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ORIGINAL ARTICLE Optimization of medium for indole-3-acetic acid production using Pantoea agglomerans strain PVM O.A. Apine and J.P. Jadhav Department of Biotechnology Shivaji University, Kolhapur, India Introduction Plant growth–promoting rhizobacteria are free living, which enhances the growth of the plant either directly or indirectly. The mechanism involves nitrogen fixation, phosphorous solubilization and production of various phytohormones (Karnwal 2009). The auxins are the group of phytohormones having indole ring compounds (Khamna et al. 2010) and which play a key role in stimu- lation of cell division and cell elongation. It also controls the lateral and adventitious root formation and mediates the tropic response to gravity and light. The lower concentrations of auxin stimulate root elongation, whereas higher concentration inhibits the root elongation (Madhaiyan et al. 2007). Diverse groups of micro-organisms, including soil, epi- phytic and endophytic bacteria and some cyanobacteria, were found to synthesize indole-3-acetic acid (IAA) in the presence of l-tryptophan (Pedraza et al. 2004). Micro- organisms from rhizospheres of various plants synthesize and release auxin as secondary metabolites because of rich substrates exuded from the roots in rhizosphere compared with nonrhizospheric soils (Ahmad et al. 2005). The release of l-tryptophan in root exudates may result in its conversion into IAA by rhizosphere microbes (Kravchenko et al. 2004). Several of these groups are implicated in the plant pathogenesis, while others stimulate plant growth (Pedraza et al. 2004). The earlier study showed that plant growth–promoting bacteria from different genera (Azospirillum, Enterobacter, Azotobacter, Klebsiella, Alcali- genes faecalis), actinomycetes (Streptomyces olivaceoviridis, Streptomyces rimosus) and fungi (Colletotrichum gloeospo- rioides, Ustilago maydis) have shown to enhance plant growth by the synthesis of IAA (Torres-Rubio et al. 2000; Reinekei et al. 2008; Khamna et al. 2010). The Streptomy- ces griseoviridis K61 and Streptomyces lydicus WYEC108 were used commercially for IAA production under the trade name Mycostop (Khamna et al. 2010). Keywords HPTLC, IAA, l-tryptophan, meat extract, Pantoea agglomerans strain PVM. Correspondence Jyoti P. Jadhav, Department of Biotechnology, Shivaji University, Vidyanagar, Kolhapur 416004, India. E-mail: jpjbiochem@gmail.com 2010 ⁄ 1969: received 2 November 2010, revised 3 February 2011 and accepted 10 February 2011 doi:10.1111/j.1365-2672.2011.04976.x Abstract Aims: To optimize the medium components for the production of indole- 3-acetic acid (IAA) by isolated bacterium Pantoea agglomerans strain PVM. Methods and Results: Present study deals with the production of an essential plant hormone IAA by a bacterial isolate P. agglomerans strain PVM identified by 16S rRNA gene sequence analysis. The medium containing 8 g l )1 of meat extract and 1 g l )1 of l-tryptophan (precursor) at optimum pH 7, 30°C and 48-h incubation gave the maximum production of IAA (2Æ191 g l )1 ). Effect of IAA synthesized on in vitro root induction in Nicotiana tobacum (leaf) explants was compared with that of control. IAA was characterized by high-performance thin-layer chromatography, high-performance liquid chromatography and gas chromatography–mass spectroscopy. Conclusions: Pantoea agglomerans strain PVM was a good candidate for the inexpensive and utmost production of IAA in short period, as it requires sim- ple medium (meat extract and l-tryptophan). Significance and Impact of the Study: The present report first time showed the rapid, cost-effective and maximum production of IAA. No