Strains Isolation, Conservation and Characterization

The bacterial strain COS14-R2 obtained from the flower known as Cosmos bipinnatus. The flower was collected from Gimji-si, Jeollabuk-do, Republic of Korea. The strain morphology and color were identified using R2A agar (Difco, Maryland, USA). Subsequently, the samples were placed in a dark environment and incubated at 28 ± 1 °C for 7–14 days. The Gram staining procedure was conducted using an optical microscope (Leica DM2500, Germany). The stock culture was conserved using a 20% glycerol solution in R2A liquid media and stored at -80 ºC. Bacterial morphology was examined using scanning electron microscopy (SEM, Carl Zeiss Gemini SEM 300).

DNA Extraction and Genome Sequencing of COS14-R2

The pure bacterial strain COS14-R2 (~ 1 × 109) was used for DNA extraction (Fig. 1A). Genomic DNA was extracted through a Macrogen MG™ genomic DNA purification kit and quantified using a fluorescence-based approach. The concentration of pure genomic DNA measured by the NanoDrop spectrophotometer and Qubit fluorometer. At the same time, RNA integrity was assessed using an Agilent Technologies 2100 Bioanalyzer (Macrogen, Republic of Korea). For PacBio RS sequencing, 8 µg of genomic DNA was used to create a 20 kb library (Covaris Inc., Woburn, MA, USA) and purified with AMPure PB magnetic beads (Beckman Coulter Inc., Brea, CA, USA), resulting in an apparent size exceeding 40 kb. A total of 10 μL of the library was created utilizing the PacBio DNA Template Prep Kit 1.0 (designed for 3–10 kb). The subsequent steps are based on the PacBio sample Net-Shared protocol, accessible at http://pacificbiosciences.com/. Each sample was prepared using the Illumina methodology and sequenced utilizing the HiSeq™ 4000 platform (Illumina, San Diego, USA). The assembly was performed using the SMRT Portal (version 2.3) de novo assembler, and error correction was executed with Pilin (v1.21). The final step of annotating all relevant genomic characteristics on those Contigs can be executed using the rapid prokaryotic genome annotation pipeline (PROKKA). The 16S rRNA nucleotide sequence of strain COS14-R2, obtained from the whole-genome sequence, was compared for similarities against the reference genome database using NCBI microbial genome BLAST. The ten most prominent sequences from the search were chosen for pairwise comparison utilizing the EzBioCloud database. The phylogenetic tree was generated with maximum likelihood and neighbor-joining methods, employing a bootstrap value 1000 with MEGA7 software.

Fig. 1

Experimental strain morphology and nostoxanthin biosynthesis pathway. A Pure colonies of Sphingomonas sp. strain COS14-R2. B Scanning electron microscope image (SEM, × 10,000). C Simplified schematic representation of nostoxanthin biosynthetic pathway in Sphingomonas species. Presumptive pathways of carotenoid biosynthesis in strains of Sphingomonas is presented in a blue doted lined. Shorthand notations of enzymes are indicated in blue and red. β-carotene (whose biosynthesis is enabled by the presence of CrtE, CrtB, CrtI, and CrtY) appears to be the branch point for three different pathways that produce nostoxanthin (using CrtG), zeaxanthin (using CrtZ) and caloxanthin (CrtG). Abbreviations: F6P–Fructose 6-phosphate; G-3-P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-d-xylulose-5-phosphate; MEP, 2-C-methyl-d-erythritol-4-phosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate, ATP–Adenosine triphosphate; ADP–Adenosine diphosphate; CTP–Cytidine monophosphate; CMP–Cytidine triphosphate. D Phylogenetic tree of strain COS14-R2 and related microorganisms created with the neighbor-joining method. Numbers indicate bootstrap values; T indicates type strain of a species

