MX2014012270A - Method of culturing algae. - Google Patents

Abstract

The present invention relates to a method of culturing algae in raceway ponds, wherein the physiological state of the algae is manipulated by altering one or more environmental parameter so as to simulate bloom forming conditions and to raceway ponds suitable for culturing algae according to the present invention. The alteration of the one or more environmental parameters in a specifically timed manner can be used to induce and maintain synchronous cell division.

Description

METHOD FOR CULTIVATING ALGAE FIELD OF THE INVENTION The present invention relates to a method for manipulating the physiological state of algae grown in raceway ponds by altering one or more environmental parameters to simulate algae flowering conditions and raceway type ponds for growing algae according to the present invention.

BACKGROUND OF THE INVENTION Algae have been grown in mass culture since the 1950s as a source of fertilizers (Burlow, 1953) and since then as a food source for both humans (Gershwin &Belay, 2007) and animals (Lundquist et al, 2010) and for use in water treatment (Oswald # Gotaas, 1957). However, the recent interest in third-generation biofuels has again increased interest in the massive cultivation of algae for the creation of a bio-handle for oil and lipid extraction (Oswald &Goluke, 1960). In addition, the potential use of algae for long-term carbon sequestration (Campbell et al, 2009) has highlighted the need for improved methods of mass algae culture (GB 2464763).

The algae require certain nutrients, including nitrate, phosphate, trace elements and bicarbonate. In its Ref. 251770 natural environment, seawater provides these nutrients to algae.

These nutrients are present in their maximum amounts during periods of upwelling, which is an oceanographic phenomenon that involves a movement driven by winds of dense water, cooler and usually rich in nutrients towards the surface of the ocean, replacing the more lukewarm surface water, generally with reduction of nutrients. During these periods of upwelling in natural seawater, the algae are exposed to a maximum of 40 m ol of nitrate per cubic meter. However, most of the time the upwelling is absent and therefore the concentration of available nutrients for the seaweed from the seawater is significantly lower.

The algae reflect the relationship of Redfield, which is the atomic ratio of carbon, nitrogen and phosphorus found in seawater (Redfield, 1934). This empirically developed stoichiometric relationship is found to be C: N: P = 106: 16: 1. Therefore, the maximum algal biomass that can be created is limited by the less abundant nutrient present in natural seawater, which is commonly nitrate. This limitation generally prevents the growth of algae beyond a biomass of several (less than ten) grams per cubic meter.

Different systems have been developed to produce algae commercially and are described below.

Initially, commercial algae were grown using the existing water treatment infrastructure, which consisted of natural ponds (eg, Dunaliella Salina, Hutt Lagoon, Australia) and outdoor circular water treatment facilities (Harder and Von Witsch, 1942). However, the growth rate of algae and biomass yield was found to be limited by factors that included sub-optimal mixing dynamics and inconsistent gas exchange. Then, high-rate oblong ponds known as raceway ponds were developed, in which algae could be grown to a higher density (Benemann &Oswald, 1996). Raceway ponds are open ponds in which algae and water circulate around a track. The paddle wheels are used to maintain the flow of water and to maintain the circulation of the algae to the surface and mix the entire mass of water to exchange gases with the atmosphere at a high rate. However, sub-optimal growth rates are still a problem and have prevented the mass culture of algae in raceway-type ponds from being commercially viable, especially at the scale required for the production of biomass to produce fuels (Sheehan et al., 1998), and for carbon sequestration (GB 2464763) and for the bulk production of biomass of raw material.

A main advantage of the raceway type pond is its simplicity, with low production costs and low operating costs. However, because raceway ponds are usually completely open to the environment, adverse weather (Vonshak et al., 2001) can prevent the growth of algae and contamination by external organisms often result in undesirable species. that monopolize the cultivated algae (Ben Amotz, 2003). Contamination can be avoided by regularly cleaning the ponds, but this interrupts the growth and harvest of the algae. Chemical signaling by algae, for example the release of extracellular polysaccharides by diatoms (Hoagland et al., 1993) as well as the release of monosaccharides and amino acids (Granum et al., 2002) can provide a fertile soil for bacteria and others to grow. Pollutants or allelopathic growth that inhibits the compounds (Prince et al., 2008) can slow down or stop the production of the desired algae. In addition, it can be very difficult to control other environmental factors such as water temperature, color of light and intensity of light, resulting in the photoinhibition of algae growth (Goldman, 1979).

Alternative methods have been developed in which environmental conditions can be controlled more than close. A photobioreactor (PBR) is a closed system in which algae are grown in a controlled environment that is isolated from the ambient atmosphere and forces algae through thin layers to maximize their solar exposure. The exchange of gases, the addition of nutrients, the dilution of algae and the removal of waste products can be easily controlled. In addition, the contamination of algae is minimized.

The primary objective of the highly controlled growth environment of a PBR is to achieve a very high density of algae in order to maximize productivity (Miyamoto &Benemann, 1988). However, the disadvantages arise due to the high density of algae. The need to constantly monitor parameters such as temperature, solar irradiation, gas exchange, the addition of nutrients and the removal of waste products requires expensive and sophisticated technology. In addition, abnormally high algal densities can lead to the formation of biofilms and surface adhesion of algae normally transported in water, which are exhibiting stress responses whereby algae form aggregates to reduce the environmental stress of the artificial growth environment (Sukenik &Shelaf, 1984). Biofilm formation can make a PBR difficult to clean and can clog the equipment (Ben Amotz, 2008), and high density of algae can actually change the physiology of algae resulting in the counterproductive reduction of product performance and product characteristics.

Controlling the growth environment of algae occurs at a significant cost. The systems of Photobioreactor Closed (PBR) operate in transparent tubing, plastic bags or other containers with walls. The physical drag of the water alone creates enough friction to negate the benefits of additional performance due to the associated cost of this friction. The energy cost of many of these systems exceeds the energy of the light captured by the photosynthetic organisms, being in this way fundamentally unsustainable (Stephenson, et al., 2010). In addition, in high isolation environments, these reactor environments are made of panels, tubes or bags that experience significant heat, causing new growth challenges.

In comparison, algae grown in open raceway type ponds are usually managed to operate at relatively lower cell densities than in PBRs, although both systems still maintain an artificially high density of algae in relation to those commonly found in nature. The cell density of algae in open raceway type ponds can often reach several million cells / ml in order to maintain a high biomass of algae to supply a continuous stream that can be harvested more effectively. The density of algae grown in raceways type ponds is often high enough that the algae respond in unforeseen harmful ways, for example through quorum sensing system where the algae join to form flocs that suddenly settle in the ponds (Taraldsvik & Myklestad, 2000).

