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Turk J Biol 34 (2010) 313-318 © TÜBİTAK doi:10.3906/biy-0812-15 Indole-3-acetic acid and gibberellic acid production in Aspergillus niger Işıl SEYİS BİLKAY1,*, Şafak KARAKOÇ2, Nilüfer AKSÖZ1 1 Hacettepe University, Faculty of Science, Department of Biology (Biotechnology), 06532 Beytepe, Ankara - TURKEY 2 MARA, National Food Reference Laboratory, Department of Biotechnology and GMO, Yenimahalle, 06171, Ankara - TURKEY Received: 19.12.2008 Abstract: The effects of incubation time, temperature, pH, and agitation on indole-3- acetic acid and gibberellic acid production in Aspergillus niger were studied. For indole-3-acetic acid production, 6 days of incubation at 25 °C and pH 6.0 was found to be optimum. Optimum conditions for gibberellic acid production were 12 days of incubation at 30 °C and pH 5.0. Agitation increased both indole-3-acetic acid and gibberellic acid production. Key words: Aspergillus niger, gibberellic acid, indole-3-acetic acid, phytohormone, plant growth regulator Aspergillus niger’den indol asetik asit ve gibberellik asit üretimi Özet: İnkübasyon süresi, sıcaklık, pH ve çalkalamanın, Aspergillus niger’den indol-3-asetik asit ve gibberellik asit üretimine etkisi araştırıldı. İndol asetik asit üretimi için 6 gün inkübasyon süresi, 25 °C sıcaklık ve pH 6,0 uygunken, gibberellik asit üretimi için 30 °C, pH 5,0 ve 12 gün inkübasyon süresinin uygun olduğu saptandı. Çalkalamanın ise hem indol-3-asetik asit hem de gibberellik asit üretimini arttırdığı gözlendi. Anahtar sözcükler: Aspergillus niger, gibberellik asit, indol-3-asetik asit, bitki hormonu, bitki büyüme düzenleyicisi Introduction Gibberellic acid is a plant growth regulator of economic and industrial importance (1). Phytohormones, mainly including auxins, cytokinins, abscisic acid, gibberellins, and ethylene, induce some important physiological responses at different stages of plant development at low concentrations (2). Various gibberellins are available and are associated with several plant growth and development processes, such as seed germination, stem elongation, flowering, and fruit development (3,4). On the other hand, auxin was first isolated and characterized as a plant hormone, and indole-3-acetic acid (IAA) is a type of auxin (5). Gibberellic acid is synthesized by Gibberella fujikuroi, Sphaceloma manihoticola, Neurospora crassa, Aspergillus niger, Sphaceloma sp., Rhizobium phaseoli, Azospirillum brasilense, Pseudomonas sp., and Phaeosphaeria sp., whereas IAA is synthesized by Pseudomonas sp., Bacillus sp., Aspergillus niger, Rhizopus, Acetobacter diazotrophicus, Herbaspirillum seropedicae, Rhizobium sp., Azospirillum sp., and Erwinia sp. (6-12). Especially in developing countries where agricultural activities play an important role, plant growth regulators as well as fertilizers should be used to maximize production efficiency. Therefore, synthetic plant growth regulators should be produced 313 IAA and GA production in A. niger economically on an industrial scale. In this respect, the effects of some physiological conditions of indole3-acetic acid and gibberellic acid production in A. niger were investigated in this study. Materials and methods Microorganism (microbial strains) A. niger was obtained from the laboratory culture collection of Hacettepe University, Department of Biotechnology. The fungus was maintained on potato dextrose agar at +4 °C and subcultured at 30-day intervals. Medium Indole-3-acetic acid and gibberellic acid were produced on synthetic Czapek-Dox broth (Difco) media, and 0.1% tryptophan was added to the IAA production medium. The media were distributed to 250 mL Erlenmeyer flasks that contained 100 mL of medium and were sterilized in an autoclave at 121 °C and 1.5 atm pressure for 15 min. Incubation A. niger (2 × 107 spores/mL) was inoculated into 100 mL of production medium and incubated at 30 °C -1 in dark conditions on a rotary shaker (150 rev min ). After the incubation, indole-3-acetic acid and gibberellic acid production and growth were measured. Determination of growth The amount of growth in cultures was calculated as dry weight with a filtration method. The growth media were filtered through preweighed filter paper, and then the biomass was dried in an incubator at 30 °C for 24 h and weighed. Indole-3-acetic determination acid extraction and Indole-3-acetic acid production was determined with the method described by Mahadevan and Chandramohan (13). Culture media were filtered, and then samples were acidified to pH 3.0 with 1 N HCl and extracted using liquid-liquid (diethyleter/ NaHCO3) extraction. The samples were concentrated and dissolved in methanol. Indole-3-acetic acid in methanol was determined with Salper reagent (1 mL of 0.5 M FeCl3 in 50 mL of 35% perchloric acid). Absorbance was read at 535 nm using a Jenway 6105 314 UV/VIS spectrophotometer. The amount of indole3-acetic acid was calculated from the standard curve. Gibberellic acid extraction and determination Culture media were filtered, and then samples were acidified to pH 2.5 with HCl and extracted using liquid-liquid (ethylacetate/NaHCO3) extraction (14). Gibberellic acid in the ethyl acetate phase was measured by UV spectrophotometer (Jenway 6105 UV/VIS) at 254 nm (15). The amount of gibberellic acid was calculated from the standard curve. Physiological production conditions affecting the A. niger was grown in a rotary shaker (150 rev -1 min ) in dark conditions at 30 °C. Incubations were carried out for 6 days and 12 days for indole-3-acetic acid and gibberellic acid, respectively. The effect of temperature was studied by incubating the media at different temperatures. To determine the optimal pH values for indole-3-acetic acid and gibberellic acid productions, media with different pH values (3.0-7.0) were used. To investigate the effect of agitation and static incubation conditions on synthesis, 2 parallel experiment sets were used. The incubations were carried out at 30 °C in both sets; the first set was agitated at 150 rpm while the second was static. GA3 and IAA Interactions In order to investigate the effect of IAA on GA3 synthesis, Czapek-Dox broth (Difco) medium containing 0.05% GA3 (Merck) was used. The effect of GA3 on IAA synthesis was determined by adding 0.05% IAA (J.T. Baker) to the medium. The growth rates and productions were compared in each case. Results IAA production in A. niger was studied for 5-15 days and maximum production was observed on day 6. When GA3 production was investigated for 5-24 days, maximum production was observed on day 12 (Figure 1). The effect of incubation temperature on IAA and GA3 production was investigated. It was observed that both GA3 production and growth were maximized at 30 °C and decreased at higher temperatures. Maximum IAA production and growth were observed at 25 °C (Figure 2). I. SEYİS BİLKAY, Ş. KARAKOÇ, N. AKSÖZ 6 100 3 indole-3-acetic acid production (mg/L) 150 20 25 6 7 10 13 incubation time (days) 15 growth (g/L) 14 250 12 10 8 150 6 100 4 50 2 6 9 12 15 18 21 24 12 250 10 200 8 150 6 100 4 50 2 0 growth (g/L) 200 300 0 incubation time (days) (b) GA 3 production Figure 1. Effect of incubation time on IAA and GA3 production. (a) IAA production Results are the mean of 3 replicates. Error bars are shown on the graph. (b) GA3 production Results are the mean of 3 replicates. Error bars are shown on the graph. It was observed that IAA production was maximized at pH 6.0 (Figure 3), while the optimum pH value for GA3 production was 5.0. As can be seen in Figure 3, growth did not change considerably at different pH values, especially for IAA. It was observed that agitation increases both IAA and GA3 production, but that growth is higher in the static case for IAA (Figure 4). 25 30 35 40 45 incubation temperature (°C) growth (g/L) gibberellic acid production (mg/L) growth (g/L) 300 5 30 35 incubation temperature (°C) 8 7 6 5 4 3 2 1 0 (a) IAA production 0 gibberellic acid production (mg/L) 5 gibberellic acid production (mg/L) gibberellic acid production (mg/L) 40 0 (a) IAA production 0 60 50 0 growth (g/L) 80 9 200 indole-3-acetic acid production (mg/L) growth (g/L) growth (g/L) Growth (g/L) indole-3-acetic acid production (mg/L) indole-3-acetic acid production (mg/L) 0 (b) GA 3 production Figure 2. Effect of incubation temperature on IAA and GA3 production. (a) IAA production Results are the mean of 3 replicates. Error bars are shown on the graph. (b) GA3 production Results are the mean of 3 replicates. Error bars are shown on the graph. GA3 and IAA interactions were analyzed. It was observed that GA3 production and growth in the absence of IAA were 238.7 mg/L and 10.2 g/L, respectively, whereas these values increased to 354.2 mg/L and 11.3 g/L in the presence of IAA. IAA production and growth in the absence of GA3 were 128.3 mg/L and 6.8 g/L, respectively, whereas these values increased to 132.7 mg/L and 8.1 g/L in the presence of GA3. Discussion The relation between incubation time and IAA and GA3 production in A. niger were investigated. In similar studies on IAA and GA3 production in various fungi, incubation times between 10 and 18 days were 315 IAA and GA