NL2011855C2 - Method for cultivating algae. - Google Patents

Description

METHOD FOR CULTIVATING ALGAE Technical field

The present invention relates to a method for cultivating algae.

Background of the invention

Cultivation of phototrophic microorganisms like algae in open pond systems is well developed but only a few species can be maintained in traditional open systems. Closed photobioreactors (PBR) provide opportunities for algae cultivation with specifically adapted culturing conditions. There are different types of photobioreactors like flat plate-PBRs, annular PBRs, e.g. bubble column PBRs and tubular PBRs. These can be further categorized according to orientation of tubes or panels, e.g. horizontal, the mechanism of circulating the culture, the method used to provide light, the type of gas exchange system, the arrangement of the individual growth units, and the materials of construction employed.

In the photobioreactor the microorganisms are suspended in a liquid medium. Photobioreactors allow the microorganisms to be illuminated either by natural sunlight or by artificial light sources.

Of the many designs of closed photobioreactors, devices with tubular solar collectors are the most promising from a commercial perspective (E. Molina et al.; Journal of Biotechnology 92 (2001); pages 113-131 and D. Briassoulis et al.: Bioresour. Technol. 101 (2010); pages 6768-6777).

However, a remaining obstacle in algae cultivation in tubular photobioreactors is that harvesting of the algae is cumbersome and costly. Currently, centrifugation is widely applied, but this approach requires the purchase of centrifuges and can hardly be automated. Another approach is to use filters which allow semi-automation, but confer a constant risk of becoming clogged.

It is an object of the present invention to overcome obstacles in algae cultivation in tubular photobioreactors.

Summary of the invention

The present inventor came up with the idea to use alkaline flocculation as the harvesting method for algae cultivation in tubular photobioreactors.

Alkaline flocculation has long been considered incompatible with bulk algae production systems such as tubular photobioreactors, because it was believed that the amount of alkali needed would increase as a linear function of the number of algal cells, and for that reason the method was considered technically infeasible as a mechanism of harvesting algae at large scale (refer e.g. Sayre, US20090181438). However, in contrast to previous beliefs, it has been found that the amount of alkali needed increases as a linear function of the logarithm of the number of cells, thus requiring much less alkali as previously expected.

The present disclosure relates to a new way of culturing algae in a tubular photobioreactor, wherein the algae are harvested by means of alkaline flocculation, thereby obviating problems associated with centrifugation and filtration.

Detailed description of the invention

As a first aspect, there is provided for a method for culturing and harvesting algae in a tubular photobioreactor, wherein algae preferably are harvested, i.e. wherein the algae culture is (substantially) dewatered, by alkaline flocculation.

Alkaline flocculation is based on the principle that algal cells generally have a more or less identical surface charge that repels them from one another. Flocculants may block this surface charge by binding, thereby allowing the particles to adhere to each other, generating floes.

Alkaline flocculation can be performed as disclosed for example in Schlesinger et al (2012, Biotechnology Advances 30, 1023-1030), e.g. by adding magnesium hydroxide and/or calcium hydroxide to the algae culture, wherein the pH preferably is at least 8, 9 or even 10 (as measured with pH electrode of Pasco Scientific, Roseville, CA). Schlesinger however does not disclose that alkaline flocculation can be used in combination with tubular photobioreactors. The present inventor found that this specific combination is unexpectedly synergistic.

Preferably, slaked lime-stones or dolomites (containing mixtures of calcium and magnesium hydroxides) can be used as a source of calcium hydroxide and magnesium hydroxide, to further reduce the cost as compared to pure calcium hydroxide and magnesium hydroxide.

Calcium hydroxide and magnesium hydroxide can be used in various weight ratios, but preferred are ratios between 5:1 and 1:5, more preferably between 5:1 and 1:1, or 3:1 and 1:3.

Once floes are formed, the algae can rapidly settle. The mixing of the alkali with the algae is preferably performed just before entering the harvesting tank of the tubular photobioreactor. In this way, the floes can rapidly sediment and removed in a continuous manner from the bottom of tank. Alternatively, dissolved airflotation may be used to direct the floes to the top of the vessel where the cells can be skimmed off.

Optionally, the algal cells can be further concentrated by low speed centrifugation (e.g. 3-10 min at 30-70 g) and/or filtration (e.g. with a filter retaining particles >15-50 pm), and can then be processed by standard techniques known to the skilled person. Flocculation may not only lower the volume of material to be further dewatered, it may also lower the required speed and time of subsequent centrifugation.

