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CultureconditionsforjasmonicacidandbiomassproductionbyBotryodiplodiatheobromaeinsubmergedfermentationARTICLEinPROCESSBIOCHEMISTRYSEPTEMBER1998ImpactFactor:2.52DOI:10.1016/S0032-9592(98)00035-1

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Process Biochemistry' Vol. 33, No. 7, pp. 715-7211. 1998 C~ 1998 Published by Ir".lsc'~'icr Science l.td, All rights reserved

Printed in Great Britain ()032-9592/0g $ - - see front manor

S 0 0 3 2 - 9 5 9 2 I 98 )000035- l

Culture conditions for jasmonic acid and biomass production by Botryodiplodia

theobromae in submerged fermentation

F. Eng, ~' M. Gut i6r rez -Ro jas b and E. Favela-Torres h*

"Divisi6n de Biotecnologia, lnstituto Cubano de lnvestigaciones de los Dcrivados de la Carla de Azficar, Vfa Blanca y Carrelera Central 8(16, A.P. 4026, Ciudad Habana, Cuba

"Departamento de Biotecnologia, Universidad Aut6noma Metropolitana, Unidad lztapalapa, A.P. 55535, C.P. 09340, M6xico D.F., Mdxicc~

(Received 12 January 1998; accepted 1 March 1998)

Abstract

Jasmonic acid (JA) is a plant growth regulator produced by Bottyodiplodia theobromae in submerged fermentation. Eight strains of B. theobromae isolated from Cuban orange waste peel were screened fl~r JA production. Strain 2434 was selected for its higher JA production (914 rag/I) and productivity (c)1.4 mg/I day). Studies carried out at different temperatures showed that the highest JA production (925 rag/I) was obtained at 30-32C. Although biomass production was enhanced under agitation (up to 100 rpm), JA production was negatively affected when the agitation was increased. Maximum JA production (900 rag/l) was obtained under non-agitated conditions. Studies concerning the effect of different carbon and nitrogen somces showed that fructose, glucose and sodium nitrate were the best sources for JA production. 1998 Elsevier Science Ltd. All rights reserved

K(vwords." Jasmonic acid, Bot~odiplodia theobromae, biosynthesis, growth, submerged fermentation, phytohormone.

Introduction

Jasmonie acid (JA) [3-oxo-2-(2'-cis-pentenyl)-ciclopen- tane-l-acctate and their derivatives are a group of native plant growth regulators called jasmonates, their major representatives are isomers cis [(+)-7-iso-JA] and trans [(--)-JA] [1]. These compounds are widely distributed in higher plants [2] and microorganisms [3,4] showing phytohormone action and playing important roles as inhibitors of plant growth, inducers of tuberization on potato, Jerusalem artichoke and yam and in senescence promotion of detached leaves, among others [5]. These compounds also induce the expression of several defensive genes in plants against pathogen attack or wounding such as the proteinase gene inhibitors in tomato and the vegetative storage protein genes in soybean [5,6].

*To whom correspondence should be addressed.

715

Many studies concerning the biosynthesis of JA in plants have been published [7-13]. However, know- ledge about JA production by microorganisms is still limited. JA is a secondary metabolite synthesized and secreted in the late growth phase or the stationary phase after 5-10 days fermentation. Bot~odiplodia the- obromae [ 14-16], mutants of Gibberella fujikuroi [ 17], Collihya conffuens, Coprinus alkalinus and Mvcena tinti- nabulum [3] have been reported as JA producers. Nevertheless, little information concerning JA produc- tion conditions is available since most of lhe studies carried out are published as patents or abstracts. JA production by B. theobromae D7/2 was higher at agita- tion rates below 190 rpm [15, 16] and at temperatures of cultivation from 27C to 30C.

Species of the genus Botryodiplodia arc able to grow in minimum defined media [16]. JA production by B. theobrornae D7/2 increased with sucrose and glucose as

716 E Eng. M. GutiOrrez-Rojas, E. Favela-Torres

Table 1. Screening of Botryodiplodia theobromae strains for jasmonic acid and biomass production after 10 days of culture

Strain Biomass (g/L) JA (mg/l)

1 9.13 457.49 1F 6.95 ND* 2 16.06 ND 2F 9.84 2.2O 489 4.35 ND 1119 6.95 ND 1368 14.83 ND 2434 11.10 914.10

*ND: not detected.

carbon sources [16]. The effect of the nitrogen source is not clear since JA production by B. theobromae D7/2 was higher with sodium nitrate, potassium nitrate or calcium nitrate [16], whilst JA production by Lasiodi- plodia theobromae $22L was similar when organic or inorganic nitrogen sources were used [18].

