WO2013121365A1

WO2013121365A1 - Methods and systems for cultivation of microalgae

Info

Publication number: WO2013121365A1
Application number: PCT/IB2013/051188
Authority: WO
Inventors: Ami Ben-Amotz
Original assignee: SEAMBIO FUEL Ltd
Current assignee: SEAMBIO FUEL Ltd
Priority date: 2012-02-14
Filing date: 2013-02-14
Publication date: 2013-08-22
Anticipated expiration: 2014-08-14

Description

METHODS AND SYSTEMS FOR CULTIVATION OF MICROALGAE

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods and systems for cultivation of microalgae.

The global carbon cycle is heavily influenced by the activities of man. For example, the combustion of fuels by man is believed to have resulted in a large increase in the amount of carbon dioxide present in the atmosphere. In the last hundred years, global fossil carbon emissions have increased by more than a factor of ten. As nations around the globe continue to become more industrialized, demands for energy are expected to increase dramatically. As such, in the absence of new technological solutions, it is believed that the trend toward increased fossil carbon emissions will continue.

Carbon dioxide is considered to be a "greenhouse" gas and is believed to have contributed to global warming trends. Carbon dioxide, along with water vapor, methane, nitrous oxide, and ozone, causes more heat to be retained by the Earth than would otherwise be captured. It is believed that this is due, at least in part, to the observed increase in greenhouse gas concentrations. Further increases in global temperatures may lead to various catastrophic effects including a rising sea level, increased extreme weather events, reduced agricultural yields, glacier retreat, and species extinction, amongst others.

In an effort to prevent catastrophic events from occurring, significant resources have been devoted to developing systems to reduce the amount of carbon dioxide emitted into the atmosphere. The various strategies pursued can be grouped into two broad categories: reduction of carbon emissions and capture of atmospheric carbon.

In nature, plants efficiently capture atmospheric carbon through the process of photosynthesis. Using sunlight as energy, plants convert carbon dioxide and water into the precursors of carbohydrates and other plant constituents. Many different types of plants and microorganisms capture considerable amounts of carbon dioxide. Algae are photosynthetic organisms that occur in most habitats. They vary from small, single- celled forms to complex multicellular forms. Algae are estimated to generate as much as 80 percent of the Earth's oxygen. It is also estimated that algae fix 90 gigatons of carbon per year.

Various attempts have been made at designing algae culture systems in order to capture carbon dioxide and to generate a significant algal biomass which can be mainly converted to biofuels such as biodiesel or bioethanol.

In general, there are two types of algae culture systems: open culture systems and closed culture systems. Open culture systems are open to the atmosphere and are equivalent to common high plants agriculture. They have the advantage of being relatively inexpensive to construct and maintain. However, open culture systems are subject to contamination issues. In contrast, closed culture systems or the photobioreactors are closed to the atmosphere and therefore provide the advantages of a controlled environment, lower evaporative water loss, and fewer contamination issues. However, many closed culture systems require relatively complex structures and therefore have substantially higher construction and operating costs. In addition, many closed culture systems have issues associated with insufficient light penetration, algae growth on walls, in- situ contamination that is difficult to clean, and poor temperature control.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of cultivating microalgae comprising:

(a) cultivating a microalgae culture under acidic pH conditions that predominantly drive carbonic acid and ammonia assimilation by the microalgae; and optionally subsequently

(b) cultivating the microalgae culture under basic pH conditions that predominantly drive carbonate and nitrate assimilation by the microalgae.

According to some embodiments of the invention, the microalgae culture is a monoculture.

According to some embodiments of the invention, the acidic pH conditions are between 4-5.

According to some embodiments of the invention, the basic pH conditions are between 9-11. According to some embodiments of the invention, a time ratio between step (a) and step (b) is predetermined according to an algal productivity of interest.

According to some embodiments of the invention, the algal productivity of interest is selected from the group consisting of biomass, carbohydrates, lipids and protein.

According to some embodiments of the invention, the cultivating is effected in open-body ponds.

According to some embodiments of the invention, the cultivating comprises enriching the microalgae culture with inorganic carbon.

According to some embodiments of the invention, the inorganic carbon is comprised in flue gas.

According to some embodiments of the invention, wherein step (a) or (b) comprises transferring the microalgae culture into a source of flue gas so as to allow flow of flue-gas into the microalgae culture, and returning the microalgae culture into the open-body pond.

According to some embodiments of the invention, the source of flue-gas comprises a power plant.

According to some embodiments of the invention, the inorganic carbon comprises calcium carbonate.

