Method for the biotechnological production of the blue-green fungus pigment xylindein

Abstract

The invention relates to a method for the biotechnological production of the blue-green fungus pigment xylindein from fungus biomass in a bioreactor, the reactor contents being inoculated with biomass that is uncoloured. The invention also relates to the use of uncoloured biomass of the fungus culture Chlorociboria sp. for inoculation in the biotechnological production of the blue-green fungus pigment xylindein.

Claims

1. A method for the biotechnological production of the blue-green fungus pigment xylindein in a bioreactor, comprising the steps of (a) bringing the reactor contents into contact with a biomass of the fungus culture Chlorociboria sp. and stirring the reactor contents to form xylindein-containing biomass, (b) separating the xylindein-containing biomass which formed in step (a), and (c) extracting the xylindein from the biomass separated in step (b) using a solvent, wherein the biomass with which contact is made in step (a) is an uncoloured biomass.

2. The method according to claim 1, wherein the solvent in step (c) is selected from the group consisting of acetone, 2-butanone and a mixture thereof.

3. The method according to claim 1, wherein after the separation of the xylindein-containing biomass in step (b), dry and/or comminution of the biomass takes place and before step (c).

4. The method according to claim 1, wherein the reactor contents in step (a) comprise a nutrient medium which contains up to 50 vol % food residues.

5. The method according to claim 1, wherein the reactor contents in step (a) comprise a nutrient medium which contains a 1 vol % to 20 vol % orange juice solution.

6. The method according to claim 1, wherein the xylindein obtained in step (c) is purified by the additional steps of (d) drying the xylindein and redissolving it in a water-soluble organic solvent, and (e) precipitating purified xylindein by adding water to the mixture from step (d).

7. The method according to claim 1, wherein further xylindein is isolated by ultrafiltration of the liquid culture supernatant remaining in step (b) after separation of the biomass.

8. The method according to claim 1, wherein the fungus culture Chlorociboria sp. is selected from the group consisting of Chlorociboria aeruginascens and Chlorociboria aeruginosa.

Description

(1) FIG. 1 shows the curve of numerous parameters, including the pH over the cultivation time, on the basis of an embodiment.

(2) FIG. 2 shows the curve of the pH in the case of inoculation (of a main culture) with coloured or uncoloured fungus biomass, on the basis of an embodiment.

(3) FIG. 3 shows, by way of example, the sequence of the method according to the invention in an embodiment, comprising precultivation (first, second and third part from the left-hand side) and main culture (right-hand part), inoculation (contact) with uncoloured biomass being carried out initially in each stage.

(4) All embodiments of the invention can be combined with one another in any way.

EMBODIMENTS

(5) The invention is illustrated by the following embodiments without being limited thereto.

Embodiment 1: Precultivation of the Xylindein-Producing Fungus Chlorociboria sp.

(6) The preculture for the process described in embodiment 1, step a. for producing blue-green fungus biomass took place in a plurality of cultivation steps. In this case, cultivation was initially carried out on a Petri dish scale in order to maintain the strain (maintenance of the fungus culture strain). A 50 vol. % orange juice agar consisting of 50 vol. % orange juice (100 vol. % orange juice is understood to be orange juice having a fruit content of 100 vol. %) and at least 30 g/L agar-agar (also referred to as agar, Chinese/Japanese gelatine or Japanese isinglass) was used for this purpose. The nutrient medium was autoclaved at 121 C. for 15 minutes. The poured agar plates were inoculated using an inoculation piece (plaque, 1 cm.sup.2) of an older or acquired strain plate and incubated at 20-22 C. From this cultivation, two plaques were transferred into the shake flask scale and cultivated in an aqueous nutrient medium consisting of 5 vol. % orange juice. A total of 200 mL of precultivated fungus biomass suspension (preculture solution) was then transferred from the shake flask culture into a 3 L batch reactor (cultivation medium: 5 vol. % orange juice solution) and further cultivated. The pH was monitored continuously, and samples of the culture solution were taken regularly to determine the colour of the fungus biomass. It was cultivated until the increase in pH lessened and the pH began to decrease again. At the same time, the L value from the colour determination approached the limit at which a change from uncoloured to coloured biomass occurred. The cultivation of this preculture was stopped at L=85 (luminance value). Table 1 shows the values of the colour determination of the biomass by means of the Lab colour space and RGB colours. The method for determining the colour of the fungus biomass and the required pre-treatment is described in the following section.

(7) During pre-cultivation, it is necessary to transfer the fungus into the next larger scale during the exponential growth phase. For this purpose, for the fungus Chlorociboria aeruginascens IHIA39, 7 days was determined for the shake flask culture and 7 days was also determined for the 5 L cultivation. This reduced the pre-cultivation time by at least 50% in comparison with the use of coloured, xylindein-containing biomass as inoculum.