production in A. niger 40 6 growth (g/L) 20 4 5 6 incubation initial pH 7 80 7.5 60 40 7 20 0 6.5 agitated incubation condition 4 (a) IAA production (a) IAA production 12 250 10 200 8 150 6 100 4 50 2 3 4 5 6 incubation initial pH 7 0 (b) GA 3 production Gibberellic acid production (mg/L) 300 0 gibberellic acid production (mg/L) growth (g/L) growth (g/L) gibberellic acid production (mg/L) gibberellic acid production (mg/L) static growth (g/L) 300 12 250 10 200 8 150 6 100 4 50 2 0 Growth (g/L) indole-3-acetic acid production (mg/L) 60 growth (g/L) growth (g/L) 8 80 0 indole-3-acetic acid production (mg/L) growth (g/L) indole-3-acetic acid production (mg/L) indole-3-acetic acid production (mg/L) 0 agitated static incubation condition Figure 3. Effect of pH on IAA and GA3 production. (a) IAA production Results are the mean of 3 replicates. Error bars are shown on the graph. (b) GA3 production Results are the mean of 3 replicates. Error bars are shown on the graph. used (6,16-19). In another study, the synthesis of IAA was maximized after 30 days of incubation (20). IAA and GA3 are secondary metabolites of fungi, excreted by a microorganism near the end of the growth phase or during the stationary phase. Therefore, it was expected that the production times of these plant regulators were long. Although GA3 production was maximized on day 12, which is parallel to the findings of similar studies in the literature, it was determined that IAA production was 316 (b) GA 3 production Figure 4. Effect of incubation condition on IAA and GA3 production. (a) IAA production Results are the mean of 3 replicates. Error bars are shown on the graph. (b) GA3 production Results are the mean of 3 replicates. Error bars are shown on the graph. maximized on day 6. Therefore, it can be concluded that IAA production in A. niger is advantageous when compared to GA3, as 2 batches of IAA can be produced in the same period required to produce one batch of GA3. The optimum incubation temperature for IAA production was found to be 25 °C, which is room temperature. It is obvious that maintaining the I. SEYİS BİLKAY, Ş. KARAKOÇ, N. AKSÖZ optimum temperature during production is an important factor in the economics of the entire process. Therefore, in a process that can be carried out at room temperature, the need for extra energy for heating or cooling is eliminated, which can be considered a major advantage. The optimum temperature for GA3 production was found to be 30 °C, which is probably due to the fact that the enzymes synthesizing GA3 are comparatively inactive at higher temperatures. In similar studies, IAA production was found to be maximized at 28 °C (21,22). In some other studies on IAA and GA3 production, optimum temperatures were in the range of 25-30 °C, which is parallel to our findings (1,6,17,23). The results of the pH analyses of GA3 production in previous studies were found to be between 5.0 and 5.5, which is similar to our findings (6,16,24,17). It was concluded that agitation during production is preferable when compared with a static condition. This was an expected result, as, in an agitated case, the production medium is more homogeneous and the oxygen supply is better, which increases the biomass in the medium as well as the production. This result also implies that IAA and GA3 synthesis is oxygendependent (25-27). Lower IAA production was probably due to the fact that, in the static case, a greater amount of biomass hindered oxygen entry, therefore limiting production. Furthermore, higher growth rates were accompanied by higher GA3 production. GA3 did not improve IAA production to the same extent, but GA3 production in the presence of IAA increased much more when compared with the production of IAA in the presence of GA3. This result implies that GA3 mainly stimulates cell growth, and IAA production did not increase, as it is a secondary metabolite. Similar results were obtained in a previous study on GA3 and IAA interaction carried out with Gibberella fujikuroi (28). In conclusion, optimum production conditions of IAA and GA3 in A. niger were slightly different. For IAA production, 6 days of incubation at 25 °C and pH 6.0 is suitable, whereas 12 days of incubation at 30 °C and pH 5.0 is suitable for GA3 production. In addition, it was concluded that IAA production at 25 °C and pH 6.0 was more efficient than production at 30 °C and pH 5.0, which were common findings of previous studies. Acknowledgements This work was supported by a grant from the Hacettepe University Academic Research Unit. 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