CROSS REFERENCE TO RELATED U.S PATENT APPLICATIONS

This patent application relates to, and claims the priority benefit, U.S. Provisional Patent Application Serial No. 61 /481 ,041 filed on April 29, 201 1 entitled METHOD TO ENHANCE GROWTH AND PIGMENTS AND

CAROTENOIDS CONTENTS OF PHOTOSYNTHETIC ORGANISMS, filed in English, which is incorporated herein in its entirety by reference.

FIELD

The present invention relates to the process of culturing species of microalgae from the genus Haematococcus with the culture circulated through a magnetic field, for the purpose of increasing biomass production and/or increasing the biomass content of components including astaxanthin, carotenoids, and chlorophylls.

BACKGROUND

The usefulness of biomass of species of microalgae from the genus Haematococcus is well-documented, this biomass has been used in aquaculture for several decades and in health applications for at least one decade. In aquaculture it provides the pigments which are necessary dietary components for the aquatic organisms to become the proper color and also increases their health, leading to more valuable product and higher production. For human health astaxanthin is known to be a potent antioxidant and has been developed into the product Cardax© which is used to speed recovery after myocardial infarction.

Haematococcus is generally grown phototrophically and is often grown in open pond configurations, photobioreactors (which are often tubular or planar in geometry) or combinations of the two systems. The typical strategies used in microalgal technology have been used to increase biomass production and modulate biomass composition with Haematococcus including: altering light levels; increased carbon dioxide partial pressure; different nutrient sources and levels; different mixing techniques; and alterations to the inorganic components or their concentrations in the growth media.

The use of magnetic fields to stimulate algal biomass production is a relatively recent innovation. It has been described only for genera Chlorella and Spirulina.

It would be beneficial to provide a process to provide a method to significantly increase the rate of biomass production from microorganisms including such as astaxanthin, chlorophyll, carbohydrates and carotenoids, all of which are commercially useful.

SUMMARY

The present disclosure discloses a process of increasing production of one or more of astaxanthin, carotenoids, carbohydrates and chlorophyll, comprising providing a photosynthetic microorganism that naturally produces astaxanthin, carotenoids, carbohydrates and chlorophyll, inoculating a light- exposed bioreactor with inoculum of the microorganism under conditions to induce growth of the microorganism in the light exposed bioreactor, subjecting at least a portion of the bioreactor to a magnetic field during the course of growth resulting in increased production of at least one of astaxanthin, carotenoids, carbohydrates and chlorophyll compared to when no magnetic field is applied; and harvesting the astaxanthin, carotenoids, carbohydrates and chlorophyll of the bioreactor.

The present disclosure describes a process to produce

Haematococcus biomass at an accelerated rate with an increased

astaxanthin and caroteniod content by periodic exposure to magnetic fields. The process can be applied to growth in both open ponds and closed photobioreactor type growth systems.

This disclosure provides a process to produce microorganism biomass at a higher rate comprising subjecting microorganisms to a magnetic field during the growth phase in combination with means of providing periodic exposure of the microorganisms to the magnetic field.

The magnetic field may be applied periodically during the course of growth or the magnetic field may applied constantly during the course of growth. The magnetic field may be applied with a magnetic field strength in a range from about 1 mT to about 100 mT during the course of growth, and more preferably between about 5 mT to about 15 mT. The magnetic field may be static or it may be variable. A mode of biomass production may be any one or combination of continuous, semi-continuous, batch, or semi-batch.

A further understanding of the functional and advantageous aspects of the invention can be realized by reference to the following detailed description.

BRIEF DESCRIPTION OF DRAWINGS

The following is a description of a process for enhancing growth of biomass by exposure to magnetic fields, reference being had to the accompanying drawings, in which:

Figure 1 shows in the upper section a raceway pond used as model for large-scale microalgal biomass production (Example 2). 1 : Paddle wheel; 2: Baffles; 3: Cylindrical Aerators; 4: Location of side-stream for magnetic field exposure. Length of the pond in the longest dimension is approximately 40 cm. The lower section of Figure 1 shows a schematic of experimental set-up (Example 2). 1 : Raceway pond; 2: Peristaltic pump; 3: Solenoid

electromagnet; 4: Array of fluorescent lights; 5: Water to keep at constant level; 6: Thermocouple; 7: Quantum Sensor; 8: Filter sterilized air source; 9: Aerator discharge into raceway; 10: DC power supply. Raceway pond was used to determine growth curves under the Earth's magnetic field (control) and 10 mT magnetic field strength, while a similar set-up except with growth in axenic Erlenmeyer flasks was used for other experiments. The

experimental set-up for Example 3 is very similar, except that the pond is replaced with the flat plate photobioreactor shown in Figure 5.

