HK1151188A - Algal culture production, harvesting, and processing - Google Patents

Description

Algal culture production, harvesting and processing

This application claims priority to U.S. provisional patent application 61/023,572 filed on 25/1/2008, incorporated herein in its entirety.

Background

Increasing global demand and environmental issues have led to the search for alternative and greener sources of fuels, animal feed, pharmaceuticals, nutraceuticals, polyunsaturated fatty acids, plant nutrients, minerals, vitamins and other products. One environmental source of these products is algae. Algae are a particularly attractive source because they can be grown using land that is not normally available for food production or other purposes. However, there are several obstacles to producing these products from algae, including selecting the appropriate algae, developing appropriate growth conditions for optimal lipid production, and preventing contamination by undesirable algae species and other organisms. When large-scale cultivation of algae in outdoor locations is sought, weather and pollution are constant threats and these obstacles are multiplied. Therefore, new algae production technologies are strongly needed.

Disclosure of Invention

The present invention provides methods for selectively culturing target algae. The method comprises, culturing a target alga in a first pond; diluting the target alga in the first pond; supplying a nutrient composition to the first pond; and maintaining culture selectivity in the first pond. This and other methods of the invention can be used to produce lipids for biofuels (e.g., biodiesel) and lipids for polyunsaturated fatty acids (e.g., omega-3 fatty acids). The method and other methods of the invention can also be used to produce feedstocks such as animal feed and aquaculture feed. This and other methods of the invention can be used to produce plant nutrients such as beta-carotene and astaxanthin.

The present invention provides a method for selectively culturing target algae of Scenedesmus (Scenedesmus). The method comprises, culturing a target alga in a first pond; diluting the target alga in the first pond; supplying a nutrient composition to the first pond; and maintaining culture selectivity in the first pond.

The present invention provides a method for selectively culturing the target alga Scenedesmus obliquus. The method comprises, culturing a target alga in a first pond; diluting the target alga in the first pond; supplying a nutrient composition to the first pond; and maintaining culture selectivity in the first pond.

The invention provides a method for selectively culturing a target alga Scenedesmus obliquus. The method comprises the following steps. And (4) culturing the target algae in the pipeline pond. Carbon dioxide is added to the pipeline pond if a pH of about 8.5 or higher is reached. If a temperature of 33 ℃ or higher is reached, a coolant is added to the pipeline pond. Approximately every 20 hours, the algae in the raceway pond is diluted by about 60%. At about the same time as the diluting step, the pipeline pond is supplied with a nutrient composition, wherein the nutrient composition comprises sodium bicarbonate, urea, trisodium phosphate, and ferrous chloride, the sodium bicarbonate concentration is at least 2mM, and the nitrogen to phosphorus ratio is at least about 15: 1. The volume of algae obtained in the dilution step is drained into a nitrogen deficient stress pond. Algae were harvested from the stress pond and dewatered. Lipids are extracted from algae.

The invention provides biofuels, feedstocks, polyunsaturated fatty acids, plant nutrients, and any other useful products produced by any of the methods of the invention.

The present invention provides selective outdoor pond algae cultures comprising a target algae. The target algae may be green algae. The green algae may belong to the genus scenedesmus. The target algae may be diatoms. The pond may be a raceway pond.

Detailed Description

According to the present invention, there is provided a method of selectively culturing a target alga for lipid production. This and other methods of the invention can be used to produce lipids for biofuels (e.g., biodiesel) and lipids for polyunsaturated fatty acids (e.g., omega-3 fatty acids). The method and other methods of the invention can be used to produce feedstocks such as animal feed and aquaculture feed. This and other methods of the invention can be used to produce plant nutrients such as beta-carotene and astaxanthin.

The target algae may be any suitable algae species or one or more strains thereof. That is, while the target alga is typically a single algal species, in some embodiments it may be a combination of two or more algal species and/or strains thereof. The target algae preferably comprises algae that are capable of producing high levels of lipids under suitable conditions.

The target algae may comprise at least one green algae. In some embodiments, the target algae is diatoms. The target algae can be obtained, isolated and acclimatized from any natural or artificial source. In some embodiments, the algae are obtained from a source local to the production site of the algae culture. In some embodiments, the target algae is obtained from louisiana, usa. In some embodiments, the target algae is obtained in or near charles lake, louisiana. The target algae may be clustered algae. Isolation and purification of the target algae can be performed by pipette, medium, light and temperature methods. In some embodiments, the isolated and purified target algal strain may survive for several days at a lower temperature (e.g., below 10 ℃). Acclimatization of the target algal strain may include treating the strain at a lower temperature, less light source and minimal nutrient medium. The purified algal strain can be cultured in 5ml of medium and then scaled up to several thousand liters of medium, natural water or treated water. Pure cultures can be prepared from clean water such as Reverse Osmosis (RO) or distilled water. The target algal strain may then be introduced into filtered or unfiltered source water or treated water for acclimation. Aliquots of the inbred cultures can be maintained in clean water as stock cultures.

In some embodiments, the target algae comprises one or more green algae of the genus scenedesmus or any combination thereof. In some embodiments, the green algae comprises scenedesmus obliquus. In some embodiments, the green algae are selected from the group consisting of: scenedesmus obliquus, Scenedesmus quadricarina (Scenedesmus quadricauda), Scenedesmus maxima (Scenedesmus maxima), Scenedesmus armatus (Scenedesmus armatus), Scenedesmus orbiculatus (Scenedesmus olivensis), Scenedesmus dimorphius, and any combination thereof. Variants of these species may be used. For example, Scenedesmus quadricarina maximus (Scenedesmus quadratus maximus) can be used. Scenedesmus obliquus may, for example, comprise Scenedesmus obliquus Texas University (UTEX) strain 1450.