According to some embodiments of the invention, the cultivating is effected at low culture flow.

According to some embodiments of the invention, the method further comprising:

(c) harvesting microalgae from the microalgae culture following step (b). According to some embodiments of the invention, the harvesting is effected by a method selected from the group consisting of flocculation, sedimentation, filtration and floatation.

According to some embodiments of the invention, the method further comprising:

(d) recycling a culture medium of the microalgae culture following step (c).

According to some embodiments of the invention, the microalgae comprises marine microalgae. According to some embodiments of the invention, the microalgae comprises a fresh water microalgae.

According to some embodiments of the invention, the microalgae culture comprises sea water.

According to some embodiments of the invention, the microalgae culture comprises fresh water, brackish water and/or wastewater.

According to some embodiments of the invention, the microalgae is selected from the group consisting of starch-producing algae; chrysolaminarin— producing algae

According to some embodiments of the invention, the microalgae comprise coccoids.

According to some embodiments of the invention, the microalgae comprise flagellates.

According to some embodiments of the invention, a culture medium of the microalgae culture of the step (a) or (b) is augmented to include the following constituent values:

(i) 10-500 mM NaCl;

(ii) 1-500 mM Mg⁺⁺;

(iii) 1-20 mM K⁺;

(iv) 0.1-5 mM Ca;

(v) Fe³⁺;

(vi) 1-500 mM S04^2";

(vii) nitrate or ammonia 1-20 mM;

(viii) 0.01-1 mM pho sphate ;

(ix) 1-5 mM total dissolved carbon (TDC).

According to some embodiments of the invention, the microalgae culture of step (a) is augmented to include the following constituent values:

0.5-5 mM TDC;

1- 5 mM ammonia.

2- 5 mM ammonia;

1-3 ppm Chlorine. According to some embodiments of the invention, the microalgae culture of step (b) is augmented to include the following constituent values:

5 mM-20 mM TDC;

nitrate 0.5-2 mM.

According to some embodiments of the invention, for lipid accumulation the microalgae culture of step (b) is augmented to include nitrate concentration of 0.5-1 mM.

According to some embodiments of the invention, for carbohydrate accumulation the microalgae culture of step (b) is augmented to include nitrate concentration of 1-5 mM.

According to some embodiments of the invention, the method further comprises monitoring in the open body ponds a parameter selected from the group consisting of medium pH, medium TDC, medium conductivity, medium salinity, depth, medium temperature, cell number, chlorophyll, carotenoids, biomass and environmental conditions.

According to an aspect of some embodiments of the present invention there is provided a cultivation system comprising a first body pond which comprises a microalgae culture having an acidic pH between 4-5 and a second open body pond which comprises a microalgae culture having a basic pH between 9-11, wherein the first body pond and the second body pond are connected via a conduit for allowing fluid communication which allows transfer of microalgae culture having the acidic pH from the first body pond to the second body pond.

According to some embodiments of the invention, the cultivation system further comprises a conduit for introducing inorganic carbon into the first open body pond and optionally the second open body pond.

According to some embodiments of the invention, the cultivation system further comprises a device for pumping microalgae culture having an acidic pH between 4-5 from the first open body pond to the second open body pond via the conduit.

According to an aspect of some embodiments of the present invention there is provided an open body pond of at least 200,000 m cultivation medium and wherein a portion of microalgae out of total organisms in the cultivation medium corresponds to at least 20 %. Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a graph showing dissolved inorganic carbon species as a function of increasing pH;

FIG. 2 shows a pond system according to some embodiments of the present invention; and

FIG. 3 shows a pond system according to some embodiments of the present invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods and systems for cultivating microalgae.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Microalgae are one of the most potential sources of bioenergy. In the reports by U.S. Department of Energy, it is mentioned that bio-diesel transformed from the oil produced using the microalgae would fully meet the demand in the American diesel market.

Extensive and intensive systems are used for the mass cultivation of microalgae, also known as open systems and closed systems, respectively. Generally, open ponds have been associated with contamination, excessive space requirements and limited location possibilities due to climate. At the same time, closed bioreactors are mainly been considered too expensive.

While reducing the present invention to practice the present inventor has devised an innovative approach for extensive microalgal cultivation which involves a growth stage under low pH conditions. This can be optionally combined with a second stage of cultivation under highly basic pH conditions. This biphasic cultivation system is done in large open body ponds and using in parallel two biological pH biochemical controlled nitrogen assimilation systems. The first, carbonic acid and ammonia assimilation at low pH and the second, carbonate and nitrate assimilation at high pH using induced carbonic anhydrase, the outer membrane enzymes of certain algae to capture C0₂ at the most efficient way with full carbon recycling and utilization. The use of two pH stages under first acidic and then most alkaline conditions allows intensive outdoor cultivation of the algae, rapid growth on the first stage with control of both carbohydrate and lipid accumulation on each stage.