Figure 2 shows the optimum magnetic field strength for growth of H. pluvialis was determined in axenic Erlenmeyer flasks cultures to be 10 mT (Example 1). Statistically significant differences in growth rate are marked with different letters (P<0.05, n=3). Mean ± one standard error is shown.

Figure 3 shows normalized biomass, as biomass divided by initial biomass, versus time for H. pluvialis cells exposed to 10 mT magnetic field and cells exposed only to Earth's magnetic field in the raceway pond (control) (Example 2). The initial biomass concentration was 0.03 ± 0.01 for the control treatment and 0.02±0.01 g/L for the 10 mT treatment. Mean ± one standard error is shown (n=3).

Figure 4 shows the final biomass, maximum growth rate, maximum daily production rate and average daily production rate are all significantly higher for the 1 0 mT treatment, compared to the control exposed only to Earth's magnetic field (Example 2). H. pluvialis cells were grown in the raceway pond. Mean ± one standard error is shown (n=3).

Figure 5 shows a schematic of photobioreactors used in Example 3. Dimensions are in inches. Thickness of the units is about 1 inch (usable volume about 3 L). Photobioreactors are constructed from polypropylene frames with transparent polycarbonate windows. The experimental set-up for the photobioreactors is similar to that shown in Figure 1 , except that the raceway pond is replaced with a photobioreactor.

Figure 6 shows a plot of normalized biomass, as biomass divided, by ^" initial biomass, versus time for H. pluvialis cells exposed to 10 mT magnetic field and cells exposed only to Earth's magnetic field in the photobioreactor (control) (Example 3). The initial biomass concentration was 0.13±0.01 for the control treatment and 0.09±0.01 g/L for the 10 mT treatment. Mean ± one standard error is shown (n=3).

Figure 7 shows the final biomass, maximum growth rate, maximum daily production rate and average daily production rate are all significantly higher for the 10 mT treatment, compared to the control exposed only to Earth's magnetic field (Example 3). H. pluvialis cells were grown in the raceway pond. Mean ± one standard error is shown (n=3).

Figure 8 shows biomass and pigment composition for control,

(exposed only to Earth's magnetic field) and 10 mT H. pluvialis cells

(Example 4). Parameters with statistically significant differences are marked with an asterisk (P<0.05, n=3). Mean ± one standard error is shown.

Figure 9 shows calculated astaxanthin production for H. pluvialis cells grown in either open raceway pond (Example 2) or closed photobioreactior (PBR) (Example 3), with or withoutI O mT magnetic field treatment

(Example 5). Astaxanthin production is calculated as described in Example 5. Note that astaxanthin production in continuous mode is inferred from batch data, as is common practice in biochemical engineering. Figure 10 shows normalized biomass, as biomass divided by initial biomass, versus time for C. kessleri cells exposed to 10 mT magnetic field and cells exposed only to Earth's magnetic field in the raceway pond (control) (Example 6). The initial biomass concentration was 0.32±0.07 for the control treatment and 0.22±0.06g/L for the 10 mT treatment. Points significantly different from the control are marked with an asterisk (P<0.05, n=3). Mean ± one standard error is shown.

Figure 11 shows final biomass, maximum growth rate, maximum daily production rate and average daily production rate are all significantly higher for the 10 mT treatment, compared to the control exposed only to Earth's magnetic field. C. kessleri cells were grown in the raceway pond

(Example 6). Statistically significant differences from the control are marked with an asterisk (P<0.05, n=3). Mean ± one standard error is shown.

Figure 12 shows biomass and pigment composition for control (exposed only to Earth's magnetic field) and 10 mT C. kessleri cells

(Example 6). Parameters with statistically significant differences are marked with an asterisk (P<0.05, n=3). Mean ± one standard error is shown.

DETAILED DESCRIPTION

Various embodiments and aspects of the disclosure will be described with reference to details discussed below. The following description and drawings are illustrative of the disclosure and are not to be construed as limiting the disclosure. Numerous specific details are described to provide a thorough understanding of various embodiments of the present disclosure. However, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of embodiments of the present disclosure.

As used herein, the terms, "comprises" and "comprising" are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in this specification including claims, the terms, "comprises" and "comprising" and variations thereof mean the specified features, steps, or components are included. These terms are not to be interpreted to exclude the presence of other features, steps, or components. As used herein, the term "exemplary" means "serving as an example, instance, or illustration," and should not be construed as preferred or advantageous over other configurations disclosed herein.

