1 Introduction

Indigo, traditionally extracted from Indigofera plants, is mainly used as a dye for denim fabric [1]. The growing demand for jeans has significantly increased the need for indigo dye, resulting in the widespread use of chemically synthesized indigo [2]. However, the production of synthetic indigo depends on petrochemicals and involves toxic reactants and catalysts, leading to considerable environmental pollution [3, 4]. Therefore, there is an increasing demand for carbon–neutral and environmentally friendly processes. Over time, several eco-friendly and bioprocess methods have been developed to replace synthetic indigo, with enzyme-mediated synthesis from biomass and bioresources being particularly notable [4,5,6,7].

The enzymatic production of indigo utilizes indole or tryptophan as substrates (Fig. 1A). Indole is converted to 3-hydroxyindole (indoxyl) through monooxygenase-mediated C-3 hydroxylation, a process facilitated by tryptophanase when tryptophan is used [7]. This indoxyl is then oxidized and dimerized to form indigo. Several enzymes, such as flavin monooxygenase, toluene monooxygenase, naphthalene dioxygenase, and cytochrome P450 monooxygenase, can catalyze the indole-3-oxidation process [8,9,10,11,12,13,14]. Additionally, indole-3-acetate monooxygenase has also been identified for this reaction [15]. However, scaling up the production of high-concentration indigo through enzymatic conversion remains challenging, and the use of recombinant strains falls under the regulatory policies for genetically modified organisms (GMOs), complicating commercialization efforts.

Fig. 1
figure 1

A Synthetic pathway of indigo from indole. B Isolation of Acinetobacter species from soil source on indole agar plates, demonstrating distinct blue coloration. C 16S rRNA sequence analysis for strain identification revealing the isolated strain as Acinetobacter sp., closely related to Acinetobacter oleivorans

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Another challenge in biological indigo synthesis is the natural production capabilities of wild-type strains. Strains such as Pseudomonas, Streptomyces, and Acinetobacter species possess monooxygenase enzymes in their genomes that can oxidize indole [15,16,17]. This capability is easily detected through the formation of blue-colored colonies when indole is used as a substrate [8, 9]. Our research group previously isolated an indole-converting microorganism from soil, identified as an Acinetobacter species, which produced up to 1.0 mg/L of indigo from 1 mM indole [15]. Sequence analysis revealed that the key enzyme involved in indole 3-oxidation was indole-3-acetate monooxygenase, encoded by the icaA gene. Heterologous expression of icaA resulted in a more than 20-fold increase in indigo production compared to the wild-type strain [15].

Despite these advancements, indigo production by wild-type strains has not exceeded 10 mg/L [15]. This low yield may be due to the intrinsic insolubility and toxic effects of indigo at high concentrations. Indigo tends to be highly insoluble, making it difficult to pass through the host membrane, often leading to truncation or aggregation between membranes [13]. Various strategies, including solubilization with detergents and reducing chemicals, have been employed to address this issue, achieving some success in increasing production yields [4]. However, there is still significant potential for improvement, particularly through host engineering strategies.

In this study, we isolated a new strain capable of converting indole into indigo from the soil and investigated its indigo production capabilities. The production varied with the nutrients present in the liquid culture. During incubation, the liquid culture exhibited abundant bubbling, identified as biosurfactant produced by Acinetobacter. We characterized the biosurfactant by measuring surface tension reduction and surfactant index. The study highlighted the relatively higher indigo production levels compared to other wild-type strains, considering the co-production of indigo and surfactant [18]. The strain's full genome was sequenced, and candidate genes responsible for indigo and biosurfactant production were identified.

Despite these promising results, several challenges remain for the industrialization of bio-based chemical production. Continuous research efforts are needed to transition from petroleum-based to bio-based eco-friendly methods. Specifically, the production of indigo and biosurfactants by microorganisms requires further advancements in host and process engineering [3, 4]. This study provides valuable insights into the co-production of biosurfactants with other valuable biochemicals, contributing to future advancements in this field.

2 Materials and methods

2.1 Bacterial strains and chemicals

Acinetobacter strains were sourced from soil samples collected from a farmland in Hamyang, Korea, following a fire incident. Indole, indigo, indirubin, ethyl acetate (EA), hexadecane, and dodecane were procured from Sigma-Aldrich Korea. M9 minimal media, Luria–Bertani (LB), and Terrific Broth were obtained from Difco™ (Difco Korea).

