Acta Biotechnologica: Volume 8, Number 4 1988 [Reprint 2021 ed.] 9783112581803, 9783112581797

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Acta Journal of microbial, biochemical and bioanalogous technology

Akademie-Verlag Berlin ISSN 0138-4988 Acta Biotechnol., Berlin 8 (1988) A, 305—392

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Acta BIlMlMliiO 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

Editori il Board: D. Meyer, Leipzig P. Moschinski, Lodz A. Moser, Graz M. D. Nicu, Bucharest Chr. Panavotov, Sofia L. D. Phai, Hanoi H. Sahm, Jülich W. Scheler, Berlin R. Schulzc, Halle B. Sikyta, Prague G. K . Skrjabin, Moscow M. A. Urrutia, Habat.a

1988

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

Number 4

Managing Editor :

L. Dimter, Leipzig

Volume 8

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

•

B E R L I N

"Acta Biotechnologica" publishes original papers, short communications, reports and reviews from t h e whole field of biotechnology. The journal is to promote t h e establishment of biotechnology as a new and integrated scientific field. The field of biotechnology covers microbial technology, biochemical technology and the technology of synthesizing and applying bioanalogous reaction systems. The technological character of t h e journal is guaranteed by t h e fact t h a t papers on microbiology, biochemistry, chemistry and physic« 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 -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): t o a bookshop or to the wholesale distributing agency K u n s t und Wissen, Erich Bieber OHG, Wilhelmstr. 4 - 6 , D-7000 S t u t t g a r t 1; — in the other Western European countries: to K u n s t und Wissen, Erich Bieber GmbH, General Wille-Str. 4, CH-8002 Zürich; — in other countries: to t h e international book- and journal-selling trade, to Buchexport, Volkseigener Außenhandelsbetrieb der D D R , P F 160, D D R - 7010 Leipzig. or to the Akademie-Verlag Berlin, Leipziger Str. 3—4, P F 1233, D D R -1086 Berlin. Acta Biotechnologica Herausgeber: I n s t i t u t für Biotechnologie der AdW der D D R , Permoserstr. 15, D D R - 7050 Leipzig (Prof. Dr. Manfred Ringpfeil) und V E B Chemieanlagenbaukombinat Leipzig—Grimma, Bahnhofstr. 3 - 5 , D D R - 7240 Grimma (Dipl.-Ing. Günter Vetterlein). Verlag: Akademie-Verlag Berlin, Leipziger Straße 3 - 4 , P F 1233, D D R - 1 0 8 6 Berlin; Fernruf: 2 2 3 6 2 0 1 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 Druekhaus „Maxim Gorki", D D R - 7 4 0 0 Altenburg. Erscheinungsweise: Die Zeitschrift „Acta Biotechnologica" erscheint jährlich in einem Band mit 6 Heften. Bezugspreis eines Bandes 192,— DM zuzüglich Versandspesen; Preis je H e f t 32,— DM. Der gültige Jahresbezugspreis f ü r die D D R ist der Postzeitungsliste zu entnehmen. Bestellnummer dieses Heftes: 1094/8/4. Urheberrecht: Alle Rechte vorbehalten, insbesondere der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translation into foreign languages). No p a r t of this issue m a y be reproduced in any form, by photoprint, microfilm or any other means, without written permission form t h e publishers. © 1988 by Akademie -Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 18520 03000

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

A Mathematical Approach for the Estimation of Biomass Production Rate in Solid State Fermentation RODBIGUEZ L E O N , J . A . 1 , SASTRE, L . 1 , ECHEVARRIA, J . 1 , DELGADO, G . 1 , BECHSTEDT, W . 2

1

ICIDCA, Apartado 4026, Ciudad de la Habana. Cuba. 2 VEB Chemieanlagenbaukombinat Leipzig—Grimma, Hauptabteilung Biotechnologie, P F 674, Leipzig 7010, G.D.R.