reports are available on the optimization of particular medium components for the production of IAA. This study demonstrates a novel approach for in vitro root induction in N. tobacum (leaf) explants. Journal of Applied Microbiology ISSN 1364-5072 ª 2011 The Authors Journal of Applied Microbiology 110, 1235–1244 ª 2011 The Society for Applied Microbiology 1235 Pantoea agglomerans is wide spread in many diverse natural and agricultural habitats, in particular it is associ- ated with many plants as common epiphytes and endo- phytes. Currently, the genus Pantoea includes seven species and two subspecies, the majority of which are associated with plant growth promotion (Barash and Manulis-Sasson 2009; Sergeeva et al. 2007). Besides extensive research on IAA production by microbial species, there is still a need to do further research in this area for process improvement. Here, we report the highest IAA production under optimized med- ium conditions using a bacterial isolate P. agglomerans strain PVM compared to earlier reports. Materials and methods Chemicals l-Tryptophan, meat extract, perchloric acid, ortho-phos- phoric acid, nitric acid and ethanol were obtained from the Himedia Laboratories, Mumbai, India. All chemicals used were of the highest purity available and of analytical grade. Screening, isolation and identification of micro-organism Isolation of the microbial strain capable of IAA produc- tion carried out from the agricultural soil by enrichment culture technique. The minimal salt medium supple- mented with l-tryptophan was used for the isolation. The 250-ml conical flasks containing the above-stated medium (100 ml) were prepared in duplicate and inoculated with 1 g of soil sample, followed by incubation at 30°C under shaking conditions at 120 rev min )l for 48 h. A loopful of sample from soil-free enrichments was streaked on agar plate having media composition as stated earlier. Morphologically distinct colonies were selected for screening of its IAA-producing ability. The most efficient bacterial isolate is selected and used for further studies. Identification of the isolate as P. agglomerans strain PVM was done by 16S rRNA gene sequence analysis at Chro- mous biotech Pvt. Ltd, Bangalore, India, and the sequence is deposited in the Gene Bank. Phylogenic analysis The partial nucleotide sequence of P. agglomerans strain PVM was obtained from Chromous biotech Pvt. Ltd. Blasted by using the NCBI server (http://ncbi.nlm.nih.gov/ Blast.cgi), the homologous species were used for phylogenic analysis. The evolutionary history was inferred using the neighbour-joining method (Saitou and Nei 1987). The optimal tree with the sum of branch length = 36Æ65209254 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein 1985). The phylogenetic tree was linearized assuming equal evolu- tionary rates in all lineages (Takezaki et al. 2004). The clock calibration to convert distance to time was 1 (time per node height). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary dis- tances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method (Tamura et al. 2004) and are in the units of the number of base substitutions per site. Codon positions included were 1st + 2nd + 3rd + noncoding. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). There are total 1393 positions in the final dataset. A phylogenetic analysis was conducted in mega4 (Tamura et al. 2004). Organism and culture conditions Bacterial strain (stock culture) was maintained routinely on nutrient agar containing the following (g l )1 ): peptone (10), yeast extract (3), NaCl (5), agar (20) and l-trypto- phan (1), stored at 4°C until used. All the experiments were carried out using above-stated medium. The experi- ments were carried out in triplicates at pH 6Æ8, 30°C and at 120 rev min )l unless otherwise stated. pH and temperature optima To enhance the reproducibility of results and to optimize the biosynthesis process as a whole, the factorial design of experiments, the ‘one factor at a time’ method, was employed in this study. Here, the experimental factors are varied one at a time with the remaining factors held constant. The pH optima for the production of IAA was determined by varying pH to 3, 4, 5, 6, 7, 8, 9 and 10. Similar experiments were performed to assess the effect of temperature by incubating flasks at 10, 20, 30, 40 and 50°C. Finally, the produced IAA and residual l-tryptophan were assayed. Effect of precursor ( L -tryptophan) on IAA production The effect of l-tryptophan concentrations on IAA production was studied using medium supplemented with l-tryptophan at concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 g l )1 followed by incubation at optimum pH and temperature. Consequence of media components on IAA production Effects of various media components on the production of IAA were assessed by altering the medium composition Biosynthesis of indole-3-acetic acid O.A. Apine and J.P. Jadhav 1236 Journal of Applied Microbiology 110, 1235–1244 ª 2011 The Society for Applied Microbiology ª 2011 The Authors accordingly. NaCl (2, 4, 6, 8, 10 g l )1 ), carbon sources (sucrose, glucose, maltose, lactose, fructose and starch) and amino acids (l-glycine, l-alanine, l-valine, l-leucine, l-isoluceine, l-phenylalanine, l-tyrosine, l-tryptophan, l-aspartic acid, l-glutamic acid, l-histidine, l-arginine, l-asparagine, l-cystein, l-methionine, l-threonine, l-pro- line, l-serine, l-glutamine and l-lysine) were used at (1 g l )1 ), while nitrogen sources (meat extract, beef extract, tryptone, yeast extract, peptone) at (10 g l )1 ) concentration was investigated. The medium components giving the maximum yield in minimum time course was preferred for further optimiza- tion by eliminating the other medium components of nutrient agar. Effect of incubation time The effect of incubation time on IAA production was assayed by incubating bacterial cultures under optimum conditions up to 168 h. Production of IAA and residual l-tryptophan was measured at every after 24 h. IAA and L -tryptophan assay The IAA produced was assayed by previously quoted method (Gordon and Weber 1951). The supernatant (1 ml) was mixed with two drops of ortho-phosphoric acid and 4 ml of Salkowski’s reagent followed by incuba- tion for 30 min in dark, at room temperature. The devel- oped pink colour was measured by recording the absorbance at 530 nm using UV–Vis spectrophotometer (Hitachi UV 2800, Tokyo, Japan). Quantification of IAA was performed using standard curve. l-Tryptophan utilized was determined by estimating the residual l-tryptophan remaining in the broth by pre- viously quoted spectrophotometric test (Hassan 1975). Briefly, 1 ml of cell-free aliquots was taken from the broth and evaporated on a boiling water bath to dryness, followed by addition of 1 ml of nitric acid (16 mol 1 )l ) and incubation at 50°C for 15 min. The contents were then cooled at room temperature following the addition of 4 ml of sodium hydroxide (5 mol l )l ) solution; ethyl alcohol was used to make the final volume of 10 ml. After mixing the contents, absorbance was recorded at 360 nm. Quantification of l-tryptophan was performed using stan- dard curve. Tryptophan aminotransferase assay Preparation of cell-free extract The bacteria grown in optimized medium at 30°C and 120 rev min )l were harvested during exponential phase after 12 h (centrifuged at 9000 g for 10 min at 4°C). The cell pellets