2.2 Strain isolation and gene annotation

The soil samples were diluted with autoclaved distilled water and allowed to settle for a day. Concurrently, M9 minimal medium agar plates supplemented with 1 mM indole were prepared. 0.1 mL of the diluted soil samples were spread on these agar plates and incubated at 30 °C for one day [15]. Blue colonies were visually identified, isolated, and streaked onto new agar plates to obtain individual colonies.

2.3 Production of indigo and biosurfactant

The initial inoculation was done in LB broth taking single colony from LB agar plate, followed by shaking at 200 rpm and 30 °C overnight. Subsequently, 1% (v/v) of the overnight culture was transferred into 25 mL portions of LB broth, M9 minimal media with 1% glucose, and M9 minimal media, each supplemented with 1 mM indole [4, 15]. These cultures were incubated for 24 h at 200 rpm and 30 °C. For biosurfactant analysis, the strain was incubated in LB medium for 24 h under the same conditions.

2.4 Extraction of indigo and biosurfactant

Samples were centrifuged at 8,500 rpm for 15 min at 4 °C. Indigo was extracted from the cells by vortexing with an equal volume of EA to the culture medium, followed by centrifugation at 8,500 rpm for 15 min at 25 °C [4, 15]. The blue supernatant was then dried in a fume hood. For the culture medium supernatant, the pH was adjusted to 2 using 6 M HCl and 5N NaOH, and incubated overnight at 4 °C. An equal volume of EA was added for extraction, followed by centrifugation at 8,500 rpm for 15 min at 25 °C. The supernatant was subsequently dried in a fume hood.

2.5 Quantitative analysis of the synthesized indigo dye

Indigo quantification was performed using high-performance liquid chromatography (HPLC) [4, 15]. The HPLC system, equipped with a UV detector and a reverse-phase C18 column (Zorbax Extend-C18; 250 mm × 4.6 mm, 3.5 µm, Agilent Technologies), utilized a gradient solvent system composed of water (solvent A), methanol (solvent B), and acetonitrile (solvent C) in a ratio of 19:80:1.

2.6 1H NMR, liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS/MS) analysis for structural identification of indigo

For qualitative analysis of indigo, both LC–ESI–MS/MS and a 600 MHz high-resolution NMR Spectrometer were used. LC–ESI–MS/MS analysis employed an Ultimate 3000 RS system (Thermo Fisher Scientific Inc.) with a binary gradient system. Chromatographic separation of the dye was carried out on a C18 column (Waters Cortecs C18; 2.1 mm × 150 mm, 1.6 µm) with a 2 µL injection volume. The solvents used were water with 0.1% formic acid and methanol with 0.1% formic acid. m/z values were detected using an LTQ mass spectrometer (Thermo Fisher Scientific Inc.). For 1H NMR spectra, an AVANCE 600 instrument was utilized.

2.7 Physical properties of biosurfactant by emulsifying activity and surface tension

The emulsifying activity of the biosurfactant was determined by calculating the emulsion index (E24) [18]. The cell-free supernatant was mixed with hexadecane in equal volumes and vortexed for 1 min. After 24 h at room temperature, the emulsification index (E24) was calculated using Eq. (1), dividing the height of the emulsion layer by the total height of the liquid column. Measurements were performed in triplicate, and results were expressed as mean ± standard deviation.

$$\begin{aligned}\text{Emulsification index E}24&=\frac{\text{Height of the emulsion layer}}{\text{Total height of the liquid column}}\\& \quad \times 100{\%}\end{aligned}$$
(1)

Surface tension of the supernatant was measured using a KSV Model Sigma 702 instrument (Biolin Scientific).