Summary The oxygen uptake rate (OUR) was studied in a solid state fermentation process of dried citrus peel with the strain Aspergillus niger QH-2 in order to obtain the growth estimation of the microorganism in the system. The relationship between OUR, the maintenance coefficient (m) and the yield for oxygen consumption ( y 0 J allows the estimation of the biomass rate if we consider that both parameters are not constants in some periods of the process. It was estimated that in the first 24h the strain has an specific growth rate of 0.174 h _ 1 with values for F 0 , a n d 1 1 1 ¡ n the order of 2.84 g-cell/g-oxygen and 0.006 g-oxygen/g-cell • h respectively.

Introduction I t was previously demonstrated [1] that the growth calculation of a microorganism in a solid state fermentation by exit gas analysis is quite feasible. At the same time these authors advised that before a general application of the method proposed it should be determined if the maintenance coefficient and the biomass yield for oxygen consumption are constant or not. Dealing with solid state fermentation of dried citrus peel with the strain Aspergillus niger QH-2 it was demonstrated [2] that a good estimate for the specific growth rate could be obtained if we consider that Y0t is not constant during the process and estimating m by external calculation, considering this last parameter constant during the whole fermentation with a value of 0.018 g-oxygen/g-cell • h. In this work we propose the estimation of the biomass production rate and specific growth rate from the original OUR data like proposed [1], but considering a mathematical optimization procedure [3] that permit the calculation of the parameters here pointed out without any other calculation of parameters out of the system. Materials and Methods Microorganism and Solid Substrate The strain Aspergillus niger and dried citrus peel were employed as reported previously [4]1*

308

Acta Biotechnol. 8 (1988) 4

Cultivation Fermentations were carried^ out in rotary fermenters containing 1.2 kg fermentation mass which was aerated with water saturated air at a rate of 1 VkgM (liters of air/kg wet substrate • min) under non-aseptic conditions. A spore inoculum was carried out with a concentration of 2 X 107 spores/g substrate and initial moisture content was 56—60% considering the optimum reported before [5]. Analytical Methods Were reported in the works just cited. Mathematical Procedure A gradient method with H O O K E - J E E V E S [6] acceleration was used to minimize the mean square desviation between the calculated amount of biomass and the experimental results. The differential equation was solved with the R U N G E - K U T T A method in each optimization step. The experimental data for 0 2 and C0 2 were smoothed bv a F O U R I E R filter [7], Results and Discussion In order to obtain the values that allow a proper estimation of and m it was decided to take several samples at previously determined times that allow a mass balance at each point selected which bring an information about the real biomass obtained in the process. The experimental biomass was calculated in each sampling point by the expression: B = 31 (P( -

Pi) (100 -

H)IPb

X 100

(1)

where B Pt Pi M PB H

biomass [g]_ protein content at time t [%] protein content at initial time [%] product weight at time t [g] protein content of the pure biomass [%] humidity [%]

Tab. 1 shows the results obtained at the different times as were reported formerly [2], The initial protein content was 5 . 7 % and the biomass obtained at 48 and 60 h were corrected by adding the biomass content of the samples. Tab. 1. Results of solid state fermentation of dried citrus peel Time

[h] 0

24 48

60 1

Mass [g] 1147 1023 877

818

63.6 61.1 66.7 68.3

[%]

Protein content

Biomass [g]

6.0 14.0 17.7 19.9

5.36 117.9 124.51 129.9 1

Corrected values considering the biomass content in the samples

J., A.

RODRIGUEZ L E O N ,

SASTKE, L .

et a)., Solid State Fermentation

309

Furthermore we used the experimental data for oxygen consumption and C0 2 production demonstrated as Fig. 1. S A T O et al. [ 1 ] , proposed the estimation of biomass growth using the following expression: 0

0

,

1

-

¿

(

N

T

*

-

1

)

solving this equation considering YQ2 and TO constant for the period evaluated. However dealing with solid state fermentation of dried citrus peel this was not the case when the whole period of fermentation is considered, that means, at times higher than 20 to 24 h when the mono- afid disaccharides were metabolized and an attack to polymers (pectin or fiber components) is considered.