The integration of two stages cultivation system using ammonia and nitrate ion species and a wide range of pH from the acidic to the alkaline together with variable total dissolve carbon (TDC) enhances algal production while eliminating predators to reach algal monoculture at high biomass and high photosynthetic productivity.

As is illustrated in the Examples section which follows (see Tables 2-3 hereinbelow), the present inventor was able to achieve controlled production of lipids and carbohydrates. As can be seen, superior biomass, carbohydrate and protein production were obtained under the first stage of acidic pH conditions, while higher lipid accumulation was evidenced following exposure to alkaline conditions (2^nd stage). These results were obtained in at least two algal species Coccomyxa and Monodus substantiating the operability of the present teachings to photosynthetic aquatic microalgae in general. Thus, according to an aspect of the invention there is provided a method of cultivating microalgae comprising:

(b) cultivating said microalgae culture under basic pH conditions that predominantly drive carbonate and nitrate assimilation by the microalgae.

As used herein the term "cultivating" refers to growth and expansion of a microalgae culture.

As used herein the term "microalgae" refers to aquatic photosynthetic

(autotrophic) unicellular microphytes. The present teachings refer to the use of any species of such microalgae. The algae used according to some embodiment of the present teachings are both fresh water and marine species including e.g., coccoides and flagellates. It will be appreciated that the microalgae can be naive or genetically modified to express a protein of interest. Methods of genetically modifying microalgae are well known in the art (some are taught in U.S. 20090010947 and 6,027,900 each of which is hereby incorporated by reference in its entirety).

According to a specific embodiment, the microalgae culture is a monoculture. As used herein, the term "monoculture" refers to a culture in which microalgae (single species or a combination of two or more species) is the dominant species. In other words, the culture comprises at least 90 % microalgae (e.g., a single species of microalgae) out of total microorganisms in the pond. The monoculture is predator-free i.e., it does not include predators of the cultivated microalgae.

Any species of microalgae may be cultivated, alone or in various combinations, such as for example Chlorella, Chlamdomonas, Chaetoceros, Spirolina, Dunaliella and Porphyridum.

The specific algal species is selected according its ability to accumulate a product of interest, such as carbohydrates and lipids, under the indicated defined conditions, as below.

Exemplary species are provided in Table 1, below. Table 1: Microalgae Species

Cultivation of the microalgae can be done is fresh water, brackish water, waste water or sea water. According to a specific embodiment, cultivation is effected in brackish water.

Water, carbon dioxide, minerals and light are all important factors in cultivation, and different algae have different requirements. The basic reaction in water is carbon dioxide + light energy + water = glucose + oxygen + water. The skilled artisan is familiar with the basic nutritional and climatic needs of the selected microalgae species. Some guidelines are provided hereinbelow.

The water must be in a temperature range that will support the specific algal species being grown.

In most algal-cultivation systems, light only penetrates the top 3 to 4 inches (76- 100 mm) of the water. As the algae grow and multiply, the culture becomes so dense that it blocks light from reaching deeper into the water. To use deeper ponds, it is required to agitate the water, circulating the algae so that it does not remain on the surface. Paddle wheels can stir the water and compressed air coming from the bottom lifts algae from the lower regions. Agitation also helps prevent over-exposure to the sun.

Nutrients such as nitrogen (N), phosphorus (P), and potassium (K) can serve as fertilizer for algae. Silica and iron, as well as several trace elements, may also be considered important marine nutrients as the lack of one can limit the growth of, or productivity in, a given area.

According to a specific embodiment, water (any water source) can be augmented to provide a basic nutritional medium which comprises NaCl, 10-500 mM, 100-300 mM or specifically, lOOmM; Mg⁺⁺ 1-500 mM, 1-100 mM or 1-10 mM or specifically 5mM; K⁺, 1-20 mM or 1-10 mM and specifically 5mM; Ca⁺², 0.1-500 mM. 0.1-100 mM, 1-100 mM or specifically 50mM; Fe³⁺ as available and specifically 2μΜ;

S0₄ , 1-500 mM, 1-100 mM or 1-10 mM and specifically 5mM; nitrogen source, nitrate or ammonia, l-20mM and preferably by stage (acidic or basic, as described below); phosphate, 0.01-1 mM and preferably by stage; total dissolved carbon (TDC) as

CC 2- "₂, HCO₃ ^" and CO₃ ^" l-5mM to be supplied by carbon gas or salt by stage.

