METHOD OF CULTURING HAEMATOCOCCUS SPECIES FOR MANUFACTURING OF ASTAXANTHIN

Abstract

A method of culturing Haematococcus species for manufacturing of astaxanthin comprising the steps of: providing a substrate, arranging the Haematococcus species on the surface of the substrate, exposing the Haematococcus species arranged on the substrate to high light intensities from the beginning of a culturing process and avoiding a two-step culturing process of the Haematococcus species with a first step which is an initial culturing taking place by exposure of the Haematococcus species to low light energy followed by a second step of subsequent culturing of the Haematococcus species by exposure of the Haematococcus species to higher light energy than applied in the first step to induce astaxanthin formation, and optionally—harvesting the cultured Haematococcus species and/or—isolating astaxanthin.

Claims

1-9. (canceled)

10. A process for an improved astaxanthin production comprising a biofilm cultivation of Haematococcus on a substrate in a one-step procedure at high light intensity to increase biomass productivity and induce and increase astaxanthin production at the same time, wherein high light intensity is 200 μmol photons m.sup.−2 s.sup.−1 or more.

11. The method of claim 10, wherein the astaxanthin is isolated from harvested Haematococcus species.

12. The method of claim 10, wherein the Haematococcus species is Haematococcus pluvialis.

13. The method of claim 10, wherein the substrate is a sheet-like material.

14. The method of claim 13, wherein the substrate is a porous sheet.

15. The method of claim 13, wherein the sheet-like material is selected from the group consisting of paper, cellulose ester, in particular cellulose acetate, mixed cellulose ester, cellulose, cellulose nitrate, polyamides, polyesters and polyolefins.

16. The method of claim 10, wherein a light/dark cycle of 14/10 hours is used in combination with 5% CO.sub.2 during the cultivation.

17. The method of claim 10, wherein the amount of astaxanthin is higher than 4 g m.sup.−2.

18. The method of claim 10, wherein a low-light exposure is not mandatory, wherein low light is 50 μmol photons m.sup.−2 s.sup.−1 to less than 200 μmol photons m.sup.−2 s.sup.−1.

Description

[0026] FIG. 1 depicts a laboratory-scale Twin-Layer test tube.

[0027] FIG. 2 shows biomass and astaxanthin production by H. pluvialis cultivated at different light intensities.

[0028] FIG. 3 compares biomass (g m-2) and astaxanthin (g m-2) productivity of H. pluvialis of a 10 days one-step process as presented here versus a hypothetical PSBR two-step process combining a “green phase” and a “red phase” of 5 days each.

DETAILED DESCRIPTION OF THE INVENTION

[0029] In WO 2005/010140 A1 a particularly useful method of cultivating microalgae in a biofilm, their growth, and harvesting of astaxanthin is described, in particular a porous substrate for use in the present invention. This reference is incorporated by reference. FIG. 1 depicts a laboratory-scale Twin-Layer test tube. alg—immobilized microalgae, pcm—polycarbonate membrane as a carrier for microalgae, gf—glass fiber mat, air membrane pump for air supply, cm—culture medium as presented by Schultze et al. (2015) to cultivate microalgae in biofilms under various light and CO.sub.2 conditions. Other laboratory-scale modifications of porous substrate bioreactors realizing the principle described in WO 2005/010140 A1 were described by Liu et al. (2013); Murphy et al. (2012); Nowack et al. (2005), incorporated by reference.

[0030] Scaled up developments of this particular technology offering up to a few square meter substrate surface for biofilm cultivation were described by Naumann et al. (2013) and Zhang et al. (2015), all incorporated by reference.

[0031] H. pluvialis was grown using porous substrate bioreactors at low light intensities using devices presented by Zhang et al. (2014), Wan et al. (2014) and Yin et al. (2015), all incorporated by reference.

[0032] Instead of cultivating H. pluvialis in these variations of porous substrate bioreactors, H. pluvialis can be grown immobilized in a biofilm on other substrate, which do not display two major surfaces. This can be plastic or concrete structures, etc., which are supplied with culture medium applied on the surface of the biofilm.

