Chemicals and organisms


All media components for the cultivations with Penicillium ochrochloron were purchased from Merck (Darmstadt, Germany) with the exception of HEPES (Roth, Darmstadt, Germany). The reference antibiotic tigecycline and the 96-well plates were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Antibacterial susceptibility testing was performed with Mueller Hinton Bouillon (MHB, Becton Dickinson, New Jersey, USA) as a medium. All solvents were obtained from VWR International (Vienna, Austria). Ethyl acetate and acetone were additionally distilled according to the ÖAB (Österreichisches Arzneibuch). Ultra pure water was obtained utilizing the Sartorius arium® 611 UV purification system (Sartorius AG, Göttingen, Germany). Silica gel 40–63 μm and pre‐packed cartridges for flash chromatography were purchased from Merck (Darmstadt, Germany) and Büchi (BÜCHI Labortechnik AG, Flawil, Switzerland), respectively. Thin-layer chromatographic analysis was performed using silica TLC plates 60 F254 (Merck, Darmstadt, Germany).


Petri dish and bioreactor batch experiments were performed with Penicillium ochrochloron CBS 123823. The bacteria used for antimicrobial testing were Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 obtained from the American Type Culture Collection (ATCC®, Virginia, USA) as well as a methicillin-resistant Staphylococcus aureus strain (MRSA, clinical isolate, wild-type), and Enterococcus faecium strain resistant to linezolid and vancomycin (LVRE, clinical isolates, wild-type). For photoantimicrobial assays, E. coli (DSM1103, DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and S. aureus DSM1104 (DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were used.

(Photo)Chemical characterisation of xanthoepocin


For the isolation process the following instruments were utilized: The mill MF10 basic (IKA Labortechnik, IKA®-Werke GmbH & Co. KG, Staufen, Germany), the balances KERN ALS 220-4 (KERN & SOHN GmbH, Balingen-Frommern, Germany) and Sartorius Cubis®-series (Sartorius AG, Göttingen, Germany), the rotary evaporators Heidolph LABOROTA 4000-efficient, Heidolph Hei-VAP Precision (Heidolph Instruments GmbH & CO. KG, Schwabach, Germany), and IKA RV 10 (IKA®-Werke GmbH & Co. KG, Staufen, Germany). Furthermore, the ultrasonic bathes Sonorex RK 106, Sonorex RK 52, and Sonorex TK 52 (BANDELIN electronic GmbH & Co. KG, Berlin, Germany) were utilized and the flash-chromatography system Reveleris® X2 (BÜCHI Labortechnik AG, Flawil, Switzerland). HPLC measurements were carried out using the modular system Agilent Technologies 1260 Infinity II and the Agilent Technologies 1200 Series from Agilent Technologies, Inc. (Santa Clara, USA). For preparative HPLC chromatography, a Dionex (Thermo Fisher Scientific Inc., Waltham, USA) HPLC-system, consisting of a Dionex UltiMate 3000 pump, an ASi-100 Automated Sample Injector, a Dionex UltiMate 3000 Column Compartment, a Dionex UVD170U, and a Gilson 206 Fraction collector in combination with a Synergi MAX-RP 80 Å column (250 × 10.00 mm, 4 micron) from Phenomenex (Aschaffenburg, Germany) was used. NMR spectra were acquired using two spectrometers from Bruker, an Avance II 600 spectrometer operating at 600 MHz (1H) and 151 MHz (13C) and an Avance III HD spectrometer operating at 400 MHz (1H) (Bruker Corporation, Billerica, USA). The spectra were recorded in deuterated solvents supplied by Euriso-Top (Cambridge Isotope Laboratories, Inc., Saint-Aubin, France). Optical rotation data was acquired on a JASCO P-2000 polarimeter (Jasco Deutschland GmbH, Pfungstadt, Germany). IR spectra were recorded on an ALPHA FT-IR apparatus (Bruker, Ettlingen, Germany) equipped with a Platinum ATR module.