These values are aimed at optimizing algal growth.

As mentioned, embodiments of the invention rely on cultivating a microalgae culture under acidic pH conditions (as a first or single stage) that predominantly drive carbonic acid and ammonia assimilation by the microalgae. Uptake of carbonic acid and ammonia by the algae, releases protons to the medium with no need for pH gradient and no need for proton pump.

The acidic conditions, according to some embodiments of the invention, comprise a pH below 5, or according to further specific embodiments, 4-5, 4.5-5 or 4- 4.5. According to a further specific embodiment, the pH range at the acidic conditions is 4-5.

The pH of the culture is predominantly acidified by adding to the culture ammonia 1-5 mM (the uptake of ammonia by the microalgae releases proton (H+) to the culture); additionally, the medium is acidified by flowing carbon dioxide (thereby also enriched with carbon) by using inorganic carbon, such as using flue gas of various sources e.g., power plants.

The present inventor has realized that cultivation under acidic pH with essentially no nitrate at this stage (below 0.1 nM) has a number of advantages:

1. it reduces the predator load, thus supporting a monoculture;

2. maintains both enzymes carbonic anhydrase and nitrate reductase latent. Methods of measuring carbonic anhydrase are well known in the art. The enzyme is analysed by simple titration and is low in algae grown under low pH on carbonic acid. Nitrate reductase assay is well known in the art and is low in enzymes grown on ammonia.

Exemplary embodiments for cultivating under acidic conditions are provided infra.

Microalgae (e.g., Table 1) are grown at low pH below 5 using carbon dioxide (CO₂) or flue gases for carbon source and ammonium ions (N¾ or NH4⁺ by any source). These conditions maintain high biomass under species selected conditions. Uptake of ammonia by the algae releases proton (H⁺) to the medium so at 5mM NH₄ ⁺ the medium is acidified. The following parameters are monitored: weather, and temperature. The following parameters are controlled: pH between 4 to 5, total dissolve carbon (TDC) 0.5-5 mM, conductivity between 1 to 20 mS/cm, salinity between 0.1 to 2 M and depth between 5 cm to 40 cm, ammonia l-5mM, and sulfate l-500mM. The following biological parameters are analyzed: cell number 1-300 million/ml, chlorophyll 0.1-30 mg/L, carotenoids 0.02-6 mg/L and biomass 0.005-1.5 g/L (Table 4, Low pH).

Various contaminant organisms and predators such as fungi, zooplankton, crustacean, etc. are mitigated under conditions of large-scale by the integration of low pH 4-5, high ammonia 2-5 mM, and addition of chlorine 1-3 ppm.

As mentioned, under low pH conditions (acidic, as defined herein), certain algal products are generated in better efficiency than under alkaline conditions.

Thus, an acidic culture will perform better than an alkaline culture in the production of total biomass (as determined e.g., by total dry weight) carbohydrates and proteins. Conversely, lipid generation is better achieved under alkaline conditions.

Thus according to an optional embodiment, the microalgae culture is cultivated under basic pH conditions that predominantly drive carbonate and nitrate assimilation by the microalgae. The extracellular induced carbonic anhydrase allows active uptake of carbon from the medium into the cells while keeping pH gradient between the medium and the intracellular space. Under neutral cellular pH nitrate reductase and protein biosynthesis proceed and function optimally.

As used herein "basic pH conditions" or "alkaline pH conditions" (also referred to herein as "2^nd stage") comprise a pH of at least 8, or according to specific embodiments at least 9, 9-11, 8-11. According to a specific embodiment, the pH range at the basic conditions is 9-11. Culturing under basic pH conditions is also advantageous in terms of medium recycling, as further described hereinbelow.

Culturing under basic conditions comprises outdoor conditions of high pH 9-11 using flue gas, bicarbonate or carbonate (any source, sodium carbonate, calcium carbonate, magnesium carbonate and others), to reach high TDC of at least about 5 mM and nitrate 0.5-2 mM (see Table 5, high pH). The alkaline conditions induce the enzyme carbonic anhydrase, an algal outer membrane for most efficient carbon capture at these high pH conditions. Carbonic Anhydrase (CA) Induction in Stage 2 (basic) - The induced enzyme CA allows algal cultivation at high TDC (at least 5mM) and high pH for cellular carbohydrates and oil controlled production.