Non-scenedesmus algae and other aquaculture microorganisms may also be employed according to the present invention. In some embodiments, the target algae comprises one or more green algae of the genus chlorella, such as chlorella minutissima (chlorella minutissima), or any combination thereof. In some embodiments, the target algae comprises one or more green algae of the genus Botryococcus (Botryococcus), such as Botryococcus braunii (Botryococcus braunii), Botryococcus suvialis (Botryococcus sueditica), or any combination thereof. In some embodiments, the target algae comprises one or more green algae of the genus Chlamydomonas (Chlamydomonas) or any combination thereof. In some embodiments, the target algae comprises one or more green algae of the genus Closterium (Closterium) or any combination thereof. In some embodiments, the target algae comprises one or more green algae of the genus pediastra (Pediastrum), or any combination thereof. In some embodiments, the target algae comprises one or more green algae of the genus linear algae (Melosira) or any combination thereof. In some embodiments, the target algae comprises one or more green algae of the genus coleus (Oedogonium), or any combination thereof. In some embodiments, the target algae comprises one or more green algae of the genus Haematococcus (Haematococcus), such as Haematococcus pluvialis, or any combination thereof. In some embodiments, the target algae comprises one or more green algae of the genus Dunaliella (Dunaliella), such as Dunaliella salina (Dunaliella salina), Dunaliella parva (Dunaliella parva), Dunaliella viridis (Dunaliella viridis), or any combination thereof. In some embodiments, the target algae comprises one or more dinoflagellates (Prymnesiophyceae) green algae of the genus Chlorella (Isochrysis) such as Isochrysis galbana (Isochrysis galpana) or any combination thereof. In some embodiments, the target algae comprises one or more than one turquoise (Prasinophycean) green algae of the genus Tetraflagellate (Tetraselmis), such as Tetraselmis subcoccus (Tetraselmis sueca), or any combination thereof. In some embodiments, the target algae comprises one or more diatoms. Examples of diatoms include, but are not limited to, those of the genus Skeletonema (skelleetonema) such as Skeletonema costatum (skelleetonema costatum), those of the genus Chaetoceros (Chaetoceros) such as Chaetoceros caldarius (Chaetoceros calceins), or any combination thereof. The methods of the invention described herein for a particular algae may also be used by replacing or adding other algae described herein or otherwise known.

In some embodiments, the target algae are produced from a substantially pure culture. In some embodiments, the target algae is selected from a population of algae cultures. The target alga in the first pond can be maintained in the first pond for any suitable time. By amplifying the seed culture of the target alga, the volume of the alga culture in the first pond can be achieved. In some embodiments, the amplification step comprises two or more successive larger volumes of the target alga.

The cultivation selectivity according to the present invention does not require the mono-cultivation of the target algae. Maintenance of culture selectivity comprises maintaining the target alga as the dominant alga in the algal culture of the first pond. There may be a temporary loss of culture selectivity, for example, when the algal culture is enlarged, or during or after weather or other events. In some embodiments, the target algae is maintained at least 50% of the total algae. In some embodiments, the target algae is maintained at least 75% of the total algae. In some embodiments, the target algae is maintained at least 90% of the total algae. In some embodiments, the target algae is maintained at least 95% of the total algae. In some embodiments, the target algae is maintained at least 99% of the total algae. The open pond culture may comprise 100% pure strains of the algae of interest, or may be at least 90% pure. In some embodiments, the open pond culture can be at least 50% pure. In some embodiments, other algal species are cultured with the target algae for research or general production purposes.

A method of selectively culturing a target alga comprises, culturing the target alga in a first pond; diluting the target alga in the first pond; supplying a nutrient composition to the first pond; and maintaining culture selectivity in the first pond. In some embodiments, the step of supplying the nutrient composition is performed substantially simultaneously with the step of diluting. In some embodiments, the pH of the culture is maintained at about pH6 to about pH 8. The method may additionally comprise the steps of: carbon dioxide is added to the first pond if a pH of about 8.5 or higher is reached. The addition of carbon dioxide to maintain the pH may be done in conjunction with or independent of using carbon dioxide as a nutrient source.

The method may additionally comprise the steps of: if a temperature of 33 ℃ or higher is reached, a cooling liquid is added to the first pond. In some embodiments, the cooling fluid comprises fresh medium. When reference is made to "a culture medium", any suitable medium or media may be employed, unless otherwise indicated. For example, a medium containing 5mM sodium bicarbonate, 1mM urea (or sodium nitrate or ammonia), 30-trisodium phosphate and 2-ferrous chloride may be used. In some embodiments, reverse osmosis water is used to prepare the culture medium.

Any suitable nutrient composition may be employed in the present invention. In some embodiments, the nutrient composition comprises sodium bicarbonate at a concentration of at least about 0.6mM, as measured after addition of the nutrient composition to the pond. In some embodiments, the nutrient composition comprises sodium bicarbonate at a concentration of at least about 2mM, as measured after addition of the nutrient composition to the pond. The nutrient composition may comprise a source of iron. In some embodiments, the iron source comprises ferrous chloride. The nutrient composition may comprise a nitrogen source and a phosphorous source. In some embodiments, the nitrogen source comprises urea and the phosphorus source comprises trisodium phosphate. In some embodiments, the nitrogen to phosphorus ratio is at least about 15: 1. In some embodiments, the ratio is at least about 29: 1. In some embodiments, the ratio is about 30: 1.

Any suitable structure or combination of structures may be used for the first pond. The first pond may be a raceway pond. The raceway pond provides a housing that allows the cultured target algae to move in a loop. Any suitable loop geometry may be employed. For example, the shape of the raceway pond can approximate the shape of a race track or runway. The pond may comprise parallel rectangular channels connected at either end by semi-circular or sufficiently curved channels to adjacent ends of the parallel rectangular channels to form a continuous channel. The raceway pond can contain one or more channels of the same or different sizes. In some embodiments, the pond is divided evenly into 2 channels, the width of each channel remaining constant throughout the pond stroke.

The first pond can comprise a transparent enclosure. The envelope may be completely or partially transparent. In some embodiments, the transparent envelope comprises an acrylic polymer. However, any suitable material that allows light to pass through may be used for the transparent envelope.

