J. Gen. Appl. Microbiol., 61, 1–9 (2015) doi 10.2323/jgam.61.1 2015 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation
Full Paper Indole-3-acetic acid production by newly isolated red yeast Rhodosporidium paludigenum (Received August 31, 2014; Accepted October 20, 2014) 1
Pumin Nutaratat, Weerawan Amsri,1 Nantana Srisuk,1,2,* Panarat Arunrattiyakorn,3 and Savitree Limtong1,2,* 2
1 Department of Microbiology, Faculty of Science, Kasetsart University, Chatuchak, Bangkok 10900, Thailand Center for Advanced Studies in Tropical Natural Resources, NRU-KU, Kasetsart University, Chatuchak, Bangkok 10900, Thailand 3 Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand
Indole 3-acetic acid (IAA) is the principal hormone which regulates various developmental and physiological processes in plants. IAA production is considered as a key trait for supporting plant growth. Hence, in this study, production of indole3-acetic acid (IAA) by a basidiomycetous red yeast Rhodosporidium paludigenum DMKU-RP301 (AB920314) was investigated and improved by the optimization of the culture medium and culture conditions using one factor at a time (OFAT) and response surface methodology (RSM). The study considered the effects of incubation time, carbon and nitrogen sources, growth factor, tryptophan, temperature, shaking speed, NaCl and pH, on the production of IAA. The results showed that all the factors studied, except NaCl, affected IAA production by R. paludigenum DMKU-RP301. Maximum IAA production of 1,623.9 mg/l was obtained as a result of the studies using RSM. The optimal medium and growth conditions observed in this study resulted in an increase of IAA production by a factor of up to 5.0 compared to the unoptimized condition, i.e. when yeast extract peptone dextrose (YPD) broth supplemented with 0.1% L-tryptophan was used as the production medium. The production of IAA was then scaled up in a 2-l stirred tank fermenter, and the maximum IAA of 1,627.1 mg/l was obtained. This experiment indicated that the obtained optimal medium and condition (pH and temperature) from shaking flask production can be used for the production of IAA in a larger size production. In addition, the present research is the first to report on the optimization of IAA production by the yeast Rhodosporidium.
Key Words: indole-3-acetic acid; one factor at a time; optimization; response surface methodology; Rhodosporidium; yeast
Introduction Indole-3-acetic acid (IAA), a plant hormone classified as an indole derivative of the auxin family, is widely studied and found as a dominant type of auxin in plants. IAA can be found in large amounts at the apical meristem, buds and young leaves which are the active growing structure of a plant (Ueda et al., 1991). This indicates that plants require IAA more than other auxin derivatives. IAA has a number of important roles in plants, such as the stimulation of cell division, cell elongation, cell differentiation, light and gravitational responses, regulating leaf fall and fruit ripening (Teale et al., 2006; Trotsenko et al., 2001), and offers increased protection to plants from external stress (Bianco and Defez, 2009). In addition, IAA serves as a regulating agent for microbial cell differentiation; for example, it stimulates spore germination and mycelial elongation in streptomycete actinomycetes (Matsukawa et al., 2007), and induces filamentation (induces invasive growth) and substrate adhesion at low concentrations of IAA in Saccharomyces cerevisiae (Prusty et al., 2004). Recently, Rao et al. (2010) showed that IAA stimulated filamentation in Candida albicans. However, a high concentration of IAA was found to inhibit cell growth. In 2011, Thailand imported about 6.3 million tons (93,844 million baht) of agrochemicals (including plant growth regulator) which is about a 2-fold increase from that imported in 2006 (http://www.nic.go.th/gsic/ uploadfile/Chemical.pdf). This indicates that the demand
*Corresponding authors: Nantana Srisuk and Savitree Limtong, Department of Microbiology, Faculty of Science, Kasetsart University, Chatuchak, Bangkok 10900, Thailand. Tel: +66 2 5625444 Fax: +66 2 5792081 E-mail:
[email protected];
[email protected] None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work.