Nitrate Reductase (NR) Induction in Stage 2 (basic). The enzyme nitrate reductase is induced on transferring the algae to medium containing nitrate (all sources) at alkaline pH.

CA and Simultaneous Induction (Stage 2 continued). Both enzymes CA and NR will be induced and synchronized under high pH, high TDC under sufficient and deficient nitrate (NO₃ ) to produce high carbohydrates and high oil, respectively.

The ability to control cellular constituents allows the controlled production of cellular products of commercial value.

Thus, the decision if to include an additional stage of culturing under basic conditions or the time in culture at each step (a) or (b) [also referred to herein as time ration between step (a) and step (b)] is governed according to an algal productivity of interest.

As used herein "an algal productivity of interest" refers to a cellular constituent or total biomass which is of commercial value to an end user. Examples of such algal products include, but are not limited to , carotenoids, antioxidants, fatty acids, enzymes, polymers polysaccharides), peptides, toxins and sterols.

For example, lipid enrichment is achieved under step (b) (basic stage). In order to obtain high content of lipids it is necessary to supply limiting concentration of nitrogen which is provided on Stage 2 by nitrate. The nitrate content should be below ImM (e.g., 0.1-1 mM) for cellular lipid enrichment. However, when the enrichment of cellular carbohydrates is desired the content of nitrate is increased above ImM.

According to a specific embodiment, cultivation at each step is effected until a concentration of about 1 gram of ash free fry weight algae per liter is obtained. The life time cycle of each stage is related to the environmental conditions and varies between 24 to 72 hours.

At each step of the cultivation the culture may be enriched with inorganic carbon such that the TDC at the first (acidic stage) is 0.5-5mM, while at the basic stage (second stage) high TDC of at least 5 mM is achieved (e.g., 3-6 mM). Any source of inorganic carbon can be used. These include, carbon dioxide, carbonic acid, bicarbonate and carbonate (see Figure 1).

According to a specific embodiment, flue gas is used as the source of inorganic carbon. According to an embodiment of the invention, flue gas is streamed into the algae culture.

Alternatively or additionally, the microalgae culture is transferred into a source of flue gas (e.g., power plant) so as to allow flow or streaming of flue-gas into the microalgae culture, and returning the microalgae culture into the open-body pond. This embodiment negates the need to transfer the flue-gas to the cultivation area.

As used herein, the phrase "flue gas" refers to the exhaust gas from any sort of combustion process (including coal, oil, natural gas, etc.). Flue gas typically includes acid gases such as C0₂, S0₂, HC1, S0₃, and NO_x.

The present invention contemplates removing at least 5 % of the C0₂ present in the flue gas by carbon dioxide fixation, more preferably at least 10 % of the C0₂ present in the flue gas, more preferably at least 20 % of the C0₂ present in the flue gas, more preferably at least 30 % of the C0₂ present in the flue gas, more preferably at least 40 % of the C0₂ present in the flue gas and even more preferably at least 50 % of the C0₂ present in the flue gas.

According to one embodiment, the flue gas is streamed (directly) into the algae culture medium. The gas may be bubbled at the bottom of the algae culture thereby creating turbulence, mixing the culture so that the algae at the bottom of the culture moves to the top. In this way an airy culture is obtained which comes in contact with the nutrients and oxygen. Further movement in the culture ensures that the algae has sufficient exposure to a light source.

Alternatively, or additionally, the flue gas may be streamed above the algae culture (i.e. in a gas headspace). As C0₂-rich gas flows in the headspace, C0₂ dissolves into the liquid medium. Misters which spray the liquid medium into the gas headspace may be used in some embodiments.

According to a specific embodiment, the culture medium of the microalgae culture of step (a) or (b) is augmented to include the following constituent values:

(i) 10-500 mM NaCl;

(ϋ) 1-500 mM Mg (iii) 1-20 niM K⁺;

(iv) 0.1-5 mM Ca;

(v) Fe³⁺;

(vi) 1-500 niM S04^2";

(vii) nitrate or ammonia 1-20 mM;

( viii) 0.01-1 mM pho sphate ;

(ix) 1-5 mM total dissolved carbon (TDC).

According to a specific embodiment the microalgae culture of step (a) is augmented to include the following constituent values:

0.5-5 mM TDC;

1-5 mM ammonia.

According to a further embodiment, the microalgae culture of step (a) is augmented to include the following constituent values:

2-5 mM ammonia;

1-3 ppm Chlorine.

