HK40002710A - Improved method for growing algae - Google Patents

Description

Method for improving algae growth

Background

As the world population continues to grow, the demand for edible protein sources continues to increase. Although the land available for planting traditional crops is limited, there is a need to meet this demand. Algae are a replacement for traditional crops. Algae have several advantages over terrestrial crops. The algae can grow very fast, and the generation time is as short as 10 hours. Many algal species are also rich in protein, and some 70% of blue-green algal species are reported to be protein (Becker, biotech. adv. (2007)25: 207). The rapid growth rate of algae and the high protein content mean that algae produce more than 10 times more protein per acre than legumes.

Algae are particularly useful for the production of dietary proteins for humans and non-ruminants because, in addition to being able to produce large quantities of protein, the protein is of high quality in terms of amino acid composition. The amino acid composition of algae is similar to that of other high quality plant sources, such as soybean, and far superior to grains such as wheat (Fabregas and Herrero, Applied Microbiol. Biotechnol. (1985)23: 110). Algal proteins have a relatively high biological value (BECKER, supra) compared to human-favored animal proteins such as eggs and casein. Furthermore, algal proteins are highly digestible. Additionally, algae can be genetically engineered to produce proteins of particular nutritional value.

In addition to being a source of nutritional proteins, algae are also used in the production of therapeutic proteins. Protein-based therapies are increasingly important in medical therapy. In recent years, interest in eukaryotic microalgae as an alternative to recombinant protein production has increased. Production of proteins in transgenic algae can have the same advantages as transgenic plants, including cost, safety, rapid scalability. Microalgae expressing a therapeutic protein can be grown under the improved conditions described herein. Expression of recombinant proteins in chloroplasts of the green alga Chlamydomonas reinhardtii has been established (Mayfield S.P., et al (2007) Curropin Biotechnol 18: 126-133). These proteins include reporter proteins (Franklin S., et al Plant J (2002)30: 733-744; Mayfield S.P. and Schultz J.plant J (2004)37:449 458; Muto M., et al BMC Biotechnol (2009)9:26), large and complex mammalian single-chain antibodies (Mayfield S.P., et al ProcNatl Acad Sci USA (2003)100: 438-. Proteins purified from algae have advantages over proteins produced by standard methods because they are free of toxins and viral agents that may be present in the preparation from bacterial or mammalian cell cultures.

An important factor that hinders the widespread use of algae as a protein source and a way of producing therapeutic proteins is the high production cost. Algae for human consumption or pharmaceutical use must be produced according to high quality standards. In most cases, algae must be grown in a closed fermentation vessel. The use of closed fermentation vessels increases production costs in terms of the large capital required to procure and install the fermenters and the high energy costs associated with operating the fermenters.

One way to reduce the production cost is to increase the growth rate of the algae. With faster growth rates, capital and operating costs amortized over a larger volume of product, unit production costs are reduced. An improved method of producing algae is provided herein that results in significantly higher growth rates and improved nutritional characteristics.

Disclosure of Invention

Several aspects and embodiments of the inventive concepts disclosed herein include: aerobic, heterotrophic process for the high density cultivation of Chlamydomonas species in Chlamydomonas species, wherein the culture reaches a target density of 50-200 g/L of dry cell weight. In some embodiments, the target density of the culture is at least 50g/L, at least 60g/L, at least 70g/L, at least 80g/L, at least 90g/L, at least 100g/L, at least 110g/L, at least 120g/L, at least 130g/L, at least 140g/L, at least 150g/L, at least 160g/L, at least 170g/L, at least 180g/L, at least 190g/L, or at least 200g/L of the stem cell weight. In other embodiments, the target density is 50g/L to 75g/L, 75g/L to 100g/L, 100g/L to 125g/L, 125g/L to 150g/L, 150g/L to 175g/L, or 175 to 200g/L of dry cell weight. In some embodiments, the density of the production culture prior to harvest is 50g/L, 55g/L, 65g/L, 70g/L, 75g/L, 80g/L, 85g/L, 90g/L, 95g/L, 100g/L, 105g/L, 110g/L, 115g/L, 120g/L, 125g/L, 130g/L, 135g/L, 140g/L, 145g/L, 150g/L, 155g/L, 160g/L, 165g/L, 170g/L, 175, 180, 185, 190, 195, or 200g/L of the stem cell weight. In some embodiments, the target density or concentration is reached within 250 hours after the start of the production culture.

The Chlamydomonas species used may be any species capable of heterotrophic or mixotrophic growth, such as Chlamydomonas reinhardtii, Chlamydomonas dysomos, Chlamydomonas chandane, Chlamydomonas debaryana, Chlamydomonas moenbauna, Chlamydomonas moewussii, Chlamydomonas culeus, Chlamydomonas noctifida, Chlamydomonas aurata, Chlamydomonas monoplanata, Chlamydomonas marvanii or Chlamydomonas probosceri. In one embodiment, the chlamydomonas species is a wild-type species, i.e. it does not contain heterologous or exogenous genes.

The method comprises obtaining an inoculum of a substantially pure culture of a species of Chlamydomonas at a concentration of 0.1g/L to 15 g/L. The inoculum is used to produce a production culture by adding it to an initial volume of fermentation medium, wherein the amount of inoculum used does not exceed 20% of the initial volume of fermentation medium. The production culture is then grown aerobically at a pH of about 6.0 to 10.0 and at a temperature of about 15 ℃ to about 37 ℃. In one embodiment, the production culture is grown in the absence of light. In one method, the production culture is fed by providing a feeding medium (100 times the concentration of the fermentation medium supplemented with glacial acetic acid). The feeding schedule is determined by the pH change of the production culture. The production culture is grown until it reaches the desired target density, at which point the algae is harvested from the culture.

In certain embodiments, the production culture is fed nutrients when the pH of the production culture increases beyond a predetermined set point. In some embodiments, feeding is started when the pH exceeds 7.5 and stopped when the pH is below 6.8. In other embodiments, feeding is used to maintain the pH of the production culture at pH 6.6. + -. 0.1, pH 6.8. + -. 0.1 or pH 7.0. + -. 0.1.

Harvesting may be accomplished by any method known in the art, including but not limited to filtration, batch centrifugation, or continuous centrifugation. In some cases, the production culture reached harvest density within 250 hours after the start of culture.

The harvested algae may be dried to a moisture content of, for example, no more than 15% by spray drying, ring drying, paddle drying, tray drying, solar or sun drying, vacuum drying or freeze drying.

Another aspect provides a nutritional supplement comprising at least 90% of at least one species of the genus chlamydomonas, wherein the nutritional supplement is no more than 15% moisture, at least 50% crude protein, at least 10% fat content, no more than 5% ash. In some embodiments, the nutritional supplement further contains omega 3 fatty acids, omega 6 fatty acids, and omega 9 fatty acids.

In one aspect, a culture of one or more species of chlamydomonas under growing conditions is provided, wherein the density of the culture increases at a rate of 50% to 300%, 50% to 100%, 100% to 150%, 150% to 200%, 200% to 250%, or 250% to 300% per 24 hour period.

In one aspect, a culture of one or more chlamydomonas species under production conditions is provided, wherein the density of the culture increases at a rate of at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 225%, at least 250%, at least 275%, or at least 300% per 24 hour period.

Also provided is a culture of one or more Chlamydomonas species under steady state conditions, wherein the culture has an algal density of at least 50g/L, at least 60g/L, at least 70g/L, at least 80g/L, at least 90g/L, at least 100g/L, at least 110g/L, at least 120g/L, at least 130g/L, at least 140g/L, at least 150g/L, at least 160g/L, at least 170g/L, at least 180g/L, at least 190g/L, at least 200g/L, and wherein steady state is defined as a state where the concentration of algae in the culture increases by about 0.1% to about 50% per 24 hour period.

Other aspects provide a method of producing a therapeutic protein from a substantially pure culture of at least one species of the genus chlamydomonas expressing at least one exogenous therapeutic protein. The method comprises inoculating a production culture with an inoculum comprising a substantially pure culture containing about 0.1g/L to about 15g/L of at least one species of genus chlamydomonas expressing at least one exogenous therapeutic protein. The amount of inoculum used does not exceed 20% of the initial volume of the production culture. The production culture is then grown under aerobic conditions at a pH of about 6.4 to 10 and a temperature of about 15 ℃ to about 37 ℃. In one embodiment, the production culture is grown in the absence of light. The production cultures were fed as planned using a feeding medium (100 times the medium concentration of the production culture supplemented with glacial acetic acid) based on the pH of the production culture. The production culture is grown until the algae reaches the desired target concentration, and then the algae is harvested from the culture medium. Harvesting may be performed by filtration, batch centrifugation or continuous centrifugation.

In a particular embodiment, feeding is determined based on an increase in the pH of the production medium. In a particular embodiment, if the pH is greater than 7.4, the production culture is fed with nutrients and is stopped when the pH reaches 6.8. In other embodiments, the feeding program is designed to maintain the pH of the production medium at pH 6.6. + -. 0.1, pH 6.8. + -. 0.1 or pH 7.0. + -. 0.1.

In some embodiments, the target concentration is at least 65g/L or at least 70 g/L. In other embodiments, the target concentration is 75g/L, 80g/L, 90g/L, 95g/L, 100g/L, 105g/L, 110g/L, 115g/L, 120g/L, 125g/L, 130g/L, 135g/L, 140g/L, 145g/L, 150g/L, 155g/L, 160g/L, 165g/L, 170g/L, 175g/L, 180g/L, 185g/L, 190g/L, 195g/L, or 200 g/L.

In some embodiments, the algae are dried post-harvest by, for example, spray drying, ring drying, paddle drying, tray drying, solar or sun drying, vacuum drying, or freeze-drawing. In other embodiments, the method further comprises isolating at least one therapeutic protein from the algae.

Drawings

These and other features, aspects, and advantages of the claimed invention will become better understood with regard to the following description, appended claims, and accompanying drawings where:

FIG. 1 is a graph of the percentage of oxygen saturation and agitation in a seed fermentor versus the batch age (batch).

FIG. 2 is a graph of pH and temperature in a seed fermentor versus batch age.

FIG. 3 is a graph of the weight concentration of stem cells and percentage of canister fill in a seed fermentor versus batch age.

FIG. 4 is a graph of the percent oxygen saturation and agitation in the production fermentor versus batch age.

FIG. 5 is a graph of pH and temperature in the production fermentor versus batch age.

FIG. 6 is a graph of the weight concentration of stem cells and the percentage of tank fill in the production fermentor versus the batch age.

Figure 7 shows the numbering and folding using filtration by microwave method to determine the weight of Dry Cells (DCW).

Fig. 8 is a photograph of a wave pocket.

FIG. 9 shows the weight and growth rate of stem cells achieved during fermentation using basal media consisting of the components in Table 4, feeder media consisting of the trace element components in Table 5 and the compounds listed in Table 6.

FIG. 10 shows the Dissolved Oxygen (DO), temperature and impeller agitation rate achieved during fermentation using basal media composed of the ingredients in Table 4, trace elements composed of the ingredients in Table 5 and nutrient media composed of the compounds listed in Table 6.

FIG. 11 shows the gas flow and pH measured during fermentation using a basal medium consisting of the ingredients in Table 4, trace elements consisting of the ingredients in Table 5 and a feeding medium consisting of the compounds listed in Table 6.

FIG. 12 shows the feed medium consumed by Chlamydomonas reinhardtii cultures during fermentation using a basal medium consisting of the ingredients in Table 4, a trace element consisting of the ingredients in Table 5 and a feed medium consisting of the compounds listed in Table 6.