Isolation of xanthoepocin

The dried bioreactor batch mycelium (see also “Submerged cultures—bioreactor batch experiments” Section) was fine-milled with an electric mill (MF10 basic, IKA, Germany) at 3750 rpm to the smallest grain size and transferred (60 g) into an Erlenmeyer flask (500 mL) covered with aluminum foil. The mycelium was first defatted with petroleum ether (3 × 200 mL, 30 min ultrasonication each) and afterward extracted with acetone (3 × 200 mL, 30 min sonication each). The acetone fractions were combined and reduced under vacuum. The resulting ochrous, viscose liquid was mixed with C18 material and submitted to flash chromatography (Reveleris®) using a C18 column (40 µm, 80 g). As the mobile phase, acidified water (H2O + 0.1% HCOOH) and acetonitrile (ACN + 0.1% HCOOH) were used. As gradient, the following system was used: t = 0 min, 10% B–> t = 10 min, 90% B–> t = 30 min, 90%B. The blue light-absorbing fractions were combined, reduced, and yielded a dark yellow solid, xanthoepocin (m = 142.6 mg). Further purification was done via preparative HPLC employing a MaxRP 80 Å (4 µm, 850 × 10 mm) column from Synergy (Phenomenex, Aschaffenburg, Germany). The following gradient system was employed: t = 0 min, 10% B –> t = 7.5 min, 90% B –> t = 20 min, 90% B. As mobile phase, water and acetonitrile were used. During the entire process, light was excluded as much as possible.

HPLC analysis of the extracts

The concentrated extract derived either from extracted cultures of the petri dish experiment (see “Solid state cultures—petri dish experiments” Section), or the bioreactor experiment (see “Submerged cultures—bioreactor batch experiments” Section) was solved in DMSO (V = 1 mL), filtered (PFTE, 0.45 µm), and submitted to an HPLC–DAD analysis utilizing a Synergi MaxRP (80 Å, 150 × 4.60 mm) column and water as well as acidified acetonitrile (ACN + 0.1% HCOOH) as mobile phase. The gradient started at 50% B, was increased to 85% in the first 7.5 min, and further increased to 98% B until t = 20 min. A needle wash and a post and pre-run washing step (5 and 10 min, respectively) was implemented between the measurements. The DAD chromatogram (λ = 396 nm) was extracted and integrated via Origin 2020 (OriginLab Corp., Northampton, USA).

Dynamic light scattering experiments

A stock solution of xanthoepocin (5 mM, DMSO) was prepared and diluted with MHB medium (1:10 and 1: 1000 yielding a c = 500 µM and c = 5 µM solution). The size distribution was measured at T = 25 °C. The medium without analyte was analyzed as blank.

To determine the critical aggregation concentration (CAC) the stock solution was diluted in PBS and the count rate was measured in triplicates at T = 25 °C. The average of each count rate was corrected by the attenuation factor and plotted against the concentration. Linear fitting was done with Origin 2020 (OriginLab Corp., Northampton, USA).

DMA assay

The DMA assay was performed utilizing 9,10-dimethylanthrance (DMA) solubilized as chemical probe in ethanol as previously described [26]. In short, xanthoepocin was solved in DMSO and diluted with ethanol. The chemical probe DMA, or the probe with ascorbic acid, or ascorbic acid alone (c = 1 g mL−1) were added as well. As a reference, berberine (c = 1 mg mL−1, 2.97 mM, DMSO, V = 10 µL) was used. Before and after each irradiation step (5 cycles, each λirr = 468 nm, H = 1.24 J cm−2 min−1, t = 5 min) the optical densities at λ = 377 nm and λ = 468 nm were recorded as technical duplicate. The observed difference in the DMA absorbance was determined for each sample set.

Emission and absorbance measurements

A xanthoepocin solution was prepared in deuterated methanol, and the absorbance coefficient calculated from five concentrations (below 150 µM). To guarantee an equal distribution, each solution was filtered (400 µm) immediately before the spectroscopic measurement. A xanthoepocin solution in methanol was prepared with an OD of approximately 0.1 at the detection wavelength of λ = 450 nm. The visible light emission of xanthoepocin and the near-infrared emission of singlet oxygen was detected at λ = 1270 nm as previously reported [46]. An absorbance measurement (200–800 nm) before and after excitation was recorded to assure the stability of xanthoepocin. As an excitation source, a 450 nm CW laser diode was utilized (0.4 W/cm2). A solution of [Ru(bpy)3]Cl2 in deuterated methanol served as reference (ΦΔ = 0.73).