resuspended in the 0Æ5 m borate buffer (pH 8Æ5), and the cells were disrupted by sonication with stroke of 40 Hz for 30 s, total seven strokes keeping 2 min of time interval after each stroke. After sonication, the homogenate was centrifuged at 14 000 g for 10 min, and the supernatant was used as a crude enzyme source. Assay The tryptophan aminotransferase activity was determined by the previously described method (Matheron and Moore 1973; Khandaswami and Vaidyanathan 1973) with some modifications. The final assay concentration contained 0Æ5 ml of enzyme extract and 2Æ5mlof0Æ5 mol borate buffer (pH 8Æ5) containing 0Æ1 lmol of pyridoxal phosphate, 40 lmol of l-tryptophan and 20 lmol of a-ketoglutarate. The reaction mixture was incubated for 30 min at 37°C, and the reaction mixture without a-keto- glutarate was used as a control. The product formed after enzymatic reaction gave absorbance at 310 nm, so DA 310 was monitored before and after the incubation. Protein content was determined by the Lowry method (Lowry et al. 1951). In vitro root induction In vitro experiment was designed to study the effect of IAA synthesized by P. agglomerans strain PVM on root induction in Nicotiana tobacum. The three different media combinations were the following: Murashige and Skoog (MS) basal medium as negative control; MS + synthetic IAA (2 mg l )1 ) as positive control; and MS + IAA synthe- sized by P. agglomerans strain PVM (cell-free broth) as test. All medium combinations were supplemented with sucrose 3% as per the requirement of the experiment. The medium was solidified with 0Æ2% clarigel (Hi-media, India). The pH of the medium was adjusted to 5Æ8±0Æ05 before autoclaving. Surface-sterilized explants were placed in test tubes (25 · 100 mm), containing 15 ml of med- ium, and culture bottles (200 ml capacity), containing 40 ml of medium, and were autoclaved at 15 psi and 121°C for 20 min. All cultures were maintained at 25 ± 2°C with 16 h light and 8 h in the dark. Analytical studies High-performance thin-layer chromatography (HPTLC) The analysis of produced IAA was performed by using HPTLC system (CAMAG, Muttenz ⁄ Switzerland). Station- ary-phase HPTLC silica gel 60 F 254 (Merck, Germany), predeveloped with methanol, was dried at 120°C for 20 min, cooled to room temperature and equilibrated O.A. Apine and J.P. Jadhav Biosynthesis of indole-3-acetic acid ª 2011 The Authors Journal of Applied Microbiology 110, 1235–1244 ª 2011 The Society for Applied Microbiology 1237 with the relative humidity of the laboratory. The sample was applied on to the plate by spray-on technique (nitrogen as spray gas). Two microlitres of standard IAA (2 mg ml )l ), and the broth before and after incubation (IAA produced) were loaded, by using TLC sample- loading instrument (CAMAG LINOMAT 5). The plate was developed by using saturated twin-trough chamber; 10 ml of developing solvent n-hexane ⁄ ethyl acetate (4 : 6) in front trough of 20 · 10 cm chamber. The chamber was saturated for 20 min prior to plate development. Developing distance is 60 mm from the lower edge of the plate. After development, the developed plate was scanned in the absorbance mode with slit dimension of 5 · 0Æ45 mm, scanning distance 5–65 mm, at 280 nm, using deuterium lamp by using TLC scanner. The results were analysed using HPTLC Win cats planar chromatography manager, ver. 1.4.4.6337 software (CAMAG Muttenz ⁄ Switzerland). High-performance liquid chromatography (HPLC) HPLC analysis was carried out (Waters model no. 2690; Waters Corp., Milford, MA, USA) on C18 column (sym- metry, 4Æ6mm·250 mm) by using HPLC-grade methanol as mobile phase (75 : 25) with flow rate of 1 ml min )1 for 10 min and UV detector at 280 nm. The standard