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Purification and biofabrication of 5-aminolevulinic acid for photodynamic therapy against pathogens and cancer cells

Purification and biofabrication of 5-aminolevulinic acid for photodynamic therapy against... Introduction red light (Yi et  al. 2021b). Since both 5-ALA and PPIX 5-Aminolevulinic acid (5-ALA), an endogenous, non- are natural metabolites in organisms, 5-ALA-aPDT is an proteinogenic amino acid, is a precursor in the biosyn- effective and non-toxic treatment to all kinds of patho - thesis of all porphyrins and tetrapyrrole compounds (Yi gens and MDR. Thus, 5-ALA is a valuable and potential et  al. 2021a). In recent decades, 5-ALA with different chemical compound in this era. purity has wide applications in agricultural (Hotta et  al. 5-ALA is produced from chemical synthesis or biofab- 1997) and the medical field (Inoue 2017; Juzeniene et al. rication. However, the chemical synthesis of 5-ALA is in 2002). 5-ALA can be used as a growth promoter or insec- low yield and high cost. Moreover, toxic compounds will ticide depending on its concentration. A low concentra- be released during the production of 5-ALA via chemi- tion of 5-ALA stimulates cell metabolism and assists the cal process (Matsumura et al. 1994; Miyachi et al. 1998). growth of plants and crops (Sasaki et  al. 2002). On the In contrast, a high-yield 5-ALA can be achieved via bio- contrary, harmful insects can be eliminated with a higher synthesis in an eco-friendly mode, making biotransfor- amount of 5-ALA on plants when the insects consume mation of 5-ALA a fascinating approach. However, the the 5-ALA. The pure 5-ALA has been used as a precur - saccharides, protein, amino acids, organic acids, and sor of photosensitizer in photodynamic therapy (PDT) to metal ions retained in the 5-ALA fermentation reduce diagnose tumor cells, treat cancer cells and cure skin dis- the efficiency of 5-ALA purification and decrease the eases (Yi et  al. 2021a). In this therapy, a photosensitizer antibacterial activity (Okada et al. 2016). Therefore, puri - protoporphyrin IX (PPIX) was produced and showed fication of 5-ALA from the broth is critical and necessary. fluorescence when a high concentration of 5-ALA accu - Generally, the 5-ALA in microbial broth has been puri- mulated in the cell. PPIX would cause reactive oxygen fied by using ion-exchange chromatography (IEC) from species (ROS) under appropriate wavelength and lead to other compounds and contaminants (Venosa et al. 2004; cell damage by generating singlet oxygen. 5-ALA-induced Din et al. 2021). The optimal condition for desorption of photodynamic therapy and diagnosis had been reported different compounds on the resin in IEC may differ from since 1990 (Kennedy et  al. 1990) and were approved by the distinct properties. Moreover, the ingredients in the FDA in 2017. Moreover, 5-ALA can be applied in anti- culture medium and the additional substrates for 5-ALA microbial PDT (aPDT) to attain non-invasive and non- production make the separation of 5-ALA in IEC diffi - toxic treatment for various wound infections. cult. Therefore, the pH value, ion strength, and the iso - The antibacterial photodynamic therapy (aPDT) has electric point of the compound should be optimized for a been recently demonstrated as one of the most outstand- high desorption rate in IEC. ing approaches to treating multi-drug resistant (MDR) In this study, 5-ALA was purified by using IEC from pathogens, containing three important elements: photo- fermentation broth. To maximize the recovery of 5-ALA, sensitizer (PS), illuminating light with fitting wavelength, the eluent buffer, ion concentration and pH were opti - molecular oxygen. As 5-ALA is converted into PPIX, a mized through desorption process from chromatography. strong PS, the accumulated PPIX in the cell will cause After preliminary purification by IEC, 5-ALA solution apoptosis by producing ROS under the illumination by was adjusted by phosphoric acid to pH 3 and stirred with L ee et al. Bioresources and Bioprocessing (2022) 9:68 Page 3 of 10 activatied carbon to remove the colored molecules. The Purification of 5‑ALA using chromatography rotary evaporator was employed to obtain a higher con- The strong acid cation exchange resin (Amberlite centration of 5-ALA before precipitation. Finally, 5-ALA IR120) was packed in a column (7.07  cm , 20 cm height). was precipitated with diethyl ether, methanol, ethanol, First, the resin was immersed in 50  mL of 1.5  M HCl and acetone. The purified 5-ALA was applied to against 2 for 1.5  h, followed by 50  mL of 1.5  M NaOH. A 50  mL different tumor cells and 4 pathogens by PDT, and elimi - of 1.5 M HCl was passed through the column to prepare nated Aeromonas hydrophila (A. hydrophila) in algal cul-an H -form condition. The resin was washed by ddH O ture to examine the antimicrobial ability of 5-ALA. once between each step. The culture broth of 5-ALA was adjusted to pH 4.2–4.8 with acetate acid before adsorp- Materials and methods tion. A 600 mL broth was applied to the column and then Chemicals100 mL ddH O passed through to wash out the residual Amberlite IR120 strong acid cation exchange resin, medium. HCl, sodium acetate buffer (SAB) and ammo - 5-ALA and acetylacetone were purchased from the nia were applied in this study to examine the efficiency of Sigma-Aldrich, USA. Hydrochloride acid was purchased 5-ALA desorption with different concentrations and dif - from Fluka, Switzerland. Ammonium hydroxide was pur- ferent pH. Finally, 85% phosphate acid was added into the chased from Thermo-Fisher, USA. Activated carbon was desorbed 5-ALA solution and adjust the pH to 3.0. ordered from Alfa Aesar, USA. Phosphoric acid was pur- chased from Merck, USA. Acetone, diethyl ether, etha- Crystallization of 5‑ALA nol, and methanol were purchased from ECHO, Taiwan. To remove the impurities in the broth, different amount 4-Dimethylaminobenaldehyde (DMAB) was ordered of activated carbon was added into the solution and from ACROS Organics . Perchloric acid and sodium stirred at 500  rpm for 30  min for decolorization. The acetate were purchased from SHOWA, Japan. solution was then concentrated in a rotary evaporator to obtain a higher concentration of 5-ALA  (i.e., 250 to Culture condition of 5‑ALA 500  g/L), which was dripped into the different organic Biofabrication of 5-ALA was carried out by culturing solvents including diethyl ether, methanol, ethanol, or strain RcI from a previous publication (Yu et  al. 2022). acetone (Tachiya 2016). Finally, the precipitate was dried RcI was precultured in Luria–Bertani (LB) medium at in the vacuum dryer (EYELA, Japan). 