The size of the first pond can be any suitable size. Providing a pond volume (capacity) capable of holding at least a volume of algal culture. The volume of the pond may contain additional volume to allow for the entry of sediment and other liquids, minimizing or eliminating overflow. For example, a 22 liter capacity pond can suitably accommodate an algal culture volume of about 18 liters. In some embodiments, the algal culture volume of the first pond is about 18 liters or more. In some embodiments, the algal culture volume of the first pond is about 600 liters or more. In some embodiments, the algal culture volume of the first pond is about 14,000 liters or more.

The depth of the algal culture in the first pond is any suitable depth. Provides a depth at which the amount of algae and the amount of algae are in close balance to sunlight. In some embodiments, the first pond comprises an average algal culture depth of about 13 to 20 centimeters. In some embodiments, the average algal culture depth is about 18 centimeters.

The target algae in the first pond can be mixed at any suitable rate. Suitable rates may be rates that allow the algal cells to approach sunlight and nutrients. In some embodiments, the target algae is mixed at a speed of about 12 cm/sec, about 15 cm/sec, or about 18 cm/sec. Mixing may be provided by any suitable means. In some embodiments, one or more paddle wheels are used to provide mixing. Fresh culture and medium were added immediately before the paddle wheel. In some embodiments, the paddle wheel has at least 6 paddles and a strut between the ends of each paddle. The paddle wheel can be positioned so that it straddles and is outside the intermediate dividing line of the pond wall. In some embodiments, the paddle wheel is positioned such that it pushes the culture the maximum distance before the channel curve. The number of paddle wheels employed may depend on the width of the pond. In some embodiments, 1 to 3 paddle wheels are employed. If more than one paddle wheel is used, they may be placed in parallel. The number and positioning of the paddle wheels may vary depending on the material used to make the paddle wheels and its strength.

The target alga in the first pond can be diluted to any suitable extent at any suitable frequency. Dilution may be continuous, substantially continuous or intermittent. In some embodiments, relatively large volumes of algal culture are removed relatively infrequently. In some embodiments, relatively small volumes of algae culture are removed relatively frequently. The target algae may be diluted with any suitable device. The algal culture may be diluted by adding a culture medium, the algal cells may be removed, or the dilution may be performed by a combination thereof. The removal of the algal culture and the addition of the culture medium need not be simultaneous. The target algae can be diluted in any suitable amount to maintain substantially stable growth of the algae in the first pond, and to utilize the algae of the first pond for other uses. In some embodiments, the growth of algae is logarithmic for at least a portion of the time spent in the first pond. In some embodiments, the diluting step comprises diluting the target alga in the first pond by a dilution of from about 35% to about 60%. In some embodiments, the dilution is about 50%. In some embodiments, the dilution is performed approximately every 20 hours. The algae concentration may be measured using any suitable method. In some embodiments, dilution is performed when the transparency (black and white) plate reads 5-6cm (when the plate is no longer visible). In some embodiments, the concentration of algae in the first pond is maintained in the range of about 2 million to about 3 million algae/ml. The volume of algal culture removed from the first pond can depend on the percent dilution and the culture volume. This volume can be more than about 20% and less than about 60% of the total culture volume of the first pond. The concentration of algae in the withdrawn volume may depend on whether the dilution is continuous or intermittent. The number of cells may be from about 2.5 million cells/ml to about 5 million or about 6 million cells/ml. The number and frequency of dilutions can be adjusted to compensate for differences in sunlight. For example, adjustments may be made based on year, season, hemisphere, and/or latitude. The particular species and strain of the target alga may also vary with these parameters. For example, one strain may be used during winter or cold seasons, and another strain may be used during summer or warm seasons.

The diluting step can comprise removing a volume of the target alga from the first pond. In some embodiments, the target algal volume from the first pond is discharged into the second pond. In some embodiments, the removal of the volume of the target alga from the first pond and its discharge into the second pond are substantially simultaneous. In other embodiments, the removal from the first pond and discharge into the second pond is separated by a suitable period of time.

The algal depth of the second pond can be any suitable depth. In some embodiments, the algal depth of the second pond is about 18 centimeters to about 30 centimeters. The residence time of the target alga once discharged into the second pond can be any suitable period of time. In some embodiments, the residence time is about 3 days. The second pond will provide sufficient capacity to hold a volume of algal culture discharged into the second pond during the retention period. For example, when the residence time is about 3 days, and the algal culture is added to the second pond daily, the pond should hold 3 days of "flowing" (added) culture, 3 times the volume of each batch of flowing culture. In some embodiments, the concentration in the second pond ranges from 5 million to 1 million cells/ml.

The second pond can comprise any suitable structure or combination of structures. Any suitable set of conditions can be maintained in the second pond. The second pond can be a stress pond. The second pond may be a settling pond. In some embodiments, the second pond is both a stress pond and a settlement pond. Stress ponds will provide an environment that causes the target algae to increase production of lipids that can be harvested for biofuel production. The stress pond environment can be achieved in many different ways. For example, the target algae may typically be starved for nutrients or deprived of one or more nutrients. In some embodiments, the stress pond is nitrogen deficient. The nitrogen deficiency may be complete or partial. In addition to nitrogen, other nutrients (including carbon dioxide) can be added to the algal culture in the second pond. Conditions can be provided in the second pond that maximize production of lipids or other desired products by the target alga. In some embodiments, culture selectivity is maintained in the second pond. In some embodiments, the second pond is a stress pond and is similar in design to the first pond, e.g., a raceway pond, but deeper. The settling pond allows the target algae to settle. In some embodiments, the settling pond is funnel-shaped. In embodiments where the stress pond and the sedimentation pond are not the same pond, the second pond can be a stress pond and the third pond can be a sedimentation pond.

The target algae can be harvested from the second pond for downstream processing, such as lipid extraction and final biofuel production. The target algae can be harvested using any suitable device, in any suitable amount, and at any suitable frequency. In some embodiments, harvesting is performed about 52 hours to about 54 hours after discharging the volume of target algae into the second pond. In some embodiments, harvesting is performed about 72 hours after discharging the volume of target algae into the second pond. In some embodiments, harvesting is performed once the lipid concentration reaches at least about 25% of the cell population. Lipid content can be determined using any suitable measurement. In some embodiments, the lipid content is measured using a fluorometer. The target reading of the fluorometer can depend on the chosen aperture and the concentration of the sample. Any suitable fluorescent dye may be used. Examples include nile red, nile blue and india blue.