Culture Conditions and Carotenoid Pigment Production

The bacterial strain was cultivated in a 100 mL baffled Erlenmeyer flask containing 50 mL of media using an orbital shaker set at 150 rpm. A defined quantity of inoculum (v/v) obtained from a culture in the exponential development stage is added to a new, sterile medium. To examine the influence of temperature on the production of yellow pigments, the incubation temperature was adjusted to different levels, ranging from 10 to 40 °C. Bacterial pigment synthesis has been enhanced to enable the adjustment of pH and the C/N ratio. To evaluate the influence of pH on pigment production, the pH levels of the culture medium were adjusted to cover a spectrum of values ranging from 5 to 11. The medium consisted of 40 g L−1 glucose, 5 g L−1 yeast extract, and 0.7 g L−1 MgSO4 (SI. Table 1). The pH of the media was initially set to 7.5 before sterilization. Batch fermentation was conducted using a 300-mL medium in 100 and 1 L baffled Erlenmeyer flasks. The flasks were incubated at 35° C on a rotary shaker set at 150 rpm for 120 h. The culture was continuously irradiated by light-emitting diodes with four combinations of monochromatic narrowband light: red, green, blue, and white light (RGB 12 V, MANI LED, South Korea). Linear LED lights were positioned underneath the culture flask in every experiment. The study assessed growth rates, biomass generation, carotenoid levels, and concentration. During incubation, samples were collected at 48 and 72 h of growth. Centrifuged cells were collected and rinsed with distilled water. The dry weight (mg/L) of the cells was determined via freeze drying (FD8508, iLShin Biobase, The Netherlands).

Table 1 Genomic features of Sphingomonas species strain COS14-R2Fed-Batch Fermentation

Fed-batch fermentation (Marado-PDA, CNS Co., Ltd., Korea) was carried out based on the batch fermentation, in duplicate, with an initial working volume of 2 L and a total volume of 2.5 L in the bioreactor. The following physical variables were controlled: pH 7.5 temperature, 35 °C; agitation speed, 200 rpm; and the dissolved oxygen concentration, 60%. Feeding solution was prepared with glucose (500 g/L) and MgSO4 (0.7 g/L) under a pH condition and fed for 72 h. The feeding strategies for the fed-batch process were based on the procedure described earlier [15, 16]. Glucose used as the sole carbon source for a fed-batch fermentation process was developed to obtain a high cell density culture with high nostoxanthin productivity and concentration.

Carotenoid Pigment Extraction and Quantification

The carotenoid content was removed from the recovered cells by employing acetone at 55° C and an ultrasonic bath (220 V and 60 Hz, JINWOO JAC Ultrasonic Device, Korea) until the cells were fully decolorized. Cells were collected from the fermented media of Sphingomonas sp. strain COS14-R2 at different time intervals. The yellow carotenoid pigment was isolated using centrifugation at 10,000 rpm, 20 min at 4 °C. The yellow supernatant was passed through a 0.2 μm nylon syringe filter (HENKE-JECT, Korea) and dried using a dust-free N2 flush (TLS Technology, TLS HPS-1, Korea). The carotenoid concentration in a batch culture was measured using high-performance liquid chromatography (HPLC) with a UV–visible photodiode detector manufactured (Agilent, USA). The study was performed on a 250 × 4.6 mm D. S-3 m YMC carotenoid column at a wavelength of 470 nm. The mobile phases consist of a solvent mixture including ethyl acetate, methanol (95:5), ammonium acetate (0.4 g/L), and acetic acid (0.1%) in methanol. There are six distinct mobile phases. The flow rate was set at a constant value of 0.6 mL/min. The detection of carotenoids was carried out utilizing a diode array detector (DAD) within the wavelength range of 300 nm to 700 nm. The retention time (tR) of specific carotenoids on HPLC system is determined by calculating the standard (Sigma-Aldrich, Germany) xanthophyll, astaxanthin, zeaxanthin, and β-carotene retention time and establishing a linear relationship. The concentration of the solution ranged from 0.49 to 125 mg/mL. The total carotenoid content (TCC) was determined following the previously published methods [17, 18].