Detailed Description

The following detailed description is provided to assist those skilled in the art in carrying out the claimed invention. However, this detailed description should not be construed to unduly limit the claimed invention since modifications and variations to the embodiments discussed herein may be made by those of ordinary skill in the art without departing from the scope of the present invention.

All publications, patents, patent applications, public databases, public database entries, and other references cited in this application are herein incorporated by reference in their entirety as if each individual publication, patent application, public database entry, or other reference were specifically and individually indicated to be incorporated by reference.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

For the numerical ranges provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or any other intervening value in that stated range is also encompassed. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, and each range where either or both endpoints are included in the smaller range or neither is included in the smaller range is also included to exclude any particular endpoint from the stated range. If a stated range includes one or both of the endpoints, ranges that do not include one or both of the included endpoints are also included. Unless otherwise indicated, culture density or concentration is based on Dry Cell Weight (DCW).

Provided herein are improved methods for heterotrophic or mixotrophic, aerobic production of algae of the genus chlamydomonas. As used herein, the terms "heterotrophic" and "heterotrophic" refer to a condition in which algae can utilize an organic carbon source as an energy source and as a carbon source. Therefore, heterotrophic algae can grow in the absence of light. As used herein, the terms "mixotrophic" and "mixotrophic" refer to the situation where algae grow under conditions where light (photosynthesis) and organic carbon can be used as an energy source. Under mixed nutrient conditions, algae can be grown by photosynthesis using light as an energy source to immobilize inorganic carbon (e.g., carbon dioxide, bicarbonate, and/or carbonate), and by heterotrophic growth using organic carbon as an energy source.

Any chlamydomonas species capable of utilizing an organic carbon source as an energy source can be used to practice the methods disclosed herein. Algae can be strictly heterotrophic in that it is genetically manipulated, either naturally or by any means available in the art, to be unable to undergo photosynthesis. By genetic manipulation is meant altering the genetic makeup of an organism by human manipulation. Genetic manipulation as used herein includes recombinant DNA techniques and traditional plant breeding techniques. If traditional plant breeding techniques are used, the photosynthetic algae can be strictly heterotrophic by, for example, selection and random mutagenesis. If recombinant DNA technology is used, algae can be rendered unable to photosynthesize by any technique known in the art, such as gene knockout or gene silencing techniques. Various methods of rendering algae photosynthetically unavailable will be readily apparent to those skilled in the art.

Alternatively, the algae may be natural or through the use of genetically manipulated mixed nutrients. Examples of naturally mixotrophic Chlamydomonas species include, but are not limited to, Chlamydomonas reinhardos, Chlamydomonas dysomo, Chlamydomonas mundane, Chlamydomonas debarkii, Chlamydomonas moewussii, Chlamydomonas noctifida, Chlamydomonas aurata, Chlamydomonas apppinata, Chlamydomonas marvanii, and Chlamydomonas proboscigera. For example, non-naturally heterotrophic photosynthetic algae can be prepared by using, for example, the application of selective pressure. Alternatively, photosynthetic algae can metabolize exogenous organic carbon sources by introducing necessary metabolic pathway genes, by using recombinant DNA techniques known in the art.

Whether the algae are grown heterotrophically or under mixed nutrient conditions, the algae are grown without photosynthesis by providing the necessary nutrients. For example, the medium in (or on) which the organism is grown can be supplemented with any desired nutrients, including organic carbon sources, nitrogen sources, phosphorus sources, vitamins, metals, lipids, nucleic acids, micronutrients and/or any organism-specific requirements. Organic carbon sources include any carbon source that algae can metabolize, including but not limited to acetate; simple carbohydrates (e.g., glucose, sucrose, lactose); complex carbohydrates (e.g., starch, glycogen); proteins and lipids. In one embodiment, the carbon source is an acetate salt provided in the form of acetic acid.

The algae used in the practice methods disclosed herein can be a single species or a mixed species of Chlamydomonas. In one embodiment, the culture contains predominantly one species of the genus chlamydomonas. As used herein, a culture is primarily a species when the species constitutes more than 50% of the algal organism. For example, a particular chlamydomonas species that contains greater than 50%, greater than 55%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% is a culture that predominantly comprises that species. In other embodiments, the culture is free of adventitious contamination with respect to species or genus. As used herein, a culture is said to be free of adventitious contamination for a genus or species if the culture is not contaminated with the deliberate organism. For example, a culture that is free of adventitious contamination for Chlamydomonas may contain one or more species of Chlamydomonas algae, but not any other species of Chlamydomonas. Likewise, cultures without adventitious contamination for Chlamydomonas reinhardtii do not include other organisms other than Chlamydomonas reinhardtii, including other Chlamydomonas species. Methods for determining whether a culture is free of adventitious contamination, such as contamination by yeast or bacteria, are known to those skilled in the art.

In one embodiment, the culture is free of fungi or bacteria as determined by standard methods. In particular embodiments, the culture is determined to be free of aerobic bacteria by a sterile Colony Forming Unit (CFU). In another embodiment, the culture is determined to be free of yeast, mold, and coliform bacteria by a colony forming unit. In other embodiments, the culture is determined to be free of Escherichia coli, Staphylococcus aureus, and Salmonella sp by being free of colony forming units.

Using the methods disclosed herein, algae are grown heterotrophically or mixotrophically in a bioreactor. The term bioreactor refers to a system that is closed to the environment and does not exchange gases and contaminants directly with the environment. Where the algae are mixed nutrients, they may be grown in a photobioreactor. Photobioreactors are bioreactors that incorporate some type of light source to input light energy to the reactor. The photobioreactor can be described as a closed, illuminated culture vessel designed for controlled biomass production of a phototrophic liquid cell suspension culture. Examples of the photobioreactor include, for example, glass containers, plastic tubes, transparent tanks, plastic sleeves, and plastic bags. Examples of light sources that can be used to provide the energy required to sustain photosynthesis include, for example, fluorescent lamps, LEDs, and natural sunlight.

In certain embodiments, the algae are grown heterotrophically in the absence of light, in which case a bioreactor other than a photobioreactor may be used. In one embodiment, the bioreactor is made of an opaque material, such as stainless steel, that does not allow light to enter the interior of the bioreactor. In another embodiment, the bioreactor is made of a material that allows light to enter the interior, such as those used in photobioreactors, but the bioreactor itself is placed in an enclosure, such as a room or cubicle, that prevents light from entering the bioreactor. In yet another embodiment, the photobioreactor is covered with an opaque material to prevent light from entering.

There are several features to be present in a bioreactor, whether or not light is allowed to enter the interior of the bioreactor. In one embodiment, the bioreactor is constructed of a material that allows for sterilization inside the bioreactor. Methods that may be used for sterilization include heat sterilization, chemical sterilization, or a combination of both. The bioreactor may also optionally have heating and cooling elements that allow the contents of the bioreactor to be maintained within a desired temperature range. Various methods for controlling the temperature of a bioreactor are known in the art.

In addition, the bioreactor may have a method of controlling the oxygen content of the culture medium contained in the bioreactor. Oxygen can be introduced into the medium using pure oxygen, ambient air, or a combination of pure oxygen and ambient air. Oxygen may be introduced into the culture medium by any known method. For example, the oxygen may be introduced by direct injection, agitation, or a combination of direct injection and agitation. When directly injected, the oxygen may be introduced in a gaseous state, a liquid state, or a combination of both.

The bioreactor may also have means to adjust the pH of the culture medium. The pH of the medium can be lowered by adding an acid or increased by adding a base. The acid or base may be introduced in liquid form, gaseous form, solid form, or some combination thereof. In controlling the pH, a strong acid may be used, a weak acid may be used, a strong base may be used, a weak base may be used, or any combination thereof. Any acid or base compatible with the algae being grown may be used. In one embodiment, the pH is controlled by the addition of glacial acetic acid. Acetic acid is useful because it can be converted to acetate, which can serve as an energy and carbon source for the algae. In one embodiment, the pH of the medium is maintained at about 6.0 to about 10.0, about 6.0 to about 9.0, about 6.0 to about 8.0, or about 6.0 to about 7.0. In other embodiments, the pH of the medium is maintained at about 6.4 to 7.2, about 6.45 to about 7.15, about 6.5 to about 7.1, about 6.55 to about 7.05, about 6.6 to about 7.0, about 6.65 to 6.95, about 6.7 to about 6.9, or about 6.75 to about 6.85. In one embodiment, the pH of the medium is maintained at pH 6.5. + -. 0.1, pH 6.8. + -. 0.1 or pH 7.0. + -. 0.1.

In one embodiment, the algae can be grown, for example, in a small-scale laboratory culture system. Small scale laboratory systems refer to culturing in a volume of less than about 6 liters. In one aspect, the small-scale laboratory culture can be 1 liter, 2 liters, 3 liters, 4 liters, or 5 liters. In another aspect of the invention, the small-scale laboratory culture may be less than 1 liter. In one aspect, the small-scale laboratory culture can be 100 milliliters or less.

It will be apparent to those skilled in the art that the volume of culture will depend on the amount of algal biomass desired. The volume is limited only by the size of the bioreactor available to the skilled person. In one embodiment, the volume of the bioreactor may be greater than about 5 liters, or greater than about 10 liters, or greater than about 20 liters. The large-scale growth may be growth of a culture in a volume of 50 liters or more, 100 liters or more, 200 liters or more, 300 liters or more, 400 liters or more, 500 liters or more, 600 liters or more, 700 liters or more, 800 liters or more, 900 liters or more, 1000 liters or more, 2000 liters or more, 3000 liters or more, 4000 liters or more, 5000 liters or more, 6000 liters or more, 7000 liters or more, 8000 liters or more, 9000 liters or more, or 10000 liters or more.

The present disclosure also discloses very large scale culture systems. In one aspect, the culture volume may be at least 20,000 liters. In another aspect, the culture volume can be up to 40,000 liters. In another aspect, the culture volume can be up to 80,000 liters, up to 100,000 liters, up to 125,000 liters, up to 150,000 liters, or up to 175,000 liters. In another aspect, the culture volume can be up to 200,000 liters. In another aspect, the culture volume can be up to 250,000 liters. In another aspect, the culture volume can be up to 500,000 liters. In another aspect, the culture volume can be up to 600,000 liters. In another aspect, the culture volume can be up to 1,000,000 liters.

At the same time, it is obvious to the person skilled in the art that when larger culture volumes are used, it may be necessary to grow the inoculum through several intermediate volumes. The following is a general discussion of the method of scale-up. It is well within the ability of one of ordinary skill in the art to modify the methods discussed herein to accommodate different sized initial cultures and different sized production cultures. For purposes of this exemplary discussion, the starting material is a TAP plate with streaking with the desired algal plant or plants. The algae were removed from the TAP plate using a sterile loop and added to a sterile, baffled 500mL shake flask containing 150mL of the appropriate medium and incubated at 150rpm for about 5 days at room temperature (about 20 ℃ C. to 28 ℃ C.) with shaking.

Alternatively, the intermediate culture may be performed in a wave bag. The bellows bag is a sterile plastic bag with one or more ports that allow gas exchange (with the atmosphere or incoming gas), sampling, and/or nutrient replenishment (see fig. 8). The culture conditions of the wave bag (including agitation) are similar to shake flasks, but may involve movement other than rotation. The skilled person will be able to envisage other suitable vessels for the intermediate culture.