[0033] By use of these cultivation procedures, H. pluvialis can be cultivated and subjected to high light intensities,

[0034] FIG. 2 shows cultivation of H. pluvialis at different light levels with 5% of supplementary CO.sub.2 in an experiment as described in Example 1 below. At low light intensities of 45 μmol m.sup.−2 s.sup.−1 (comparable to the green phase of suspension cultivation) no astaxanthin production and only a moderate biomass productivity is reached. High light intensities of about 530 and 785 μmol m.sup.−2 s.sup.−1 resulted in an increased biomass and astaxanthin productivity in parallel, showing that (i) astaxanthin induction by high light does not impair growth as observed in suspension-type bioreactors, and (ii) production of astaxanthin can be performed in a continuous one step process, which does not suffer from non-productive (green) growth phases. Highest astaxanthin productivities were reached at 785 μmol m.sup.−2 s.sup.−1. High light induced a total astaxanthin productivity of about 0.39 g astaxanthin m.sup.−2 d.sup.−1, which was linear during the experimental period and reached 3.3 g m.sup.−2 after 10 d of growth. At longer cultivation periods, astaxanthin can be higher than 4 g m.sup.−2, and at higher light intensities of 1,013 μmol m.sup.−2 s.sup.−1, astaxanthin productivity is still as high as 0.32 g m.sup.−2 d.sup.−1 (data not shown). A hypothetic two-stage scenario was constructed to compare productivity to the one-stage system according to the invention.

[0035] FIG. 3 compares biomass (g m.sup.−2±SD, n=3; FIG. 3A) and total astaxanthin (g m.sup.−2±SD, n=3; FIG. 3B) productivity of H. pluvialis of a 16 days one-step process as presented here versus a PSBR two-step process combining a “green phase” and a “red phase” of 8 days each in order to compare the efficiency of one and two-phase approaches when light is the only applied stress factor. The one-phase approach consisted of exposure to 1,000 μmol photons m.sup.−2 for 16 days (black squares), whereas the two-phase consisted of 8 days at 90 μmol photons m.sup.−2 s.sup.−1 and 8 days at 1,000 μmol photons m.sup.−2 (grey squares). Vertical dotted line indicates when light intensity was switched for the two-phase approach. Light/dark cycle was 14/10 hours and aeration was supplemented with 5% CO.sub.2 during the whole cultivation period. Biomass increased linearly in each phase of cultivation, but at very different rates (FIG. 3A). A growth rate of 5.4 g m.sup.−2 d.sup.−1 was observed at low light and it increased to 17.2 g m.sup.−2 d.sup.−1 when switched to high light. This value was similar to that obtained during the first 8 days of cultivation directly at 1,000 μmol photons m.sup.−2 s.sup.−1 in the one-phase approach. Also, at day 8, standing crop was three fold higher in the one-phase approach, with the value of 146 g m.sup.−2, when compared to 48.2 g m.sup.−2 of the two-phase. Similar trends were observed when analysing total astaxanthin values: Accumulation in the two-phase group only occurred at high light, in a similar rate to that observed for the first eight days of the one-phase: 0.34 and 0.35 g m.sup.−2 d.sup.−1, respectively (FIG. 8B). At day 8, astaxanthin yield was 2.5 g m.sup.−2 at 1,000 μmol photons m.sup.−2s.sup.−1 and 0.08 g m.sup.−2 at 90 μmol photons m.sup.−2 s.sup.−1, which results in a 32-fold increase. This difference was abolished after the exposure to high light and astaxanthin production reached 2.8 g m.sup.−2 in both groups at day 16. However, in the two-phase system it required twice as long as in the one-phase approach. The effect of the high irradiance was, therefore, independent of the beginning of the exposure. That is, the use of a low light green phase hindered the high productivities, requiring a longer cultivation period to reach similar yields, which increases costs and contamination risks.

[0036] According to the invention, after cultivation, H. pluvialis can be loosened from the support, in particular a perforated support, by the effect of mechanical forces such as scraping or detachment by an air blade or other suitable tools to blow off materials, or, by chemical treatment such as a treatment with surfactants and/or organic solvents.

[0037] In another embodiment, the H. pluvialis can be harvested together with the perforated support, This may be practical if the H. pluvialis are decomposed remaining on the support so as to obtain ingredients by extraction, for example. The extracted H. pluvialis or cellular debris can be separated mechanically from the extract together with the support.