According to another or additional embodiment, the microalgae culture of step (b) is augmented to include the following constituent values:

5 mM-20 mM TDC;

nitrate 0.5-2 mM.

According to an embodiment, for lipid accumulation said microalgae culture of step (b) is augmented to include nitrate concentration of 0.5-1 mM.

According to an embodiment, for carbohydrate accumulation said microalgae culture of step (b) is augmented to include nitrate concentration of 1-5 mM.

As mentioned, cultivation is effected in an open body pond, such as raceway- type ponds. According to a specific embodiment the pond may be enclosed with a transparent or translucent barrier. The open-body pond system comprises a plurality of ponds (e.g., 5. 10, 50 or 100 ponds) of basic and acidic ponds (having the above-defined pH conditions) that are interconnected as further described hereinbelow. For example, the open ponds may be each divided outside and inside by ground dikes where each unit of operation is 1-2 hectare in step wise down ground level. The pond is about 5-40 cm deep to ensure maximal photosynthesis (deeper water require agitation as described hereinabove). The pond may be of 1, 2 5, 10 or 20 hectares. Typically, water flow (between the pools) is achieved by leveling down with no agitation or mixing that are typical for an intensive cultivation.

The ponds may be lined with clay or calcium carbonate, under acidic conditions the latter is used as a carbon dioxide source.

Throughout cultivation i.e., at step (a), step (b) or both steps, monitoring a various abiotic/biotic parameters is effected. Examples of such parameters include, but are not limited to, culture medium pH, culture medium TDC, culture medium conductivity, culture medium salinity, depth, culture medium temperature, cell number, chlorophyll, carotenoids and biomass.

Once a productivity of interest is obtained, the microalgae culture is harvested.

Any method of harvesting known in the arts can be used. These include inorganic low cost flocculation, sedimentation, filtration or floatation as common in waste water treatment.

Cultures can be harvested by continuous centrifugation in a batch-centrifuge or in a "de-sludger" centrifuge at about 3000 g. Generally it is advantageous to harvest each day about half the cells, the remaining culture being diluted with fresh medium. One liter contains about 1 gram ash free dry weight (AFDW).

Algae can be harvested by gravitational sedimentation. The culture is transferred to a conical tank where they settle out. About 30 % of the daily growth settles out by gravitation and there a concentration factor of about 25 is attained. Centrifugation of this sediment yields fresh algal paste.

The algae may be flocculated from the culture medium by the addition of certain salts, such as ferric chloride, aluminum chloride or aluminum sulfate. A concentration of about 0.1 mM to 0.5 mM ferric chloride, or about 0.1 to 0.5 mM aluminum chloride or about 0.1 mM to 0.5 mM aluminum sulfate results in the flocculation of the microalgae and the settled out cells may be harvested after about one hour. The sediment is centrifuged to yield algal paste.

The algae can be concentrated by cross-flow filtration through a cross-flow filtration system of the type supplied by A. T. Ramot Plastics Ltd., Tel-Aviv, which comprises a plurality of porous plastic tubings assembled in a modular filtration unit containing bundles of many tubes. About 75 liters of algae culture can be concentrated per hour yielding a concentration by a factor of 15. Centrifugation yields algal paste.

According to a specific embodiment, when using fresh water high pH medium, it is possible to use low concentration of flocculants and therefore high efficiency of algae separation with almost full recycling of the culture medium back for algae re- growth.

The algae biomass is further pressed and partially dehydrated according to the specifications of the end user for biofuel (biodiesel and bio-ethanol) downstream processing.

After centrifugation or collection of the algae by other means, there is obtained a paste which contains about 50 percent by weight of water. The dry algae contains at optimum conditions about 2-3 folds higher protein content than lipids when produced under acidic conditions (see Table 2) and about the same or 20-30 % higher lipid content than protein content, when grown in stage 2.

The product of interest can be extracted using any method known in the art.

Some are described below.

Glycerol extraction can be effected using any method known in the art, such as described in details in U.S. 4,115,949 which is hereby incorporated by reference in its entirety.

Proteinous residue can be extracted using cyclohexane (1: 1 by volume) as further detailed in 4,115,949 (supra).

The present invention also contemplates a cultivation system.

Thus according to a specific embodiment there is provided cultivation system 10 (Figure 2). Cultivation system 10 comprises a first body pond 12 which comprises a microalgae culture 14 having an acidic pH between 4-5 and second open body pond 16 which comprises a microalgae culture 18 having a basic pH between 9-11, wherein first body pond 12 and second body pond 16 are connected via conduit 20 allowing fluid communication which allows transfer of culture 14 from first body pond 12 to second body pond 14.

