Significant medium components for enhanced Jasmonic acid production by Lasiodiplodia theobromae using Plackett-Burman Design

P. C. Dhandhukia and V. R. Thakkar*

Lab#302B, BRD School of Biosciences, Sardar Patel Maidan, Vadtal Road, Satellite Campus, Post Box No. 39, Sardar Patel University, Vallabh Vidyanagar-388120, (Gujarat), India.

* For correspondance: vasuthakkar @ gmail.com

Current Trends in Biotechnology and Pharmacy, Vol.1 (1) 79-86 (2007) ISSN:0973 - 8916

Abstract

A Plackett-Burman design and statisticalscreening was employed to optimize medium components for jasmonic acid production by Lasiodiplodia theobromae. The seven medium components malt extract, sucrose, NaNO₃, yeastextract, FeSO₄ 7H₂O, MnSO₄ H₂O, and MgSO₄7H₂O were screened using a Plackett-Burman design to optimize medium component forjasmonic acid production using L. theobromae.When sucrose was used alone as a carbon sourcewith these medium components, the jasmonicacid production was found to be 80 mg/l. Increasein jasmonic acid production (225.3 mg/l) wasobserved with augmentation of malt extract andchanging other medium componentsconcentration based on statistical screening. Itwas found that malt extract, sucrose, NaNO₃,MnSO₄ 7H₂O, and MgSO₄ 7H₂O were significantcomponents influencing jasmonic acidproduction. Yeast extract and FeSO₄ 7H₂O alsoshowed significantly negative effect on thejasmonic acid production. Therefore significantly positive factors could be increased andsignificantly negative factors could be decreasedfor higher jasmonic acid production.

Keywords

Jasmonic acid, Lasiodiplodia theobromae,Plackett-Burman design, Significant factors.

Introduction

Jasmonic acid (JA) (3-oxo-2-(2’-cis-pentenyl)cyclopantane-1-acetate) and methyl jasmonatesare of importance in perfumery industries andbeing used world wide in various products like,toilet soaps, chewing gum, even in smokingpreparations (1). JA and its derivatives are alsovery important secondary metabolites in the plantdefense system (2, 3). JA was first isolated fromthe cultural filtrate of Lasiodiplodia theobromae(the synonym of Botryodiplodia theobromae)and its role was found as a plant growth regulator(4). In spite of high demand, very fewfermentation studies were carried out for JAproduction using L. theobromae (5-7). This isthe first report on screening of mediumcomponents for JA production using statisticalexperiment design.

The relevant literature and “prior art” serve onlyas starting point for the development offermentation. By manipulating nutritionalrequirements, physical parameters and geneticmakeup of the producing strain increase inproductivity of microbial metabolite can beachieved. A fermentation improvement programmay begin by measuring product yield as aresponse to factors like strength of mediumcomponents. Nutritional requirement can bemanipulated either by the conventional or statistical approach. Conventional methodsinvolve changing one independent variable at atime (OVAT) while others are kept at fixed level.However statistical methods offer severaladvantages over conventional methods beingrapid and reliable, shortlists significant nutrients,helps understanding the interaction among thenutrients at various concentrations and reducestotal number of experiments tremendouslyresulting in saving time and cost of glassware,chemicals and manpower (8, 9). Screening isthe first phase of an experimental study toeliminate non-significant factors so that effortsmay be concentrated upon most significantingredients selected for further optimization.The selection of the medium components for thisstudy was carried out based on the literaturesurvey and our preliminary laboratory work. Asystematic experiment was then carried out bysetting the independent variables according to aPlackett-Burman design (10) at two levels andJA produced was measured in each batch,followed by statistical analysis in order tointerpret the significant medium components.Such approach is a useful screening processemployed to identify the contribution of eachmedium component to the response of the systemand thus allows for a reduction in the number ofvariables that need to be considered (11).

The Plackett-Burman design is highlyrecommended when more than five factors haveto be investigated. These designs are very usefulfor economically detecting large main effects,assuming all interactions are negligible whencompared with the few important main effects.Two level factorial Plackett-Burman design, wasselected because it screens up to v variable injust ‘v+1’ experimental run (10, 12). Theseexperimental designs are available in multipleof four runs (13) and advantageous over number of variable). Higher-order linear fullfactorial and quadratic Box-Behnken designswould have required 66 and 52 experimentalbatches, respectively, which would have beenprohibitively uneconomical (14). In this study,the aim was to screen the effects among sevenindependent variables (Table 1) in an economicalmanner (13). Due to orthogonal nature of thePlackett-Burman Design, it gives only pure effectof each variable not confounded with interactionamong variables (15).