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for agrochemicals has been increasing and continues to grow in recent years. As a result, the production cost of agricultural crop has increased. Synthetic IAA is expensive and less stable than synthetic auxin analogs such as indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) (Nissen and Sutter, 1990). Therefore IBA and NAA are widely imported and used in horticulture rather than IAA. However, the uses of synthetic chemicals have now been found to cause the toxic contamination of soil, natural water reservoir, air and, finally, crops. This is obviously due to the use of chemicals in large quantities, and for long periods of time, decreasing soil quality which therefore becomes unsuitable for re-plantation. An alternative to synthetic IAA is the use of biosynthetic IAA, which may be more stable than synthetic IAA. This is due to the fact that the most IAA found in plants and Pseudomonas savastanoi pv. savastanoi (Glass and Kosuge, 1986, 1988) are in conjugated form (IAA-linked to sugar or amino acid). The roles of the IAA-conjugated form are involved in the transport, storage and protection of IAA from enzymatic degradation (Cohen and Bandurski, 1982). However, the optimization of IAA production in plants is more difficult and slower than in microorganisms. The biosynthetic IAA production by microorganisms is therefore of interest to investigate. IAA biosynthesis pathways in bacteria and plants have a high degree of similarity, although some intermediates can differ. Two major pathways for IAA biosynthesis have been proposed, such as tryptophan-independent and tryptophan-dependent pathways. The indole-3-pyruvic acid, indole-3-acetamide and indole-3-acetonitrile pathways were considered as main IAA biosynthesis through a tryptophan-dependent pathway (Duca et al., 2014). IAA can be produced by various kinds of microorganisms, such as bacteria, fungi, yeast and algae. Research on the IAA-producing capacity of yeasts have been conducted for a long time, i.e. Pichia spartinae (Nakamura et al., 1991), Candida valida, Rhodotorula glutinis and Trichosporon asahii (El-Tarabily, 2004), Cyberlindnera (Williopsis) saturnus (Nassar et al., 2005), Rhodotorula graminis and Rhodotorula mucilaginosa (Xin et al., 2009), Candida tropicalis (Amprayn et al., 2012), Candida maltose (Limtong and Koowadjanakul, 2012), Cryptococcus sp. (Deng et al., 2012) and Rhodosporidium fluviale (Limtong et al., 2014). The large-scale production of fermentation products by yeasts is well-developed. The presence of a thick cell wall protects yeast cells from physical damage during fermentation (Johnson and EchavarriErasun, 2011). In addition, yeast cells harvest technology has been shown to be simpler than that used for bacteria, due to the large cell size of yeast. Therefore, yeast is one of the more interesting IAA producers. Furthermore, only a few reports on the optimization of yeast IAA production have been published. This study, therefore, investigates the optimization of IAA production in yeast by both the one factor at a time (OFAT) approach and the statistical (RSM) method. The factors previously described in bacteria (Apine and Jadhav, 2011; Balaji et al., 2012; Khamna et al., 2010; Patil et al., 2011; Sadeghi et al., 2012) were investigated to evaluate their effects on IAA production
by the red yeast Rhodosporidium paludigenum DMKURP301, the highest IAA producer obtained from the previous work of Nutaratat et al. (2014). Material and Methods The microorganism. Rhodosporidium paludigenum DMKU-RP301 (AB920314) was isolated from rice phyllosphere and grown on yeast extract peptone dextrose (YPD) agar (10 g/l yeast extract, 20 g/l peptone, 20 g/l dextrose and 15 g/l agar). Optimization of IAA production by Rhodosporidium paludigenum DMKU-RP301 using one factor at a time (OFAT). Yeast inoculum was prepared in the same manner throughout the study unless otherwise stated, i.e. 50 ml YPD culture was set up in a 250 ml Erlenmeyer flask and incubated on an orbital shaker (Jeio Tech, South Korea) at 150 rpm at 30°C for 16–18 hr. Inoculum was transferred into the 250 ml Erlenmeyer flask containing of 50 ml YPD broth amended with 0.1% (w/v) L-tryptophan. The medium initial pH was adjusted to 6, whereas an initial OD 600 was adjusted to 0.1 prior to incubation on an orbital shaker at 170 rpm at 30°C. Samples were taken daily for 9 days. The supernatant was collected by centrifugation for 5 min at 4427 × g. The supernatant was analysed for IAA by HPLC (Agilent Technologies, USA) equipped with a Cosmosil SC18-MS-II column (Nacalai Tesque, Japan) and UV detector (Agilent Technologies, USA) at 280 nm. Forty percent of solution A (methanol: acetic acid: water; 10:0.3:89.7 v/v/v) and 60% of solution B (methanol: acetic acid: water; 90:0.3:9.7 v/v/v) were used as a mobile phase with a flow rate of 0.5 ml/min as described by Kim et al. (2006); however, isocratic elution was used instead of gradient elution. Authentic IAA (Sigma, USA) was used as a standard. The effects of the media components and environmental conditions on IAA production were studied. Effects of various media components on IAA production were assessed by altering the medium composition accordingly. Carbon sources (arabinose, dextrose, fructose, galactose, glycerol, lactose, maltose, mannitol, mannose, my-inosital, raffinose, sorbitol, sorbose, starch, sucrose, xylitol and xylose) were used at 1%. Concentrations of the optimal carbon source (0.1, 0.5, 1, 1.5, 2 and 2.5%) were then investigated. Similarity, nitrogen sources (ammonium chloride, potassium nitrate, corn steep liquor and peptone) were used at 0.1% and optimum concentration was studied at 0.05, 0.1, 0.5, 1, 1.5 and 2%. Growth factors (beef extract, yeast extract and malt extract) were used at 0.1% and optimum concentration was studied at 0.05, 0.1, 0.5 and 1%. L-tryptophan concentration (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6%) was investigated. The medium initial pH was adjusted to 6, whereas an initial OD600 was adjusted to 0.1 prior to incubation on an orbital shaker at 170 rpm at 30°C. The supernatant was analysed for IAA. The effect of environmental conditions on IAA production was studied by cultivating yeast in the optimal medium of an initial pH adjusted to 4–7 and incubated on an orbital shaker at 100, 150 and 200 rpm at different temperatures (25, 30 and 35°C). The supernatant was analysed for IAA.
