Acta Biotechnologica: Volume 9, Number 5 1989 [Reprint 2021 ed.] 9783112581926, 9783112581919

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Volume 9 • 1989 • Number 5

Journal of microbial, biochemical and bioanalogous technology

Akademie-Verlag Berlin ISSN 0138-4988 Acta Biotechnol., Berlin 9 (1989) 5, 3 9 3 - 4 9 6

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Acta Biotictanloiia Journal of microbial, biochemical and bioanalogous technology

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

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

1989

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 L. P. Jones, El Paso F. Jung, Berlin H. W . D. Katinger, Vienna K . A. Kalunyanz, Moscow J. M. Lebeault, Compiègne

Number 5

Managing Editor:

L. Dimter, Leipzig

Volume 9

A K A D E M I E - V E R L A G

B E R L I N

"Acta Biotechnologica" publishes original papers, short communications, reports and reviews from t h e whole field of biotechnology. The journal is t o promote t h e establishment of biotechnology as a new and integrated scientific field. The field of biotechnology covers microbial technology, biochemical technology and t h e technology of synthesizing and applying bioanalogous reaction systems. The technological character of t h e journal is guaranteed b y t h e fact t h a t papers on microbiology, biochemistry, chemistry and physics must clearly have technological relevance. Terms of subscription for t h e journal "Acta Biotechnologica" Orders can be sent — in the GDR: to Postzeitungsvertrieb or to the Akademie-Verlag Berlin, Leipziger Str. 3—4, P F 1233, D D R - 1 0 8 6 Berlin; — in the other socialist countries: to a bookshop for foreign languages literature or t o the competent news-distributing agency; — in the FRG and Berlin (West): to a bookshop or to t h e wholesale distributing agency K u n s t u n d Wissen, Erich Bieber oHG, Postfach 102844, D-7000 S t u t t g a r t 10; — in the other Western European countries: to K u n s t und Wissen, Erich Bieber GmbH, General Wille-Str. 4, CH-8002 Zürich; — in other countries: to t h e international book- and journal-selling trade, to Buchexport, Volkseigener Außenhandelsbetrieb der D D R , P F 160, D D R - 7 0 1 0 Leipzig, or to t h e Akademie-Verlag Berlin, Leipziger Str. 3—4, P F 1233, D D R - 1 0 8 6 Berlin.

Acta Biotechnologica Herausgeber: I n s t i t u t f ü r Biotechnologie der AdW der D D R Permoserstr. 15, D D R - 7 0 5 0 Leipzig (Prof. Dr. Manfred Ringpfeil) und V E B Chemieanlagenbaukombinat Leipzig—Grimma Bahnhofstr. 3 - 5 , D D R - 7 2 4 0 Grimma, (Dipl.-Ing. Günter Vetterlein). Verlag: Akademie-Verlag Berlin, Leipziger Straße 3 - 4 , P F 1233, D D R - 1 0 8 6 Berlin; F e r n r u f : 2236201 und 2 2 3 6 2 2 9 ; Telex-Nr.: 114420; B a n k : Staatsbank der D D R , Berlin, Konto-Nr.: 6836-26-20712. Redaktion: Dr. Lothar Dimter (Chefredakteur), Martina Bechstedt, K ä t h e Geyler, Permoserstr. 15, D D R - 7 0 5 0 Leipzig; Tel.: 2392255. Veröffentlicht unter der Lizenznummer 1671 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: V E B Druckhaus „Maxim Gorki", D D R - 7 4 0 0 Altenburg. Erscheinungsweise: Die Zeitschrift „Acta Biotechnologica" erscheint jährlich in einem B a n d mit 6 Heften. Bezugspreis eines Bandes 198,— DM zuzüglich Versandspesen; Preis je H e f t 33,— DM. Der gültige Jahresbezugspreis f ü r die D D R ist der Postzeitungsliste zu entnehmen. Bestellnummer dieses Heftes: 1094/9/5. Urheberrecht: Alle Rechte vorbehalten, insbesondere der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner F o r m — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translation into foreign languages). No p a r t of this issue m a y be reproduced in any form, b y photoprint, microfilm or any other means, without written permission from t h e publishers. © 1989 b y Akademie-Verlag Berlin. Printed in the German Democratic Republic. A N (EDV) 18520 03000

Acta Biotechnol. 9 (1989) 5, 3 9 5 - 4 1 9

Akademie-Verlag Berlin

Importance of Bacteriophages in Fermentation Processes WÜNSCHE, L .

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

Summary Any bacterial strain can be infected by virulent phages or harbour one or more prophages. Therefore, bacteria-phage interactions are to be regarded as fundamental properties of bacteria. In current industrial fermentation processes phages can be advantageously employed for the identification of bacterial production strains (phage typing). In some cases phages are involved in the production of enzymes and special substances. The fundamental importance of phages in any technical fermentation process, however, is based on the peculiarities of their obligately parasitic life cycle. The propagation of phages in fermentation processes can cause complete (or at least partial) lysis of the production strains and, consequently, serious disturbances in the production process and considerable economic losses. The phage problem in the fermentation industry has not yet been completely solved. For the protection of technical processes against virulent phages five measures are discussed: phage-protected sterile fermentation, employment of alternative cultures, employment of phage-resistant mutants, employment of phage inhibitors, and employment of immobilized bacterial cells. The problem of the protection of bacterial production strains from prophage induction is more difficult and practically unsolved. Two possibilities to minimize the process risk due to temperate phages, the elimination of inducing factors during the fermentation process, and the selection of production strains which are difficult to induce, are discussed. Introduction Viruses are known t o be obligate parasites of higher living systems which are able to cause numerous diseases in plants, animals and men. Virus-like particles in prokaryotic cells were first described by TWORT [1] in 1915. Independently of these findings d'HERELLE [2] in 1917 characterized a lytic agent of bacterial cells as a virus, terming it bacteriophage. Recently a great number of phages have been described for bacterial species which have been comprehensively investigated in regard to their relations t o bacteriophages. Escherichia coli m a y be quoted as an illustration in this respect: m a n y morphologically different virulent and temperate phages containing in most cases double stranded D N A , in some cases single stranded D N A or R N A , can infect distinct strains of this bacterial species, e.g. the T-even and T-odd phages, the phages X, Mu, 0 X 174, C-l and C-2, fd, f 2, MS 2, M 13, R 17 etc. A comparable situation is observed in other common and well investigated bacterial genera, e.g. Bacillus, Clostridium, Salmonella, Streptococcus, Streptomyces etc. Therefore, it is not surprising that during studies of bacteria with 1*

