (1989) 6

3. There can be formed zones of insufficient oxygen mass transfer. The role of the factor upon changes in medium stirring rate will be discussed below. In apparata with turbine stirrers high medium Stirring rates cause an uneven distribution of introduced energy (e) in the working volume of the fermenter [10]. This is very expressed at high sucrose concentrations, when medium viscosity increases (Fig. 7). Besides, in the peripheral 1.18-

1.02 •

0-9Ai 5

i 10 SL%1

Fig.7. Dependence of cultivation medium viscosity on sucrose concentration

i 15

zone of fermenter the value of medium microstirring (KR) sharply decreases (Fig. 8). It can be assumed that in the peripheral zone there can appear an insufficient oxygen mass transfer in the system liquid-cell, since it may cause a decrease in the activity of AC — the key enzyme of aerobic metabolism of Brevibacterium flavum (Fig. 9). Using data, demonstrating the solubility of oxygen in media with sucrose concentrations ranging from 1 to 16% (from 2.32 • 10" 4 M to 1.85 • 10" 4 M) [20], we have established that upon biomass content 30 g/1 and p02 = 30% 0 2 concentration may fall to nil in 2—3 sec. Our calculations as to equation 3 show that the time of cell passage through all fermenter zones is notably shorter (0.1—0.5 sec). Consequently, it can be assumed

SC%] Pig. 8. Changes in the value of criterion KR (1) in 5 • 10~3 m distance from the wall of the apparatus depending on sucrose concentration in the medium; 2 — AC activity in Brevibacterium flavum 2 2 L D under given cultivation conditions ( p 0 2 = 30%, n = lOOOrpm)

573

Ruklisha, M. P., Vanags, J. J. et al., Medium Stirring Intensity

sr/.j Fig. 9. Changes in AC activity upon various rates of stirrer revolution (rpm): 1 — 500, 2 — 600, 3 — 700, 4 — 800, 5 - 1000. Experimental conditions: p02 = 30%, S - from 2.5 to 15%

that the appearance of the Crabtree effect under the above conditions is not connected with an insufficient oxygen mass transfer in the cells, situated in the peripheral zone of the fermenter. Thus, the most probable reason for the appearance of the Crabtree effect upon intensive stirring regimes is membrane changes at increased values of the kinetic energy e of medium flow fluctuations. To support the assumption we studied the distribution of kinetic energy e in fermenter volume at various stirring regimes. e max (2), W (3) and FstT (7) values, determined from energy distribution e, were compared with the activity of TCA-cycle enzyme — aconitase (Fig. 10). The figure shows that upon ~ 700 rpm the activity of aconitase drastically decreases, and the slope of curve emax(w) abruptly changes ((5emax/ e crit . To determine the maximum value of dissipated energy emax, the energy e must be measured close to the stirrer in 2—3 points. Experiments, carried out with lysine producers Brevibacterium flavum show, that by a gradual increase of energy introduced into stirrer zone there can be achieved several stages of the physiological state of cells: 1. Cells with a complete TCA-cycle, characterized by high qp and Fp/a values (Fig. 12); 2. Cells with a marked Crabtree effect, characterized by an incomplete TCA-cycle, increased GLDH and glycolysis enzyme activities, increased UNA and protein content

575

RTJKLISHA, M. P . , VANAGS, J . J . e t al., M e d i u m Stirring I n t e n s i t y

Glucose Glucose-

GLUCOSE

I

6-

1

phosphate

Î

Ribose-5^phospha

PEP

GLUCOSE-6-PHOSPHATE

te

\

1 Pyruva

1

PYRUVATE

te

*• LACTATE

1

ACETYL-CoA

. - A ,

Citrate

Aspartate i

1

RIBOSE-S-PHOSPHATE

+ PEP

ASPARTATE

-OAA

-

~CITRATE \

T

J_

MALATEtC

Glyoxylate

LYSINE

I iso- Ci träte

Succinate

oi-Oxoglutarate

LYSINE

f

iso-CITRATE GLYOXYLATE\ -x-OXOGLUTARATE

SUCCINATE' GLUTAMATE

Glutamate

Fig. 12. A scheme of the functioning of central metabolic pathway enzymes in Brevibacterium flavum upon normal (A) and critical (B) values of energy introduced into the central zone of the fermenter