Indole-3-acetic acid production by newly isolated red yeast Rhodosporidium paludigenum Table 1.
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Effect of incubation time, carbon source and its concentration, nitrogen source and its concentration, growth factor and its concentration, tryptophan concentration, shaking speed, temperature and pH on IAA production by Rhodosporidium paludigenum DMKU-RP301. IAA is expressed as means ± standard deviations of values obtained from triplicate experiments.
Factors Incubation time (days) 1 2 3 4 5 6 7 8 9 Carbon sources Arabinose Dextrose Fructose Galactose Glycerol Lactose Maltose Manitol Mannose My-inositol Raffinose Sorbitol Sorbose Starch Sucrose Xylitol Xylose Sucrose concentration (%) 0.1 0.5 1 1.5 2 2.5 Nitrogen sources NH4Cl Corn steep liquor KNO 3 Peptone
IAA (mg/l) 0±0 21.2 ± 1.9 119.6 ± 10.3 301.7 ± 28.9 308.5 ± 21.0 303.5 ± 28.2 321.7 ± 15.1 294.0 ± 21.6 309.0 ± 7.1
103.3 ± 3.5 151.9 ± 14.4 165.4 ± 13.9 161.1 ± 9.1 13.9 ± 1.8 73.1 ± 5.5 54.0 ± 3.0 153.8 ± 14.3 166.7 ± 14.1 79.6 ± 6.1 123.2 ± 0.1 142.5 ± 13.2 47.5 ± 7.2 90.2 ± 5.0 201.6 ± 8.3 55.0 ± 1.6 120.8 ± 5.0
47.4 ± 3.4 157.3 ± 3.6 201.6 ± 8.3 58.0 ± 3.2 56.8 ± 2.2 56.4 ± 1.6
195.8 ± 8.3 314.8 ± 3.0 280.3 ± 5.7 271.0 ± 2.7
Optimization of IAA production using response surface methodology (RSM). Preliminary study on a combinatorial effect of nitrogen source and growth factor: Yeast inoculum was transferred into a test tube (16 × 150 mm) containing 5 ml of medium broth consisting of 1% sucrose amended with 0.1% (w/v) L -tryptophan. Various organic nitrogen compounds (peptone, tryptone, corn steep liquor, skimmed milk, soy isolate), inorganic nitrogen compounds (ammonium chloride, ammonium sulfate, potassium nitrate, sodium nitrite, urea, monosodium glutamate) and organic compounds contain-
Factors
IAA (mg/l)
Corn steep liquor concentration (%) 0.05
310.2 ± 14.9
0.10 0.5
314.8 ± 13.3 271.6 ± 6.3
1.00
271.5 ± 0.3
1.50
270.6 ± 1.1
2.00
238.0 ± 1.6
Growth factor sources Beef extract
214.7 ± 7.1
Yeast extract Malt extract
304.6 ± 8.8 222.9 ± 7.6
Yeast extract (%) 0.05 0.10
236.2 ± 0.7 289.0 ± 3.9
0.30
387.1 ± 8.5
0.50 1.00
502.3 ± 11.6 565.4 ± 33.8
Tryptophan concentration (%) 0.10 0.20
465.7 ± 3.7 670.8 ± 4.8
0.30 0.40
861.3 ± 9.8 1,056.4 ± 6.1
0.50 0.60
1,014.8 ± 3.0 1,042.3 ± 12.5
Shaking speed (rpm) 100 150 200
953.2 ± 5.9 996.5 ± 6.5 1,194.6 ± 8.8
Temperature (°C) 25 30 35 pH 4 5 6 7
1,207.6 ± 5.9 1,287.9 ± 8.2 304.0 ± 9.4
983.8 ± 12.5 1,333.6 ± 11.1 1,360.5 ± 2.5 1,464.7 ± 4.5
ing growth factors (beef extract, malt extract and yeast extract) were screened (0.1% w/v) and their effects on IAA production were investigated. The initial OD600 was adjusted to 0.1 prior to incubation on a reciprocal shaker (MM-10, Taitec, Japan) at 180 strokes/min at 30°C for 7 days. The IAA in culture supernatants was analysed by HPLC. Screening of influence factors for IAA production by the Plackett-Burman experimental design: The PlackettBurman design was employed in this study to identify factors that affect IAA production by the red yeast R.
