EJBR2017v7i4art315 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2017; 7 (4): 315-323 Statistical optimization as a powerful tool for indole acetic acid production by Fusarium oxysporum Ghada Abd-Elmonsef Mahmoud 1 *, Hassan H. A. Mostafa 2 1 Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut 71516, Egypt 2 Central Laboratory of Organic Agriculture, Agricultural Research Center, Giza 12619, Egypt *Corresponding author: Dr. Ghada Abd-Elmonsef Mahmoud; Tel.: 20 1010711661; Fax: 20 88 2342708; E-mail: ghada_botany@yahoo.com; ghada.moukabel@science.au.edu.eg ABSTRACT Crop production is challenged in our world by increasing food demands, decrease natural resource bases and climatic change. Nowadays plant growth regulators works like fertilizers in increasing plant growth production efficiency and needed to produce in large industrial scale. Fermentation condition and medium constituents can significantly affect on the product production and designing an acceptable fermentation medium is critical importance. In this paper Fusarium sp. could be considered as promising indole-3-acetic acid producers with the ability to improve the production using statistical methods. The results showed that fermentation type, incubation temperature and L-tryptophan were the most influencing parameters on the production. Maximum IAA production by Fusarium oxysporum was 300.4 mg/l obtained under the fermentation conditions: temperature at 25oC, incubation period 5 days, pH 7, inoculums size 2%, shaking rate at 150 rpm and medium constituents: Glucose 40 g/l, yeast extract 3 g/l, L-tryptophan 1 g/l, KH2PO4 2 g/l, NaNO3 4 g/l, MgSO4·7H2O 0.1 g/l with regression analysis (R2) 99.67% and 2.12-fold increase in comparison to the production of the original level (142 mg/l). Keywords: Auxin; Production; Plackett-Burman; Fusarium. Abbreviations: Indole-3-acetic acid (IAA), potato dextrose agar medium (PDA). 1. INTRODUCTION In the last few years by increasing population number every year the fulfilling food requirement remains a challenging task as climate changes affected on the agricultural production systems and there has been a growing interest in increasing crop plant yield [1]. Phytohormones which could produce by microorganisms are known to play vital roles in plant growth and establishment by helping plants to acclimatize to varying environments [2]. Several phytohormones control many physio- logical and bio-chemical processes like abscisic acid, gibberellins, ethylene, auxins, cytokinins, and brassinosteroids [3]. Indole-3-acetic acid extensi- vely was the first identified plant hormone and the most important member of the auxins family of phytohormones [4]. Its play a vital role in physiological processes e.g. root initiation, pro- duction of longer roots, tissue differentiation, increase number of root hairs and lateral root which are involved in nutrient uptake [5-7]. In recent paper Received: 18 August 2017; Revised submission: 04 October 2017; Accepted: 20 October 2017 Copyright: © The Author(s) 2017. European Journal of Biological Research © T.M.Karpiński 2017. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. DOI: http://dx.doi.org/10.5281/zenodo.1012348 316 | Mahmoud & Mostafa Indole acetic acid production by fungi European Journal of Biological Research 2017; 7 (4): 315-323 by Takahashi [8] revealed that indole-3-acetic acid intracellular plant concentration is controlled by the biosynthesis and degradation process. The presence of several microorganisms synthesizes IAA as secondary metabolites through tryptophan pathway are very important factor in soil fertility [9]. Several bacterial isolates could produce IAA however; most of the previous studies do not take into account of IAA production by filamentous fungi. Indole-3-acetic acid was produced by fila- mentous fungi like Colletotrichum gloeosporioides, Colletotrichum acutatum, Dibotryon morbosum, Fusarium, Rhizopus suinus, Phoma glomerata, Penicillium, Taphrina deformans, Ustilago esculenta and Ustilago zeae [10-18]. A classical method of optimizing the fermentation conditions and medium constituents depends on single parameter whilst all the other factors are