As used herein, the terms "about" and "approximately", when used in conjunction with ranges of dimensions of particles, compositions of mixtures, or other physical properties or characteristics, are meant to cover slight variations that may exist in the upper and lower limits of the ranges of dimensions so as to not exclude embodiments where on average most of the dimensions are satisfied but where statistically dimensions may exist outside this region. It is not the intention to exclude embodiments such as these from the present disclosure.

The present invention provides a novel process which can be applied to microorganisms, preferably Haematococcus, to increase the rate of biomass production and can increase the astaxanthin content within

Haematococcus.

The following definitions are used in the description:

Batch production: Bioreactor is inoculated and left for a finite period of time with no flows in or out. At the end of the this time, referred to as the growth time or growth period in this disclosure, the reactor contents are harvested. Semi-batch production: The same as batch production, except that nutrients can be added continuously or periodically to the bioreactor.

Continuous production: The bioreactor is inoculated as in batch mode. Once the desired cell density is reached this density is maintained by continuously drawing off liquid containing cells and diluting with water which may contain nutrients. In this case the growth period or growth time is the entire period of time for which the bioreactor is operated at or near the desired cell density.

Semi-continuous production: The same as continuous production except that a portion of the culture is harvested and replaced periodically instead of continuously.

One aspect of this disclosure provides a process to produce microorganism biomass at a higher rate comprising generation of an inoculum comprising microorganisms, preparation of a suitable bioreactor for inoculation, whereby the bioreactor provides means for continuous flow, inoculation of the bioreactor with the inoculum, whereby microorganisms subject to continuous flow within the bioreactor are subjected to a magnetic field during the growth time.

This disclosure provides a process to produce green algae biomass at a higher rate with a higher astaxanthin content comprising exposure of green algae to a magnetic field during the growth phase.

This disclosure provides a process to produce green algae with a higher astaxanthin content comprising exposure of green algae to a magnetic field during the growth phase.

This disclosure provides a process to produce green algae biomass at a higher rate with a higher astaxanthin content comprising periodic exposure of green algae to a magnetic field during the growth phase.

Another aspect of the disclosure provides a process to produce Haematococcus biomass at a higher rate with a higher astaxanthin content comprising exposure of Haematococcus to a magnetic field during the growth phase.

Another aspect of this disclosure to provide a method to produce Haematococcus with a higher astaxanthin content comprising exposure of Haematococcus to a magnetic field during the growth phase.

Another aspect of the invention provides process to produce

Haematococcus biomass at a higher rate with a higher astaxanthin content comprising periodic exposure of Haematocaccus to a magnetic field during the growth phase.

Another aspect of the invention provides process to produce

Haematococcus biomass at, a higher rate with a higher astaxanthin content comprising generation of a Haematococcus inoculum ; inoculation of said bioreactor with said inoculum in which the Haematococcus are subjected to conditions appropriate for growth and subjection of all or a portion of the bioreactor volume of Haematococcus to a magnetic field for all or a portion of the growth time.

Another aspect of this disclosure provides a process to produce Haematococcus biomass at a higher rate with a higher astaxanthin concentration comprising generation of a Haematococcus inoculum, preparation of a suitable bioreactor for inoculation; inoculation of said bioreactor with said inoculum in which the microalgae are subjected to conditions appropriate for growth; subjection of all or a portion of the bioreactor volume for all or a portion of the growth time to a magnetic field, wherein said process may include separation of astaxanthin from

Haemaotococcus biomass.

Another aspect of the disclosure provides a process to produce Haematococcus biomass at a higher rate with a higher carbohydrate concentration comprising generation of a Haematococcus inoculum, preparation of a suitable bioreactor for inoculation; inoculation of the bioreactor with the inoculum in which the microalgae are subjected to conditions appropriate for growth; subjection of all or a portion of the bioreactor volume for all or a portion of the growth time to a magnetic field, wherein the process may include separation of carbohydrates from

Haemaotococcus biomass.

Another aspect of the invention provides a process to produce Haematococcus biomass at a higher rate providing for a higher chlorophyll concentration comprising generation of a Haematococcus inoculum, preparation of a suitable bioreactor for inoculation; inoculation of said bioreactor with said inbculum in which the microalgae are subjected to conditions appropriate for growth; subjection of all or a portion of the bioreactor volume for all or a portion of the growth time to a magnetic field, wherein said process may include separation of chlorophyll from

Haemaotococcus biomass.