Time

Ch]

Fig. 1. Production of C0 2 and consumption of 0., during the fermentation

This equation was solved for solid state fermentation of dried citrus peel [2] considering the maintenance coefficient for oxygen consumption constant for the whole process and it was estimated from data f6r different solid, state fermentations w ith' Aspergillus niger QH-2. The value obtained was 0.0018 g-oxygen/g-cell • h. Now we state the possibility of making a mathematical optimization in which Y 0l and TO are considered variables in the whole period and constant for the specific ones (which were previously selected). In solid state fermentation of dried citrus peel it seems appropriate to choose a period in which all the available simple sugars are metabolized, and two periods more in which the possible attack of pectin and cellulose is assumed. This periods were choosen at 24, 48 and 60 h. The optimization basis is to obtain the proper values of YQi and m that via the expression (2) allows at the end of the period a calculated value for the biomass that was close to that value analytically determined by the mass balance. Tab. 2 offers the results of this procedure. Comparing these results with those obtained whenTOwas calculated out of the process and constant for the whole period it was observed that both parameters are not constant. However the maintenance coefficient has a very good agreement when it was calculated by the optimization procedure for the period between 24—48 h. This can be due to the fact that when it was calculated whith data outside the system, the points that were

310

Acta Biotechnol. 8 (1988) 4 Tab. 2. Predicted values by the mathematical method Time range

m

[h]

1

2

[h-i)

Experimental biomass [g]

4-60 4-24 24-48 48-60

2.77 2.84 2.00 1.98

0.011 0.006 0.017 0.010

NS 0.174 0.008 0.004

5.43 117.9 124.5 129.9

1 2

F0,

g-cell/g-oxygen g-oxygen/g-cell • h

3

¡J-

Estimated biomass [g] 5.43 115.6 122.5 127.6

values at 4h

selectioned belong basically to tsiij region, or that maintenance coefficient is affected by the variations in the process (temperature, p H etc.) and that in this period (24 —48 h) these variations are not so pronounced. I t must be kept in mind that this is a period in which growth is greatly affected. In the initial period selected, 0—24 h, the growth is vigorous due to sugars consumption and in the last one, after 48 h, the growth is fairly poor. With the estimation of biomass content at different times in which oxygen or carbon dioxide were determined by exit gas analysis (each hour) it was calculated the maximum specific growth rate for biomass and oxygen consumption and the agreement between these two procedures was exact for the first period (0.175 h _1 ). After this initial period the estimation of the specific growth rate from 0 2 or C0 2 rates is not possible because they show a decreasing pattern. I t must be pointed out that the respiratory quotient was nearly 1 in the whole process. I t will be very usefull to consider a process in which the substrate is not so heterogeneous as dried citrus peel in order to determine if these facts are like as in our process. In other words the possible variation of Y0s and m during solid state fermentation can not be misunderstood. Received October 12, 1987

References [1] SATO, K . , NAGATANI, M., NAKAMURA, K . , SATO, S . : J . F e r m e n t . T e c h n o l . GL ( 1 9 8 3 ) 6, 6 2 3 . [ 2 ] RODRIGUEZ LEON, J . A . , BECHSTEDT, W . , ECHEVARRÍA, J . , SIERRA, N . , DELGADO, G . :

Proceedings of 1 th International Cuban Seminar on Biotechnology, Havana, Cuba, 1986 (in press). [3] BEVERIDGE, G., SCHECHTER, R.: Optimization, Theory and Practice, La Habana, Cuba: Edición Revolucionaria (1970). [ 4 ] RODRIGUEZ, J . A . , ECHEVARRÍA, J . , RODRIGUEZ, E . J . , SIERRA, N . , DANIEL, A . , MARTINEZ,

O.: Biotechnol. Lett. 7 (1985) 8, 577. [ 5 ] RODRIGUEZ, J . A . , BECHSTEDT, W . , ECHEVARRÍA, J . , SIERRA, N . , DELGADO, G., DANIEL, A . ,

MARTINEZ, O.: Acta Biotechnol. 6 (1986) 3, 253. [6] MUGELE, R . : I B M S y s t e m s J . 1 ( 1 9 6 2 ) 2. [ 7 ] AUBANEL, E . , MYLAND, J . , OLDHAM, K . , ZOSKI, C.: J . E l e c t r o a n a l . C h e m . 1 8 4 ( 1 9 8 5 ) 2 3 9

Acta Biotechnol. 8 (1988) 4, 311-318

Kinetic Study of pTG201 Plasmid Stability in Escherichia coli MAKIN-INIESTA, F .