and a high value of cell TEA, increased LDH activity. The value of ¡x is close to its maximum; 3. Cells with a low activity of metabolic enzymes, as well as with decreased values of qs> qp, ft and others, i.e., cells in the state of "turbohypobiosis" [21]. I t can be assumed that the regulatory activity of medium stirring rate upon the rate of biochemical reactions in Brevibacterium, flavum cells is caused by changes in the electrochemical potential of membranes (A/jtYL+) upon e > e c r i t in the zone of the stirrer. Upon a certain decrease of zJi«~H+ the rate of cell growth is maintained by an additional substrate uptake and by changes in enzyme activities according to the Crabtree effect. A further increase of e and, obviously, a decrease of Zl1a~H+ lead to more essential changes of cell structure, which disable them to maintain a normal physiological state. Consequently, in order to ensure a certain rate of cell growth or product synthesis it is important to maintain a corresponding value of e in the stirrer zone during the process of fermentation. The abovesaid is especially valid in mycelial cultures, for example Trichoderma viride a.o., and in media of increased viscosity [22]. Conclusion The classical concept that for certain microorganisms there exists a critical concentration of oxygen [23], has become absolute [6]. In fact, within certain limits the oxygen concentration and stirring intensity are interchangeable parameters [2]. Moreover, stirring intensity can be a reason of turbohypobiosis [21] and, as demonstrated in the present article, it can create (by a constant p02) complicated biochemical reactions of the population. Detailed studies of flow structure and energy distribution in bioreactors [9] have revealed a considerable stirring unevenness in the apparata, expressed as FUt. The main

576

Acta Biotechnol. 8 (1989) 6

reason for the complicated morphological [14], physiological and biochemical changes, and for the onset of turbohypobiosis is the unevenness of stirring (at constant j>02) or high specific energy introduced into the bioreactor. Abbreviations and Symbols Metabolic coefficients qp — lysine synthesis rate [g/g • h] q, — substrate uptake rate [g/g • h] Enzymes [katals • 10~ n /mg protein] AC GLDH DAP-DC LDH MDH F-1,6-DPA PFK SDH TEA DNA RNA Qo. c Kr n I W T V Q d F sit h Oi ô

— aconitase — glutamate dehydrogenase — diaminopimelate decarboxylase — lactate dehydrogenase — malate dehydrogenase — fructose-l,6-diphosphate aldolase — phosphofructokinase — succinate dehydrogenase — total reducing activity of cells [¡xg TTC-F/100 mg cells] — desoxyribonucleic acid [%] — ribonucleic acid [%] — rate of oxygen uptake by cells [mmol/g • h] — kinetic energy of medium flow fluctuations [cond. units] — criterion of microstirring [relative units] — velocity of stirrer revolution [rpm] — distance of the point under study from stirrer [m] — possibility of cell presence in the critical zone — duration of cell presence in bioreactor [s] — working volume of stirred medium [m3] — stirrer productivity [m3 • s _ 1 ] — stirrer diameter [m] — stress factor [relative units] — height of stirrer blade [m] — angle of outlet jet [grad] — a degree in (7)

Received May 11, 1988 Revised September, 9, 1988 References [ 1 ] RXTKLISHA, M . P . , JAKOBSONE, M . A . , VIESTUKS, U . E . , MEZHINYA, G . R . , SKLGA, S . E . :

Prikl. Biokhim. Mikrobiol., 2 (1976) 518 (in Russian). [2] VIESTUKS, U. E., RUKLISHA, M. P. — In: Abstr., 5th Intern. Ferment. Symp., Berlin (1976), 98. [3] VIESTUKS, U. E., KUZNETSOV, A. M., SAVENKOV, V. V. — In: Fermentation Systems. Riga: Zinatne, 1986, 368 p. (in Russian).

RUKLISHA,

M. P.,

VANAGS,

J . J . et al., Medium Stirring Intensity

577

[4] B U K I N , V . K . , B E K E R , M . J . , RUTSEVA, L . S . , BAZDYREVA, N . M . , LATSAR, A . A . , KALVANE, I . J . , SEDYALDS, A . K . , B E K E R , A . F . , VIESTURS, U . E . , LIEFINSH, G . K . , VALDMAN, A . VIESTURE, Z . A . , LUZHKOV, A . M . , ARESHKINA, L . J . :

USSR Author's Certif. No.