The objective of this work was to study the effect of temperature, agitation rate and carbon and nitrogen sources on JA and biomass production by B. theo- bromae in submerged fermentation.

Materials and methods

Microorganisms

Eight strains of B. theobromae (Table 1) from the lnsti- tuto Nacional de Investigaciones Fundamentales de la Agricultura Tropical (Cuba) isolated from Cuban Citrus cinensis Osbeck cv Valencia, were used. The strains were stored on malt agar extract slants at 4C and subcultured every 2 months.

Culture techniques

A sample of the stock culture was transferred to malt agar extract plates and incubated for three days at 30C. Five loops of mycelium (7 mm diameter) were used for inoculation of 25 ml of culture medium in 100ml Erlenmeyer flasks and incubated at different temperatures (25-35C) and agitation rates (0-150 rpm) as indicated.

Media composition

Culture media with the following basal salt composi- tion were used (in g/l): sucrose, 50; NaNO3, 7.5; KHzPO4, 2.0; KCI, 0.3; MgSO4.7H20, 0.6; FeSO4.7H20, 0.6; ZnSO4.7H20, 0.03; MnSOa.7H20, 0.003; CuSO4.7H20, 0.003; NazMoO4.2H20, 0.003; yeast extract, 1.0. After autoclaving initial pH was adjusted to 5.5-5.6 with NaOH (1 M). The effect of different carbon sources [sucrose, glucose, fructose,

lactose, sorbitol, starch and starch:sucrose (l:l)] and nitrogen sources (NH4CI, (NH4)zHPO4, NH4NO~, NaNO3, (NH4)2SO4, urea and yeast extract) on growth and jasmonic acid production was carried out using 21.0 g carbon/I and 1.24 g nitrogen/l, respectively. Cul- tures were carried out over a 10 days period in static submerged cultures at 30C.

Analytical methods

Biomass concentration was determined by dry weight after broth filtration on filter paper Whatman 41 followed by drying at 60C for 24 h. Glucose and fruc- tose were determined by HPLC (Binary LC Pump 250, Perkin Elmer) with a refraction index detector (LC-30 RI, Perkin Elmer) [19]. Sulphuric acid 30 mM at flow rate of 0.6 ml/min was used as mobile phase through a Rezek Organic Acid (Phenomenex) column. Glucose (Sigma) and Fructose (Supelco) were used as stand- ards. JA concentration was determined by HPLC as described by Koda [4]. Determinations were made with an ultraviolet detector (UV/Vis Spectrometric Detector LC 290, Perkin Elmer) using methanol: acetic acid (60:0.1) as mobile phase at 0.85ml/min through a Spherisorb ODS-2 (Phase Sep) column. (+)-JA (Sigma) was used as standard.

Results and discussion

Strain screening

Botryodiplodia theobromae is a phytopathogenic fungus common in tropical countries, capable to produce JA and JA-like substances at commercial levels [3]. Eight strains of B. theobromae isolated from cuban citrus peel were screened for their capacity to produce JA on submerged static culture at 30C. JA and biomass pro- duction are shown in Table 1. Although all the strains tested grew at significant levels (4.3-16.1 g/l) JA was only produced by strains 1, 2F and 2434. Strains with lower growth (489 and 1119) and strains with higher growth (2 and 1368) did not produce JA. Since strain 2434 produce the highest titers of JA after 10 days of culture (Table 1) it was selected for further studies. Previous studies also showed that JA production by different B. theobromae strains was strain dependent [2, 14] under the same culture conditions. Maximal JA concentrations of 500 [2] and 800mg/l [14] were reported.

Effect of temperature

Temperature is an important factor for growth and secondary metabolites production by microorganisms. However, the maximum temperature for growth does

Jasmonic acid production hv B. theobromae 717

1000 . . . . . . . . . . . . . . .

