A process for cultivating microalgae

Abstract

A method for cultivating microalgae comprises the steps of inoculating a culture media with microalgae and adding a substrate into the inoculated culture media; and cultivating the microalgae under heterotrophic growth conditions and periodically introducing microscopic air bubbles and the substrate into the inoculated culture media.

Description

[0001] A PROCESS FOR CULTIVATING MICROALGAE

[0002] Field of Invention

[0003] The invention relates to the field of microalgae cultivation. Particularly, the invention relates to a process of cultivating microalgae with increased yield through intermittent air supply and intermittent substrate feeding.

[0004] Background of Invention

[0005] Microalgae are unicellular microorganisms that can grow rapidly in saltwater, freshwater and even wastewater such as domestic sewage water and palm oil milling effluents . Their high photosynthesis efficiency helps with reduction of atmospheric carbon dioxide through photosynthesis. Besides, microalgae contain a large amount of valuable bioproducts within their cells including, but not limited to, pigments such as lutein and phycobilin, antioxidants such as astaxanthin, polysaccharides, vitamins such as P-carotene, lipids such as polyunsaturated fatty acids (PUFAs), and protein. Hence, microalgae have been cultivated and harvested for many industrial applications such as wastewater treatment, carbon sequestration, and production of food supplements, organic chemicals, pharmaceuticals, biofuel and fodder.

[0006] However, a major bottleneck in large-scale microalgae production is limited growth rate and biomass production. Different technologies have been developed to increase microalgae production. For example, United States Patent No. US9670454B2 disclosed a method of cultivating microalgae that provides increased production of microalgae biomass, protein or lipids. Particularly, the method involves delivering oxygen gas into a culture media inoculated with microalgae, wherein the oxygen gas comprises between 80% to 100% oxygen. In addition, Ronda and colleagues (2012) disclosed increased aeration rate with periodic sparging improves growth rate of Spirulina platensis and its y-Linolenic acid production in Spirulina platensis. Moreover, Mitra and colleagues (2023) disclosed that an intermittent feeding of medium supplemented with bicarbonate increases biomass yield as well as biomass productivity rate. Nevertheless, the total microalgae cultivation time to achieve a desired yield typically requires 7 days or more.

[0007] The present invention provides a method for cultivating microalgae, which increases the yield of microalgae. In more particular, the method increases the growth rate of microalgae while reducing cell breakage of microalgae during cultivation.

[0008] Summary of Invention

[0009] An object of the invention is to provide a method for cultivating microalgae which increases the growth rate of microalgae. Particularly, the method involves intermittently supplementing the culture media inoculated with microalgae with substrates at the beginning of microalgae cultivation and during the log phase of microalgae growth. The supplementation of substrates is eliminated during the lag phase of microalgae growth.

[0010] Another object of the invention is to provide a method for cultivating microalgae which increases the microalgae production. Particularly, the method involves cultivating microalgae in a culture media with periodic aeration of microscopic air bubbles. Periodic aeration of microscopic air bubbles into the culture medium eliminates recirculation of contents in the culture bioreactors, thereby reducing the risk of cell breakage and increasing microalgae production.

[0011] At least one of the preceding objects is met, in whole or in part, in which the embodiment of the present invention describes a method for cultivating microalgae comprises inoculating a culture media with microalgae and adding a substrate into the inoculated culture media; and cultivating the microalgae under heterotrophic growth conditions and periodically introducing microscopic air bubbles and the substrate into the culture media. Preferably, the microscopic air bubbles are introduced into the culture media for 4 min to 8 min every hour. Preferably, the substrate is introduced daily into the culture media when the cultivated microalgae enters logarithmic phase of microalgae growth. In a preferred embodiment of the invention, the microalgae are heterotrophic microalgae or autotrophic microalgae adapted to heterotrophic conditions. Preferably, the microalgae are selected from species Euglena, Chlorella, Haemaiococcus. Dunaliellci, Spirulina, Botryococcus, Phaeodactylum, Porphyridium, Chaetoceros, Crypthecodinium, Isochrysis, Nannochloris, Nitzschia, Schizochy trium, Aphanizomenon, Odontella, Lyngbya, Scennedesmas, and Tetraselmis.

