Use of photosynthetic organisms for metabolite production

Abstract

A method of increasing the heme content of heme oxygenase containing photosynthetic organisms by cultivating the photosynthetic organism in the presence of one or more metalloporphyrins (MPPs).

Description

Use of photosynthetic organisms for metabolite production TECHNICAL FIELD

[0001] The present disclosure relates to the use of the heme oxygenase containing photosynthetic organisms of the phyla Cyanophyta, Rhodophyta, or Cryptophyta, for the production of heme. BACKGROUND ART

[0002] Heme (heme b or protoheme) is generally the most versatile and plentiful heme in living cell. It is an iron-containing tetrapyrrole molecule, ubiquitously distributed in nature and involved in various biological processes like respiration, oxygen metabolism, oxygen transfer, photosynthetic electron transportation, oxidative stress responses across all domains of life. Heme is the major source of functional iron in humans and animals and is required for carrying out critical intracellular activities such as DNA synthesis and cellular respiration.

[0003] Iron deficiency results in a range of acute and chronic human health consequences such as iron deficiency anaemia, fatigue, fainting, heart palpitations, impaired learning and concentration. Iron deficiency anaemia is currently the most common mineral deficiency throughout the world and affects approximately 25% of the global human population. In Western countries, iron deficiency is most commonly seen in people who follow restricted energy diets to lose weight and in those who choose a vegetarian or vegan diet. However, it is in developing economies where the major need lies.

[0004] Dietary sources of iron come in two forms: heme and non-heme iron. Heme iron is naturally abundant in red meat, chicken, seafood and fish and in humans, heme can constitute 75 % of the average person’s iron intake. It is also the most bio-available form of iron known and is usually diet derived from red meat. Non-heme iron is generally found in plant-based foods like vegetables, fruits, nuts and beans. In meat, heme iron is in the ferrous form, but in non-heme iron sources it occurs in the oxidized Fe3+form. As meat is a major source of heme iron, people who cannot access sufficient heme from animal sources in their daily meals are at increased risk of iron deficiency. However, meat production is associated with environmental concerns relating to unacceptable levels of global greenhouse gas emissions, excessive water consumption, and land degradation. Current Fe supplements have significant side effects and suffer from low bioavailability.

[0005] There have been previous attempts to produce animal-free heme. Recombinant production of haemoglobin has attempted in yeast and bacteria. However, this is a technically difficult approach due to the tight regulation of the heme biosynthesis pathway and the downstream processing / degradation of heme into pigments.

[0006] New sources of bioavailable heme iron are required to help combat this ubiquitous mineral deficiency. There is a need for effective, bioavailable heme iron supplementation for people who have iron deficiency anaemia, who are vegetarian or vegan, or who are trying to reduce their red meat consumption.

[0007] There is a need to find new sources of heme iron; or at least the provision of heme iron sources to compliment the previously known sources of nutritionally available iron. The present disclosure seeks to provide an improved or alternative method for the production of nutritionally available iron.

[0008] The previous discussion of the background art is intended to facilitate an understanding of the present disclosure only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application. SUMMARY OF INVENTION

[0009] The present disclosure provides a method of increasing the heme content of heme oxygenase containing photosynthetic organisms, the method comprising the step of: i) cultivating the heme oxygenase containing photosynthetic organism in the presence of one or more metalloporphyrins.

[0010] Optionally, the metal element of the metalloporphyrin (MPP) is chosen from: zinc, tin or chromium.

[0011] Optionally, the metalloporphyrins are introduced to the cultivation medium at a rate of from 0.01 µmole of MPP.g-1of biomass to 40 µmole MPP.g-1of biomass.

[0012] Optionally, the metalloporphyrins are introduced to the cultivation medium for at least 24 hours.

[0013] Optionally, the heme oxygenase containing photosynthetic organism is chosen from: Cyanophyta, Rhodophyta, or Cryptophyta.

[0014] The method of increasing the heme content of heme oxygenase containing photosynthetic organisms using cultivation in the presence of metalloporphyrins may be accompanied by a further method of increasing heme content such as carrying the cultivation out under specific light cycles, inducing oxidative stress and / or exposing the heme oxygenase containing photosynthetic organism to ultraviolet radiation to enhance the production or retention of heme.

[0015] Optionally, the cultivating of a heme oxygenase containing photosynthetic organism to increase heme content is carried out using a paddle wheel or jet driven raceway pond cultivation system.

[0016] The present disclosure further provides a composition comprising heme, wherein the heme is generated by a heme oxygenase containing photosynthetic organism. Optionally, the heme composition is produced by the following steps: i) cultivating the heme oxygenase containing photosynthetic organism in the presence of one or more metalloporphyrins. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which: Figure 1 is a schematic of the general biosynthetic pathway of heme in Cyanophyta. The top portion of the pathway shows the synthesis of ALA precursor. The middle portion of the biosynthesis pathway is common to all organisms. The lower section represents the fate of protoporphyrin IX in plants and algae. Figure 2 is a schematic of the metabolism of heme to phycobiliproteins in Cyanophyta. Figure 3 is a graph of the heme content of A. platensis in response to different zinc protoporphyrin IX (ZnPP) concentrations at different time intervals. (* indicates outlier). Figure 4 is a graph of the heme content (relative to control) of Arthrospira at 0.4 μmole ZnPP g-1increase over time, allowing prediction of maximum heme increase (rate = Δ Heme rise max) and optimal harvesting time (Δ Hour opt). The equation used for curve fitting is y = a(1-e-bx) where y = Δ Heme enhanced and x = Time. Figure 5 is a graph of the growth of A. platensis in response to different concentrations of ZnPP. Figure 6 is a graph of the effective quantum yield (Fq’ / Fm’) of A. platensis after receiving various concentration of ZnPP. Figure 7 is a graph showing the relationship between Δ heme (A) and Δ phycocyanin (B) when ZnPP doses applied to inhibit HO enzyme activity.DESCRIPTION OF INVENTION Detailed Description of the Invention

[0018] Some photosynthetic organisms could be utilized as an alternative source of heme as the molecule is already found in the biosynthetic pathway for accessory pigment production via a precursor, protoporphyrin IX, that is also found part of chlorophyll biosynthesis. Generally, in photosynthetic organisms, the heme branch of the pathway receives a lower quantity of protoporphyrin IX than the chlorophyll branch. However, the relative distribution of protoporphyrin IX into the heme and chlorophyll branches is more likely equal in Cyanophyta because of the high production level of light-harvesting phycobiliproteins, e.g. biliverdin IX and phycocyanin, that are derived from heme. The conversion of heme to these light harvesting proteins is controlled by the enzyme heme oxygenase (HO).

[0019] In the biosynthetic pathway of Cyanophyta, Cryptophyta and algae (including red and green algae), there are two areas that are particularly critical in terms of manipulating heme production. For green algae, it is the branching of protoporphyrin IX between the insertion of Fe to form heme and Mg to ultimately form chlorophyll. In Cyanophyta, Cryptophyta and Rhodophyta (red algae), it is the conversion of heme into biliverdin by the action of the heme oxygenase enzyme.

[0020] Many physical and chemical factors (e.g. light, temperature, pH, salinity, nutrients, inorganic carbon, CO2, and O2), are known to have a significant effect on the overall growth and accumulation of bioproducts in Cyanophyta, Rhodophyta, or Cryptophyta. These factors have been manipulated to increase the bioaccumulation of several natural products including proteins, carbohydrates, lipids, pigments, and vitamin content.