(8) Method for Determining the Colour of the Fungus Biomass

(9) After a sample of the liquid culture was taken, the biomass contained was separated off by filtration, washed using distilled water and dried (directly on the filter paper) at 60 C. for 24 hours in a drying oven. On the basis of the dried biomass, the colour in the Lab colour space was determined by means of a spectrophotometer (Datacolor ELREPHO). The determined values are average values of 5 measured values. A conversion into, for example, RGB colours is possible using one of the available databases (for example http://www.cielab-farben.de/farbdatenbank.html).

(10) TABLE-US-00001 TABLE 1 Determination of the colour of the biomass in different stages Values are listed for uncoloured biomass (for inoculation), for biomass when the colour changes (from uncoloured biomass to coloured biomass) and for strongly blue-green coloured biomass. R G B L a b Inoculation 215 212 194 85 1 9 Colour change 160 165 126 67 7 20 Blue-green 109 144 116 56 16 11 colouration 85 111 97 44 11 5 An L value (luminance = lightness) of approx. 70 was determined as the limit for distinguishing between coloured and uncoloured biomass.

Embodiment 1Step a: Producing Blue-Green Fungus Biomass on a 70 L Scale

(11) For the main cultivation for producing blue-green fungus biomass containing xylindein, a 70 L bioreactor (Applikon Biotechnology B.V.) was supplied with 55 L of 5 vol. % orange juice in water. The nutrient medium was heat-sterilized at 123 C. for 30 min (Sterilization in Place, SIP) in the bioreactor, and the process parameters (as listed in Table 2) were set. 5 L of preculture with uncoloured biomass (from embodiment 1, precultivation) were transferred to the reactor and cultivated for 14 days. By means of process monitoring, the point in time of the colour change of the fungus biomass from uncoloured to blue-green was determined by online measurement of the pH. During the cultivation, the pH increased from pH 4 to pH 4.2. From about day 4, the pH decreased slightly to 4.1 and increased again to pH 4.5 from day 5 to day 6. This pH behaviour indicates the colour change of the culture.

(12) The cultivation parameters used are listed in Table 2.

(13) TABLE-US-00002 TABLE 2 cultivation parameters of a preculture and the main culture of Chlorociboria sp. for the biotechnological production of the blue-green fungus pigment xylindein. Production of the preculture Precultivation Precultivation Precultivation Main step 1 in step 2 in step 3 in cultivation Description baffled flask 3 L bioreactor 7 L bioreactor 70 L bioreactor Volume 500 mL 3 L 7 L 70 L Working volume 200 mL 2 L 5.5 L 55 L H/D ratio 1.5 1.8 2.2 Stirrer type Orbital Disc stirrer Disc stirrer Disc stirrer Number of stirrers 2 3 3 Stirrer diameter 4.8 cm 4.9 cm 10 cm Stirrer speed 120 rpm 150 rpm 150 rpm 100 rpm Internal fittings 3 baffles 3 flow 3 flow 4 flow disrupters disrupters disrupters Sparging type Membrane Ring sparger Ring sparger Ring sparger Sparging rate 0.5 vvm 0.5 vvm 0.27 vvm Temperature control Shaker Rod/heating Double Double casing casing casing Temperature 22 C. 22 C. 22 C. 22 C. Inoculum 2 1 cm.sup.2 200 ml 500 ml 5 L KLa value 4.5 h.sup.1 7.9 h.sup.1 4.7 h.sup.1 Tip speed 0.38 m/s 0.46 m/s 0.52 m/s Power requirement 0.02 kW/m.sup.3 0.04 kW/m.sup.3 0.02 kW/m.sup.3

(14) TABLE-US-00003 TABLE 3 comparison of the results of the biotechnological production of the blue-green fungus pigment xylindein in the case of inoculation with coloured or uncoloured biomass Comparative example Embodiment Inoculation with Coloured biomass Uncoloured biomass Source Literature Own research (Boonloed et al. 2016). Volume 0.25 L 55 L Cultivation time 10 weeks 2 weeks (main culture) Productivity 3.5 mg/L/d 4.8 mg/L/d Amount of xylindein 62 mg 3.7 g produced

(15) The productivity indicates the amount of xylindein that was produced per litre of reactor contents per day. It is determined by weighing the xylindein produced in the method and dividing by the volume of the culture medium and the number of days of contact and stirring (step a.).

(16) Accordingly, the yield is defined as the mass of xylindein produced divided by the volume of the reactor contents.

Comparative Example 1bInoculation with Coloured Biomass

(17) All cultivations were also carried out in parallel using coloured, i.e. xylindein-containing biomass. These comparative examples were carried out analogously to the above regulations, with the difference that, after the first inoculation with the inoculation piece, it was only inoculated with coloured biomass.