The target algae can be dehydrated. Dewatering may be performed as part of the harvesting step or as a separate step. In some embodiments, the dewatering step comprises using at least one of a belt press and a dewatering apparatus (dewaterer). In some embodiments, the harvesting step comprises dewatering the target alga by suctioning the settled target alga from the second pond. Aluminum sulfate (e.g., 50-100ppm), or ferric chloride (e.g., 10-30ppm) can be used to help settle the algae. The polymer (e.g. 0.5% algal biomass) can be used to promote algal coagulation prior to use of the belt press. Any suitable polymer or combination of polymers may be employed. In some embodiments, an emulsion polymer is used. Examples of emulsion polymers include Flopam EM 640, Flopam EM 840, and combinations thereof. In some embodiments, a solution polymer is employed. More solution polymer may be required than when emulsion polymer is used. Additionally, or in the alternative, the coagulation promoting agent may include one or more of clay, pH adjustment (e.g., pH increase), nutrient deficiency, and charged electrode.

In some embodiments, after reaching densities of 3 million cells/ml and above, the algae in the outdoor pond are harvested and passed through a filter with a 30 micron or higher sieve size, depending on the filtration rate. The filtered product is an algal paste, which can be treated with a solvent (e.g., methanol, chloroform, acetone, ethanol, hexane, etc.) to extract lipids and purified to obtain a biofuel. Omega-3 fatty acid extracts, animal feeds such as aquaculture feeds, beta-carotene, vitamins, etc., can also be extracted from different algae species, including microalgae. The pond water after filtering the algal mass can be treated by ultraviolet fluorescence exposure for 60 minutes or more. In some embodiments, the extended duration is up to 3 hours or more. The uv treated water can be pumped back into the pond and added with different nutrients such as nitrogen, phosphate and carbon dioxide. Fresh inoculum can be pumped into the pond for algal growth. In some embodiments, nutrients of carbon, nitrogen, phosphorus, minerals, vitamins are added. In some embodiments, the primary nutrient is added separately to the culture pond.

Lipids can be extracted from the target algae. Can be used forTo employ any suitable lipid extraction device. In some embodiments, the extracting step comprises at least one of chloroform methanol extraction and hexane extraction. The algal material can be treated with a solvent, such as the methods of Bligh and Dyer, Fajardo and supercritical CO₂Process to extract lipids. Lipids can be processed into biodiesel using transesterification methods using bases, such as described by Holup and Skeaff. In addition or in the alternative, bioethanol, biohydrogen, biomethanol and other products may be produced.

Byproducts of biodiesel production processes, such as omega-3 fatty acids and other groups of polyunsaturated fatty acids (PUFAs), are extracted from algal paste. These desired lipids can be obtained even without biodiesel production and therefore need not be considered as by-products. The major omega-3 fatty acids include alpha-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Omega-3 fatty acids and PUFAs may be used in pharmaceutical and nutraceutical applications. Omega-3 fatty acids can be obtained as a by-product of the lipid extraction process by treating the lipids at different temperatures of processing. In some embodiments, all of these reactions are performed in an anoxic environment. In some embodiments, the target algal strain produces approximately greater than 22% omega-3, greater than 29% PUFAs, greater than 20% monounsaturated fats, and greater than 27% saturated fats. In some embodiments, the algal lipid product may include about 26.1% omega C18-3 fat, 20% monounsaturated fat, 26.4% polyunsaturated fat, 25% saturated fat, and 2.5% trans fat. The carbon chain may include, but is not limited to, different percentages of C12 to C24 chains. The actual lipid profile may vary with the increase or decrease in one or more components, depending on the algae growth conditions. Other methods may also be employed. Omega-3 fatty acids can be used for a variety of health uses, such as the prevention or treatment of medical conditions common to the heart and circulatory system, inflammatory diseases, and cancer. Algae also have vitamin resources including: a, C, E, which can be obtained from microalgae using a vitamin extraction process.

The production of the algal meal feedstock may include the following steps. The paste obtained after extraction was treated with an anti-solvent and washed, washed with deionized water, air-dried, and sterilized at about 60 ℃ for about 12 hours. The biomass can then be comminuted and packaged into suitable containers according to the supplier's requirements. In some embodiments, the algal meal product comprises 3% crude fiber, 0.1% calcium, 39% protein, 0.2% monounsaturated fat, 0.2% omega-3 fat, 0.2% polyunsaturated fat, 0.2% saturated fat, 0.1% trans fat, and 1% other fats.

The biomass can also be used to produce ethanol. Biogas can be produced from the anaerobic digestion of biomass. The feedstock of the present invention may contain varying amounts of protein, lipids, carbohydrates, fiber, minerals, vitamins and other nutrients. The process of the present invention can be adjusted to produce these various amounts.

The lipid content may be equal to or greater than 10%, 20%, 25%, 30%, 35%, 40%, or 50% of the algal paste. In some embodiments, the lipid content is 26.3% of the algal paste. The raw material (meal) may be equal to or greater than 10%, 20%, 25%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the algal paste. The protein content may be equal to or greater than 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the starting material (meal). In some embodiments, the protein content is 39% protein.

According to the present invention, there is provided a method for selectively culturing a target alga of the genus Scenedesmus. The method comprises, culturing a target alga in a first pond; diluting the target alga in the first pond; supplying a nutrient composition to the first pond; and maintaining culture selectivity in the first pond.

The invention provides a method for culturing a target alga Scenedesmus obliquus. The method comprises, culturing a target alga in a first pond; diluting the target alga in the first pond; supplying a nutrient composition to the first pond; and maintaining culture selectivity in the first pond.