[0012] In a preferred embodiment of the invention, the substrate is a carbon source selected from molasses, dextrose, glucose, fructose, sucrose, galactose, xylose, mannose, rhamnose, N- acetylglucosamine, glycerol, floridoside, glucuronic acid, and lactose.

[0013] In a preferred embodiment of the invention, the logarithmic phase of microalgae growth begins after three days of cultivation.

[0014] In a preferred embodiment of the invention, the method further comprises a step of removing hydrogen gas from the culture media through nitrogen bubbling on first day of cultivation until four days after cultivation.

[0015] In a preferred embodiment of the invention, the method further comprises a step of harvesting the cultivated microalgae after five days of cultivation.

[0016] In a preferred embodiment of the invention, the microalgae are cultivated in a plurality of bioreactors.

[0017] Preferably, the bioreactors are bubble column bioreactors.

[0018] Preferably, the method further comprises a step of combining microalgae cultivated in two bioreactors into one bioreactor after each day of cultivation.

[0019] Still other objects and advantages of the present invention will become readily apparent to those skilled in the art from the following detailed description, wherein the preferred embodiments of the invention are shown and described, simply by way of illustration of the best mode contemplated of carrying out the invention. As will be realized, the invention is capable of other and different embodiments, and its several details are capable of modifications in various obvious aspects, all without departing from the invention. Accordingly, the drawings and description thereof are to be regarded as illustrative in nature, and not as restrictive.

[0020] Brief Description of Drawings

[0021] For the purpose of facilitating an understanding of the invention, there are illustrated in the accompanying drawings the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

[0022] Figure 1 illustrates microalgae cells are cultivated in bioreactors of increasing sizes which are arranged in parallel fashion, in which microalgae cells cultivated in two smaller bioreactors are combined into one larger bioreactor after each day of cultivation.

[0023] Figure 2 shows the optical density of Euglena over a 7 days growth cycle with and without substrate addition on Day 3 after inoculation.

[0024] Figure 3 shows the biomass dry weight of Euglena over a 7 days growth cycle with and without substrate addition on Day 3 after inoculation.

[0025] Detailed Description of Invention

[0026] Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. However, it is to be understood that limiting the description to the preferred embodiments of the invention is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim. The embodiment described herein is not intended as limitations on the scope of the invention.

[0027] The term “cultivate” as used herein refers to intentional fostering of growth and proliferation of one or more cells by culturing under suitable conditions.

[0028] The terms “culture media”, “media” and “growth media” can be used interchangeably unless otherwise mentioned. These terms refer materials that provide one or more essential nutrients or materials that support algal growth.

[0029] The present invention describes a method for cultivating microalgae, which is suitable for use in bioreactor systems. Advantageously, the method disclosed herein increases the yield of microalgae. More particularly, this method increases the growth rate of microalgae cells while reducing microalgal cell breakage during cultivation, thereby increasing the production of microalgal biomass.

[0030] In the preferred embodiment of the invention, the method for cultivating microalgae comprises a step (a) of inoculating a culture media with microalgae cells and a step (b) of cultivating the microalgae. One or more microalgae species can be cultivated in the culture media. The method disclosed herein is suitable for cultivating heterotrophic microalgae and autotrophic microalgae adapted to heterotrophic conditions. Particularly, the method of the invention is suitable for cultivating microalgae selected from the species Euglena, Chlorella, Haematococcus, Dunaliella, Spirulina, Botryococcus, Phaeodactylum, Porphyridium, Chaetoceros, Crypthecodinium, Isochrysis, Nannochloris, Nitzschia, Schizochytrium, Aphanizomenon, Odontella, Lyngbya, Scennedesmas, and Tetraselmis. Preferably, the microalgae cells are selected from Euglena gracilis, Chlorella vulgaris, Haematococcus pluvialis, Dunaliella salina, Spirulina platensis, Botryococcus hraunii, Phaedactylum tricomutum, Porphyridium cruentum, Chaetoceros calcitrans, Crypthecodinium cohnii, Isochrysis galhana, Nannochloris oculate, diatom Nitzschia laevis, Schizochytrium limacinum, Aphanizomenon flos-aquae, Odontella aurita, Lyngbya majuscule, Scenedesmus almeriensis, and Tetraselmis tetrathele. More preferably, the microalgae cells are selected from Euglena gracilis, Chlorella vulgaris, Haematococcus pluvialis, Dunaliella salina, and Spirulina platensis.