Usually, the culture is carried out in the absence of light, but this is not essential. Approximately 2 days before the next magnification, a sterile sample was taken to check for contamination and proper morphology of the algae. After 5 days of culture, the Dry Cell Weight (DCW) of the shake flask is usually 1.0g/L to 3.0 g/L. Methods for determining a DCW value are provided herein. About 20% of the contents of the 500mL shake flask (in this case 120mL) were then added to a sterile, baffled 3L shake flask containing about 1050mL of media, with a final volume of about 1200mL or a dilution ratio of 1: 10. 1200mL of culture was cultured in the same manner as 150mL of culture, including morphological and contamination checks. The shake flask process is continued until at least 10% of the volume of culture used in the production bioreactor is obtained.

Once enough algae has grown in the shake flask, the algae can be used to inoculate the bioreactor. For the purposes of this disclosure, a bioreactor culture differs from a shake flask culture in that in the bioreactor culture, the culture is fed by the addition of a concentrated medium. The bioreactor is inoculated with a culture volume that is no more than about 10% of the initial culture medium volume in the bioreactor. The concentration of algae used to inoculate the bioreactor should be in the range of about 0.1g/L to about 15g/L Dry Cell Weight (DCW). In particular embodiments, the transferred algae concentration is from about 0.1g/L to about 10g/L, from about 0.1g/L to about 5g/L, from about 0.1g/L to about 4g/L, from about 0.1g/L to about 3g/L, from about 0.1g/L to about 2g/L, from about 0.1g/L to about 1g/L, from about 0.1g/L to about 0.9g/L, from about 0.1g/L to about 0.8g/L, from about 0.1g/L to about 0.7g/L, from about 0.1g/L to about 0.6g/L, from about 0.1g/L to about 0.5g/L, from about 0.1g/L to about 0.4g/L, from about 0.1g/L to about 0.3g/L, and from about 0.1g/L to about 0.2 g/L. In one embodiment, the concentration of algae used to inoculate the bioreactor is calculated using the following formula:

wherein, C_inIs the concentration of the inoculum, V_brIs the initial medium volume in the bioreactor, C_brIs the initial concentration of algae in the bioreactor immediately after inoculation.

The medium used to grow the algae in the bioreactor (fermentation medium) can be any medium suitable for heterotrophic culture of one or more algae plants grown in the bioreactor. In one embodiment, the components of the fermentation medium are set forth in Table 1.

TABLE 1 fermentation Medium A

The composition of the trace element solution is shown in table 2.

TABLE 2 microelement solution A

The fermentation medium and the trace element solution are sterile solutions so that the algae culture in the bioreactor is not contaminated by other organisms. The fermentation medium may be used in the form as given in table 1, table 4 and table 7, or may be used in a more concentrated form. In some embodiments, the fermentation medium is used at 2-fold, 3-fold, 4-fold, or 5-fold the concentration of the fermentation medium in tables 1, 4, and 7.

The temperature of the culture within the bioreactor is maintained at a temperature of from about 15 ℃ to about 26 ℃ or from about 26 ℃ to about 37 ℃. In another embodiment, the temperature is maintained at about 26.5 ℃ to about 29.5 ℃. In other embodiments, the temperature is maintained from about 27 ℃ to about 29 ℃ or from about 27.5 ℃ to about 28.5 ℃. In a specific embodiment, the temperature of the bioreactor contents is maintained at about 28 ℃.

The percent oxygen saturation in the culture medium is maintained between about 5% and 100% during fermentation in the bioreactor. In other embodiments, the oxygen saturation percentage is maintained at about 5% to about 90%, about 5% to about 80%, about 5% to about 70%, about 5% to about 60%, or about 5% to about 50%. In other embodiments, the oxygen saturation percentage is maintained at about 5% to about 55%, about 10% to about 50%, about 15% to about 45%, about 20% to about 40%, or about 25% to about 35%. In another embodiment, the percent oxygen saturation in the culture medium is maintained at 30% ± 2.5%. The percent oxygen saturation in the medium is maintained using any of the methods described herein or known in the art.

The range over which the percent oxygen saturation is allowed to vary during fermentation (culture) depends in part on the configuration of the bioreactor, particularly its ability to introduce oxygen. Typically, the operator will determine the set point and the associated upper and lower ranges. The set point may be between 5% and 100%. The allowable ranges may be within any of the ranges provided herein.

The method of the present invention is characterized in that the concentration of the feeding medium used for feeding the algae during the fermentation process is 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 110 times or 120 times the concentration of the medium. Typically, a feeding medium with a carbon source is supplemented to support a high growth rate under heterotrophic conditions. Thus, in one embodiment, the feeding medium is 100 times the concentration of the fermentation medium provided herein, supplemented with glacial acetic acid as a carbon source. The composition of a specific feeding medium is given in table 3.

TABLE 3 feed Medium A

Chemical name	Chemical formula (II)	Content (wt.)	Unit of
				Ammonium hydroxide	NH4OH	25	g·L-1
Ammonium dihydrogen phosphate	NH4H2PO4	10	g·L-1
				Potassium dihydrogen phosphate	KH2PO4	1.4	g·L-1
Calcium chloride	CaCl2	0.9	g·L-1
				Magnesium sulfate heptahydrate	MgSO4·7H2O	5.63	g·L-1
Sodium hydroxide	NaOH	4	g·L-1
				Solution of trace elements	Mixing	40	mL·L-1
(glacial) acetic acid	CH3CO2H	510	g·L-1

Feeding of the culture was triggered by a change in pH. Since cultures use acetate to meet their energy requirements, a decrease in the amount of acetate in the medium results in an increase in the pH of the medium. The increase in pH is used to trigger the addition of feeding medium to restore the pH to the desired set point. Use of this method allows for rapid growth and higher culture densities than previous culture systems. Feeding is determined by the operator determining the pH set point and the range over which feeding medium is started and stopped. The feeding of the nutrient medium is started when the pH exceeds a set point by a certain value, and the feeding of the nutrient medium is stopped when the pH is lower than the set point by the selected value. As understood by those skilled in the art, the exact set point and range will depend on the particular species of algae being grown. In the case of chlamydomonas species, the pH range should not be below pH 5.5 or above pH 10. The set point of the pH is usually in the range of 6.5 to 7.5, fluctuating within a range of + -1.0 pH units. One skilled in the art will appreciate that the pH range may not be symmetrical, which may allow the pH to rise to a greater extent than the decrease. For example, feeding may be started when the pH exceeds the set point by 0.5pH units, but stopped when the pH drops below the set point by 0.2pH units.

One skilled in the art will appreciate that the production of one bioreactor can be used to inoculate another bioreactor. In the case of very large bioreactors, it is not feasible to grow the inoculum only in flasks or wave bags. In this case, a smaller bioreactor is used to produce the amount of inoculum needed to inoculate the final production bioreactor.

In another embodiment, the composition of the fermentation medium is set forth in Table 4.

TABLE 4 fermentation Medium B

In another embodiment, the composition of the trace elements is shown in table 5.

TABLE 5 microelement solution B

Chemical name	Chemical formula (II)	Content (wt.)	Unit of
				Disodium EDTA anhydrate	Na2EDTA·2H2O	21.50	g·L-1
Sodium carbonate	Na2CO3	3.320	g·L-1
				Ferric ammonium citrate	(NH4)5Fe(C6H4O7)2	7.1	g·L-1
Zinc sulfate monohydrate	ZnSO4·H2O	0.449	g·L-1
				Manganese chloride tetrahydrate	MnCl·4H2O	6.15	g·L-1
Blue vitriod	CuSO4·5H2O	0.4995	g·L-1
				Ammonium molybdate tetrahydrate	(NH4)6Mo7O24·4H2O	0.0352	g·L-1
Selenium sulfide	SeS2	0.014	g·L-1

In another embodiment, the composition of the feeding medium is given in table 6.

TABLE 6 feeding Medium B

Chemical name	Chemical formula (II)	Content (wt.)	Unit of
				Ammonium hydroxide	NH4OH	4.6	g·L-1
(glacial) acetic acid	CH3CO2H	195.4	g·L-1

In another embodiment, the composition of the fermentation medium is given in table 7.

TABLE 7 fermentation Medium C

In another embodiment, the composition of the trace elements is set forth in table 8.

TABLE 8 microelement solution C

Chemical name	Chemical formula (II)	Content (wt.)	Unit of
				Disodium EDTA anhydrate	Na2EDTA·2H2O	21.50	g·L-1
Sodium carbonate	Na2CO3	3.320	g·L-1
				Ferric ammonium citrate	(NH4)5Fe(C6H4O7)2	7.1	g·L-1
Zinc sulfate monohydrate	ZnSO4·H2O	0.449	g·L-1
				Manganese chloride tetrahydrate	MnCl·4H2O	6.15	g·L-1
Blue vitriod	CuSO4·5H2O	0.4995	g·L-1
				Ammonium molybdate tetrahydrate	(NH4)6Mo7O24·4H2O	0.0352	g·L-1
Selenium sulfide	SeS2	0.014	g·L-1

In another embodiment, the composition of the feeding medium is given in table 9.

TABLE 9 feeding Medium C

Chemical name	Chemical formula (II)	Content (wt.)	Unit of
				Ammonium hydroxide	NH4OH	4.6	g·L-1
(glacial) acetic acid	CH3CO2H	195.4	g·L-1
				Potassium dihydrogen phosphate	KH2PO4	1.25	g·L-1

The fermentation medium and the trace element solution are sterile solutions so that the algae culture in the bioreactor is not contaminated by other organisms. The fermentation medium may be used in the form given in the table herein (1-fold concentration) or may be used in a more concentrated form. In particular embodiments, the fermentation medium is used at 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold the concentration of the fermentation medium provided in the tables. The feeding medium can be used in the form given in the table herein (1-fold concentration) or in a more concentrated form. In particular embodiments, the feeding medium is used at 2-fold, 3-fold, 4-fold, or 5-fold the concentration of the feeding medium in the table. Preferably, media or solutions having the same name are used together, for example fermentation medium a together with trace element solution a and feeding medium a.

Using the methods described herein, very high density algal cultures can be produced. Accordingly, the present disclosure provides an algal culture having an algal concentration of at least 50g/L dry cell weight under steady state conditions. In other embodiments, a composition having a stem cell weight of at least 55g/L, at least 60g/L, at least 65g/L, at least 70g/L, at least 75g/L, at least 80g/L, at least 85g/L, at least 90g/L, a steady state concentration of algal culture of at least 95g/L, at least 100g/L, at least 105g/L, at least 110g/L, at least 115g/L, at least 120g/L, or at least 125g/L, at least 150g/L, at least 175g/L, or at least 200g/L dry cell weight. In a particular embodiment, the culture is a culture of a species of Chlamydomonas. In a specific embodiment, the culture is a culture of chlamydomonas reinhardtii. As is known in the art, a plot of the mass or concentration of an organism versus time will generally form a sigmoidal curve. Steady state is a period of relatively mild growth after the inflection point of the sigmoidal growth curve. For purposes of this disclosure, an algal culture is considered to be in a steady state when the concentration of algae increases by about 0.1% to about 10% within 24 hours. In other embodiments, steady state refers to a state where the concentration of algae in the culture is increased by about 0.1% to 50%, about 0.1% to 25%, about 0.1% to 7%, about 0.1% to about 5%, 0.1% to 3%, or 0.1% to 2%. When the culture concentration is decreased, the culture is not considered to be in a steady state.

Once the culture reaches the desired concentration, the culture can be propagated to additional bioreactors or harvested or both. If propagated to additional bioreactors, the inoculation process is the same as described herein. The additional bioreactor may be the same size as the bioreactor from which the inoculum was obtained, or may be larger or smaller. It will be apparent to those skilled in the art that propagation and/or harvesting may be continuous, batch or semi-batch. In batch mode, the entire culture is either harvested or used for propagation. In continuous mode, a small amount of culture is continuously removed for harvesting or propagation, while a small amount of medium is added to replace the removed medium. Semi-batch is between batch and continuous. In semibatches, a portion, but not all, of the culture is periodically removed for propagation or harvesting. As with the continuous mode, a corresponding amount of medium was added to the semibatch to replace the removed medium.