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paludigenum DMKU-RP301. Environmental factors, i.e. temperature, pH and shaking speed, and the consequence of medium components i.e. concentration of sucrose, yeast extract, NaCl and L-tryptophan, were investigated. Each factor was represented at two levels; –1 represented a low level and +1 represented a high level in 12 treatments (Plackett and Burman, 1946). All experiments were carried out in 250 ml Erlenmeyer flasks containing 50 ml of medium, and in triplicate. Culture supernatants were analysed for IAA by HPLC. The data obtained were analysed by SPSS software version 16.0 (SPSS Inc., USA) to assess the influence of the factors on IAA production. Once the significant factors were sought, the next step was to optimize the actual values of these process factors by the Box-Behnken design (BBD). Optimization for IAA production by the Box-Behnken design: The Box-Behnken design was used for the investigation of the interaction between the influencing factors and optimization of IAA production. The influenced factors were selected for the study with each factor represented at three levels; –1 represented a low level, 0 represented a middle level and +1 represented a high level in 54 treatments (Box and Behnken, 1960). All experiments were carried out in 250 ml Erlenmeyer flasks containing 50 ml of medium in triplicate. Supernatants were analysed by HPLC. The data was subjected to multiple linear regression analysis using SPSS software version 16.0 and Statistica Software version 5.0 (StatSoft Inc, USA). The model coefficients and their standard errors were calculated and the model obtained was tested for lack-of-fit using the SPSS program. Then, a contour plot was drawn to explain the interactions of the variables on IAA yield, and determine their optimum concentrations for maximum IAA production using Statistica Software version 5.0. Finally, the model and values obtained were validated in triplicate. After optimization of IAA production in shaking flask culture, a 1.5 L working volume of optimal medium was used for IAA production in a 2-l stirred tank fermenter (BIOSTAT B, B. Braun Biotech International, Germany). The fermenter was operated at a temperature of 30°C, agitation speed of 200 rpm, aeration at 3.0 l/min (2 vvm), and the pH was uncontrolled during fermentation. Samples were taken daily for 7 days. The supernatant was collected by centrifugation and analysed for IAA by HPLC. Results and Discussion Optimization of IAA production by Rhodosporidium paludigenum DMKU-RP301 using OFAT When cultivated in YPD broth supplemented with 0.1% L -tryptophan, R. paludigenum DMKU-RP301 started to produce IAA (21.2 mg/l) after the second day of incubation and rapidly increased (301.7 mg/l) after the fourth day until the maximum IAA (321.7 mg/l) was reached after seven days incubation (Table 1). The result was consistent with Xin et al. (2009) that the maximum IAA production by the yeast Rhodotorula graminis WP1, Rh. mucilaginosa PTD2 and Rh. mucilaginosa PTD3 were obtained when cultivation proceeded to the seventh day and declined to 309.8 mg/l at the eighth day of incubation. The decline of IAA production was due to the re-
Fig. 1. Preliminary study on the combined effects of nitrogen source and growth factor on IAA production by Rhodosporidium paludigenum DMKU-RP301. The error bars represent standard deviations of three replicated assays.
lease of IAA degrading enzymes such as IAA oxidase and peroxidase (Datta and Basu, 2000). Xin et al. (2009) indicated that Rh. graminis WP1 showed the highest IAA production as 39 mg/g dry cell when grown in YPD broth supplemented with 0.1% L-tryptophan. However, it should be noted that the yeast R. paludigenum is a perfect stage of the yeast Rh. graminis, and their ability to produce IAA was therefore compared. Our results indicated that R. paludigenum DMKU-RP301 produced a higher amount of IAA (53.6 mg/g dry cell) than that reported for Rh. graminis WP1. Among all the carbon sources studied, the highest amount of IAA (201.6 mg/l) was observed when sucrose was used. However, other carbon sources, i.e. mannose, fructose, galactose, mannitol and dextrose, also resulted in relatively high amounts of IAA, viz. 166.7, 165.4, 161.1, 153.8 and 151.9 mg/l, respectively, under the conditions studied. Sucrose was therefore chosen as the best carbon source for IAA production by R. paludigenum DMKURP301, as it gave the highest level of IAA and is a lowcost substrate compared with the others. Subsequently, the optimum sucrose concentration was studied and the highest amount of IAA (201.6 mg/l) was observed when 1% sucrose was used (Table 1). Among all the nitrogen sources tested, R. paludigenum DMKU-RP301 exhibited the highest IAA production (314.8 mg/l) when corn steep liquor, a low-cost substrate, was supplied as a nitrogen source. Corn steep liquor was therefore selected as the best nitrogen source for IAA production by R. paludigenum DMKURP301 and a concentration of 0.05% was found to be optimal for IAA production (310.2 mg/l) by this yeast under the conditions studied. Ammonium chloride, peptone and potassium nitrate showed a lower amount of IAA production of 195.8, 271.0 and 280.3 mg/l, respectively. It may be noted that a slightly higher IAA production (314.8 mg IAA/l) was found when yeast was grown in the medium supplemented with 0.1% corn steep liquor (Table 1). As no significant difference of IAA yield was found between the two concentrations, we therefore selected 0.05% corn steep liquor for IAA production by R. paludigenum DMKU-RP301 to reduce the production cost. Among all the growth factors tested, the highest IAA production (304.6 mg/l) was observed when an optimal concentration of 1% yeast extract was supplemented into a culture
Indole-3-acetic acid production by newly isolated red yeast Rhodosporidium paludigenum Table 2.