[0031] Particularly, in step (a) of the method disclosed herein, an inoculum of a microalgae species or inocula of two or more microalgae species are introduced into the culture media. Following the inoculation (Day 0), the microalgae cells in the inoculated culture media enters a lag phase of growth. Any inoculation method can be used to introduce microalgae cells into the culture media, as long as the viability of the microalgal cells is not compromised. The culture media suitable for use in the method disclosed herein can be a basic culture medium containing a nitrogen source, a phosphorus source, a calcium source, a potassium source, a magnesium source, microelements, and vitamins. The nitrogen source can be ammonia, nitrate or urea. In an exemplary embodiment, the microalgae are inoculated in Conway’s medium. Alternatively, enrichment media such as modified Bristol medium (MBM), modified Zarrouk medium, Walne medium and the Guillard's F / 2 medium are used for the inoculation of microalgae.

[0032] Step (a) of the method further comprises adding a substrate into the inoculated culture media prior to step (b). Preferably, the substrate is a carbon source selected from molasses, dextrose, glucose, fructose, sucrose, galactose, xylose, mannose, rhamnose, N-acetylglucosamine, glycerol, floridoside, glucuronic acid, and lactose. More preferably, the substrate is molasses. It is preferred that the volume of substrate added into the culture media is about 1% of the working volume of the culture media.

[0033] Pursuant to the preferred embodiment of the invention, step (b) of the method involves cultivating the microalgae under heterotrophic growth conditions. In particular, the cultivation of microalgae cells occurs in environments with limited or no light. The other heterotrophic growth conditions including, but not limited to, temperature, pH level, carbon dioxide levels, rate of oxygen delivery and nutrients concentrations in the culture media are adjusted based on the species of the microalgae cells inoculated in the culture media, in which the adjusted conditions favour the growth of the microalgae cells in the culture media. The method may comprise maintaining pH of the inoculated culture media between a pH of 3.5 and a pH of 4.5. The method may also comprise maintaining a temperature of the inoculated culture media between 25 °C and 35 °C.

[0034] In the preferred embodiment of the invention, step (b) further comprises periodically introducing microscopic air bubbles or microbubbles into the inoculated culture media. Preferably, the microscopic air bubbles contain oxygen gas. The introduction of microscopic air bubbles can be done by using micro spargers or microbubble diffusers. Introduction of microscopic air bubbles provides a gentle mixing of the microalgae cells and growth nutrients in the culture media, providing the microalgae cells with adequate exposure to growth nutrients while reducing the shear stress on the surface of the microalgae cells. Accordingly, the probability of microalgal cell breakage during mixing of the contents in the culture media is significantly reduced, hence increasing the final microalgae yield. Microbubbling eliminates the use of pumps for mixing the contents in the culture media which will cause significant shear stress on the microalgae cells and eventually damage the cells.

[0035] Preferably, the microscopic air bubbles are introduced into the inoculated culture media for 4 minutes to 8 minutes every hour, or more preferably 6 minutes every hour. The inventors found that introducing air bubbles into the culture media every hour for only 4 minutes to 8 minutes is sufficient to achieve optimal microalgal growth. Periodic introduction of microscopic air bubbles reduces the exposure of the microalgae cells to shear stress generated by the mixing of the contents in the culture media, further reducing the chance of microalgal cell breakage. Advantageously, the combination of periodic aeration and microscopic air bubbles increases the final microalgae yield.

[0036] In the preferred embodiment of the invention, step (b) further comprises periodically introducing the substrate into the inoculated culture media during cultivation of the microalgae. Preferably, the substrate is a carbon source selected from molasses, dextrose, glucose, fructose, sucrose, galactose, xylose, mannose, rhamnose, N-acetylglucosamine, glycerol, floridoside, glucuronic acid, and lactose. More preferably, the substrate is molasses. It is preferred that the volume of substrate added into the culture media is about 1% of the working volume of the culture media. Preferably, the substrate is introduced daily into the culture media when the cultivated microalgae enters logarithmic (log) phase of microalgae growth.

