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Indole-3-Acetic Acid and Gibberellic Acid Production in Aspergillus Niger

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Indole-3-Acetic Acid and Gibberellic Acid Production in Aspergillus Niger 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 1Hacettepe University, Faculty of Science, Department of Biology (Biotechnology), 06532 Beytepe, Ankara - TURKEY 2MARA, 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 synthesized by Gibberella Gibberellic acid is a plant growth regulator of fujikuroi, Sphaceloma manihoticola, Neurospora economic and industrial importance (1). crassa, Aspergillus niger, Sphaceloma sp., Rhizobium Phytohormones, mainly including auxins, cytokinins, phaseoli, Azospirillum brasilense, Pseudomonas sp., abscisic acid, gibberellins, and ethylene, induce some and Phaeosphaeria sp., whereas IAA is synthesized by important physiological responses at different stages Pseudomonas sp., Bacillus sp., Aspergillus niger, of plant development at low concentrations (2). Rhizopus, Acetobacter diazotrophicus, Herbaspirillum Various gibberellins are available and are associated seropedicae, Rhizobium sp., Azospirillum sp., and with several plant growth and development processes, Erwinia sp. (6-12). such as seed germination, stem elongation, flowering, Especially in developing countries where and fruit development (3,4). On the other hand, auxin agricultural activities play an important role, plant was first isolated and characterized as a plant growth regulators as well as fertilizers should be used hormone, and indole-3-acetic acid (IAA) is a type of to maximize production efficiency. Therefore, auxin (5). synthetic plant growth regulators should be produced 313 IAA and GA production in A. niger economically on an industrial scale. In this respect, UV/VIS spectrophotometer. The amount of indole- the effects of some physiological conditions of indole- 3-acetic acid was calculated from the standard curve. 3-acetic acid and gibberellic acid production in A. Gibberellic acid extraction and determination niger were investigated in this study. Culture media were filtered, and then samples were acidified to pH 2.5 with HCl and extracted using Materials and methods liquid-liquid (ethylacetate/NaHCO3) extraction (14). Microorganism (microbial strains) Gibberellic acid in the ethyl acetate phase was measured by UV spectrophotometer (Jenway 6105 A. niger was obtained from the laboratory culture UV/VIS) at 254 nm (15). The amount of gibberellic collection of Hacettepe University, Department of acid was calculated from the standard curve. Biotechnology. The fungus was maintained on potato dextrose agar at +4 °C and subcultured at 30-day Physiological conditions affecting the intervals. production Medium A. niger was grown in a rotary shaker (150 rev min-1) in dark conditions at 30 °C. Incubations were Indole-3-acetic acid and gibberellic acid were carried out for 6 days and 12 days for indole-3-acetic produced on synthetic Czapek-Dox broth (Difco) acid and gibberellic acid, respectively. The effect of media, and 0.1% tryptophan was added to the IAA temperature was studied by incubating the media at production medium. The media were distributed to different temperatures. To determine the optimal pH 250 mL Erlenmeyer flasks that contained 100 mL of values for indole-3-acetic acid and gibberellic acid medium and were sterilized in an autoclave at 121 °C and 1.5 atm pressure for 15 min. productions, media with different pH values (3.0-7.0) were used. To investigate the effect of agitation and Incubation static incubation conditions on synthesis, 2 parallel A. niger (2 × 107 spores/mL) was inoculated into experiment sets were used. The incubations were 100 mL of production medium and incubated at 30 °C carried out at 30 °C in both sets; the first set was in dark conditions on a rotary shaker (150 rev min-1). agitated at 150 rpm while the second was static. After the incubation, indole-3-acetic acid and GA and IAA Interactions gibberellic acid production and growth were 3 measured. In order to investigate the effect of IAA on GA3 synthesis, Czapek-Dox broth (Difco) medium Determination of growth containing 0.05% GA3 (Merck) was used. The effect The amount of growth in cultures was calculated of GA3 on IAA synthesis was determined by adding as dry weight with a