[0038] In yet another embodiment, (low) volumes of liquid (e.g. water, culture medium) can be used to wash off the immobilized H. pluvialis from the perforated support, obtaining a dense suspension of H. pluvialis for further processing (concentration, drying, and/or extraction of astaxanthin) or H. pluvialis may be obtained by collecting loosened biomass in flowing culture medium.

[0039] In particular, H. pluvialis can be loosened from the support after drying and may then be collected.

[0040] In another embodiment, H. pluvialis can be loosened from the support and used after drying or without drying, or without extraction of astaxanthin,

[0041] In still another embodiment, extraction of astaxanthin can be performed by treatment with chemicals such as solvents in particular organic solvents, when H. pluvialis remains on the substrate and astaxanthin is removed from H. pluvialis with the chemicals.

[0042] In particular, extraction of astaxanthin from dried or concentrated H. pluvialis cells can be performed by methods utilizing organic solvents (Dong et al., 2014) such as hydrochloric acid pretreatment followed by acetone extraction, hexane/isopropanol mixture solvent extraction, methanolic extraction followed by acetone extraction, or by other natural oils such as soy-oil or palm oil. Astaxanthin furthermore can be obtained by supercritical extraction using carbon dioxide (e.g. Nobre. et al., 2006) following to crushing of H. pluvialis cells.

[0043] The invention is described by the following non-limiting examples.

EXAMPLE 1

[0044] Experimental Setup

[0045] Bench Scale Biofilm Photobioreactors

[0046] The bench scale Twin-Layer photobioreactor (PBR) used was described by Schultze et al (2015), incorporated by reference. Briefly, the system consists of a glass fibre mat (50×10 cm) placed vertically inside a transparent PMMA tube (50 cm long, 12 cm diameter) on a polyvinylchloride (PVC) support, Culture medium is constantly circulated by a peristaltic pump. It is applied at the top of the glass fibre, spreading down with gravity and returning to the medium reservoir, placed inside the PVC support. The system is supplied with 1 L of culture medium which is exchanged every 2-3 days to avoid nutrient limitation for algal growth. Aeration is supplied inside the PVC tube.

[0047] For the inoculation of the bioreactor, H. pluvialis suspension cultures were concentrated and then filtrated onto polycarbonate membranes (PC40, 0.4 μm pore size, 25 mm diameter, Whatman, Dassel, Germany) as described by Naumann et al (2013), incorporated by reference, to obtain an initial biomass density of 5 g m.sup.−2, Filters were then placed on the wet glass fibre mats on the PBRs.

[0048] For the one-step approach, nutrient-rich culture medium was used throughout the experimental period and different light intensities were evaluated. For comparison, a two-step experiment at low light was performed. Complete culture medium was initially used for growth. After 6 days the stress was induced by changing the medium, namely leaving out the nitrogen source and/or additional salt (0.8%).

[0049] Sampling and Determination of Dry Weight

[0050] At each sampling point, at least three filters were collected from each PBR. Biomass that had overgrown the inoculation area was removed and the filter was freeze dried to constant weight. Dry weight was determined gravimetrically and biomass was stored at −20° C. until astaxanthin analysis.

[0051] Astaxanthin Determination

[0052] Astaxanthin was determined spectrophotometrically as described by Li et al (2012). Freeze dried biomass samples were extracted with Dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany), incubated at 70° C. for 5 minutes then centrifuged at 4000 g for 5 minutes. Extraction was repeated until a colourless pellet was obtained. Supernatants were collected and the OD was measured at 530 nm (Infinite M200 plate reader, pecan, Mannedorf, Switzerland). When necessary for complete extraction, cells were broken by grinding with sand. Astaxanthin concentration was determined based on a calibration curve constructed with astaxanthin standard (98.6% purity, Dr. Ehrenstorfer, Ausburg, Germany) dissolved and diluted in DMSO.

[0053] Furthermore, other artificial or natural culture media which are suitable to promote microalgal growth can be used for H. pluvialis cultivation.

[0054] Supplementary CO.sub.2 is advantageous to promote biomass growth in particular under high light conditions, however, is not essential.

[0055] Light sources, in general can be artificial illumination and use of natural sunlight, whereas the use of sunlight may be preferred for economic reasons (in particular if high intensities are required).

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