In certain embodiments, some or all of the material in the bioreactor may be harvested. In one embodiment, all material is harvested from the bioreactor once the culture has reached a desired stage of growth, such as the log or steady state stage of growth. In another embodiment, continuous harvesting can be from the growth or steady state culture of algae. In one aspect, the removal of algae maintains the culture in a logarithmic growth phase. In another aspect, the removal of algae maintains the culture in a steady state stage. The determination of growth rate and microalgae growth stage is known in the art. For example, in Sode et al, J.Biotechnology (1991)21: 209-.

After removing a portion or all of the algae containing the culture medium from the bioreactor, it is desirable to separate the algae from the culture medium (dewatering). In one embodiment, harvesting comprises separating at least 90% of the microalgae from the culture medium to produce a liquid depleted of microalgae. In another embodiment, at least 95% of the microalgae is removed from the culture medium. In another embodiment, at least 97% of the microalgae is removed from the culture medium. In another embodiment, at least 99% of the microalgae are removed from the culture medium. In other embodiments, more than 50% of the microalgae are removed. In another embodiment, more than 75% of the microalgae are removed from the culture medium. In another embodiment, more than 80% of the microalgae is removed from the culture medium. In yet another embodiment, less than 30% of the microalgae may remain in the culture medium after harvesting. In another embodiment, less than 25% of the microalgae remains in the culture medium after harvesting. In another embodiment, less than 5% of the microalgae remains in the culture medium after harvesting. In another embodiment, less than 2.5% of the microalgae remains in the culture medium after harvesting. In one embodiment, less than 1% of the microalgae remains in the culture medium after harvesting.

The separation of the microalgae from the liquid may be accomplished by methods known to those of ordinary skill in the art. In one embodiment, the microalgae may be allowed to settle by gravity and the overlying liquid removed. In another embodiment, the microalgae can be harvested by centrifuging the culture containing the microalgae. In one embodiment, centrifugation of the liquid culture can be performed in batch mode using a fixed volume centrifuge. In a different embodiment, batch harvesting of microalgae can be accomplished using continuous flow centrifugation. In another embodiment, microalgae can be continuously harvested from a growing culture by continuous flow centrifugation. In other embodiments, dewatering can be accomplished by filtration, such as tangential flow filtration. The filtration can be performed in batch or continuous harvest mode. In other embodiments, dewatering can be accomplished by electrophoretic techniques, such as electrolytic coagulation and electrolytic flocculation. In other embodiments, dewatering can be accomplished by flocculation. Flocculation may be accomplished by chemical flocculation using synthetic or natural flocculants or by auto-flocculation. Methods of inducing flocculation include those that may be found in U.S. patent No.8,969,066 and U.S. patent publication No. us 2015/0284673 (application No.14/649524), each of which is incorporated herein by reference in its entirety. The flocs may be separated from the broth by gravity, centrifugation, Dissolved Air Flotation (DAF), or any other method known to those skilled in the art. If chemical flocculation is used, it may be desirable in some cases to use a food grade flocculant. Food grade flocculants are commercially available from a variety of sources.

After dewatering, the harvested algae may still contain a large amount of liquid. To facilitate long term storage, it is desirable to remove the additional liquid by drying. In some embodiments, the algal biomass is dried such that the resulting material is at least 75% dry cell weight, at least 80% dry cell weight, at least 85% dry cell weight, at least 90% dry cell weight, or at least 95% dry cell weight. In other embodiments, the algae are dried such that the resulting material contains less than 25% moisture, less than 20% moisture, less than 15% moisture, less than 10% moisture, or less than 5% moisture. Drying of the algal biomass may be accomplished by any method known in the art. Exemplary drying methods include spray drying, ring drying, paddle drying, tray drying, solar or sun drying, vacuum drying, and freeze drying.

After drying, the algae can be suitable for human consumption or as a nutritional supplement or additive to animal feed. Thus, provided herein is a nutritional supplement or feed additive comprising at least 90%, at least 95%, at least 98%, or at least 99% chlamydia species and about 80% ± 5% or 90% ± 5% dry cell weight, said nutritional supplement or feed additive further having greater than about 50% ± 5% crude protein, about 10% ± 5% crude fat, and less than 5% ash. In some embodiments, the nutritional supplement or additive further comprises at least 0.4% (w/w), at least 0.5% (w/w), at least 0.75% (w/w), at least 1.0% (w/w), or at least 1.25% (w/w) omega-3 fatty acids. In other embodiments, the nutritional supplement or additive contains at least 0.25% (w/w), at least 0.75%, at least 1.25%, at least 1.5%, or at least 1.75% omega-6 fatty acids. In another embodiment, the nutritional supplement or additive comprises at least 1.25% (w/w), at least 1.75%, at least 2.25%, or at least 2.75% omega-9 fatty acids. The nutritional supplement or additive is also characterized by being free of microbial contamination as determined by the criteria in table 10.

Watch 10

Because the methods disclosed herein provide such high density cultures, the methods can be used to produce algae that have been genetically modified to produce proteins of therapeutic value. In some embodiments, the therapeutic protein is a naturally occurring protein. In other embodiments, the therapeutic protein is a foreign protein. An exogenous nucleic acid, nucleotide, polypeptide, or protein as described herein is defined as associated with a host. The exogenous nucleic acid, nucleotide, polypeptide, or protein is a nucleic acid, nucleotide, polypeptide, or protein that is not naturally present in the host or at a different location in the host.

Therapeutic proteins are useful for treating or preventing a disease or disorder. The therapeutic protein may be a mammalian protein, such as a human protein. The therapeutic protein may be used for veterinary care or for human care. The therapeutic proteins are useful for treating companion animals, domestic animals, exotic animals, wild animals, and production animals. Therapeutic proteins may be involved in, for example, cell signaling and signal transduction.

Examples of therapeutic proteins are antibodies, transmembrane proteins, growth factors, enzymes, immunomodulating proteins or structural proteins. The therapeutic protein may be a protein present in an animal or human, or a derivative of a protein present in an animal or human. Examples of the production of therapeutic proteins using algae can be found in, for example, in proc.natl.acad.sci.usa (2003)100: 438-42; plant Biol. (2004)7: 159-65; vaccine (2005)23: 1828-32; curr, opin, biotechnol, (2007)18: 1-8; expert Opin biol Ther. (2005)5: 225-35; biotechnol.lett. (2010)32: 1373-83; and international patent application publication WO 2001/063,284.

The nucleotide sequence encoding the therapeutic protein of interest may be a naturally occurring or wild-type sequence, or may be a modified sequence. Types of modifications include deletion of at least one nucleic acid, addition of at least one nucleic acid, or substitution of at least one nucleic acid. One skilled in the art will know how to modify nucleotide sequences.

One particular type of modification that can be made to a nucleotide sequence is codon optimization. As known in the art, one or more codons of an encoding polynucleotide may be "biased" or "optimized" to reflect codon usage of the host. For example, one or more codons of the encoding polynucleotide can be "biased" or "optimized" to reflect chloroplast codon usage or nuclear codon usage. Most amino acids are encoded by two or more different (degenerate) codons, and it is well recognized that various organisms utilize certain codons instead of others. Throughout the specification, "bias" or codon "optimization" may be used interchangeably. Codon bias can be biased differently in different plants, including, for example, in algae as compared to tobacco. In general, the codon bias selected reflects the codon usage of the organism (or organelle therein) engineered with the nucleic acid. Polynucleotides that favor a particular codon usage can be synthesized de novo, or can be genetically modified using conventional recombinant DNA techniques, e.g., by site-directed mutagenesis methods, to alter one or more codons such that they favor chloroplast codon usage. The use of such preferential codons used in chloroplasts is referred to herein as "chloroplast codon usage". Examples of chloroplast and nuclear codon usage for chlamydomonas reinhardtii can be found in the art, for example, in U.S. patent application publication No.: 2004/0014174 and international patent publication No. WO2011/063,284.

Expression of the therapeutic protein in algae is achieved by using an expression vector. An expression vector is a vector designed such that a coding sequence inserted at a specific site will be transcribed and translated into a protein. The expression vector or linearized portion thereof may comprise one or more exogenous nucleotide sequences encoding a therapeutic protein of interest. Examples of exogenous nucleotide sequences that may be transformed into a host include nucleic acid sequences encoding mammalian proteins. In some cases, the exogenous sequence is flanked by two sequences having homology to sequences contained in the host to be transformed.

A homologous sequence is, for example, one having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a reference amino acid sequence or nucleotide sequence, e.g., an amino acid sequence or nucleotide sequence in a host cell from which the protein is naturally derived or derived. The homologous sequences enable recombination of the exogenous sequence into the nuclear or plastid genome of the host algae to be transformed. In some embodiments, the expression vector comprises a polynucleotide, such as a promoter and/or transcription terminator, operably linked to one or more control elements. A nucleic acid sequence is operably linked when the nucleic acid sequence is in a functional relationship with another nucleic acid sequence. For example, if the DNA of the presequence or secretion leader is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably linked to the DNA of that polypeptide; a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or, if the ribosome binding site is positioned so as to facilitate translation, it is operably linked to a coding sequence. Typically, the operably linked sequences are contiguous and, in the case of secretory leaders, contiguous and in the reading phase. Ligation is achieved by ligation at restriction enzyme sites. If appropriate restriction sites are not available, the instant invention may be usedSynthetic oligonucleotide adaptors (adaptors) or linkers known to those skilled in the art. Sambrook et al, Molecular Cloning, A Laboratory Manual,2^ndEd, Cold Spring Harbor Press, (1989) and Ausubel et al, Short Protocols in Molecular Biology,2^ndEd.,John Wiley&Sons(1992)。

As used herein, a regulatory or control element broadly refers to a nucleotide sequence that regulates transcription, translation of a polynucleotide, or the positioning of a polypeptide operably linked thereto. Examples include, but are not limited to, RBSs, promoters, enhancers, transcription terminators, hairpin structures, RNase stability elements, splice signals for initiation (start) codon, intron excision and maintenance of the correct reading frame, stop codons, amber or ochre codons, and IRES. Regulatory elements may include promoters and transcriptional and translational stop signals. To introduce specific restriction sites to control the ligation of sequences to the coding region of a nucleotide sequence encoding a polypeptide, elements may be provided with linkers. In addition, a sequence comprising a cellular compartmentalization signal (i.e., a sequence that targets the polypeptide to the cytoplasm, nucleus, chloroplast membrane, or cell membrane) can be linked to a polynucleotide encoding a protein of interest. Such signals are well known in the art and have been widely reported.

In an expression vector, the nucleotide sequence of interest is operably linked to a promoter recognized by the host cell to direct mRNA synthesis. Promoters are untranslated sequences, usually located 100 to 1000 base pairs (bp) upstream of the start codon of a structural gene, that regulate the transcription and translation of nucleic acid sequences under their control. The promoter may be a constitutive promoter or an inducible promoter. An inducible promoter is a promoter that, under its control, initiates an increase in the level of DNA transcription in response to certain changes in the environment, such as the presence or absence of nutrients or changes in temperature. In contrast, constitutive promoters maintain relatively constant transcription levels.