Materials and Methods

Microorganism

L. theobromae (MTCC 3068) strain was obtainedfrom Microbial Type Culture Collection (MTCC)Institute of Microbial Technology, Chandigarh,India. This strain was maintained on potatodextrose agar (Hi-Media, Mumbai, India) slants.After inoculation, slants were incubated at 30°Cfor three to four days for obtaining growth andlater stored at 4°C. Strain was sub-cultured everymonth.

Chemicals and solvents

(±) JA, a racemic mixture, was purchased fromSigma, Bangalore, India and used as a standardreference compound for quantification of JA. Alldehydrated media components were purchasedfrom Hi-media, Mumbai, India. All solvents(99.9%) were purchased from Qualigens,Mumbai, India. Ammonia (25% w/v) was usedfresh.

Cultural technique

Sample of stock culture was transferred fromworking slants to potato dextrose agar plates andincubated for three days at 30°C. The basalmedium contained (g/l) KH₂ PO₂ , 2.0; KCl, 0.3;ZnSO₄ 7H₂O, 0.03; CuSO₄ 7H₂O, 0.003; andNa₂MoO_4. 2H₂O, 0.003. The pH was maintained5.5 (6) through out the experiment and the concentrations of other medium componentsmalt extract, sucrose, NaNO₃ , yeast extract, FeSO₄ 7H₂ O; MnSO₄ 7H ₂O and MgSO₄ 7H₂ O were as per the combination given in Table 1and 2. Agar plug (eight mm diameter) was cutwith sterile cork borer and used for inoculationof 100 ml of the broth culture in 250 mlErlenmeyer flask and incubated for eight daysat 30°C in static condition.

Screening of important nutrient components

Total seven medium components (independentvariables, v= 7) were selected for the study witheach variable represented at two levels, high (+)and low (-) concentrations also four dummyvariables were kept at two concentrations. Theinput factor and their corresponding upper andlower level values are shown in Table 1. A twolevel design only minima and maxima of inputvariables were used to form a design space. The resulting twelve experimental cases for seveninput parameters (v+1+ four dummy variables)are shown in Table 2 with the number of positivesigns and negative signs. Each column containedequal number of positive and negative signs.Thus each row represented a trial batch and eachcolumn represented an independent (assigned)or dummy (unassigned) variable. The effect ofeach variable was determined by the followingequation:

E( xi) = ( JAi + - JAi - ) / N - (1)

Where E₍ xi) is the concentration effect of the testedvariable. JAi+ and JAi- are the jasmonic acidproduction from the trials where the variable (xi)measured was present at high and lowconcentrations, respectively; and N is the numberof trial divided by two. Experimental error wasdetermined by calculating the variance amongthe dummy variables as follows:

V eff = (E d ) 2 _{/ n} - (2)

Where V_eff is the variance of the concentrationeffect, E d is the concentration effect for thedummy variable and n is the number of dummyvariables. The standard error (SE) of theconcentration effect was calculated by takingsquare root of the variance of an effect and thesignificance level (p-value) of each concentrationeffect was determined using student’s t-test:

t ₍ xi ) = E ₍ xi ) / SE - (3)

Cultural filtrate of L. theobromae afteracidification to pH 3 with 6 M HCl, was extractedwith equal volume of ethyl acetate. Extract wasconcentrated to 100 times. JA measurement wascarried out using high performance thin layerchromatography (HPTLC). Concentrated extractwas loaded on silica gel 60 F254 aluminum foils(Merck, Germany) along with standard JA usingLinomate-5 spray on applicator (Camag,Switzerland) of HPTLC under the flow of N₂.Foils were ran with iso-propanol: ammonia:water (10: 1: 1 (v/v)) (17). After running, foilswere dried in air and scanned with Scanner-3(Camag, Switzerland) and quantified with thehelp of winCATS software ver. 1.2.2 bymeasuring density of the JA band separated onthe TLC foils (18).

Statistical analysis of data

Yields of JA obtained in experiments werecalculated for determination of variablesignificance using Microsoft Excel regressioncoefficient and statistical t-values for equalunpaired samples. Ingredient with highest t-valuewas considered as best nutrient and thus selectedfor further optimization studies. A main effectwith a positive sign indicates that the highconcentration of this variable is near to optimumand a negative sign indicates that the low concentration of this variable is nearer to optimum.