The invention provides a method for selectively culturing a target alga Scenedesmus obliquus. The method comprises the following steps. And (4) culturing the target algae in the pipeline pond. Carbon dioxide is added to the pipeline pond if a pH of about 8.5 or higher is reached. If a temperature of 33 ℃ or higher is reached, a coolant is added to the pipeline pond. Approximately every 20 hours, the target alga in the raceway pond is diluted by about 60%. At about the same time as the diluting step, the pipeline pond is supplied with a nutrient composition, wherein the nutrient composition comprises sodium bicarbonate, urea, trisodium phosphate, and ferrous chloride, the sodium bicarbonate concentration is at least 2mM, and the nitrogen to phosphorus ratio is at least about 15: 1. The target algal volume obtained in the dilution step is discharged into a nitrogen-deficient stress pond. The target algae are harvested from the stress pond and dewatered. Extracting lipid from target algae.

Any of the methods of the present invention may additionally comprise the steps of: producing a biofuel from the lipids produced by the target alga. Any suitable method may be employed. For example, transesterification may be employed. In some embodiments, the biofuel is biodiesel. In some embodiments, the biofuel is bio-coal essence (bio-jet). Biofuels produced by any of the methods of the invention are also an aspect of the invention.

The invention provides a biofuel produced by any of the methods of the invention.

Any of the methods of the present invention may additionally comprise the steps of: polyunsaturated fatty acids are produced from the target algae. In some embodiments, the polyunsaturated fatty acids include omega-3 fatty acids. In some embodiments, the omega-3 fatty acids comprise alpha-linolenic acid (ALA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or any combination thereof. The present invention provides polyunsaturated acids produced by any of the methods of the present invention.

Any of the methods of the present invention may additionally comprise the steps of: producing a feedstock from the target alga. The raw material may be an animal feed, an aquaculture feed, or any combination thereof. The present invention provides a feedstock produced by any of the methods of the present invention.

Any of the methods of the present invention may additionally comprise the steps of: plant nutrients are produced from the target algae. The plant nutrient may be a carotenoid. In some embodiments, the carotenoid is astaxanthin, beta-carotene, or any combination thereof. The present invention provides plant nutrients produced by any of the methods of the present invention.

According to the present invention there is provided a selective outdoor pond algae culture comprising a target algae of the genus scenedesmus. The culture may be in a pond. The pond may be a raceway pond. As described above with respect to the methods of the invention, the selective algae culture need not be a mono-culture. In some embodiments, the target algae is at least 50% of the total algae. In some embodiments, the target algae is at least 75% of the total algae. In some embodiments, the target algae is at least 90% of the total algae. In some embodiments, the target algae is at least 95% of the total algae. In some embodiments, the target algae is at least 99% of the total algae. The selective outdoor pond algae culture can comprise scenedesmus obliquus. The target algae may be selected from: scenedesmus obliquus, Scenedesmus quadricaudatus, Scenedesmus maximus, Scenedesmus oboensis, Scenedesmus tundifolius, Scenedesmus obliquus and any combination thereof. Variants of these species may be used. For example, the largest variety of Scenedesmus tetracaudatus can be used. In some embodiments, the scenedesmus obliquus comprises scenedesmus obliquus UTEX strain 1450.

Selective outdoor pond algae cultures comprising non-scenedesmus target algae and/or other aquaculture microorganisms may also be employed according to the present invention. In some embodiments, the culture comprises one or more green algae of the genus chlorella, such as chlorella minutissima, or any combination thereof. In some embodiments, the culture comprises one or more green algae of the genus botryococcus, such as botryococcus braunii, botryococcus suvialis, or any combination thereof. In some embodiments, the culture comprises one or more green algae of the genus chlamydomonas, or any combination thereof. In some embodiments, the culture comprises one or more green algae of the genus closterium or any combination thereof. In some embodiments, the culture comprises one or more green algae of the genus spirulina, or any combination thereof. In some embodiments, the culture comprises one or more green algae of the genus diatom, or any combination thereof. In some embodiments, the culture comprises one or more green algae of the genus coleus, or any combination thereof. In some embodiments, the culture comprises one or more green algae of the genus Haematococcus, such as Haematococcus pluvialis, or any combination thereof. In some embodiments, the culture comprises one or more green algae of the genus dunaliella, such as dunaliella salina, dunaliella palustris, dunaliella viridis, or any combination thereof. In some embodiments, the culture comprises one or more dinoflagellates of the genus chrysophyceae, such as dinoflagellates like Strongylocentrotus, or any combination thereof. In some embodiments, the culture comprises one or more green algae of the genus tetradinoflagellate, such as Platymonas subcordiformis, or any combination thereof. In some embodiments, the diatoms are target algae, or are used in culture in combination with one or more green algae. Examples of diatoms include, but are not limited to, those of the genus skeletonema, such as skeletonema costatum, those of the genus chaetoceros, such as chaetoceros calcoaceticus, or any combination thereof. The culture may be in a pond. The pond may be a raceway pond. In some embodiments, the target algae is at least 50% of the total algae. In some embodiments, the target algae is at least 75% of the total algae. In some embodiments, the target algae is at least 90% of the total algae. In some embodiments, the target algae is at least 95% of the total algae. In some embodiments, the target algae is at least 99% of the total algae.

The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the cultivation of a green algae culture according to the invention while maintaining culture selectivity. Scenedesmus obliquus culture (university of Texas) was used. To increase the volume, the slant culture (20mL, 50 ten thousand cells/mL) was subcultured into 6 tubes (50mL culture until a concentration of 100 ten thousand cells/mL was reached) using a UTEX nutrient medium, although other suitable media could be used. The UTEX nutrient medium was peptone medium of Bristol medium, containing 1g/L peptone. The Bristol medium was 2.94mM NaNO₃，0.17mM CaCl₂·2H₂O，0.3mM MgSO₄·7H₂O，0.43mM K₂HPO₄，1.29mM KH₂PO₄And 0.43mM NaCl. Once growth was established, the cultures were transferred to 250ml Erlenmeyer flasks, starting at this time with the nutrient concentrations, which are described below. When the cell density increased (to a concentration of 1 million cells/ml in 200ml culture), the culture was transferred to a 1.5 liter bubble column (1.25L culture grown up to 2 million cells/ml) and the same nutrient treatment was continued. The culture was then transferred into outdoor pipeline ponds (each pond having a capacity of about 22 liters, containing about 18 liters of algal culture). The concentration of cells in the pond is maintained between 2 million and 3 million cells/ml. An acrylic pond was used to ensure sufficient light, the mixing speed being about 15 cm/s.