Acta Biotechnol. 9 (1989) 5

396

unconventional and extreme properties phages have also been found. Several phages of methylotrophic bacteria, both methanotrophic [3—6] and methanol utilizing [7—9] strains have been detected in the last years. Bacteriophages and virus-like particles have also been found in all main groups of archaebacteria [10—21]: whereas the known phages of extremely halophilic and of the methanogenic archaebacteria seem to be very similar to the tailed phages of eubacteria, the phages of extremely thermophilic, sulfurdependent archaebacteria are characterized by an unusual morphology and extraordinary properties, e.g. resistance to extremely high temperatures [20]. If any group of bacteria has been investigated intensively in respect to lysogeny, temperate phages have been detected in a relatively high percentage of the examined strains. By way of example, it was found [22] t h a t 43% of 113 investigated strains belonging t o three Streptococcus species released phages either spontaneously or after induction which formed plaques on sensitive indicator strains. As specific indicator strains are not available in many cases, electron-microscopic examination usually shows a significantly higher percentage of bacterial strains to be lysogenic: apparently intact phage particles were detected in suspensions of 28 out of 50 strains of Streptococcus lactis after UV-irradiation; in another 13 strains incomplete phages could be proved [23]. I n conclusion of all these findings it can be postulated t h a t — a t least one virulent phage should exist for any bacterial strain — any bacterial strain can harbour one or more different prophages. Therefore, the interactions between bacteria and phages must be considered as basic properties of all bacterial systems. Distribution of Bacteriophages in Natural

Biotopes

Similar to bacteria, bacteriophages are widely spread in nature: basically bacteriophages can be found in the same biotopes as are settled by their host bacteria. Preferred sources of bacteriophages are soil, water and, especially, waste water. The persistence of free bacteriophages in nature seems to be largely dependent on their adsorption on solids, from which they can be eluted and/or disaggregated by a change of ion concentration [24], Solid associated phages are less sensitive to environmental conditions t h a n freely suspended phages [25]. Another, basically different and effective strategy of persistence of phages is based on the principle of lysogeny. As dormant prophages bacteriophages survive adverse environmental conditions within their bacterial host cells. The very fragmentary knowledge of the occurrence and distribution of bacteriophages in natural (and technical) biotopes is ultimately due to the general problems of microbial ecology, especially to the difficulties involved in enumerating and identifying microorganisms in a particular bio tope. To concentrate and detect phages from relatively large volumes of water, the virus adsorption-elution (viradel) procedure utilizing microporous filters seems to be preferred by most investigators [26]. Subsequent to enrichment the phages can be enumerated b y electron microscopy and, if a sensitive host strain for propagation is available, identified by the methods of phage taxonomy. Phage

Taxonomy

The principal criteria used b y the International Committee on Taxonomy of Viruses for the classification of bacteriophages are [27]: — Nucleic acid: nature, mass and composition — Virion: symmetry, dimensions, mass and gross composition — Antigenic relationships, host range and resistance to the environment (chiefly ether, chloroform and heat).

WÜNSCHE, L., Bacteriophages

397

According to these properties 10 phage families (Fig. 1) and a small special group (F 3 group, represented by phage TTV 1, consisting of three members) within the classification system of viruses are generally accepted [27, 28], possibly the new group of rodshaped archaebacteriophages must be regarded as an additional phage family [27]. In contrast to the situation in the taxonomy of higher living systems the species issue in virology has been contentious und unclarified for many years. According to the currently accepted rules of virus nomenclature a species name should consist of a single word or, if essential, a hyphenated word followed by numbers or single letters. Names of phage "species" formed by letters, numbers, or combinations of these already have wide usage for viruses [28], Hitherto more than 2,900 phages have been investigated electronmicroscopically, about 95% display the typical morphology of tailed phages. Defective phages, mutants, and phage-like bacteriocins are not included in this number. The number of phage descriptions was about 150 in 1978, currently about 100 phages are characterized anually [28], preferably phages of technologically important bacterial strains. Detection of

Bacteriophages

Virulent bacteriophages are directly traceable under the electron-microscope or indirectly by propagation on the host strain using the double agar layer method developed by A D A M S [29]: After spreading of a soft agar layer containing the mixture of host cells and phages, each in suitable dilution, on a normal agar layer, a dense bacterial lawn is formed within the top agar layer. The multiplication of each phage on this lawn results in a small clear lysis spot (plaque). Occasionally, the clear plaques may be surrounded by a turbid halo caused by the action of phage lysozyme. By counting the plaques the titer of active phages may be determined quantitatively as plaque forming units (p.f.u.). Approximately 102 p.f.u./ml -1 can still be proved by this method without enrichment. Depending on the specificity of the phage-host systems to be tested (e.g. strictly anaerobic bacteria) this basic method is to be modified and standardized for routine application. The detection of temperate phages is more difficult because the host cells are basically resistant to their own spontaneously or inductively released phages. In most cases the phage titer is too low ( < 106 phage particles per ml) for detection under the electron microscope. The sure detection is possible by plaque formation on closely related but phage-sensitive indicator strains. Such indicator strains, however, are not available in all cases. Prophage cured strains proved to be susceptible indicator strains for their own temperate phages. General Survey of the Role of Bacteriophages

in Biotechnology

The fundamental importance of bacteriophages in biotechnology is well-founded by the ubiquitous distribution of phages in nature, their host specificity, and the different interactions between phages and their bacterial host cells. In this review it can only be suggested and not discussed in detail that bacteriophages may be used in many respects in the broad field of the genotypic optimization of bacterial production strains. They are valuable tools for the investigation of the structure of the genetic material (genetic mapping) as well as vectors for the gene transfer (e.g. transduction). In this connection the multifarious possibilities to use particular sequences of phage genomes (e.g. the generally strong promotors, cos sites, etc.) for the in-vitro construction of efficient cloning vectors should be mentioned. In industrial fermentation processes bacteriophages can be applied directly to the identification of production strains (or, if necessary, for other components of a fermenter