Many promoters are active in algae, including promoters that are endogenous to the algae being transformed, and promoters that are not endogenous to the transformed algae (i.e., promoters from other algae, promoters from higher plants, and promoters from plant viruses or algal viruses). Exogenous and/or endogenous promoters active in algae, as well as antibiotic resistance genes functional in algae, include, but are not limited to, those described in, for example, the following documents: curr. Microbiol. (1997)35(6):356-62(Chlorella vulgaris); marine Biotechnol. (NY.) (2002)4(l):63-73(Chlorella ellipsoidea); Gen.Genet. (1996)252(5):572-9(Phaeodactylum tricornutum); plant MoI. biol. (1996)31(1):1-12(Volvox cateri); proc.Natl.Acad.Sci.U S A (1994)91(24):11562-6(Volvox carteri); falciatore A, Castotti R, Leblanc C, Abresia C, Bowler C, PMID:10383998, (1999)1(3) 239-; plant Physiol (2002)129(1) 7-12 (Porphyridium sp.); proc.Natl.Acad.Sci.USA, (2003)100(2):438-42.(Chlamydomonas reinhardtii); Natl.Acad.Sci.USA, (1990)87(3) 1228-32 (Chlamydomonas reinhardtii); nucleic Acids Res. (1992)20(12):2959-65Marine Biotechnol. (NY): 2002)4(1):63-73 (Chlorella); biochem. MoI. biol. int. (1995)36(5) 1025-35(Chlamydomonas reinhardtii); microbiol. (2005)43(4):361-5 (Dunaliella); marine Biotechnol (NY) (1999)1(3): 239-; appl.Microbiol.Biotechnol. (2002)58(2) 123-37 (variaus species); gene, genomics (2004)271(1):50-9(Thermosynechococcus elongates); bacteriol. (2000),182, 211-; FEMS microbiol.lett. (2003)221(2) 155-9; plant Physiol (1994)105(2): 635-41; plant MoI.biol. (1995)29(5) 897-907(Synechococcus PCC 7942); marine pollut. Bull. (2002)45(1-12):163-7(Anabaena PCC 7120); proc. Natl. Acad. Sci. USA (1984)81(5) 1561-5(Anabaena (variaous strains)); Proc.Natl.Acad.Sci.USA (2001)98(7):4243-8 (Synechocystis); Gen.Genet. (1989)216(1):175-7 (variaus species); moi. microbiol. (2002)44(6) 1517-31; plasmid (1993)30(2):90-105 (Freumyca diplisophon); gene (1993)124:75-81(Chlamydomonas reinhardtii); current Micro (1991)22: 15-20; current Genet (1991)19: 317-. Additional promoters can be found in table 1 of us patent 6,027,900.

The polynucleotide or recombinant nucleic acid molecule encoding the therapeutic protein can be introduced into the algal cells using any method known in the art. Polynucleotides can be introduced into cells by a variety of methods that are well known in the art and are selected, in part, based on the particular host cell. For example, polynucleotides may be introduced into cells using direct gene transfer methods, such as electroporation or particle-mediated (biolistic) transformation using a particle gun, "glass bead" or liposome-mediated transformation.

Microparticle-mediated transformation utilizes microparticles, such as gold or tungsten, which are coated with the desired polynucleotide by precipitation with calcium chloride, spermidine, or polyethylene glycol. The microparticle particles are accelerated into the cells at high speed using a device such as the BIOLISTIC PD-1000 particle gun (BioRad; Hercules CA). Methods for transformation using biolistic methods are well known in the art (e.g., as described in Christou, Trends in Plant Science (1996)1: 423-. Exemplary methods for transforming algae can be found in international patent application publication nos. WO 2011/034,863, WO2011/063,284, and biosci.biotechnol.biochem. (2014)78: 812-7; J.biosci.Bioeng. (2013)115: 691-4; proc.Natl.Acad.Sci.USA (2011)108: 21265-9; and Plant Physiol (2002)129: 7-12; adv.explore.med.biol. (2007)616: 1-9; molec. biotechnol. (2005)30: 185-91; science (1988)240: 1534-38; folia Microbiol. (2000)45: 496-504; plant Physiol (2002)129: 7-12; from Molec.Gen.genetics (2000)263:404-10.J.biosci.Bioeng. (1999)87: 307-14; proc.Natl.Acad.Sci.USA (1990)87: 2087-90; plant Cell (1989)1: 123-32; plantatibotechnol.j. (2007)5: 402-12; and J.Biotechnol. (2013)163: 61-8.

When nuclear transformation is used, proteins can be modified for plastid targeting by using a Plant cell nuclear transformation construct (where the DNA coding sequence of interest is fused to any available transit peptide sequence capable of facilitating transport of the encoded protein into the Plant plastid) and by driving expression using an appropriate promoter.targeting of the proteins can be achieved by fusing DNA encoding plastids (e.g., chloroplasts, transit peptide sequences) to the 5' end of the DNA encoding the protein.sequences encoding the transit peptide regions can be obtained, for example, from Plant nuclear-encoded plastid proteins, where Plant nuclear-encoded plastid proteins such as the small subunit of ribulose bisphosphate carboxylase (SSU), EPSP synthase, Plant fatty acid biosynthesis-related genes (including fatty acyl-ACP thioesterases, Acyl Carrier Proteins (ACPs), stearoyl-ACP desaturase, β -ketoacyl synthase and acyl-ACP thioesterases or LHCPII gene, etc.) plastid transit peptide sequences can also be obtained from nucleic acid sequences encoding carotenoid biosynthetic enzymes such as GGPP synthase, phytoene and phytoene desaturase and other phytoene mature peptide sequences disclosed in the Plant mature protein transport sequence (1988. phytotoxin) and further, for the delivery of phytotoxin transport proteins, such as a phytotoxin, a protein, a phytoalexin, a protein, a phytotoxin, such as a protein, a phytotoxin, a protein, a phytotoxin, a protein.

Once the therapeutic protein is expressed, it can be administered to a subject. In some embodiments, the therapeutic protein is administered to a subject consuming algae. In other embodiments, the therapeutic protein is purified or isolated from the algae prior to administration. There are several methods available for purifying or isolating proteins, which are known to those skilled in the art. These methods include chromatography by, for example, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, agglutination chromatography, High Performance Liquid Chromatography (HPLC), electrophoresis under native or denaturing conditions, isoelectric focusing, and immunoprecipitation.

As will be appreciated by those skilled in the art, the exact route of administration will vary depending upon factors such as the particular therapeutic protein used, the condition to be treated, and the subject to be treated. Exemplary methods of administration include, but are not limited to, oral, enteral, mucosal, transdermal, or parenteral. Examples of methods of administration include oral, intranasal, intratracheal, intravenous, intramuscular, subcutaneous, intraperitoneal, intraarterial, intrasternal, intralesional, topical, transdermal, inhalation, and iontophoresis. The most suitable route of administration can be readily determined by one of ordinary skill in the art based on generally known factors, such as those described herein.

Examples

The following examples are intended to provide examples of the application of the present invention. The following examples are not intended to be exhaustive or to limit the scope of the invention as claimed.

Example 1Preparation of the culture Medium and feeding

All media and culture materials are sterile unless otherwise indicated. For purposes of the following examples, sterility is defined as at least a log16 reduction of geobacillus stearothermophilus spores. All materials used are suitable for human consumption (food grade) unless otherwise indicated.

Solution of Trace Elements (TE)The TE solution was prepared as two separate solutions, called TE_A(Table 11) and TE_B(table 12) and then they were mixed in equal proportions. TE_AIs a clear orange solution, and TE_BIs a clear blue solution. When mixed, the resulting solution (TE) was clear green. All solutions were prepared by adding the solid reagents to sterile deionized water. Takedown table 11 and tableThe reagents were added to the solution in the order listed in 12.

TABLE 11TE_A

Chemical name	Chemical formula (II)	Content (wt.)	Unit of
				Disodium EDTA dihydrate	Na2EDTA·2H2O	23.00	g·L-1
Sodium carbonate	Na2CO3	4.64	g·L-1
				Ferric ammonium citrate	(NH4)5Fe(C6H4O7)2	10.47	g·L-1

TABLE 12TE_B

Chemical name	Chemical formula (II)	Content (wt.)	Unit of
				Disodium EDTA dihydrate	Na2EDTA·2H2O	20	g·L-1
Zinc sulfate monohydrate	ZnSO4·H2O	0.899	g·L-1
				Manganese chloride tetrahydrate	MnCl·4H2O	2.376	g·L-1
Blue vitriod	CuSO4·5H2O	0.999	g·L-1
				Sodium carbonate	Na2CO3	2.00	g·L-1
Ammonium molybdate tetrahydrate	(NH4)6Mo7O24·4H2O	0.070	g·L-1
				Sodium selenite	Na2SeO3	0.034	g·L-1

To prepare the final TE solution, an equal volume of TE was added_AAnd TE_BThe solutions were mixed together. To reduce the bioburden of the overall process, the resulting solution was sterile filtered through a 0.22 μm pore size membrane. The final composition of the TE solution is shown in Table 2. Any unused TE solution was stored in an opaque container at 2-8 ℃.

Preparation of the MediumThe medium, seed fermentation and production fermentation (fermentation medium) used for the two flask stages are simple formulations of salts with added solutions of trace elements (table 2). The pH medium was adjusted to about pH 6.0 using a stock solution of 98% glacial acetic acid. The pH of the medium before the addition of acetic acid was about 10.5. The medium was sterilized by autoclaving. In this example, the composition of the medium is shown in Table 1.F111-GF AntiFoam is an optional ingredient that is not normally added to the medium for flask growth.

The feeding (feeding medium) of the seeds and production fermentations was a concentrated form (100-fold) of the fermentation medium of table 1, wherein a large amount of acetic acid was added as the main carbon source. The evaporation loss of acetic acid was mitigated by filter sterilization through a membrane filter having a pore size of 0.22 μm without high-pressure sterilization. The components of the feeding are shown in Table 3.

Example 2Flask cell mass propagation

The following procedure details the making of 1 stage I flask with a total effective volume of 150mL, which resulted in 1 stage II flask with a total effective volume of 1,200 mL. More flasks can be made as needed to meet seed fermentor inoculation requirements. The general criteria for flask requirements is that there is 1 liter of stage II flask broth per 9 liter volume of broth in the seed fermentor. The initial flask inoculum can be from a streaked TAP plate, or from a 1mL cryovial (cryopellet).

Stage ITo initiate propagation of phase I flask cell mass, 150mL of sterile fermentation medium was added to a sterile baffled 500mL shake flask with vented closure in a biosafety cabinet. For inoculation from the plates, appropriate TAP plates streaked with the desired Chlamydomonas reinhardtii strain were obtained and streaked 4cm using a sterile loop in a biosafety cabinet and then used in stage I flasks of the inoculation preparation.

This typically increases the phase I propagation time by 192 hours to 240 hours if cryopellets are used. The desired pellet count was removed from the-80 ℃ freezer and immediately thawed in 500mL beaker of water at ambient temperature. Typically 2 cryopellets are used per 50mL of fermentation medium. The beaker containing the cryopellet was quickly placed in a water bath at about 35 ℃ to thaw the material in the cryopellet. Once the material in the cryopellet has thawed, the cryopellet is gently shaken to ensure that all of the material has liquefied. After thawing, the cryopellet is sprayed and/or wiped with 70% ethanol and the contents of the pellet are transferred to a sterile baffled 500mL shake flask containing 150mL sterile fermentation medium. After transferring the contents of the cryopellet to the flask, the flask was kept undisturbed for 1 hour. After a waiting time of 1 hour, with material (e.g. of) The shake flask was wrapped to reduce exposure to light and transferred to an orbital shaker at ambient temperature (about 25 ℃) and 150RPM, with a suitable light source of about 100 to about 500 micro einstein. After 4 days on the shaking table, the exposure limiting material was removed and the flask was incubated until a green color was observed, which was typically 10 days after the start of the process.