The nutrient concentrations employed to maintain the pond culture as described above include sodium bicarbonate, urea, trisodium phosphate, and ferrous chloride. The concentrations stated are those obtained after addition of nutrients to the pond. Sodium bicarbonate was used at a concentration of 2 mM. A nitrogen to phosphorus ratio (N: P) of about 30: 1 was used at 0.75mM N and 20. mu.MP. Ferrous chloride was used at about 2 μ M. Scenedesmus obliquus grows well in the pH range of 6-8. For this purpose, carbon dioxide was periodically bubbled throughout the day once the pH reached 8.5.

Scenedesmus obliquus has a doubling rate of about 20 hours. By maintaining the cell residence time at about 20 hours, consistent growth of algae can be maintained, while other organisms with longer residence times are washed out. To achieve this residence time, the culture was diluted 60% per day.

The optimal growth temperature range of Scenedesmus obliquus is 20-30 ℃. However, at 35 ℃, growth declined dramatically. In order to maintain the temperature in the optimum range, the pond is maintained at a minimum depth of 18cm and the mixing speed is 15 cm/s. The temperature was monitored hourly and when 33 ℃ was exceeded, the culture was diluted with fresh medium.

To increase lipid content, excess biomass from daily dilution was transferred to deeper stressed ponds where a culture was grown to deplete essentially all nitrogen. Because the nutrient concentration provided in the pipeline pond is nitrogen sufficient for 24 hours of growth, the stressed culture is depleted of nitrogen in about 4-6 hours. The cultures were then maintained under nitrogen stress for an additional 48 hours and then harvested.

Lipid analysis was performed using fluorescence and total lipid extraction. Fluorescence may be a lipid measurement method. The dye nile red is highly fluorescent in the presence of lipids for achieving a reading. A Turner 110 type fluorometer with a F4T5/d lamp was used. The emission filter used was 420-470nm and the excitation filter used was > 520 nm. For this operation, the culture was diluted to a biomass of 3 ppm. The dye was then added at a concentration of 1 ppm. The solution was mixed using a vortex mixer for 5 minutes and then the results were read at 5 minute intervals for 1 hour. The results were compared to a standard solution of 1ppm triolein (with 1ppm nile red).

Total lipid extraction was performed using a modified Bligh and Dyer method. The lipids useful for biodiesel production were extracted using chloroform and methanol in a 1: 1 ratio. The target algae were first dewatered and the slurry was then dried overnight using a bench top dewatering apparatus. The algae pieces were then weighed and an equal amount of chloroform methanol solution was added. The slurry was then mixed using a vortex. After 30 minutes, the tube cap was removed and the solvent was allowed to evaporate. After evaporation was complete, the contents were filtered and measured.

The method of harvesting scenedesmus obliquus can vary. To produce biodiesel, the algae slurry is dewatered and insufficiently dried. An inexpensive and reasonably efficient dewatering method is the use of settling ponds, which also serve as stress ponds. The dual-purpose pond allows algae to accumulate lipids while providing a storage site for harvesting. Scenedesmus obliquus maintains a negative charge around the cell wall during the growth phase. This charge causes the cells to repel each other. After the cell ages, it no longer rapidly photosynthesizes, loses charge, and can aggregate with other cells. These lumps become larger and eventually sink to the bottom of the pond, allowing for the withdrawal of a thicker slurry. In stressed ponds, cells reach this resting phase. As the cells accumulate lipids, they also begin to clot and settle.

Example 2

This example demonstrates the cultivation of a culture of a target alga for the production of beta-carotene according to the invention. Beta-carotene is a lipid and oil soluble product that has antioxidant, free radical trapping properties and cancer preventing activity. Different algae species can be cultured to obtain beta-carotene pellets. For example, seawater, and sometimes fresh water, algae of the genus dunaliella, such as dunaliella salina, dunaliella palustris (d.para), dunaliella viridis (d.viridis), and any combination thereof, may be employed in the basal medium. Dunaliella is a unicellular, biflagellated, naked green alga. Pakaea ducreyi (d.parva) and dunaliella salina can accumulate a large amount of beta-carotene. These algae can grow in the range of 20 to 40 ℃, but can also tolerate much lower temperatures.

The following substances can be used for preparing the culture medium for the production of algal beta-carotene: 2.14M NaCl, 4.81. mu. MFeCl₃，1.82μM MnCl₂，0.13mM NaH₂PO₄And 1.18mM NaNO₃Sea water and other minerals may also be used. Can reach 30-40gm dry weight/m²Production capacity per day. Harvesting was performed by high pressure filtration device using diatomaceous earth as filter material. Harvested biomass may also be dried and sold for consumption. In some cases, the algal mass is centrifuged or filtered, NaCl is added, and then centrifuged for several cycles. Cells can be disrupted by osmotic pressure, but the beta-carotene remains bound to the membrane. During this step, the beta-carotene globules are released from the membrane to the supernatant, existing as a suspension. The suspension was mixed with a solution containing 50% sucrose and Tris HCl and the preparation was centrifuged. Purified beta-carotene pellets were collected from the upper layer while a chlorophyll-containing film was precipitated at the bottom.

Example 3

This example demonstrates the cultivation of diatom or chlorella cultures for use in aquaculture feed according to the invention. Can be used in outdoor pondCulturing diatoms, skeletonema costatum, chaetoceros calcaratus, dinoflagellates such as dinoflagellates (Prymnesiophyceae Isochrysis galpana) and Platymonas subcordiformis (Prasinophyces tetraselmis sueca) to produce aquaculture feed. The stock culture was maintained under constant illumination at 2000 lux (lux) at a temperature of 22-24 ℃. In the presence of NaNo₃、NaH₂PO₄、Na₂SIO₃、FeCl₃And Na₂Diatoms were cultured in seawater media of EDTA. For green algae, the silicate solution is omitted. The storage culture was maintained in the laboratory and the culture was inoculated into an outdoor pond. The optimum temperature is 20 to 33 ℃. Algae were harvested using a 20 micron filter, and the biomass was air dried to serve as feed for shrimp, mussels and other fish larvae. The product includes not only aquaculture feed, but also typically includes protein and fiber.