(18) TABLE-US-00004 TABLE 4 comparison of the results in the complete cultivation chain from the shake flask to the 70 L bioreactor. Comparative example Embodiment Inoculation with coloured uncoloured biomass biomass Source Own research Own research Process time 6 weeks 4 weeks Preculture (shake flask) 2 weeks 1 week Preculture (7 L bioreactor) 2 weeks 1 week Main culture (70 L bioreactor) 2 weeks 2 weeks Onset of colouration of the main 6 d 4 d culture after Absorption values of the 0.063 0.115 supernatant of the main culture at 640 nm after the end of cultivation (correlated with the xylindein concentration)

(19) As can be seen in Table 4, as a result of the inoculation with uncoloured biomass, the process time of 6 weeks could be reduced by a third to 4 weeks, while the onset of colouration occurred more quickly at the same time. The absorption values of the supernatant at 640 nm after the end of cultivation (correlate with the xylindein concentration in the supernatant) show that more xylindein was produced when inoculating with uncoloured biomass than when inoculating with coloured biomass. This advantage is also evident with regard to the amount of xylindein contained in the fungus biomass.

Embodiment 1Step b: Preparing the Xylindein-Containing Moist Biomass for Producing Blue-Green Xylindein-Containing Dry Biomass

(20) When the 70 L bioreactor was harvested, the blue-green, xylindein-containing moist biomass was separated from the liquid culture supernatant by filtration. A filter bag made of polypropylene woven fabric having a mesh size of 80 m was used for this purpose. The liquid culture supernatant was also coloured blue-green. Xylindein diffused into the medium and residual, particularly small biomass particles were additionally produced from said liquid culture supernatant by means of ultrafiltration (10 kDa membrane).

(21) The blue-green xylindein-containing moist biomass was dried at a maximum of 70 C. for 24 hours by drying in a planar manner on a metal sheet in an oven with a maximum load of 350 g.sub.moist biomass/dm.sup.2. The dried blue-green biomass was processed into powder (particle size<0.5 mm) in an ultracentrifugal mill.

Embodiment 1Step c: Extracting the Xylindein

(22) The powdered fungus biomass was mixed with the extraction agent 2-butanone (MEK). The blue-green fungus pigment xylindein dissolved in the extraction agent. The extract solution was separated from the extraction residue by filtration. The extractant was removed from the extract solution by rotary evaporation. The extract produced in this way was redissolved in 15 mL of solvent 2-butanone (MEK) and diluted with distilled water in a volume ratio of 1:10 (1 part MEK and 10 parts distilled water). The fungus pigment xylindein was precipitated and filtered out. The xylindein was then washed a plurality of times using distilled water and dried at 103 C. for 24 h.

LITERATURE

(23) Stange, S., Steudler, S.; Delenk, H.; Werner, A.; Walther, T.; Bley, T.; Wagenfhr, A. Optimierung der Pigmentbildung vom holzverfrbenden Pilz Chlorociboria aeruginascens, part 1: Biomasseund Pigmentsbildung auf Agar und in Flssigmedien Holztechnologie, 2018, 59, 1, 52-60 Stange, S., Steudler, S.; Delenk, H.; Stange, R., Werner, A.; Walther, T.; Bley, T.; Wagenfhr, A. Optimierung der Pigmentbildung des holzverfrbenden Pilzes Chlorociboria aeruginascens, Teil 1: Pigmentbildung im Holzsubstrat Holztechnologie, 2018, 59, 2, 47-54 Harrison, R.; Quinn, A.; Weber, G.; Johnson, B.; Rath, J.; Remcho, V.; Robinson, S.; Ostroverkhova, O. Fungi-derived pigments as sustainable organic (opto) electronic materials Proceedings of SPIE, 2017, 10101 Weber G. L.; Boonloed A.; Naas, KM.; Koesdjojo M. T.; Remcho V. T.; Robinson S. C. A method to stimulate production of extracellular pigments from wood-degrading fungi using a water carrier Curr. Res. Environm. Appl. Mycol., 2016, 6, 218-230 Weber, G.; Chen, H. L.; Hinsch, E.; Freitas, S.; Robinson S. Pigments extracted from the wood-staining fungi Chlorociboria aeruginosa, Scytalidium cuboideum, and S. ganodermophthorum show potential for use as textile dyes Color. Technol., 2014, 130, 445-452 Robinson, S. C.; Hinsch, E.; Weber, G.; Freitas, S. Method of extraction and resolubilization of pigments from Chlorociboria aeruginosa and Scytalidium cuboideum, two prolific spalting fungi Color. Technol., 2014, 130, 221-225 Boonleod, A.; Weber, G. L.; Ramzy, K. M.; Dias, V. R.; Remcho, V. T. Centrifugal partition chromatography: A preparative tool for isolation and purification of xylindein from Chlorociboria aeruginosa Journal of Chromatography A, 1478 (2016) 19-25 Saikawa, Y.; Watanabe, T.; Hashimoto, K.; Nakata, M. Absolute configuration and tautomeric structure of xylindein, a blue-green pigment of Chlorociboria species Phytochemistry 55 (2000) 237-240