[0021] In Cyanophyta, Rhodophyta, or Cryptophyta, the biosynthesis of heme is a step in the synthesis of the water-soluble, light-harvesting, protein-associated pigments known as phycobiliproteins. The synthesis of phycobiliproteins from heme relies on heme oxygenase (HO).

[0022] Without being held to any particular theory, it is believed that blocking the action of HO in microalgae known to accumulate high levels of phycobiliprotein pigments should lead to an accumulation of heme in these cells. Metalloporphyrins (MPPs) are competitive inhibitors of the HO enzyme. The present disclosure proposes that application of MPPs will lead to a reduction in the activity of HO, and therefore allow increased retention and accumulation of heme.

[0023] Porphyrins are a class of tetrapyrrole macrocycles with a skeleton of 16-atom rings containing four nitrogen atoms. The porphyrin free base has 11 double bounds and can easily be transformed into an MPP by replacing the inner two pyrrole protons with a metal ion.Method of increasing heme content

[0024] The present disclosure therefore provides a method of increasing the heme content of heme oxygenase containing photosynthetic organisms, the method comprising the step of: i) cultivating the heme oxygenase containing photosynthetic organism in the presence of one or more metalloporphyrins.

[0025] The increased heme content of the heme oxygenase containing photosynthetic organism may be due to increased retention and accumulation of heme. However, the increased heme content may also be due to increased heme production. Metalloporphyrins

[0026] In one aspect, the metalloporphyrin of the present disclosure has a stronger affinity for the HO enzyme than ‘native’ heme, resulting in competitive inhibition of the enzyme. Optionally, the metalloporphyrin of the present disclosure is one that contains a metal element from the fourth or fifth periods of the Periodic Table. For example, the metal element may be chosen from the following: fourth period - scandium (Sc), titanium (Ti), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), and zinc (Zn); fifth period - yttrium (Y), zirconium (Zr), niobium (Nb), molybdenum (Mo), technetium (Tc), ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), and cadmium (Cd). In one example, the metal element of the metalloporphyrin is chosen from: zinc, tin or chromium. Optionally, the metal element of the metalloporphyrin is not iron (Fe).

[0027] Optionally, the metalloporphyrin is chosen from the list comprising: zinc protoporphyrin (ZnPPIX), tin protoporphyrin (SnPPIX), chromium protoporphyrin (CrPPIX), zinc mesoporphyrin (ZnMPIX), mesoporphyrin (SnMPIX), chromium mesoporphyrin (CrMPIX) and zinc deuteroporphyrin IX bis glycol (ZnBG) and synthetic analogues of the natural metalloporphyrin ferroporphyrin (heme). In one example, the metalloporphyrin for use in the present disclosure is zinc protoporphyrin (ZnPPIX). Optionally, the metalloporphyrin is not haemoglobin or myoglobin.

[0028] Metalloporphyrins (MPPs) are structural homologues of heme and potent competitors of HO, the critical enzyme in the catabolism of heme. Their action is due to the catalytic site of HO recognizing metalloporphyrins with central metal ions other than iron. HO favours some of these metalloporphyrins over heme as a substrate, sometimes by a large factor. If a non-heme MPP binds the HO, HO cannot then bind heme and convert the heme to biliverdin IXa (the next step in converting heme to phytocyanin). As the heme is not converted to biliverdin IXa, it is able to build up in the cell. The potency of metalloporphyrins is influenced by the central metal cation and the nature of the side chains. Traditionally, tin porphyrins have been seen as more potent than zinc or cobalt porphyrins. With side chains composed of ethyl groups, mesoporphyrins are traditionallyseen as more potent and stable than the other forms. They are not oxidatively degraded because they have no oxygen-binding capacity.

[0029] Optionally, the metalloporphyrins are introduced to the cultivation medium at a rate of from 0.01 µmole of MPP.g-1of biomass to 40 µmole MPP.g-1of biomass. The rate may be 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 35, or 40 µmole MPP.g-1of biomass.

[0030] The rate may range from a lower value selected from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 35, or 39 µmole MPP.g-1of biomass to a higher value selected from 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 35, or 40 µmole MPP.g-1of biomass. The rate may be from 0.01 to 40 µmole MPP.g-1of biomass, from 0.01 to 20 µmole MPP.g-1of biomass, from 0.01 to 10 µmole MPP.g-1of biomass or from 0.04 to 4 µmole MPP.g-1of biomass.

[0031] For example, the metalloporphyrin may be zinc metalloporphyrin (ZnPP) and may be introduced to the cultivation medium at a rate of from 0.01 µmole of ZnPP.g-1of biomass to 40 µmole ZnPP.g-1of biomass. The rate may be 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 35, or 40 µmole ZnPP.g-1of biomass.

[0032] The rate may range from a lower value selected from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 35, or 39 µmole ZnPP.g-1of biomass to a higher value selected from 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 35, or 40 µmole ZnPP.g-1of biomass. The rate may be from 0.01 to 40 µmole ZnPP.g-1of biomass, from 0.01 to 20 µmole MPP.g-1of biomass, from 0.01 to 10 µmole ZnPP.g-1of biomass.

[0033] Optionally, the metalloporphyrins are introduced to the cultivation medium for at least 1 hour. For example, the metalloporphyrins may be present in the cultivation medium for at least 1h, 2h, 3h, 4h, 5h, 6h, 10h, 12h, 24 h, or 36 h. The metalloporphyrins may be present in the cultivation medium for 1 day, 2 days, 3 days, 4 days, 5 days 6 days, 7 days or more.

[0034] The metalloporphyrins may be present in the cultivation medium for the entire time the Cyanophyta, Rhodophyta, or Cryptophyta is being cultivated. Alternatively, the metalloporphyrins may be introduced into the medium after the heme oxygenase containing photosynthetic organisms has been allowed to grow to a specified density. The heme oxygenase containing photosynthetic organisms may be allowed to grow to a specified density in the absence of an MPP, then the organisms may be harvested from the cultivation medium on a semi-continuousbasis (or batch basis) and the harvested organisms may then be exposed to MPP for a period of time to induce heme production. Alternatively, the metalloporphyrins may be slowly removed from the cultivation medium by controlled replacement of spent medium containing the metalloporphyrins with fresh medium that is metalloporphyrin-free, or slowly added to the cultivation medium by controlled replacement of spent metalloporphyrin-free medium with fresh metalloporphyrin containing medium.

[0035] Optionally, the metalloporphyrins are introduced to the cultivation medium at the start of the dark cycle for organism growth. MPP compounds have been reported to be potential photosensitisers because of their porphyrin structure. Heme Oxygenase Containing Photosynthetic Organism

[0036] Cyanophyta, Rhodophyta, and Cryptophyta contain heme oxygenase (HO) to convert heme to the water-soluble, light-harvesting, protein-associated pigments known as phycobiliproteins. This pathway is not available in organisms such as green algae, as they do not contain HO.

[0037] The organism for use in the present disclosure may be a heme oxygenase containing photosynthetic organism. Optionally, the heme oxygenase containing photosynthetic organism is chosen from the following: Cyanophyta, Rhodophyta, or Cryptophyta.

[0038] Optionally, the heme oxygenase containing photosynthetic organism produces at least one chlorophyl compound. For example, the heme oxygenase containing photosynthetic organism may produce chlorophyll a, chlorophyll b, chlorophyll c, or chlorophyll d, and combinations of the same. The heme oxygenase containing photosynthetic organism may produce only one chlorophyll type, or more than one. Optionally, the Cyanophyta used to produce the heme is able to perform oxygenic photosynthesis.