R.,

502019,

Byull. Izobret. 5 (1976), 83 (in Russian). M. B . , D A L K A , R. B . , U D R O V S K I S , G . A.: Mikrobiol. Prom-st. 3 ( 1 9 7 9 ) , 1 0 (in Russian). V I E S T U R S , U. E., K R I S T A P S O N S , M. Z., B Y L I N K I N A , J . S. — I n : Cultivation of Microorganisms. Moscow: Pisch. Prom-st., 1980, 231 p. (in Russian). R U K L I S H A , M . P . , S H V I N K A , J . E . , V I E S T U R S , U . E . , S A K S E , A. K . , L E I T E , M . P . : Izv. Akad. Nauk Latv. SSR 3 (1984), 85 (in Russian). KUN, E., A B O O D , L. G . : Science 109 (1949), 144. R I K M A N I S , M. A., V A N A G S , J . J., V I E S T U R S , U. E.: Biotekhnologiya 1 (1987), 72 (in Russian). V A N A G S , J . J . , B E R Z I N S H , A. J . , Z E L T I N A , M. 0., R I K M A N I S , M. A. — I n : Abstr., I V AllUnion Conference "Monitored Cultivation of Microorganisms". Puschino: USSR Acad. Sci., 1986, 146 (in Russian). E G L A J S , V. 0 . : Izv. Akad. Nauk Latv. SSR 4 (1980), 107 (in Russian). B R A G I N S K Y , L. N., B E G A T C H E V , V . N., B A R A B A T C H I N , V . M. — In: Stirring in Liquid Media. Leningrad: Khimia, 1984, 335 p. (in Russian). D E S O U R A , A . , P I N E , R . W . : Can. J . Chem. Eng. 5 0 ( 1 9 7 2 ) , 1 5 . R U K L I S H A , M . P., A P S I T E , A . F . , O S E , V . P., S A V E N K O V , V . V . — In: Selection and Cultivation of Amino Acid and Enzyme Producers. Riga: Zinatne, 1979, 7 (in Russian).

[5] SHKAGALE,

[6] [7]

[8] [9] [10] [11] [12] [13] [14]

[ 1 5 ] BEZBORODOV,

[16] [17] [18] [19]

[20] [21]

[22] [23]

A. I.,

RUKLISHA,

M. P.,

MEZHINA,

G. R.,

SELGA,

S. E . ,

VIESTURS,

U.

E.:

Mikrobiol. Prom-st. 1 (1972), 36 (in Russian). R U K L I S H A , M . , M A R A U S K A , D., S H V I N K A , J., T O M A , M . , G A L Y N I N A , N . : Biotechnol. Lett. 3 (1981), 465. T O M A , M. K., S H V I N K A , J . E., R U K L I S H A , M. P., S A K S E , A. K., B A B U R I N , L. A.: Prikl. Biokhim. Mikrobiol. 20 (1984), 95 (in Russian). D O E L L E , H . W., E W I N G S , K. N., H O L L Y W O O D , N. W.: Adv. Biochem. Eng. 23 (1983), 1. M A R A U S K A , V . F . , R U K L I S H A , M. P . , G A L Y N I N A , N . I . : Mikrobiologiya 5 0 ( 1 9 8 1 ) , 7 6 3 (in Russian). B A B U R I N , L. A., S H V I N K A , J . E., V I E S T U R S , U. E.: Eur. J . Appl. Microbiol. Biotechnol. 13 (1981), 15. T O M A , M . K . , R U K L I S H A , M . P . , V I E S T U R S , U . E . , R I K M A N I S , M . A . , V A N A G S , J . J . : Izv. Akad. Nauk Latv. SSR 5 6 ( 1 9 8 7 ) , 2 1 (in Russian). Z E L T I N A , M. O., L E I T E , M. P., V A N A G S , J . J., A P I N E , A. J., V I E S T U R S , U . E.: Acta Biotechnol. 7 (1987), 157. C R U E G E R , W., C R U E G E R , A. — In: Biotechnology: A Textbook of Industrial Microbiology. B R O O K , T. D. (ed.). Madison: Science Techn. Inc., 1984, 308 p.

Book Review A.

MOSER

Bioprocess Technology Kinetics and Reactors Revised and Expanded Translation Wien, New York: Springer-Verlag, 1988. 451 pp., DM 1 9 8 , - , oS 1 3 9 0 , - , ISBN 3-211-96603-X Intended primarily for industrial microbiologists and chemical and biochemical engineers, this monograph covers in great detail bioprocess kinetics and related mathematical modeling. This book bridges the gap between basic principles and applied engineering practice. After defining bioprocess technology and describing its engineering principles (according to a strategy of integrating biology and physics) in the initial two chapters, bioreactors are treated in depth in Chapter 3. The core of the book is in Chapters 4 and 5 , in which Professor M O S E R provides first an analysis of process kinetics and then presents detailed mathematical models for all situations. Finally, formal kinetics are combined with bioreactor models in Chapter 6 to achieve estimates of real bioreactor performance. Numerous examples of computer simulations are reproduced in the Appendix. Consistent with the author's belief, the theoretical presentations in this volume make it eminently practical. 6

Acta Biotechnol. 9 (1989) 6

RUKLISHA,

M. P.,

VANAGS,

J . J . et al., Medium Stirring Intensity

577

[4] B U K I N , V . K . , B E K E R , M . J . , RUTSEVA, L . S . , BAZDYREVA, N . M . , LATSAR, A . A . , KALVANE, I . J . , SEDYALDS, A . K . , B E K E R , A . F . , VIESTURS, U . E . , LIEFINSH, G . K . , VALDMAN, A . VIESTURE, Z . A . , LUZHKOV, A . M . , ARESHKINA, L . J . :

USSR Author's Certif. No.