These commercial systems aim to maintain high biomasses of tens of grams of algae per cubic meter (compared with natural biomasses of less than 10 grams per cubic meter). In order to achieve the above, algae culture will be performed at abnormally high nitrate nutrient levels of 400-800 mmol or higher based on which higher nutrient concentrations will allow higher cell densities and therefore higher production rates per unit area. However, the disadvantage of these systems is the high cost, which is a combination of the cost of the nutrients added to the cost of the land and equipment used. In systems, the nutrients required to create a ton of dry algal biomass can cost $ 440 / ton or more.

However, high cell densities provide additional disadvantages that need to be addressed in these systems. One of the disadvantages in treating with the oxygen produced. While photosynthetic algae produce oxygen and at high cell densities, the concentration of oxygen produced during daylight hours quickly becomes toxic to cells and needs to be actively removed. In addition, during daylight hours, carbon dioxide is consumed during photosynthesis and needs to be actively replenished, which in turn creates the need for pH control. Therefore, there is a need, when using high cell densities, to actively manage the growth environment of the algae.

An additional disadvantage is the greater absorption of light per unit of water resulting from the use of high cell densities. This leads to rapid increases in temperature and requires exposure of the alga to light in thin layers to ensure that enough light penetrates the water to support photosynthesis. Therefore, a cooling system is required to treat the increase in temperature, which adds to the cost of these high cell density methods.

To date, all artificial attempts to create more efficient algal growth have not come close to the efficiency of light, nutrients and exploitation of resources or quantum yield of photosynthesis recorded during these blooms of natural algae. We believe that the reason is that these systems have been developed to optimize environmental parameters that are not related to these flowering conditions. Instead, much consideration has been given to developing growth systems that address other constraints. For example, systems have been designed to grow algae with high lipid content, on expensive land (eg, California) or high isolation environments or with low water levels (eg, Arizona), in wastewater, produced water ( saline water from mining activities or fresh water (Sheehan et al., 1998) High cell densities to reduce the cost of harvest, very low tanks to avoid the own shade and promote the exchange of gases, conditions of continuous growth , continuous harvesting to effectively use the harvesting equipment and the addition of titrated nutrients have been fully developed by engineering to maintain control of these growth systems.In addition, these growth systems are populated with few sets of resistant organisms (eg , Dunaliella salina, Haematococcus sp.) That can tolerate high temperature and / or hypersaline conditions that occur in these systems. two.

Thus, there remains an urgent need to develop a method and apparatus for the efficient mass cultivation of algae that is commercially viable and both simple and inexpensive.

BRIEF DESCRIPTION OF THE INVENTION The present inventor has concluded that many problems that are associated with the present methods and apparatus for growing algae can be overcome by cultivating the algae under specifically controlled conditions in which the algae are subject to physiological changes such as those experienced during natural algal blooms.

In a first aspect, the present invention provides a method of algae culture, wherein the physiological state of the algae is manipulated by altering one or more environmental parameters in order to stimulate the conditions of flowering formation. Therefore, the method manipulates the physiological state of algae grown in raceway ponds by altering one or more environmental parameters to simulate flowering formation conditions. In particular, the methods of the invention simulate the quantum yield of photosynthesis observed in natural algal blooms.

This is done by changing the behavior of algal cells based on certain stimuli, for example, nutrient levels, light, color of light, temperature and gas composition signals. Raceway ponds and the way they are operated represent a mechanism to create external signals that allow algae cells to feel, balance and acclimate to these signals. When handling the physiological state of the algae in this way, acclimation results in beneficial states of exponential cell growth and its cellular aggregation corollary. The growth state of the algae is artificially controlled by manipulating the physiological state of the algae as a result of the rhythmic "variability" of the environmental signals that the algae receive.

The approach of the present invention is to manipulate the behavior of algae and their response to external stimuli within a single species to allow the effective use of resources (yield) as well as to induce a state that leads to aggregation. This is achieved primarily by manipulating the physiological (biochemical) response to the inputs (nutrients and physical stimuli) available (regardless of how these environmental factors are controlled). The invention recognizes that each species of alga is soaked in cellular machinery (such as light harvesting complexes, carbonate import, nutrient transport, circadian cycle control and others) that has slight differences and that can be activated or deactivated within a single cell or an assembly of a species. This cellular machinery is flexible and dynamic and can create a "gap" in the state where the speed of growth - and the efficiency of the "quantum performance of photosynthesis" of machinery - changes the behavior of each cell so that Collectively, cells outperform competition with other organisms. The invention provides a platform where environmental conditions (although controlled) induce these state changes, from stationary to flowering or high yield growth. An advantage of the method of the invention is that the generation and maintenance of exponential and synchronized growth can be achieved without the addition of supplementary nutrients, with the additional nutrients being those already present in the algae culture medium provided (such as water from sea). Because no supplemental nutrient is required, the cost of this method of algae culture is significantly less than that associated with traditional algal culture methods.

The cell culture of algae can be separated into four phases: adaptation, stationary, exponential and senescence. During the adaptation phase, the cells may be alive, but in a state of inactivity where their low metabolism is transformed to a very low level to maintain basic cellular functions during unfavorable conditions. Cells do not multiply and often exist in stored reserves or have formed protective cysts to transit suboptimal conditions. Even when the conditions are sufficient to support the culture again, the cells are inactive in their response, because they have to synthesize the components necessary cell phones to allow them to photosynthesize and divide again.

During the stationary phase, cell deaths are roughly equivalent to cell replication and the total number of cells remains more or less stationary because the cells maintain a small and resilient population. Because seasonal and species successional events are usually short-term, most algae spend most of the year in this stationary phase. An advantage of this phase is that the cells have the necessary machinery to feel the "ideal" conditions for their growth. When these ideal conditions are present, some species of algae can transition to a phase of accelerated growth, where the cells divide as often as possible and the cellular machinery focuses predominantly on cell replication.

It is during this period of exponential growth that the population "blooms" and achieves a temporary imbalance where the prey of the predator or the dynamics of competition inter species for example are deactivated. The cells replicate faster than their predators can consume them and the spice begins to dominate the water column. For example, Irigoien (2005) interpreted flowering conditions as physical or chemical disturbances that they interrupt the predator-prey controls that normally operate at the level of the microbial ring, opening 'gaps' in which some populations of phytoplankton species can be fired. This 'pond' means that algae cells are growing faster than predators or competitors can feed on them or interrupt their growth. During this temporary population explosion, cells that have been present in small numbers mixed with other species, grow rapidly and dominate the local environment, overtaking other organisms in competition and numerically exceeding other species in the water column.