Another aspect of the invention provides a process to produce Haematococcus biomass at a higher rate providing for a higher carotenoid concentration comprising generation of a Haematococcus inoculum, preparation of a suitable bioreactor for inoculation; inoculation of said bioreactor with said inoculum in which the microalgae are subjected to conditions appropriate for growth; subjection of all or a portion of the bioreactor volume for all or a portion of the growth time to a magnetic field, wherein said process may include separation of carotenoids from

Haemaotococcus biomass

In another aspect of the invention the bioreactor comprises a magnet or electromagnet assembly which provides a magnetic field, preferably static and preferably uniform, over a portion of the culture, preferably between 0.1 and 5 % of the total volume, most preferably 1 % of the total volume.

The magnet or electromagnet may be inside the main reactor but preferably is outside and connected by a slip-stream. Preferably the culture is circulated periodically through the magnet or electromagnet. This can be achieved by placing the magnet or electromagnet in the path of fluid flow if it is inside the reactor, or preferably it can be achieved by pumping a slipstream through an external vessel which is exposed to the magnet, preferably using a peristaltic pump or another pump design which causes low shear forces and does not contaminate the growth media. If an electromagnet is used it can be a solenoid, an H-frame design, or any other design which can provide regions of fairly uniform magnetic field strength. A low frequency current is used, preferably a static current. If magnets are used then a Halbach array configuration, such as a Halbach cylinder, or another configuration which produces a fairly uniform magnetic field strength can be used.

In the process disclosed herein, microalgae of the genus

Haematococcus are grown and magnetic field exposure for part of the period of growth or for the whole period of growth, preferably for the entire period of growth, is implemented according to the description set forth in this section. This results in increased biomass production and/or increased astaxanthin content and therefore an increased astaxanthin production rate.

In another aspect of the invention, green algae, cyanobacteria, or diatoms are exposed to static magnetic fields during the microorganism growth phase to increase the rate of biomass production.

In another aspect of the invention, green algae, cyanobacteria, or diatoms are periodically exposed to static magnetic fields during the microorganism growth phase to increase the rate of biomass production.

Broadly, the present disclosure discloses a process of increasing production of one or more of astaxanthin, carotenoids, carbohydrates and chlorophyll biomass components. A photosynthetic microorganism that naturally produces astaxanthin, carotenoids, carbohydrates and chlorophyll is provided and a light-exposed bioreactor is innoculated with inoculum of the microorganism under conditions to induce growth of the microorganism in the light exposed bioreactor. At least a portion of the bioreactor is exposed to a magnetic field during the course of growth resulting in increased production of at least one of astaxanthin, carotenoids, carbohydrates and chlorophyll compared to when no magnetic field is applied; and harvesting the

astaxanthin, carotenoids, carbohydrates and chlorophyll of the bioreactor.

More specifically, in a non-limiting and exemplary example, the process described herein comprises growth of microalgae from the genus

Haematococcus to generate an inoculum which is used to inoculate a reactor followed by subjection of that reactor and its contents to conditions

appropriate for the phototrophic growth of microalgae, as defined below in detail, and exposure of the the Haematococcus within the reactor to a magnetic field during their growth phase.

The microorganisms can be grown in any suitable container, including a photobioreactor which can be of the open-pond type or of the closed photobioreactor type.

Conditions appropriate for the phototrophic growth of microalgae are known to those skilled in the art. Microalgae of the genus Haematococcus are grown according to standard methods used for the growth of green microalgae. This includes exposure to reasonable light levels (50-3000 micromoles of photosynthetically active radiation per square meter per second); growth in a medium containing an inorganic nitrogen source and inorganic phosphate source at concentrations on the order of 1 g/L and 0.1 g/L or higher; providing carbon dioxide in a gaseous stream with 360 ppm w/w carbon dioxide concentration or more; providing adequate mixing by means of bubbling or mechanical agitation; providing trace metals, a source of magnesium, and other nutrients; taking the usual steps employed by one skilled in the art to ensure no contamination in closed photobioreactor configurations and minimize contamination in open pond systems.

The process provides for exposure of the microorganisms within the reactor to a magnetic field. The exposure of the microorganisms to the magnetic field may be periodic. Further, the exposure may be either constant or periodic over the growth phase. The periodic subjection of the reactor contents to a magnetic field is preferably only a small fraction, on the order of 1 % at any one time, preferably a static magnetic field, preferably of high uniformity, with strength on the order of 1 -1 00 mT, preferably about 10 mT, for part of the growth period, preferably the entire growth period.