[0039] The heme oxygenase containing photosynthetic organism may further contain carotenoids, phycoerythrins and / or phycobilins. • Cyanophyta

[0040] Cyanophyta, also called cyanobacteria, are a phylum of Gram-negative bacteria that obtain energy via photosynthesis. Cyanophyta are variable in morphology, ranging from unicellular and filamentous to colonial forms. The cyanophytes used in the present disclosure preferably produce at least one chlorophyl compound. Optionally, the cyanophyte used to produce the heme is able to perform oxygenic photosynthesis. The cyanophytes may further contain carotenoids, phycoerythrins and / or phycobilins.

[0041] The Cyanophyta may be chosen from the following genera: Acaryochloris, Amorphonostoc, Anabaena, Anabaenopsis, Anathece, Aphanizomenon, Aphanocapsa,Aphanothece, Archaeosphaeroides, Arthrospira, Aulosira, Borzia, Calothrix, Chamaesiphon, Chlorogloea, Chlorogloeopsis, Chroococcidiopsis, Chroococcus, Coelomoron, Coelosphaerium, Cuspidothrix, Cyanobacterium, Cyanodictyon, Cyanonephron, Cyanothece, Cylindrospermopsis, Cylindrospermum, Dolichospermum, Eucapsis, Fischerella, Geitlerinema, Gloeobacter, Gloeocapsa, Gloeocapsopsis, Gloeothece, Gloeotrichia, Gomphosphaeria, Heteroleibleinia, Homoeothrix, Hydrocoryne, Jaaginema, Jakutophyton, Johannesbaptistia, Kamptonema, Komvophoron, Lemmermanniella, Leptolyngbya, Limnococcus, Limnothrix, Loefgrenia, Lyngbya, Merismopedia, Microcystis, Nodularia, Nostoc, Oscillatoria, Ozarkcollenia, Palaeolyngbya, Petalonema, Phormidium, Planktothrix, Prochlorococcus, Prochloron, Radaisia, Richelia, Rivularia, Roseofilum, Rothpletzella, Scytonema, Spirulina, Synechococcus, Synechocystis, Trichodesmium, Wollea, and Woronichinia. For example, the cyanobacteria may be from the genus Arthrospira.

[0042] Optionally, the cyanophytes of the present disclosure is chosen from the list comprising: Arthrospira platensis and Arthrospira maxima. • Rhodophyta

[0043] Rhodophyta (also known as rhodophytes or red algae), are eukaryotic algae. The Rhodophyta form a distinct group characterized by having eukaryotic cells without flagella and centrioles, chloroplasts that lack external endoplasmic reticulum and contain unstacked (stroma) thylakoids, and use phycobiliproteins as accessory pigments, which give them their red colour. Rhodophytes may be unicellular or multicellular. The Rhodophytes used in the present disclosure may produce at least one chlorophyl compound. Optionally, the Rhodophytes used to produce the heme are able to perform oxygenic photosynthesis. The Rhodophytes may further contain carotenoids, phycoerythrins and / or phycobilins.

[0044] There are about 155 genera of Rhodophyta. For example, the Rhodophyta used in the present disclosure may be chosen from the following genera: Cyanidium, Rhodella, Porphyra.

[0045] Optionally, the Rhodophyta used in the present disclosure is chosen from: Cyanidium caldarium, Rhodella violacea, Porphyra purpurea. • Cryptophyta

[0046] Cryptophyta (also known as cryptomonads) are a class of algae distinguished by the presence of characteristic extrusomes called ejectosomes, which consist of two connected spiral ribbons held under tension. If the cells are irritated either by mechanical, chemical or light stress, the ejectosomes act like flagella to propel the cell in a zig-zag course away from the disturbance. Cryptophytes are unicellular. The cryptophytes used in the present disclosure may produce at least one chlorophyl compound. Optionally, the cryptophytes used to produce the heme is ableto perform oxygenic photosynthesis. The cryptophytes may further contain carotenoids, phycoerythrins and / or phycobilins.

[0047] The cryptophyte may be chosen from the following genera: Chroomonas, Cryptomonas, Cyathomonas, Falcomonas, Geminigera, Goniomonas, Guillardia, Hemiselmis, Isoselmis, Plagioselmis, Protocryptomonas, Pseudocryptomonas, Pyrenomonas, Rhinomonas, Rhodomonas, Storeatula, and Wallaceina. Optionally, the cryptophyte is not Chilomonas, which does not contain chlorophyll. Additional methods for inducing increased heme retention

[0048] The use of metalloporphyrins to induce increase retention of heme may be accompanied by another cultivation technique used to increase heme production or retention. For example, cultivation of the heme oxygenase containing photosynthetic organism with a metalloporphyrin to retain heme may be carried out under specific light cycles, oxidative stress and / or exposure to ultraviolet radiation to enhance the production or retention of heme. Light cycling

[0049] Without being held to any theory, it is believed that the protoporphyrin IX in heme oxygenase containing photosynthetic organisms is directed towards the metabolic pathway when exposed to light but is channelled towards the heme metabolic pathway after transition from light to dark.

[0050] The ratio of ATP and ADP in the chloroplast is an essential factor that regulates the activation of the Mg-chelatase and Fe-chelatase enzymes. Due to photosynthetic reactions during the day ATP levels increase in the cell, which then activates the Mg-chelatase enzyme to utilize that energy by inserting Mg2+into protoporphyrin IX. This increased level of ATP also inhibits the Fe-chelatase enzyme and thus heme synthesis. Insertion of Fe2+into protoporphyrin IX by Fe- chelatase does not require ATP and therefore heme production is favoured in dark conditions. Furthermore, ADP has a strong inhibitory effect on Mg2+chelation. Thus, manipulating light-dark cycles to regulate ATP and ADP may be used as a switch between chlorophyll and heme synthesis.

[0051] Obviously, in photosynthetic organisms, it is important to ensure that any reduction in light does not result in detrimental growth and productivity of the culture. However, a balance of the light required for chlorophyll activity and the heme-favouring dark conditions may increase heme production by protoporphyrin IX being redistributed away from the Mg-protoporphyrin IX / chlorophyll pathway to the heme biosynthesis pathway. A combination of decreasing Mg- protoporphyrin IX production combined with blocking HO may lead to increased accumulation of heme.Induction of oxidative stress

[0052] Induction of oxidative stress may also be a tool that could be utilized in microalgal cultivation to increase heme content. It has previously been shown that increasing oxidative stress in Arabidopsis thaliana leads to inducing expression of HEMA2 and FC1 gene analogues which then upregulates heme production. Without being held to any theory, it is believed that the same application of oxidative stress can be used to increase heme product in the present case. For example, the heme oxygenase containing photosynthetic organisms may be exposed to ozone, hydrogen peroxide, or reduced O2levels, to induce oxidative stress. Exposure to ultraviolet radiation

[0053] Exposure to UV-A and UV-B radiation may increase fatty acid and pigment production in heme oxygenase containing photosynthetic organisms. The UV exposure may lead to increased accumulation of pigment as the organism produces increased pigments as a protective mechanism to absorb the radiation before it affects the cell. As these pigments may include heme on their generative pathway, increasing pigment production activity, combined with blocking HO, may lead to increased accumulation of heme.

[0054] Exposure to UV radiation may further decrease chlorophyll a and / or b production as radiation (particularly UV-B) can lead to oversaturation of the light reactions of photosynthesis. In that case, reduction of the biochemical pathway to chlorophyll production may result in protoporphyrin IX being redistributed away from the Mg-protoporphyrin IX / chlorophyll pathway to the heme biosynthesis pathway. Decreasing Mg-protoporphyrin IX production combined with blocking HO may lead to increased accumulation of heme. Cultivation methods

[0055] A number of parameters such as the type of cultivation system, and the cultivation system pH, temperature and CO2 concentration may significantly affect the growth rate and nutrient utilisation capabilities of heme oxygenase containing photosynthetic organism cultivation.