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Analysis of variance (ANOVA) of Plackett–Burman design for IAA production by Rhodosporidium paludigenum DMKU-RP301.
Source
Type III Sum of squares
Corrected model Intercept Temperature NaCl L-tryptophan pH Shaking speed Yeast extract Sucrose Error Total Corrected total
Degree of freedom
507336.04* 1849138.18 441.05 2735.82 98712.44 64013.72 70562.47 215780.31 55090.24 13242.44 2369716.65 520578.47
7 1 1 1 1 1 1 1 1 4 12 11
Mean square
F-value
p-value
72476.58 1849138.18 441.05 2735.82 98712.44 64013.72 70562.47 215780.31 55090.24 3310.61
21.89 558.55 0.13 0.83 29.82 19.34 21.31 65.18 16.64
0.005 0.000 0.734 0.415 0.005 0.012 0.010 0.001 0.015
*R2 = 0.98 (adjusted R 2 = 0.93).
Table 3.
Model Regression Residual Total Lack of fit Pure error
Analysis of variance (ANOVA) in the regression model for optimization of IAA production by Rhodosporidium paludigenum DMKU-RP301.
Sum of squares
Degree of freedom
Mean square
F-value
p-value
1.80E7 1867290.01 1.99E7 1286787.32 98880.70
27 134 161 36 5
666222.92 13935.00
47.81
0.000
35744.09 19776.14
1.81
0.265
medium, which yielded 565.4 mg of IAA/l. A lower production of IAA (214.7 and 222.9 mg/l) was observed when beef extract and malt extract were used, respectively. Therefore, 1% yeast extract was selected for further studies (Table 1). Moreover, R. paludigenum DMKU-RP301 produced the highest amount of IAA (1,056.4 mg/l) with 0.4% L-tryptophan supplementation. We observed that a higher concentration of L-tryptophan (0.5 and 0.6%) did not increase IAA production by this yeast (Table 1). Results indicated that optimal tryptophan concentration improved IAA yield up to 3.3 fold of the preliminary observed level when R. paludigenum DMKU-RP301was cultivated in YPD supplemented with 0.1% L-tryptophan, or up to 1.9–2.3 fold when compared with the production in the corn steep liquor-sucrose-yeast extract broth supplemented with 0.1% L-tryptophan. This suggested, therefore, that L-tryptophan had a significant importance to IAA production by R. paludigenum DMKU-RP301. R. paludigenum DMKU-RP301 showed the highest amount of IAA (1,194.6 mg/l) when cultivated using an orbital shaker with a shaking speed of 200 rpm. However, it was found that lower shaking speeds (100 and 150 rpm) resulted in slightly lower levels of IAA (953.2 and 996.5 mg IAA/l, respectively) compared to the level observed at 200 rpm (Table 1). Statistical analysis showed a significant difference between IAA production at 200 rpm and the two lower speeds. Thus, we note that shaking speed had a slight effect on IAA production by R. paludigenum DMKU-RP301 under all other conditions studied here. IAA production of R. paludigenum DMKU-RP301 grown in 1% sucrose—0.05% corn steep liquor—1% yeast extract broth supplemented with 0.4% L-tryptophan under different incubation temperatures were compared and the
Table 4.
Regression analysis of the second order polynomial model for IAA production by Rhodosporidium paludigenum DMKU-RP301.
Model
Coefficient
Standard error
t-value
p-value
−8272.78 546.17 −141.22
2954.76 193.36 1502.51
−2.80 2.83 −0.09
0.006 0.005 0.925
X3 X4 X5
133.97 15.04 −551.62
214.26 5.06 663.53
0.63 2.98 −0.83
0.533 0.003 0.407
X6
−379.47 −9.74 −1140.96
298.29 3.40 531.27
−1.27 −2.86 −2.15
0.206 0.005 0.034
−25.55 −0.02
9.45 0.01
−2.71 −1.84
0.008 0.068
−977.32 5.06 74.18 3.17 −0.34 56.17 −0.67
104.94 21.25 48.19 6.43 0.14 21.42 9.64
−9.31 0.24 1.54 0.49 −2.52 2.62 −0.07
0.000 0.812 0.126 0.622 0.013 0.010 0.945
43.92 −2.97 1741.22 −417.47 −0.52
80.32 2.41 189.32 120.48 0.32
0.55 −1.23 9.20 −3.47 −1.60
0.585 0.220 0.000 0.001 0.111
72.09 60.17 1.74 0.82 −173.14
35.70 11.36 1.07 0.48 53.55
2.02 5.30 1.63 1.70 −3.23
0.045 0.000 0.106 0.091 0.002
(Constant) X1 X2
X 12 X 22 X 32 X 42 X 52 X 62 X 1X 2 X 1X 3 X 1X 4 X 1X 5 X 1X 6 X 2X 3 X 2X 4 X 2X 5 X 2X 6 X 3X 4 X 3X 5 X 3X 6 X 4X 5 X 4X 6 X 5X 6
X 1 = Temperature, X 2 = L-tryptophan, X3 = pH, X 4 = Shaking speed, X 5 = Yeast extract, X6 = Sucrose.