37  °C, 200  rpm for 16  h. The preculture cell was inocu - lated with 2% (v/v) into 300  mL MM9 medium contain- HPLC analysis ing (NH ) SO (16  g/L), N a HPO •12H O (16  g/L), The high-performance liquid chromatography (HPLC, 4 2 4 2 4 2 KH PO (3  g/L), yeast extract (2  g/L), M gSO •7H O Hitachi, Japan) was employed to analyze the purity of 2 4 4 2 (0.5 g/L), MnSO •7H O (0.01 g/L), glucose (20 g/L), and 5-ALA precipitate. Derivatization of samples were per- 4 2 glycerol (10  g/L) in a 1-L bioreactor at 37  °C, 300  rpm formed by the reaction consisting of 680 μL of 0.05  M with 1  vvm aeration. The final concentration of 0.1  mM borate buffer (pH 9), 480  μL of 100% methanol, 12  μL IPTG, 0.4 mM ferric citrate, 4 g/L glycine, 1 g/L succinate sample and 30  μL of 200  mM diethyl ethoxymethylen- and 30  μM PLP were added in cultivation when OD emalonate (DEEMM). The samples were heated at 70 °C reached 0.6–0.8 and shifted the culture to 30  °C and for 2  h to complete the degradation of excess DEEMM 500  rpm until 24  h. Substrates including 3  g/L glycine, and derivatization. Afterward, the samples were placed 1.5 g/L glucose, and 2 g/L succinate were fed at 12 h. The into HPLC with a quaternary pump, an inline degas- cell concentration  was measured by a spectrometer ser, an autosampler, and a column thermostat. Chroma- (SpectraMax 340, Molecular Devices, USA) with an opti- tographic separation was carried out by reverse-phase cal density at 600 nm (OD ). chromatography on a C18 column (YMC-C18 column, 4.6 × 250  mm, 5  μm particle size), maintained at 35  °C. Ehrlich assay for quantification of 5‑ALA Mobile phase A was composed of 100% acetonitrile, and A 200 μL 5-ALA sample was mixed with 200 μL sodium B was made up of 25 mM aqueous sodium acetate buffer acetate (pH 4.6) and 40  μL acetylacetone, then the mix- (pH 4.8). The flow rate of 0.8  ml/min was used, with ture was heated at 100  °C for 10  min to accelerate the the following gradient program: 0–2  min, 20–25% A; reaction (Yu et al. 2022). After cooling to room tempera- 2–32  min, 25–60% A; 32–40  min, 60–20% A. Detection ture, the mixture was mixed and reacted with the same was carried out at 284 nm (Xue et al. 2020). volume of Ehrlich’s reagent for 10  min in dark. Finally, the solution was analyzed by optical density at wave- Cancer cell culture and photodynamic therapy length 553 nm by using a spectrophotometer. Human lung adenocarcinoma cells (A549 cells) and melanoma skin cancer cells (A375 cells) were purchased Lee et al. Bioresources and Bioprocessing (2022) 9:68 Page 4 of 10 Table 1 The absorption, desorption, and recovery of 5‑ALA from cation ion‑ exchange chromatography using different eluents Eluents Conc. (M) pH Applied volume Adsorbed 5‑ ALA (g) Desorbed 5‑ ALA (g) Recovery (%) (mL) HCl 1.5 ND 110 1.24 0.101 8.1 HCl 3.0 ND 110 1.24 0.149 12.0 CH COONa 0.74 3.1 110 1.24 0.78 62.9 CH COONa 0.74 3.1 250 1.24 0.79 63.7 NH OH 1.0 11.5 250 1.25 1.15 92.0 All the chromatography is injected by 300 mL 5-ALA at 4.16 g/L for each batch. ND means not determined incubated at 37 °C for 3 h to metabolize 5-ALA to PPIX. from Bioresource Collection and Research Center Subsequently, it was illuminated with or without LED red (BCRC, Taiwan), and cultured in Dulbecco’s modified light at a wavelength of 635 nm for 30 min, correspond Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) - supplemented with 10% (v/v) fetal bovine serum (FBS, ing to power density of 100 J/cm for aPDT (HUA YANG Invitrogen, Carlsbad, CA, USA). All cells were incubated Precision Machinery Co., Taiwan). Finally, to calculate in 10-cm tissue culture dishes at 37 °C and 5% (v/v) CO . the colony-forming unit (CFU), a 20-µL aliquot with a –1 –5 The cancer cells were seeded in 96-well plates using a dilution rate of 10 to 10 was spread onto an agar plate fresh DMEM culture medium, then incubated under and incubated at 37 °C for 16 h. The A. hydrophila, Bacil - 37 °C and 5% (v/v) CO for 24 h before being treated by lus cereus (B. cereus), Staphylococcus aureus (S. aureus) 5-ALA-PDT. The cells were incubated for 3 h with differ - were tested by following the procedure as aforemen- ent concentrations of 5-ALA (0, 5, 10 g/L). Thereafter, the tioned. Each experiment was carried out in triplicate. cells were exposed to the red light source at 635 nm with power density 100 J/cm for 15 min (HUA YANG Preci- aPDT in the algal culture sion Machinery Co., Taiwan). After PDT treatment, the Chlorella sorokiniana (Cs) was precultured in TAP cells were incubated at 37  °C and 5% (v/v) CO for dif- medium for 2 days to reach OD at 0.8 (Lin et al. 2021). 2 680 ferent time (0, 2, 4, 12  h) to make reactive oxygen spe- cies attack cells. Finally, the CCK-8 assay was applied to identify the viability of the cells. Before performing the cell counting kit-8 (CCK-8) assay, the culture medium consisting of 5-ALA was removed due to the background value. 10  µL of the CCK-8 reagent (MedChemExpress Ltd.) and 100 µL of the DMEM were added to each well, incubated the cells were at 37  °C and 5% (v/v) CO for 1  h, and optical density at 450  nm was measured using a spectrophotometer. Statistical analysis was performed using GraphPad Prism software version 8.0 (GraphPad Prism software, San Diego, USA). Differences in cell via bility among the groups were analyzed using a t-test, and the values of p < 0.05 were statistically significant. Antibacterial photodynamic therapy (aPDT) against pathogens The elimination of P. hauseri by aPDT was carried out with minor modifications from a previous study (Yi et al. 2021b). The pathogen was incubated in a 10 mL LB medium at 37 °C and 175 rpm for 16 h. The concentration of P. hauseri was adjusted to OD at 0.2 (approximately 10  cells/mL) and injected 180 µL into 96-well plates. An Fig. 1 Eec ff t of sodium acetate (a, b) and ammonia (c, d) for elution of 5‑ALA from IEC. Recovery by using (a) different concentration of appropriate amount of 5-ALA solution (i.e., 0.25%, 0.5% sodium acetate at pH 3.5, and (b) 2 M CH COONa with different pHs. and 1%) was added to the cell sequentially. The plates Recovery by using (c) different concentration of ammonia at pH 11 were wrapped with aluminum foil to avoid the light and and (d) 1 M NH OH with different pHs 4 L ee et al. Bioresources and Bioprocessing (2022) 9:68 Page 5 of 10 Fig. 2 (a) Decolorization of 5‑ALA after IEC using 0.5%, 1% and 2% activated carbon. (b) The HPLC result for 5‑ALA precipitation from different organic solvents including methanol (10:1), ethanol (10:1), acetone (5:1) and acetone (20:1). The volume ratio of solvent to 5‑ALA (v/v) is shown in the parentheses Table 2 The purity of 5‑ALA from different solvents extraction OD =0.08 for Cs, and 0.2, 0.3, or 0.4 of OD   for A. 680 600 and precipitation hydrophila, while the 5-ALA concentrations were used by 0.05%, 0.1% and 0.2%, respectively. Afterward, a 20-µL Organic Solvent 5‑ ALA Crude 5‑ ALA Purity (%) solvents volume volume product content aliquot was dropped on the TAP plate for a 3-day cultiva- (mL) (mL) (g) (g) tion to observe the growth of Cs and A. hydrophila. Diethyl 20 2 0.001 ND ND ether Result and discussion Methanol 20 2 0.12 0.005 4.17 Optimization of chromatography for 5‑ALA Ethanol 20 2 0.30 0.007 2.33 To maximize the recovery of 5-ALA from the desorption Acetone 10 2 0.36 0.13 36.1 step, the desorption efficiency