This explosion of population is characterized in the present by three phases of growths which are growth phases artificially induced in the method of the present invention. 1) Exponential growth - where algae cells divide regularly near their maximum growth rate for 10-40 generations. This is a specific relative measurement of the species and refers to the difference in the growth rate, in relation to the stationary growth phase or adaptation for the particular species. Some species will have growth rates of several divisions per day, while others will divide only once every 2-3 days. During the flowering events, the species of algae that is blooming is usually growing faster (dividing more frequently) than most of the other species present. Most of the species considered here will have an exponential growth rate of 0.8 - 3 divisions per day. 2) Synchronized growth - where most algae cells make the transition through the cycle states of the cells in parallel and divide approximately at the same time. Entering synchronization usually requires that cells be temporarily stopped at a specific stage of the cell cycle dependent on an external environmental signal. When the signal is communicated, the cells simultaneously advance to the next stage of the cell cycle and the cell division becomes synchronized where the cells divide into a very narrow time slot (for example, within one hour, two hours before the sunset). In the context of the present invention, "synchronized growth" means that most cells divide and align through the different stages of cell division. The "majority of cells" means that preferably 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 95% -99% or more of the cells are aligned through the different stages of cell division. Alignment through the different stages of cell division means that the cells are divided same time, considering the "same time" as the one that falls within the period of approximately 10% of the time taken for a growth cycle. For example, algae cells with a 24-hour growth cycle are considered to be aligned through different stages of cell division and to be divided within approximately 2-3 hours of each. Taking the same example, that most cells are synchronized means that at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 95% -99% or more cells are split within 2-3 hours of each in a 24-hour growth cycle. Synchronization in an active process and requires cell cycle signaling and control mechanisms and the ability to sense the environmental factors that result in synchronization. This is fundamentally different for a cell population that is passively responding to the availability of external resources (such as light or nutrients) where the stages of cellular cycles essentially form a Gaussian distribution and the cells are divided over a wide period of time throughout the growth cycle. 3) Flowering phase - is the stage where the population "explodes" and grows from a small original population mixed with other species, to dominate the water column and interrupt the normal predator-prey relationships and resource competition. The above is important for three reasons, 1) during the flowering phase, algae cells exhibit the highest quantum yield of photosynthesis or convert solar energy into chemical energy in the most efficient way; 2) during the flowering phase, the use of nutrients is mainly channeled towards growth and cell growth is relatively more efficient than during the stationary growth or adaptation phase; and 3) during the flowering stage, algae cells grow more, compete with other species and predators and dominate the water column reducing the need to control other species in the open-flow, open-pond system, which operates with natural sea water.

Achieving the flowering phase, with exponential and synchronized growth, becomes a key differentiator of the present invention and allows a commercially significant advantage compared to other algal culture methodologies. The invention replicates, under artificial conditions, this state of "flowering", deliberately inducing the flowering state (and optionally, the transition to cellular aggregation), where the state of exponential growth achieved is a unique condition that is a high yield and overwhelms pollutants and predators through a high rate of algae cell growth.

Preferably, according to the invention, the Maintenance of exponential and synchronized growth can be achieved without the addition of supplemental nutrients, namely nutrients that are additional to those already present in an algae culture medium. Because no supplemental nutrient is required, the cost of this method of algae culture is significantly less than that associated with traditional algal culture methods.

The natural blooms of algae on a large scale are well known and regular phenomena in highly diverse aquatic environments. A flowering of algae occurs when a species of algae increases drastically in numbers so that large areas of the ocean are numerically dominated by algae. In comparison with the PBR cultivation systems and artificially controlled raceway-type pond described here, these natural algal blooms are formed in complex and highly dynamic environments, in a scenario of many other competing species and predators. Even when conditions are favorable, algae grows exponentially, often doubling daily for two or three weekly periods (Platt &Subba Rao, 1970) and results in a "bloom" of specific species of algae above and beyond their abundance normal According to the invention described herein, it is It is important to understand how these natural events occur. There is a small but it means iva variability in the ocean on a scale of centimeters or less (in addition to the obvious big currents). These small niches provide an opportunity for some algae to grow free of predators, competitors or diseases (eg, viruses or bacteria). In a sense, individual seaweed species are waiting to operate in a stable survival mode until they feel a balance of multiple environmental factors to which they are adapted to exploit (here adaptation means selection by evolution, not acclimation to variables such as light conditions high or low). When the appropriate conditions are met, then the algae completely change their cellular machinery, behavior, growth rate and even structures to exploit these conditions. For example, after the diatoms have removed the nutrients from the surface waters, the flowering dinoflagellates will actively migrate between the mixed water depth (relatively dark) where the nutrients / are higher and absorb the nutrients at night, then migrate (swimming or floating) to surface waters to make photosynthesis during the day. These dinoflagellates do not respond simply to low nutrient conditions, but that instead respond to a balance of low nutrients, water column mixture (turbulence), temperature, light availability and, in some cases, chemicals emitted by the previous species. So it would not achieve much by adding more or less nutrients in this situation.

There are many "transactions" like this: they depend on the appropriate use of light, temperature, nutrient, turbulence signals to achieve the desirable state of flowering induction. What the present invention provides is a system that reproduces these favorable conditions for algae; We are looking for both a state of reducing pollutants and high performance.

It should be noted that the period of exponential cell growth results from fundamental physiological changes in algae cells. During these flowering events, cellular physiology optimizes growth and maximizes the quantum yield of photosynthesis, the efficiency of converting light into biomass at near its theoretical maximum level of 8% for a prolonged period during the day (Maranon 2005). This temporary effectiveness of converting solar energy into chemical energy is a key component that allows algae to grow exponentially. During the time of exponential growth, these algae grow at a faster rate than at any other time in their life cycle (Barber et al., 1996). These reach the high rate of photosynthesis during a given moment of the day where the algae clearly distinguish the stages of photosynthesis, growth and synchronized cell division during the daylight cycle. The algae exhibit a rate close to the maximum quantum yield of photosynthesis, throughout the morning and early afternoon (Babin et al, 1995), while during the afternoon and at night, the cellular machinery is geared towards the growth and cellular duplication. Therefore, during the exponential growth phase, algae cell division is actively aligned to induce diurnal rhythmic behavior (Bruyant et al., 2005) where photosynthesis is maximized during the most powerful hours of daylight and daylight hours. The cells synchronize both cell division and their photosynthetic machinery. In this study, cells achieve maximum photosynthesis from dawn to 2 PM in the afternoon. After the period, photosynthesis declined as the cells prepared for cell division within the hours before sunrise the next morning.