Departamento de Genetica y Microbiologia Universidad de Murcia 30071 Murcia, Spain

Summary Recombinant pTG201 plasmid coming from pBR322 plasmid, has been incorporated into Escherichia coli K12 to determine the influence of several culture conditions on the variation of the copy number. Continuous and batch cultures on LB medium without antibiotic selection and different oxygen tensions (21% and 100%) have been tested. The expression of pTG201 encoded genes and the kinetics of plasmid loss differ significantly from the behaviour of pBR322 plasmid.

Introduction Plasmids are useful instruments for Genetic Engineering so nowadays it is possible the cloning and expression of foreign DNA in bacteria for objectives such as industrial production of plasmid encoded proteins. However, after overcoming the difficulties of cloning and expression, the problem of instability of the recombinant plasmids appears and the use at industrial level of such systems can be infeasible. This is obvious with pTG201 plasmid that was used as experimental model to study the instability of recombinant plasmids. This pTG201 plasmid derivates from pBR322 where genetic material from X phage and Pseudomonas putida was inserted [1, 2], As a consequence of foreign DNA insertion, pTG201 becomes more unstable than pBR322, when assayed in the same conditions and strains of E. coli [3, 4], Recently several authors [3, 5] showed, that the immobilization of E. coli cells, carrying pTG201 plasmid, can improve the plasmid stability and the expression of cloned genes in relation to free cell cultures. This phenomenon shows good perspectives on its practical use but we know little about the mechanism that determines the different plasmid stability kinetics between free and immobilized cells of E. coli. On the other hand, the culture of immobilized cells in presence of pure oxygen has been assayed by several authors to ameliorate the cell growth in support of immobilization [6]. Consequently we are interested in knowing the kinetic behaviour of pTG201 unstability in free cells cultures in presence of different oxygen tensions.

Acta Biotechnol. 8 (1988) 4

312

Materials and Methods Bacterial

Strain and

Plasmid

The strain used was E. coli K 1 2 and the plasmid was pTG201 (6.44 kilobases) that contains the gene X y l E from P. putida which codes for catechol 2—3 dioxygenase (cat-02-ase) and /S-lactamase activities, both used as plasmid markers. The gene X y l E is under control of 1 P R promoter which1 is repressed by CI8B7, pTG201 plasmid is not conjugative and was constructed by a siijiilar method to that described by ZUKOWSKI et al. [1] and generously gifted by TRANSGENE S . A . (France). K12/pTG201 strain was obtained by S . S A Y A D I and M. NASRI (personal communication). Medium and Culture

Conditions

B L growth medium contained (per litre of deionized water) 10 g tryptone (Difco) 5 g yeast extract (Difco) and 5 g NaCl, pH was adjusted to 7.2. Continuous cultures were carried out in a little chemostat already described [5], modified to take samples of beads during the culture. Conditions were the following: 45 ml working volume, a oxygen supply rate equal to 3 times working volume per minute. Oxygen was supplied at 2 1 % (air) and 100% respectively. Temperature was 3 7 °C and dilution rates (D) according to DE T A X I S DU P O E T et al. [ 5 ] were adjusted to a value of 0.7 times the maximal growth rate of the strain (¡¿max). The number of cell generations were determined from t X ¡¿/In 2, where I is the time from the beginning of the experiment and JA the growth rate. Cell Counting and Stability of

Plasmid

Samples from effluent of cultures were plated on AL medium (BL medium additioned of 15 g/1 agar). The presence of cat-02-ase carrying plasmid was detected spraying the colonies with 0.5 M catechol [1, 2], the clones carrying cat-02-ase activity (P-\- cells) turned yellow and the remainder stayed white (P— cells). Antibiotic resistance was assayed in AL plates with 50 mg/1 ampicillin. Assay of Cat-02-ase

Activity

Conversion of catechol into 2-hydroxymuconic semi-aldehyde was measured spectrophotometrically and cat-02-ase activity was determined as increment of OD 375 nm/min as already described [2]. Enzyme activity' was expressed for 100 ¡J.1 of cell culture. Enzyme activity per biomass unit was determined as enzyme activitv/OD 660 nm of the culture'. Plasmid