Cultivation system 10, may further comprise conduit 22 for introducing inorganic carbon from inorganic carbon supply vessel 46 into first open body pond 12 and optionally second open body pond 16. Cultivation system 10 may further comprise pump 24 for pumping microalgae from first open body pond 12 to second open body pond 16 via the conduit 20.

Cultivation system 10 may further comprise a conduit 11 leading water from source of water 26 via water softener 28, (if seawater is used), into first open body pond 12. Nutrients are supplied into first open body pond 12 or second open body pond 16 via conduit 32 from liquid nutrient supply vessel 30. Part of the liquid nutrient medium is returned via conduit 34 to first open body pond 12, while part of culture 14 flows via conduit 20 to second open body pond 16. Basic culture 18 is flowed from second open body pond 16 via conduit 36 to separator 38 and via conduit 40 to product reservoir 42, which is an algae paste of about 50 % water content. Part of the nutrient liquid is returned to second open body pond 16 via conduit 44.

Cultivation system 100 shows another embodiment to the transfer of algae into the flue gas source and its returned upon TDC enrichment (Figure 3).

Cultivation system 10 comprises a first body pond 112 which comprises a microalgae culture 114 having an acidic pH between 4-5 and a second open body pond 116 which comprises a microalgae culture 118 having a basic pH between 9-11, wherein first body pond 112 and second body pond 114 are connected via conduit 120 allowing fluid communication which allows transfer of microalgae culture 114 from first body pond 112 to second body pond 114.

Cultivation system 110, further comprises at least one conduit 122 for transferring microalgae culture 114 or 118 into a source of flue gas such that flue gas is streamed into the cultivation medium and further cultivation system 110 comprises at least one conduit 124 for returning microalgae culturell4 or 118 being TDC rich into first body pond 112 or second body pond 114.

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 ; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

The results of five-years outdoor experiments are summarized below with respect to various products in the indicated microalgae. The experiments were done in marine enriched media.

Table 2: Content of Cellular Ingredients under the Two Biphasic Stages in Starch Algae and in Chrysolaminarin Algae

Stage 1 Sta g l

Ingredient Coccomyxa Monodus Coccomyxa Monodus

Chlorophyll (pg/cell) 0.1-0.2 0.1-0.2 0.05-0.1 0.05-0.1

Carotenoids (pg/cell) 0.02-0.04 0.02-0.04 0.01-0.02 0.01-0.02

Starch (mg/g dry weight) 400-500 none 250-300 none

Chrysolaminarin (mg/g none 400-500 none 250-300 dry weight)

Lipids (mg/g dry weight) 100-200 100-200 200-350 300-350

Protein mg/g dry weight 300-400 300-400 250-300 250-300

Table 3: Aerial Productivity of Carbohydrates, Lipids and Protein

Table 4 - Low pH Ammonia

Physical Parameters

5

Table 5- Chemical and Biological Parameters

Nutrients

We Cells Algal Chi Car Car Chi Contami Chlori ath lOVm Specie mg/ mg/ / pg/cel Biomas nation N0₃ HP0₄ NH₄ ⁺ ne er 1 s L L Chi 1 s g/L #/ml niM niM Mg⁺⁺ niM SO₄ ^" ppm

1.0 0.1 0 0.1 0.100 0.005 0.5 add add add

30 6 0.20 0.10 1.5 0.5 add add add

5

Table 6 - High pH Nitrate

Chemical & Biological Parameters

Nutrients

Chlo NH₄ Contami Biom Algal

Weat +

rine Mg⁺ HP0₄ N0₃ nation ass Chi Car/ Car Chi Sped Cells her ppm so₄ ^" mM + ^" mM mM #/ml g/L pg/cell Chi mg/L mg/L es lOVml add add 0.5 0.5 0.005 0.100 0.02 0.1

0.2 1.0

add add 0.5 2 1.5 0.03 0.2 2 10 300

Table 7 - Physical Parameters

co₂ Cond

TD Depth Depth Depth Tem Wate Cond am

Weat C Vol. after after before p pm Temp pH pH r Salt pm mS/c her mM m³ Harv. cm cm °C am °C pm am NaCl (m³) M mS/cm m

ambi ambie

5 5 5 5 ent nt 10 9 Add 0.1 1.0

ambi ambie

5 40 40 40 ent nt 11 10 Add 2 20 Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A method of cultivating microalgae comprising:

2. The method of claim 1, wherein said microalgae culture is a monoculture.

3. The method of claim 1, wherein said acidic pH conditions are between 4-

5.

