Continuous production of indole-3-acetic acid by immobilized cells of Arthrobacter agilis
Indole acetic acid (IAA) is a plant growth-promoting hormone used in agriculture; therefore, its continuous production is of paramount importance. IAA-producing eight bacteria were isolated from the rhizosphere of Verbascum vulcanicum. Among them, Arthrobacter agilis A17 gave maximum IAA production (75 mg/L) and this strain was used to immobilization studies. The A. agilis A17 cells were immobilized in calcium alginate for the production of IAA. Optimization of process parameters for IAA production was carried out to enhance IAA production using immobilized cells. The maximal production of IAA was 520 mg/L under the following optimal conditions: 1% mannitol, 30 °C, pH 8.0, and 24 h incubation. It was determined that the immobilized cells could be reused (13 times) for the production of IAA.
KeywordsIndole acetic acid Immobilized cells Fermentation Arthrobacter agilis
Indole acetic acid (IAA) is a natural auxin which is produced by plants, bacteria and fungi. In plants, IAA is critical for plant growth and development. Most of the bacteria isolated from the rhizosphere can produce IAA (Duca et al. 2014; Patten and Glick 1996). Many researchers reported that plant growth-promoting bacteria from different genera (Azospirillum, Bacillus, Enterobacter, Azotobacter, Klebsiella, Pseudomonas, and Arthrobacter), Actinomycetes (Streptomyces olivaceoviridis, S. rimosus) and fungi (Colletotrichum gloeosporioides, Ustilago maydis) enhance plant growth by the synthesis of IAA (Khamna et al. 2010; Reineke et al. 2008).
IAA can be synthesized via either tryptophan-dependent pathway or tryptophan-independent pathway. The indole-3-acetamide, indole-3-pyruvate, tryptamine, tryptophan side-chain oxidase and indole-3-acetonitrile pathways are considered as main IAA biosynthesis through a tryptophan-dependent pathway in bacteria (Spaepen et al. 2007). Forni et al. (1992) and Yadav et al. (2015) reported that cells of Arthrobacter species (A. sulfonivorans, A. sulfureus, A. globiformis, A. nicotianae, A. crystallopoietes) produced IAA in the culture medium when precursor l-tryptophan was present in the medium. However, some bacteria have the tryptophan-independent pathway (starting from indole or indole-3-glycerol phosphate) to produce IAA (Arora and Bae 2014; Arora et al. 2015a).
Immobilized biocatalysts are widely used to produce different types of products and enzymes. They provide many benefits, such as higher stability, lower operational costs, continuous use of the biocatalysts, higher resistance to contamination, easier separation from the production medium and enhanced reaction yield (Kurbanoglu et al. 2010a, b; Okay et al. 2013). Different support materials (carrageenan, polyurethane, polyethylene glycol, and alginate) have been suggested for cell immobilization. Among them, sodium alginate is the most commonly used system because it is a rapid, nontoxic, low cost, and an easy method (Singh et al. 2012; Silbir et al. 2014; Yewale et al. 2016).
According to our knowledge, there is no earlier study using Ca-alginate immobilized A. agilis cells for IAA production. Considering the economic importance of IAA, this study aimed to continuous production of IAA with immobilized cells of A. agilis A17.
Materials and methods
Isolation of IAA producing bacteria from rhizospheric soil
Soil sample was collected from the rhizosphere of Verbascum vulcanicum from Palandöken Mountain, Turkey (39°49′15N, 41°17′34E). The isolation of microorganisms was done according to Mohite (2013). One gram of rhizospheric soil sample was suspended in 10 mL of sterile physiological water. It was incubated on rotary shaker at 150 rpm for 10 min. One mL of sample was serially diluted up to 10−7. One hundred microliter of diluted sample was plated on sterile Luria–Bertani (LB) agar medium containing 1 g/L l-tryptophan (Sigma) and incubated for 3 days at 30 °C. Single colonies were picked up and streaked on sterile LB agar plates to get pure culture. Total eight isolates were obtained from the rhizospheric soil and they were screened out for the production of IAA.
Screening of isolates for indole acetic acid production
The isolates were grown in tryptic soy broth (TSB) on a rotary shaker at 150 rpm for 24 h at 30 °C. Bacterial isolates were evaluated for IAA production by inoculating 1 mL of each of cells suspension (1 × 106 CFU/mL) in 50 mL of TSB (containing 1 g/L l-tryptophan) kept in 250-mL Erlenmeyer flasks. The flasks were incubated in the dark at 30 °C, 150 rpm, for 72 h.
Determination of IAA production
After incubation periods, the culture media were centrifuged (4000 rpm for 10 min). One mL of supernatant was combined with 2 ml of Salkowski’s reagent (49 mL 35% perchloric acid and 1 mL 0.5 M FeCl3) and incubated for 30 min at room temperature. The production of IAA was determined by colorimetric measurement at 530 nm using Salkowski’s reagent as described by Patten and Glick (2002). The quantity of IAA was determined by comparison with a standard curve using pure IAA.