Acta Biotechnol. 9 (1989) 5

398

Family

Morphology I 1 1=

Typ of nucleic acid

100nmi

Plasmavirldae

ds \

/

Surface

view

Corticoviridae

Myovirida

e

* < >

view

4

<
" 2 g « M A S S O ce o í ü

h o "" fi O O

« u «3 © ® -2 - § 3 œ tí

-s JS

O

, o tí

5

à i

2 d l

s

I o 00 Ci C5 O N

« L

á

&

3

•a

s

p

a

O .2 O

O Ci C5 ^ Oi

Ethanol

D

concentration

m T'max

P[g/l]

Pig. 2. Changes of the characteristic temperatures of the temperature-profile curve of ethanol production with increasing ethanol concentration (Saccharomyces cerevisiae IBT H 191 (symbols see Fig. 1)

434

Acta Biotechnol. 9 (1989) 5

significantly with increasing alcohol concentration. Predominantly the relationships existing in this context are linear with the exception of the function AT'^* = f(P). Compared to it, the upper temperature limit of optimum ethanol formation T" ptl keeps constantly over the entire ethanol concentration interval (Fig. 2). 2. Essentially the 5 ranges of the temperature-profile curve of ethanol formation are preserved, but their proportions are significantly influenced by the ethanol concentration (Fig. 3). So one can state a linear decreasing of both — the interval of thermal non-retarded ethanol formation in the suboptimal temperature range, < T < and — the first superoptimal temperature range characterized by a dominating reversible thermal hindrance of ethanol formation, T'0'ptt < T < with increasing ethanol concentration.

Ethanol

concentration

P[g/l]

Pig. 3. Changes of the separate temperature intervals of the temperature-profile curve of ethanol production of Saccharomyces cerevisiae IBT H 191 with increasing ethanol concentration o range of thermally non-retarded ethanol formation in the suboptimal temperature interval, • transition range in the suboptimal temperature interval, • optimal temperature range, • first range of thermal deactivation in the superoptimal temperature interval, • second range of thermal deactivation in the superoptimal temperature interval

The second superoptimal temperature range the irreversible deactivation of cells dominates within, < T < decreases in a nonlinear way and it disappears almost entirely at ethanol concentrations above P = 100 g/1. Against it the optimal temperature range of ethanol formation, 7" ptl < T < Topt,» shows a linear increase with increasing ethanol concentration and the suboptimal transition range, T'^ < T < T'aptl, remains unchanged only. 3. The maximum possible values of the specific ethanol formation rate v'mtLX decrease linearly with increasing ethanol concentration according to the inhibitory equation C i = n — aP, which was derived for the used yeast in an earlier paper [30].

RICHTER, E., BECKER, U., Ethanol Production

435

Using the obtained values listed in Tab. 1 the suboptimal A B R H E N I U S plots for the ethanol concentrations mentioned above were formed in Fig. 4. One can see that the slopes of the presented straight lines are not affected by the ethanol concentration. This means that the activation energy of ethanol formation must be also independent on the ethanol concentration. Assuming the nonappearance of significant deviations Tab. 1. Some maximum possible values of the specific ethanol formation rate obtained in discontinuous and continuous fermentation experiments performed at several suboptimal temperatures and under condition of different ethanol concentrations (data dimension: mmol/gh) Temperature [K] 283.16 288.16 289.16 290.16 291.16 292.16 293.16 297.16 298.16 300.16

Ethanol concentration Omol/1

0.54 mol/1

3.70 6.52

3.26 5.87

—

—

—

1.09 mol/1 2.72 5.00

—

—

—

—

11.30 17.61

10.22 15.66 17.39 21.85

8.70 13.48

3.35

3.W

3.U5

0.87 1.55 1.74 1.96

—

-

103-(1jT)

2.17 3.91

—

—

24.13

2.17 mol/1

—

—

—

1.63 mol/1

5.43 6.10 6.74

— — —

—

—

—

—

—

—

—

—

[1/K] 3.50

Pig. 4. Suboptimal ABRHENIUS plots of experimental specific ethanol formation rates of Saccharomyces cerevisiae I B T H 191 for different ethanol concentrations P 1. P = 0.00 mol/1 2. P = 0.54 mol/1 3. P = 1.09 mol/1 4. P = 1.63 mol/1 5. P = 2.17 mol/1

436

Acta Biotechnol. 9 (1989) 5

from the standard state (P = 0) in both the metabolism and the biomass composition over the whole first suboptimal temperature range the following equation was applied for calculating the mean activation energy of ethanol formation: t

.JMnfc)

t

kE(él

+

J_

J_ T!

él)

T,

where k is the number of value combinations (vl5 T1lv2> T2) used in each run of calculation and Av corresponds to the absolute error of measurement for the specific ethanol formation rate, estimated at Av = 0.005 g/gh and Av = 0.11 mmol/gh, respectively. Indeed, the activation energies calculated for the used ethanol concentrations can be considered as equal among one another (Tab. 2). There are no significant differences between the calculated mean values and the previously published value of AE^toh = (78.5 i 2.2) kJ/mol • deg. found for the ethanol-free state [26]. The great error limits at higher ethanol concentrations are caused by the fact that under those conditions the Av

ratio — attains an unfavourable dimension. v

Tab. 2. Mean values of the activation energy of ethanol formation calculated for different ethanol concentrations Ethanol concentration, P [mol/1]

Number of used value combinations k

Mean activation energy, zl-B^tOH [kJ/mol • deg.)]