Example 4

This example demonstrates the production of astaxanthin by culturing a culture of an algal of interest according to the present invention. Haematococcus pluvialis was cultured in the laboratory and tested for astaxanthin content. Astaxanthin is a carotenoid pigment used for various pharmaceutical and nutraceutical purposes. The algae is initially a green, bi-flagellated member of the green alga, and is typically grown in freshwater environments. Each cell has a single goblet chloroplast containing many starch nuclei. When cells are stressed by factors such as high light intensity, nutrient depletion, direct exposure to sunlight, etc., they form cysts, which appear red, which makes them viable for a long time. The cysts accumulate a large amount of the haematochrome astaxanthin in their cells, which can amount to 4% of its dry weight. Laboratory cultured haematococcus pluvialis is stressed by high temperature and nutrient deficiency. The cysts are allowed to settle by gravity and supercritical CO is used₂Treated to disrupt their cells. The ruptured cells released accumulated astaxanthin, dried them gently at room temperature, and packaged.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) is to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims (88)

1. A method of selectively culturing a target alga, the method comprising:

culturing a target alga in a first pond;

diluting the target alga in the first pond;

supplying a nutrient composition to the first pond; and

culture selectivity was maintained in the first pond.

2. The method of claim 1, wherein the method further comprises the steps of: carbon dioxide is added to the first pond if a pH of about 8.5 or higher is reached.

3. The method of claim 1, wherein the method further comprises the steps of: if a temperature of 33 ℃ or higher is reached, a cooling liquid is added to the first pond.

4. The method of claim 3, wherein the cooling fluid is fresh medium.

5. The method of any one of claims 1-4, wherein the step of supplying the nutrient composition is performed at about the same time as the step of diluting.

6. The method of any one of claims 1-5, wherein the nutrient composition comprises sodium bicarbonate at a concentration of at least about 0.6mM, as measured after addition of the nutrient composition to the pond.

7. The method of any one of claims 1-6, wherein the nutrient composition comprises sodium bicarbonate at a concentration of at least about 2mM, as measured after addition of the nutrient composition to the pond.

8. The method of any one of claims 1-7, wherein the nutrient composition comprises a source of iron.

9. The method of claim 8, wherein the source of iron comprises ferrous chloride.

10. The method of any one of claims 1-9, wherein the nutrient composition comprises a nitrogen source and a phosphorous source.

11. The method of claim 10, wherein the nitrogen source comprises urea and the phosphorous source comprises trisodium phosphate.

12. The method of claim 10 or 11, wherein the nitrogen to phosphorus ratio is at least about 15: 1.

13. The method of claim 12, wherein the ratio is about 29: 1.

14. The method of claim 12, wherein the ratio is about 30: 1.

15. The method of any one of claims 1-14, wherein the first pond is a raceway pond.

16. The method of any one of claims 1-14, wherein the first pond comprises a transparent enclosure.

17. The method of claim 15, wherein the transparent envelope comprises an acrylic polymer.

18. The method of any one of claims 1-17, wherein the algal culture volume of the first pond is about 18 liters or more.

19. The method of any one of claims 1-17, wherein the algal culture volume of the first pond is about 600 liters or more.

20. The method of any one of claims 1-17, wherein the algal culture volume of the first pond is about 14,000 liters or more.

21. The method of any one of claims 1-20, wherein the first pond comprises an average algal culture depth of about 13-20 centimeters.

22. The method of claim 21, wherein the average algal culture depth is about 18 centimeters.

23. The method of any one of claims 1-22, wherein the target alga is mixed at a speed of about 12 cm/sec, about 15 cm/sec, or about 18 cm/sec.

24. The method of any one of claims 1-23, wherein the diluting step comprises diluting the target alga in the first pond by a dilution of from about 35% to about 60%.

25. The method of any one of claims 1-25, wherein the dilution is performed about every 20 hours.

26. The method of any one of claims 1-25, wherein the dilution is performed when the transparency plate reads 5-6 cm.

27. The method of any one of claims 1-25, wherein the dilution is continuous.

28. The method of claim 27, wherein the concentration of algae in the first pond is maintained in a range from about 2 million to about 3 million algae/ml.

29. The method of any one of claims 1-28, wherein the diluting step comprises removing a volume of the target alga from the first pond.

30. The method of claim 29, wherein the method further comprises:

the target algal volume from the first pond is discharged into the second pond.

31. The method of claim 30, wherein the algal depth of the second pond is about 18 to about 30 centimeters.

32. The method of claim 30 or 31, wherein the second pond is a stress pond.

33. The method of claim 32, wherein the stress pond is nitrogen deficient.

34. The method of any one of claims 30-33, wherein the method further comprises:

harvesting the target alga from the second pond.

35. The method of claim 34, wherein harvesting is performed from about 52 hours to about 54 hours after discharging the volume of the target alga into the second pond.

36. The method of claim 34, wherein harvesting is performed about 72 hours after discharging the volume of the target alga into the second pond.

37. The method of claim 34, wherein harvesting is performed once the lipid concentration reaches at least about 25% of the cell population.

38. The method of any one of claims 34-37, wherein the method further comprises:

dewatering the target algae harvested from the second pond.

39. The method of claim 38, wherein said dewatering step comprises using at least one of a belt press and a dewatering apparatus.

40. The method of any one of claims 34-37, wherein the harvesting step comprises dewatering the target alga by suctioning the settled target alga from the second pond.

41. The method of any one of claims 1-40, wherein the method further comprises:

extracting lipid from target algae.

42. The method of claim 41, wherein the extracting step comprises at least one of: chloroform: methanol extraction and hexane extraction.

43. The method of any one of claims 1-42, wherein the method further comprises:

amplifying the seed culture of the target alga to realize the culture of the target alga in the first pond.

44. The method of claim 42, wherein the step of amplifying comprises two or more steps of successively larger volumes of the target alga.

