AIP Conference Proceedings 2563, 080001 (2022); https://doi.org/10.1063/5.0103220 2563, 080001
© 2022 Author(s).
Streptomyces hygroscopicus subspecies hygroscopicus strain I18: Incubation time and tryptophan concentration effects
on Indole-3-acetic acid (IAA) hormone production
Cite as: AIP Conference Proceedings 2563, 080001 (2022); https://doi.org/10.1063/5.0103220 Published Online: 31 October 2022
Mia Fitriani, Achmad Arifiyanto, Sumardi, et al.
Streptomyces hygroscopicus Subspecies hygroscopicus Strain I18: Incubation Time and Tryptophan Concentration Effects on Indole-3-Acetic Acid (IAA) Hormone Production
Mia Fitriani
1, Achmad Arifiyanto
1, a), Sumardi
1, Martha Lulus Lande
1, Christina Nugroho Ekowati
1, Titik Nur Aeny
2, Hapin Afriyani
31Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Lampung, Jl. Prof. Dr. Ir. Sumantri Brojonegoro No.1, Rajabasa, Bandar Lampung, Lampung 35141, Indonesia
2Department of Plant Protection, Faculty of Mathematics and Natural Sciences, Universitas Lampung, Jl. Prof. Dr. Ir. Sumantri Brojonegoro No.1, Rajabasa, Bandar Lampung, Lampung 35141, Indonesia
3Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Lampung, Jl. Prof. Dr. Ir. Sumantri Brojonegoro No.1, Rajabasa, Bandar Lampung, Lampung 35141, Indonesia
a)Corresponding author: [email protected]
Abstract. Plant hormones are chemical messengers that regulate a wide range of physiological activities in plants. Microbes need a definite time to synthesize a compound. The microbial life cycle consists of several phases, often requiring a shorter incubation time than in complex organisms such as plants, animals, and humans. This study aimed to determine the optimum incubation time and the best tryptophan levels of Streptomyces hygroscopicus subsp. hygroscopicus strain i18 in Indole-3-acetic acid (IAA) hormone. IAA has synthesized on liquid ISP4 medium using L-tryptophan supplementation as a precursor. Cell density was measured every day for 4-11 days using the spectrophotometric approach to track the incubation time. The doses of L-Tryptophan were 0, 1, 2, 3, 4, 5, and 6 mg/mL, respectively. The levels of IAA produced by bacteria were measured and compared with standard IAA. Streptomyces hygroscopicus subsp. hygroscopicus strain i18 was obtained the optimum incubation time at 9th days with an IAA level of 12.30 μg/mL. Supplementing with 5 mg/mL L-tryptophan resulted in the highest IAA level of 17.90 μg/mL. This study would be worthwhile in assisting agricultural product quality improvement through field application for further research.
INTRODUCTION
Plant hormones have required as chemical messengers that impact a plant's ability to respond to the environment and speed up its growth [1,2]. Rhizosphere bacteria can create plant hormones, also known as phytohormones. Because they dwell in plant roots, these bacteria include plant growth promoter rhizobacteria (PGPR). They live in colonies and aid in the growth of plants as well as the prevention of illness. Auxin-producing rhizobacteria include Actinomycetes, Azospirillum, Rhizobium, Pseudomonas, Bacillus, Micrococcus, Kocuria, and Enterobacter [3].
Streptomyces was a soil bacteria member of Actinomycetes whose capable of producing plant hormones, including Indole-3-acetic acid (IAA) [4]. This hormone belongs to the auxin family and served as a signaling molecule in plant development, root elongation, and enzyme activation. It improved nutrient intake by developing longer roots and increased lateral root hairs. It also has a significant impact on plant development[5].
Several factors, such as incubation duration and Tryptophan content, can alter the amount of IAA generated by Actinomycetes [6]. In maize and peas, supplementing precursors in the form of Tryptophan to the environment enabled seed germination and root elongation [7]. Streptomyces sp has potentially produced IAA that helped plant growth.
Streptomyces sp had generated biosurfactants and antimicrobial performances from the rhizosphere of the Sidoarjo mud area [8].
Streptomyces sp. strain i18 had identified as Streptomyces hygroscopicus subsp. hygroscopicus and was isolated from pineapple plantation soil [9]. Metabolite extracted from this strain contained saponins, triterpenoids, and
anthraquinone. It showed the potential to inhibit the growth of Plasmodium parasites [10]. Unfortunately, information regarding the ability of this strain to produce IAA was not yet available. This study intended to determine i18 bacteria' ability to produce IAA at the optimal time and the effect of the dose of tryptophan amino acid supplementation.