IAA and sample IAA produced in broth were prepared in HPLC-grade water and used for further analysis. Gas chromatography–mass spectroscopy (GC–MS) Extraction of sample The broth (incubated for 48 h) was centrifuged at 6600 g for 15 min, and the supernatant was collected. The super- natant was extracted with double volume of ethyl acetate in separating funnel. The ethyl acetate fraction was recov- ered into a new flask and evaporated. The residue was dissolved in methanol and used for analysis. GC–MS analysis A GC–MS analysis of sample was carried out using a Shimadzu (Nakagyo-Ku, Kyoto, Japan) 2010 MS Engine, equipped with integrated gas chromatograph with a Res- tek column (0Æ25 mm, 60 m; XTI-5). Helium was used as carrier gas at a flow rate of 1 ml min )1 . Injector and detector temperatures were both set to 280°C. The oven temperature was held at 80°C, for 2 min, then pro- grammed to rise from 80 to 200°Cat10°C min )1 and then finally programmed from 200 to 280°Cat 20°C min )1 rate for 7 min. The compounds were identi- fied on the basis of mass spectra and by using the NIST library. Results Identification and phylogenic analysis The potent IAA-producing bacterial species, which was iso- lated, was identified as P. agglomerans strain PVM. The phylogenic position of P. agglomerans strain PVM in relation to other species of this genus is illustrated in Fig. 1a; the digits adjacent to nodes are the statistical frequency of the indicated species. The numbers shown in parentheses are accession numbers of different species. The strain is deposited in GenBank under the accession number GU929212. pH and temperature optima It was observed that P. agglomerans strain PVM showed maximum IAA production of 1Æ441 g l )1 utilizing 0Æ752 g l )1 l-tryptophan at pH 7 (Fig. 1b). Similar study with temperature optimization showed that 1Æ53 g l )1 IAA was produced by utilizing 0Æ804 g l )1 of l-tryptophan (Fig. 2a), when incubated at 30°C. Optimum l-tryptophan concentration The maximum production of IAA 158% (1Æ577 g l )1 ) was achieved by utilizing 78% (0Æ783 g l )1 ) l-tryptophan, when medium was supplemented with 1 g l )1 concentra- tion of l-tryptophan as a precursor (Fig. 2b). Effect of media components The effect of various concentrations of NaCl on IAA production was evaluated. The higher amount of IAA production (1Æ176 g l )1 ) was achieved by utilizing (0Æ780 g l )1 ) l-tryptophan in NaCl (4 g l )1 )-supplemented medium (Fig. 2c). Sucrose was best utilized by the organism and gave 1Æ308 g l )1 of IAA with 0Æ738 g l )1 utilization of l-trypto- phan (Fig. 2d). On the other hand, carbon sources like glucose and fructose gave the moderate IAA production 0Æ985 and 0Æ945 g l )1 , respectively. When the effect of various nitrogen sources on IAA production with respect to tryptophan utilization was studied (Fig. 3a), it was concluded that meat extract gave higher production of IAA (2Æ104 g l )1 ) by utilizing (0Æ803 g l )1 ) l-tryptophan as compared to other nitrogen sources. Hence, it was further optimized, which gave (2Æ191 g l )1 ) IAA utilizing (0Æ826 g l )1 ) l-tryptophan at 8gl )1 meat extract. Also, when the effect of amino acids on IAA produc- tion was studied in the presence of l-tryptophan (Fig. 3c), it was observed that l-tryptophan was solely the Biosynthesis of indole-3-acetic acid O.A. Apine and J.P. Jadhav 1238 Journal of Applied Microbiology 110, 1235–1244 ª 2011 The Society for Applied Microbiology ª 2011 The Authors best source for IAA production in comparison with the combinations with the other amino acids tested. Optimum incubation time The effect of incubation time was studied under opti- mized cultural conditions. The production of IAA and utilization