maintained at a fixed level [19]. However, statistically based experimental designs proved to be most popular for production optimization as it enables us to obtain the physicochemical and factors influencing on the production process with less number of planned experiments. Plackett-Burman design is practical efficient when we screening large number of factors to produce optimal response [20]. The main objective of this paper is to test the ability of different Fusarium isolates to produce indole-3-acetic acid on glucose medium, secondly to improve IAA production by investigating the effect of several parameters on the production process and found the optimum fermentation conditions and medium constituents for the highest IAA production using statistical approach (Plackett-Burman design). 2. MATERIALS AND METHODS 2.1. Fusarium sp. isolation and identification Fusarium species, isolated from different parts of Egyptian clover, faba bean, garlic, maize and onion plants on potato dextrose agar medium (PDA). The pure cultures were maintained aerobically on the same medium and stored at 4±1°C until using [21]. Fusarium sp. identified based on their macroscopic and microscopic characteristics [22]. 2.2. Inoculums preparation Prior to indole-3-acetic acid production experiments, Fusarium sp. were grown aerobically on potato dextrose agar medium at 28±1°C for 4 days. Homogeneous spore suspension of Fusarium sp. was prepared by scraping fungal hyphae from culture plates and suspended in sterilized distilled water containing 0.01% (v/v) tween 80 (2 × 106 spore/ml) and stirred for 30 min. One ml of the inoculums was transferred to an Erlenmeyer flask 250 ml containing 100 ml of the production medium. 2.3. Screening for IAA production by Fusarium sp. Czapek's dextrose liquid medium supple- mented with 0.2 g/l L-tryptophane, was used as production medium containing (g/l): glucose, 30.0; yeast extract, 5; NaNO3, 3.0; KH2PO4, 1.0; MgSO4·7H2O, 0.5; KCl, 0.5 and FeSO4·7H2O, 0.01. These contents were dissolved in 1000 ml distilled water with initial pH adjusted to 5.5 before autoclaving. After sterilization in an autoclave at 121°C and 1.5 atm pressure for 20 min. chloramphenicol, 250 mg/ml was sterilized sepa- rately by membrane filtration, using a membrane of pore size 0.22 mm and added as bacteriostatic agent. Incubation was carried out at 28±1°C on a rotary shaking (150 rpm) for 7 days. All the experiments were carried out independently in triplicates. 2.4. Optimization using Plackett-Burman design Plackett-Burman design was used to screen the fermentation parameters that influenced indole- 3-acetic acid (IAA) production with respect to their main effect and without interaction effects between various constituents of the medium [23]. Eleven trails carried out by Plackett-Burman design for screening the fermentation parameters under investigation is shown in Table 1. Each independent variable was tested at two levels, high (+1) and low (−1). In each column and row should contain equal number of negative and positive signs. The program Sigma XL (Version 6.12) was used to analyze this experiment. 317 | Mahmoud & Mostafa Indole acetic acid production by fungi European Journal of Biological Research 2017; 7 (4): 315-323 Table 1. Plackett-Burman design for different variables screening in IAA production by Fusarium oxysporum (I). Variable code Variable Unit Level Low (-1) (0) High (+1) A Incubation temperature C° 25 30 35 B Incubation time D 5 7 9 C Fermentation type Shaking Shaking Static D Inoculums size % 0.5 1 2 E Initial pH 5 6 7 F Glucose gl-1 20 30 40 G Yeast extract gl-1 3 5 7 H L-tryptophan gl-1 0.1 0.5 1 J KH2PO4 gl -1 0.5 1 2 K MgSO4·7H2O gl -1 0.1 0.5 1 L NaNO3 gl -1 1 2 3 Plackett-Burman design was used to screen and evaluate the important medium components that influence the response. Indole-3-acetic acid yields are explained by the following polynomial equation: Y=bo + ∑biXi + ∑bijXiXj + Ei (1) Where, Y: the variable dependent response; i: the regression coefficient; X: the independent variable level and E: the experimental error. The experimental data were statistically analyzed to determine the significant difference (p≤0.05) in response under different conditions. The response surface graphs were also plotted using the same software. The quality of fit for the regression model equation was expressed as R2. 