800

600 ,~

"-~ 400

200

0

25 27 29 31 33 35

Temperature ( o C)

Fig. 1. Effect of temperature on jasmonic acid production bv Bot~odiplodia theobromae strain 2434 after 10 days of culture.

glucose and fructose and completely depleted after three days of cultivation (data not shown). In all cases, JA production started once that growth reached the statkmary phase [Fig. 2(b)]. Two different profiles of JA production were observed. In the static submerged culture (0 rpm) JA production started after 4 days of cultivation reaching a maximum value of 900 mg/I after 12 days o! cultivation. Agitated cultures presented a different JA production profile. JA concentration reached maximal values of 351}, 300 and 200 mg/l at 50, 11}0 and 151} rpm, respectively after 8 days of culture then JA concentrations strongly decreased at concen- trations lower than 51) mg/I at 12 clays. Reduction in JA concentration might be related to the consumption of JA due to carbon limitation or to changes on pH in thc culture medium (see below). However, since maximum biomass concentration at 0 and 150 rpm was similar,

not always correspond to the maximum for secondary metabolitcs production [2[)]. Fig. 1 presents the maximum JA concentration values obtained at dif- ferent cultivation temperatures (25-35C). JA produc- tion occurred at all range of temperature assayed. However, maximal JA concentrations were produced at 30-32C. Studies carried out on surface culture demonstrated that the maximum growth of B. theo- bromae 2434 was attained at 30C (results not shown). Therefore, maximal biomass and JA production was obtained at the same temperature (30C). This result agrees with previous studies. JA production by B theo- bromae D7/2 was maximum at 27-30C [16], whilst maximal biomass and JA production were obtained at 25C by L. theohromae cultured in surface culture [18].

Eff ect ql" agitation

Even though the effect of aeration-agitation is pro- nounccd in JA production in submerged culture, kinetic studies related to the JA production are not available [ 15, 16]. A typical time course cultivation of B. theobromae 2434 in shake flasks at different agitation rates (0-150 rpm) for biomass, JA production and pH evolution is shown in Fig. 2. In all cases, growth reached a stationary phase after 4 days of culture, followed by a decrease in the biomass concentrations [Fig. 2(a)]. Biomass production increased with agita- tion rate up to 100 rpm. At 150 rpm maximum biomass concentration (11 g/l) corresponded to the 65% of the biomass produced at 100rpm. The reduction in biomass concentration after 4 days of culture was enhanced bv the stirring speed. Although biomass con- centrations at 4 days of culture were strongly dependent on agitation (12.5 g/1_+26%), similar values (10.84g/1+12%) were obtained at 12 days of cultiva- tion. The decrease in biomass concentration was con- siderably low at () and 15(I rpm. Sucrose was invcrtcd to

2O

.=

s

4

0 m

A

. : " . _ _ .AAA O O

i o

1000

800

- 6 O0

E 400 <

200

B 0 0 0

0

10 c

8

6

4 i i

2

O 0

0 0

o ~ I~,a | A

0

0 2 4 6 8 10 12

T ime (days)

Fig. 2. Kinetics of cell growth (A), jasmonic acid production (B) and pH (C) by Botryodiplodia theobromae strain 2434 grown in shake flasks at different agitation rates: 0 rpm ( ) , 5(I rpm (tt), 1!)0 rpm (A) and 1511 rpm (e).

718 E Eng, M. Guti&rez-Rojas, E. Favela-Torres

disappearance of JA from the culture broth could be related to final pH. Profiles of pH at different agitation rates are shown in Fig. 2(c). pH decreased from 5.5 to 3.75 after 2 days of cultivation. Thereafter it slowly increased throughout the cultivation up to 6 and 7 for culture with agitation and to 8.8 for culture without agitation. Increase of pH could be related to the deple- tion of nitrate ion by B. theobromae 2434 in the culture medium generating an alkaline pH by ionization of sodium ion. B. theobromae only biosynthesizes the cis (+)-7-iso-JA isomer, 3 however, at pH values higher than 7 the cis ( +)-7-iso-JA isomerizes to the trans( - ) - JA isomer, resulting in an equilibrium of about 95:5 [trans(-)-JA: cis( +)-7-iso-JA] [5]. Additionally, in the cis(+)-7-iso-JA the side-chains are oriented being more unstable than the trans-oriented isomer. Thus, at pH values below 7 the cis( + )-7-iso-JA might be assimi- lated by B. theobromae. This approach might be of interest for JA production in bioreactor under con- trolled conditions (pH, stirring and aeration rate) because biomass production can be favoured during the first step of the culture (high aeration rate) and JA production enhanced by reducing the aeration rate and controlling pH at alkaline values to maintain the trans- oriented isomer.