Copy (PNC)

Determination

Bacterial cells were lysed by the method of ECKHARDT [ 7 ] and P R O J A N et al. [8]. 250 ¡¿1 of cell lysate were prepared for electrophoresis by addition of 50 (j.m of 5 0 % glycerol, 0.02% xylene cyanol, and 0 . 0 2 % bromophenol blue. Horizontal electrophoresis was performed in 1 % agarose slab gels (4 mm thick), which were run at 2.5 V/cm for 12 to 16 h in Tris-borate-EDTA buffer (89 mM Tris base, 2.5 mM disodium E D T A , and 8.9 mM boric acid). After electrophoresis, gels were stained with 1 |j.g/ml of ethidium bromide for 30 min. Plasmid bands in the gel were irradiated by an Ultra-Violet Transilluminator and photographed using a 665 Polaroid film (Polaroid Corp., Cambridge MA). The quantification of plasmid bands in the negatives was made according to PRTJNELL

MARIN-INIESTA, F., Plasmid Stability in E. coli

313

e t al. [9] scanning t h e negatives along a perpendicular direction t o t h e lane axis with a scanning densitometer (Mod. R F T I I , Transidyne General Corporation). The ratio of integrated plasmid and chromosomal D N A peaks were used for PCX determination according t o PKOJAN et al. [8]. Statistical

Calculations

E x p e r i m e n t a l d a t a were f i t t e d t o one or t w o lines by least square method using a suitable program [11] in a n M24 OLIVETTI microcomputer. Results Batch Culture Fig. 1 shows the g r o w t h curve of E. coli K12/pTG201 in b a t c h culture supplied with 2 1 % oxygen where ii. shows the m a x i m a l value (¡¿max), a t t h e exponential phase, equal to 2.08 h _ 1 . A generation n u m b e r equal to 6.5 between inoculation a n d stationary phase was determined a n d , during this period, t h e fraction of cells carrying t h e plasmid (F = P — / ( P r P - - ) ) ranged f r o m an initial value of F = 0.97"to F = 0,90. I n Fig. 1 we can also observe t h e evolution of PCN, we can see t h a t there is an inverted relation between PCX and a values. W e notice in f a c t a n i m p o r t a n t PCX decrease during t h e exponential phase (fi = ¡¿max), a n d later, when ¡j. decreases, PCX increases up t o i t s m a x i m u m value in t h e stationary phase. The cat-02-ase activity reaches it m a x i m u m value a t t h e early stationary phase (12 hours of culture) and later diminished quickly. Concerning b a t c h cultures supplied with 100% oxygen we find a ¡¿max value equal to 1.63 h r 1 , lower t h a n in 2 1 % oxygen, b u t the behaviour of PCX a n d cat-02-ase activity is t h e same. On t h e other hand, in our experimental conditons no significant difference in specific growtli rates between cells with a n d without plasmid has been observed and "consequently t h e relation ¡xp—/¡xp+ (a) was equal to 1 either a t 2 1 % or 100% oxygen. -|0,60

- 0,30

-A-

Time [h] Fig. 1. Batch culture supplied with 21% oxygen — «— Biomass (OD 660 nm) — A— Enzyme activity (cat-02-ase activity/100 ¡xl of culture) — A — Enzyme activitity/OD 660 nm — • — Plasmid copy nxlmber per chromosome (PCN) X 10 -2

314

Acta Bioteohnol. 8 (1988) 4

Continuous

Culture

Biomass production by the chemostat in presence of 21% oxygen showed an average value in the equilibrium of 6.2 X 10~8 cells/min. A decrease in biomass production for cultures in 100% oxygen, with average value of 3.4 X 10~8 cells/min, reflecting the minor fi. above mentioned was noted. Culture effluents were tested for enzyme activity (measured as cat-02-ase activity/ 100 [i.1 of culture). During the first 50 generations average values of enzyme activity equal to 3.6 X 10" 2 for cultures at 21% oxygen and 3.3 X 10" 3 at 100% oxygen were abtained. If enzyme activity per biomass unit is considered we obtain values of 2.0 X 10 - 2 ot 21% oxygen and 4.7 X 10~3 at 100% oxygen. 60

100

200 Generations