[0056] Solar radiation (light), temperature, and pH (to control the CO2 availability) are the most critical factors that affect the productivity and growth of heme oxygenase containing photosynthetic organisms. • Cultivation System

[0057] The heme oxygenase containing photosynthetic organisms of the present disclosure may be grown in a cultivation system selected from the list comprising: paddle wheel or jet driven raceway ponds, inclined thin layer ponds, or closed tubular (biocoil) photobioreactors. The skilled person would understand which system would be most appropriate for each of Cyanophyta, Rhodophyta, or Cryptophyta. For example, the main cultivation system for the cyanophyteSpirulina is raceway pond. Cyanophyta or Cryptophyta microalgae can be cultivated using open ponds when grown in liquid. Alternatively, Cyanophyta or Cryptophyta microalgae can also be grown in biofilms. Rhodophyta may preferably be grown in open ocean systems, open ponds or closed photobioreactors (plate, airlift and tubular).

[0058] The cultivation system may be an open system or a closed system. Raceway pond cultivation systems are generally open systems and inclined thin layer photobioreactor cultivation systems and tubular (biocoil) photobioreactors are generally closed systems.

[0059] The cultivation system may be an outdoor set up (with the light for heme oxygenase containing photosynthetic organism growth being provided by the sun) or an indoor setup (the light for growth being provided by electrical lighting and / or sunlight through glass panels). To be able to use the natural sun light for growth of heme oxygenase containing photosynthetic organisms and reduce the overall cost of production, the cultivation system is an outdoor cultivation system. Optionally, the outdoor set up is an open pond system (raceway ponds or inclined ponds).

[0060] The cultivation system may be a batch system, a fed-batch system (continuous or semi- continuous) or a continuous cultivation system. In a batch process, all nutrients are provided at the beginning of the cultivation, without adding any more in the subsequent culturing cycle. Additional CO2 may be provided to the batch system to increase heme oxygenase containing photosynthetic organism growth and to control pH. The entire heme oxygenase containing photosynthetic organism biomass is harvested at the end of a cultivation cycle. In a fed batch system, nutrients are added in a constant supply during cultivation. The entire heme oxygenase containing photosynthetic organism biomass is harvested at the end of a cultivation cycle. In a continuous cultivation system, nutrients are added in a constant supply during cultivation accompanied by a concomitant removal of medium such that one or more of the elements is held at a steady state. When medium is removed, heme oxygenase containing photosynthetic organism biomass is also removed in a continuous harvest process. Optionally, the cultivation system of the present disclosure is a fed-batch semicontinuous system. When operated semi- continuously, a known portion of cultivation is removed at regular time intervals based on biomass growth and nutrient removal rate and an equal volume of fresh cultivation medium is added to the cultivation.

[0061] Light delivery, distribution and utilization are critical parameters for the design of heme oxygenase containing photosynthetic organism cultivation systems due to the photosynthetic behaviour of such organisms. Utilization of photosynthetic active radiation (PAR) by heme oxygenase containing photosynthetic organisms in outdoor cultivations is affected by (a) seasonality (diurnal irradiance, variable cloudiness), (b) geographical location and latitude (solarelevation from sunrise to sunset), (c) geometry of cultivation systems design, (d) rate at which cultivation is diluted and (e) higher light scattering and diminution in turbulent flows.

[0062] A characteristic strength of a closed tubular (biocoil) photobioreactor system is that it is self-supporting, arising from the coiled nature of the structure. The coiling of a biocoil is advantageous in setting up relatively lengthy tubes in a small surface area. The configuration of a biocoil creates a large surface area to volume ratio, which may be many times higher than a paddle wheel driven raceway pond with significant improvement on light conditions. This improvement on light distribution in a biocoil is obvious from its higher volumetric productivity than the pond, especially on days with lower sunshine. The higher volumetric productivity achieved in a biocoil demonstrates that closed PBRs are less susceptible to negative effects of meteorological conditions in the austral winter season. This is because a biocoil warms up faster than an open raceway pond (due to large surface area to volume ratio) and rapidly attains optimum temperature condition for photosynthesis to begin. A biocoil system has shorter light path compared to a raceway pond, which provides higher biomass concentration. However, a biocoil may incur oxygen build-up in the coil, which inhibits heme oxygenase containing photosynthetic organism photosynthesis. Removal of oxygen build-up from closed heme oxygenase containing photosynthetic organism cultivation systems is a critical engineering challenge limiting heme oxygenase containing photosynthetic organisms’ productivity in closed PBRs.

[0063] A cultivation system that may be used in the methods of the present disclosure is an inclined thin layer pond (ITLP). Though the areal productivity achieved by an ITLP is comparable to those attained in regular raceway ponds, ITLP use may result in water and downstream processing cost savings compared to open raceway ponds.

[0064] Preferably, if the photosynthetic organism of the present disclosure is a cyanophyte (such as Spirulina), the cultivation system is an open pond system, a closed photobioreactor or a hybrid membrane photobioreactor system. • Light

[0065] Mean irradiance in heme oxygenase containing photosynthetic organism cultivation systems is a very important factor to consider in cultivation and design of efficient systems. There is a strong correlation between growth rates and mean irradiance in cultivation as it more accurately describes the availability of light to the cells through the depth of the cultivation column.

[0066] The depth of a raceway pond or thin layer inclined photobioreactor must be optimised to account for irradiance (which if provided by the sun will depend on the season and the latitude) and the environmental temperature (which may depend on the season). High irradiance and / or high temperatures increase heme oxygenase containing photosynthetic organism growth, but also increase evaporation which affects the concentration of elements such as nitrogen andphosphorus and the cultivation system salinity. As these chemicals become more concentrated, they may inhibit growth of the heme oxygenase containing photosynthetic organisms. To achieve the maximum biomass productivity and highest heme production, the best cultivation depths for paddle wheel driven raceway ponds are 15 cm to 30 cm deep (for example 20 cm to 25 cm deep), and inclined ponds are 0.5 cm top 2 cm deep (for example 1.0 to 1.5 cm deep). When grown outdoors, heme oxygenase containing photosynthetic organism cultivations of relatively average densities are challenged by supra-optimal irradiance and that approximately 90% of the incident photons could be absorbed in the first 0.01 m of the cultivation column, leaving only a small region of the cultivation in optimal irradiation conditions.

[0067] The use of open ponds (raceway and inclined) may be advantageous where the solar irradiance is high. In areas of low solar irradiance, the irradiance of the raceway ponds or thin layer inclined photobioreactors may be supplemented by artificial lighting. If the raceway ponds or thin layer inclined photobioreactors are maintained under cover or inside a structure, the irradiance may be provided by windows or clear panelling to allow sunlight to penetrate and / or by artificial lighting.

[0068] Optionally, the irradiance is provided at from 100 W.m-2to 1500 W.m-2. The daily irradiance in an outdoor cultivation system may vary considerably, particularly between seasons. The heme oxygenase containing photosynthetic organisms in the cultivation system can be grown in outdoor conditions with solar irradiances from 2.0 MJ m−2to 35 MJ m−2with air temperatures from 5oC to 40oC or indoors using an irradiance of 150 μmol photons m−2s-1to 220 μmol photons m−2s-1and at constant growth temperate from 23oC to 27oC. Warm dry temperate climactic conditions such as those found in Western Australia are preferred for optimal heme oxygenase containing photosynthetic organism growth in outdoor systems.