of l-tryptophan was directly proportional to the incubation time; after 48-h incubation IAA produc- tion enhanced by twofold (2Æ059 g l )1 ) utilizing (0Æ865 g l )1 ) l-tryptophan (Fig. 3d). Tryptophan aminotransferase activity The crude tryptophan aminotransferase activity was found to be 312Æ44 U mg )l of enzyme after 12 h of incu- bation, and thereafter enzyme activity slightly decreased. In vitro root induction Pantoea agglomerans strain PVM showed more induc- tion of roots compared with the leaf explants grown on the MS medium supplemented with synthetic IAA (positive control). The negative control (MS basal) does not show the induction of roots in the N. tobacum leaf explants (Fig. 4). This confirms the produced IAA will be better option for the in vitro root induction in plants. Analysis of IAA HPTLC analysis showed the same peak profile for the broth after incubation and standard IAA (Fig. 5e). The Rf value of standard IAA was 0Æ57. However, the broth obtained after incubation showed the Rf 0Æ57 value 0 0·2 0·4 0·6 0·8 1 1·2 1·4 1·6 345678910 pH IAA produced and L -tryptophan used (g l –1 ) 432 0 53 Enterobacter sp. GIST-OutAn2 (EF429007·1) Enterobacter sp. AN2 (GQ451698·1) Enterobacter sp. CO 8-9 (EU181139·1) Enterobacter sp. rif200834 (FJ527677·1) Enterobacter sp. E4M-U (GQ478275·1) Enterobacter sp. KK1 (GQ871449·1) Enterobacter sp. pp9c (GQ360072·1) Enterobacter sp. WAB1938 (AM184277·1) Acinetobacter radioresistens strain Philippines-11 (EF446895·1) Pantoea sp. P102 strain P102 (AF394539·1) Enterobacter sp. SPi (FJ405368·1) Endophytic bacterium EH68 (GU339293·1) Enterobacter sp. DHM-1T (FJ745300·1) Enterobacter sp. R4M-Q (GQ478271·1) Pantoea agglomerans strain P29 (DQ356903·1) Pantoea agglomerans strain PVM (GU929212·1) Proteobacterium symbiont of Nilaparvata lugens clone TM58 (FJ774962·1) Enterobacter cloacae strain IHB B 1374 (GU186117·1) Pantoea sp. M3 (EF192586·1) Enterobacteriaceae bacterium GIST-WP2s1 (EF428984·1) 20 0 0 3 0 8 0 1 1 432 01 5 (a) (b) Figure 1 (a) Phylogenic tree of the Pantoea agglomerans strain PVM and related organ- isms were aligned based on 16S rRNA sequences (neighbour-joining tree). Scale bar – number of nucleotide changes per sequence position. The number at nodes shows the bootstrap values obtained with 1000 resembling analysis. (b) Optimization of pH for IAA production; IAA produced ( ), L -tryptophan used ( ). IAA, indole-3-acetic acid. O.A. Apine and J.P. Jadhav Biosynthesis of indole-3-acetic acid ª 2011 The Authors Journal of Applied Microbiology 110, 1235–1244 ª 2011 The Society for Applied Microbiology 1239 similar to that of standard IAA. Broth before incubation do not show the presence of prominent peak. The HPLC elution profile of standard IAA showed major peak at retention time 2Æ86 min, while elution profile of the broth after incubation (IAA produced) confirmed the production of IAA with peak at retention time 2Æ85 min (Fig. 5f). GC–MS analysis of the extracted samples after incuba- tion revealed presence of IAA. The gas chromatogram showed presence of a major peak at retention time 11Æ9 min, which was analysed by monitoring the proton- ated molecular ion of methyl IAA molecular ion m ⁄ z 189 together with the major fragment ion at m ⁄ z 130, and our results are in good agreement with previously described GC–MS analysis by Muller et al. 2002 (Fig. 5g). Discussion Present work mainly concerned with the IAA production potential of P. agglomerans strain PVM, optimization of medium components and in vitro root induction in N. tobacum. Pantoea agglomerans strain PVM used in this study produced different levels of IAA in the presence of l-tryptophan under various physicochemical conditions. Acidic and alkaline