Decomposition studies

Initially, a solution of xanthoepocin was placed at the window and submitted to a HPLC measurement after t = 5 min and ten days. Having observed considerable effects of sunlight already after five minutes, a more thorough experiment was conducted: A solution of xanthoepocin in d6 DMSO (39.3 mg mL−1) was irradiatated with blue light (λ = 455 ± 30 nm, 0.64 mW cm−2) in a 96 well plate for 20 min. Samples for the HPLC-analysis (V = 200 µL) were withdrawn every minute for the first five minutes, then every second minute until 10 min, and finally after 15 and 20 min. The samples were anylzed by HPLC–DAD with the established method (see HPLC analysis).

Light stability measurement

Light stability of xanthoepocin was measured in deionized water. A xanthoepocin stock solution (c = 1 mg mL−1) was prepared. A part of the stock solution (V = 0.30 mL) was diluted with water (V = 9.7 mL) to a final concentration of c = 30 µg mL−1. The sample (final xanthoepocin concentration c = 49 µM) was deoxygenated with argon for 20 min prior to the measurements. The samples were stirred under argon and kept at 297 K for the duration of the experiment. Irradiation was performed using a custom 413 nm LED (0.86 mW, 95 min, 5 J cm−2), and UV–Vis absorbance spectra were measured every 30 s. In parallel, an identical solution was studied in the dark.

Solid state cultures—petri dish experiments


To perform the explorative screening experiments, agar plates with 20 mL of either a glucose-limited, ammonium-limited, or phosphate-limited medium were prepared according to Additional file 1: Table S1. Glucose and salt solutions were autoclaved separately to avoid the formation of toxic compounds. All media were set to pH 7 before autoclaving and were combined under sterile conditions after cooling down to room temperature. The trace element solution (composition see [31]) was added sterile filtered.

Cultivation and illumination conditions

For the screening experiment, each petri dish was inoculated three times with a needle, which was dipped into a spore suspension with a spore density of 5 × 108 spores. Afterward, each inoculated petri dish was placed in separate light-impermeable carton boxes (Additional file 1: Fig. S12) and continuously irradiated with one light-emitting diode (LED; Additional file 1: Fig. S13A) for seven days in a climate chamber at 25 °C. Spectrum (Additional file 1: Fig. S13B) and intensity (Additional file 1: Table S2) of the illumination conditions was determined with a radiometer (Thorlabs PM100D, Silicon Power Head 200 nm–1100 nm) and a spectrometer (Ocean Optics Maya 2000 Pro, Grating HC-1 200 nm to 1050 nm, Optical Fiber 600 µm), respectively. After seven days of incubation, the cultures were photo-documented, and the colony diameters were determined before the colonies were extracted.

Xanthoepocin extraction of fungal colonies from solid media

Variations in colony morphology, (i.e., glucose-limited grown cultures grew flat and loose, whereas ammonium- and phosphate-limited cultures grew dense and slightly elevated), made it necessary to adjust the way the colonies were detached from the agar plates. Glucose-limited grown colonies were cautiously scratched from the surface of the agar plate with a scalpel. This was not possible with ammonium- or phosphate-limited colonies, which had to be excised from the agar plate with the scalpel and any agar remnants carefully removed afterward. Independent of the medium, the single colonies were then transferred to 10 mL amber-colored glass vials, and 5 mL acetone was added for xanthoepocin extraction. For additional light protection during the extraction step, the vials were covered with aluminium foil and ultrasonicated for 15 min. Afterward, the extract was filtered through a cotton-filled Pasteur pipette into a second amber-colored glass vial, which was also covered in aluminium foil. Before the subsequent evaporation step, the foil was permeated with a needle. The acetone was evaporated in a constant air stream overnight until only the pure extract remained in the vial.

With this method, the whole colony had to be extracted, and thus the obtained xanthoepocin values could not be attributed to a normalized biomass value. Nevertheless, the obtained data allowed valuable first insights into a potential nutrient- and light-dependent xanthoepocin production since ammonium- and phosphate-limited grown cultures showed cultures with comparable sizes (Additional file 1: Fig. S14) and the xanthoepocin levels of glucose-limited cultures were significantly lower (Fig. 2).