2.8 Dodecane biodegradability

For inoculum preparation, the strain was initially inoculated in LB broth and shaken at 200 rpm and 30 °C overnight. 1% (v/v) of the overnight culture was transferred to 100 mL of TB medium with 1 mM indole and incubated for 48 h at 200 rpm and 30 °C. After centrifugation (8500 rpm, 4 °C, 15 min), the cells were washed twice with 0.1 M potassium phosphate buffer (pH 7.0). Cell density was adjusted to OD 20, and cells were resuspended in the same buffer containing 1% (v/v) dodecane and 1 mM indole, then incubated at 30 °C and 200 rpm. Samples were collected at 0 and 24 h, vortexed with an equal volume of EA, and centrifuged. The organic solvent supernatant was analyzed using a 6500 GC gas chromatography system (Younglin, Anyang, South Korea), with separation on an Agilent J&W GC column (CP-Sil 8 CB amine, 30 m, 0.25 mm i.d., 0.25 μm film thickness). The column temperature was initially set at 130 °C, raised to 230 °C at 20 °C/min, and held for 3 min. Nitrogen served as the carrier gas at 2 mL/min flow, with a 10:1 split ratio. Injection and detection temperatures were set at 330 °C and 300 °C, respectively.

3 Results and discussion

3.1 Screening and isolation of indigo and biosurfactant co-producing bacteria

Microorganisms were screened based on colony color, specifically targeting those the colonies that exhibited a blue hue. The strains displaying blue hue coloration were isolated and re-cultivated on indole-containing plates to confirm their capability to convert indole (Fig. 1B). The isolated strain exhibited a pronounced dark blue color, and a single colony was selected for further examination. 16S rRNA sequencing revealed that this strain belongs to the Acinetobacter genus, closely related to Acinetobacter oleivorans (Fig. 1C).

3.2 Structural analysis of isolated blue coloration dye

The structure of the blue dye was identified using LC–ESI–MS/MS and 1H NMR analyses. LC–ESI–MS/MS results showed 5 to 6 major metabolites in the full scan chromatogram. A peak with a retention time of 30.2 min corresponded to a mass value of 263.5 m/z, matching the molecular weight of indigo (Fig. 2A). Comparative analysis with chemically synthesized indigo showed a mass value of 263.4 m/z and fragment ion patterns at 239.7 and 217.6 m/z (Fig. 2B), confirming that the indole metabolite produced by the isolated Acinetobacter sp. was indigo dye.

Fig. 2
figure 2

A Separation of indigo in LC–ESI–MS/MS analysis. The blue dye was observed at a retention time of 30.2 and showed an exact match with the standard indigo chemical. B Mass analysis of the blue dye showed a distinct 263.5 m/z value which corresponds well with that of the indigo standard chemical. C H-NMR analysis of the produced indigo. LC–ESI–MS/MS: liquid chromatography electrospray ionization mass spectrometry

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1H NMR analysis further confirmed the indigo structure (Fig. 2C). Peaks at 2.50 ppm and 2.52 ppm were associated with dimethyl sulfoxide (DMSO) solvent, while peaks in the ranges of 6–7 ppm and 10 ppm indicated resonances for the N–H in indole and a 4-spin AA’BB’ spectrum of indigo, respectively. The proton of the N–H in indigo appeared as a single peak at 10.5 ppm. Peaks at 7.51 ppm corresponded to protons H4 and H4' in the indole structure, and peaks at 7.28 ppm were attributed to protons H7 and H7', both doublets. Peaks at 7.47 and 7.02 ppm corresponded to protons H5, H5' and H6, H6', respectively, both triplets. These 1H NMR results were consistent with previously obtained data, confirming the blue dye as indigo synthesized by the wild-type Acinetobacter sp.

3.3 Production of indigo dye in M9 minimal and LB complex medium

The indigo production by wild-type Acinetobacter sp. was evaluated in indole-supplemented M9 minimal medium with and without 1.0% glucose, as well as in LB complex medium. The highest indigo production titer was observed at 24 h and gradually decreased thereafter (Fig. 3A). In LB medium, indigo production did not exceed 3.5 mg/L, with a yield of around 2.5%, whereas in M9 medium without glucose, the highest indigo production of 45.5 mg/L was achieved (Fig. 3B). The addition of glucose reduced indigo production by 8.9 mg/L, potentially due to lower indole uptake or repression of indole-3-oxidase expression despite the stable supply of NADPH from glucose metabolism. The blue coloration of the culture supernatant after centrifugation is shown in Fig. 4A (insert). Additionally, the cell pellet also exhibited blue coloration, suggesting partial secretion and intracellular accumulation of indigo.