Not all algae cells will transition to the exponential growth phase under ideal conditions. The transition to the exponential growth phase is an active process that requires a change in cellular physiology, for example, changes in the photosynthetic machinery.

During flowering formation conditions, algae experience exponential growth and are growing in synchronization - referred to in the present "synchronous growth". The above can be seen in diatoms that form chains where the adjacent cells are tightly aligned during the different stages of cell division. This synchronization persists while the algae are in the exponential phase (that is, during the flowering period). Because the individual cells in these chain-forming organisms are bound together, it can safely be conjectured that all of the clonal cells have experienced the same environmental conditions, including light, nutrients and temperature. There is no difference how the cells have been cultivated in relation to the others.

The above can be seen in the samples of March 14, 2013, in the Bay of St. Helena in South Africa, with a magnification of 160 times. The photomicrographs (Figures 3 and 4) show two different chain-forming diatoms where all the cells have been subjected to exactly the same environmental conditions by virtue of being connected to each other. In the first image (Figure 3), Chaetoceros sp. of a cosmopolitan chain-forming diatom are in a variety of clearly visible cell division stages. While this seaweed is By growing, the individual cells are not synchronized or growing exponentially which is consistent with the growth of the stationary phase. During the sampling period of two days, these cells were declining numerically in relation to other cells in the water column. It should be noted that some indicators of physiological status (eg, fluorescence versus pigmentation) and growth phases can be measured by automated underwater measurements in individual cells, in itself. In addition, this is a large and robust candidate species with long intercalated beards (curved silicate spines protruding from the axis of the cell chain) and an organism that forms transparent exopolymer particles (TEP), where both they could be used to assist in the harvesting of algal biomass because of the numerous junctions and the ability to form marine snow.

In the second image (Figure 4), the cells of Pseudo-nitzschia pungens, another chain-forming diatom, also with a 160-fold enlargement of the same sample, taken at the same time, is demonstrating both synchronized growth and numerically abundant. These cells were increasing in the water column at a rate that was commensurate with the exponential growth in the early stages of a small local bloom (during the sampling period of two days, cells increased 4-6 times). This algae can form domoic acid (a potent neurotoxin) in the stationary growth phase and the mobile chains can physically bind to substrates and slide along these substrates, making them unattractive species in the context of animal feed production . What can be seen clearly in the photograph is the symmetry of the two chloroplast plaques in each cell, which lie along the ribbon, where each lies on each side of the median transapical plane, indicating the same stage in the cell cycle, while these cells are in a synchronized life cycle and were not in a variety of cell division cycle like the other organisms.

What these images show is an example of how different algae species can coexist and how they can experience the same environmental conditions and as a result of subtle differences in their physiological state, they can respond in very different ways. The Chaetoceros sp. is surviving, while the Pseudo-nitzschia sp is struggling and its photosynthetic machinery is geared towards maximum growth.

As illustrated in this example, when these organisms are not flourishing, cell division is not synchronized. The observations show that during the rest of the year, when the conditions are not ideal, the cells are divided individually and different parts of the chain are dividing at different times, or in other words, the cells are not synchronized. This is an indicator that the cells are in a different growth mode, like the stationary phase. This stationary phase is important from the point of view of keeping the cells in the water column through unfavorable seasons, in advance of the appropriate environmental conditions to present themselves.

Instead of being a passive process that simply happens because it happens to coincide with the timing of inputs, the cellular synchronization in an active process that requires the cells to feel the ideal conditions, including the concentration of nutrients, light and mixing regime. The cells actively maintain the machinery to sense the signals and then change their biochemical composition in order to grow and compete differently. The invention exploits these physiological mechanisms to achieve the synchronous growth of algae.

For example, three different mechanisms (and costly from the cellular perspective) (only for the sensitivity of light) have been developed to help measure and respond to the available light field. In nature, the total photoperiod (the daily hours during which the cells) depends on the geographical location of the cells, weather conditions, water turbulence, season, depth, water clarity and cell density. Some are even sensitive to the variation in the light of the moon. Therefore, if cells are simply cultured in the same photoperiod, they will not automatically fall into synchronous growth; the opposite may occur. For example, if the algae are grown for an inappropriate amount of time, with an inappropriate amount of light or an inappropriate light color, they will desynchronize and return to stationary growth, where the cells are divided at different rates, some are inactive, some continue to grow and some grow much in advance of a better environment to divide; in this way, the cells are no longer synchronized. So that more or less light provided at the same time does not necessarily result in the same cell growth; Active steps are required to achieve synchronous growth. In the system of the present invention, a system is described that changes the rate of growth, changes the physiological state, changes the concentration and proportion of biomass, to intentionally cause repeated undulations in the temperature and nutrient regime in order to induce a behavior favorable in the algae. In one method, fresh and cold seawater is used from the deep sea (for example, from a depth under the depth of the mixed layer, the nutrilin, the pienoclina or the euphoric zone depending on the local hydrography), to mix it with warm surface water to achieve this effect.

As the expert reader will appreciate, this cell synchronization process differs between different types of algae. For example, in some algae, proportions of red or faraway red light, or proportions of blue to green light indicate the appropriate seasonal conditions to induce synchronization. However, in most algae species, forced exposure to light (the process of exposing all cells to the same light: dark cycle) is not sufficient to induce synchronization. In these algae, forced exposure to light is necessary, but it is not sufficient to induce synchronization.

Synchronization requires a complex machinery that works within and between individual algal cells. Complex dynamics, as population cycles, may arise when the individual members of a population become synchronized. Even cycles driven by synchronization in unstructured microbial populations may occur. Some unicellular phytoplankton species exhibit regular, inducible and reproducible population oscillations. In these organisms, measurements of the size distribution of the cells reveal that the progress Through the mitotic cycle it is synchronized with the population cycles. A study by Massie et al.2010 indicates a strong connection between complex processes within cells and population dynamics. Massie's mathematical model, which represents both the cell cycle process and the population level, suggests that cycles occur because individual cells become synchronized by interacting with each other through a set of common nutrients. An external disturbance by direct manipulation of available nutrients results in a phase readjustment, unmasking intrinsic oscillations and producing a transient collective cycle as individuals gradually distance themselves. Precisely, they are manipulations of this type, such as through the programmed and dosed addition of seawater, which allows the method of the present invention to deliberately induce, maintain and stop cellular synchronization. By achieving these effects, this method allows the control of cellular physiology in such a way that a highly productive growth is achieved. For example, this can be achieved by using cells to inoculate that have passed through a programmed nitrogen depletion that stops their cell progression in the nitrogen-sensitive phase of their life cycle, in this way it is possible to start the production phase with cells that They are already in sync.