conditions seemed to hamper the production of IAA as well as growth of organism also, elevated or decreased temperatures from 30°C seemed to affect IAA production. Literature survey revealed that Klebsiella strain K8 produces 0Æ0169 g l )1 IAA at pH 8 while 0Æ0179 g l )1 of IAA produced at 37°C (Sachdev et al. 2009). The pH does not affect IAA production in Azospirillum brasilense SM. It will grow in the tempera- ture range of 25–37°C with the optimum being 30°C. Deviations from the optimum temperature allowed signif- icant improvement in IAA biosynthesis (Malhotra and Srivastava 2009). Industrially important white-rot fungus Lentinus sajor-caju gave 0Æ18 g l )1 of IAA production at pH 7Æ5 and 30°C in dark (Yurekli et al. 2003). The IAA production and utilization of l-tryptophan decreased with increasing l-tryptophan concentration. The earlier 0 0·2 0·4 0·6 0·8 1 1·2 1·4 1·6 1·8 Temperature (°C) IAA produced and L -tryptophan used (g l –1 ) 0 20 40 60 80 100 120 140 160 180 L -tryptophan (g l –1 ) IAA produced and L -tryptophan used (%) 0 0·2 0·4 0·6 0·8 1 1·2 1·4 NaCl (g l –1 ) IAA produced and L -tryptophan used (g l –1 ) 0 0·2 0·4 0·6 0·8 1 1·2 1·4 10 20 30 40 50 1234 56 7 8910 24 6810 Sucrose Glucose Maltose Lactose Fructose Starch Carbon source (1g l –1 ) IAA produced and L -tryptophan used (g l –1 ) (a) (c) (d) (b) Figure 2 (a) Optimization of temperature for IAA production; IAA produced ( ), L -tryptophan used ( ). (b) Effect of L -tryptophan on IAA production; IAA produced ( ), L -tryptophan used ( ). (c) Effect of NaCl on IAA production; IAA produced ( ), L -tryptophan used ( ). (d) Effect of carbon sources on IAA production; IAA produced ( ), L -tryptophan used (h). IAA, indole-3-acetic acid. Biosynthesis of indole-3-acetic acid O.A. Apine and J.P. Jadhav 1240 Journal of Applied Microbiology 110, 1235–1244 ª 2011 The Society for Applied Microbiology ª 2011 The Authors reported production in Bacillus megaterium MiR-4, Bacillus sp. NpR-1, Bacillus subtilis TpP-1 and Bacillus licheniformis FiR-1 that produced 0Æ0927, 0Æ0681, 0Æ0654 and 0Æ0626 g l )1 , respectively, at 1 or 1Æ2gl )1 l-trypto- phan (Ali et al. 2010). Rhizobium sp. isolated from root nodules of Dalbergia lanceolaria and Roystonea regia also 0 0·5 1 1·5 2 2·5 Nitrogen source (1%) IAA produced and L -tryptophan used (g l –1 ) 0 0·5 1 1·5 2 2·5 Meat extract (g l –1 ) IAA production and L -tryptophan used (g l –1 ) 0 0·2 0·4 0·6 0·8 1 1·2 1·4 1·6 L -glycine L -alanine L -valine L -leucine L -isoleucine L -phenylalanine L -tyrosine L -tryptophan L -aspartic acid L -glutamic L -histidine L -arginine L -aspargnine L -cystein L -methionine L -threonine L -proline L -serine L -glutamine L -lysine Amino acids (g l –1 ) IAA produced and L -tryptophan used (g l –1 ) 0 0·5 1 1·5 2 2·5 3 Meat ext. Beef ext. Tryptone Yeast ext. Peptone 14523 678910 24 48 72 96 120 144 168 Time (h) IAA produced and L -tryptophan used (g l –1 ) (a) (b) (c) (d) Figure 3 (a) Effect of nitrogen sources on the IAA production; IAA produced ( ), L -tryptophan used (h). (b) Optimization of meat extract for IAA production; IAA produced ( ), L -tryptophan used ( ). (c) Effect of amino acids on IAA production; IAA produced ( ), L -tryptophan used ( ). (d) Effect of incubation time on IAA production; IAA produced ( ), L -tryptophan used ( ). IAA, indole-3-acetic acid. (a) (b) (c) Figure 4 In vitro root induction (a) negative control (MS basal medium), (b) positive control (MS basal + synthetic IAA) and (c) test (MS basa- l + IAA synthesized by Pantoea agglomerans strain PVM). MS, Murashige and Skoog; IAA, indole-3-acetic acid. O.A. Apine and J.P. Jadhav Biosynthesis of indole-3-acetic acid ª 2011 The Authors Journal of Applied