System and method for producing phycocyanin

Abstract

The invention discloses microorganism cell culture conditions that result in increased cellular and media concentrations of a biological pigment. The invention has applications in use as a natural food coloring, as antioxidants in the food supplement industries, in the nutraceutical, pharmaceutical, and cosmeceutical industries, and a non-toxic ink. The method results in pigment that is relatively easy to separate from the microorganism culture.

Claims

1. A system for producing increased levels of phycocyanin, the system comprising a vessel and a lamp, wherein the lamp provides a source of light, wherein the light consists of electromagnetic radiation consisting of a wavelength of between 670 and 690 nm, and wherein the vessel further comprises a cyanobacteria in a growth medium, and wherein the cyanobacteria synthesizes increased levels of phycocyanin compared to that of cyanobacteria separately cultured in the presence of white light.

2. The system of claim 1, wherein the cyanobacteria is Arthrospira or Spirulina.

3. The system of claim 1, wherein the electromagnetic radiation comprises a maximum wavelength emission of 680 nm.

4. The system of claim 1, wherein the increased levels of phycocyanin are at least 4.5-fold greater than levels of synthesized phycocyanin in the same cyanobacteria separately cultured in the presence of white light.

5. The system of claim 1, wherein the vessel is selected from the group consisting of a tank, a bioreactor, a pond, and an open raceway.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Schematic of the energy conversion process in photosynthesis. P680 and P700 represent the reaction centre Ch1 a of Photosystem II and Photosystem I respectively. Phycocyanin (PC) (610-620 nm) is present in a complex with Phycoerythrin (PE) (540-570 nm) and Allophycocyanin (APC) for the phycobilisome light harvesting apparatus which absorb specific wavelengths of light for use in photosynthesis.

(2) FIG. 2. The Infors stirred-tank photobioreactor shown with red (left) and white (bottom right) LED jackets and closed jacket during normal operation (top right). Technical data: Infors cell culture system impeller (1 pitched blade impeller, 3 blades), flow direction: upwards, Angle: 45 degrees, Dimensions: A diameter, B height, C length across blade: A=65 mm, B=52 mm, C=72 mm Vessel: Total volume 3.6 liters, Inner diameter: 115 mm, Height 370 mm.

(3) FIG. 3. Typical emission spectra comparing typical white LED, typical red LED (typically around 620-640 nm) and EPITEX 680 nm LED light. Right shows emission spectra of EPITEX 680 nm LED emitting narrow intense light with an optimum emission wavelength of 680 nm.

(4) FIG. 4. Growth curves for separate batch runs of A. platensis culture (error bars represent standard error of the mean) with table showing average growth rate of A. platensis under 680 nm LEDs compared to typical white LEDs, with no significant difference in growth under the two light conditions.

(5) FIG. 5. Absorbance spectra of Phycocyanin extracts from A. platensis, normalized at 678 nm, cultured under 680 nm LEDs compared to typical while LEDs. Light dashed lines represent error (s.e.m., standard error of the mean). A larger absorption peak representing Phycocyanin can be seen at around 620 nm in the extract from A. platensis cultured under 680 nm LEDs.

(6) FIG. 6. Average Phycocyanin yield (mg/g) from A. platensis cultured under 680 nm LEDs compared to typical white LEDs. Error bars represent standard error of the mean. A large significant increase in Phycocyanin levels can be seen in A. platensis through culturing under 680 nm LEDs.

(7) FIG. 7. Mass spectra for A. platensis cultures cultured under 680 nm LEDs compared to typical white LEDs show differences in abundant proteins under the two light conditions.

(8) FIG. 8. Left shows A. platensis culture (top) and extract (bottom) from culturing under typical white LED. Right shows A. platensis culture (top) and extract (bottom) from culturing under 680 nm LED.

(9) FIG. 9. Average Phycocyanin yield (mg/g) for separate batches of A. platensis, inoculated from the previous batch, cultured under 680 nm LEDs shows an increasing yield of Phycocyanin with each subsequent run, likely indicating A. platensis is undergoing continual adaptation to enable utilization of 680 nm light more efficiently in photosynthesis.