The inoculated stage I flask was then wrapped with aluminum foil or some other opaque material from top to bottom and placed on an orbital shaker set at 150RPM with a shaker value of 1.9cm and conducted at ambient temperature (typically about 25 ℃). The inoculated phase I flask was kept on an orbital shaker for about 120 hours. If the stage I flask is started with a cold pellet, the total shaker time is about 312 hours to 360 hours (120 hours plus an additional 192 hours to 240 hours).

For contamination examination, samples were aseptically removed from the stage I flasks in a biosafety cabinet using a sterile pipette 48 hours prior to inoculation of the stage II flasks, visually examined by microscopy and streaked on LB plates (Luria-berthipllate, see "Molecular Cloning, a Laboratory Manual," Sambrook and Russell, 2001). LB plates should be streaked in a volume of 1mL and should be incubated at 37 ℃ for 24 hours. After 120 hours of phase I propagation, 1mL of sample was aseptically removed from the phase I flask in a biosafety cabinet using a sterile pipette for quantification of optical density at 750nm and dry cell weight concentration (DCW). Methods of determining DCW values are provided herein. The final DCW value of the typical stage I flask was 1.8 g.L^-1～2.5g·L^-1But at a concentration of 3.2 g.L^-1Is acceptable.

Stage IIA stage II flask was prepared by adding 1,050mL of sterile fermentation medium to a sterile, baffled, 3,000mL shake flask with a vent in a biosafety cabinet. After addition of the stage I inoculum, the initial effective volume of the stage II flask was 1,200 mL. The volume of the final phase I broth was equal to 10% of the effective volume of the desired initial phase II (in this case150mL) was used to inoculate the prepared stage II flask in a biosafety cabinet.

Incubation of the stage II flasks was performed in a similar manner as the stage I flasks. The inoculated stage II flask was then wrapped with aluminum foil or other opaque material from top to bottom from the lid and placed on an orbital shaker set at 150RPM, with a shaker amount of 1.9cm, and at ambient temperature (typically about 25 ℃). The inoculated phase I flask was kept on an orbital shaker for about 120 hours.

As a contamination check, samples were aseptically removed from the stage II flasks in a biosafety cabinet using a sterile pipette 48 hours prior to inoculation of the seed fermentor for visual inspection by microscope and streaking of LB plates. LB plates should be streaked in a volume of 1mL and should be incubated at 37 ℃ for 24 hours. The final DCW value of the general stage II flask is 1.8 g.L-1 to 2.5 g.L^-1But at concentrations as high as 3.2 g.L^-1Is also acceptable.

Example 3Seed fermentation

Seed fermentation was used to produce larger quantities of cells at higher DCW concentrations than in simple shake flasks to inoculate larger production fermentors to the desired initial DCW concentration. The aim of seed fermentation is to produce at least 30-40 g DCW.L within 144-182 h^-1And no detectable contaminants.

Preparing a sterile fermentation medium in a sterile fermentation vessel. The volume of culture medium prepared should be equal to 90% of the desired initial volume of the seed fermentor to allow for the addition of 10% by volume of inoculum. After 120 hours of propagation in the phase II flask and confirmation of no contamination, the sample from the phase II flask was removed aseptically with a sterile pipette in a biosafety cabinet and used for OD₇₅₀And quantification of DCW. As previously mentioned, the final DCW for a typical stage II flask was 2.5. + -. 0.7 g.L^-1. Once it was confirmed that the DCW of the stage II flask was within the desired range, inoculation of the seed fermentor was possible.

The required inoculation volume of the seed fermentor was equal to 10% of the effective volume of the initial seed fermentor, so the initial dry cell weight concentration was 0.25. + -. 0.07 g/L. The seed inoculum was aseptically transferred from the flask to a sterile seed inoculation vessel in a biosafety cabinet and then to a seed fermentor. Addition of inoculum typically results in an increase in pH above the set point of pH 6.8. If the pH controller is not properly adjusted, an excessive amount of acidic feed may be added to the fermentor during this period, resulting in a severe pH drop. A pH below 5.5 may adversely affect fermentation performance and should be avoided. To prevent this, the pH control may be temporarily disabled prior to inoculation. If pH control is disabled, the pH value is manually adjusted to the set point directly after inoculation and pH control is re-enabled. After successful inoculation and activation of pH control, an increase in pH should be observed, indicating metabolic activity, more specifically, consumption of acetate.

Seed fermentation was carried out aerobically and the process parameters and control strategy are listed in table 13. In this example, the seed fermentation is performed in the absence of light, which means that any viewing port is covered during the fermentation.

Table 13 parameters of exemplary seed fermentations

Parameter(s)	Control strategy	Set point	Plus or minus deviation	Unit of
					Total fermentation time(TFT)	n/a	168	24	H
Temperature of	Set point	28	0.5	℃
					pH	Set point	6.8	0.2	pH
Air flow	Set point	1	0.1	Vvm
					pO2	Set point	30	3	% air state
Pressure of	Set point	0	0.01	Bar (barg)
					Speed of feeding	Feedback (pH value)	Variables of	n/a	g feed/liter/hour
Stirring the mixture	Feedback (pO)2)	Variables of	n/a	RPM

In this example, the feed stream was a mixture of concentrated medium and glacial acetic acid (see table 3) that was filter sterilized using a 0.22 μm pore filter. The feed stream was fed to the fermentor by a peristaltic pump. The acidic feeding stream is used to adjust the pH and the dose is automatically given by the pH controller when the pH increases beyond its set point (one-sided "pH-state"). Dissolved Oxygen (DO) was controlled at a set point of 30% air saturation (at atmospheric pressure) by adjusting the stirring rate. The spray rate can also be adjusted if stirring alone is insufficient to maintain the DO set point. In the case of significant FOAM formation, it may be equal to 0.06mL FOAM per liter of fermentation brothA sterile antifoam bolus of F111-GF antifoam was added to the fermentor. For the purposes of this example, "significant foam" is defined as the amount of foam sufficient to "hold up" the acid added from the top of the fermentor. If the accumulator/mass flow meter indicates that acid is being fed, but no decrease in pH is observed, a "significant foam" may form. In this case, the presence of "significant foam" should be verified by visual inspection through a sight glass (which is temporarily uncovered). If the foam is allowed to form for too long, breaking the foam may pour excess "blocked" acid into the liquid medium, resulting in death of the culture. Therefore, the foam formation should be solved as soon as possible.

For OD₇₅₀And DCW at 0 hr, 24 hr, 48 hrThe seed fermentors were sampled at Total Fermentation Times (TFT) of hour, 72 hours, 96 hours, 120 hours, 144 hours and 168 hours. Exemplary results obtained for various parameters measured are shown in fig. 1-3.

48 hours before inoculation of the production fermentor, samples were aseptically removed from the seed fermentor through a sterile sampling port for visual inspection by microscope and streaking on LB plates (incubation at 37 ℃ for 24 hours) for further inspection for contamination. The harvest criterion for seed fermentation was a DCW of 30 g.L^-1～40g·L^-1But at concentrations as high as 50.0 g.L^-1Is acceptable.

Example 4Production fermentation

Production fermentation operates similarly to seed fermentation; the main difference between the two processes is the initial concentration of the cell mass, and for the production fermentation the inoculum may come from another fermenter instead of from the shake flask. The aim of the production fermentation is to produce at least 60g of DCW.L, usually in 90-110 h^-1No detectable contaminants.

The sterile fermentation medium may be prepared in a fermentation vessel. Alternatively, the sterile fermentation medium can be produced outside the fermentation vessel and then introduced into the sterile fermentation vessel. The volume of the medium should be equal to 90% of the desired initial volume of the seed fermentor to allow for the addition of 10% by volume of inoculum.

After completion of the seed fermentation and confirmation of no contamination, the final sample was aseptically taken through an aseptic sampling port to quantify the OD₇₅₀And DCW. Typically the final DCW for seed fermentation is between 30.0g/L and 40.0g/L, but concentrations as high as 50.0g/L are acceptable. Once it was confirmed that the DCW of the seed fermentor was within the desired range and no contamination occurred, the production fermentor was inoculated.

The required inoculum volume of the production fermentor was 10% of the initial volume of the production fermentor, so the initial DCW concentration was 3.0g/L to 4.0 g/L. Particularly for the inoculation of production fermenters, it is preferred that the seed inoculum is exposed to hypoxic conditions for no more than 15 minutes. Process control (e.g., agitation and sparging) should be maintained in the seed fermentor during inoculum transfer, and residence time in the inoculum transfer line should be minimized as much as possible. If the organism undergoes oxygen starvation prior to inoculation, the treatment performance is significantly reduced.

Addition of inoculum typically results in an increase in pH above the set point. If the pH controller is not properly adjusted, an excess of acidic feed may be added to the fermentor during this period, resulting in a severe pH drop. pH values below 5.5 may adversely affect fermentation performance and should be avoided. Thus, pH control may be temporarily disabled prior to inoculation. After inoculation, the pH should be manually adjusted to the set point before re-enabling pH control. After successful inoculation and activation of pH control, an increase in pH should be observed, indicating metabolic activity, in particular, consumption of acetate. Production fermentations were carried out under aerobic conditions, with process parameters and control strategies listed in table 14. Production fermentations are usually carried out in the absence of light, covering all the viewing ports during the fermentation.

TABLE 14 production fermenter Process parameters

Parameter(s)	Control strategy	Set point	Error of + -1	Unit of
					Total fermentation time	n/a	100	10	h
Temperature of	Set point	28	0.5	℃
					pH	Set point	6.65	0.2	pH
Air flow	Set point	1	0.1	vvm
					pO2	Set point	30	3	% air state
Pressure of	Set point	0	0.01	Bar (barg)
					Speed of feeding	Feedback (pH value)	Variables of	n/a	g feed/liter/hour
Agitation	Feedback (pO)2)	Variables of	n/a	RPM

The feeding stream (feeding medium a, table 3) is a mixture of concentrated medium and glacial acetic acid, which is filter sterilized at 0.22 μm and fed into the fermenter by means of a peristaltic pump. The acidic feeding stream is used to adjust the pH and is automatically dosed by the pH controller when the pH increases beyond its set point (one-sided "pH-state"). The percent saturation of dissolved oxygen (abbreviated herein as DO) was controlled at a set point of 30% air saturation (at atmospheric pressure) by adjusting the agitation rate. The spray rate can also be adjusted if stirring alone is insufficient to maintain the DO set point.

In the case of significant FOAM formation, it may be equal to 0.06mL FOAM per liter of fermentation brothA sterile antifoam bolus of F111-GF antifoam was added to the fermentor. For the purposes of this disclosure, "significant foam" is defined as an amount of foam sufficient to "retard" the addition of acid from the top of the fermentor. If the accumulator/mass flow meter indicates that acid is being fed, but no decrease in pH is observed, a "significant foam" may form. In this case, the presence of "significant foam" should be verified by visual inspection through a viewing port and taking appropriate action. If the foam is allowed to form for too long, breaking the foam may pour excess "blocked" acid into the liquid medium, resulting in death of the culture. Therefore, the foam formation should be solved as soon as possible.