Flocculation may remove up to 90% of external water (surrounding the algae cells) in as little as 15 min, resulting in a floe that can be further concentrated easily by low cost sedimentation and/or filtration.

Moreover, preferably, all or part of the (substantially) cell-free medium after flocculation can be recycled back to culture facility, i.e. the at least one transparent tube, ideally after the pH is lowered with e.g. carbon dioxide and/or phosphoric acids.

In a preferred embodiment, the algae are cultured until a cell density of 106, 107, or 108 cells per ml_ is reached, before being harvested.

As a further step of the method, the harvested algae can be subjected to hexane extraction to extract valuable hydrophobic components such as lipids, omega-3 fatty acids, eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, beta-carotene, and/or gamma-linolenic acid.

Any suitable method of hexane extraction can be employed, but preferably it is carried out as for example described in Halim et al (2012, Biotechnology Advances 30, 709-732). For this, the algal mass can be treated with hexane to extract the lipids. Hexane extraction is a process which involves extracting hydrophobic components from material bearing such by treating the material with hexane as opposed to e.g. extracting the lipids by mechanical pressing methods (such as expellers, hydraulic presses, etc.).

The hexane extraction method may recover almost all the lipids and leaves behind only 0.5% to 0.7% residual lipids in the algal material. In the case of mechanical pressing the residual lipids left in the material may be anywhere from 6% to 14%. The hexane extraction method can be applied directly to any lipid containing materials such as harvested algae material.

In a nutshell, the extraction process may consist of treating the algal material with hexane and recovering the hydrophobic components (e.g. lipids) by distillation from the resulting solution of the components in hexane (miscella). Evaporation and condensation from the distillation of miscella recovers the hexane which can be reused for extraction. The low boiling point of hexane (67°C / 152°F) and the high solubility of lipids in it are the properties exploited in the extraction process. For more details on this process reference is made to Halim et al (2012, Biotechnology Advances 30, 709-732).

As mentioned above, the present disclosure concerns (use of) a tubular photobioreactor (PBR), i.e. a so-called closed system, wherein microalgae can be circulated through transparent tubes generally made of polyethylene, acrylic or glass (Brentner et al, 2011, Environmental Science & Technology 45, 7060-7067). Of course, the term “tubular photobioreactor” is meant to mean a photobioreactor of the tubular type and does not require that the whole photobioreactor is of a tubular shape, e.g. the harvesting tank, the pump and/or the degassing unit can have different shapes.

In view thereof, as a further aspect, there is provided for a tubular photobioreactor, suitable for cultivating algae, wherein the tubular photobioreactor preferably is combined with (connected to) a harvesting tank comprising magnesium hydroxide and calcium hydroxide, preferably both at a concentration of between 0.001 to 0.35 g/ml_, more preferably between 0.005-030, or 0.01-0.20 g/ml_.

Preferably the tubes of the PBR according to the present disclosure have a light transparency of at least 60, 70, 80, 90% in the 400-700 nm range (NEN 2675). Further, ideally, a pump is used for the circulation such that enough liquid velocity is created for turbulent flow which is advantageous to mix the algae cells between lighter and darker zones within the tube (Norsker et al, 2011a Biotechnol Adv 29, 24-7), for a homogenous mixture of nutrients and C02, and further to prevent algae settling on the bottom of the tubes.

In a further preferred embodiment of the PBR, the algae stream passes a degassing unit that withdraws the accumulated oxygen using for example an air stream. Removal of oxygen can prevent that the oxygen concentration in the culture medium becomes to high which may inhibit growth of the algae. In addition, cooling can be done by evaporative cooling with e.g. sprinklers (Lundquist et al 2010, “A realistic technology and engineering assessment of algae biofuel production”, Luque, R).

So a preferred tubular photobioreactor according to the present disclosure comprises at least one transparent tube, a harvesting tank, a pump (for circulation of the algae culture), and/or a degassing unit (for withdrawing accumulated oxygen). There may be at least 10, 20, 50, 100, 500, or more transparent tubes wherein the algae are cultured.

Preferably, the at least one or more transparent tube is positioned substantially horizontally along its longitudinal direction, i.e. having at most 20, 10, or 5 cm height difference per meter along its longitudinal direction. Furthermore, the at least one transparent tube can be mounted to movable, preferably wheeled, structure (supported by the floor) that can be moved in and out of for example a greenhouse.