The process provides for an increase in the rate of biomass production (Figure 7), an increase in astaxanthin concentration within the biomass, an increase in carbohydrate concentration within the biomass, an increase in caretenoid concentration within the biomass, and an increase in chlorophyll concentration within the biomass (Figure 8).

The astaxanthin produced is suitable for applications in human health, aquaculture, agriculture, and other applications. The astaxanthin produced may be in free alcohol, mono-ester, or di-ester form or a mix of these three forms in varying ratios, all of which are suitable for the above-mentioned applications. Depending on the desired application the biomass may be used in its whole form, in a disrupted form (e.g. by sonication or other methods to improve bioavailability of astaxanthin), or astaxanthin can be purified from the biomass to varying degrees, including the isolation of pure astaxanthin from the produced biomass.

In one embodiment of the invention the photobioreactor referred to in (ii) is an open-pond as shown in Figure 1. Preferably the pond can be of the paddle-wheel circulated raceway-type. In this embodiment a side-stream is moved from the reactor, by the pressure drop imparted by the paddle-wheel or using a peristaltic pump, through a magnet or electromagnet, preferably a solenoid electromagnet, with a magnetic field strength between 1 and 100 mT, preferably a static field with a strength of 10 mT. In this embodiment the entire culture volume is cycled through the magnet-exposed volume at a frequency between 1 and 0.01 /min, preferably around 0.1 /min, and the volume exposed to the electromagnet comprises between 0.1 and 100 % of the reactor volume, preferably about 1 % of the reactor volume. In this embodiment the race-way pond can be operated in either batch mode; fed- batch mode; semi-continuous mode; or continuous mode. In this embodiment an increase in growth rate by a factor of about 2.5 can be achieved relative to a non-magnetized culture; and astaxanthin content can be increased by about 66 %. In batch mode final biomass produced can be increased by a factor of about 2.5, resulting in almost 5 times the astaxanthin production per batch compared to non-magnetized cultures. If operated in continuous or semi- continuous mode biomass production can be increased by about a factor of 4, resulting in more than 6 times the daily astaxanthin production compared to non-magnetized cultures. Magnetic field exposure resulted in similar increases in carotenoid and total chlorophyll production, with about a 33 % increase in total chlorophyll content and a 25 % increase in total carotenoid content.

In another embodiment of the invention the photobioreactor is preferably a closed photobioreactor, preferably the flat-planar type of photobioreactor as shown in Figure 5. With this type of photobioreactor the contents are mixed by bubbling or mechanical pumping, preferably by bubbling to create an air-lift or bubble-column effect. In this embodiment a side-stream is moved from the reactor, by the pressure drop imparted by the reactor mixing or using a peristaltic pump, through a magnet or

electromagnet, such as for example a solenoid electromagnet, with a magnetic field strength between 1 and 100 mT, preferably a static field with a strength of 10 mT. In this embodiment the entire culture volume is cycled through the magnet-exposed volume at a frequency between 1 and 0.01 /min, preferably around 0.1 /min, and the volume exposed to the electromagnet comprises between 0.I and 100 % of the reactor volume, preferably about 1 % of the reactor volume. In this embodiment the photobioreactor can be operated in either batch mode; fed-batch mode; semi-continuous mode; or continuous mode. In this embodiment an increase in growth rate by about 8 % can be achieved relative to a non-magnetized culture; and astaxanthin content can be increased by about 66 %.

In batch mode final biomass produced can be increased by about 5 %, resulting in about 70 % more astaxanthin production per batch compared to non-magnetized cultures. If operated in continuous or semi-continuous mode biomass production can be increased by about a factor of 2, resulting in more than 4 times the daily astaxanthin production compared to non-magnetized cultures.

In any of these embodiments once the biomass is produced it can be harvested by known methods including filtration, flocculation, sedimentation, and centrifugation. This biomass concentrate can be dried by freeze-drying or other methods of drying. The dried biomass itself can be the final product. Alternatively astaxanthin can be separated from the biomass by known lipid extraction techniques including Folch extraction, Bligh-Dwyer extraction, or extraction using ethanol, isopropanol, or other solvents [Griffiths MJ, Hille RP, Harrison STL. 2010. Selection of Direct Transesterification as the

Preferred Method for Assay of Fatty Acid Content of Microalgae. Lipids 45: 1053-1060.] Astaxanthin can be further purified from the lipid extract by chromatographic methods including liquid chromatography and high- performance liquid chromatography.