[0037] In an exemplary embodiment of the invention, the microalgae cells in the inoculated culture media remain in the lag phase after the first two days of cultivation (Day 1 and Day 2). The log phase of microalgae growth begins after three days of cultivation (Day 3) where the microalgae cells begin to grow and propagate exponentially. Substrate is added into the culture media daily since Day 3 until the microalgae cells enter a stationary phase or steady state where the microalgae cell growth begins to decline and are ready to harvest. Preferably, the cultivation period is completed after 5 days, which is shorter than the typical 7-day cultivation period of conventional cultivation methods. Advantageously, supplementation of culture media with a carbon source before and during cultivation, except during the lag phase of microalgal growth, leads to an increase of microalgae yield.

[0038] In a preferred embodiment of the invention, the method disclosed herein further comprises a step of removing hydrogen gas from the culture media through nitrogen bubbling in order to maintain the pH of the culture media. Preferably, nitrogen bubbling is conducted on first day of cultivation until one day before harvesting. More preferably, nitrogen bubbling is conducted on first day of cultivation until four days after cultivation.

[0039] In a preferred embodiment of the invention, step (b) of the method disclosed herein comprises cultivating the microalgae in a plurality of bioreactors. Vertical column bioreactors such as bubble column bioreactors (BCRs) can be employed to cultivate the microalgae cells. The method may further comprise a step (c) of combining microalgae cultivated in two bioreactors into one bioreactor as the cultivation progresses. In an exemplary embodiment of the invention, the microalgae are cultivated in a plurality of bioreactors of increasing sizes which are arranged in a parallel fashion. Preferably, the contents in two smaller bioreactors are combined into a larger bioreactor after each day of cultivation.

[0040] In a preferred embodiment of the invention, the method further comprises a step (d) of harvesting the cultivated microalgae. Preferably, the cultivated microalgae cells are harvested after five days of cultivation (Day 5) when the cells enter stationary phase. The dry cell weight of the cultivated microalgae cells begins to decrease if the cultivation is continued for more than five days. Preferably, nitrogen enrichment of the microscopic air bubbles is stopped one day before harvesting. Advantageously, the overall calorific value of the harvested microalgae cells is maintained at range of 4000 kcal / kg to 5000 kcal / kg after the elimination of nitrogen bubbling at the end of the cultivation cycle.

[0041] Advantageously, as illustrated in Figure 3, the method disclosed herein is able to increase the microalgae yield by 77%. The increase in microalgae yield is attributed to the combined effects of periodic aeration, microscopic air bubbles and supplementation of culture media with a carbon source before and during cultivation, except during the lag phase of microalgal growth.

[0042] Below is an example to illustrate the aspects and embodiments of the invention. The example is not intended to limit the disclosed invention, which is limited only by the claims.

[0043] Comparative Analysis and Justification for Substrate Addition in Euglena

[0044] Cultivation

[0045] The impact of substrate addition timing on the growth and biomass productivity of Euglena was investigated. Two cultivation strategies were employed: (1) a single substrate addition on Day 0 (after inoculation) at 1% of the total culture volume, and (2) substrate addition on Day 0 (after inoculation) and continued daily from Day 3 (after three days of cultivation) onward at 1% of the total culture volume. Figures 2 and 3 clearly demonstrated that strategy (2) where substrate was added starting on Day 3 greatly enhanced biomass production and productivity compared to the single-addition condition.

[0046] In the condition where substrate was added starting on Day 3, the dry cell weight (DCW) reached 22 g / L on Day 5, a significant increase compared to the single-addition condition, which achieved only 14.47 g / L on Day 5. This enhanced growth can be attributed to the availability of nutrients during the exponential phase, which began on Day 3, as shown by the increase in optical density (OD) data (Figure 1). Regular substrate supplementation provided sufficient nutrients to sustain metabolic activity and biomass accumulation throughout this phase.