of HCl, sodium acetate Acetone 20 2 0.50 0.25 50.0 buffer (SAB) and ammonia were compared. As shown Acetone 40 2 0.45 0.21 46.7 in Table  1, the lowest recovery of 12.1% was acquired by 3  M HCl while the 92% recovery was achieved by 1  M 5-ALA is used at concentration of 500 g/L. ND means not determined ammonia at pH 11.5 among all conditions. Since 5-ALA was adsorbed on the strong acid cation resins, the alka- line eluents could reduce the affinity of substrate and A. hydrophila was precultured in LB medium at 37  °C, exchange 5-ALA from the resin (Moreira and Gando-Fer- 200  rpm for 16  h. The mixture of 5-ALA, A. hydrophila reira 2012). Afterwards, the optimal recovery of SAB and and Cs were prepared in a 10 mL TAP medium and cul- ammonia in different pH and concentrations were exam - tured under white light (100  µmol/m /s) with shaking ined and shown in Fig. 1. The high concentration of both at 150  rpm. The final   concentration  was adjusted to 600 Lee et al. Bioresources and Bioprocessing (2022) 9:68 Page 6 of 10 Fig.4 Taguchi L9 experimental results for 5‑ALA aPDT against the pathogen of Proteus hauseri. K1, K2 and K3 mean the average of killing rate for 3 levels of each parameter. The three levels of each factor: illuminating time (15, 30, 45 min); culture time (2, 3, 4 h); 5‑ALA concentration (1%, 2%, 3%). R is the divergence which is indicated by Fig. 3 Cell viability with purified 5‑ALA treatment among different red dot incubated time. Killing rate of (a) 0.5% and (b) 1% 5‑ALA treatment to A549 human lung cancer cells. Killing rate of (c) 0.5% and (d) 1% 5‑ALA treatment to A375 melanoma skin cancer cells purification from IEC due to the isoelectric point at pH 5.69. When ammonia was applied, 5-ALA possessed neg- ative charges and left the resin at pH higher than 5.69 in SAB and ammonia reached high recovery, which may this study. Therefore, 92% of 5-ALA recovery was carried result from the intensive molecular collision of cation out by 1 M ammonia at pH 9.5 (Fig. 1c, d), which was 1.3- and the adsorption site. However, the recovery remained folds higher than using 1 M sodium acetate buffer at pH 60% with 2 M to 4 M SAB (Fig. 1a), while 85 ± 5% recov- 4.67 and followed by pH 3.8 condition in the previous ery was accomplished by 0.5 M to 1 M ammonia (Fig. 1c). report (Tripetch et al. 2013). Patrickios and his colleagues reported that the chemicals are electrically neutral and the affinity between resins and molecular are lost at isoelectric point, enabling the acqui- Decolorization, crystallization and purity of 5‑ALA sition of compounds from IEC (Patrickios and Yamasaki As the pigments in the fermentation broth would 1995). 5-ALA was more stable in an acidic surrounding decrease the purity of 5-ALA during the process, a differ - (pH 2—4) than that in the alkaline condition (Bunke et al. ent amount of activated carbon was employed for decol- 2000), but the alkaline eluent was more favorable for  its orization. The solution after decolorization with 0.5%, 1% and 2% activated carbon was clear as shown in Fig.  2a. Subsequently, the solution was adjusted to pH 3 with 85% phosphoric acid for 5-ALA stability. To obtain a high con- Table 3 Taguchi L9 experiment design for optimization of 5‑ALA aPDT against the pathogen of Proteus hauseri centration of 5-ALA up to 500 g/L before crystallization, the solution after decolorization was concentrated by a No. Illumination Culture 5‑ ALA Killing rate (%) rotary evaporator at 65 °C. Afterward, the poor solvents: time (min) time (h) conc. (%) diethyl ether, methanol, ethanol, and acetone, were used 1 15 2 1 10.3 in the crystal method and dehydrated the 5-ALA solu- 2 15 3 2 100 tion. The result indicated that the ketones and alcohols 3 15 4 3 100 were better solvents for dehydration of 5-ALA (Table  2). 4 30 2 2 99.5 The most precipitates were obtained from acetone with 5 30 3 3 99.9 a volume ratio of 10:1, reaching 50% purity of 5-ALA. 6 30 4 1 98.4 However, there was only oily liquid occurred in the solu- 7 45 2 3 99.8 tion and no precipitates from diethyl ether. As shown 8 45 3 1 92.3 in HPLC analysis (Fig.  2b), acetone was the best solvent 9 45 4 2 100 for 5-ALA precipitation when a higher concentration of L ee et al. Bioresources and Bioprocessing (2022) 9:68 Page 7 of 10 (p = 0.0003) and 83% (p = 0.0002) under 12  h incubation for A549 cells and A375 cells, respectively (Fig.  3b, d). Previous studies have suggested the potential of 5-ALA- PDT in treating cancer cells and the inhibition of cell sur- vival activity significantly depend on both dose and time (Cai et al. 2018; Teijo et al. 2016; Wachowska et al. 2011). The results verified the cell necrosis of A549 and A375 cells when 5-ALA was administered externally under light exposure or dark. Antibacterial activity against pathogens using aPDT Wound infection is a perilous defect of skin or soft tis- sue caused by pathogenic organisms which triggers the immune response in human bodies. Depending on the etiology and severity of the microbial invasion, the infections range from minor superficial to fatal symp - toms (Simões et  al. 2018). Traditionally, antibiotics such as beta-lactams, glycopeptides, quinolones, sul- phonamides and tetracyclines were developed for med- ical applications to eliminate the pathogens for wound recovery. Among all the antibiotics, penicillin has been the most prevalent against various pathogens in wound infection (Shukla et  al. 2020). Penicillin was a revolu- tionary discovery to fight against bacterial infections in the first half of the twentieth century, and the antibi - otics developed from penicillin reduced the mortality caused by infectious diseases effectively (Sarmah et  al. 2018). However, the misuse and abuse had a serious Fig. 5 The plate assay of aPDT using 5‑ALA from (a, c, e) purified consequence: the generation of multi-drug resistant (NCKU) and (b, d, f) commercial (Sigma A3785) against pathogen (MDR). According to WHO’s estimations, approxi- Aeromonas hydrophila (a, b) Bacillus cereus (c, d), Staphylococcus mately 700,000 deaths are caused by MDR infections aureus (e, f). The left and right sides of plate represented control every year (Klausen et al. 2020). Owing to the problems (without 5‑ALA) and experiment group (with 1% or 0.25% 5‑ALA) above, the aPDT is known as a new therapeutic assay. purified 5-ALA was obtained at the volume ratio of 20:1 Four pathogens, P. hauseri, A. hydrophila, B. cereus, (acetone/5-ALA). and S. aureus, were selected for 5-ALA-aPDT in this study. Proteus species are widespread in the environ- 5‑ALA‑PDT treatment on A549 and A375 cancer cells ment which causes the urinary tract infections (UTIs) in In this section, we focused on the effectiveness of 5-ALA- human by spreading from the rectum to the periurethral PDT for cancer cells, A549 and A375 cells. When treated and bladder (Armbruster and Mobley 2012). A. hydroph- with 5-ALA-PDT, the viability of the cells showed differ - ila is one of the most common bacteria isolated in fresh- ences