R.,

502019,

Byull. Izobret. 5 (1976), 83 (in Russian). M. B . , D A L K A , R. B . , U D R O V S K I S , G . A.: Mikrobiol. Prom-st. 3 ( 1 9 7 9 ) , 1 0 (in Russian). V I E S T U R S , U. E., K R I S T A P S O N S , M. Z., B Y L I N K I N A , J . S. — I n : Cultivation of Microorganisms. Moscow: Pisch. Prom-st., 1980, 231 p. (in Russian). R U K L I S H A , M . P . , S H V I N K A , J . E . , V I E S T U R S , U . E . , S A K S E , A. K . , L E I T E , M . P . : Izv. Akad. Nauk Latv. SSR 3 (1984), 85 (in Russian). KUN, E., A B O O D , L. G . : Science 109 (1949), 144. R I K M A N I S , M. A., V A N A G S , J . J., V I E S T U R S , U. E.: Biotekhnologiya 1 (1987), 72 (in Russian). V A N A G S , J . J . , B E R Z I N S H , A. J . , Z E L T I N A , M. 0., R I K M A N I S , M. A. — I n : Abstr., I V AllUnion Conference "Monitored Cultivation of Microorganisms". Puschino: USSR Acad. Sci., 1986, 146 (in Russian). E G L A J S , V. 0 . : Izv. Akad. Nauk Latv. SSR 4 (1980), 107 (in Russian). B R A G I N S K Y , L. N., B E G A T C H E V , V . N., B A R A B A T C H I N , V . M. — In: Stirring in Liquid Media. Leningrad: Khimia, 1984, 335 p. (in Russian). D E S O U R A , A . , P I N E , R . W . : Can. J . Chem. Eng. 5 0 ( 1 9 7 2 ) , 1 5 . R U K L I S H A , M . P., A P S I T E , A . F . , O S E , V . P., S A V E N K O V , V . V . — In: Selection and Cultivation of Amino Acid and Enzyme Producers. Riga: Zinatne, 1979, 7 (in Russian).

[5] SHKAGALE,

[6] [7]

[8] [9] [10] [11] [12] [13] [14]

[ 1 5 ] BEZBORODOV,

[16] [17] [18] [19]

[20] [21]

[22] [23]

A. I.,

RUKLISHA,

M. P.,

MEZHINA,

G. R.,

SELGA,

S. E . ,

VIESTURS,

U.

E.:

Mikrobiol. Prom-st. 1 (1972), 36 (in Russian). R U K L I S H A , M . , M A R A U S K A , D., S H V I N K A , J., T O M A , M . , G A L Y N I N A , N . : Biotechnol. Lett. 3 (1981), 465. T O M A , M. K., S H V I N K A , J . E., R U K L I S H A , M. P., S A K S E , A. K., B A B U R I N , L. A.: Prikl. Biokhim. Mikrobiol. 20 (1984), 95 (in Russian). D O E L L E , H . W., E W I N G S , K. N., H O L L Y W O O D , N. W.: Adv. Biochem. Eng. 23 (1983), 1. M A R A U S K A , V . F . , R U K L I S H A , M. P . , G A L Y N I N A , N . I . : Mikrobiologiya 5 0 ( 1 9 8 1 ) , 7 6 3 (in Russian). B A B U R I N , L. A., S H V I N K A , J . E., V I E S T U R S , U. E.: Eur. J . Appl. Microbiol. Biotechnol. 13 (1981), 15. T O M A , M . K . , R U K L I S H A , M . P . , V I E S T U R S , U . E . , R I K M A N I S , M . A . , V A N A G S , J . J . : Izv. Akad. Nauk Latv. SSR 5 6 ( 1 9 8 7 ) , 2 1 (in Russian). Z E L T I N A , M. O., L E I T E , M. P., V A N A G S , J . J., A P I N E , A. J., V I E S T U R S , U . E.: Acta Biotechnol. 7 (1987), 157. C R U E G E R , W., C R U E G E R , A. — In: Biotechnology: A Textbook of Industrial Microbiology. B R O O K , T. D. (ed.). Madison: Science Techn. Inc., 1984, 308 p.