In a further preferred embodiment, the pump and/or the degassing unit, and preferably the harvesting tank of the tubular photobioreactor are comprised in a vehicle such as a van or truck. In this way, the system is easily movable, so that after harvesting the system can be dismantled and moved in one day. A particular advantage of the present tubular photobioreactor system is that it can achieve higher algae densities, higher productivity, better control, and lower risk for contamination as compared to open systems (Batan et al, 2010, Environmental Science & Technology 44, 7975-7980). Moreover, less C02has to be added because it cannot escape into the air (Lundquist et al 2010, “A realistic technology and engineering assessment of algae biofuel production”, Luque, R).

The present disclosure is not limited to the cultivation of any particular species of (green) (autotrophic) algae. However, preferably the algae are microalgae, preferably microalgae of a genus chosen from the group consisting of Skeletonema, Aphanizomenom, Nostoc, Odontella, Schyzochytrium (known for the production of docosahexaenoic acid), Ulkenia (produces docosahexaenoic acid), Porphyridium (produces arachidonic acid), Thalassiosira, Phaeodactylum, Chaetoceros, Navicula, Nitzschia, Amphora, Isochrysis, Pavlova,

Tetraselmis, Pyramimonas, Rhodamonas, Dunaliella (salina) (produces beta-carotene), Chlorella, Scenedesmus, Nannochloropsis (oculata) (produces eicosapentaenoic acid), Crypthecodinium (cohnii) (produces docosahexaenoic acid), Haematococcus (pluvialis), Chlorella (vulgaris), and Arthrospira (produces gamma-linolenic acid).

More preferably, the microalgae is of the genus Nannochloropsis (oculata) for producing eicosapentaenoic acid and/or docosahexaenoic acid.

The (micro) algae can be cultivated (i.e. cultured/grown) in salt or freshwater, where the preference is to cultivate in salt water, in particular in the case of the use of Nannochloropsis (oculata) for producing eicosapentaenoic acid and/or docosahexaenoic acid.

Algae are prokaryotic or eukaryotic photosynthetic (autotrophic) microorganisms. Microalgae grow using photosynthesis to convert sun (or artificial) light energy into chemical energy. They live in (salt or fresh) water, require light, C02, and nutrients for growth. Important parameters for algae production are light, temperature, pH, mixing grade, salinity, and nutrients.

When illuminated, photosynthesis occurs in the algae, enabling the production of carbohydrates from C02 and H20. The carbohydrates thus formed enables growth of the algae. This is called autotrophic growth. Due to the photosynthesis process C02 is consumed, while 02 is produced. The withdrawal of C02 by the algae from the liquid phase causes an increase of the pH of the liquid phase, while the production of oxygen causes an increase of the level of dissolved oxygen in the liquid phase.

In order to control pH and dissolved oxygen level of the liquid phase, transfer of 02 and C02 from the liquid phase to the gas phase and vice versa is preferred. Therefore algae cultures in photobioreactors are often aerated. In the case of upright liquid containers this is done by injecting gas at the bottom of the reactor. In tubular photobioreactors as according to the present disclosure, aeration and degassing can be achieved in a degassing unit. C02 can be fed to by injecting C02 or C02 enriched air directly into the liquid medium, preferably before the circulation pump.

Most algae have a temperature optimum between 20-30 °C, and an optimum pH range between 7 and 9 which can be achieved by the addition of C02 (Lavens and Sorgeloos, 1996, FAO Fisheries technical paper 361, University of Ghent, Ghent). Further, degassing of the produced 02 is important because high concentrations reduce algal productivity (Norsker et al, 2011a Biotechnol Adv 29, 24-7).

Microalgae are rich in many specific and attractive compounds, some of which are very interesting as nutritional supplements, such as long-chain polyunsaturated fatty acids including omega-3. Other nutriceuticals derived from microalgae are vitamins and antioxidants, such as beta-carotene and astaxanthin. As well as important applications in the food industry, microalgae are also used in the pharmaceutical market as they contain sterols, which can be used as building blocks for pharmaceuticals (hormones).

The culturing of the algae according to the present disclosure is preferably performed in a greenhouse, wherein the greenhouse preferably comprises plants such as at least 100, 200, 500, 1000 or more plants.

In this embodiment, at least one transparent tube of the tubular photobioreactor can be comprised in a greenhouse, wherein the greenhouse preferably comprises plants.

Many similarities exist between the cultivation of crop plants in greenhouses and the cultivation of algae in photobioreactors. For example, crop plants as well as algae require light, water, nutrients, C02 and an optimal climate to be able to grow with good quality. In view thereof, combining the two is synergistic, and allows for example for more efficient energy usage.