MATERIALS AND METHODS Bacterial culture
Bacteria were cultured on International Streptomyces Project medium 4 (Inorganic salts-starch) and incubated at room temperature for 4-7 days. The composition of the medium consisted of 10 g Soluble Starch, 1 g MgSO4 x 7H2O, 1 g NaCl, 2 g (NH4)2SO4, 2 g CaCO3, 1 L distilled water, 1 mL Trace Salts solution (0.1 g FeSO4 x 7H2O, 0.1 g MnCl2 x 4H2O, 0.1 g ZnSO4 x 7H2O, 100 mL distilled water), and 20 g agar. A loop of inoculum was taken and put in a liquid inorganic salts-starch medium, incubating for 7th days at room temperature for a 10 mL starter [11].
Incubation time
In 90 mL liquid inorganic salts-starch media, Tryptophan 2 mg/mL was added together with a 10 mL starter and shaken at 10,000 rpm in the dark. The production medium was taken 2 ml and centrifuged at 3000 rpm for 30 minutes.
The separated supernatant was taken 1 ml and reacted with 2 ml Salkowski solution, incubated for 60 minutes in the dark, and observed for a pink to red color change. It was measured on days 4-11 with a spectrophotometer with a wavelength of 530 nm. The data obtained were analyzed using ANOVA and BNT follow-up test [12].
Tryptophan supplementation
The starter was taken in 10 mL and placed in 90 mL of liquid inorganic salts-starch media with 1, 2, 3, 4, 5, and 6 mg/mL of Tryptophan, respectively. They had incubated on a shaker incubator for the optimum incubation time previously achieved. Culture media containing bacteria without Tryptophan was assigned as a control. The production medium was taken 2 ml and centrifuged at 3000 rpm for 30 minutes. One milliliter of the separated supernatant was mixed with two milliliters of Salkowski solution and incubated in the dark for 60 minutes. The data were analyzed using the non-parametric Kruskal-Wallis test after measurements using a spectrophotometer with a wavelength of 530 nm [13].
RESULTS AND DISCUSSIONS
The presence of IAA can be detected by reacting with the supernatant of the Salkowski reagent. The interaction between IAA and Fe causes free electrons from the ligand to fill the "d" orbital in Fe3+ with IAA, resulting in the formation of a complex bond [Fe2 (OH) 2 (IA)4]. The color change indicates the high content of IAA produced is proportional to the density of the red color. It had seen in Figure 1.
FIGURE 1. Red color change on IAA production with the addition of Tryptophan 0-6 mg/mL
During the incubation time of days 4-11, Strain i18 was adequate to produce the IAA hormone. The optimum incubation time for strain i18 occurred on day 9 with a value of 12.30 μg/mL. The culture began to enter the optimal production phase of IAA after day 7, Table 1.
TABLE 1. Production of IAA on days 4-11 by Streptomyces hygroscopicus subsp. hygroscopicus strain i18 Day IAA concentration (μg/mL)
4 3.90 ± 0.01
5 6.90 ± 0.09
6 8.00 ± 0.02
7 8.20 ± 0.09
8 8.60 ± 0.04
9 12.30 ± 0.10
10 11.00 ± 0.06
11 8.00 ± 0.10
Tryptophan administration on IAA production had a significant effect, indicated by the Asymp value. In a non- parametric test using the Kruskal Walis method, the Significant value is 0.008. The Tryptophan concentration of 5 mg/mL resulted in IAA levels of 17.90 g/mL, which was the highest among other Tryptophan dosing treatments (Table 2).
TABLE 2. Tryptophan supplementation effect at IAA content produced by Streptomyces hygroscopicus subsp. hygroscopicus strain i18
Tryptophan concentration (mg/mL) IAA concentration (μg/mL)
0 2.90 ± 0.01
1 8.60 ± 0.01
2 11.20 ± 0.01
3 14.80 ± 0.01
4 16.30 ± 0.06
5 17.90 ± 0.06
6 16.80 ± 0.03
The ability of rhizobacterial isolates to produce IAA was considered a beneficial method for identifying helpful microbes, and they have a significant impact on plant growth [14]. IAA was an inorganic-volatile compound produced by Streptomyces spp [15]. The production of secondary metabolites was the ability of each organism. Secondary metabolites biosynthesis began at the end of the logarithmic or exponential phase until the end of the stationary phase, after cell division and multiplication stop [16].