between the cells incubated with increasing con- water, seawater, and sewage environments. In addition, centrations of 5-ALA from 0.5 to 1  g/L, and the control A. hydrophila is the cause of zoonotic diseases such as group (p < 0.0001). As the incubation time increased, gastrointestinal disease, sepsis, and aquatic wound infec- the inhibition of both cancer cells also increased, and tion (Kussovski et  al. 2009). B. cereus is associated with the enhancement in killing rate with time was more sig- foodborne illness and food spoilage, provoking vomit- nificant when 1% 5-ALA was used and shown in Fig.  3 ing, and diarrhea to humans (do Prado-Silva et al. 2021). (p = 0.0053 for A549 cells and p < 0.001 for A375 cells). S. aureus is the most epidemic pathogen, which is often Cell viability was significantly lower in the treatment found in human skin, mucous membranes, and purulent groups than in the control groups under 1% 5-ALA treat- wounds, causing vomiting, abdominal pain, diarrhea, and ment. As a result, 56% and 43% killing rates for A549 cells fever (Pérez-Laguna et al. 2018). and A375 cells were obtained by 5-ALA-PDT treatment To optimize the condition of 5-ALA-aPDT, the Tagu- with 0.5% purified 5-ALA under 4  h incubation. After chi L9 method was designed with 3 factors: illuminat- increasing to 1% 5-ALA, the cell killing rate reached 74% ing time, culture time and 5-ALA concentration. The Lee et al. Bioresources and Bioprocessing (2022) 9:68 Page 8 of 10 Fig. 6 (a) Schematic of experiment design for 5‑ALA against A. hydrophila, a major fish and algae pathogen. (b) The antibacterial activity on TAP plate assay with different concentrations of 5‑ALA against different OD of A. hydrophila in microalgae C. sorokiniana culture at 30 °C for 3 days 600nm under 100 µmol/m /s light intensity. C indicates the commercial 5‑ALA from Sigma (Sigma A3785) and P is the purified 5‑ALA in this study. (c) The cultivation of C. sorokiniana with 0.05% or without 5‑ALA and different biomass of A. hydrophila in terms of OD at 0.2, 0.3 and 0.4 after 3‑ day culture experiments were carried out on P. hauseri and the effi - As shown in Fig.  5a–f, the 100% killing rates were ciency of each group was shown in Table  3. The K val - achieved with 1% and 0.25% of 5-ALA for A. hydrophila, ues indicated that the optimal illuminating time was B. cereus or S. aureus, though the optimal 5-ALA concen- 30  min (Fig.  4). Since  5-ALA would  be degraded under tration was 3% for P. hauseri (Table  3). The four harmful strong light exposure, resulting in low killing rate. On the pathogens were eliminated by 5-ALA-aPDT, verifying the other hand, the killing rate showed a positive correlation feasibility of purified 5-ALA, which showed impressive with culture time and 5-ALA concentration. From the antibacterial activity against pathogens as pure 5-ALA R-value, the concentration of 5-ALA was the dominant from commercial. factor in 5-ALA-aPDT treatment, while the illuminat- ing time and culture time had similar influences on the Growth improvement and A. hydrophila elimination result. Therefore, the optimal condition for the elimina - in algae culture tion of P. hauseri by 5-ALA-aPDT was acquired by 3% Microalgae is an innovative biofuel producer in the next- of 5-ALA with 30-min illumination and 4-h cultivation. generation bioenergy. However, scaling-up of microal- Although it showed better efficiency of aPDT with higher gae in the open-pond is still challenge due to bacterial concentration of 5-ALA (i.e., 2 to 3%) from the Taguchi’s contamination (Wang et  al. 2013). Currently, chemical L9 experiment, using 1% of 5-ALA still eliminated P. pesticides are extensively used to protect the microal- hauseri effectively. Taking the cost effect on the process, gae system, such as trichlorfon, methyl parathion, and using 1% of 5-ALA was economically efficient to further optimize antibacterial activity. L ee et al. Bioresources and Bioprocessing (2022) 9:68 Page 9 of 10 experiment of aPDT with algae. YCL, CCC, JHH: performed cancer cell experi‑ diazinon (Zhu et  al. 2020). Although the chemical con- ments. ISN: conceptualization, investigation, resources and writing—review trol showed effective prevention of contamination in and editing. All authors read and approved the final manuscript. microalgae culture, the reagents would inhibit the growth Funding of microalgae (Chen and Jiang 2011). 5-ALA has been The authors are grateful for the financial support received from the Ministry applied to kill bacteria and improve the plant growth of Science and Technology (MOST 110–2221‑E‑006–030‑MY3 and MOST (Wang et  al. 2021), as well as trigger tolerance to cold, 108–2221‑E‑006–004‑MY3) in Taiwan. high salt conditions (Zhang et al. 2006), and enhance pig- Availability of data and materials ment production (Lyu et  al. 2022). Therefore, 5-ALA is The authors approved the availability of data and materials for publishing the a promising natural compound to resolve the biological manuscript. contamination of microalgae. The schematic diagram of 5-ALA-aPDT in algae– Declarations bacteria co-culture (AB culture) is shown as Fig.  6a. Ethics approval and consent to participate To confirm the effectiveness of 5-ALA-aPDT against Not applicable. A. hydrophila which is a famous pathogen in aquatic Consent for publication condition for Cs culture, 0.05%, 0.1% or 0.2% 5-ALA All the authors have read and approved the manuscript before the submission were applied to the AB culture. The growth of Cs was to Bioresources and Bioprocessing. inhibited with A. hydrophila without 5-ALA (Fig.  6b, Competing interests 0.2A) compared to the control strain, while the growth The authors declare that they have no competing interests. was improved with 0.05% 5-ALA in the absence of A. hydrophila (Fig. 6b, 0.05% P). Moreover, the cultivation Author details Department of Chemical Engineering, National Cheng Kung University, of Cs showed better growth with 5-ALA and lower con- Tainan, Taiwan. Institute of Clinical Medicine, College of Medicine, National centration of A. hydrophila (Fig.  6c), ascertaining the Cheng Kung University, Tainan, Taiwan. Department of Ophthalmology, elevation of cell growth with 5-ALA (Jiao et  al. 2017). National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Notably, the 5-ALA concentration should be increased when more A. hydrophila cell was added (Fig.  6c). The Received: 12 April 2022 Accepted: 3 June 2022 results showed the outstanding performance of purified 5-ALA to eliminate pathogen in AB culture. References Armbruster CE, Mobley HL (2012) Merging mythology and morphology: the Conclusion multifaceted lifestyle of Proteus mirabilis. Nat Rev Microbiol 10:743–754 Due to the imperative demand of 5-ALA, the produc- Bunke A, Zerbe O, Schmid H, Burmeister G, Merkle HP, Gander B (2000) Deg‑ tion and purification is exigent in recent years. 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Purification and biofabrication of 5-aminolevulinic acid for photodynamic therapy against pathogens and cancer cells