0 0.54 1.09 1.63 2.17

10 12 6 9 4

78.5 78.4 79.6 78.4 79.0

± ± ± ± ±

2.2 2.7 3.5 10.3 27.6

Conclusions With increasing ethanol concentration the temperature-profile curve of ethanol production changes in such a way that its integral function value (corresponding to the area between the curve and the abscissa) yf;

B

/ f(T) • dT = lim ZvTi-ATi T" n—Hx i=l min J

decreases gradually down to nought. Since an explicit equation of the function v = f(T) being valid for the entire ethanoldependent temperature-profile curves was not available an exact calculation of the integral was not possible. But a sufficient approximation to the true value can be obtained if AT( is equated to 1 K: / f(T) • d Te*

X

K)/> • deg.

RICHTER, K., BECKER, U., Ethanol Production

437

On this base one can evaluate some tendencies and limits in the thermally affected ethanol-forming performance of the used yeast. As the plot in Fig. 5 shows, the thermally-limited overall ethanol-forming performance decreases linearly with increasing ethanol concentration and the resulting straight line intersects the abscissa at P = 107 g/1. The latter is identical with the maximum ethanol concentration for ethanol formation -Pmax found for the used yeast strain in other experiments previously [30].

Kg. 5. Decrease of the approximated integral function value of the temperatureprofile curve of Saccharomyces cerevisiae IBT H 191 with increasing ethanol concentration rpft ip/f 1

max

max

/ f(T).AT*> iptt m/f 1

min

I>r-deg.

rain

In this context a characteristic phenomenon is the shifting of the quantities T^',» T'0'fti, T'max, and vT to lower values. As an explanation for these effects, it can be assumed that in the presence of ethanol the reversible thermal deactivation of the viable cells starts previously at lower temperatures, compared to alcohol-free conditions. Therefore, the suboptimal temperature range, in which the ethanol formation is thermal activated only, decreases with increasing ethanol concentration without affecting the activation energy of ethanol formation. Since the thermal deactivation of the cells occurs but in a small extent first of all the microorganisms are able to compensate it by the simultaneously operating thermal activation over a greater temperature interval. From it follows that the optimal temperature range of ethanol formation increases with increasing alcohol concentration as it is shown in Fig. 1. In the superoptimal temperature range the thermal deactivating effects are forced by ethanol in such an extent

438

Acta Biotechnol. 9 (1989) 5

that the ethanol formation is stopped at lower temperatures and in extreme case the second superoptimal temperature interval is almost completely eliminated. Under these conditions the reversible thermal deactivation potentiated by ethanol seems to be the reason for the cessation of the ethanol formation.

Symbols V vT

"o Av T P ± m> maxi ± T'maxi î'opt mu -1 min mit 1 D

l

Jm"

opti» ±m"opta. n. x

rprr

•*• max R a

specific ethanol formation rate specific ethanol formation rate at the temperature T maximum possible value of v at the temperature T maximum specific ethanol formation rate obtained in the absence of ethanol at optimum temperature absolute error of measurement for v temperature ethanol concentration final maximum temperature of growth initial maximum temperature of growth optimum temperature of growth minimum temperature of ethanol formation temperature at which the reversible deactivation of ethanol formation begins temperature limits of optimum ethanol formation temperature at which the irreversible deactivation of ethanol formation begins maximum temperature of ethanol formation activation energy of ethanol formation gas constant inhibitory coefficient

g/gh, mmol/gh g/gh, mmol/gh g/gh, mmol/gh g/gh, mmol/gh g/gh, mmol/gh °C, K g/1, mol/1 °C, K °C,K °C, K °C, K °C, K °C, K °C, K °C, K kj/mol • deg. J/mol • deg. 1/gh

Further symbols are explained in the text. Received August 22, 1988

References [1] NAQODAWITHANA, T . W . , CASTELLANO, C., STEINKRATTS, K . H . : A p p i . Microbiol. 2 8 (1974),

383. [2] V a n UDEN, N . , MADEIRA-LOPES, A . : B i o t e c h n o l . Bioeng. 1 8 (1976), 791 [3] EROSHIN, V. K . , UTKIN, I . S., LADYNICHEV, S. V., SAMOYLOV, V. V., KTJVSHINNIKOV, V. D . ,

SKBYABIN, G. K . : Biotechnol. Bioeng. 18 (1976), 289. [4] NAVARRO, J . M., DTTRAND, G. : Ann. Microbiol. (Inst. Pasteur) 129 B (1978), 215. [5] KROTJWEL, P . G., BRABER, L . : B i o t e c h n o l . L e t t . 1 (1979), 403.

[6] LEE, J. H., WILLIAMSON, D., ROGERS, P. L. : Biotechnol. Lett. 2 (1980), 83. [7] ZIFFBR, J., IOSIF, M. I.: Biotechnol. Lett. 4 (1982), 809. [8] BROWN, S. W . , OLIVER, S. G . : B i o t e c h n o l . L e t t . 4 (1982), 269. [9] HUGHES, D . B . , TUDBOSZEN, N . J . , MOYE, C. J . : B i o t e c h n o l . L e t t . 6 (1984), 1.

[10] MAVRINA, L., POZMOGOVA, I., SCHADE, W . : Z. Allgem. Mikrobiol. 23 (1983), 571, 581. [11] SA-COBREIA, I . , V a n UDEN, N . : B i o t e c h n o l . B i o e n g . 2 5 (1983), 1665.

[12] GIBSON, B.: J. Appi. Bacteriol. 86 (1973), 365. [13] CORBY, J. R.: J. Appi. Bacteriol. 40 (1976), 269. [14] VEBRIPS, C. T., GLAS, R., KWAST, R. H.: J. Appi. Microbiol. Biotechnol. 8 (1979), 299.