The effect of agitation rate on biomass yield produc- tion (Yx/s, g biomass/g sucrose) and JA production related to biomass (Y,~A/x, g JA/g biomass) is shown in Fig. 3. Yx/s reached a maximum value of 0.32 g/g at 100 rpm. However, it decreased below 0.22 g/g at 150. YJA/x decreased from 0.018 to 0.006g/g at agitation rates ranging from 0 to 150 rpm, attaining a minimum of 0.004g/g at 100rpm. Reduction in the Yx/s at 150rpm might be related to the production of an extracellular polysaccharide produced at high agitation rates, probably due to high dissolved oxygen concentra- tions. Increase in the viscosity of the broth in the agi- tated cultures was also observed in this work, Gfinther

0.40 0.02

0.30

0.20

0.10

t3

0.00 I ~ 0

0 50 tO0 150 rpm

0.016

0.012

0.008

0.004

Fig. 3. Effect of the agitation rate on y,.~, (I) and YJA/x (D) in jasmonic acid production during the growth of Botryodiplodia theobromae strain 2434 in shake flasks.

Table 2. Effect of different carbon sources on jasmonic acid production, final pH and biomass concentration by Botryodi- plodia theobromae strain 2434

Carbon source Biomass (g/l) JA (mg/l) Final pH

Dextrose 10.73 1136.6 8.40 Fructose 15.54 1273.2 7.51 Lactose 13.06 125.6 5.96 Sorbitol 23.23 173.6 6.79 Starch 17.38 403.7 7.99 Starch + sucrose 13.40 914.0 7.63 Sucrose 9.06 910.0 7.21

et al. [15, 16] reported that JA production with b. theo- bromae D7/2 was reduced when the culture was agi- tated above 190 rpm by the simultaneous synthesis of an extracellular polysaccharide. The decrease of Y~/s at 150 rpm might also be related to the synthesis of other metabolites such as indoles [2], curcubic acid, (+)-9,10- dihydro-7-isojasmonic acid and (+)-l l ,12-didehydro- 7-isojasmonic acid [21].

Effect of carbon sources

The influence of different carbon sources on the pro- duction of biomass and JA is shown in Table 2. All the carbon sources tested were used for growth by B. theo- bromae 2434 and JA was produced at different levels. There was an inverse correlation between biomass and JA production. Low growth and high JA production was obtained with glucose and sucrose as carbon sources. The lowest JA production was obtained with carbon sources (sorbitol and starch) that allowed better growth. The highest and lowest JA production were obtained with fructose and lactose as carbon source. It was previously reported that glucose or sucrose could be used as sole carbon source for JA production with B. theobrornae D7/2 [16]. Moreover, Broadbent et al. [18] reported that sucrose, glucose, glycerol or a mixture of these as carbon sources allowed higher JA production by L. theobromae strain $22L.

The high JA production observed in this work with fructose and dextrose could be related to the fact that in order to be assimilated, these substrates do not require previous enzymic hydrolysis as required for sucrose, starch or lactose assimilation. Novaratnam et al. [22] working with B. theobromae IMI 334891 in manioc starch medium supplemented with salts found that maximum glucoamylase activity was obtained in shake flasks at 160 rpm and pH 6.0. Nevertheless, a combination of two carbon sources with different uptake rate as starch/sucrose yielded higher JA concen- tration. Cultures with final pH values below 7.0 (lactose and sorbitol) exhibited the lowest JA produc- tion (125.6 and 173.6 g/I, respectively).

Table 3. Effect of nitrogen sources on jasmonic acid produc- tion, final pH and biomass concentration by Botryodiplodia theobromae strain 2434

Nitrogen source Biomass (g/l) JA (rag/l) Final pH g g

720 E Eng, M. Guti6rrez-Rojas, E. Favela- Torres

tuto Nacional de Investigaciones Fundamentales de la Agricultura Tropical) for providing us with strains of B. theobromae.

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