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highest amount of IAA (1,287.9 mg/l) was observed when the yeast was cultivated at 30°C. A lower level of IAA (1,207.6 mg/l) was found when the culture was incubated at 25°C (Table 1), whereas at 35°C incubation, R. paludigenum DMKU-RP301 produced the lowest amount of IAA as a result of a decrease in yeast growth. It could thus be concluded that cultivation at 30°C was preferable for growth, and for IAA production of R. paludigenum DMKU-RP301. Finally, we found that R. paludigenum DMKU-RP301 produced the highest amount of IAA (1,464.7 mg/l) when cultivated in a 1% sucrose—0.05% corn steep liquor—1% yeast extract broth supplemented with 0.4% L-tryptophan and adjusted to an initial pH of 7, although the yeast generally grew well in slightly acidic conditions (pH of 5–6). At an initial pH of 4, R. paludigenum DMKU-RP301 grew poorly and resulted in a low level of IAA production (Table 1). The overall results of OFAT optimization of IAA production by R. paludigenum DMKU-RP301 revealed that the optimal medium compositions and culture conditions obtained in this study increased the IAA production up to 4.6-fold, compared to the level observed in YPD broth supplemented with 0.1% L-tryptophan. In addition, the maximum amount of IAA found in this study was higher than that reported in some bacteria, such as Acetobacter diazotrophicus L1 (26.3 mg/l) (Patil et al., 2011), Pantoea rodasii (229 mg/l) (Walpola et al., 2013) or actinomycetes such as Streptomyces viridis CMUH009 (300 mg/l) (Khamna et al., 2010) and S. atrovirens (about 190 mg/l) (Abd-Alla et al., 2013). However, the amount of IAA produced by R. paludigenum DMKU-RP301 was still lower than that produced by Pantoea agglomerans PVM (2,191 mg/l) under its optimized media components and growth conditions (Apine and Jadhav, 2011). Optimization of IAA production by Rhodosporidium paludigenum DMKU-RP301 using RSM Preliminary study on the combined effects of nitrogen source and growth factor. Some organic nitrogen sources used for yeast cultivation could be a source of growth factors that best promote yeast growth and cell activities. The combined effect of various nitrogen sources and growth factors contained inside were evaluated with respect to the IAA production of R. paludigenum DMKU-RP301. Results indicated that three types of nitrogen source, i.e. potassium nitrate, yeast extract and tryptone, promoted IAA production to 257.9 mg/l, 281.8 mg/l and 260.5 mg/l, respectively (Fig. 1). However, when production cost is important, an expensive nitrogen source such as tryptone may not be a desirable choice. To minimize production cost a cheaper nitrogen source such as potassium nitrate was determined. Although potassium nitrate is cheap and resulted in a high IAA concentration (257.9 mg/l) it supplied only nitrogen and potassium sources. In contrast with potassium nitrate, yeast extract is more expensive, but the highest level of IAA production (281.8 mg/l) was obtained when yeast extract was supplied to a yeast culture. This may be due to the versatile organic contents of yeast extract, such as vitamins and growth factors that support yeast growth. In addition, yeast extract also increase tryptophan availability to IAA production by yeast. Moreover,
when the nitrogen content in 0.1% potassium nitrate, 0.1% yeast extract and 0.1% tryptone were compared, it was found that yeast extract gave the highest nitrogen content. Yeast extract was therefore selected as the desired choice of nitrogen and growth factor. Ammonium chloride, ammonium sulfate and urea were found not preferable for IAA production by R. paludigenum DMKU-RP301 as a lower level of IAA was observed. The present results are consistent with the report of Trotsenko et al. (2001), which showed that indole synthesis was strongly inhibited by ammonium ions. In addition, the substitution of ammonium sulfate with potassium nitrate led to a 2- to 15-fold increase in the rate of indole synthesis. This is possibly due to the competition of ammonium ions with the amino groups of tryptophan that are eliminated during the biosynthesis of indoles (Trotsenko et al., 2001). In a comparison of yeast extract and corn steep liquor, yeast extract resulted in a higher IAA yield than corn steep liquor. However, when corn steep liquor was used (as a nitrogen source) in combination with yeast extract (as a source of growth factor), a slightly higher IAA (304.6 mg/l) was observed, whereas an IAA production of 281.8 mg/l was shown when yeast extract was solely used. To reduce the production cost, we therefore decided to use yeast extract as both sources of nitrogen and vitamin or growth factor for IAA production by R. paludigenum DMKU-RP301. Evaluation of influencing factors for