RICHTER, K., BECKER, U., Ethanol Production

439

[15] TÖRÖK, T., REICHABT, 0 . : E u r . J . Appl. Microbiol. Biotechnol. 17 (1983), 191. [16] [17] [18] [19] [20]

V a n UDEN, N . , ABBANCHES, P . , CABECA-SILVA, C . : A r c h . Microbiol. 6 1 (1968), 381. V a n UDEN, N . , MADEIBA-LOPES, A . : Z. A l l g e m . Mikrobiol. 1 0 (1970), 515. OLIVEIBO-BAPTISTA, A., V a n UDEN, N . : Z. A l l g e m . M i k r o b i o l . 1 1 (1971), 59. V a n UDEN, N . , MADEIRA-LOPES, A . : A r c h . Microbiol. 1 0 4 (1975), 23. SIMOES-MENDES, B . , MADEIRA-LOPES, A., V a n UDEN, N . : Z. A l l g e m . Mikrobiol. 18 (1978), 275. [21] V a n UDEN, N . , DA CRUZ DUARTE, H . : Z. A l l g e m . Mikrobiol. 2 1 (1981), 743.

[22] LOTTKEIRO, V., Van UDEN, N.: Biotechnol. Bioeng. 24 (1982), 1881. [23] L E I O , C., V a n UDEN, N . : B i o t e c h n o l . Bioeng. 2 4 (1982), 1581. [24] SA-CORREIA, I . , V a n UDEN, N . : B i o t e c h n o l . B i o e n g . 2 8 (1986), 301.

[25] SA-CORREIA, I.: Biotechnol. Bioeng. 28 (1986), 761.

[26] RICHTER, K . , BECKER, U . : A c t a B i o t e c h n o l . 7 (1987), 87.

[27] RICHTER, K . : Acta Biotechnol. 7 (1987), 127.

[28] RICHTER, K., BECKER, U.: Acta Biotechnol. 8 (1988), 29. [29] RICHTER, K., BECKER, U.: 3rd Symp. of Socialist Countries on Biotechnology. Bratislava, 1983. [30] RICHTER, K . , BECKER, U . : A c t a B i o t e c h n o l . 5 (1985), 145.

Book Review Verzeichnis faktographischer Datensammlungen und Datenbasen zu Grundlagen- und Anwendungsgebieten der Biotechnologie Berlin: Wissenschaftliches Informationszentrum der AdW der DDR, 1988. 114 S.

The results of an analysis of the current situation regarding factographic biotechnology information in the GDR have been compiled in a directory. The aim of this survey is to inform on factographic data bases in the field of biotechnology that are available in the GDR. It includes information on small factographic data collections and large factographic data bases relevant to basic and applied research in the fields of biotechnology. Each data base is described by the following details : data base producer, availability in the GDR, contents, size, updating, software, etc. This directory is the first issue of the "Biotechnology Information Service of the GDR". This new information service is designed to become a practical tool for the provision, exchange and mediation of information relevant to biotechnology. I t will help users to inform rapidly and comprehensively on the existing information possibilities. The next issue of the "Biotechnology Information Service" will contain information on bibliographic data bases in the field of biotechnology that are available in the GDR. This directory is obtained from: Scientific Information Centre of the Academy of Sciences of the GDR, Schiffbauerdamm 19, DDR-1040 Berlin, GDR. E . POETZSCH

RICHTER, K., BECKER, U., Ethanol Production

439

[15] TÖRÖK, T., REICHABT, 0 . : E u r . J . Appl. Microbiol. Biotechnol. 17 (1983), 191. [16] [17] [18] [19] [20]

V a n UDEN, N . , ABBANCHES, P . , CABECA-SILVA, C . : A r c h . Microbiol. 6 1 (1968), 381. V a n UDEN, N . , MADEIBA-LOPES, A . : Z. A l l g e m . Mikrobiol. 1 0 (1970), 515. OLIVEIBO-BAPTISTA, A., V a n UDEN, N . : Z. A l l g e m . M i k r o b i o l . 1 1 (1971), 59. V a n UDEN, N . , MADEIRA-LOPES, A . : A r c h . Microbiol. 1 0 4 (1975), 23. SIMOES-MENDES, B . , MADEIRA-LOPES, A., V a n UDEN, N . : Z. A l l g e m . Mikrobiol. 18 (1978), 275. [21] V a n UDEN, N . , DA CRUZ DUARTE, H . : Z. A l l g e m . Mikrobiol. 2 1 (1981), 743.

[22] LOTTKEIRO, V., Van UDEN, N.: Biotechnol. Bioeng. 24 (1982), 1881. [23] L E I O , C., V a n UDEN, N . : B i o t e c h n o l . Bioeng. 2 4 (1982), 1581. [24] SA-CORREIA, I . , V a n UDEN, N . : B i o t e c h n o l . B i o e n g . 2 8 (1986), 301.

[25] SA-CORREIA, I.: Biotechnol. Bioeng. 28 (1986), 761.

[26] RICHTER, K . , BECKER, U . : A c t a B i o t e c h n o l . 7 (1987), 87.

[27] RICHTER, K . : Acta Biotechnol. 7 (1987), 127.

[28] RICHTER, K., BECKER, U.: Acta Biotechnol. 8 (1988), 29. [29] RICHTER, K., BECKER, U.: 3rd Symp. of Socialist Countries on Biotechnology. Bratislava, 1983. [30] RICHTER, K . , BECKER, U . : A c t a B i o t e c h n o l . 5 (1985), 145.

Book Review Verzeichnis faktographischer Datensammlungen und Datenbasen zu Grundlagen- und Anwendungsgebieten der Biotechnologie Berlin: Wissenschaftliches Informationszentrum der AdW der DDR, 1988. 114 S.

The results of an analysis of the current situation regarding factographic biotechnology information in the GDR have been compiled in a directory. The aim of this survey is to inform on factographic data bases in the field of biotechnology that are available in the GDR. It includes information on small factographic data collections and large factographic data bases relevant to basic and applied research in the fields of biotechnology. Each data base is described by the following details : data base producer, availability in the GDR, contents, size, updating, software, etc. This directory is the first issue of the "Biotechnology Information Service of the GDR". This new information service is designed to become a practical tool for the provision, exchange and mediation of information relevant to biotechnology. I t will help users to inform rapidly and comprehensively on the existing information possibilities. The next issue of the "Biotechnology Information Service" will contain information on bibliographic data bases in the field of biotechnology that are available in the GDR. This directory is obtained from: Scientific Information Centre of the Academy of Sciences of the GDR, Schiffbauerdamm 19, DDR-1040 Berlin, GDR. E . POETZSCH

Acta Bioteohnol. 9 (1989) 5, 440

Akademie-Verlag Berlin

Book Review William W.