2.5. Analytical analysis After the incubation period, Fusarium mycelium was recovered by filtration through dried and weighed Whatman filter paper (No. 113), washed with distilled water three times and then dried at 70°C overnight for dry mass (DM) determination. The supernatants were centrifuged at 4,000 rpm for 15 min. and sterilized by membrane filtration, using a membrane of pore size 0.22 mm to remove any remaining spores for quantitative determination of indole-3-acetic acid (IAA). Indole-3-acetic acid was determined spectro- photometerically (Fig. 1) using Salkowski reagent containing 1ml of 0.5 M FeCl3·6H2O dissolved in 50 ml of 35% HClO4 [24]. Two ml of Salkowski reagent was added to one ml of culture supernatant in 10 ml test tube leaves it in room temperature and read the color after 25 min. but before 3 h at 535 nm. The developed pink color measured using T60 UV with a split beam UV visible spectrophotometer covers a wavelength range of 190-1100 nm. The amount of IAA in the supernatant was measured quantitatively at 535 nm against substrate-free blank. The standard curve was prepared using pure IAA (1-100 mg/l). Control IAA Figure 1. Different pink color degrees of IAA production after the reagent added in comparison with control (free IAA). 318 | Mahmoud & Mostafa Indole acetic acid production by fungi European Journal of Biological Research 2017; 7 (4): 315-323 3. RESULTS 3.1. Isolation and identification of Fusarium sp. Ten Fusarium isolates were isolated from different parts of Egyptian clover, faba bean, garlic, maize and onion plants on PDA medium. Based on Fusarium growth on the plate and the microscopic characters, Fusarium sp. were identified into seven species. Fusarium solani, F. oxysporum, F. chlamy- dosporum, F. camptoceras, F. incarnatum, F. verti- cilloides and F. nygami. The purified isolates were screened for their ability to produced indole-3-acetic acid (IAA) on fermented medium. Only one isolate was selected for further experiments based on the highest indole-3-acetic acid (mg/l) production. 3.2. Indole-3-acetic acid production by Fusarium sp. All the isolates grown on the production medium and showed various degrees of dry mass and indole-3-acetic acid production. A wide variation in IAA production on the screening medium ranged from 18.37±1.04 to 142±6.46 mg/l and dry mass varied between 1.2±0.2 and 6.1±0.53 g/l. The highest fungus dry mass and indole-3-acetic acid producer was Fusarium oxysporum (I) isolated from onion rhizoplane giving 142±6.46 mg/l IAA (with productivity 23.14 mg/l/day) and 6.1±0.53 g/l dry mass so it was selected for the further experiments, the overall measurement results are summarized in Fig. 2. Figure 2. Screening for IAA production on glucose medium by different isolates of Fusarium species. Brief description of indole-3-acetic acid highly producer Fusarium oxysporum (Schlech- tendal) emend. Snyder & Hansen; Growth on PDA medium 50 mm in one week, Texture floccose becoming felted, Color white to pale apricot, usually with a purple tinge, Reverse purple. Conidiophores hyaline, simple, short, bearing spore masses at the apexes; two kinds of conidia: macroconidia boat- shaped, with slightly tapering apical cells and hooked basal cells, 4-celled; and microconidia ellipsoidal, 1-celled. Chlamydospores globose and usually solitary (Fig. 3). 3.3. Optimization of IAA production using Plackett-Burman design The Plackett-Burman design was an effective way to improve IAA production. The highly IAA producer (Fusarium oxysporum (I)) was chosen for screening the effects of different parameters on IAA production using Plackett-Burman design. Each variable was studied at two levels (-1, 1) as declared in Table 1. Relationship between the response and the screened variables was expressed by the following polynomial equation: IAA (mg/l) = (91.483) + (-40.02) * A: Incu- bation temperature + (-22.15) * B: Incubation time + (-49.82) * C: Fermentation type + (6.82) * D: Inoculum size + (2.95) * E: Initial pH + (18.35) * F: Glucose + (-1.75) * G: Yeast extract + (26.48) * H: L-tryptophane + (0.95) * J: KH2PO4 + (-13.78) * K: MgSO4 + (17.65) * L: NaNO3 (2) The results obtained in Table 2 indicated that there was a wide variation in IAA production from (13.6 to 300.4 mg/l) and dry mass varied between 3 and 9.1 g/l. This indicates the important effect of the medium components and environmental factors on growth and production of IAA. The ANOVA results are shown in Tables 3 showed that among the eleven variables, G (yeast extract) and J (KH2PO4) were found to be non-significant (p>0.05). Among the tested parameters, fermentation type, incubation temperature and L-tryptophan were the most effective parameters plays a crucial role in IAA production with 49.82%, 40% and 26.48% coefficient effect as shown in Pareto-Plot (Fig. 4). All the predicted values of Plackett-Burman design were located in close proximity to the experimental values. This supports the hypothesis 319 | Mahmoud & Mostafa Indole acetic acid production by fungi European Journal of Biological Research 2017; 7 (4): 315-323 that the model Eq. (2) is sufficient to describe the response of the experimental observations of IAA production (Fig. 5). Three-dimensional response surface curves were generated to study the interaction between each two variables (Fig. 6A-F). The Model F value of 324.6 (p<0.05) implies that the model is significant. Model F value is calculated as ratio of mean square regression and mean square residual due to the real error. The R2 value was 99.67% indicated that the entire variation was explained by the model. The adjusted R2 value was 99.36%. Maximum IAA production (300.4 mg/l) by Fusarium oxysporum (I) obtained under the fermen- tation conditions: temperature at 25oC, incubation period 5 days, pH 7, inoculums size 2%, shaking rate at 150 rpm and medium constituents: Glucose 40 g/l, yeast extract 3 g/l, L-tryptophan 1 g/l, KH2PO4 2 g/l, NaNO3 4 g/l, MgSO4·7H2O 0.1 g/l. Figure 3. Fusarium oxysporum (I) Sch., A: Chlamysospores (Ch); B: Monophilaidic conidiogenous cell (Ph) and hypha (Hy); C: Macroconidia (Ma); Bars, 10 µ m; D: Fungus growth on potato dextrose agar medium. Figure 4. Pareto-Plot for Plackett-Burman parameter estimates the effect of each parameter on IAA produced by Fusarium oxysporum (I). Figure 5. Comparison between IAA (mg/l) experimental and predicted values of the Plackett-Burman design. D C B A 320 | Mahmoud & Mostafa Indole acetic acid production by fungi European Journal of Biological Research 2017; 7 (4): 315-323 Table 2. Plackett-Burman design variables with IAA production by Fusarium oxysporum (I) as response. Trials A B C D E F G H J K L IAA (mgl-1) Dry mass (gl-1) 1 -1 -1 1 1 1 -1 1 1 -1 1 -1 95.6 4.8 2 -1 1 1 1 -1 1 1 -1 1 -1 -1 46 6.82 3 1 1 -1 1 -1 -1 -1 1 1 1 -1 65.6 3.66 4 1 -1 1 1 -1 1 -1 -1 -1 1 1 24.8 6 5 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 146.8 4 6 1 1 -1 1 1 -1 1 -1 -1 -1 1 75.6 9.1 7 1 1 -1 1 1 -1 1 -1 -1 -1 1 70 8.4 8 1 1 1 -1 1 1 -1 1 -1 -1 -1 15.6 3.88 9 1 -1 1 -1 -1 -1 1 1 1 -1 1 50.8 3.6 10 -1 1 -1 -1 -1 1 1 1 -1 1 1 188 7.99 11 1 1 1 -1 1 1 -1 1 -1 -1 -1 19.2 3.81 12 1 -1 1 1 -1 1 -1 -1 -1 1 1 23.6 5.4 13 1 1 -1 1 -1 -1 -1 1 1 1 -1 59.2 3.68 14 -1 1 1 1 -1 1 1 -1 1 -1 -1 55.2 8.83 15 -1 -1 -1 1 1 1 -1 1 1 -1 1 284 4.8 16 -1 1 1 -1 1 -1 -1 -1 1 1 1 13.6 5.22 17 -1 1 -1 -1 -1 1 1 1 -1 1 1 202.8 8.21 18 1 -1 -1 -1 1 1 1 -1 1 1 -1 78.4 6.8 19 -1 -1 1 1 1 -1 1 1 -1 1 -1 79.6 4.4 20 1 -1 1 -1 -1 -1 1 1 1 -1 1 54.8 4.2 21 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 144.8 3 22 1 -1 -1 -1 1 1 1 -1 1 1 -1 80 7.2 23 -1 -1 -1 1 1 1 -1 1 1 -1 1 300.4 4.6 24 -1 1 1 -1 1 -1 -1 -1 1 1 1 21.2 5.51 The sign +1 and −1 represent the two different levels (high and low) of the independent variable under investigation. A: Incubation temperature, B: Incubation time, C: Fermentation type, D: Inoculums size, E: Initial pH, F: Glucose, G: Yeast extract, H: L-tryptophan, J: KH2PO4, K: MgSO4·7H2O and L: NaNO3. Table 3. Statistical analysis of Plackett-Burman design of each variable at two levels for IAA production by Fusarium oxysporum (I). Variable code Variable Coefficient t value P value A Incubation temperature 91.48 69.58 <0.0001* B Incubation time -40.02 -30.44 <0.0001* C Fermentation type -22.15 -16.85 <0.0001* D Inoculums size -49.82 -37.89 0.0002* E Initial pH 6.82 5.19 0.0445* F Glucose 2.95 2.24 <0.0001* G Yeast extract 18.35 13.96 0.2079 N H L-tryptophan -1.75 -1.33 <0.0001* J KH2PO4 26.48 20.143 0.4838 N K MgSO4.7H2O 0.95 0.72 <0.0001* L NaNO3 -13.78 -10.48 <0.0001* t – student's test, p – corresponding level of significance,* Significant at p ≤0.05, N, non-significant at p≥0.05. 