One embodiment of the present invention provides a process comprised of (i) the growth of microalgae from the genus Haematococcus to generate an inoculum, ii) the design and construction of a photobioreactor for the growth of Haematococcus, which can be of the open-pond type or of the closed photobioreactor type, iii) the inoculation of the reactor referred to in (ii) with the prepared inoculum according to (i) and the subjection of that reactor and its contents to conditions appropriate for the phototrophic growth of microalgae, and iv) the periodic subjection of the reactor contents, preferably only a small fraction, on the order of 1 % at any one time, to a magnetic field, preferably a static magnetic field, preferably of high uniformity, with strength on the order of 1 -100 mT, preferably about 1 0 mT, for part of the growth period preferably the entire growth period.

In one embodiment, the present invention incorporates i) periodic exposure at a defined ratio and frequency ii) within a scalable industrially relevant bioreactor iii) at a useful light level close to the average natural light level over a day iv) with a static magnetic field on the order of 5- 15 mT and v) that the magnetic field is applied during the whole duration of biomass growth, or at least a portion of this period. Only a small portion of the reactor volume must be magnetized, greatly reducing the cost. The use of conditions and reactors similar to those used in industry means that the present invention can be used on a larger scale more readily than the subject matter preceding this invention.

The present invention is further illustrated by the following non-limiting examples. EXAMPLES

Example 1. Inoculum Production And Optimal Static Magnetic Field Strength

Haematococcus pluvialis var. Flotow was grown in 0.22 pm filter sterilized Bold's Basal freshwater media, made with glass-distilled deionized water (DDIW). All chemicals were analytical grade and purchased from the Sigma-Aldrich Corporation (MO, USA). H. pluvialis was grown in this media for 3 months for acclimation, and the inoculum was taken from cultures in the exponential growth phase. Innoculum cultures were grown in 2-L Erlenmeyer flasks, bubbled with 0.5 volumes/volume/minute of filter-sterilized air. It was verified each week that the cultures were axenic using a 10 μΙ inoculating loop to streak three Petri dishes of Difco™ nutrient agar which were incubated at 25 °C. No bacterial colonies grew, verifying that the cultures were axenic.

These cultures were also pumped through a static magnetic field generated by a solenoid electromagnet, similar to the experimental set-up shown in Figure 1 , except with flask cultures instead of a race-way pond. The magnet was set to a field strength of 0 mT (off), 5 mT, 10 mT, and 15 mT to identify an optimal magnetic field strength for growth enhancement.

The optimum magnetic field strength was found to be around 1 0 mT

(Figure 2). This led to a more than 2-fold increase in specific growth rate. Specific growth rate was significantly increased with 5 mT and, 1 5 mT magnetic field exposure, however the increase was less than for 10 mT exposed cultures.

Example 2. Growth In Open Raceway Pond With And Without 10 mT Static Magnetic Field Exposure

Growth and biomass production were measured in a small scale raceway pond with an effective volume of 3.6 I. The pond features an oval channel, with a usable depth of 6 cm and is circulated by a paddle wheel. Filter sterilized (0.22 μηι) air is bubbled into the reactor via four perforated stainless steel tubes to provide carbon dioxide (388 ppmv). A schematic generated using Solidworks 2009 v.sp3.0 is shown in Figure 1. Although a small-scale pond was used the design could easily be scaled-up to hundreds of cubic litres in volume, which is typical of the largest race-way ponds.

The pond was operated in a bio-safety cabinet to minimize

contamination. DDIW was added daily to maintain constant volume. The aeration rate was 0.5 I air/min and the paddle wheel was set at 20 RPM, providing a surface velocity of about 25 cm/s. All experiments were run at 22±1 °C. Artificial light was provided by a 1 :1 mix of GE Ecolux wide spectrum and Phillips Plant and Aquarium fluorescent tubes for 1 6 hours each day. A Li-Cor LI190 quantum sensor (NE, USA) was used to measure photosynthetically active radiation (PAR), which was set at 200±10 μηιοΙ photons/m2s. The pond was sterilized with 70 % v/v aqueous isopropanol and washed with DDIW.

A side-stream from the reactor was exposed to a uniform 10 mT SMF, generated by a water-cooled solenoid, which caused no measurable heating, facing magnetic North. Temporal and spatial homogeneity of the SMF were measured with a gauss meter (model 5180, Cole-Parmer, IL, USA) and varied by less than 1 % and 10 %, respectively. The side stream was run by a peristaltic pump at 200 ml/min, with 1 % v/v of the reactor volume exposed at any time. As a control the same set-up was used with the electromagnet shut off. A 15 % v/v inoculum from axenic flasks was used to give an initial cell concentration between 5 and 15 x 10 ⁶ cells/ml.