CHRISTIE

High-Performance Liquid Chromatography and Lipids A Practical Guide Oxford, New York, Beijing, Frankfurt, Sao Paulo, Sydney, Tokyo, Toronto: Pergamon Press, 1987. 272 pp., 63 fig., 21 tab., $ 38.00, ISBN 0-08-034212-4

Lipid analysts have been slow to take up high-performance liquid chromatography (HPLC) largely because of the lack of an all-purpose detection system suited to lipids. In the meantime, some considerable advances have been made, and a substantial body of published work now exists. The subject is not suited to "re'cipe" treatment, as the wide variation in the equipment available to analysts, especially the detectors, means that some developmental work may be necessary to reproduce published separations. The practice of HPLC is thus a skilled occupation, in which the analyst can take some pride. The study of those compounds, that are included under the diffuse generic term of "lipids", has assumed considerable importance in recent years with the recognition that they are involved in many vital biological processes in animals, plants and microorganisms. I t has long been known, for example, that lipids serve as a major storage form for energy in animal and plant tissues, and that they are responsible for maintaining the structural integrity of cells as the principal components of the membranes. Methods for the analysis of lipids are therefore of great importance for many research, clinical and quality control applications. During the last decades, gas-liquid chromatography (GLC) and thin-layer chromatography (TLC) provided the knowledge in the fields of lipid chemistry and biochemistry. In the last years, the situation has been changing rapidly, and many separations of lipids by means of HPLC have been described that cannot be raivalled by other methods. New detectors have been developed, and are now available commercially, that have the potential to overcome many of the former problems. The author gives a comprehensive survey on modern lipid research by means of HPLC for both analysts specialized on HPLC and lipid biochemists which is based on the evaluation of 916 references and many years of own experience in lipid analysis. After introductory chapters dealing with HPLC (theoretical considerations and equipment) and lipids (their structures and occurence), the important steps of extraction, storage and preliminary fractionation of lipids are treated. Two thirds of the book are devoted to HPLC separations of lipid classes and individuals: methods for the separation of simple lipids, and of both simple and complex lipid classes in a single step (chapter 5), methods for the separation of individual phospholipid classes (6), the analysis of fatty acids (7), the separation of molecular species of glycerolipids (8), separation of sphingolipids (9), and some miscellaneous separations (10). An excellently written practical guide. As stated earlier by the author, the book should "remain on the laboratory bench, not on the library shelf". CHRISTIE'S book can be recommended to all those who are concerned with separation, identification and structure research of lipids and related compounds, particularly to all chromatography laboratories in medicine, agriculture, life science and biotechnology. B . NAGEL

Acta Biotechnol. 9 (1989) 5, 4 4 1 - 4 4 5

Akademie-Verlag Berlin

Irradiation Treatment of Straw for its Microbiological Conversion — A Method for the Production of Feed Protein PETBYAEV, E . V . 1 , VETROV, V . S . 1 , PAVLOV, A . V . 1 , STAKHEEV, I . V . 2 , BABITSKAYA, Y . G . 2 , GORBACHEV, V . M . 2 , GLTJSHONOK, T . G . 2 1

2

V. I. Lenin Byelorussian State University Research Institute of Physico-chemical Problems 220080 Minsk, U.S.S.R. Byelorussian Academy of Sciences Institute of Microbiology Minsk, U.S.S.R.

Summary A method for improving the nutritional value of straw as a substrate for fungal growth by radiation is described. The conditions for fungal growth on a medium containing irradiated straw of winter rye were studied. I t was found that the straw substrate improves both the quantity of carbohydrates and the quality of their composition in the nutrient medium. Easier accessibility of the irradiated substrate to enzymatic hydrolysis allows the time bioconversion of cellulose into protein to be reduced and the protein yield to be increased, thus improving the qualitative composition of the fungal biomass. The data obtained suggest a possibility of converting straw into a fodder product by substituting the method of using reagents for pretreatment by a simpler and less labour-consuming method. A most complete utilization of raw plant materials in the microbiological industry requires their pretreatment. Some methods were developed for hydrolysis of plant substrates [1]. In spite of the great diversity of procedures used for hydrolysis of lignocellulosic substrates, the problem remains far from being efficiently solved. The search and development of more perfect methods for treating lignocellulose-containing raw materials are an urgent problem. The irradiation pretreatment of materials is one of such methods. I n previous investigations [2, 3] it was shown t h a t ionizing radiation m a y be an effective method t o substitute alkaline or acid pretraetment. I n this paper the conditions are investigated for the mycelial growth on a substrate containing irradiated winter-rye straw.

Experimental Winter-rye straw irradiated in a 20 kW 1.8—2 MeV electron accelerator ILU-6 was used as a component of the nutrient medium. In the present work we used the strains Penicillum verruculosurn and Tyromyces lacteus. The mycelial growth was carried out in submerged cultivation [4], The contents of reducing substances (RS), easily and not easily hydrolyzable polysaccharides (EHP and NEHP), degree of polymerization (DP) of the straw cellulose in the substrates of study were determined using procedures suggested in [5]. The qualitative composition of sugars in the irradiated substrates was determined using gas-liquid chromatography [6]. 4

Acta Biotechnol. 9 (1989) 5

442

Acta Biotechnol. 9 (1089) 5

Results and Discussion D a t a given in Tab. 1 suggest t h a t in straw treated by ionizing irradiation at a dose of 0 to 1.54 MGr R S content increases from 1.2 to 6.5%, percentage of E H P , from 21.2 to 40.0%, while D P of straw cellulose is markedly reduced, N E H P content (of cellulose) also diminishes from 42.0 to 18%. All the above physicochemical changes produce favourable conditions for the growth of microorganisms. Tab. 1. Dependence of RS and components of straw cellulose on absorbed doses Dose [MGr]