The present disclosure is not limited to any particular species of (crop) plants), but preferred (crop) plants are chosen from the group consisting of pepper, cucumber, bean, eggplant, melon, squash, tomato, carrot, lettuce, and radish. Alternatively, the plants are chosen from the group consisting of orchid, lily, rose, Chenille plant, Chinese Hibiscus, and African Violet.

In a last aspect, the present disclosure relates to the use of alkaline flocculation for harvesting algae cultured in a tubular photobioreactor.

Clauses 1. Method for culturing and harvesting algae in a tubular photobioreactor, wherein algae are harvested by alkaline flocculation. 2. Method according to clause 1, wherein the alkaline flocculation is performed by adding magnesium hydroxide and/or calcium hydroxide to the algae culture. 3. Method according to any of the previous clauses, wherein hydrophobic components are extracted from the harvested algae by hexane extraction. 4. Method according to any of the previous clauses, wherein the algae are microalgae, preferably microalgae of a genus chosen from the group consisting of Skeletonema, Aphanizomenom, Nostoc, Odontella, Schyzochytrium, Ulkenia, Porphyridium, Thalassiosira, Phaeodactylum, Chaetoceros, Navicula, Nitzschia, Amphora, Isochrysis, Pavlova, Tetraselmis, Pyramimonas, Rhodamonas, Dunaliella, Chlorella, Scenedesmus, Nannochloropsis, Crypthecodinium, Haematococcus, Chlorella, and Arthrosplra. 5. Method according to any of the previous clauses, wherein the culturing of the algae is performed in a greenhouse, wherein the greenhouse preferably comprises plants. 6. Method according to any of the previous clauses, wherein the tubular photobioreactor comprises at least one transparent tube, a harvesting tank, and preferably a pump, and/or a degassing unit. 7. Method according to clause 6, wherein the at least one transparent tube is assembled to a movable, preferably wheeled, structure. 8. Method according to any of clauses 6-7, wherein the pump and/or the degassing unit, and preferably the harvesting tank, are comprised in a vehicle. 9. Tubular photobioreactor, wherein the tubular photobioreactor is combined with a harvesting tank comprising magnesium hydroxide and calcium hydroxide, preferably both independently at a concentration of between 0.001 and 0.50 g/ml_. 10. Tubular photobioreactor according to clause 9, wherein the algae are microalgae, preferably microalgae of a genus chosen from the group consisting of Skeletonema, Aphanizomenom, Nostoc, Odontella, Schyzochytrium, Ulkenia, Porphyridium, Thalassiosira, Phaeodactylum, Chaetoceros, Navicula, Nitzschia, Amphora, Isochrysis, Pavlova, Tetraselmis, Pyramimonas, Rhodamonas, Dunaliella, Chlorella, Scenedesmus, Nannochloropsis, Crypthecodinium, Haematococcus, Chlorella, and Arthrospira. 11. Tubular photobioreactor according to any of clauses 9-10, wherein the tubular photobioreactor comprises at least one transparent tube, a harvesting tank, a pump, and/or a degassing unit. 12. Tubular photobioreactor according to any of clauses 11, wherein the at least one transparent tube is assembled to a movable, preferably wheeled, structure. 13. Tubular photobioreactor according to any of clauses 10-11, wherein the at least one transparent tube is comprised in a greenhouse, wherein the greenhouse preferably comprises plants. 14. Tubular photobioreactor according to any of clauses 11-13, wherein the pump and/or the degassing unit, and preferably the harvesting tank, are comprised in a vehicle. 15. Use of alkaline flocculation for harvesting algae cultured in a tubular photobioreactor. Brief description of the figures

Figure 1: Typical design of a tubular photobioreactor according to the present disclosure (from Leijdekkers, 2013, Msc thesis; Hemming et al 2012, “Algenteeltsystemen voor de tuinbouw, Rapport GTB-1221 DLO, Wageningen UR Glastuinbouw).

Example

An example of the present disclosure is obtained by placing a tubular photobioreactor in a greenhouse together with pot plants, wherein the photobioreactor has 48 transparent PVC tubes of 2.44 m length and 30 mm internal diameter.