[0069] High and low temperatures above or below the tolerance limits of heme oxygenase containing photosynthetic organisms affect its performance. Passive evaporative cooling systems are used in closed PBRs for temperature control. The challenge of such a system is economics and sustainability due to freshwater limitations. Paddle wheel driven open raceway ponds do not need evaporative cooling systems but still require freshwater addition to compensate for water loss by evaporation.

[0070] One of the basic factors for obtaining maximal productivity for high density cultivations is that the average irradiance in the cultivation (Imean) should fall at the linear phase of the PI curve, hence around optimum irradiance (Ioptimum) and optimum rETRmax. The FrETR describes the actual functional max rETR of the cultivation, averaging out the spatial rETR across the depth of the cultivation. The FrETR-ratio on the other hand, describes the capacity of a cultivation to perform in relation to its maximal rETR, giving an indirect quantification of how much of the cultivation is in optimal irradiance and hence optimal rETR.

[0071] The proportion of irradiance delivered to the heme oxygenase containing photosynthetic organisms in the cultivation system may be increased in the red wavelengths (500-600 nm; the optimal wavelengths for photosynthesis) by use of a light diffusing luminescent solar concentrator (LSC). The LSC may be used to both (i) increase the proportion of irradiance in the 500-600 nm wavelengths as light is absorbed by luminescent particles when it hits the surface of an LSC and the absorbed light will be emitted from the edges at a longer wavelength when reflected internally, and (ii) increase the amount of the 500-600 nm wavelengths which penetrate the depths of the cultivation system where light penetration is decreased due to turbidity. • Depth and Flow rate

[0072] The optimum depth should be chosen at the point where the trade-off between higher growth rates (and consequently biomass yield, g L−1), and harvested volume (represented in areal productivity) is not disadvantageous.

[0073] If a raceway pond cultivation system is used, the raceway ponds may have a depth of from 10 cm to 30 cm. Optionally, the raceway ponds have a flow rate of from about 0.05 m.s-1to 0.5 m.s-1. A paddle wheel may be utilised to control flow.

[0074] The use of thin layer inclined open ponds may be advantageous when it comes to growth of heme oxygenase containing photosynthetic organisms in locations with high rain fall.

[0075] Optionally, the thin layer inclined photobioreactors have a depth of from 0.5 cm to 2 cm. Optionally, the thin layer inclined photobioreactors have a flow rate of from about 50 L.min-1to 100 L.min-1. A pump may be utilised to control flow.

[0076] A significant contributor to the cost of culturing heme oxygenase containing photosynthetic organisms is the costs involved in mixing of the water of the cultivation system. Paddle wheels are the most common mixing mechanism used in large scale heme oxygenase containing photosynthetic organism cultivation systems. Despite their simplicity in design and low capital cost, the major disadvantage of paddlewheels is their operating cost which is seen to significantly contribute to the overall heme oxygenase containing photosynthetic organism production cost.

[0077] The cultivation system may be continuously mixed or mixed only for specific periods. For example, the cultivation system may be stirred for 12 h of a 24 h period, or all 24 h. Alternatively, the cultivation system may be continuously mixed, but the mixing rate may be reduced for some of each 24 h period, for example during the night period. Optionally, the cultivation system is operated with a velocity of paddle wheels in raceway ponds at 0.25 m.s-1during the day and night. Alternatively, the cultivation system is operated with a pump flow rate in an inclined thin layer photobioreactor at 0.25 m.s-1during the day (14 h) which is reduced to 0.20 m.s-1for 10 h during the night.• Carbon Dioxide and pH

[0078] Optionally, the heme oxygenase containing photosynthetic organisms are cultivated in the presence of additional CO2, beyond that available from the atmosphere. The presence of additional CO2 provides two advantages: it increases the carbon available for the heme oxygenase containing photosynthetic organism growth, and it maintains the pH of the cultivation solution at about pH 6.5. However, it is important to use as little CO2 as possible, as much will be lost to the atmosphere, resulting in environmental pollution. Using a Proportional-integral + dead- zone control strategy can reduce the CO2 usage, resulting in a reduction in CO2 associated costs and most importantly, reduction in the amount of greenhouse gas lost to the atmosphere.

[0079] Optionally, the additional CO2 is provided using 1% CO2 at a flow rate of 0.5 L.min−1or equivalent (e.g.2% at a flow rate of 0.25 L.min−1or 0.5% at a flow rate of 1.0 L.min−1). The addition of 1% CO2 or equivalent favourably controls the pH of cultivations (kept below pH 9) and results in significantly higher heme oxygenase containing photosynthetic organism biomass productivity than control cultivations with no CO2 supplementation.

[0080] Optionally, the additional CO2 is only added during the light illumination period (e.g. during the day).

[0081] One preferred method of controlling pH is on / off control technology that helps to regulate the pH level in the pond. That is, when the pH level in a heme oxygenase containing photosynthetic organism cultivation system rises above a certain point due to heme oxygenase containing photosynthetic organism photosynthetic activities, CO2 is injected into the cultivation system until the pH drops below a certain point.

[0082] Alternatively, the CO2 utility and pH control can be controlled for heme oxygenase containing photosynthetic organism production with the use of a proportional integral (PI) + deadzone control system as described in Isiramen et al (2002) Improving pH control and carbon dioxide utilisation efficiency yin microalgae cultivation systems with the use of a Proportional- integral + dead-zone control strategy Bioresource Tech Reports 17: 100917.

[0083] Optionally, the cultivation solution of the present disclosure is maintained by addition of CO2at a pH of from 5.0 to 8.0, for example from 5.5 to 7.5, 6.0 to 7.0 or from pH 6.0 to 7.0 during culturing of the heme oxygenase containing photosynthetic organisms. For example, the pH of the cultivation solution may be maintained at a pH of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0 during culturing of the heme oxygenase containing photosynthetic organisms. Optionally, the pH is maintained at approximately pH 6.5 during culturing of the heme oxygenase containing photosynthetic organisms. A pH of about 6.5 ensures effective use of CO2in the cultivation solution.• Temperature

[0084] The temperature of the cultivation system has a significant effect on the growth of the heme oxygenase containing photosynthetic organisms. Regulation of the temperature of the cultivation solution results in improvements in the heme oxygenase containing photosynthetic organism biomass yield. Despite the increased energy requirements associated with temperature regulation, the increases in biomass results in an overall advantage. The temperature regulation may be heating of the cultivation system, or cooling of the cultivation system, depending on the environmental conditions.

[0085] Optionally, the temperature of the cultivation solution is maintained at from 150C to 200C for at least 12 h or up to 24 h each day.

[0086] As there is no available light for photosynthesis during the night, it is preferable that the temperature regulation is only performed during the daylight or illuminated hours. Thus, the temperature regulation may be performed for from 1 h to 12 h during each daylight period. Compositions

[0087] The present disclosure further provides heme generated by a heme oxygenase containing photosynthetic organism. Optionally, the heme is produced by the following steps: i) cultivating the heme oxygenase containing photosynthetic organism in the presence of one or more metalloporphyrins.

[0088] The present disclosure further provides a composition comprising heme, wherein the heme is generated by a heme oxygenase containing photosynthetic organism. Optionally, the heme composition is produced by the following steps: i) cultivating the heme oxygenase containing photosynthetic organism in the presence of one or more metalloporphyrins.

[0089] Optionally, the metalloporphyrin is chosen from the list comprising: zinc protoporphyrin (ZnPPIX), tin protoporphyrin (SnPPIX), chromium protoporphyrin (CrPPIX), zinc mesoporphyrin (ZnMPIX), mesoporphyrin (SnMPIX), chromium mesoporphyrin (CrMPIX) and zinc deuteroporphyrin IX bis glycol (ZnBG) and synthetic analogues of the natural metalloporphyrin ferroporphyrin (heme).