45. The method of any one of claims 1-44, wherein the target alga is at least one green alga.

46. The method of claim 45, wherein said green algae are algae of the genus Scenedesmus.

47. The method of claim 45 or 46, wherein the green alga is Scenedesmus obliquus.

48. The method of claim 45 or 46, wherein the green algae are selected from the group consisting of: scenedesmus obliquus, Scenedesmus quadricaudatus, Scenedesmus maximus, Scenedesmus oboensis, Scenedesmus tundifolius, Scenedesmus obliquus and any combination thereof.

49. The method of any one of claims 46-48, wherein the Scenedesmus obliquus is Scenedesmus obliquus UTEX strain 1450.

50. The method of claim 45, wherein said green algae belong to a genus selected from the group consisting of: scenedesmus, chlorella, botryococcus, chlamydomonas, crescentella, discoreana, linear algae, coleoptera, haematococcus, dunaliella, chrysophyceae, tetragonia, and any combination thereof.

51. The method of any one of claims 1-44, wherein the target alga is at least one diatom.

52. The culture of claim 52, wherein said diatoms belong to a genus selected from the group consisting of: skeletonema costatum, chaetoceros, and any combination thereof.

53. A method of selectively culturing a target alga of the genus scenedesmus, the method comprising:

culturing a target alga in a first pond;

diluting the target alga in the first pond;

supplying a nutrient composition to the first pond; and

culture selectivity was maintained in the first pond.

54. The method of claim 53, wherein said green algae are selected from the group consisting of: scenedesmus obliquus, Scenedesmus quadricaudatus, Scenedesmus maximus, Scenedesmus oboensis, Scenedesmus tundifolius, Scenedesmus obliquus and any combination thereof.

55. A method of selectively culturing a target alga scenedesmus obliquus, the method comprising:

culturing a target alga in a first pond;

diluting the target alga in the first pond;

supplying a nutrient composition to the first pond; and

culture selectivity was maintained in the first pond.

56. A method of selectively culturing a target alga scenedesmus obliquus, the method comprising:

culturing algae in the pipeline pond;

adding carbon dioxide to the pipeline pond if a pH of about 8.5 or greater is reached;

adding a cooling liquid to the pipeline pond if the temperature reaches 33 ℃ or higher;

about every 20 hours, diluting the algae in the raceway pond by about 60%;

adding a nutrient composition to the pipeline pond at about the same time as the diluting step, wherein the nutrient composition comprises sodium bicarbonate, urea, trisodium phosphate, and ferrous chloride, the sodium bicarbonate concentration is at least 2mM, and the nitrogen to phosphorus ratio is at least about 15: 1;

discharging the volume of algae obtained in the dilution step into a nitrogen deficient stress pond;

harvesting algae from the stress pond and dewatering; and

lipids are extracted from algae.

57. The method of any one of claims 1-56, wherein the target alga is maintained at least 50% of total alga.

58. The method of any one of claims 1-56, wherein the target alga is maintained at least 75% of total alga.

59. The method of any one of claims 1-56, wherein the target alga is maintained at least 90% of total alga.

60. The method of any one of claims 1-56, wherein the target alga is maintained at least 95% of total alga.

61. The method of any one of claims 1-56, wherein the target alga is maintained at least 99% of total alga.

62. The method of any one of claims 1-61, further comprising:

using lipids produced from the target algae, biofuels are produced.

63. The method of claim 62, wherein the biofuel is biodiesel.

64. The method of claim 62, wherein the biofuel is a bio-coal extract.

65. A biofuel produced by the method of any one of claims 1-64.

66. The method of any one of claims 1-59, further comprising:

polyunsaturated fatty acids are produced from the target algae.

67. The method of claim 66, wherein the polyunsaturated fatty acid is an omega-3 fatty acid.

68. The method of claim 67, wherein said omega-3 fatty acid is selected from the group consisting of: alpha-linolenic acid (ALA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and any combination thereof.

69. A polyunsaturated acid produced by the method of any one of claims 1-59 or 66-68.

70. The method of any one of claims 1-59, further comprising:

producing a feedstock from the target alga.

71. The process of claim 70, wherein the feedstock is selected from the group consisting of: animal feed, aquaculture feed, and any combination thereof.

72. A feedstock produced by the method of any one of claims 1-59, 70, or 71.

73. The method of any one of claims 1-59, further comprising:

plant nutrients are produced from the target algae.

74. The method of claim 73, wherein said plant nutrient is a carotenoid.

75. The method of claim 74, wherein said carotenoid is selected from the group consisting of: astaxanthin, beta-carotene, and any combination thereof.

76. A plant nutrient produced by the method of any one of claims 1-59 or 73-75.

77. A selective outdoor pipeline pond algae culture comprising a target algae of the genus scenedesmus.

78. The culture of claim 77, wherein the target algae is at least 50% of the total algae.

79. The culture of claim 77, wherein the target algae is at least 75% of the total algae.

80. The culture of claim 77, wherein the target algae is at least 90% of the total algae.

81. The culture of claim 77, wherein the target algae is at least 95% of the total algae.

82. The culture of claim 77, wherein the target algae is at least 99% of the total algae.

83. The culture of any one of claims 77-82, wherein the target alga is Scenedesmus obliquus.

84. The culture of claims 77-82, wherein the target alga is selected from the group consisting of: scenedesmus obliquus, Scenedesmus quadricaudatus, Scenedesmus maximus, Scenedesmus oboensis, Scenedesmus tundifolius, Scenedesmus obliquus and any combination thereof.

85. The culture of claim 83 or 84, wherein said Scenedesmus obliquus is Scenedesmus obliquus UTEX strain 1450.

86. The culture of claims 77-82, wherein the target alga belongs to a genus selected from the group consisting of: scenedesmus, chlorella, botryococcus, chlamydomonas, crescentella, discoreana, linear algae, coleoptera, haematococcus, dunaliella, chrysophyceae, tetragonia, and any combination thereof.

87. The culture of claims 75-80, wherein the target algae comprises one or more diatoms.

88. The culture of claim 87, wherein said diatoms belong to a genus selected from the group consisting of: skeletonema costatum, chaetoceros, and any combination thereof.