Column Chromatography

Silica gel column chromatography was used to purify the total carotenoid from COS14-R2. Silica gel (200–200 mesh) was activated at 110 °C for 4 h to remove the moisture using a wet packing column (600 mm × 500 mm). The column was equilibrated with petroleum ether and acetone (6:4, v/v). Prior to sample separation, the level of liquid was lowered to equal the stationary phase. Cell biomass was harvested from a fed-batch fermenter and washed twice using deionized water. The samples were freeze-dried and kept at – 80 °C before nostoxanthin extraction. Yellow colored freeze-dried biomass 5.33 g was soaked in acetone (1:10) in closed screw flask bottle. The extraction was conducted at a room temperature, with continuous magnetic stirring at a rate of 600 rpm. The subsequent extracts were dried in rotary evaporator (EYELA, Japan), One gram of sample was blended with silica gel, and loaded in the column. The elution was performed with a gradient of petroleum ether and acetone at a constant flow rate of 50 mL/min, and the fractions were screened using HPLC and UV. Based on the HPLC retention time (tR) and UV absorption (λmax) of specific carotenoids combined together. Totally 104 fractions were collected and similar characteristics fractions were concentrated by rotary evaporator. Further, the colored samples were air-dried in a dust-free N2 flesh (TLS HPS-1, Republic of Korea). Dry weight of the samples was determined after freeze drying. Each individual fractions were dissolved in methanol and filtered (0.22 µm) for further characterization. The retention time (tR) of specific carotenoids on a HPLC calculated by standard retention time and linear relationship. Four analytical standards of astaxanthin, lutein, zeaxanthin and β-carotene (Sigma-Aldrich, Germany) and F6 were dissolved in methanol at a concentration of 0.78–100 mg/mL (standard), 0.025–3.2 mg/mL (F6) and a standard calibration curve was constructed for quantification.

Chromatographic Analysis for Nostoxanthin Identification

The purity of F6 was assessed using thin-layer chromatography (TLC) compared to reference standards (1/10 mg/mL, zeaxanthin, astaxanthin, and β-carotene). Each fraction was applied to a TLC Silica Gel 60 F254 plate (Merck, Darmstadt, Germany). The plates were developed with petroleum ether, with a separation achieved using a mixture of acetone in a ratio of 6:4 (v/v). A distance of 7.8 cm was completed after 16 min of development at a temperature of 22 ºC. The plates were dried using a hair dryer and cool air. The values were recorded for both the samples and standards. The purified nostoxanthin fraction was identified and characterized by liquid chromatography coupled with MS. The freeze-dried F6 sample was suspended in methanol and underwent water bath sonication. The known concentration of methanol solution was examined in an LC system (ACQUITY I-Class plus FTN) and coupled to the electron spray-ion trap mass spectrometer XEVO G2-XS QTof (Waters Corporation, Milford, MA) [16]. The purified fractions were examined using FT-IR spectroscopy (Perkin Elmer, Germany).

In vitro Free-Radical Scavenging Assay

The radical scavenging activity test was conducted using the methodology published by Xiao et al. [19]. Serial dilutions of the column-purified nostoxanthin (1 mg/mL) were made using ethanol as the solvent. Ascorbic acid and quercetin used as a standard (0.1 mg/mL). Each 1 mL portion of DPPH solution was promptly combined and thoroughly blended with various concentrations of nostoxanthin (ranging from 100 to 0.8 µg/mL) and ascorbic acid, quercetin evaluated with different concentration gradients.

Statistical Analysis

All experiments were performed in triplicate. The means and standard deviation of means were calculated by Microsoft Excel 2018 (Microsoft Office). Duncan's multiple range tests were used for conducting pairwise or individual (one-to-one) comparisons in the analysis of variance (ANOVA). A significance level of p < 0.05 was used to determine the individual differences. The statistical analyses were conducted using the IBM SPPS statistical program (Version 27, SPPS Inc., Chicago, USA).

Genome Sequence Accession Numbers

The genome sequence of strain COS14-R2 was deposited in the GenBank database under the accession number PRJNA1132764. The strain has also been deposited in Korean Agricultural Culture Collection (KACC) with the accession number KACC 23802. In addition, culture was deposited in the Korean Collection for Type Cultures (KCTC), and the accession number was KCTC 8638.