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Abstract

Introduction red light (Yi et  al. 2021b). Since both 5-ALA and PPIX 5-Aminolevulinic acid (5-ALA), an endogenous, non- are natural metabolites in organisms, 5-ALA-aPDT is an proteinogenic amino acid, is a precursor in the biosyn- effective and non-toxic treatment to all kinds of patho - thesis of all porphyrins and tetrapyrrole compounds (Yi gens and MDR. Thus, 5-ALA is a valuable and potential et  al. 2021a). In recent decades, 5-ALA with different chemical compound in this era. purity has wide applications in agricultural (Hotta et  al. 5-ALA is produced from chemical synthesis or biofab- 1997) and the medical field (Inoue 2017; Juzeniene et al. rication. However, the chemical synthesis of 5-ALA is in 2002). 5-ALA can be used as a growth promoter or insec- low yield and high cost. Moreover, toxic compounds will ticide depending on its concentration. A low concentra- be released during the production of 5-ALA via chemi- tion of 5-ALA stimulates cell metabolism and assists the cal process (Matsumura et al. 1994; Miyachi et al. 1998). growth of plants and crops (Sasaki et  al. 2002). On the In contrast, a high-yield 5-ALA can be achieved via bio- contrary, harmful insects can be eliminated with a higher synthesis in an eco-friendly mode, making biotransfor- amount of 5-ALA on plants when the insects consume mation of 5-ALA a fascinating approach. However, the the 5-ALA. The pure 5-ALA has been used as a precur - saccharides, protein, amino acids, organic acids, and sor of photosensitizer in photodynamic therapy (PDT) to metal ions retained in the 5-ALA fermentation reduce diagnose tumor cells, treat cancer cells and cure skin dis- the efficiency of 5-ALA purification and decrease the eases (Yi et  al. 2021a). In this therapy, a photosensitizer antibacterial activity (Okada et al. 2016). Therefore, puri - protoporphyrin IX (PPIX) was produced and showed fication of 5-ALA from the broth is critical and necessary. fluorescence when a high concentration of 5-ALA accu - Generally, the 5-ALA in microbial broth has been puri- mulated in the cell. PPIX would cause reactive oxygen fied by using ion-exchange chromatography (IEC) from species (ROS) under appropriate wavelength and lead to other compounds and contaminants (Venosa et al. 2004; cell damage by generating singlet oxygen. 5-ALA-induced Din et al. 2021). The optimal condition for desorption of photodynamic therapy and diagnosis had been reported different compounds on the resin in IEC may differ from since 1990 (Kennedy et  al. 1990) and were approved by the distinct properties. Moreover, the ingredients in the FDA in 2017. Moreover, 5-ALA can be applied in anti- culture medium and the additional substrates for 5-ALA microbial PDT (aPDT) to attain non-invasive and non- production make the separation of 5-ALA in IEC diffi - toxic treatment for various wound infections. cult. Therefore, the pH value, ion strength, and the iso - The antibacterial photodynamic therapy (aPDT) has electric point of the compound should be optimized for a been recently demonstrated as one of the most outstand- high desorption rate in IEC. ing approaches to treating multi-drug resistant (MDR) In this study, 5-ALA was purified by using IEC from pathogens, containing three important elements: photo- fermentation broth. To maximize the recovery of 5-ALA, sensitizer (PS), illuminating light with fitting wavelength, the eluent buffer, ion concentration and pH were opti - molecular oxygen. As 5-ALA is converted into PPIX, a mized through desorption process from chromatography. strong PS, the accumulated PPIX in the cell will cause After preliminary purification by IEC, 5-ALA solution apoptosis by producing ROS under the illumination by was adjusted by phosphoric acid to pH 3 and stirred with L ee et al. Bioresources and Bioprocessing (2022) 9:68 Page 3 of 10 activatied carbon to remove the colored molecules. The Purification of 5‑ALA using chromatography rotary evaporator was employed to obtain a higher con- The strong acid cation exchange resin (Amberlite centration of 5-ALA before precipitation. Finally, 5-ALA IR120) was packed in a column (7.07  cm , 20 cm height). was precipitated with diethyl ether, methanol, ethanol, First, the resin was immersed in 50  mL of 1.5  M HCl and acetone. The purified 5-ALA was applied to against 2 for 1.5  h, followed by 50  mL of 1.5  M NaOH. A 50  mL different tumor cells and 4 pathogens by PDT, and elimi - of 1.5 M HCl was passed through the column to prepare nated Aeromonas hydrophila (A. hydrophila) in algal cul-an H -form condition. The resin was washed by ddH O ture to examine the antimicrobial ability of 5-ALA. once between each step. The culture broth of 5-ALA was adjusted to pH 4.2–4.8 with acetate acid before adsorp- Materials and methods tion. A 600 mL broth was applied to the column and then Chemicals100 mL ddH O passed through to wash out the residual Amberlite IR120 strong acid cation exchange resin, medium. HCl, sodium acetate buffer (SAB) and ammo - 5-ALA and acetylacetone were purchased from the nia were applied in this study to examine the efficiency of Sigma-Aldrich, USA. Hydrochloride acid was purchased 5-ALA desorption with different concentrations and dif - from Fluka, Switzerland. Ammonium hydroxide was pur- ferent pH. Finally, 85% phosphate acid was added into the chased from Thermo-Fisher, USA. Activated carbon was desorbed 5-ALA solution and adjust the pH to 3.0. ordered from Alfa Aesar, USA. Phosphoric acid was pur- chased from Merck, USA. Acetone, diethyl ether, etha- Crystallization of 5‑ALA nol, and methanol were purchased from ECHO, Taiwan. To remove the impurities in the broth, different amount 4-Dimethylaminobenaldehyde (DMAB) was ordered of activated carbon was added into the solution and from ACROS Organics . Perchloric acid and sodium stirred at 500  rpm for 30  min for decolorization. The acetate were purchased from SHOWA, Japan. solution was then concentrated in a rotary evaporator to obtain a higher concentration of 5-ALA  (i.e., 250 to Culture condition of 5‑ALA 500  g/L), which was dripped into the different organic Biofabrication of 5-ALA was carried out by culturing solvents including diethyl ether, methanol, ethanol, or strain RcI from a previous publication (Yu et  al. 2022). acetone (Tachiya 2016). Finally, the precipitate was dried RcI was precultured in Luria–Bertani (LB) medium at in the vacuum dryer (EYELA, Japan). 