The synchronization of cells, achieved in accordance with the teachings herein can be evaluated through the determination of what is the state of the cell cycle in which the cells of the algae are. Individual states can be determined through methods such as specific DNA stains (eg, Highest PointGreen®) in conjunction with flow cytometry (Hy a et al., 2012). If it is found that most of the cells are in the same state of the cell cycle, then the cells can be considered to be synchronized. As stated above, the "majority of cells" means that preferably 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 95% -99% or more of the cells are aligned through the different stages of cell division. Alignment through the different stages of cell division means that cells divide at the same time, considering the "same time" as falling within the period of approximately 10% of the time taken for a growth cycle. For example, algae cells with a 24-hour growth cycle are considered to be aligned through different stages of cell division and to be divided within approximately 2-3 hours of each. These measurements can also be automated and conducted in situ (inside the pond) using submersible flow cytometers for individual cell imaging as FlowCytobot (Olson and Sosik, 2007) that after the classification can differentiate the growth stages of the algae optically. In addition to the light field, other environmental signals may indicate that growth conditions are ideal, and species will transition to exponential phase growth. These include the availability of nutrients, temperature and turbulence, for example. When all these conditions are correct, the cells respond to these signals and grow at an exponential rate, as opposed to survival in the stationary or adaptive phase. It is crucial to note that these conditions differ between algae species. While some species bloom early in the season when storms mix deep nutrients to the surface, other species follow later, when the water column is still rich in nutrients but more stable. Some species can fight under conditions where in the surface water the nutrients are depleted and actively recover nutrients from the depth at which they arrive at night swimming or submerging at the appropriate speed. These differences in growth conditions create common patterns of species succession (Smayda 1980). What the present invention achieves is the ability to recreate these conditions on land, that is, in culture ponds, to replicate the conditions that induce the cells to grow exponentially.

For some species, such as diatoms, algae cells undergo ocean-water column mixtures driven by winds, while for other species, such as dinoflagellates, these flowering events are combined with daily migrations to deeper, nutrient-rich waters , during the night (Epplcy &Harrison 1975), followed by migration to lower depths where there is greater light availability for more effective photosynthesis (Kiefer &Lasker 1975). This has important consequences, since the cells can be impelled to have an asynchronous vertical migration that maintains the flowering growth for a prolonged period of time. This cellular migration also positions the cells in the light field and nutrient regime optimally appropriate for a physiological state of a low nitrogen-to-carbon ratio that maximizes cell mass while maintaining exponential growth (Ralston et al., 2007) . Eventually, these favorable natural conditions change and the blooms collapse due to nutrient limitation, a change in environmental conditions or predators.

The response to external stimuli such as salinity, temperature, nutrients, light or population dynamics change the physiology and behavior of algae. The The answers depend directly on the intensity, frequency and combination of these environmental factors (Feng et al., 2008). Flowering-forming algae have the ability to integrate the signals they receive from these environmental signals and acclimate to flowering support conditions (Sanudo-Whilhemy et al., 2004). For example, the nutrient physiology of flowering-forming diatoms (Pseudo-nitzschia sp.), Rafidophytes (Heterosigma akashiwo) and dinoflagellates (Alexandrium sp. Ceratium sp.), Favors the rapid intake of nutrients (species with high affinity for nitrogen). ) unlike many similar non-flowering species in the same genus (Kudela et al., 2010). Between blooms, these flowering species can change to a completely different physiological mode, where these species have a much slower kinetic of nutrient intake, similar to that of other non-flowering species in the same genus.

In one embodiment, the timing of the alteration of one or more environmental parameters is programmed to coincide with a particular point in the algal cell cycle in order to stimulate the conditions of flowering formation. Under the conditions of flowering formation, the growth of the algae is approximately exponential. Under conditions, the remote population is preferably growing and dividing in a synchronic way - this is called in the present synchronous growth. As used herein, "synchronous growth" means that most cells divide and align through the different stages of cell division. The "majority of cells" means that preferably 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 95% -99% or more of the cells are aligned to through the different stages of cell division. Alignment through the different stages of cell division means that cells divide at the same time, considering the "same time" as falling within a period of about 10% of the time taken for a growth cycle. For example, algae cells with a 24-hour growth cycle are considered to be aligned through different stages of cell division and to be divided within approximately 2-3 hours of each. Taking the same example, most cells that are synchronized means that at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 95% -99% or more than cells) within 2-3 hours of each in a 24-hour growth cycle. The synchronous growth of the algae is discussed above and can be achieved and monitored as described herein or by any method known in the art (Tamiya et al., 1953, Cattolico et al., 1976). Specific examples of conditions Suitable embodiments are provided in the detailed works of the invention described herein, generated by modeling. Other equivalent mechanisms will be apparent to the skilled reader, imbued with the teachings of the present invention.

For example, the alternation of one or more environmental parameters can be programmed to coincide with a particular point in the algal cell cycle by timing the alteration to coincide with a particular time of day, as described herein. The alteration of one or more environmental parameters in a specifically programmed manner can be used to induce synchronous cell division. The algae can then be cultured to maintain synchronization of cell division by providing programmed alteration of one or more environmental parameters at regular intervals to retain the physiological state of choice.

Regular intervals can be programmed to coincide with points in the day, such as sunrise, sunset or a predetermined time before or after sunrise or sunset. In an alternative mode, regular intervals can be programmed to coincide with particular points in the cell cycle or a particular growth state. In one embodiment, the growth state is characterized by the carbon: nitrogen cellular relationship.

In specific modalities, one or more of the following parameters are altered: to. intensity of light; b. light color relation (ie ratio of the wavelength of light); c. Nutrient concentration; d. nutrient balance, - e. water temperature; F. density of algae cells; g. concentration of dissolved gases; I h. dissolved gases ratio.