Submerged cultures—bioreactor batch experiments

Preculture and bioreactor batch medium

To obtain a filamentous mycelium in the preculture, P. ochrochloron was cultivated in a Glucose-HEPES medium with 1 M HEPES (pH 7.3) for 72 h at 30 °C on a rotary shaker at 350 rpm as described earlier [29]. Each bioreactor batch cultivation was inoculated with three precultures.

Since the screening experiments revealed that the xanthoepocin content was highest under ammonium-limited conditions, this nutrient limitation was chosen for the bioreactor batch experiments. Similar to the petri dish experiments, glucose and salt solutions (composition ammonium limited medium see [29]) were autoclaved separately and combined under sterile conditions after reaching room temperature. 10 mL trace element solution per liter medium (composition see [31]) was filtrated (0.2 µm) and added under aseptic conditions.

Cultivation in bioreactors and illumination conditions

All bioreactor batch experiments were performed in a Biostat B bioreactor (Braun Sartorius, Germany) with a working volume of 4.6 L at 25 °C, 890 rpm, and at an aeration rate of 0.56 vvm as previously described [29]). The pH was kept constant at pH 7 with sterile 5 M NaOH.

The bioreactor batch experiments were performed with four individual illumination settings, meaning three different irradiation scenarios and one cultivation in the absence of light as control. Each light setting varied in intensity and spectral composition, as it is shown in Additional file 1: Fig. S16. White light conditions were realized with the existing laboratory light (fluorescent tubes, Philips TLD 18 W/33–640). Blue light together with red light conditions were achieved with single color high flux light-emitting diodes (blue: Osram Opto Semiconductors LD CQDP-2U3U-W5-1 and red: Osram Opto Semiconductors LH CPDP-2T3T-1–0). Details of the used light sources and prevailed intensities on the outside of the bioreactor vessel are summarized in the Additional file 1: Table S3.

To ensure constant irradiation conditions, an efficient thermal management of the LEDs was necessary. Therefore, the LEDs were placed on an aluminum core circuit board to guarantee sufficient heat dissipation. To avoid any unwanted light contamination, the bioreactor vessel was covered with fabrics impermeable to light (Additional file 1: Fig. S16A). Light intensity was measured with a radiometer (Thorlabs PM100D, Silicon Power Head 200 nm–1100 nm, wavelength settings according to weighted response curve were 451 nm, 570 nm, and 660 nm for blue, white, and red light conditions, respectively) at different positions outside the vessel and was determined as the mean value of each measurement point. The spectral composition was gained with a modular spectrometer (Ocean Optics Maya 2000 Pro, Grating HC-1 200 nm to 1050 nm, Optical Fiber 600 µm). The corresponding values of the photon flux density were calculated whilst taking the spectral composition of the light source into account.

Sampling and harvesting

The bioreactors were sampled regularly, whereby for each sampling time point, triplicate samples were withdrawn from the bioreactor. The triplicate samples (5 mL each) were filtered with a vacuum pump (Millipore, Darmstadt, Germany) through a preweighed cellulose acetate filter (Braun Sartorius, Germany) with a pore size of 0.45 µm. Afterward, the obtained filtrate was aliquoted into 1.5 mL tubes and stored at -20 °C until later nutrient analytics. In addition to the triplicate samples, 10–15 mL of the culture broth was filled in 15 mL falcons and also stored at -20 °C until later analysis. Residual nutrient concentrations were measured as described elsewhere [47]. The filters with the biomass were dried at 105 °C to estimate the dry weight.

After five days of cultivation (approximately 92 h), the bioreactor batch cultivations were stopped. The culture broth was harvested and filtered through a dish towel or fabric sheet into a bucket. The resulting mycelium was wrung by hand to remove most liquid, then laid out flat on a tablet and dried at 40 °C overnight. The dried mycelium and the filtrate were stored separately at -20 °C until further use.

Xanthoepocin analytic of dried mycelia

The dried mycelium was ground with an electric mill (MF10 basic, IKA, Germany) at 3750 rpm to the smallest grain size before it was sieved to 0.5 mm, which provided a homogeneous grain size. Approximately 25 mg (exact weight noted) of the ground mycelium was filled in 1.5 mL amber-colored eppendorf tubes in triplicates.