37  °C, 200  rpm for 16  h. The preculture cell was inocu - lated with 2% (v/v) into 300  mL MM9 medium contain- HPLC analysis ing (NH ) SO (16  g/L), N a HPO •12H O (16  g/L), The high-performance liquid chromatography (HPLC, 4 2 4 2 4 2 KH PO (3  g/L), yeast extract (2  g/L), M gSO •7H O Hitachi, Japan) was employed to analyze the purity of 2 4 4 2 (0.5 g/L), MnSO •7H O (0.01 g/L), glucose (20 g/L), and 5-ALA precipitate. Derivatization of samples were per- 4 2 glycerol (10  g/L) in a 1-L bioreactor at 37  °C, 300  rpm formed by the reaction consisting of 680 μL of 0.05  M with 1  vvm aeration. The final concentration of 0.1  mM borate buffer (pH 9), 480  μL of 100% methanol, 12  μL IPTG, 0.4 mM ferric citrate, 4 g/L glycine, 1 g/L succinate sample and 30  μL of 200  mM diethyl ethoxymethylen- and 30  μM PLP were added in cultivation when OD emalonate (DEEMM). The samples were heated at 70 °C reached 0.6–0.8 and shifted the culture to 30  °C and for 2  h to complete the degradation of excess DEEMM 500  rpm until 24  h. Substrates including 3  g/L glycine, and derivatization. Afterward, the samples were placed 1.5 g/L glucose, and 2 g/L succinate were fed at 12 h. The into HPLC with a quaternary pump, an inline degas- cell concentration  was measured by a spectrometer ser, an autosampler, and a column thermostat. Chroma- (SpectraMax 340, Molecular Devices, USA) with an opti- tographic separation was carried out by reverse-phase cal density at 600 nm (OD ). chromatography on a C18 column (YMC-C18 column, 4.6 × 250  mm, 5  μm particle size), maintained at 35  °C. Ehrlich assay for quantification of 5‑ALA Mobile phase A was composed of 100% acetonitrile, and A 200 μL 5-ALA sample was mixed with 200 μL sodium B was made up of 25 mM aqueous sodium acetate buffer acetate (pH 4.6) and 40  μL acetylacetone, then the mix- (pH 4.8). The flow rate of 0.8  ml/min was used, with ture was heated at 100  °C for 10  min to accelerate the the following gradient program: 0–2  min, 20–25% A; reaction (Yu et al. 2022). After cooling to room tempera- 2–32  min, 25–60% A; 32–40  min, 60–20% A. Detection ture, the mixture was mixed and reacted with the same was carried out at 284 nm (Xue et al. 2020). volume of Ehrlich’s reagent for 10  min in dark. Finally, the solution was analyzed by optical density at wave- Cancer cell culture and photodynamic therapy length 553 nm by using a spectrophotometer. Human lung adenocarcinoma cells (A549 cells) and melanoma skin cancer cells (A375 cells) were purchased Lee et al. Bioresources and Bioprocessing (2022) 9:68 Page 4 of 10 Table 1 The absorption, desorption, and recovery of 5‑ALA from cation ion‑ exchange chromatography using different eluents Eluents Conc. (M) pH Applied volume Adsorbed 5‑ ALA (g) Desorbed 5‑ ALA (g) Recovery (%) (mL) HCl 1.5 ND 110 1.24 0.101 8.1 HCl 3.0 ND 110 1.24 0.149 12.0 CH COONa 0.74 3.1 110 1.24 0.78 62.9 CH COONa 0.74 3.1 250 1.24 0.79 63.7 NH OH 1.0 11.5 250 1.25 1.15 92.0 All the chromatography is injected by 300 mL 5-ALA at 4.16 g/L for each batch. ND means not determined incubated at 37 °C for 3 h to metabolize 5-ALA to PPIX. from Bioresource Collection and Research Center Subsequently, it was illuminated with or without LED red (BCRC, Taiwan), and cultured in Dulbecco’s modified light at a wavelength of 635 nm for 30 min, correspond Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) - supplemented with 10% (v/v) fetal bovine serum (FBS, ing to power density of 100 J/cm for aPDT (HUA YANG Invitrogen, Carlsbad, CA, USA). All cells were incubated Precision Machinery Co., Taiwan). Finally, to calculate in 10-cm tissue culture dishes at 37 °C and 5% (v/v) CO . the colony-forming unit (CFU), a 20-µL aliquot with a –1 –5 The cancer cells were seeded in 96-well plates using a dilution rate of 10 to 10 was spread onto an agar plate fresh DMEM culture medium, then incubated under and incubated at 37 °C for 16 h. The A. hydrophila, Bacil - 37 °C and 5% (v/v) CO for 24 h before being treated by lus cereus (B. cereus), Staphylococcus aureus (S. aureus) 5-ALA-PDT. The cells were incubated for 3 h with differ - were tested by following the procedure as aforemen- ent concentrations of 5-ALA (0, 5, 10 g/L). Thereafter, the tioned. Each experiment was carried out in triplicate. cells were exposed to the red light source at 635 nm with power density 100 J/cm for 15 min (HUA YANG Preci- aPDT in the algal culture sion Machinery Co., Taiwan). After PDT treatment, the Chlorella sorokiniana (Cs) was precultured in TAP cells were incubated at 37  °C and 5% (v/v) CO for dif- medium for 2 days to reach OD at 0.8 (Lin et al. 2021). 2 680 ferent time (0, 2, 4, 12  h) to make reactive oxygen spe- cies attack cells. Finally, the CCK-8 assay was applied to identify the viability of the cells. Before performing the cell counting kit-8 (CCK-8) assay, the culture medium consisting of 5-ALA was removed due to the background value. 10  µL of the CCK-8 reagent (MedChemExpress Ltd.) and 100 µL of the DMEM were added to each well, incubated the cells were at 37  °C and 5% (v/v) CO for 1  h, and optical density at 450  nm was measured using a spectrophotometer. Statistical analysis was performed using GraphPad Prism software version 8.0 (GraphPad Prism software, San Diego, USA). Differences in cell via bility among the groups were analyzed using a t-test, and the values of p < 0.05 were statistically significant. Antibacterial photodynamic therapy (aPDT) against pathogens The elimination of P. hauseri by aPDT was carried out with minor modifications from a previous study (Yi et al. 2021b). The pathogen was incubated in a 10 mL LB medium at 37 °C and 175 rpm for 16 h. The concentration of P. hauseri was adjusted to OD at 0.2 (approximately 10  cells/mL) and injected 180 µL into 96-well plates. An Fig. 1 Eec ff t of sodium acetate (a, b) and ammonia (c, d) for elution of 5‑ALA from IEC. Recovery by using (a) different concentration of appropriate amount of 5-ALA solution (i.e., 0.25%, 0.5% sodium acetate at pH 3.5, and (b) 2 M CH COONa with different pHs. and 1%) was added to the cell sequentially. The plates Recovery by using (c) different concentration of ammonia at pH 11 were wrapped with aluminum foil to avoid the light and and (d) 1 M NH OH with different pHs 4 L ee et al. Bioresources and Bioprocessing (2022) 9:68 Page 5 of 10 Fig. 2 (a) Decolorization of 5‑ALA after IEC using 0.5%, 1% and 2% activated carbon. (b) The HPLC result for 5‑ALA precipitation from different organic solvents including methanol (10:1), ethanol (10:1), acetone (5:1) and acetone (20:1). The volume ratio of solvent to 5‑ALA (v/v) is shown in the parentheses Table 2 The purity of 5‑ALA from different solvents extraction OD =0.08 for Cs, and 0.2, 0.3, or 0.4 of OD   for A. 680 600 and precipitation hydrophila, while the 5-ALA concentrations were used by 0.05%, 0.1% and 0.2%, respectively. Afterward, a 20-µL Organic Solvent 5‑ ALA Crude 5‑ ALA Purity (%) solvents volume volume product content aliquot was dropped on the TAP plate for a 3-day cultiva- (mL) (mL) (g) (g) tion to observe the growth of Cs and A. hydrophila. Diethyl 20 2 0.001 ND ND ether Result and discussion Methanol 20 2 0.12 0.005 4.17 Optimization of chromatography for 5‑ALA Ethanol 20 2 0.30 0.007 2.33 To maximize the recovery of 5-ALA from the desorption Acetone 10 2 0.36 0.13 36.1 step, the desorption efficiency of HCl, sodium acetate Acetone 20 2 0.50 0.25 50.0 buffer (SAB) and ammonia were compared. As shown Acetone 40 2 0.45 0.21 46.7 in Table  1, the lowest recovery of 12.1% was