As microalgal culture Nannochloropsis oculata can be selected because of its high content of an essential PUFA, eicosapentaenoic acid (EPA). Nannochloropsis oculata can be circulated through the tubes by a centrifugal pump via a buffer tank such that the circulation velocity is about 1 m/s corresponding to a flow rate of 340 l/min. The algae stream further can pass a degassing unit to withdraws accumuted oxygen. Such degassing units are known to the skilled person, and one that uses an air stream can be used. Cooling can be done when appropriate by evaporative cooling with sprinklers. A controller unit can continuously record culture temperature and pH measured by sensors submersed in the buffer tank. The pH can be regulated by C02 dosing into an outlet tube from the buffer tank to maintain the culture within set limits, preset in between pH 7.3/7.8 for Nannochloropsis production. This enables C02to be dosed on demand, i.e. during growth periods and disabled during night respiration in the culture. As a nutrient feed, a combination of agricultural fertilizers and urea can be used that comprises 1 g-l Red Superba (Norsk Hydro 7% N, 4% P, 21% K), 1 g/l urea and Guillard’s f/2 vitamins and trace metals, 1 and 0.5 ml/l respectively.

Algae can be harvested in a harvesting tank when reached their maximal culture density while still in late exponential growth. Cell densities of 6x107cell/ml cultures may be used. Calcium hydroxide can be added as a fine suspension of particles in water containing 0.15 g/ml_ Ca(OH)2. NaOH, Mg(OH)2 and KOH can be added as 0.15 g/ml_ solutions. NH4OH can be added as a 30% solution in water. The algae culture can be mixed during the addition of the alkali. The onset of flocculation can be visually determined by a “grainy” appearance of the culture. pH can be continuously measured during flocculation with a pH electrode (Pasco Scientific, Roseville, CA), using DataStudio software. Once floes are visually observed, they can be allowed to settle and supernatants can be decanted thereafter. For example, the floes can rapidly sediment and removed in a continuous manner from the bottom of harvesting tank.

Claims (15)

A method for cultivating and harvesting algae in a tubular photobioreactor, wherein algae are harvested by alkaline flocculation. The method of claim 1, wherein the alkaline flocculation is performed by adding magnesium hydroxide and / or calcium hydroxide to the algae culture. A method according to any of the preceding claims, wherein hydrophobic components are extracted from the harvested algae by extraction with hexane. A method according to any of the preceding claims, wherein the algae are microalgae, preferably microalgae of a genus selected from the group consisting of Skeletonema, Aphanizomenom, Nostoc ,, Odontella, Schyzochytrium, Ulkenia, Porphyridium, Thalassiosira, Phaeodactylum, Chaetoceros, Navicula, Nitzschia, Amphora, Isochrysis, Pavlova, Tetraselmis, Pyramimonas, Rhodamonas, Dunaliella, Chlorella, Scenedesmus, Nannochloropsis, Crypthecodinium, Haematococcus, Chlorella, and Arthrospira. A method according to any of the preceding claims, wherein algae cultivation is carried out in a greenhouse, wherein the greenhouse preferably comprises plants. Method according to any of the preceding claims, wherein the tubular photobioreactor comprises at least one transparent tube, a harvesting tank, and preferably a pump and / or a degassing unit. The method of claim 6, wherein the at least one transparent tube is mounted on a movable, preferably machined, structure. Method according to any of the claims 6-7, wherein the pump and / or the degassing unit and preferably the harvesting tank are included in a vehicle. A tubular photobioreactor, wherein the tubular photobioreactor is combined with a harvesting tank comprising magnesium hydroxide and calcium hydroxide, preferably both independently at a concentration between 0.01 and 0.50 g / ml. The tubular photobioreactor according to claim 9, wherein the algae are microalgae, preferably microalgae of a genus selected from the group consisting of Skeletonema, Aphanizomenom, Nostoc ,, Odontella, Schyzochytrium, Ulkenia, Porphyridium, Thalassiosira, Phaeodactylum, Chaetoceros, Navicula, Nitzschia , Amphora, Isochrysis, Pavlova, Tetraselmis, Pyramimonas, Rhodamonas, Dunaliella, Chlorella, Scenedesmus, Nannochloropsis, Crypthecodinium, Haematococcus, Chlorella, and Arthrospira. A tubular photobioreactor according to any of claims 9-10, wherein the tubular photobioreactor comprises at least one transparent tube, a harvesting tank, a pump and / or a degassing unit. The tube-shaped photo bioreactor according to claim 11, wherein the at least one transparent tube is mounted on a movable, preferably machined structure. A tubular photobioreactor according to any of claims 10-11, wherein the at least one transparent tube is included in a greenhouse, the greenhouse preferably comprising plants. A tubular photobioreactor according to any of claims 11-13, wherein the pump and / or the degassing unit and preferably the harvesting tank are included in a vehicle. 15. Use alkaline flocculation for harvesting algae grown in a tubular photo bioreactor.