Microbiology 110, 1235–1244 ª 2011 The Society for Applied Microbiology 1241 produced IAA at 2Æ5 and 3 g l )1 l-tryptophan concentra- tion, respectively (Basu and Ghosh 2001; Ghosh and Basu 2002). Curtobacterium plantarum 6-I, Streptomyces sp. 3s and Pseudomonas fluorescens 540 produced IAA 0Æ078, 0Æ020 and 0Æ067 g l )1 , respectively, at 0Æ4gl )1 l-tryptophan (Merzaeva and Shirokikh 2010). Pantoea agglomerans strain PVM reported here gave maximum IAA production at lower l-tryptophan concentration compared with ear- lier reports. Abiotic stress like the presence of salt in the medium hampers the production of IAA. Increasing salt stress will reduce the production of IAA and the utilization of the precursor molecule. Pantoea agglomerans strain PVM reported here gave relatively highest yield of IAA in the presence of sucrose as carbon source. Earlier studies regarding Rhizobium strains vary in their utilization and production of IAA with different carbon sources. The Rhizobium strains 12, 16 and 18 require sucrose and Rhizobium strain 13 and Rhizobium sp. from Cajanus cajan require glucose for maximum production of IAA (Datta and Basu 2000; Shridevi and Konada 2007). However, white-rot fungus Lentinus sajor-caju showed 0Æ18 g l )1 of IAA production in glucose-containing medium, but use of sucrose instead of glucose resulted in a substantial decrease in IAA biosynthesis (Yurekli et al. 2003). Meat extract gave the maximum IAA production among the various nitrogen source tested. Hence, the meat extract was further optimized at 8 g l )1 to give highest IAA production (2Æ191 g l )1 ) (Fig. 3b). In the presence of l-tryptophan, only P. agglomerans strain PVM A a B b c (f) (e) abc (g) 900·0 All trade at 254 nm 700·0 600·0 500·0 400·0 300·0 200·0 100·0 0·0 0·00 2·00 2·00 3·00 4·00 5·00 Minutes 6·00 7·00 8·00 9·00 10·00 1·50 1·00 1·00 2·00 2·841 2·860 3·00 4·00 5·00 Minutes 6·00 7·00 8·00 9·00 10·001·00 0·50 0·00 0·25 0·20 0·10 0·15 0·05 0·00 1·0 0·5 0·0 50 51 77 103 146 189 130 100 150 200 250 300 350 400 450 500 550 AUAU 0·10 0·20 0·30 0·40 0·50 0·60 0·70 0·80 1·00 0·0 5·0 10·0 15·0 20·0 25·0 30·0 35·0 40·0 50·0 0·0 100·0 200·0 300·0 400·0 500·0 600·0 700·0 900·0 (AU) (mm) (Rf) (AU) Figure 5 (e) Three-dimensional graph generated after scanning of high-performance thin-layer chromatography plate: (a) standard IAA, (b) broth before incubation and (c) broth after incubation. (f) HPLC elution profile of (A) standard IAA and (B) HPLC elution profile of broth after incubation. (g) Gas chromatography–mass spectroscopy analysis of extracted broth after incubation showed protonated molecular ion of methyl IAA molecu- lar ion m ⁄ z 189 together with the major fragment ion at m ⁄ z 130. IAA, indole-3-acetic acid; HPLC, high-performance liquid chromatography. Biosynthesis of indole-3-acetic acid O.A. Apine and J.P. Jadhav 1242 Journal of Applied Microbiology 110, 1235–1244 ª 2011 The Society for Applied Microbiology ª 2011 The Authors gave the maximum production of IAA. The earlier study revealed that Rhizobium strain 16 showed maximum growth and higher IAA production, when medium supplemented with l-glutamic acid as nitrogen source (Shridevi and Konada 2007). Pantoea agglomerans strain PVM reported here gave the maximum production of IAA within short incubation period. The synthesized IAA was characterized by various analytical techniques like HPTLC, HPLC and GC–MS. For the first time, in vitro root induction was observed by using IAA. 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Jadhav Department of Biotechnology. by Pantoea agglomerans strain PVM) . MS, Murashige and Skoog; IAA, indole -3- acetic acid. O.A. Apine and J.P. Jadhav Biosynthesis of indole -3- acetic acid