acquired by 3  M HCl while the 92% recovery was achieved by 1  M 5-ALA is used at concentration of 500 g/L. ND means not determined ammonia at pH 11.5 among all conditions. Since 5-ALA was adsorbed on the strong acid cation resins, the alka- line eluents could reduce the affinity of substrate and A. hydrophila was precultured in LB medium at 37  °C, exchange 5-ALA from the resin (Moreira and Gando-Fer- 200  rpm for 16  h. The mixture of 5-ALA, A. hydrophila reira 2012). Afterwards, the optimal recovery of SAB and and Cs were prepared in a 10 mL TAP medium and cul- ammonia in different pH and concentrations were exam - tured under white light (100  µmol/m /s) with shaking ined and shown in Fig. 1. The high concentration of both at 150  rpm. The final   concentration  was adjusted to 600 Lee et al. Bioresources and Bioprocessing (2022) 9:68 Page 6 of 10 Fig.4 Taguchi L9 experimental results for 5‑ALA aPDT against the pathogen of Proteus hauseri. K1, K2 and K3 mean the average of killing rate for 3 levels of each parameter. The three levels of each factor: illuminating time (15, 30, 45 min); culture time (2, 3, 4 h); 5‑ALA concentration (1%, 2%, 3%). R is the divergence which is indicated by Fig. 3 Cell viability with purified 5‑ALA treatment among different red dot incubated time. Killing rate of (a) 0.5% and (b) 1% 5‑ALA treatment to A549 human lung cancer cells. Killing rate of (c) 0.5% and (d) 1% 5‑ALA treatment to A375 melanoma skin cancer cells purification from IEC due to the isoelectric point at pH 5.69. When ammonia was applied, 5-ALA possessed neg- ative charges and left the resin at pH higher than 5.69 in SAB and ammonia reached high recovery, which may this study. Therefore, 92% of 5-ALA recovery was carried result from the intensive molecular collision of cation out by 1 M ammonia at pH 9.5 (Fig. 1c, d), which was 1.3- and the adsorption site. However, the recovery remained folds higher than using 1 M sodium acetate buffer at pH 60% with 2 M to 4 M SAB (Fig. 1a), while 85 ± 5% recov- 4.67 and followed by pH 3.8 condition in the previous ery was accomplished by 0.5 M to 1 M ammonia (Fig. 1c). report (Tripetch et al. 2013). Patrickios and his colleagues reported that the chemicals are electrically neutral and the affinity between resins and molecular are lost at isoelectric point, enabling the acqui- Decolorization, crystallization and purity of 5‑ALA sition of compounds from IEC (Patrickios and Yamasaki As the pigments in the fermentation broth would 1995). 5-ALA was more stable in an acidic surrounding decrease the purity of 5-ALA during the process, a differ - (pH 2—4) than that in the alkaline condition (Bunke et al. ent amount of activated carbon was employed for decol- 2000), but the alkaline eluent was more favorable for  its orization. The solution after decolorization with 0.5%, 1% and 2% activated carbon was clear as shown in Fig.  2a. Subsequently, the solution was adjusted to pH 3 with 85% phosphoric acid for 5-ALA stability. To obtain a high con- Table 3 Taguchi L9 experiment design for optimization of 5‑ALA aPDT against the pathogen of Proteus hauseri centration of 5-ALA up to 500 g/L before crystallization, the solution after decolorization was concentrated by a No. Illumination Culture 5‑ ALA Killing rate (%) rotary evaporator at 65 °C. Afterward, the poor solvents: time (min) time (h) conc. (%) diethyl ether, methanol, ethanol, and acetone, were used 1 15 2 1 10.3 in the crystal method and dehydrated the 5-ALA solu- 2 15 3 2 100 tion. The result indicated that the ketones and alcohols 3 15 4 3 100 were better solvents for dehydration of 5-ALA (Table  2). 4 30 2 2 99.5 The most precipitates were obtained from acetone with 5 30 3 3 99.9 a volume ratio of 10:1, reaching 50% purity of 5-ALA. 6 30 4 1 98.4 However, there was only oily liquid occurred in the solu- 7 45 2 3 99.8 tion and no precipitates from diethyl ether. As shown 8 45 3 1 92.3 in HPLC analysis (Fig.  2b), acetone was the best solvent 9 45 4 2 100 for 5-ALA precipitation when a higher concentration of L ee et al. Bioresources and Bioprocessing (2022) 9:68 Page 7 of 10 (p = 0.0003) and 83% (p = 0.0002) under 12  h incubation for A549 cells and A375 cells, respectively (Fig.  3b, d). Previous studies have suggested the potential of 5-ALA- PDT in treating cancer cells and the inhibition of cell sur- vival activity significantly depend on both dose and time (Cai et al. 2018; Teijo et al. 2016; Wachowska et al. 2011). The results verified the cell necrosis of A549 and A375 cells when 5-ALA was administered externally under light exposure or dark. Antibacterial activity against pathogens using aPDT Wound infection is a perilous defect of skin or soft tis- sue caused by pathogenic organisms which triggers the immune response in human bodies. Depending on the etiology and severity of the microbial invasion, the infections range from minor superficial to fatal symp - toms (Simões et  al. 2018). Traditionally, antibiotics such as beta-lactams, glycopeptides, quinolones, sul- phonamides and tetracyclines were developed for med- ical applications to eliminate the pathogens for wound recovery. Among all the antibiotics, penicillin has been the most prevalent against various pathogens in wound infection (Shukla et  al. 2020). Penicillin was a revolu- tionary discovery to fight against bacterial infections in the first half of the twentieth century, and the antibi - otics developed from penicillin reduced the mortality caused by infectious diseases effectively (Sarmah et  al. 2018). However, the misuse and abuse had a serious Fig. 5 The plate assay of aPDT using 5‑ALA from (a, c, e) purified consequence: the generation of multi-drug resistant (NCKU) and (b, d, f) commercial (Sigma A3785) against pathogen (MDR). According to WHO’s estimations, approxi- Aeromonas hydrophila (a, b) Bacillus cereus (c, d), Staphylococcus mately 700,000 deaths are caused by MDR infections aureus (e, f). The left and right sides of plate represented control every year (Klausen et al. 2020). Owing to the problems (without 5‑ALA) and experiment group (with 1% or 0.25% 5‑ALA) above, the aPDT is known as a new therapeutic assay. purified 5-ALA was obtained at the volume ratio of 20:1 Four pathogens, P. hauseri, A. hydrophila, B. cereus, (acetone/5-ALA). and S. aureus, were selected for 5-ALA-aPDT in this study. Proteus species are widespread in the environ- 5‑ALA‑PDT treatment on A549 and A375 cancer cells ment which causes the urinary tract infections (UTIs) in In this section, we focused on the effectiveness of 5-ALA- human by spreading from the rectum to the periurethral PDT for cancer cells, A549 and A375 cells. When treated and bladder (Armbruster and Mobley 2012). A. hydroph- with 5-ALA-PDT, the viability of the cells showed differ - ila is one of the most common bacteria isolated in fresh- ences between the cells incubated with increasing con- water, seawater, and sewage environments. In addition, centrations of 5-ALA from 0.5 to 1  g/L, and the control A. hydrophila