321 | Mahmoud & Mostafa Indole acetic acid production by fungi European Journal of Biological Research 2017; 7 (4): 315-323 A B C D E F Figure 6. Response surface plots of IAA production by Fusarium oxysporum (I) showing the effect of two variables (other variables were kept at zero in coded unit): (A) Glucose and yeast extract, (B) Glucose and L-tryptophane, (C) Glucose and KH2PO4, (D) Glucose and NaNO3, (E) Fermentation type and incubation time, (F) Fermentation type and inoculums size. 4. DISSCUSSION Indole-3-acetic acid produced by all Fusa- rium isolates on the production medium with various degree of production giving maximum value by Fusarium oxysporum. Lynch [25] suggested that indole-3-acetic acid is a common product of L-tryptophan metabolism which produced by several microorganisms including plant growth- promoting rhizobacteria, other bacterial types and fungi. Hasan [15] found that all isolates of Fusarium oxysporum which isolated from different plant seeds could produce IAA (100-140 mg/l). After screening the effect of eleven parameters on IAA we found that fermentation type, incubation temperature and L-tryptophan were the most effective parameters play a crucial role in IAA production. Thuler [26] revealed that IAA produc- tion is oxygen dependent, so agitation during production seems to be preferable when compared with a static condition. When incubation performed by agitation the production medium homogeneous better and the oxygen supplies increase, which increase both biomass and production in the medium [27]. From later researches, the optimum incubation temperature for IAA production was 322 | Mahmoud & Mostafa Indole acetic acid production by fungi European Journal of Biological Research 2017; 7 (4): 315-323 range of 25-30°C by several microorganisms [15, 28, 27]. Maximum IAA production (300.4 mg/l) by Fusarium oxysporum (I) obtained under the fer- mentation conditions: temperature at 25oC, incu- bation period 5 days, pH 7, inoculums size 2%, shaking rate at 150 rpm and medium constituents: Glucose 40 g/l, yeast extract 3 g/l, L-tryptophan 1 g/l, KH2PO4 2 g/l, NaNO3 4 g/l, MgSO4·7H2O 0.1 g/l. In agreement with our results, indole-3-acetic acid synthesis by ectomycorrhizal fungi was maximized after 30 days of incubation [29]. Indole- 3-acetic acid production by Fusarium oxysporum maximized on 15 days and 10 for mycelium [15]. Indole-3-acetic acid production Aspergillus niger give maximum production after 6 days of incubation [27]. From the outcome of our investigation it is possible to conclude that genus Fusarium can be highly recommended in industrial production of indole-3-acetic acid. Also using statistical method in optimization for improving the production has a great potential for applications and was very effective in our study as the production of IAA (300.4 mg/l) in this paper increase with 2.12-fold in comparison to the production of original level (142 mg/l) using Plackett-Burman design. 5. CONCLUSION From the outcome of our investigation it is possible to conclude that genus Fusarium can be highly recommended in industrial production of indole-3-acetic acid. Also using statistical method in optimization for improving the production has a great potential for applications and was very effective in our study as the production of IAA (300.4 mg/l) in this paper increase with 2.12-fold in comparison to the production of original level (142 mg/l) using Plackett-Burman design AUTHORS’ CONTRIBUTION Both authors contributed in the success of this research and the final manuscript has been read and approved by both authors. 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