By manipulating the physiological state of the algae, it is meant that the alteration of one or more of the light intensity, wavelength ratio of light, concentration of nutrients, nutrient balance, water temperature, cell density of algae, concentration of dissolved gases and / or dissolved gas ratio alters the physiology of algae. The term "one or more" includes, 1, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or all of the 8 parameters listed herein. The physiological characteristics that can be altered include growth rate, cell division rate, photosynthesis rate, cell cycle position, protein synthesis rate, nitrogen fixation rate and synchrony of cell division in a population of algae cells .

Specifically, the desired physiological state is the maximum yield of the photosynthetic quantum, low nitrogen-to-carbon ratio, exponential growth rate temporarily experienced by flowering-forming algae during natural flowering conditions.

Many types of algae can be grown using the methods of the invention. Thus, algae can be grown in any type of water. In specific modalities, the algae are cultivated in seawater, hypersaline water, brine for desalination, brackish water, wastewater or fresh water. The term "water" covers all types of water. The term "freshwater" (as opposed to watermelon) is used to refer to water that has not previously been used as an algae culture medium in the methods of the invention and encompasses fresh seawater, hypersaline water, brine for desalination , brackish water, wastewater or fresh water. Thus, fresh water has not been recovered or recirculated from any other part of the pond system after harvesting or any other operation of the ponds. According to one embodiment of the present invention, the dilution of cultured algae preferably involves the use of fresh water (including any of sea water, hypersaline water, brine for desalination, brackish water, waste water or fresh water). In this way, the environmental parameter of the cellular density of algae can be manipulated In the present invention, the algae are grown in raceways type ponds, which can be completely open to the environment. Modifications to the ponds can be made to alter the algae growth environment in order to recreate the environmental conditions experienced by the algae during natural blooms, including the intensity of light and color, the color or type of pond lining raceway type, mixing regimes, variability and availability of nutrients, water temperature, cell density of algae, availability and variability of dissolved gases and other conditions that stimulate an environment formed by flowering.

In one embodiment, the modification made to the raceway type pond is to the depth and / or width of the channels of the raceway type pond. For example, the depth and / or width of raceway type ponds may be non-uniform in order to modify the rate of flow and mixing in the raceway type pond. The variable depth can also be used to alter the penetration of light of different wavelengths of light, thus altering the intensity of the light and / or the wavelength ratio of light experienced by the algae.

In another modality, the rate and moment of the addition of Fresh water can be controlled, and in particular deep, cold, fresh seawater, to replicate the fluctuations of nutrients and temperature experienced by natural algae while they mix or migrate between the cold waters replete with nutrients and the surface waters with their exhausted nutrients.

The raceway ponds in the invention can be coated and / or covered. A coating or coatings used in the present invention can be of different colors to manipulate the wavelength ratio of the light by the algae. A cover or coatings used in the present invention may be of different colors and / or opacity to manipulate the intensity of the light and / or wavelength ratio of the light experienced by the algae.

In the context of the present invention, the monitoring and manipulation of nutrient availability and the ratio of particular nutrients is important to evaluate and modify the physiological state of algae. The carbon to nitrogen ratio in the cells is a useful indicator of the ability to fix new carbon. Thus, the term "nutrient balance" includes the carbon to nitrogen ratio in the algal cells.

In the context of the present invention, the monitoring and manipulation of the concentration and relation of dlSUeltOS gases in the culture water of the algae is also important. In particular, the rates of growth and photosynthesis are strongly affected by the ratio of oxygen (O2) and carbon dioxide (CO2). Thus, the term dissolved gases includes dissolved O2 and dissolved CO2 among others. The ratio of dissolved gases can be the ratio of O2 to dissolved CO2.

Nutrient concentration, nutrient balance, water temperature, algae cell density, concentration of dissolved gases and / or dissolved gas ratio can be manipulated by adding fresh water to raceways. The moment and rate of addition of fresh water is important in the manipulation of the physiological state of the algae.

The timing of the changes for either light intensity, light wavelength ratio, nutrient concentration, nutrient balance, water temperature, algae cell density, concentration of dissolved gases and / or gas ratio dissolved can be fixed in relation to a particular hour of the day, for example, dawn; dusk, a predetermined number of hours before or after sunrise or sunset; the highest point of solar irradiation; or a predetermined number of hours before or after the highest point of solar irradiation time. Alternatively, the moment of the changes to any of the intensity of the light, ratio of wavelength of light, concentration of nutrients, nutrient balance, water temperature, cell density of algae, concentration of dissolved gases and / or ratio of dissolved gases can be related to a point in the cycle cellular of the algae or in relation to a particular physiological state such as the ratio of carbon to nitrogen or rate of photosynthesis.

Thus, the algae grown according to the method of the present invention dynamically transitions between multiple physiological states to achieve a high yield of the quantum of photosynthesis, high use of nutrients and light, high efficiency in growth, prevention of soiling , resilience to competing organisms and ease in the harvest. In contrast, current methods of algae cultivation aim to maintain the algae in a constant and static productive state, to allow the high density growth and continuous harvest of the resulting algae.

The use of cells for inoculation that are already in synchronization of growth is achieved through the deliberate cultivation of these cells so that the cells become synchronized when nitrogen depletion is used to stop cell progression in the nitrogen-sensitive phase in their cellular cycle. The above can be achieved by increasing the cell density to a level that exceeds the availability of nitrate in natural seawater. Then, these cells are diluted with fresh seawater in a rhythmic and regular pattern, for example, by the scheduled addition of seawater 1-22 hours before cell division to maintain synchronization.

In a specific embodiment of the present invention, the algae are grown in a series of connected raceway ponds arranged in stages, where the highly productive exponential growth phase of the algae is maintained by successively diluting the growing algal population either before or when it is transferred between the stages in the series of raceway type ponds. These repeated dilutions maintain a low cellular density of the algae, overcoming the problems associated with traditional high-density algal culture methods and maintaining the growth of algae in a series of batches.

The timing of the dilution of the algae and the time of the transfer of the algae to each subsequent stage of the ponds in the series can be timed to coincide with a particular hour of the day, a particular phase in the algae's cell cycle or an indicator such as cell size in relation to a particular physiological stage of algae. Timing the dilution of algae in this manner and / or timing the transfer of algae in this manner can be used to initiate or maintain cell division synchronous The structure of the ponds can thus be a vehicle to achieve the desired state transitions (stationary to exponential growth, exponential growth to aggregation and senescence). The structural characteristics of the ponds provide a mechanism to achieve the purpose of inducing the state of flowering (efficient in terms of photosynthesis).