(10) FIG. 10. Growth curves for A. platensis batch cultures under 680 nm light. Dashed line shows A. platensis culture undergoing acclimatization for utilization of 680 nm light. A lag phase where acclimatization is occuring, is present up to day 14.

(11) FIG. 11. Top left: Flocculation of higher Phycocyanin-yielding A. platensis cultured under 680 nm LEDs. Microscope images show presence of crystals on A. platensis trichomes in flocculated cultures (bottom), absent in non-flocculating culture, indicating increase in extracellular polysaccharide, possibly as a stress response.

GENERAL DISCLOSURES

(12) The embodiments disclosed in this document are illustrative and exemplary and are not meant to limit the invention. Other embodiments can be utilized and structural changes can be made without departing from the scope of the claims of the present invention.

(13) As used herein and in the appended claims, the singular forms a, an, and the include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to a particle includes a plurality of such particles, and a reference to a surface is a reference to one or more surfaces and equivalents thereof, and so forth.

(14) The symbol when used in the context of the wavelength of electromagnetic radiation, means greater than or equal to; the term 640 nm means electromagnetic radiation having a wavelength of at least or greater than 640 nm, for example, 640 or 641 nm.

(15) The term about when used in the context of electromagnetic radiation wavelength means a wavelength of within 2 nm of the wavelength as written; therefore the term a wavelength of about 640 nm means the electromagnetic wavelength is between 638 nm and 642 nm.

DETAILED DESCRIPTION OF THE INVENTION

(16) The invention herein disclosed provides for devices and methods that may be used for the synthesis of phycocyanins. The method results in a greater than 4.5-fold .sub.[CMB1] in phycocyanin levels, a clear improvement over the prior art .sub.[CMB2]. The devices herein disclosed may be used in many applications, including, but not limited to, use as a natural food colouring, as an antioxidant in the food supplement industries, in the nutraceutical, pharmaceutical, and cosmeceutical industries, and as a non-toxic ink. The invention provides improved methods for the synthesis and commercial production of phycocyanins.

(17) In an exemplary embodiment, the method includes providing a microorganism capable of synthesizing phycocyanins, providing a suitable culture and growth medium, illuminating the microorganism in culture with red and/or near-infrared light, and in the alternative, illuminating the microorganism in culture with red and/or near-infrared monochromatic light. In an alternative embodiment, the method also provides illuminating the microorganism in culture with white light.

(18) In one embodiment the red light consists of electromagnetic radiation having wavelengths between about 640 nm and about 720 nm. In another embodiment the red light consists of electromagnetic radiation having wavelengths between 640 nm and 1000 nm. In another embodiment the red light consists of electromagnetic radiation having a maximum wavelength emission of 680 nm. In an alternative embodiment the red light consists of electromagnetic radiation having a wavelength of 678 nm. In another alternative embodiment the red light consists of electromagnetic radiation having a wavelength of 682 nm. In another alternative embodiment the red light consists of electromagnetic radiation having a wavelength of 690 nm. In another alternative embodiment the red light consists of electromagnetic radiation having a wavelength of 670 nm. In an alternative embodiment the red light consists of electromagnetic radiation having a mean wavelength of 680 nm, wherein the wavelength is within a 95% confidence interval of 640-720 nm. In one embodiment the white light consists of electromagnetic radiation having wavelengths between 350 nm and 760 nm.

(19) Culturing under red 680 nm LED light compared to white was shown to increase PC production in A. platensis by an average of 5-fold and these effects could be seen visually in the cultures. Mass spectral analysis has shown some major differences and changes on the protein level through culturing under the two different light sources. .sub.[CMB3] No significant difference was seen in growth rate under the two light sources .sub.[CMB4].

(20) The invention will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and not as limitations.