IAA production by Plackett-Burman experimental design. Factors that influenced IAA production were evaluated using the PlackettBurman experimental design. Results showed a wide range of IAA production, i.e. 116.8 ± 11.0 to 856.5 ± 11.3 mg/l. Experimental results were analysed and are summarized in Table 2. These reveal that sucrose, yeast extract and Ltryptophan concentrations, pH and shaking speed significantly (p ≤ 0.05) affected IAA production, whereas temperature and NaCl showed a non-significant effect on IAA production by R. paludigenum DMKU-RP301. However, we re-investigated the effect of temperature on IAA production by incubating a culture medium at different temperatures (24, 25, 26, 27, 28, 29, 30, 31 and 32°C). The result indicated that temperature had an influence on IAA production (data not shown). In the case of NaCl, increased salt stress in the medium was shown to reduce the production of IAA and the utilization of IAA precursor ( Ltryptophan) (Apine and Jadhav, 2011). Therefore, the study on the optimization of IAA production by R. paludigenum DMKU-RP301 focused on six factors (sucrose, yeast extract and L-tryptophan concentrations, pH, temperature and shaking speed) and the Box-Behnken experimental design was used. Optimization of IAA production using the Box-Behnken design. The average IAA production from 68.0 ± 3.6 to 1,602.9 ± 11.1 mg/l was obtained from the Box-Behnken experimental design. The experimental data were subjected to multiple linear regressions as shown in Table 3. The pvalue for the model (p = 0.000) and for lack-of-fit (p = 0.265) suggest that the obtained experimental data fitted well with the model. The regression equation obtained after ANOVA also suggested that IAA production is a function of tempera-
Indole-3-acetic acid production by newly isolated red yeast Rhodosporidium paludigenum
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Fig. 2. Interaction of X 1 (temperature) and X 4 (shaking speed) (A), X1 (temperature) and X 5 (yeast extract) (B), X 2 ( L-tryptophan) and X 5 (yeast extract) (C), X2 (L-tryptophan) and X6 (sucrose) (D), X 3 (pH) and X 5 (yeast extract) (E), X 3 (pH) and X 6 (sucrose) (F) and X 5 (yeast extract) and X 6 (sucrose) (G) on IAA production by the yeast Rhodosporidium paludigenum DMKU-RP301 using Box-Behnken experimental design.
ture, L-tryptophan concentration, pH, shaking speed and yeast extract and sucrose concentrations (Table 4). The final response equation that represented a second-order polynomial model for IAA production is as follows: Y = –8272.78 + 546.17X1 – 141.22X2 + 133.97X3 + 15.04X4 – 551.62X5 – 379.47X6 –9.74X12 – 1140.96X22 – 25.55X32 – 0.02X42 – 977.32X52 + 5.06X62 + 74.18X1X 2 + 3.17X1X3 – 0.34X1X4 + 56.17X1X5 – 0.67X1X6 + 43.92X2X3 – 2.97X2X4 + 1741.22X2X5 – 417.47X2X6 – 0.52X3X4 + 72.09X3X5 + 60.17X3X6 + 1.74X4X5 + 0.82X4X6 – 173.14X5X 6, where Y is indole-3-acetic acid (mg/l), X1 is temperature,
X2 is L-tryptophan concentration, X3 is pH, X4 is shaking speed, X5 is yeast extract concentration and X 6 is sucrose concentration. R2 is the coefficient of response variance under test and whose values are always between 0 and 1. The more closely the value of R2 is to 1, the stronger the statistical model and the better response prediction obtained (Myers and Montgomery, 1995). However, a regression model with an R2 value higher than 0.9 could be considered as having a high correlation (Gao et al., 2009). The model obtained in this work showed the determination coefficient value (R2) of 0.9060 for IAA production, indicating that the constructed statistical model could explain up to 90.60% of variability in the response. The adjusted deter-
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mination coefficient value (adjusted R2) corrects the R2 value for the sample size and for the number of terms in the model (Cochran and Cox, 1957). An adjusted R2 of 0.8870 indicates a high degree of correlation between the observed and predicted values, and supports the significance of this developed model. For IAA production by R. paludigenum DMKU-RP301, terms of linear (X1 and X4), quadratic (X12, X22, X32 and X52) and interactions (X1X 4, X1X5, X2X5, X2X6, X3X5, X3X6 and X5X6) were found to be a significant model. A contour plot was drawn to illustrate the interactions between the variables (temperature, L-tryptophan concentration, pH, shaking speed and yeast extract and sucrose concentration) on IAA yield. Variables were determined on their optimum values to maximize IAA production of the red yeast R. paludigenum DMKU-RP301 (Fig. 2). The terms X1X4, X1X 5, X2X5, X2X6, X3X5, X3X6 and X 5X6, indicated interactions of temperature and shaking speed (Fig. 2A), temperature and yeast extract (Fig. 2B), L-tryptophan and yeast extract (Fig. 2C), L-tryptophan and sucrose (Fig. 2D), pH and yeast extract (Fig. 2E), pH and sucrose (Fig. 2F) and yeast extract and sucrose (Fig. 2G), respectively. These variable interactions were also shown to significantly (p ≤ 0.05) affect the IAA production by the yeast R. paludigenum DMKU-RP301 (Table 4). The results showed that a high level of temperature, L-tryptophan, pH and yeast extract enhanced the yield of IAA. In contrast, a low level of shaking speed and sucrose enhanced the yield of IAA. A high level of temperature, L-tryptophan concentration, pH and yeast extract concentration, but low levels of shaking speed and sucrose concentration are considered to be the optimal conditions for the production of IAA by R. paludigenum DMKU-RP301. This result agrees with that reported by Ona et al. (2005) who found that limitation of the carbon source induced growth arrest required for IAA biosynthesis. As IAA is a secondary metabolite, a similar feature of secondary metabolites produced by microorganisms was therefore exhibited as the carbon limit condition and reductions in growth rate. To obtain an optimum IAA production, the temperature, concentrations of L -tryptophan, pH, shaking speed, concentrations of yeast extract and sucrose were therefore kept as 29.75–30.5°C, 0.45– 0.55%, 7.2–7.5, 90–100 rpm, 0.85–1.2% and 0.8–0.9%, respectively. Validation of the experimental model was confirmed when the influenced variables were kept as 0.9% (sucrose concentration), 0.9% (yeast extract concentration), 0.45% (L-tryptophan concentration), 7.2 (pH), 30°C (temperature) and 100 rpm (shaking speed). The results show that the observed value (1,623.9 ± 4.4 mg/l) of IAA produced was close to the predicted value (1,652.5 mg/l), which proves the validity of the model. The comparison of predicted and observed values show a good correlation between them, implying that the empirical model derived from RSM can be used to adequately describe the relationship between the influenced factors and IAA production. Maximum IAA production of 1,623.9 mg/l was obtained using the optimized culture condition, i.e. a medium containing 0.9% sucrose, 0.9% yeast extract, 0.45% L-tryptophan, adjusted pH to 7.2, and the culture was incubated at 30°C for 7 days on an orbital shaker at 100 rpm. These optimal
Fig. 3. Profiles of IAA yield (mg/l), cell growth (OD600) and pH in batch fermentation by Rhodosporidium paludigenum DMKURP301 in a 2-l stirred tank fermenter. The fermenter was operated at a temperature of 30°C, agitation speed of 200 rpm, aeration at 3.0 l/min (2 vvm), and the pH was uncontrolled during fermentation. Error bars represent the mean standard error for three determinations.
mediums and conditions increased IAA production by R. paludigenum DMKU-RP301 up to about 5-fold when compared to the production in YPD broth supplemented with 0.1% L-tryptophan, adjusted pH to 6 and with the culture incubated at 30°C using an orbital shaker at 170 rpm. In addition, the maximum amount of IAA was found after optimization using RSM (1,623.9 mg/l). When the production of IAA was scaled up in a 2-l stirred tank fermenter, the maximum IAA production was 1,627.1 mg/ l with a productivity of 9.7 mg/l/h at 168 h cultivation (Fig. 3). This result indicated that the optimal medium components and growth conditions (pH and temperature) obtained from RSM under shaking flask cultivation can be applied for IAA production in a larger production volume. Fed-batch fermentation will be of interest to perform and present as a further study. Acknowledgments This work was supported by a Thailand Research Fund/TRF ResearchTeam Promotion Grant (RTA 548009) under the title “Biodiversity and ecology of endophytic and epiphytic yeasts from leaves of agronomic crops in Thailand and production of plant growth promoting auxins by the selected promising strain with the elucidation of its biosynthetic pathway”. References Abd-Alla, M. H., El-Sayed, E. A., and Rasmey, A. M. (2013) Indole-3acetic acid (IAA) production by Streptomyces atrovirens isolated from rhizospheric soil in Egypt. J. Biol. Earth. Sci., 3, 182–193. Amprayn, K-o., Rose, M. T., Kecskes, M., Pereg, L., Nguyen, H. T. et al. (2012) Plant growth promoting characteristics of soil yeast (Candida tropicalis HY) and its effectiveness for promoting rice growth. Appl. Soil. Ecol., 61, 295–299. Apine, O. A. and Jadhav, J. P. (2011) Optimization of medium for indole-3-acetic acid production using Pantoea agglomerans strain PVM. J. Appl. Microbiol., 110, 1235–1244. Balaji, N., Lavanya, S. S., Muthamizhselvi, S., and Tamilarasan, K. (2012) Optimization of fermentation condition for indole acetic acid production by Pseudomonas species. Int. J. Adv. Biotechnol. Res.,
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