[0090] Optionally, the metalloporphyrins are introduced to the cultivation medium at a rate of from 0.01 µmole of MPP.g-1of biomass to 40 µmole MPP.g-1of biomass.

[0091] Optionally, the metalloporphyrins are introduced to the cultivation medium for at least 24 hours.

[0092] Optionally, the heme oxygenase containing photosynthetic organism is chosen from the following: Cyanophyta, Rhodophyta, or Cryptophyta. General

[0093] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[0094] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

[0095] Any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0096] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0097] The invention described herein may include one or more range of values (eg. size, displacement and field strength etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Hence “about 80 %” means “about 80 %” and also “80 %”. At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0098] Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribedto them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0099] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. The term “active agent” may mean one active agent, or may encompass two or more active agents.

[0100] The following examples serve to more fully describe the manner of using the above- described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these methods in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. References Isiramen et al (2002) Improving pH control and carbon dioxide utilisation efficiency yin microalgae cultivation systems with the use of a Proportional-integral + dead-zone control strategy Bioresource Tech Reports 17: 100917 Moheimani et al. (2013) Standard methods for measuring growth of algae and their composition In: Algae for Biofuels and Energy Springer pp.265-284EXAMPLES

[0101] Further features of the present invention are more fully described in the following non- limiting Examples. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad description of the invention as set out above. Example 1 Heme formation in Arthrospira

[0102] This study aimed to investigate the effect of zinc-protoporphyrin IX (ZnPP) on A. platensis to block the downstream processing of heme to phycocyanin pathway by inactivating the action of HO enzyme and increase heme concentration within the individual cell. ZnPP was chosen instead of other MPPs because of its efficiency to supress HO activity. Materials and methods Preparation of Zinc-protoporphyrin IX (ZnPP)

[0103] Zinc-protoporphyrin IX (ZnPP) with 95% purity was purchased form Sapphire Bioscience (producer- Enzo life Science, Farmingdale, New York, USA, lot No.08271813). ZnPP solution was made right before application to A. platensis by dissolving 31.3 mg of ZnPP with 10 ml of 0.1 N NaOH, and the final volume was adjusted up to 50 ml to get a final concentration of 1mmol ZnPP / L. Cyanobacterial cultivation and cultivation condition

[0104] The alkaliphilic cyanobacteria Arthrospira platensis (also known as Spirulina) MUR- 126 was sourced from the cultivation collection of the Algae Research and Development Centre, Murdoch University, Australia. A. platensis cultivation was cultivated using Zarrouk medium and pH of the medium was 9.5. Cultivation of A. platensis was conducted indoor at temperature 25o± 2oC, light intensity of 120 µmole photons m−2s−1with 12:12-h light / dark cycle. Light irradiance was measured using a PHOTPBIO LGBQM2 advanced quantum PAR meter (China). Cultivations were continuously stirred using 7 cm magnetic stirrer at 400 rpm for uniform mixing.

[0105] A. platensis biomass was grown in a 10L glass carboy with a working volume of 7 L and initial biomass concentration was 0.44 ± 0.1 g.L-1. The carboy was fitted with an inlet gas tube with an air stone at the opposite end, and a harvest tube. Cultivation was aerated with sterile atmospheric air at a flow rate of 0.4 Lmin-1and continuously stirred with a 7.5 cm magnetic stirrer at 400 rpm resulting a mixing speed 1.2 min-1measured by the time required for India ink to mix completely.

[0106] At the early log phase of growth (at day five after inoculation) A. platensis cultivation was distributed separately into 500 mL Erlenmeyer flasks with a working volume of 350 mL to assess the effect of ZnPP on the cyanobacterial cell. Cultivations were continuously stirred using 40 mm magnetic stirrer at 100 rpm for uniform mixing and the mixing speed was 17 s-1. Experimental design for the application of Zinc-protoporphyrin IX (ZnPP)

[0107] Zinc-protoporphyrin IX (ZnPP) was applied to the A. platensis cells at early log phage stage of growth, where the maximal rate of cell division happens.

[0108] The initial rate of Zinc-protoporphyrin IX (ZnPP) applied to A. platensis was 0.04 µmole of ZnPP.g-1of biomass. To identify the effectiveness of Zinc-protoporphyrin IX on HO enzyme inhibition of A. platensis, various doses up to 1000 times were tested. The effect of ZnPP at various concentrations (between 0.04 to 40 µmole ZnPP.g-1of biomass) were tested and exposure period of ZnPP was 72 hours.

[0109] MPP compounds are potential photosensitizers because of their porphyrin structure, although ZnPP has been reported as inert to light and has little or no photosensitizing effect. However, to avoid any photochemical reaction, ZnPP was applied to the cultivations immediately at the beginning of the dark cycle and heme content was measured against the negative control where no ZnPP was applied. All studies were carried out with a minimum of four replicates. Cultivation sampling

[0110] A 30 mL sample was collected every 12 hours (at the changeover between the light and dark period) for 72 hours including samples before and after ZnPP addition (0 hour). Harvested biomass was used to determine biomass growth, productivity, effective quantum yield, phycocyanin, heme, chlorophyll a (the only chlorophyll present in A. platensis) and total protein. Determination of cyanobacterial growth

[0111] Biomass productivity was measured by measuring dry weight (DW) and ash-free dry weight (AFDW) based on the method described in Moheimani et al. (2013) Standard methods for measuring growth of algae and their composition In: Algae for biofuels and energy Springer pp. 265-284.5 mL of cultivation was filtered through pre-combusted, pre-weighed glass filter papers (Whatman GF-C, diameter 2.5 cm, pore size 0.45 µm). The filters containing cyanobacterial biomass were dried in an oven at 90oC overnight, cooled in a desiccator, and reweighed. Filters with dry biomass were ashed in a furnace at 4500C overnight and cooled in a desiccator before weighing again. Dry weight was determined by subtracting the weight of the filter from the total dry weight, and ash-free dry weight was calculated by subtracting the ash weight from the total dry weight.

[0112] The specific growth rate and productivity of A. platensis cultivations were determined based on the methods described in Moheimani et al. (2013). Estimation of effective quantum yield (Fqʹ / Fmʹ)

[0113] Effective quantum yield (Fqʹ / Fmʹ) measures the photochemistry efficiency of PSII and is regarded as the most useful parameter for assessing the physiological status of cyanobacterial cells under light- and dark-adapted conditions. Effective quantum yield determines PSII operating efficiency under different environmental conditions, therefore is considered as a typical tool to evaluate cyanobacterial responses towards stress.

[0114] Effective quantum yield (Fqʹ / Fmʹ) was measured before and after the application of ZnPP at 12-hour intervals until the end of experiment to evaluate the responses of the cyanobacterial cell towards the chemical (Zinc-protoporphyrin IX). A portable chlorophyll a fluorometer (AquaPen-C, Czech Republic) was used at the maximum 3000 μmol photons m−2s−1saturation pulse intensity of blue light (450 nm). Pigment Analysis

[0115] For chlorophyll a and heme, cyanobacterial pellets were harvested by centrifugation and washed with deionized water and the pellets were collected after discarding the supernatant and finally stored at -600C until further analysis. The cyanobacterial pellets were subjected to three freeze-thaw cycles at room temperature in the dark to rupture the cell wall before they were crushed. However, for phycocyanin measurement, samples were harvested by filtration through glass microfiber filters (Whatman GF-C), washed with distilled water, and stored at -600C until further analysis. All pigments were extracted under dim light, to avoid pigment degradation. Chlorophyll a

[0116] The cells were crushed and extracted three times with 5 mL of ice-cold acetone containing 1% ammonia (9:1, v / v) and 1 % 2,6-ditert-butyl-a-dimethylamino-p-cresol and the supernatant separated by centrifugation 4000 rpm for 10 min. All extracts were combined and the amount of chlorophyll a content was measured spectrophotometrically (BioMate 3S UV–vis spectrophotometer) at 664 nm and 647 nm using equation detailed in Moheimani et al. (2013). Heme

[0117] Following removal of chlorophyll a, heme was extracted twice by suspending the pellet in 1.5 mL of acetone containing 2% (v / v) concentrated HCl. The acidic acetone extracts were combined and 6 mL of peroxide-free diethyl ether was added, to remove any free peroxides, followed by addition of 2 mL of saturated sodium acetate and the mixture gently swirled.