DP

RS [%]

EHP [%]

NEHP[%]

0 0.14 0.28 0.42 0.56 0.70 0.98 1.54

1300 450 390 280 240 200 155 110

1.2 2.0 2.9 2.8 4.9 5.6 6.0 6.5

21.2 22.8 25.2 28.0 30.1 32.0 36.4 40.0

42.0 39.2 37.1 33.0 30.0 28.5 22.6 18.0

When Penicillum verruculoçum fungi were grown in a medium containing 3% of irradiation-modified straw, R S contents increased from 0.45 mg/ml (native straw) to 0.84 mg/ml a t a dose of 1.54 MGr. I t should be noted t h a t protein content in the biomass rose from 10—12 to 20%. If 2 % of straw was added to the substrate, R S content increased from 0.42 to 0.77 mg/ml, and protein percentage in the biomass rose from 12 to 23—25%, respectively. The studies carried out have shown t h a t the highest protein increment in biomass was observed with straw irradiated at doses of up to 0.5 MGr. Further increase of irradiation doses does not raise the protein content in biomass. I n view of this, 0.3 MGr doses were subsequently used for modification of the substrate. This dose was chosen because both of essentially higher growth parameters of the fungal mass against the control and of economic considerations, since application of high irradiation doses would make the irradiation method too expensive and consequently inapplicable.* Pre-irradiation of the substrate influenced the qualitative and quantitative compositions of sugars in the nutrient media (Tab. 2). Due to the destruction of cellulose component of the irradiated straw, the highest carbohydrate yield in irradiated straw-based biomass was 25.07 mg/ml against 7.33 mg/ml in native straw-based biomass. Glucose and ribose prevailed in the native straw-based medium, whereas in the medium containing irradiated straw, glucose and galactose were predominant. I t is noteworthy t h a t in the medium with irradiated straw glucose amount was four times t h a t contained in the medium with native straw. Improved qualitative and quantitative compositions of carbohydrates in irradiated straw-based nutrient medium, as well as easier accessibility of the substrate to enzymatic hydrolysis reduced the time of fungal cultivation and accumulation of the end product by fungi (Tab. 3). The biomass and protein yields expressed in g/g substrate were 0.73 and 0.11 for native straw and 0.90 and 0.14 for irradiation-modified straw. * Irradiation treatment of 1 ton of straw at a dose of 0.01 MGr in an ILU-6 electron accelerator costs 2.5 Roubles.

PETRYAEV, E . V . , VETROV, V .

S. et al., Irradiation Treatment of Straw

443

Tab. 2. Carbohydrate composition of substrates Carbohydrate

Native straw

Accelerated electrontreated straw, 0.3 MGr

Ribose Xylose Arabinose Fructose Galactose Glucose Sucrose Cellobiose

0.83 trace trace 0.27 tracei 5.80 0.24 0.19

trace 0.56 trace 0.21 2.80 21.50 trace trace

Total

7.33

25.07

[mg/ml]

Thus, the treatment of straw by accelerated electrons reduces the time of the fermentation process, simultaneous increasing the protein yield and improving the qualitative composition of the fungal biomass produced. The sum of indispensable amino acids was 43.9% for accelerated electron-irradiated straw, being 38.6% for untreated straw. Tab. 3. Product versus substrate modification Treatment

Cultivation time [h]

Biomass yield, g/g substrate

Total protein yield, g/g substrate

Native straw 0.3 MGr

48 24

0.73 0.90

0.11 0.14

When Penicillum verruculosum was cultivated in a native straw-containing substrate, the lipid content in the product was 2.3%, whereas in the straw treated by accelerated electrons it was 2.5%. Irradiation of the substrate does not change the intracellular fat composition. In fungus-fermented substrate 18 amino acids were identified, 57.5 to 64.2% being unsaturated ones. Among unsaturated and saturated acids, oleic and linoleic, on the one hand, and palmitic acids, on the other, contributed the highest percentage to the sum of fatty acids. The ratio of the sums of unsaturated and saturated f a t t y acids was between 1.4 and 1.8. When solid-phase fermentation technique (SPF) was used and under submerged fermentation (SF) the irradiation treatment of straw increased the protein content in biomass. The value of products of microbial origin depends not only on the protein content but also on the qualitative composition of protein. Plant proteins may be subdivided into protoplasma proteins (albumins and globumins) and reserve proteins (prolamins and gluteins). Fractionation results have shown that in proteins of Penicillum, verruculosum and Tyromyces lacteus cultivated both on native and irradiated straw, protoplasma proteins appeared to be prevailing, their percentage being 53 to 56%. Unlike fungal biomass, the content of protoplasma proteins in the substrate was as low as 32.9%, that of reserve proteins amounted to 67.1% (Tab. 4). Cultivation procedure had some effect upon protein distribution among the fractions. Under surface cultivation straw enriched with Penicillum verruculosum mycelium appears to have much 4«

Acta Biotechnol. 9 (1989) 5

444

Tab. 4 Fractional composition of proteins in mycelial fungi biomass % of the total protein amount Variant

Albumins

Globulins

Prolamins

Gluteins

Native straw SF SPF

36.9 37.9

18.0 14.8

11.9 16.6

33.2 30.7

0.3 MGr SF SPF

37.12 41.3

17.9 14.6

10.5 14.3

34.48 29.8

SPF Native straw: 41.3 0.3 MGr 34.7

13.4 18.4

12.9 12.7

32.4 34.2

Substrate (control)