Fig. 3
figure 3

A Time-dependent bio-indigo production profile by wild-type Acinetobacter strain. 1.0 mM indole was used for substrate. B Production of indigo using Acinetobacter strain from the biotransformation of 1.0 mM indole in M9 and LB medium

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Fig. 4
figure 4

A Culture broth of Acinetobacter sp. and bubbles observed during incubation. The pellet collected by centrifugation and supernatant after separation showed distinct indigo coloration. The biosurfactant was isolated from LB broth culture. B Measurement of emulsifying index by the generated Acinetobacter surfactant. C Dodecane degradation by isolated Acinetobacter strain

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3.4 Co-production of biosurfactant by isolated Acinetobacter sp. and biosurfactant characterization

In addition to indigo production, abundant foam formation in the culture broth suggested biosurfactant production (Fig. 4B). Previous studies have reported biosurfactant production by Acinetobacter strains isolated from petroleum-contaminated soils with production around 0.52 g/L [18, 19]. These biosurfactants can decrease surface tension, potentially affecting the production of hydrophobic metabolites by altering cell membrane permeability. The isolated Acinetobacter sp. reduced surface tension from 72.7 mN/m to 56.4 mN/m ± 5.3, and exhibited an emulsification index of 52.2 ± 7.5% in hexadecane (Fig. 4B). This strain exhibited higher indigo production compared to other wild-type strains, possibly due to simultaneous biosurfactant production, which could solubilize indole and indigo through micelle formation, facilitating indole uptake and indigo release via permeabilized membranes.

3.5 Alkane biodegradability by Acinetobacter sp.

The alkane biodegradability of the strain was investigated, given that biosurfactant-producing strains often degrade crude oil. A. oleivorans DR1, a closely related species, is known for degrading crude oil and alkanes with carbon chain lengths from C12 to C30 [20]. The Acinetobacter sp. successfully degraded 1% dodecane with 35.5% efficiency (Fig. 4C). During the whole-cell reaction, biosurfactants generated during growth were almost completely utilized, suggesting that the biosurfactant enhances alkane solubility, improving uptake and degradation by the strain.

3.6 Whole genome sequencing of isolated Acinetobacter sp. and candidate genes involved in indigo and surfactant synthesis

The whole genome of the strain was sequenced, identifying key genes potentially involved in surfactant production and alkane biodegradation (Fig. 5). Genes related to aminolipids (phosphate reductase, glutamate dehydrogenases) and glycolipids (trehalose phosphate synthase, phosphoenolpyruvate synthetase) were predicted (Table 1). Acinetobacter species typically produce rhamnolipids [21, 22]. According to a previous report, the biosurfactant produced by indigenous Acinetobacter junii was identified as a rhamnolipid type with C26H48O9, C28H52O9, and C32H58O13 as the dominant components [21].

Fig. 5
figure 5

Full genome sequencing was completed and genes were annotated completely

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Table 1 Genes involved in the bio-surfactant production in Acinetobacter strains
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Genes encoding alcohol dehydrogenases, alkane-1-monooxygenases, flavin-dependent oxidoreductases, and alkanesulfonate monooxygenases, potentially responsible for alkane degradation, were also predicted (Table 2). Enzymes such as indole-3-acetate monooxygenase and alkane 1-monooxygenase are being investigated through heterologous expression in Escherichia coli.

Table 2 Genes involved in the alkane metabolism of Acinetobacter
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4 Conclusion

The screening of Acinetobacter sp. capable of producing blue dye was performed using indole agar plates, identifying the strain's dual ability to produce indigo and biosurfactant. The wild-type Acinetobacter sp. strain produced approximately 6.8 mg/L of indigo from 1 mM indole in LB media, while in M9 minimal media, the yield significantly increased to 45.5 mg/L. A notable observation was the vigorous bubbling exhibited by the strain during growth, potentially facilitating the transport of indole and indigo dye, both of which have low solubility, across cell membranes. Additionally, the strain demonstrated the capability to degrade C12 medium chain alkane.

In conclusion, the utilization of a wild-type strain for indigo production and biosurfactant supply during biotransformation offers a promising platform for alternative chemical processes, addressing concerns related to GMOs in future applications. This finding holds significant potential for the large-scale production of indigo and biosurfactant, paving the way for commercialization in various biochemical production applications. [23].