3 4 5 6 7 8 9 10 11 12
0 2 4 6 8 10 12
IAA concentration (ug/mL)
Incubation time (day)
IAA concentration (ug/mL)
FIGURE 2. The relation of incubation time to IAA concentration.
0 1 2 3 4 5 6 0
2 4 6 8 10 12 14 16 18 20
IAA concentration (ug/mL)
Tryptophan concentration (mg/mL)
IAA concentration (ug/mL)
FIGURE 3. The relation of tryptophan doses to IAA concentration.
IAA hormone production was increased by nutrient sources available during the incubation phase, while IAA production had decreased by nutrients that began to deplete during the incubation period [17]. Types of nutrients that influenced IAA production were carbon, nitrogen, and precursor type. The oxidase and peroxidase enzymes were released, which lowered the amount of IAA produced [18]. Thus, due to the availability of sufficient nutrients, there is a pattern of increases from the beginning of the 4th day to the peak on the 9th day (Figure 2).
TABLE 3. Summary of bacterial species, ideal incubation time, total incubation time, liquid medium, IAA levels produced by dosages, and L-tryptophan doses in a variety of comparative sources
No Strain
The peak period of output (day;
hour)
Duration of incubation
(days)
Liquid Media
Optimal IAA content
(μg/mL)
L -tryptophan optimal doses
(mg/mL) Sources 1
Streptomyces
fradiae NKZ-259 6; 144 14 Gause’s No.1 20.46 2 .00 [4]
2
Streptomyces hygroscopicus subsp.
hygroscopicus strain i18
9; 216 11 ISP 4 17.90 5.00 result This
3 Streptomyces sp. MS1 5; 120 7 Yeast
malt extract
(YM)
125.48 2 .00
[19]
4 Streptomyces sp. BR27 5; 120 7 104.13 1 .00
5 Streptomyces sp
VSMGT1014 5: 120 8 ISP 2 15.96 2.00 [20]
6 Streptomyces sp. PT2 5; 120 10 extract-Yeast
tryptone 3.63 5.00 [6]
At the beginning of the logarithmic stage, the yield of IAA produced was lower, presumably because the content of enzymes utilized to convert Tryptophan to IAA remained low (Figure 3). The yield of IAA produced was lower at the beginning of the logarithmic stage due to a lack of enzyme concentration and bacterial cells. In contrast, at the end of the logarithmic phase, IAA production entered following bacteria increasing growth. Monooxygenase, IAM hydrolase, indole pyruvate decarboxylase and IAAld dehydrogenase were utilized during Tryptophan bioconversion into IAA. They were produced quite a lot and were active in line with the growth rate [21].
Unfortunately, the cell density data at the time of the IAA measurement has not been measured in this study. These data help reveal that bacterial cell growth is closely related to the production of IAA as a metabolite. Table 3 informs us that the type of production media affects the amount of IAA produced. Likewise, the dose of tryptophan as a precursor in each strain of Streptomyces spp gave different results in producing IAA.
Tryptophan has been identified as a major precursor of IAA and plays an important role in modulating the level of IAA biosynthesis in bacteria. Tryptophan in the rhizosphere was obtained from the degradation of root cells, microbial cells, and root exudates. IAA produced by bacteria stimulates the growth of a plant. On the other hand, IAA controlled microbial antibiotic synthesized [22]. This growth triggers the exudation of root organic compounds which are used for the growth of these bacteria [23]. Tryptophan is used as a source of carbon and energy, which will break down into several compounds involved in metabolic pathways. This provides a mutually beneficial role between the bacteria and the plant itself [24].
CONCLUSION
On the 9th day of testing, with an IAA concentration of 12.30 μg/mL, Streptomyces hygroscopicus subsp.
hygroscopicus strain i18 produced the most IAA. The addition of 5 mg/mL of Tryptophan produced an IAA of 17.90 μg/mL as the optimum amount of Tryptophan. It was required to count the number of bacterial cells to assess the relationship between IAA production and cell growth, and as well as to quantify the amount of IAA tested with various strains for field applications.
ACKNOWLEDGMENT
We'd like to thank everyone who contributes to this research. This work was partly supported by grants from Universitas Lampung, Indonesia (1661/UN26.21/PN/2021).
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