When the TFT is 0 hour, 24 hours, 48 hours, 72 hours,Production fermentors were sampled at 96 hours and 100 hours to determine OD₇₅₀And DCW. Data and trends for typical 0.5L production fermentations are shown in figures 4-6. After about 100 hours, seen in FIG. 4, the decrease of pO2 below the set point was a result of the inability to increase the liquid culture medium aeration rate, since both agitation and sparging reached their maximum values in laboratory scale fermenters. By increasing the injection rate after agitation reaches its maximum (cascade control), ideally, the process control will maintain the value of DO at its set point throughout the fermentation process.

As with seed fermentation, production fermentation operates as a one-sided (acid) "pH-state". As a result of this mode of operation, fluctuations in pH with respect to the set point are generally observed throughout the fermentation process (see fig. 5). As culture density increases, the total acetate consumption rate increases and the size of the pH "package" decreases to a minimum of about 0.05pH units. In order to maintain fermentation performance, it is preferred that the deviation in pH should not exceed 0.5pH unit, especially in the early stages of fermentation. The pH control loop should be adjusted to minimize pH drift, if possible.

Signaling the end of production fermentation has no hard target. However, the overall goal of production fermentation is to produce as much of the algal cytoplasm as possible to specification given the time/equipment constraints. A concentration of at least 60g/L of microwave Dry Cell Weight (DCW) was used as the primary standard for harvesting production fermenters.

Example 5Harvesting and downstream processing

After the production fermentation is complete, the liquid fermentation broth is transferred from the fermentor for use in downstream processing unit operations. The final liquid medium should not be inactivated by heating. The algal cell mass was first separated from the liquid by centrifugation (about 3000g) at ambient temperature (typically about 25 ℃) and the resulting cell mass was then transferred for spray drying at a solids concentration of about 20 ± 5%. The precise conditions for spray drying vary depending on the dryer used and the condition of the material. The skilled person will be able to readily determine the optimum conditions for spray drying. The spray-dried cell mass was evaluated according to the instructions and, if deemed acceptable, packaged in a plastic-lined bucket. The target criteria for the final spray-dried algal cell mass product are detailed in table 15.

Watch 15

Vision	Target	Deviation (+/-)	Unit of
				Exterior surface	Powder of	n/a	n/a
Colour(s)	Dark green	n/a	n/a
				Contaminants	Target	Deviation (+/-)	Unit of
Aerobic Plate Counter (APC)	<10000	n/a	CFU·g-1
				Escherichia coli (general type)	n.d.	0	CFU·g-1
Yeast	<10	n/a	CFU·g-1
				Total coliform group	n.d.	0	CFU·g-1
Number of moulds	<30	n/a	CFU·g-1
				Staphylococcus aureus	n.d.	0	CFU·g-1
Salmonella	Negative of	0	org·(25g)-1
				Composition (I)	Target	Deviation (+/-)	Unit of
Moisture content	15	5	％
				Protein (coarse)	50	5	％
Fat (coarse)	10	5	％
				Ash content	<5	n/a	％
Nitrogen is present in	10	2	％
				Phosphorus (Total amount)	1	0.5	％
Sodium (Total amount)	0.5	0.3	％
				Potassium (total amount)	0.15	0.1	％
Magnesium (Total amount)	0.15	0.1	％
				Calcium (Total amount)	0.15	0.1	％
Sulfur (Total amount)	0.035	0.01	％
				Iron (Total amount)	0.015	0.01	％
Manganese (Total)	0.0035	0.001	％
				Copper (Total amount)	0.003	0.001	％
Zinc (Total amount)	0.002	0.001	％

Example 6Determination of Stem cell weight (DCW)

The glass microfiber filter membrane was marked below near the edge using permanent markings. The labeled filter membrane is then weighed on an analytical balance and measured in grams (mass)_{Initial, g}) The weight was recorded. The pre-weighed filter membrane is then placed in a filtration apparatus and a vacuum is applied. The entire filter membrane was wetted starting from the filter membrane edge and moving with 2g/L ammonium bicarbonate solution. Using a pipette, a known volume of culture medium (volume) containing algae_{Filtered, ml}) Adding into a filter membrane. The volume chosen should not be too large to prevent clogging of the filter membrane or media flowing out of the filter membrane edge. The pipette was then rinsed with 2g/L ammonium bicarbonate and the rinse added to the filter membrane. The filtration membrane is then washed with 2g/L ammonium bicarbonate in an amount equal to the volume_{Filtered, ml}3 times of the total weight of the product. The filter membrane was carefully removed from the filter unit and folded so that the number was at the 7 o' clock position (see FIG. 7). The filter membrane is then folded in four and the two corners are bent downwards to form legs. The filter membrane was then placed on a microwave oven and dried by microwaves at 70% power for 10 minutes. The filter membrane is cooled and then used for quality_{Initial, g}On the same balance to obtain the mass_{Finally, g}. The stem cell weight (DCW) was calculated as follows:

example 6Seed fermentation Using fermentation Medium B

Sterile high-concentration fermentation medium B was prepared in a sterile fermentation vessel (table 4). The volume of culture medium prepared should be equal to 90% of the desired initial volume of the seed fermentor to allow for the addition of 10% by volume of inoculum. After 120 hours of propagation of the phase II flasks and verification that no contamination occurred, the phase II flasks were aseptically removed with a sterile pipette in a biosafety cabinetSamples and for OD₇₅₀And quantification of DCW. Typically the final DCW in the stage II flask was 2.5. + -. 0.7 g/L. Once it was confirmed that the DCW of the stage II flask was within the desired range, inoculation of the seed fermentor was possible.

The required inoculation volume of the seed fermentor was equal to 10% of the effective volume of the initial seed fermentor, so the initial dry cell weight concentration was 0.25. + -. 0.07 g/L. The seed inoculum was aseptically transferred from the flask to a sterile seed inoculation vessel in a biosafety cabinet and then to a seed fermentor. Addition of inoculum typically results in an increase in pH above the set point of pH 6.8. If the pH controller is not properly adjusted, an excessive amount of acidic feed may be added to the fermentor during this period, resulting in a severe pH drop. pH values below 5.5 may adversely affect fermentation performance and should be avoided. To prevent this, the pH control may be temporarily disabled prior to inoculation. If pH control is disabled, the pH value is manually adjusted to the set point directly after inoculation and pH control is re-enabled. After successful inoculation and activation of pH control, an increase in pH should be observed, indicating metabolic activity, more specifically, consumption of acetate.

Seed fermentation was carried out aerobically and the process parameters and control strategy are listed in table 13. In this example, the seed fermentation is performed in the absence of light, meaning that any viewing port is covered during the fermentation process.

The feed stream was a mixture of glacial acetic acid and ammonia (feed medium B, table 6), which was filter sterilized using a 0.22 μm millipore filter. The feed stream was fed to the fermentor by a peristaltic pump. The acidic feeding stream is used to adjust the pH and the dosage is automatically supplied by the pH controller when the pH increases beyond its set point (one-sided "pH-state"). Dissolved Oxygen (DO) was controlled at a set point of 30% air saturation (at atmospheric pressure) by adjusting the stirring rate. The spray rate can also be adjusted if agitation alone is insufficient to maintain the DO set point.

In the case of significant FOAM formation, it may be equal to 0.06mL FOAMA sterile antifoam bolus of antifoam per liter of fermentation broth of F111-GF was added to the fermentor. For the purposes of this example, "significant foam" is defined as the amount of foam sufficient to "retard" the addition of acid from the top of the fermentor. If the accumulator/mass flow meter indicates that acid is being fed, but no decrease in pH is observed, a "significant foam" may form. In this case, the presence of "significant foam" should be verified by visual inspection through a sight glass (temporarily uncovered). If the foam is allowed to form for too long, breaking the foam may pour excess "standing" acid into the liquid medium, resulting in death of the culture. Therefore, the problem of foam formation should be solved as soon as possible. For OD₇₅₀And DCW, the seed fermentors were sampled at Total Fermentation Times (TFT) of 0 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours and 168 hours.

48 hours before inoculation of the production fermentor, samples were aseptically removed from the seed fermentor through a sterile sampling port for visual inspection by microscope and streaking on LB plates (incubation at 37 ℃ for 24 hours) for further inspection for contamination. The harvest criteria for seed fermentation is 40g/L to 60g/L DCW, but concentrations as high as 80.0g/L are acceptable.

Example 7Seed fermentation with fermentation Medium B

Production fermentation using high concentration fermentation medium B operates similarly to seed fermentation; the main differences between the two processes are the components of the fermentation medium, the trace elements and the feeding medium. The aim of the production fermentation using high concentration fermentation medium B is to produce at least 60g DCW/L, usually in the range of 90 to 140h, without detectable contaminants.

Sterile fermentation medium B may be prepared in a fermentation vessel using the formulations detailed in table 4. Alternatively, the sterile fermentation medium can be produced outside the fermentation vessel and then introduced into the sterile fermentation vessel. The volume of culture medium prepared should be equal to 90% of the desired initial volume of the seed fermentor to allow for the addition of 10% by volume of inoculum.

After completion of seed fermentation using high concentration fermentation medium and confirmation of no contamination, the final sample was aseptically removed through sterile sampling port to control OD₇₅₀And DCW quantification. The final DCW for seed fermentation is typically 40.0g/L to 60.0g/L, but concentrations as high as 80g/L are acceptable. Once it was confirmed that the DCW of the seed fermentor was within the desired range and no contamination occurred, the production fermentor was inoculated.

The inoculation volume required for the production fermentor was 10% of the initial volume of the production fermentor, so the initial DCW concentration was 4g/L to 6 g/L. Particularly for the inoculation of production fermenters, it is preferred that the seed inoculum is exposed to hypoxic conditions for no more than 15 minutes. Process control (e.g., agitation and sparging) should be maintained in the seed fermentor during inoculum transfer, and residence time in the inoculum transfer line should be minimized as much as possible. If the organisms undergo oxygen starvation prior to inoculation, the treatment performance is significantly reduced.

Addition of inoculum typically results in an increase in pH above the set point. If the pH controller is not properly adjusted, an excessive amount of acidic feed may be added to the fermentor during this period, resulting in a severe pH drop. pH values below 5.5 may adversely affect fermentation performance and should be avoided. Thus, pH control may be temporarily disabled prior to inoculation. After inoculation, the pH should be manually adjusted to the set point before re-enabling pH control. After successful inoculation and activation of pH control, an increase in pH should be observed, indicating metabolic activity, in particular, consumption of acetate. Production fermentations were carried out under aerobic conditions, with process parameters and control strategies listed in table 14. Production fermentations are usually carried out in the absence of light, covering all the viewing ports during the fermentation.

The feed stream (feed medium B, Table 6) was a mixture of glacial acetic acid and ammonia, which was filter-sterilized at 0.22 μm and fed to the fermenter by means of a peristaltic pump. The acidic feeding stream is used to adjust the pH and is automatically dosed by the pH controller when the pH increases beyond its set point (one-sided "pH-state"). The percent saturation of Dissolved Oxygen (DO) was controlled at a set point of 30% air saturation (at atmospheric pressure) by adjusting the agitation rate. The spray rate can also be adjusted if stirring alone is insufficient to maintain the DO set point.

In the case of significant FOAM formation, 0.06mL FOAM can be addedA sterile antifoam bolus of antifoam for F111-GF per liter of liquid medium was added to the fermentor. For the purposes of this disclosure, "significant foam" is defined as an amount of foam sufficient to "retard" the addition of acid from the top of the fermentor. If the accumulator/mass flow meter indicates that acid is being fed, but no decrease in pH is observed, a "significant foam" may form. In this case, the presence of "significant foam" should be verified by visual inspection through a viewing port and taking appropriate action. If the foam is allowed to form for too long, breaking the foam may pour excess "blocked" acid into the liquid medium, resulting in death of the culture. Therefore, the problem of foam formation should be solved as soon as possible.