Results and Discussion

Selection of appropriate carbon source, nitrogensource and other nutrient is one of the mostcritical stages for the development of an efficientand economic fermentation process. In this studya Plackett-Burman design was employed toevaluate the main effect of the mediumcomponents for the JA production by L.theobromae at optimized physical parameters.Table 1 represents the independent variables andthe respective high and low concentrations ofseven factors used in the optimization study. Maltextract was selected as it contains up to 3.4% oflipids in its dry matter with linoleic acid as theirmajor constituent (50–60% of total lipids) (19).Sucrose was used previously in our study as wellas elsewhere as main carbon source for samefungi and found significant, therefore included in the study (6). NaNO₃ was also selected based on our study for the screening of the carbon andnitrogen source for the JA production (data notshown). NaNO₃ was reported to be preferrednitrogen source either with glucose (1) orfructose (6) for JA production using L.theobromae. Manganese and iron salts wereempirically used in media and they were alsoimportant as cofactors for one of the enzyme(lipoxygenase) in the pathway of JA production(20).

The twelve trial Plackett-Burman experimentaldesign, with two levels of concentrations for eachvariable, was followed for the optimization ofmedium components for JA production is shownin Table 2. The variables X1-X7 represent themedium constituents and D1-D4 represent thedummy (unassigned) variables. Highest JA wasproduced in batch 2 of the design followed bybatch 7, 5, and 1 (Table 2).Table 3 represents the result of effect of eachmedium component on JA production as well as the standard error, t(xi)^,p, confidence level and ranking of each component. The componentswere screened at the confidence level of 95% onthe basis of their effects. That component, whichshowed significance at or above 95% confidencelevel and its effect was positive, was interpretedthat it was required in higher concentration thanthe indicated high value (+). However if its effectwas found negative, then it indicated that thecomponent was effective in JA production butthe amount required was lower than the indicatedas low (-) concentration in Plackett-Burmandesign. All factors in this study have showninfluence on the JA production with confidencelevel at or above 95% confidence limit and wereconsidered to be significant for JA productionby L. theobromae (Table 3).

The main effects ofthe components in the medium for JA productionare presented in the Fig 1. The malt extractshowed the maximum positive effect on JAproduction, followed by MnSO₄ 7H₂O, MgSO₄7H₂ O, Sucrose and NaNO₃ . The effect of FeSO₄7H₂ O and yeast extract were negative whichsuggested that these components are required inthe medium for JA production but in lower concentration than the low level.Malt extract was found better medium component than yeast extract. As shown in Table2, in batches 6 and 10, where yeast extract wassupplied in higher concentration but malt extractwas not given, the yield of JA was reduceddrastically and in batches 7, 8 and 9, highconcentration of malt extract and lowconcentration of yeast extract resulted in higherproduction of JA. Low concentration of yeastextract may be required to meet requirement ofvitamins for general metabolism as well as forsome enzymes of JA pathway (for example, 12-oxo-phytodienoic acid reductase) (21).Supplementation of one or more vitamins of “B”group either as separate or in form of yeast extractimproves the JA production (1). Yeast extractserve as dual-purpose nitrogen source as well asvitamin source but it is an expensive componentfor designing an economical medium (15).

From the results of the Plackett-Burman design it couldbe concluded that yeast extract is probably required for the growth but not for the JAproduction and should be supplied in lowconcentration. Sucrose can not replace the malt extract, as thehighest JA (225 mg/l) was produced in batch 2does contain low level of malt extract and whenonly sucrose was used in previous experiment,it produced only 80 mg/l of JA (7). Moreover,malt extract is useful as it contain linoleic acidas its major constituent (which is the precursorof JA). (19).The manganese showed positive effect and ironshowed negative effect might be related with themetal ion required for the catalytic center of the 7H ₂O is also lipoxygenase enzyme (20). MgSO 4 very important medium component showingmain effect same as sucrose. The requirementfor Mg is evident as Mg is required as a catalystfor many intracellular enzymatic reactions. Mgsalts have been found most useful in othermetabolite and enzyme production as well (21,22).By selection of medium components usingPlackett-Burman design in this study, aboutthree-fold increase in the JA production wasincreased compared to earlier experiment wherein BSB medium, which supported the maximumJA production as well as growth of L.theobromae, 80 mg/l of JA was produced (7).Present study has short-listed the few significantnutrients useful in increasing the yield of JA andhas also identified the nutrients, which shouldbe used in less concentration.

Acknowledgement

Authors are thankful to University GrantCommission, India for financial support.

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Table 1:Medium components (Variables) and their respective high (+) and low (-) concentration levels used in Plackett-Burman Design

Table 2:Plackett-Burman design generated by fractional rotation of full factorial design where X1 to X7 are independent variables and D1 to D4 are dummy variables

Table 3:t-value, probability, confidence level and ranking of variables for JA production

Figure. 1: Effect of media components on JA production in batch culture