The desired growth of the algae can be achieved by diluting the ponds that support the first 12-22 cell divisions with natural seawater that has not been enriched with nutrients. This can only be achieved if the biomass of the algae is relatively low because otherwise the growth of algae becomes limited in nutrients. In low concentrations of algae, the concentrations of nitrate and carbonate environment found in deep sea water are sufficient to maintain the exponential, synchronized, high-quantum growth. To exploit these natural nutrients, algae can be grown without addition of nutrients for the first 12-22 cycles of growth while diluting with fresh seawater at a rate programmed for the cell division cycle. For example, with an initial load of 25,000 cells / ml, at a growth rate of 0.9 divisions cell phones per day, and a growth rate of 0.5 cell divisions per day and a maximum nitrate load of 40 millimoles per m3 found in deep seawater (practically 35 millimoles of nitrate per m3 can be pumped profitably) and a carbonate load of 2,120 millimoles per cubic meter, to water of algae culture already in the ponds, this system avoids the need for addition of nutrients until the division stages before harvest, resulting in a general reduction of the cost of nutrients to < $ 5 per ton of biomass produced. In comparison, current commercial systems require approximately $ 440 or more per tonne of biomass.

In a specific embodiment of the invention, the algae are grown in a series of connected raceway ponds arranged in stages, where the series of connected ponds comprise - first, one or more stages of covered raceway ponds; Y - second, one or more stages of open raceway ponds; where at each successive stage in the series, raceway ponds have a larger collective volume than, for example, at least twice, at least three times or at least four times greater than, the collective volume of raceway ponds of the previous stage; and where the raceway type ponds in each successive stage of the series are sown by transferring algae grown in the raceway type ponds of the previous stage of the series so that the algae are diluted when transferred between stages in the series. In specific modalities, the algae are diluted by a factor of at least two, at least 3 or at least 4 when transferred between stages.

In a second aspect, the present invention provides a series of raceway-type ponds connected to cultivate algae arranged in stages, where the series of connected ponds comprises - first, one or more stages of covered raceway ponds; Y - second, one or more stages of open raceway ponds; where at each successive stage in the series, raceway ponds have a larger collective volume than, for example, at least twice, at least three times or at least four times greater than, the collective volume of raceway ponds of the previous stage.

In this embodiment of the invention, the methods and series of connected raceway ponds benefit from the entire growth cycle of the algae. The algae are first cultivated in covered raceway ponds, where the algae are diluted successively and where they make their transition to the exponential and synchronous growth phase.

The covered ponds allow the control of the algae culture medium and the environment, for example, temperature, light intensity and / or wavelength ratio, adverse climate and gas exchange. Once the algae are in this phase of highly productive exponential growth they are transferred to open raceway ponds where the algae grow rapidly. In order to keep the exponential phase highly productive, the algae are diluted successively. Successive dilution can be achieved by increasing the volume of water in the current stage of the raceway ponds or by transferring the algae to the next stage of the ponds in the series, thus diluting the algae by sowing a larger volume of ponds and filling the ponds to its capacity by adding fresh water. This maintains a low algae cell density in raceway ponds which allows algae to sustain much higher growth rates than traditional systems, and preferably allows algae to remain in the exponential growth phase. Alteration of algal cell density can also be programmed to mimic the conditions found in natural oceanic blooms.

The present invention may also comprise a step of harvesting the algae. Once the algae have reached maturity or the stage in which the harvest of algae is required, the environment of the algae is modified to induce a change in the algae from the exponential growth phase to the stationary phase. This is achieved by imitating the environmental conditions under which a flowering of natural algae collapses. The present invention further provides methods for harvesting the algae once they are no longer in the exponential growth phase.

The present invention solves various problems suffered by the current methods of cultivating algae. By avoiding the high artificial growth density environments of traditional algae culture methods, the complex and expensive systems required to maintain the high algal cell densities are not required. Furthermore, by not creating the artificially high growth density environments, the algae grown using the method of the present invention and the series of connected raceway type ponds of the present invention can sustain much higher growth rates than in traditional systems. The methods of the present invention require low capital expenditure and have low operating costs, while producing significant amounts of algae so as to make large-scale algae culture commercially viable.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: A raceway type pond with four channels.

The features include: 1) Controlled gate with gate for entering the pond either from a seawater channel and / or previous ponds 2) Service island and flow diverter 3) Variable speed and depth blade wheels 4) Cross-linking of CO2, countercurrent sink, or Air Sparged Hydrocylone covering both channels of each pond 5) Plastic walls or berms covered or reinforced to form the walls of the pond and channel divider 6) Island for flow diversion to maintain laminar flow 7) Changing from pond to pond controlled by door for drainage and cleaning 8) Tank discharge through gate-controlled gate Figure 2: a series of connected raceway ponds arranged in stages according to the present invention. The outline summarizes: • Seawater pipeline - to have access to deep water offshore full of nutrients • Elevated inlet channel filled from the seawater pipe with high speed, low load pumps • Covered sequential seeding ponds filled with filtered seawater to grow inocula for culture ponds • Secondary culture ponds, not covered connected to and filtered from previous ponds plus fresh seawater from the intake channel • Land, desert or coastal land near the unused ocean.

• Harvest ponds filled with the middle of growth ponds • Low elevation discharge channel at a maximum distance from the entrance Figure 3: Photomicrograph of cells from a cosmopolitan chain-forming diatom (Chaetoceros sp.) In a variety of clearly visible stages of cell division, with a 160-fold increase.

Figure 4: Photomicrograph of cells of a chain-forming diatom (Pseudo-nitzschia pungens) demonstrating synchronized growth, with a 160-fold increase.

DETAILED DESCRIPTION OF THE INVENTION Raceway type ponds The present invention takes advantage of raceway type ponds. A typical raceway type pond is illustrated in Figure 1. The raceway-type pond is oblong in shape, with a partial division in the middle of the pond (5) (along the longitudinal axis) to create a circuit with at least two channels. At one end of this division, there is a service island (2) that serves as both an anchor for the paddle wheels, the platform for the CO2 equipment and as a flow diverter. At the other end of the division is a second flow diverter (6) to ensure laminar flow along the track. The paddle wheels (3) maintain the flow of water and seaweed around the circuit.

Preferably, the invention takes advantage of open and / or covered raceway ponds. A covered raceway-type pond may be covered by any appropriate means, such as a plastic cover or membrane. In one embodiment, the cover is translucent to maximize solar irradiation for algae. In other embodiments, the opacity of the cover is increased to obscure 10-90%, 20-80%, 30-70%, or 40-60% incident light. For example, the cover may darken to 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the incident light.