Production fermentors were sampled at TFT 0 hours, 20 hours, 43 hours, 68 hours, 96 hours, 116 hours, and 140 hours to determine OD₇₅₀And DCW. Data and trends for typical 0.5L production fermentations are shown in figures 9-12. It can be seen in FIG. 10 that after about 80 hours, the decrease of pO2 below the set point is a result of the inability to increase the liquid culture medium aeration rate, since both agitation and sparging reach their maximum values in laboratory scale fermenters. By increasing the injection rate after agitation reaches its maximum (cascade control), the ideal process control will maintain the value of DO at its set point throughout the fermentation.

As with seed fermentation, production fermentation operates as a one-sided (acid) | pH-state ". As a result of this mode of operation, fluctuations in pH with respect to the set point are generally observed throughout the fermentation process (see fig. 11). As culture density increased, the total acetate consumption increased (fig. 12). The signal of the end of the production fermentation has no hardness index. However, the overall goal of production fermentation is to produce as much of the algal cell mass as possible in compliance with specifications given time/equipment constraints. As shown in FIG. 9, the microwave Dry Cell Weight (DCW) concentration exceeded 80.0 g/L.

It is to be understood that the claimed invention has been described in detail by way of illustration and example, so as to enable others skilled in the art to become familiar with the claimed invention, its principles and practical application. The specific formulations and methods of the claimed invention are not to be limited by the description of the specific embodiments presented, but rather the description and examples should be viewed in light of the appended claims and their equivalents. While some of the examples and descriptions above include some conclusions as to the manner in which the claimed invention may function, the inventors do not intend to be bound by those conclusions and functions, but rather propose them only as possible explanations.

It should be further understood that the above-described specific embodiments are not intended to be exhaustive or to limit the claimed invention and that many alternatives, modifications, and variations will be apparent to those of ordinary skill in the art in light of the foregoing examples and detailed description. Accordingly, the claimed invention is intended to embrace all such alternatives, modifications and variances which fall within the scope of the following claims.

Claims (45)

1. A method for the aerobic, heterotrophic culture of a high density culture of Chlamydomonas sp, which method comprises,

obtaining a substantially pure inoculum of a culture of a species of Chlamydomonas having a concentration of 0.1g/L to 15 g/L;

producing a production culture by inoculating an initial volume of fermentation medium, wherein the inoculated volume is no more than 20% of the initial volume of fermentation medium;

keeping the production culture under aerobic conditions with pH value of 6.0-10.0 and temperature of 15-37 ℃;

feeding the production culture by providing to the production culture an amount and a planned feeding medium determined by a change in pH of the production culture;

culturing the production culture under sterile conditions until a target density of at least 60g/L dry cell weight is reached; and

harvesting the Chlamydomonas species from the production culture;

wherein the Chlamydomonas species does not contain a heterologous gene.

2. The method of claim 1, wherein the production culture is maintained in the absence of light.

3. The method of claim 1, wherein feeding is determined by an increase in pH.

4. The method of claim 1, wherein the production culture is provided with a feeding medium to maintain the pH of the production medium at pH 6.8 ± 0.5.

5. The method of claim 1, wherein the feeding medium is provided to the production culture at any time that the pH of the production culture exceeds a culture set point and is stopped at any time that the pH of the production culture is below a pH set point.

6. The method of claim 1, wherein the pH set point is 6.0.

7. The method of claim 1, wherein the pH set point is 6.2.

8. The method of claim 1, wherein the pH set point is 6.5.

9. The method of claim 1, wherein the pH set point is 6.8.

10. The method of claim 1, wherein the pH set point is 7.0.

11. The method of claim 1, wherein the target density is at least 50g/L dry weight, at least 55g/L dry weight, at least 60g/L dry weight, at least 65g/L dry weight, at least 70g/L dry weight, at least 75g/L dry weight, at least 80g/L dry weight, at least 85g/L dry weight, at least 90g/L dry weight, at least 95g/L dry weight, at least 100g/L dry weight, at least 105g/L dry weight, at least 110g/L dry weight, at least 115g/L dry weight, at least 120g/L dry weight, at least 125g/L dry weight, at least 130g/L dry weight, at least 135g/L dry weight, at least 140g/L dry weight, at least 145g/L dry weight, at least 150g/L dry weight, at least 155g/L dry weight, At least 160g/L dry weight, at least 165g/L dry weight, at least 170g/L dry weight, at least 175g/L dry weight, at least 180g/L dry weight, at least 185g/L dry weight, at least 190g/L dry weight, at least 195g/L dry weight, or at least 200g/L dry weight.

12. The method of claim 1, wherein the target density is 50-75 g/L, 75-100 g/L, 100-125 g/L, 125-150 g/L, 150-175 g/L, or 175-200 g/L of stem cell weight.

13. The method of claim 1, wherein the target density is 50g/L, 55g/L, 65g/L, 70g/L, 75g/L, 80g/L, 90g/L, 95g/L, 100g/L, 105g/L, 110g/L, 115g/L, 120g/L, 125g/L, 130g/L, 135g/L, 140g/L, 145g/L, 150g/L, 155g/L, 160g/L, 165g/L, 170g/L, 175, 185, 190, 195, or 200g/L of the stem cell weight.

14. The method of claim 1, wherein the target density is reached within 250 hours after inoculating the production culture.

15. The method of claim 1, wherein the Chlamydomonas species is harvested by filtration or centrifugation.

16. The method of claim 15, wherein the Chlamydomonas species is harvested by batch centrifugation.

17. The method of claim 15, wherein the Chlamydomonas species is harvested by continuous centrifugation.

18. The method of claim 1, further comprising drying the harvested Chlamydomonas species.

19. The method of claim 18, wherein the drying is performed by spray drying, ring drying, paddle drying, tray drying, solar or sun drying, vacuum drying, or freeze drying.

20. The method of claim 18, further comprising drying the Chlamydomonas species to a moisture content of less than 15%.

21. The method of claim 1, wherein the Chlamydomonas species is one or more of Chlamydomonas reinhardtii, Chlamydomonas dysomos, Chlamydomonas brandane, Chlamydomonas debaryana, Chlamydomonas moewussi, Chlamydomonas culeus, Chlamydomonas noctinoctiga, Chlamydomonas aulata, Chlamydomonas applanata, Chlamydomonas marvanii, and Chlamydomonas probosclera.

22. The method of claim 21, wherein the Chlamydomonas species is Chlamydomonas reinhardtii.

23. A nutritional supplement comprising greater than 90% or at least one chlamydomonas species prepared by the method of claim 1, the nutritional supplement comprising:

composition (I) Content (wt.) Deviation (+/-) Moisture content 20％ 1 Protein (coarse) 70％ 1 Fat (coarse) 20％ 1 Ash content <5％ n/a Nitrogen is present in 20％ 1 Phosphorus (Total amount) 5％ 0.001 Sodium (Total amount) 2％ 0.001 Potassium (total amount) 1％ 0.001 Magnesium (Total amount) 1％ 0.001 Calcium (Total amount) 1％ 0.001 Sulfur (Total amount) 1％ 0.001 Iron (Total amount) 1％ 0.001 Manganese (Total) 1％ 0.001 Copper (Total amount) 1％ 0.001 Zinc (Total amount) 1％ 0.001

24. The nutritional supplement of claim 23, wherein the supplement further comprises omega 3 fatty acids, omega 6 fatty acids, and omega 9 fatty acids.

25. An algal culture comprising one or more species of Chlamydomonas under growth conditions, wherein the culture comprises the algae at a concentration of at least 50g/L dry cell weight, and wherein, under the growth conditions, the density of the culture increases at a rate of 50% to 300% per 24 hour period.

26. An algal culture according to claim 19, wherein, under steady state conditions, the density of the culture increases at a rate of 0.1% to 50% per 24 hour period.

27. A method of producing a therapeutic protein, the method comprising:

obtaining an inoculum of a substantially pure culture of a Chlamydomonas species at a concentration of 0.1g/L to 15g/L, the Chlamydomonas species expressing at least one exogenous therapeutic protein;

producing a production culture by inoculating an initial volume of fermentation medium, wherein the inoculated volume is no more than 20% of the initial volume of fermentation medium;

keeping the production culture under aerobic conditions with pH value of 6.0-10.0 and temperature of 15-37 ℃;

feeding the production culture by providing to the production culture an amount and a planned feeding medium determined by a change in pH of the production culture;

culturing the production culture under sterile conditions until a target density of at least 50g/L dry cell weight is reached; and

harvesting the Chlamydomonas species from the production culture.

28. The method of claim 26, wherein the production culture is maintained in the absence of light.

29. The method of claim 26, wherein feeding is determined by an increase in pH.

30. The method of claim 26, wherein the production culture is provided with a feeding medium to maintain the pH of the production medium at pH 6.8 ± 0.5.

31. The method of claim 26, wherein the feeding medium is provided to the production culture at any time that the pH of the production culture exceeds a culture set point and is stopped at any time that the pH of the production culture is below the pH set point.

32. The method of claim 26, wherein the pH set point is 6.0.

33. The method of claim 26, wherein the pH set point is 6.2.

34. The method of claim 26, wherein the pH set point is 6.5.

35. The method of claim 26, wherein the pH set point is 6.8.

36. The method of claim 26, wherein the pH set point is 7.0.

37. The method of claim 26, wherein the target density is at least 50g/L dry weight, at least 55g/L dry weight, at least 60g/L dry weight, at least 65g/L dry weight, at least 70g/L dry weight, at least 75g/L dry weight, at least 80g/L dry weight, at least 85g/L dry weight, at least 90g/L dry weight, at least 95g/L dry weight, at least 100g/L dry weight, at least 105g/L dry weight, at least 110g/L dry weight, at least 115g/L dry weight, at least 120g/L dry weight, at least 125g/L dry weight, at least 130g/L dry weight, at least 135g/L dry weight, at least 140g/L dry weight, at least 145g/L dry weight, at least 150g/L dry weight, at least 155g/L dry weight, At least 160g/L dry weight, at least 165g/L dry weight, at least 170g/L dry weight, at least 175g/L dry weight, at least 180g/L dry weight, at least 185g/L dry weight, at least 190g/L dry weight, at least 195g/L dry weight, or at least 200g/L dry weight.

38. The method of claim 26, wherein the target density is 50-75 g/L, 75-100 g/L, 100-125 g/L, 125-150 g/L, 150-175 g/L, or 175-200 g/L stem cell weight.

39. The method of claim 26, wherein the target density is 50g/L, 55g/L, 65g/L, 70g/L, 75g/L, 80g/L, 90g/L, 95g/L, 100g/L, 105g/L, 110g/L, 115g/L, 120g/L, 125g/L, 130g/L, 135g/L, 140g/L, 145g/L, 150g/L, 155g/L, 160g/L, 165g/L, 170g/L, 175, 185, 190, 195, or 200g/L of the stem cell weight.

40. The method of claim 26, wherein the target density is reached within 250 hours after inoculating the production culture.

41. The method of claim 26, wherein the Chlamydomonas species is harvested by filtration or centrifugation.

42. The method of claim 40, wherein the Chlamydomonas species is harvested by continuous centrifugation.

43. The method of claim 26, further comprising drying the harvested Chlamydomonas species.

44. The method of claim 42, wherein the drying is by spray drying, ring drying, paddle drying, tray drying, solar or sun drying, vacuum drying, or freeze drying.

45. The method of claim 26, further comprising isolating at least one therapeutic protein from a Chlamydomonas species.