Samples were removed aseptically for biomass determination by measuring turbidity at 750 nm, which correlated well with gravimetrically measured biomass for samples exposed to the same SMF (R ² =0.99), in a UV- VIS spectrophotometer (DU 520, Beckman-Coulter, CA, USA). Biomass was also estimated in a haemocytometer (Brightline, Hausser Scientific, PA, USA), using 20 0.0025 mm ² squares, which correlated well with gravimetric measurements at the same SMF (R ² =0.98). The average biomass between the two methods is reported. Final biomass concentration was measured gravimetrically after washing with DDIW and lyophilizing.

The growth rate, μ, was calculated daily using the measured biomass concentration, C

ln(C _¾m2 ) -ln(C _¾ml )

f2 ^{~ f} l (1 ) The daily production rate, P, was calculated from the following equation:

From Figure 3 it can be seen that cultures exposed to 10 mT static magnetic field grew significantly faster and to higher biomass concentrations than cultures exposed only to Earth's magnetic field. Final biomass concentration and maximum growth rate were both increased significantly by about 2.5 times; the average daily yield of biomass was increased by about a factor of 3 times; and the maximum biomass production, which is equal to the biomass production that could be achieved in continuous or semi-continuous operation, was increased by more than 4 times (Figure 4).

Example 3. Growth In Closed Flat Plate Photobioreactor With And Without 10 mT Static Magnetic Field Exposure

Haematococcus pluvialis was also grown in a planar photobioreactor, both with 10 mT magnetic field exposure and with no magnetic field applied. These reactors had a volume of about 3 L, a light-path length of about 1 inch, and a height and width of about 40 cm. Air was provided by a stainless-steel tube at the bottom of the reactor with 1 mm diameter holes every 2 cm. A schematic of these reactors is shown in Figure 5. These reactors could be scaled up by orders of magnitude in terms of volume by increasing the height or length of the units. Further many reactors could be operated in parallel to allow for even larger volumes (see Figure 5).

The growth conditions and magnetic field exposure were identical to the conditions in the raceway pond with the following exceptions.

1 . Light level was higher at about 800 (600-900) micromoles of

photosynthetically active radiation per square meter per second, provided by high pressure sodium lamps. 2. Mixing was provided solely by bubbling of air, preferably at a rate of about 1 volume/volume per minute (between 0.4 and 2.5 volumes/volume per minute). 3. Growth media had triple the nitrogen source to allow for a higher biomass density to be reached.

4. The cultures were grown in a non-sterile environment, but in the photobioreactor so that the cultures remained axenic for the entire growth period.

Cell biomass was determined by exactly the same methods described in Example 2.

From Figure 6 it can be seen that cultures exposed to 10 mT static magnetic field grew significantly faster than cultures exposed only to Earth's magnetic field. Final biomass concentration and maximum growth rate were increased only slightly by less than 1 5 %; but the average daily yield of biomass was increased significantly by about a factor of 2; and the maximum biomass production, which is equal to the biomass production that could be achieved in continuous or semi-continuous operation, was increased by almost 3 fold (Figure 7).

Example 4. Biochemical Analysis

Biochemical composition was determined using biomass harvested at the end of the exponential growth phase. Ash content was determined gravimetrically by burning oven-dried samples at 550 °C for 4 hours.

Carbohydrates were extracted by sonication of lyophilized samples for 5 minutes at 60 W using a Misonix XL-2000 sonication probe (NY, USA) in 1 M acetic acid [Mecozzi M, Dragone P, Amici M, Peitrantonio E. 2000.

Ultrasound assisted extraction and determination of the carbohydrate fraction in marine sediments. Organic Geochemistry, volume 31 , pages 1797-1803]. The supernatant was subjected to the phenol sulphuric acid method, using glucose as a standard.

Proteins were extracted by sonication for 3 minutes at 60 W in 1 % wlv sodium dodecyl sulphate [Meijer EA, Wijfels RH. 1998. Development of a fast, reproducible and effective method for extraction and quantification of proteins of micro-algae. Biotechnol Tech 12: 353-358.]. The supernatant was diluted 10 times and protein quantified by the Bradford method with bovine serum albumin as standard (Bio-rad, CA, USA).