is the cause of zoonotic diseases such as group (p < 0.0001). As the incubation time increased, gastrointestinal disease, sepsis, and aquatic wound infec- the inhibition of both cancer cells also increased, and tion (Kussovski et  al. 2009). B. cereus is associated with the enhancement in killing rate with time was more sig- foodborne illness and food spoilage, provoking vomit- nificant when 1% 5-ALA was used and shown in Fig.  3 ing, and diarrhea to humans (do Prado-Silva et al. 2021). (p = 0.0053 for A549 cells and p < 0.001 for A375 cells). S. aureus is the most epidemic pathogen, which is often Cell viability was significantly lower in the treatment found in human skin, mucous membranes, and purulent groups than in the control groups under 1% 5-ALA treat- wounds, causing vomiting, abdominal pain, diarrhea, and ment. As a result, 56% and 43% killing rates for A549 cells fever (Pérez-Laguna et al. 2018). and A375 cells were obtained by 5-ALA-PDT treatment To optimize the condition of 5-ALA-aPDT, the Tagu- with 0.5% purified 5-ALA under 4  h incubation. After chi L9 method was designed with 3 factors: illuminat- increasing to 1% 5-ALA, the cell killing rate reached 74% ing time, culture time and 5-ALA concentration. The Lee et al. Bioresources and Bioprocessing (2022) 9:68 Page 8 of 10 Fig. 6 (a) Schematic of experiment design for 5‑ALA against A. hydrophila, a major fish and algae pathogen. (b) The antibacterial activity on TAP plate assay with different concentrations of 5‑ALA against different OD of A. hydrophila in microalgae C. sorokiniana culture at 30 °C for 3 days 600nm under 100 µmol/m /s light intensity. C indicates the commercial 5‑ALA from Sigma (Sigma A3785) and P is the purified 5‑ALA in this study. (c) The cultivation of C. sorokiniana with 0.05% or without 5‑ALA and different biomass of A. hydrophila in terms of OD at 0.2, 0.3 and 0.4 after 3‑ day culture experiments were carried out on P. hauseri and the effi - As shown in Fig.  5a–f, the 100% killing rates were ciency of each group was shown in Table  3. The K val - achieved with 1% and 0.25% of 5-ALA for A. hydrophila, ues indicated that the optimal illuminating time was B. cereus or S. aureus, though the optimal 5-ALA concen- 30  min (Fig.  4). Since  5-ALA would  be degraded under tration was 3% for P. hauseri (Table  3). The four harmful strong light exposure, resulting in low killing rate. On the pathogens were eliminated by 5-ALA-aPDT, verifying the other hand, the killing rate showed a positive correlation feasibility of purified 5-ALA, which showed impressive with culture time and 5-ALA concentration. From the antibacterial activity against pathogens as pure 5-ALA R-value, the concentration of 5-ALA was the dominant from commercial. factor in 5-ALA-aPDT treatment, while the illuminat- ing time and culture time had similar influences on the Growth improvement and A. hydrophila elimination result. Therefore, the optimal condition for the elimina - in algae culture tion of P. hauseri by 5-ALA-aPDT was acquired by 3% Microalgae is an innovative biofuel producer in the next- of 5-ALA with 30-min illumination and 4-h cultivation. generation bioenergy. However, scaling-up of microal- Although it showed better efficiency of aPDT with higher gae in the open-pond is still challenge due to bacterial concentration of 5-ALA (i.e., 2 to 3%) from the Taguchi’s contamination (Wang et  al. 2013). Currently, chemical L9 experiment, using 1% of 5-ALA still eliminated P. pesticides are extensively used to protect the microal- hauseri effectively. Taking the cost effect on the process, gae system, such as trichlorfon, methyl parathion, and using 1% of 5-ALA was economically efficient to further optimize antibacterial activity. L ee et al. Bioresources and Bioprocessing (2022) 9:68 Page 9 of 10 experiment of aPDT with algae. YCL, CCC, JHH: performed cancer cell experi‑ diazinon (Zhu et  al. 2020). Although the chemical con- ments. ISN: conceptualization, investigation, resources and writing—review trol showed effective prevention of contamination in and editing. All authors read and approved the final manuscript. microalgae culture, the reagents would inhibit the growth Funding of microalgae (Chen and Jiang 2011). 5-ALA has been The authors are grateful for the financial support received from the Ministry applied to kill bacteria and improve the plant growth of Science and Technology (MOST 110–2221‑E‑006–030‑MY3 and MOST (Wang et  al. 2021), as well as trigger tolerance to cold, 108–2221‑E‑006–004‑MY3) in Taiwan. high salt conditions (Zhang et al. 2006), and enhance pig- Availability of data and materials ment production (Lyu et  al. 2022). Therefore, 5-ALA is The authors approved the availability of data and materials for publishing the a promising natural compound to resolve the biological manuscript. contamination of microalgae. The schematic diagram of 5-ALA-aPDT in algae– Declarations bacteria co-culture (AB culture) is shown as Fig.  6a. Ethics approval and consent to participate To confirm the effectiveness of 5-ALA-aPDT against Not applicable. A. hydrophila which is a famous pathogen in aquatic Consent for publication condition for Cs culture, 0.05%, 0.1% or 0.2% 5-ALA All the authors have read and approved the manuscript before the submission were applied to the AB culture. The growth of Cs was to Bioresources and Bioprocessing. inhibited with A. hydrophila without 5-ALA (Fig.  6b, Competing interests 0.2A) compared to the control strain, while the growth The authors declare that they have no competing interests. was improved with 0.05% 5-ALA in the absence of A. hydrophila (Fig. 6b, 0.05% P). Moreover, the cultivation Author details Department of Chemical Engineering, National Cheng Kung University, of Cs showed better growth with 5-ALA and lower con- Tainan, Taiwan. Institute of Clinical Medicine, College of Medicine, National centration of A. hydrophila (Fig.  6c), ascertaining the Cheng Kung University, Tainan, Taiwan. Department of Ophthalmology, elevation of cell growth with 5-ALA (Jiao et  al. 2017). National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Notably, the 5-ALA concentration should be increased when more A. hydrophila cell was added (Fig.  6c). The Received: 12 April 2022 Accepted: 3 June 2022 results showed the outstanding performance of purified 5-ALA to eliminate pathogen in AB culture. References Armbruster CE, Mobley HL (2012) Merging mythology and morphology: the Conclusion multifaceted lifestyle of Proteus mirabilis. Nat Rev Microbiol 10:743–754 Due to the imperative demand of 5-ALA, the produc- Bunke A, Zerbe O, Schmid H, Burmeister G, Merkle HP, Gander B (2000) Deg‑ tion and purification is exigent in recent years. 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Journal

Bioresources and Bioprocessing – Springer Journals

Published: Jun 16, 2022

Keywords: 5-Aminolevulinic acid; Ion exchange chromatography; Photodynamic therapy; Antimicrobial; Cancer cell; Microalgae

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