[0118] Peroxide free diethyl ether was produced by adding 60 g of FeSO4.7H2O, 6 mL concentrated HCl, and 110 mL water per L of diethyl ether. Any peroxide free diethyl ether that was not immediately used was stored in at 40C in the dark and checked with KI before use.

[0119] The acetone-ether layer was washed three times with 5 ml of deionized water resulting in heme being partitioned to the ether phase which was immediately evaporated at 300C under a stream of ultra-pure N2gas.

[0120] After evaporation of ether with N2gas, heme samples were dissolved in 1.5 mL of N,N- dimethylformamide (Chem-Supply, ≥99.8% purity) and stored at -25oC before analysis. All samples were analysed within 24 hours. Samples were filtered through 0.45 µm PTFE syringe filters, as previous research had shown no loss of heme after filtration. All extracts were analysed using gradient elution on a Shimadzu HPLC instrument with an analytical scale Apollo-C18column (Grace Discovery Sciences; 4.6 mm × 150 mm, 5 µm silica particle size) and a PDA detector. Heme was monitored at wavelength 398 nm. Mobile phases were methanol:water:trifluoracetic anhydrite (50:50:0.1, v / v / v, solvent A) and methanol:acetone:trifluoracetic anhydrite (80:20:0.1, v / v / v, solvent B). Heme was eluted with 100% A followed by a linear gradient to 50% B at 5 minutes, then linear gradient to 55% B at 15 minutes. Re-equilibration of the HPLC column consisted of running a linear gradient to 100% of A at 18 minutes hold for 7 minutes. The flow rate was 0.5 mL / min and column temperature set at 30oC. A 10 µL sample was injected and total time injection to injection was 25 min.

[0121] Heme was identified and quantified using commercially available hemin or ferriprotoporphyrin IX chloride (Sigma Aldrich, ≥96.0% purity). The retention time of the hemin standard for HPLC was consistently 12.98 minutes from injection. A calibration curve using the hemin standard peak area was used to quantify heme present in A. platensis samples. C phycocyanin (C-PC)

[0122] The C phycocyanin (C-PC) content of the A. platensis biomass was determined using 0.1M sodium phosphate buffer with repeated freezing and thawing, based on the protocol of Bennett and Bogorad (1973) Complementary chromatic adaptation in a filamentous blue-green alga The Journal of Cell Biology, 58(2): 419. Ruptured cells were resuspended in 5 mL phosphate buffer (pH 7.0) and the mixture was subjected to one cycle of freezing at -60 °C and thawing at room temperature in the dark. After that samples were placed in the refrigerator overnight. Ruptured cells were crushed and the phycocyanin containing supernatant was collected after centrifugation at 4000 rpm for 10 min. Phycocyanin concentration in the sample was determined spectrophotometrically (UV–vis, BioMate 3S) at 615 and 652 nm. The C-PC concentration was then calculated from the following equation:^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ( ^^ ^^. ^^ ^^−^^ ^^615 − 0.474( ^^ ^^652) ^^ ^^ℎ ^^1) = 5.34Protein content analysis

[0123] Samples were homogenized in 5mL Buiret reagent (Na2CO3: 0.1 g / L, NaOH: 0.02 g / L, NaK tartrate: 0.01 g / L, CuSO4.4H2O: 0.0025 g / L) in a glass tube. The homogenized mixtures were heated in a 100˚C water bath for 60 mins.0.5 mL of Folin-phenol reagent was added to the and mixed with a vortex mixer. All mixtures were placed in a water bath for 15 – 20 mins to return to room temperature and then centrifuged at 1000 – 2000 g for 10 mins. The absorbance of the supernatant was recorded at 660nm and protein content calculated using a standard curve described in Moheimani et al. (2013). Statistical analysis

[0124] In all cases, the results were expressed as mean values ± standard error of the mean. A two-way ANOVA and post-hoc multiple comparisons Holm-Sidak method were used to determine significant difference between the control and treatment groups (P <0.05). When normality and equal variance test failed, the Tukey pairwise comparison method based on ranks was applied using SigmaPlot software (version 14). In each case, P value <0.05 was considered significant with a sample size of (n=) 4. Results Effect of ZnPP on the heme of A. platensis

[0125] A. platensis typically produces heme to facilitate the synthesis of the natural blue phycobiliprotein pigment phycocyanin (PC) by the action of the heme oxygenase (HO) enzyme. HO enzyme inhibition of Arthrospira was tested by applying ZnPP at four different concentrations (0.04, 0.4, 4.0, and 40 µmole.g-1of biomass). The amount of heme increased was measured against the negative control where no ZnPP were applied, shown in (Figure 3).

[0126] Out of the four concentrations of ZnPP assessed, treatment with 0.4 µmole ZnPP.g-1had the greatest effect on heme content compared with control conditions. When 0.4 µmole ZnPP.g-1was applied, not only heme content increased significantly at the end of hour 72, but heme content also increased almost 3-fold over the 72 hours cultivation period and was significantly different from control at 24, 36, 48, and 72 hours after initial dose (Two-way ANOVA, p<0.05). The largest difference in heme yield between treatment and control (38% cf control) increase was recorded after 48 hours. Highest heme productivity was calculated when the A. platensis was dosed with 0.4 µmole ZnPP.g-1(Table 1).Table 1: Specific growth, biomass, and heme productivity of Arthrospira cultures with ZnPP dose and control Treatments Specific growth rate Maximum biomass Average heme (hour-1) productivity productivity (mg.L-1. hour1) (μg.L-1. hour1) Control 0.0094 ± 0.0005b0.0077 ± 0.0004b0.395 ± 0.056b0.04 μmole.g-10.0117± 0.0009a0.0113 ±0.0005a0.564 ± 0.013b0.4 μmole.g-10.0137 ± 0.0004a0.0129 ± 0.0005a0.854 ± 0.57a4.0 μmole.g-10.0131 ± 0.0004a0.012 ± 0.0039a0.454 ± 0.031b40 μmole.g-10.0076 ± 0.0012b0.0067 ± 0.0009b0.373 ± 0.099bDifferent letters show a significant difference in each row. Date are value ± SE (One way ANOVA P < 0.05)

[0127] There was a statistically significant increase in the heme content in control compared to the beginning and end of the experiment was observed (Two-way RM ANOVA, P<0.05) as culture matures with the time. However, there were no other periods during cultivation where the application of 0.04, 4.0, and 40 μmole ZnPP.g-1showed no improvement in the heme content was statistically significantly different to the control (Two-way ANOVA, P>0.05, and Figure 3A, C and D). When Arthrospira was dosed with 4.0 µmole of ZnPP.g-1, the data at 12 hours were omitted from the graph (Figure 3C, marked with an asterisk). We regarded this data point to be an outlier.