Lipid content was determination gravimetrically by sonication of the lyophilized biomass for 3 min at 60 W in 2:1 v:v chloroform/methanol then Soxhlet extraction [Folch J, Lees M, Stanley GHS. 1957. A simple method for the isolation and purification of total lipides from animal tissues. Journal of Biological' Chemistry, volume 226, pages 497-509].

Pigments were extracted by sonication in 80 % v/v aqueous acetone. Chlorophyll a, chlorophyll b, and total carotenoid concentration were determined by UV-VIS spectrometry from absorbance at 663, 645, and 480 nm [Amon Dl. 1949. Copper enzymes in isolated chloroplasts, polyphenol oxidase in Beta vulgaris L. Plant Physiology, volume 24, pages1 -15.; Kirk JTO, Allen RL. Dependence of chloroplast pigment synthesis on protein synthesis: Effect of actidione. Biochemical and Biophysical Research Communications, volume 21 , pages 523-530].

Astaxanthin content was determined for biomass from both Examples 2 and 3 harvested at the end of the exponential growth phase by using

Lababpour and Lee's first-order derivative Ultraviolet-Visible

Spectrophotometry approach (Lababpour, A. & Lee, C. (2006) Simultaneous Measurement of Chlorophyll and Astaxanthin in Haematococcus pluvialis Cells by First Order Derivative Ultraviolet-Visible

Spectrophotometry. Journal of Bioscience and Bioengineering, volume 101 , pages 106-1 10). Similar procedures to the pigment extraction were performed for astaxanthin except with 100% acetone. Spectrophotometry readings were taken from 400nm to 750nm. The first derivative of absorbance at 451 nm, the chlorophyll absorbance maximum, was used to calculate astaxanthin content.

As shown in Figure 8 astaxanthin content was increased by about 70 % by 10 mT static magnetic field treatment, up to 4.9 mg astaxanthin per gram of algal biomass (dry weight). Total chlorophyll concentration and the concentration of other carotenoids in the biomass were both increased by more than about 30 % in 10 mT treated cells harvested at the end of the exponential growth phase. Carbohydrate content was also significantly increased by 10 mT treatment. Example 5. Total Astaxanthin Production For Example 2 And 3

Astaxanthin production rates in Examples 2 and 3 were calculated by multiplying the astaxanthin content by the biomass production rates. Then astaxanthin production rates are compared between 10 mT and non- magnetized cultures from the same example (Figure 9).

As shown in Figure 9 the total yield of astaxanthin was significantly increased by 10 mT static magnetic field treatment under all conditions investigated. For the raceway pond operating in either batch or continuous mode astaxanthin production could be increased by about 5-6 times by 10 mT treatment. For the closed planar photobioreactor astaxanthin production in batch or continuous mode could be increased by about 3-4 times with 10 mT treatment.

Example 6. Application Of Technology To Other Species Of Microalgae Chlorella kessleri was grown exactly as Haematococcus pluvialis ;was grown in Example 2, except that BG-1 1 growth media was used instead of Bold's Basal Media. Biochemical analysis was performed on C. kessleri as detailed in Example 4.

Results are presented in Figures 10, 11 , and 12, it can be seen from Figures 10 and 11 that the effect on growth rate, biomass production, and final biomass was similar to the effect on Haematococcus pluvialis. In fact the increase in growth rate with 10 mT treatment was approximately 100 %.

From Figure 12 it can be seen that biomass composition was changed in Chlorella. Both. Chlorella andHaernatococcus had an increase in total chlorophyll and carbohydrate content with 10 mT static magnetic field treatment, although the other changes were different between the two species. The useful component of proteins in Chlorella was also increased, although protein content was decreased in Haematococcus.

Because of these new examples in this disclosure and because all green algae have very similar photosynthetic apparatuses it is contemplated that the application of static magnetic fields during the microorganism growth phase to increase the rate of biomass production could apply to green algae in general, and further, to cyanobacteria and diatoms, as they also use a very similar photosynthetic apparatus. Very surprisingly, it was observed that flowing the microorganisms from the bioreactor through the magnetic field of the magnet, and then returning the microorganisms through the flow system outside of the magnetic field back into the bioreactor induced a memory effect in the microorganism exposed to the magnetic field that lasted several generations of the exposed microorganisms, so that constant exposure is not required, thus allowing for a relatively simple approach to process scale up with considerable economic benefits.

The specific embodiments described above have been shown by way of example, and it should be understood that these embodiments may be susceptible to various modifications and alternative forms. It should be further understood that the claims are not intended to be limited to the particular forms disclosed, but rather to cover all modifications, equivalents, and alternatives falling within the spirit and scope of this disclosure.