EXAMPLES

Example I: Batch Cultures

(21) F/2 sterile medium (CCAP [Culture Collection of Algae and Protozoa] recipe) supplemented with 2.5 g/l NaNO.sub.3 (pH 8) was inoculated under aseptic conditions at 20% (v/v) with Arthrospira platensis (CCMP [Culture Collection of Marine Phytoplankton] 1295/Bigelow Laboratory US) (OD 0.11-0.12) in logarithmic growth phase. A stirred tank photobioreactor (STPBR) (Infors Labfors 4 benchtop modified bioreactor) with either white (Lumitronix Barre LED High-Power SMD 600 mm, 12 V) or 680 nm Red LEDs (FIGS. 2 and 3) was operated with 2.75 l of culture at 30 C. and 45 mol.sup.1 m.sup.2 light intensity with 18:6 light:dark cycle and impeller speed 200 rpm with natural compressed air (0.03% CO.sub.2) supplied at 0.08 LPM (VVM (volume of air per volume of culture per minute) 0.03 liters air per liters medium per minute, LPM) through a gauzed ring sparger. pH and dissolved oxygen was recorded online in 10 minute periods (Mettler Toledo probes). 8 ml samples were taken aseptically on days 1 (inoculation), 3, 6, 7, 10, 13, and 14 for analysis.

Example II: Growth Measurement

(22) Optical density (OD) was used alongside chlorophyll autofluorescence (CF) and direct cell counts as a proxy for growth. OD was measured in triplicate at 750 nm Griffiths et al. (2011) [14] using a Cary 100 UV/Vis Spectrophotometer (Varian) corrected with F/2 medium. CF was analysed in three triplicate 300 l samples divided into individual wells of a black 96 well plate. Samples were excited at 430 nm and emission measured at 690 nm using a FLUOstar OPTIMA fluorescence plate reader (BMG LABTECH). Readings were taken against blank samples of F/2 medium and the average values in arbitrary fluorescence units used for statistical analysis. Cell counts were performed using a Sedgewick rafter counting cell and using Leitz Dialux 20 light microscope. Triplicate 10 random sample counts were taken for 1 l of culture.

Example III: Morphological Assessment

(23) The total length and width of the spirals of 20 cyanobacteria were measured to assess any changes in the morphological features of the trichomes. Images were taken using Leitz Dialux 20 light microscope and EasyGrab software with analysis performed using Image J. Image size was calibrated using graticules at 630 pixels mm.sup.1.

Example IV: Phycocyanin Analysis

(24) PC extraction was based on the method by Zhang and Chen (1999) [15]. 5 mL samples were harvested by centrifugation at 3000 g/10 minutes (Sigma 3K18C centrifuge) in pre-weighed glass tubes. Cells were washed once in deionized water and the wet biomass weighed. The pellet was then resuspended in 3 mL 0.05 M sodium phosphate buffer (pH7). Cells were disrupted by a freeze/thaw cycle (20 C.) over 1 hour and sonicated for 3 minutes at 6 microns amplitude (Soniprep 150, MSE). Samples were then centrifuged at 10,000 g, 30 minutes (Sigma 1-15 microcentrifuge) and the absorbance of the supernatant scanned over 200-800 nm by spectrophotometer (Cary 100 UV-Vis spectrophotometer, Varian) using a quartz cuvette. Sodium phosphate buffer (0.05 M) was used as a blank and the PC concentration and purity calculated using the method by Bennet and Bogorad (1973) [10] (Equation 1) and Boussiba and Richmond (1979) [16] (Equation 2) respectively. Extraction yield was calculated as below in Equation 3. PC concentration was analysed at day 14 (or when growth reached OD 0.33) as three replicates.

(25) 1. PC (mg/mL)=(A620-0.474 (A655))/5.34.

(26) 2. PC purity=A620/A280.