[0128] Our data show that dosing Arthrospira with 0.4 µmole ZnPP.g-1results in a significant increase in heme content compared to the control. Variation of heme (0.4 µmole of ZnPP.g-1of biomass) enhanced compared to control (Δ heme = heme treatment – heme control) at each sampling time point were modelled and fitted with a best-fit curve through an exponential rise to maximum function. This led to results in a logarithmic curve that can be used to estimate the maximum achievable enhancement of heme content and an optimal harvesting time (Figure 4). The R2 value of the fitted curve (0.948) suggests 95% set of the experimental data is described by the equation for the fitted curve. The model suggests that the maximum enhancement in heme yield occurs at approximately 48 hrs. Effect of ZnPP on the growth of A. platensis

[0129] Biomass yield in all Arthrospira cultures, including control, increased over the cultivation period (Figure 5). Cultures that received a dose of 0.04 µmole of ZnPP.g-1showed the highest biomass yield, while the lowest was observed in the control. Overall, addition 0.04 and0.4 µmole of ZnPP.g-1resulted in significantly higher biomass yield than control after 72 hours incubation (Two-way ANOVA, P <0.05). No statistically significant difference was found for the biomass yield between control and treatment with 4 and 40 µmole of ZnPP.g-1after 72 hours of incubation (Two-way ANOVA, P >0.05).

[0130] There is a diurnal pattern observable in the biomass yield data (Figure 5). Due to night respiration during the dark period, it is expected that some biomass is lost during the dark period. This is clearly visible in the data for the control cultures. This is less evident in the cultures dosed with ZnPP.

[0131] The specific growth rate and biomass productivity of Arthrospira cultures exposed to 0.04, 0.4 and 4.0 μmole ZnPP.g-1were significantly increased compared to control and ZnPP doses at μmole ZnPP.g-1(One-way ANOVA, P<0.001, Table 1). Effect of ZnPP on the effective quantum yield (Fqʹ / Fmʹ) of A. platensis

[0132] Fq′ / Fm′ values of all treated Arthrospira, including control declined gradually over the cultivation time after the addition of the ZnPP chemical compared to time 0 (Two-way RM ANOVA, P<0.05, Figure 6). At each sampling point, Fq′ / Fm′ value of control cultures were statistically greater than those of cultures that received ZnPP doses 0.04, 0.4 and 4.0 μmole ZnPP.g-1. However, the overall trend shows addition of ZnPP leads to a significantly lower effective quantum yield, that suggests mild stress in photosystem II (Two-way ANOV, P<0.05)

[0133] The quantum yield data also exhibited a diurnal pattern (Figure 6). Fq′ / Fm′ value dropped at each dark period for both ZnPP dosed and control cultures. HO enzyme inhibition of ZnPP

[0134] Control-adjusted changes in heme (Δ heme) content of Arthrospira (Figure 7A) that received a dose of ZnPP at 0.4 µmole.g-1, demonstrated an increasing trend up to 48 hours before a plateau at the end of the experiment. However, following the administration of 0.04, 4.0, and 40 μmole ZnPP.g-1, the level of heme rises varied at various time intervals.

[0135] Plotting phycocyanin compared to control (Δ phycocyanin = phycocyanin treatment - phycocyanin control) showed varied levels of dropdown in phycocyanin content for (Figure 5 B) all ZnPP doses all the way through the experimental period. At a ZnPP dose of 0.4 μmole g-1, there was a decrease in phycocyanin along with an increase in heme content, although this decrease did not exactly follow the opposite trend of the heme increase. However, ZnPP doses of 0.04, 4.0, and 40 μmole.g-1showed dropdowns in phycocyanin at all those points where heme content increased.

[0136] The results indicate that ZnPP can inhibits HO enzyme activity by limiting the conversion of heme to phycocyanin, that leads to heme accumulation within the microalgal cell.

[0137] Here we have shown that heme content of Arthrospira can be significantly enhanced with a single dose addition of ZnPP. Indeed, a maximum 38% increase in heme content was seen when 0.4 µmole of ZnPP.g-1was added. Comparison of control corrected change in heme and change in phycocyanin content data indicated that, at this dose, phycocyanin content decreases as heme content increases. This is consistent with the hypothesis that the ZnPP is inhibiting the activity of the HO enzyme resulting in restricting heme availability to the downstream biosynthetic pathway for the production of phycobilins. Consequently, there is a build-up of heme in the cell.

[0138] The study also demonstrates that the application of ZnPP did not inhibit the growth of Arthrospira. Indeed, a higher biomass yield was seen when Arthrospira was dosed with the 0.04 and 0.4 µmole ZnPP.g-1 of biomass. Zinc is an important trace element that promotes the growth of Arthrospira at low concentrations, but inhibits growth at high concentrations. However, in this study, the maximal concentration of ZnPP (40 μmole.g-1of biomass; actual zinc concentration 2.65 mg / L) did not appear to have any effect on growth. Further, iron is an indispensable micronutrient for microalgal growth due to its critical role in numerous metabolic processes. Not allowing cells to convert heme to phycocyanin may be a positive signal to cells to grow faster as they see the iron availability due to the high level of internal heme.

[0139] Effective quantum yield (Fqʹ / Fmʹ) is regarded as an indicator of the health of microalgae, and it measures the efficiency of PSII. Lower Fqʹ / Fmʹ values were observed in this study, suggesting that application of ZnPP affects the effective quantum yield, but that effect was not strong enough to significantly affect algal growth.

[0140] It is to be noted that the application of addition ZnPP does not need to be lengthy. The ZnPP compound can be added a few hours prior to the full harvest of the culture. This means that the seed culture would not need to be affected at all through the cultivation.

[0141] Furthermore, the application of ZnPP does not need to be continuous. The increase in heme from a single dose is at its maximum in around 48 hours and is waning by 72 hours, suggesting that the effect is inducible and reversible. A culture system can be designed to grow A. platensis where the ‘stock culture’ could be cultivated semi-continuously under optimised conditions for maximum biomass growth. At each harvest for stock culture maintenance, the biomass removed could be treated with ZnPP and then the ZnPP treated biomass utilised as the heme-enriched product.

Claims

CLAIMS 1. A method of increasing the heme content of heme oxygenase containing photosynthetic organisms, the method comprising the step of: cultivating the heme oxygenase containing photosynthetic organism in the presence of one or more metalloporphyrins (MPPs).

2. The method of claim 1, wherein the metal element of the metalloporphyrin is chosen from: zinc, tin or chromium.

3. The method of claim 1, wherein the metalloporphyrins are introduced to the cultivation medium at a rate of from 0.01 µmole of MPP.g-1of biomass to 40 µmole MPP.g-1of biomass.

4. The method of claim 1, wherein the metalloporphyrins are introduced to the cultivation medium for at least 24 hours.

5. The method of claim 1, wherein the heme oxygenase containing photosynthetic organism is chosen from: Cyanophyta, Rhodophyta, or Cryptophyta.

6. The method of claim 1, comprising the step of cultivating the heme oxygenase containing photosynthetic organism in the presence of a second method of increasing heme content.

7. The method of claim 6, wherein the second method is chosen from: carrying the cultivation out under specific light cycles; inducing oxidative stress; and / or exposing the heme oxygenase containing photosynthetic organism to ultraviolet radiation.

8. The method of claim 1, wherein the cultivating of a heme oxygenase containing photosynthetic organism to increase heme content is carried out using a paddle wheel or jet driven raceway pond cultivation system.

9. Heme generated by the method of claim 1.

10. A composition comprising heme, wherein the heme is generated by the method of claim 1.

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