The bacterial isolates were preliminarily characterized by Gram’s staining. The selected bacterium producing the highest IAA was then identified based on the 16S rDNA sequencing using universal primer set 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1102R (5′-GAGGTTCTGTGCTCCTCAGC-3′) (Gur et al. 2014). The isolate identification was verified by the analysis of 16S rDNA sequence (RefGen Life Sciences, Ankara, Turkey) which was compared with the National Center for Biotechnology Information (NCBI) database using the BLAST search on the web site (http://www.ncbi.nlm.nih.gov/BLAST) and submitted to GenBank. This bacterium was also identified according to its cytological and metabolic features, such as pigment formation, motility, spore formation, Gram staining, nitrate reduction, catalase and oxidase tests, and starch hydrolysis. These tests were carried out according to the Harley and Prescott (2002).
Immobilization of Arthrobacter agilis A17 cells
Arthrobacter agilis cells were immobilized in calcium alginate gel beads as described previously (Kurbanoglu et al. 2010a; Okay et al. 2013). Arthrobacter agilis A17 was grown in Nutrient Broth (Merck) at 28 °C for 24 h. The culture was centrifuged at 5000 rpm for 15 min and the biomass was washed two times with sterile 0.85% (w/v) saline solution (SSS). Wet cells (4 g) were thoroughly resuspended in 40 mL of SSS and the total volume was completed to 50 ml with SSS. Sodium alginate solution (3%, w/v) was prepared by dissolving sodium alginate in SSS at 70 °C. The cell suspension was mixed with an equal volume of sodium alginate solution and stirred for 5 min. The mixture was dropped into a well stirred sterile CaCl2 solution (3.5%, w/v) using a syringe. Each alginate drop solidified upon contact with CaCl2 and formed beads that encapsulated the Arthrobacter agilis A17 cells. The beads were left to harden for 30 min at room temperature after they were washed with SSS to remove excess calcium ions and un-encapsulated cells. The average bead diameter was approximately 2–3 mm.
Optimization of reaction parameters
In dark conditions, the immobilized cells (3.5 g) were incubated in 50 mM Tris buffer containing 1 g/L l-tryptophan on a rotary shaker at 150 rpm. Optimization of all variables was performed with 25 mL of reaction mixture in a 250-mL Erlenmeyer flask. To optimize temperature, the reaction was performed at pH 7.2 and 150 rpm with temperatures varying between 18 and 36 °C. For pH optimization, the conditions were 30 °C, 150 rpm, and Tris buffer pH 6–9. Different carbon sources (glucose, lactose, sucrose, maltose, mannitol, and fructose) were screened for IAA production using immobilized beads. Effect of incubation period (6–36 h) on IAA production was studied by immobilized cells.
The statistical analyses of the data were performed using one-way analysis of variance (ANOVA). The level of significance was p < 0.05. Statistical analyses were conducted using the SPSS 20.0 software program.
Results and discussion
Isolation and screening of the bacteria for IAA production
Results of morphological and biochemical tests for the isolate A17
Growth at 5% NaCl
Arthrobacter species (pigmented Gram-positive bacteria) have been isolated from different sources, such as soil, water, foods, radioactive waste and arctic ice. It appears that beta carotene offers protection for A. agilis in the natural environment (Dieser et al. 2010; Sutthiwong et al. 2014). Many species of Arthrobacter have been reported to produce IAA in the presence of l-tryptophan. Arthrobacter globiformis and A. nicotianae produced 10.1 and 4.4 mg/L IAA (Forni et al. 1992). Yuan et al. (2011) reported that Arthrobacter sp. produced 87.7 mg/L IAA. Similarly, Yadav et al. (2015) showed that A. sulfonivorans, A. sulfureus and Arthrobacter sp. produced 27.6, 48.2 and 28.2 mg/L IAA, respectively.
Optimization of IAA production under immobilization conditions
Reaction conditions are significant for the successful production of IAA and optimization of parameters, such as temperature, pH, media composition and process time are essential in developing the process.
Effect of temperature on IAA production
Effect of buffer pH on IAA production
Effect of carbon sources on IAA production
Effect of fermentation time on IAA production
Reusability of immobilized Arthrobacter agilis cells
Screening experiments were carried out to select the most active bacterial isolate for IAA production. In this work, we successfully produced IAA using immobilized cells of A. agilis A17. The highest IAA was 490 mg/L for 24 h, at 1% mannitol concentration, 30 °C and pH 8. Moreover, our results showed that immobilized cells offers repeated use of the biocatalysts in the production of IAA. In subsequent studies, large scale application for IAA production will be developed with cells entrapped. In addition, high IAA-producing species (belonging to the genus Enterobacter, Pantoea, Klebsiella) should be investigated for overproduction of IAA with immobilized cells. The results obtained in the study suggested that production of IAA by immobilized A. agilis A17 appeared to be feasible method to continuous production of IAA.
Compliance with ethical standards
Conflict of interest
The authors declare that there is no conflict of interests regarding the publication of this paper.
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