Book Review A.

MOSER

Bioprocess Technology Kinetics and Reactors Revised and Expanded Translation Wien, New York: Springer-Verlag, 1988. 451 pp., DM 1 9 8 , - , oS 1 3 9 0 , - , ISBN 3-211-96603-X Intended primarily for industrial microbiologists and chemical and biochemical engineers, this monograph covers in great detail bioprocess kinetics and related mathematical modeling. This book bridges the gap between basic principles and applied engineering practice. After defining bioprocess technology and describing its engineering principles (according to a strategy of integrating biology and physics) in the initial two chapters, bioreactors are treated in depth in Chapter 3. The core of the book is in Chapters 4 and 5 , in which Professor M O S E R provides first an analysis of process kinetics and then presents detailed mathematical models for all situations. Finally, formal kinetics are combined with bioreactor models in Chapter 6 to achieve estimates of real bioreactor performance. Numerous examples of computer simulations are reproduced in the Appendix. Consistent with the author's belief, the theoretical presentations in this volume make it eminently practical. 6

Acta Biotechnol. 9 (1989) 6

Acta Biotechnol. 8 (1989) 6, 578

Akademie-Verlag Berlin

Obituary I t is with deep sorrow that we inform you of the death of Academician Georgy K. Skryabin who passed away on March 26, 1989 after a long and serious disease. Academician G. K. Skryabin was among the founders of the Institute of Biochemistry and Physiology of Microorganisms in Pushchino and has been its Director for nearly two decades. After his retirement in 1987 Academician Skryabin continued as Honorary Director of the Institute. Academician's Skryabin devotion to science won him the regard of all who knew him and the admiration of all who worked with him. We deplore the irreparable loss of a scholar whose work has contributed so much to the development of science. He worked in research on biochemistry and physiology of microorganisms, molecular biology, environmental biotechnology, microorganism/plant interaction and others. Since 1980, the year of foundation of „Acta Biotechnologica" Prof. Skryabin was an active member of its Editorial board. M . RINGPFEIL

Ada Biotechnologicu Volume 9

Number 6

1989

Contents U.; SCHÖN, G.; J E O H O B E K , M., R O S T , H.-J.: Apparatus System for Control in Fermentation Processes Using Mass Spectrometry (in German) 499 ISKE,

KOPP, TH.

504

: Bubble-Free Aeration (in German)

THALMANN,

E.: Biological Experiences in a Bubble-Free Aeration System (in German). . . 511

D.; KLAPPAOH, G.; H Ü H N , J . ; SCHUBIG, K.-H.: Engineering Analysis for Maximum Possible Productivity of Microbial Upper Limits (in German) 517

PÖHLAND,

GBOSSE, H . H . ; H I L L I G E B , M., BOBMANN, E . - J . : Process Control of Dissolved Oxygen Level in Aerobic Microbial Fermentations (in German) 522

B.; L I N D N E B , U . ; MATHISZIK, B . ; T I L L E B , V . : Modelbase Biotechnology — a Software Package for Seleotion of Models, Optimization, and Simulation of Biotechnological Processes (in German) 529 GOLDSCHMIDT,

L I E B S , P . ; B I E D E L , K . ; P F E I F F E B , D . ; ZIMABE, U . ; L I E T Z , P . ; SOHELLEB,

F.: Biosensor

Application in Food Industry (in German)

534

G E P P E B T , G.; THIELEMANN, H.; LANGKOPF, G.: Industrial Instruments for Determination of Turbidity in Liquid Media (in German) 541 G E P P E B T , G.; HANSOHMANN, G.; THIELEMANN, H . : Quasicontinuous Determination of Alcoholic Concentration in Fermentation Media (in German) 545

Characterization of Products of the Staroh Hydrolysis Obtained after Action of a Thermostable oc-Amylase (in German) 549 DOBBEVA, E . ; IVANOVA, V . :

J . : Simultaneous Saccharification and Fermentation of Cellulose: Effect of Ethanol and Cellulases on Particular Stages 555 SZCZODBAK,

R U K L I S H A , M . P . ; VANAGS, J . J . ; R I K M A N I S , M . A . ; TOMA, M . K . ; VIESTXJBS, U . E . :

Bio-

chemical Reactions of Brevibaeterium flavum Depending on Medium Stirring Intensity and Flow Structure 565 Book Reviews

554, 564, 577

Acta Biotechnologioa is indexed or abstracted in Current Contents/ET & AT; Chemical Abstracts; Biological Abstraots; Biotechnology Abstracts; Excerpta Medioa