[0047] In contrast, the condition with a single substrate addition on Day 0 resulted in slower growth, with DCW peaking at 17.98 g / L on Day 3 and declining thereafter. This growth limitation was due to nutrient depletion during the exponential phase, which restricted further biomass accumulation. The absence of nutrient supplementation after Day 0 was insufficient to support the metabolic demands of the rapidly dividing cells during the exponential phase.

[0048] The exponential growth phase, beginning on Day 3, represents the period of maximum cellular division and metabolic activity. Nutrient supplementation during this phase is critical to sustaining high growth rates. In the condition with substrate addition starting on Day 3, the daily addition of 1% substrate aligned with the exponential phase's nutrient demands, enabling continuous biomass accumulation. This approach not only delayed nutrient depletion but also maximized biomass yield. Conversely, in the single -addition condition, nutrients were exhausted early in the exponential phase, leading to suboptimal growth and earlier transition to the decline phase.

[0049] This demonstrated that the timing and frequency of substrate addition were critical factors in optimizing Euglena cultivation. Supplementing nutrients during the exponential phase ensures that the cells’ metabolic needs are met, significantly enhancing productivity.

[0050] Furthermore, the productivity (Day 0 to Day 5) for the condition with substrate addition starting on Day 3 was 4.445 g / L / day, representing a 77% improvement compared to the single -addition condition, which achieved only 2.511 g / L / day. This significant increase underscored the critical role of nutrient supplementation during the exponential growth phase in maximizing productivity.

[0051] The data suggested that the optimal cultivation cycle for Euglena should be 5 days. This recommendation was based on the growth phase observed during the study. For the condition with substrate addition starting on Day 3, DCW peaked at 22 g / L on Day 5 but began to show signs of decline afterward. Similarly, in the single-addition condition, DCW reached 14.47 g / L on Day 5 and started to decrease. The decline phase, characterized by reduced metabolic activity and nutrient depletion, typically began around Day 5. Consequently, the 5 -day cultivation cycle is justified as it maximized biomass yield and ensured the highest productivity before the onset of the decline phase, when further growth was limited.

Claims

Claims1. A method for cultivating microalgae, comprising: inoculating a culture media with microalgae and adding a substrate into the inoculated culture media; and cultivating the microalgae under heterotrophic growth conditions and periodically introducing microscopic air bubbles and the substrate into the culture media, wherein the microscopic air bubbles are introduced into the culture media for 4 min to 8 min every hour; and the substrate is introduced daily into the culture media when the cultivated microalgae enters logarithmic phase of microalgae growth.

2. The method according to claim 1, wherein the microalgae are heterotrophic microalgae or autotrophic microalgae adapted to heterotrophic conditions.

3. The method according to claim 1 or 2, wherein the microalgae are selected from species Euglena, Chlorella, Haemaiococcus. Dunaliellci, Spirulina, Botryococcus, Phaeodactylum, Porphyridium, Chaetoceros, Crypthecodinium, Isochrysis, Nannochloris, Nitzschia, Schizochytrium, Aphanizomenon, Odontella, Lyngbya, Scennedesmas, and Tetraselmis.

4. The method according to any one of claims 1 to 3, wherein the substrate is a carbon source selected from molasses, dextrose, glucose, fructose, sucrose, galactose, xylose, mannose, rhamnose, N-acetylglucosamine, glycerol, floridoside, glucuronic acid, and lactose.

5. The method according to any one of claims 1 to 4, wherein the logarithmic phase of microalgae growth begins after three days of cultivation.

6. The method according to any one of claims 1 to 5, further comprising a step of removing hydrogen gas from the culture media through nitrogen bubbling on first day of cultivation until four days after cultivation.

7. The method according to any one of claims 1 to 6. further comprising a step of harvesting the cultivated microalgae after five days of cultivation.

8. The method according to claim any one of claims 1 to 7, wherein the microalgae are cultivated in a plurality of bioreactors.

9. The method according to claim 8, wherein the bioreactors are bubble column bioreactors.

10. The method according to claim 8 or 9, further comprising a step of combining microalgae cultivated in two bioreactors into one bioreactor after each day of cultivation.

Images