To each tube, 1 mL acetone was added. In the next step, all tubes were exposed to ultrasonication for 15 min and then centrifuged (5804 R, Eppendorf, Germany) at 14,000 rpm for 10 min at 4 °C. The resulting supernatant, which contained xanthoepocin, was decanted into amber-colored glass vials and covered with perforated aluminum foil. This ultrasonic-assisted acetone extraction was again repeated twice with the mycelial pellet in the tubes. All three resulting supernatants of one mycelium were collected in the same glass vial (i.e., each vial contained eventually 3 mL acetone extract) before it was placed under an air stream until the acetone was evaporated entirely.

Photo-documentation of the culture broth

To photo-document pigmental changes during the cultivation process, the culture broth of each sampling time point was thawed, and one milliliter was transferred into micro cuvettes. The micro cuvettes of each bioreactor batch experiment were then arranged in the order of the sampling times and photographed in a standardized setting.

Confocal microscopy

A suspension of P. ochrochloron in PBS was analyzed under the confocal microscope (Leica TCS SPE5 II) utilizing an excitation beam fitting the absorbance range (i.e. λ =  ± 390 nm) and a emission filter fitting the emission range (i.e. λ =  ± 516 nm) of xanthoepocin respectively. An 40 × magnification was utilized, and the pictures were taken shortly after focusing the sample to avoid a photobiological reaction.

Antibacterial susceptibility tests

All bacteria for susceptibility testing were grown overnight on BD Columbia Agar plates (Becton Dickinson, New Jersey, USA) at 37 °C.

The evaluation of the MIC of xanthoepocin and tigecycline was performed by a serial dilution according to the Clinical and Laboratory Standards Institute (CLSI, 2012), which defines the MIC as the lowest concentration of a compound leading to a visible growth inhibition after overnight incubation. For this purpose, all tested bacterial cultures were adjusted to 0.5 McFarland standard turbidity in saline, which corresponds to a bacterial suspension with a concentration of 1.5 × 108 colony-forming units per milliliter (CFU mL−1). Subsequently, 50 µl of the bacterial suspension was added to 10 mL MHB.

The antibacterial activity of xanthoepocin was tested against the antibiotic tigecycline. The preparation of the stock solutions was done by dissolving xanthoepocin or tigecycline in DMSO at a concentration of 1 mg mL−1 and subsequent 1:10 dilution in sterile water. Afterward, a serial dilution was conducted in a flat-bottomed 96-well tissue plate with concentrations ranging from 10 to 0.01 µg mL−1. After the addition of 90 µl of the bacterial suspension into each well, the plates were incubated at 35 ± 2 °C overnight.

Prior to the determination of the optical density (OD), the plates were shaken at 250 rpm for 15 min. The OD was measured at 490 nm using the Bio-Rad 680 microplate reader (Hercules, California, USA). Growth inhibition in the presence of xanthoepocin and tigecycline, respectively, was assumed in case growth was reduced for at least 5 log units. Diluted DMSO (1:10, v/v) without xanthoepocin or tigecycline, respectively, served as growth control.

All procedures for bacterial susceptibility testing were performed twice under analogous conditions for each bacterial strain.

Photoantimicrobial susceptibility testing

The photoantimicrobial experiments were carried out as described previously [30]. In brief, a dilution series of xanthoepocin was prepared in MHB according to the CLSI guidelines (CLSI, 2012), and transferred into two identical 96-well plates. The 96-well plates for the microdilution tests were always prepared freshly. The inoculum was prepared from an overnight culture of S. aureus (DSM1104) or E. coli (DSM1103) and adjusted to a McFarland standard of 0.5 via turbidity measurement (λ = 600 nm). After dilution by 1:100 with MHB, 50 µL of inoculum was given to 100 µl medium and 50 µl of diluted fraction into each well.

One plate served as dark condition control. After ten minutes of incubation, the second plate was irradiated with blue light (λ = 428 ± 15 nm, H = 30 J cm−2 or 9.3 J cm−2). Afterward, the plates were incubated at T = 37 °C in the dark for 24 h. Assessment of the experiment was done by correlating the turbidity of the treated wells to the uninhibited growth control. Irradiated and dark samples were evaluated separately.

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