filtration method. The growth 0.05% IAA (J.T. Baker) to the medium. The growth media were filtered through preweighed filter paper, rates and productions were compared in each case. and then the biomass was dried in an incubator at 30 °C for 24 h and weighed. Results Indole-3-acetic acid extraction and determination IAA production in A. niger was studied for 5-15 days and maximum production was observed on day Indole-3-acetic acid production was determined 6. When GA production was investigated for 5-24 with the method described by Mahadevan and 3 days, maximum production was observed on day 12 Chandramohan (13). Culture media were filtered, and (Figure 1). then samples were acidified to pH 3.0 with 1 N HCl and extracted using liquid-liquid (diethyleter/ The effect of incubation temperature on IAA and NaHCO3) extraction. The samples were concentrated GA3 production was investigated. It was observed that and dissolved in methanol. Indole-3-acetic acid in both GA3 production and growth were maximized at methanol was determined with Salper reagent (1 mL 30 °C and decreased at higher temperatures. of 0.5 M FeCl3 in 50 mL of 35% perchloric acid). Maximum IAA production and growth were Absorbance was read at 535 nm using a Jenway 6105 observed at 25 °C (Figure 2). 314 I. SEYİS BİLKAY, Ş. KARAKOÇ, N. AKSÖZ indole-3-acetic acid production (mg/L) growth (g/L) indole-3-acetic acid production (mg/L) growth (g/L) 80 8 9 200 7 60 6 5 150 40 4 roduction 6 p 3 20 2 growth (g/L) production (mg/L) 100 indole-3-acetic acid 1 (mg/L) 0 0 3 (g/L) Growth 25 30 35 50 incubation temperature (°C) (a) IAA production indole-3-acetic acid 0 0 5 6 7 10 13 15 incubation time (days) gibberellic acid production (mg/L) growth (g/L) 300 12 (a) IAA production 250 10 gibberellic acid production (mg/L) growth (g/L) 200 8 300 14 150 6 (mg/L) 100 4 growth (g/L) 12 250 50 2 gibberellic acid production 10 0 0 200 25 30 35 40 45 8 incubation temperature (°C) 150 (b) GA 3 production 6 100 (g/L) growth Figure 2.Effect of incubation temperature on IAA and GA3 4 production. 50 2 (a) IAA production gibberellic acid production (mg/L) Results are the mean of 3 replicates. Error bars are 0 0 shown on the graph. 5 6 9 12 15 18 21 24 (b) GA3 production incubation time (days) Results are the mean of 3 replicates. Error bars are shown on the graph. (b) GA3 production Figure 1. Effect of incubation time on IAA and GA3 production. GA3 and IAA interactions were analyzed. It was (a) IAA production observed that GA3 production and growth in the Results are the mean of 3 replicates. Error bars are absence of IAA were 238.7 mg/L and 10.2 g/L, shown on the graph. (b) GA3 production respectively, whereas these values increased to Results are the mean of 3 replicates. Error bars are 354.2 mg/L and 11.3 g/L in the presence of IAA. IAA shown on the graph. production and growth in the absence of GA3 were 128.3 mg/L and 6.8 g/L, respectively, whereas these It was observed that IAA production was values increased to 132.7 mg/L and 8.1 g/L in the maximized at pH 6.0 (Figure 3), while the optimum presence of GA3. pH value for GA3 production was 5.0. As can be seen in Figure 3, growth did not change considerably at Discussion different pH values, especially for IAA. The relation between incubation time and IAA It was observed that agitation increases both IAA and GA3 production in A. niger were investigated. In and GA3 production, but that growth is higher in the similar studies on IAA and GA3 production in various static case for IAA (Figure 4). fungi, incubation times between 10 and 18 days were 315 IAA and GA production in A. niger indole-3-acetic acid production (mg/L) growth (g/L) indole-3-acetic acid production (mg/L) growth (g/L) 80 7.5 80 8 60 60 40 7 40 6 (mg/L) growth (g/L) growth (g/L) 20 20 indole-3-acetic acid production indole-3-acetic acid production (mg/L) 0 6.5 0 4 agitated static 4567 incubation condition incubation initial pH (a) IAA production (a) IAA production gibberellic acid production (mg/L) growth (g/L) gibberellic acid production (mg/L) growth (g/L) 300 12 300 12 250 10 250 10 200 8 200 8 150 6 150 6 (mg/L) 100 4 growth (g/L) 100 4 Growth (g/L) 50 2 gibberellic acid production 0 0 50 2 34567 Gibberellic acid production (mg/L) incubation initial pH 0 0 agitated static (b) GA 3 production incubation condition Figure 3.
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