(27) 3. Extraction yield (mg PC/g biomass)=(PC concentration*volume of solvent (mL)/wet biomass (g)

Example V: Mass Spectrometry (MS) Analysis

(28) Matrix Preparation:

(29) 20 mg alpha-Cyano-4-hydroxycinnamic acid (HCCA) (Brucker Daltonics) was mixed with 1 ml 50% acentonitrile: 2.5% TFA solution and saturated by 30 minutes incubation at 25 C. in an ultrasonic water bath (Grant instruments, Cambridge), vortexed at 15 minutes. Matrix was centrifuged (14,000 g, 1 minutes) (Sigma 1-15K microcentrifuge) and 50 l aliquots prepared fresh for use.

(30) Sample Preparation:

(31) 1 ml samples were centrifuged (14,000 g, 5 minutes) (Sigma 1-15K microcentrifuge) and the pellet washed twice in fresh deionized water (fdw) and stored frozen at 80 C. Pellets were thawed on ice and resuspended in 50 l fdw before spotting. Samples were mixed 1:1 with HCCA matrix and 4 l duplicate samples spotted onto a steel target plate (MTP 384 target plate ground steel, Brucker) along with 1 l bacterial standard (Brucker) layered with 1 l HCCA matrix as a calibrant. Samples then underwent MS analysis (Bruker ultraflex II MALDI-TOF). Spectra were analysed using flexAnalysis software package (Bruker).

Example VI: Population Analysis

(32) Samples were frozen in 15% sterile Glycerol and frozen at 80 C. for population analysis (Dr Andrew Free and Rocky Kindt, Edinburgh University).

Example VII: Statistical Analysis

(33) Data analysis was performed using Microsoft Excel 2007 and Graphpad Prism 5.

Example VIII: Results: Growth

(34) No significant difference in growth of cultures was observed under red 680 nm compared to white LED light (FIG. 4). Note the large acclimatization lag period in batch Red 2 (FIG. 4). The culture required a period to acclimatize to be able to utilize the red 680 nm light in photosynthesis (from observation), and this acclimatization was reversible.

Example IX: Results: PC Analysis

(35) Phycocyanin absorbs at 620 nm. The presence of PC in the extracts of red 680 nm LED batches compared to white LED was much higher (FIGS. 5 & 6). An interesting blue-shift was observed in the second Chlorophyll a peak around 670-680 nm where the peak red 680 nm extract absorption is 677-678 nm and the peak white extract absorption is 673-674 (FIG. 6).

(36) PC concentration was increased 5-fold on average (at least nine samples) through culturing under red 680 nm compared to white LED light and there was a slightly higher PC purity under red 680 nm LED light compared to white (FIG. 7 table). Visual colour differences were observed in the culture most likely resulting from increased PC content of the cells cultured under red 680 nm light (FIG. 7). In another experiment, we found that the PC concentration increased more than 10-fold (data not shown).

Example X: Results: MS Analysis

(37) Whole cell MALDI spectra showed differences in abundant proteins from culturing under red 680 nm compared to white LED light (FIG. 8).

Example XI: Results: Leaching Differences

(38) When discarding the samples prepared for MS analysis, a high concentration of PC had leached into solution in the red 680 nm samples (FIG. 9). By eye the colour difference in leached PC was substantially higher in the red 680 nm culture compared to white LED light, looking much greater than a 5-fold increase. This indicated a possible difference in the PC leaching characteristics in the red 680 nm culture, which may be beneficial to downstream processing (DSP). Culturing under 680 nm light may increase the leaching of PC from the biomass. This is clearly an unexpectedly superior result that could not have been predicted by one of skill in the art.

Example XII: Results: Culture Aggregation

(39) Culturing under 680 nm light may also increase aggregation of the culture, with benefits to DSP. Aggregation may be a result of increased production of extracellular polysaccharide (EPS) as a stress response. This is clearly an unexpectedly superior result that could not have been predicted by one skilled in the art. Those skilled in the art will appreciate that various adaptations and modifications of the just-described embodiments can be configured without departing from the scope and spirit of the invention. Other suitable techniques and methods known in the art can be applied in numerous specific modalities by one skilled in the art and in light of the description of the present invention described herein. Therefore, it is to be understood that the invention can be practiced other than as specifically described herein. The above description is intended to be illustrative, and not restrictive. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the invention should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

REFERENCES

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