17.10

12.8

54.3

15.8

more fractions I and III proteins than it has under submerged fermentation. It should be emphasized that in the irradiated substrate the contents of biologically most valuable fraction of albumin were 41.3 against 37.9% and 37.12 against 36.9%. There were some differences in the fractional composition depending on the product used (microor macromycete). Mycelial fungi-fermented straw was enriched not only with protein but also with easily assimiliable carbohydrates (alcohol- and water-soluble fractions) (Tab. 5). As compared with substrates themselves (native straw), in the end products the contents of cellulose, hemicellulose and lignous fraction are considerably reduced. Moreover, irradiation of straw facilitated the obtaining of products with lower contents of cellulose. Tab. 5. Fractional carbohydrate composition of fermented substrate Variant

Alcoholsoluble

Watersoluble

Hemicellulose

Cellulose

[%]

[%]

[%]

SF 0.3 MGr

0.31

0.67

23.9

17.6

10.Ò

SPF Native straw 0.3 MGr

0.63 2.04

3.34 2.67

18.3 20.7

19.7 17.3

9.0 11.0

SPF Native straw 0.3 MGr Substrate

3.03 2.54 0.20

6.38 4.29 0.86

22.6 11.3 30.8

18.5 15.8 38.9

8.4 9.6 17.5

[%]

[%]

Lignocellulose

Comparison of parameters characterizing the production of microbial protein on irradiation-modified straw, on partially delignified [7] and native straw has shown that, as regards the end effect (the total protein yield), the treatment of the substrate by accelerated electrons is equivalent to pretreatment by 1% sodium-hydroxide solution and essentially exceeds the results for native straw.

PETRYAEV, E. V., VETEOV, V. S. et al., Irradiation Treatment of Straw

445

To summarize what was said above, it may be noted that the irradiation pretreatment of straw results in the destruction of cellulose components of the substrate, leading to a considerable increase in carbohydrates. This, in turn, permits a 50% reduction of the time of cellulose-to-protein bioconversion, improves the composition of fungal biomass, and increases the yields of the end product and protein, etc., which makes it possible to transform straw into a valuable fodder product, with pretreatment by reagents being substituted by a simpler and less energy-consuming method. Received July 14, 1988 Revised October 18, 1988

References [ 1 ] OGARKOV, V . I . , K I S E L E V , O . I . , BYKOV, V . A . : Biotechnologiya ( 1 9 8 5 ) 3 , 1 . [ 2 ] V E T E O V , V . S., VYSOTSKAYA, N . A., D M I T R I E V , A . M . : Radiation Treatment [3]

of Wastes for Agricultural Utilization. Moscow: Energoizdat, 1984, 152 (in Russian). P E T R Y A E V , E . P . , PAVLOV, A. V., GLTJSHONOK, T. G . , et al. — I n : Abstracts of All-Union Conference "Chemistry, Technology and Application of Cellulose and its Derivatives". Vladimir, pt. 1, p. 11.

[4] STAKHEEV, I . V., KOSTINA, A . M., BABITSKAYA, V . G., e t a l . : M i k r o b i o l o g i y a 5 5 (1986) 1, 66.

P., A K I M , G . A . : Practical Work in Wood and Cellulose Chemistry. Moscow: Lesnaya Promyshlennost Pubi., 1965, 411.

[ 5 ] OBOLENSKAYA, A . V . , SHCHEGOLEV, V .

[6] YABOVENKO, V . L . , NAKHMANOVICH, B . M., MAKEEV, D . M., e t al. : F e r m e n t n a y a i S p i r t o v a y a

Promyshlennost (1977) 1, 6. [7] STAKHEEV, I . V., SHCHERBA, V. V., BABITSKAYA, V. G., e t a l . : P r i k l . B i o k h i m . Mikrobiol. 2 (1986) 3, 363.

Acta Biotechnol. 9 (1989) 5, 446

Akademie-Verlag Berlin

Book Review C. J . MCNEAL

The Analysis of Peptides and Proteins by Mass Spectrometry Proceedings of the Fourth Texas Symposium on Mass Spectrometry Chichester, New York, Brisbane, Toronto, Singapore: John Wiley & Sons, 1988. 322 pp., 177 fig., £ 39.95, ISBN 0-471-92062-2

There are several reasons why mass spectrometry (MS) has become more relevant to problems involving peptides and proteins. The mass range has been extended to 35,000 ¡x, sensivity has been improved to the pmol level and meaningful sequences are being developed using the MS/MS protocol. These developments are a direct response to the requirements of the life sciences for analytical MS: to bridge the analytical gap between protein chemistry and molecular genetics. The organizers of the Symposium selected as invited speakers a mix of mass spectroscopists working in an active biomedical environment and researchers in the life sciences mainly concerned with peptide and protein chemistry. Thus, the close interaction between the analytical requirements of protein chemistry and the development of analytical methods is reflected in the contributions to the proceedings book: the application of several MS techniques such as plasma desorption MS (PDMS), fast atom bombardment MS (FABMS), secondary ion MS (SIMS) and laser desorption MS (LDMS) to thermally labile compounds, e.g.' peptides, proteins and other biomacromolecules, and to the identification of products and parts thereof during chemical synthesis. I n most cases, MS serves as one of several analytical methods, sometimes coupled with separation methods (thermospray LC-MS, TLC-MS). The contributions cover a wide field of MS methods, of compounds (peptides, proteins, glycopeptides, oligosaccharides, glycopeptidolipids, drugs) and problems (chemical synthesis, molecular weight determination, enzyme kinetics, structure analysis). The authors focus on the application of MS methods more than on experimental details. The well illustrated articles contain numerous unpublished results which demonstrate the wide applicability of MS to basic problems in life sciences. Thus, the book has been written not only for mass spectroscopists, it addresses all those who are concerned with research in peptide and protein chemistry, in genetic engineering, and analytics of biomacromolecules. B . NAGEL

Acta Biotechnol. 9 (1989) 5, 447 - 4 5 1

Akademie-Verlag Berlin

New Synthetic Route to 9a-Hydroxy-4-androsten-3,17-dione U s z y c k a - H o r a w a , T., S k i b i n s k a , M., J a k u b o w s k i , W.

Institute of Pharmaceutical Industry ul. Rydygiera 8, 01-793 Warsaw, Poland

Summary The new synthesis of 9