4. The method of claim 1, wherein said basic pH conditions are between 9-

11.

5. The method of claim 1, wherein a time ratio between step (a) and step (b) is predetermined according to an algal productivity of interest.

6. The method of claim 5, wherein said algal productivity of interest is selected from the group consisting of biomass, carbohydrates, lipids and protein.

7. The method of claim 1, wherein said cultivating is effected in open-body ponds.

8. The method of claim 1, wherein said cultivating comprises enriching the microalgae culture with inorganic carbon.

9. The method of claim 8, wherein said inorganic carbon is comprised in flue gas.

10. The method of claim 8, wherein step (a) or (b) comprises transferring said microalgae culture into a source of flue gas so as to allow flow of flue-gas into said microalgae culture, and returning said microalgae culture into said open-body pond.

11. The method of claim 10, wherein said source of flue-gas comprises a power plant.

12. The method of claim 8, wherein said inorganic carbon comprises calcium carbonate.

13. The method of claim 1, wherein said extensive cultivating is effected at low culture flow.

The method of claim 1, further comprising:

harvesting microalgae from said microalgae culture following step (b).

15. The method of claim 14, wherein said harvesting is effected by a method selected from the group consisting of flocculation, sedimentation, filtration and floatation.

16. The method of claim 14, further comprising:

(d) recycling a culture medium of said microalgae culture following step (c).

17. The method of claim 1, wherein said microalgae comprises marine microalgae.

18. The method of claim 1, wherein said microalgae comprises a fresh water microalgae.

19. The method of claim 1, wherein said microalgae culture comprises sea water.

20. The method of claim 1, wherein said microalgae culture comprises fresh water, brackish water and/or wastewater.

21. The method of claim 1, wherein said microalgae is selected from the group consisting of starch-producing algae; chrysolaminarin— producing algae

22. The method of claim 1, wherein said microalgae comprise coccoids.

23. The method of claim 1, wherein said microalgae comprise flagellates.

24. The method of claim 1, wherein a culture medium of said microalgae culture of said step (a) or (b) is augmented to include the following constituent values:

(i) 10-500 mM NaCl;

(ii) 1-500 mM Mg⁺⁺;

(iii) 1-20 mM K⁺;

(iv) 0.1-5 mM Ca;

(v) Fe³⁺;

(vi) 1-500 mM S04^2";

(vii) nitrate or ammonia 1-20 mM;

(viii) 0.01-1 mM pho sphate ;

(ix) 1-5 mM total dissolved carbon (TDC).

25. The method of claim 24, wherein said microalgae culture of step (a) is augmented to include the following constituent values:

0.5-5 mM TDC;

1-5 mM ammonia.

26. The method of claim 25, wherein said microalgae culture of step (a) is augmented to include the following constituent values:

2-5 mM ammonia;

1-3 ppm Chlorine.

27. The method of claim 24, wherein said microalgae culture of step (b) is augmented to include the following constituent values:

5 mM-20 mM TDC;

nitrate 0.5-2 mM.

28. The method of claim 27, for lipid accumulation said microalgae culture of step (b) is augmented to include nitrate concentration of 0.5-1 mM.

29. The method of claim 27, for carbohydrate accumulation said microalgae culture of step (b) is augmented to include nitrate concentration of 1-5 mM.

30. The method of claim 1 further comprises monitoring in said open body ponds a parameter selected from the group consisting of medium pH, medium TDC, medium conductivity, medium salinity, depth, medium temperature, cell number, chlorophyll, carotenoids, biomass and environmental conditions.

31. A cultivation system comprising a first body pond which comprises a microalgae culture having an acidic pH between 4-5 and a second open body pond which comprises a microalgae culture having a basic pH between 9-11, wherein said first body pond and said second body pond are connected via a conduit for allowing fluid communication which allows transfer of microalgae culture having said acidic pH from said first body pond to said second body pond.

32. The cultivation system of claim 31, further comprising a conduit for introducing inorganic carbon into said first open body pond and optionally said second open body pond.

33. The cultivation system of claim 31, a device for pumping microalgae culture having an acidic pH between 4-5 from said first open body pond to said second open body pond via said conduit.

34. The cultivation system of claim 31, further a conduit for transferring said microalgae culture into a carbon dioxide source and another conduit for returning said microalgae culture into first open-body pond or said second open body pond.

35. An open body pond of at least 200,000 m cultivation medium and wherein a portion of microalgae out of total organisms in said cultivation medium corresponds to at least 20 %.