In one embodiment, a covered raceway type pond is a raceway-type pond covered by a greenhouse. The primary purpose of the greenhouse is to protect the seedbed algae from being contaminated with pollutants transported by wind or pollutants carried by birds. Second, the greenhouse is used to raise the temperature of the growth of algae; first to increase the growth rate of the algae and second to inactivate competing or harmful organisms that may otherwise contaminate the algae or soil the equipment. The greenhouse can also be used to selectively shade or change the color of the algae lighting to induce the desirable physiological state by altering the wavelength relationships of the light. In a specific modality, each covered raceway-type pond is covered by a greenhouse.

An open raceway type pond has no external cover, and as such, is completely exposed to the ambient atmosphere, while a covered raceway-type pond (which may be fully or partially covered) allows a partial or complete control of the temperature and light environment .

In one embodiment, each covered and / or open pond has two channels. As the raceway ponds increase in volume, they have a greater width and / or length and / or number of channels. In a specific modality, as the raceway ponds increase in volume, they have greater width and length, and the number of channels also increases. An open raceway type pond of the present invention may have 2, 4, 6, 8, 12, 14, 16, 18 or 20 channels (not including the return channel).

In a specific modality, both ponds type raceway, whether covered or open, have an aspect ratio of width to length between 1: 4 and 1:12, preferably, 1: 8. With this relationship, there is a relatively low head loss at each paddle wheel pumping station as water circulates around curves, favorable economy of construction materials (straight walls are easier to construct than curves) , the reduction of the influence of the winds as it blows through the pond (to avoid potentially unmixed areas). The width of approximately 30 meters per channel also reduces the meandering flow to avoid unmixed areas. The cross-linking of CO2 can be provided at regular intervals (Weissman and Goebel, 1987, Benemann and Oswald, 1996).

Paddle wheels can be used to maintain the flow of algae and water around covered and open raceway ponds. This is energy efficient while ensuring complete mixing and exposure of the algae to relatively low shear. This allows an effective exchange of gases within the ambient air with the algae culture medium. According to the cultivated species, paddle wheels of different speeds can be used. The speed of the paddle wheels can be 5-40 cm / sec, e.g.10-30 cm / sec, or 15-25 cm / sec. For example, cells with faster cell division they require a higher speed of pallet wheels of up to 30 cm / sec, while cells like flagellates, with a slower growth rate, prefer a slower speed of around 20 cm / sec. In one embodiment, each covered or open raceway type pond preferably has 1, 2, 3, 4, paddle wheels in the required degree of agitation. Preferably, each raceway type pond has a paddle wheels. The paddlewheel can be positioned in any section of the raceway-type pond, but is preferably positioned near the narrowest end of a raceway-type pond.

In one embodiment, one or more paddle wheels maintain the flow of algae and water within a covered and / or open raceway-type pond at a speed of about 5-40 cm / sec, for example 10-30 cm / sec , or 15-25cm / sec or 20 cm / sec, 25cm / sec or 30 cm / sec.

In one embodiment, a covered raceway-type pond has a volume of between 501 and 15,000,0001, for example, between 50 1 and 50,000 1. In a particular embodiment, the volume of raceway-type ponds covered in the series increases so that there is minus one pond in the series with a volume of (a) 50-1,0001; at least one covered raceway-type pond in the series with a volume of (b) 100 1-5,000 1; and at least one pond in the series with a volume of (c) 5,0001-50,000 1. In one embodiment, these ponds are connected in a linear way, so that there is a pond in each stage in the series.

In one modality, a raceway type pond has a volume between 1,500,000 1 and 3,000,0001, in another modality between 6,000,000 1 and 12,000,0001. In yet another modality, the volume of raceway ponds opened in the series increases so that there is at least one open raceway-type pond in the series with a volume of (a) 360,000 - 720,000 1; At least one pond in the series with a volume of (b) 1, 500,000 - 3,000,0001; and at least one pond in the series with a volume of (c) 6,000,000 - 12,000,0001.

The depth of a raceway type pond affects solar irradiation for algae. The ratio of both the intensity of the light and the wavelength of the light are altered by increasing the depth of the water. In general, the distant red, red and ultraviolet light are the ones absorbed more quickly by the water and a blue and green light penetrate as far as possible. In one embodiment, a covered or open pond has a depth between 0.05 m and 10 m, for example, 0.05 m, 0.10 m, 0.20 m, 0.30 m, 0.40 m, 0.50 m, 0.60 m, 0.70 m, 0. 80 m, 0.90 m, lm, 1.5m, 2 m, 3 m, 5 m, or 10 m depth. In one embodiment, a covered raceway type pond has a depth of 0.lm - lm, for example, 0. 1m, 0.2m, 0.3m, 0.4m, or 0.5m deep. In one modality, an open raceway type pond has a depth of 0.25 or 0.3 m - 1 m, for example 0.25 m, 0.3 -0.4 m, 0.5 m, 0.75 or 1 m depth. Preferably, a raceway-type pond has a depth of less than 1 m. At this depth there is sufficient degassing of 02 to help reduce oxidative stress and, furthermore, at this depth there is sufficient exposure to dissolve the atmospheric CO2.

The open raceway ponds of the present invention can be further modified to maintain the algae in their exponential growth phase. The open raceway type ponds are usually symmetric with channels of identical size. However, the present inventor has discovered that if the width of the channel is significantly different from its adjacent channel, very different flow rates result in the adjacent channels and turbulence within the flow in the channels. In one embodiment of the present invention, the width of the adjacent channels of an open raceway-type pond is not uniform, resulting in a change in the flow rate through the non-uniform section of the raceway-type pond. This change in flow rate mimics the transition of algae in natural blooms from the depth of mixed layers to conditions under the pinocline and back again. This increased agitation of algae also changes the abundance of dissolved gases in the culture medium, encouraging CO2 of the atmosphere to dissolve in the water, and the O2 dissolved in the water to be released into the atmosphere, thus sustaining the growth of the algae.

The raceway type ponds known in the art also have a uniform depth. However, the present inventor has discovered that if the depth of the raceway type pond varies along the length of the channel or between the adjacent channels, a change in the flow rate results. In addition, the exposure of algae to solar irradiation varies, with algae in lower areas of the raceway type pond experiencing greater light intensity and a greater proportion of red light than algae in deeper areas. This variation in depth mimics the natural transition of algae from deep sea water rich in nutrients to provide the appropriate signals to maintain the physiological productive state and to maximize the yield of photosynthesis. The yield of photosynthesis can be affected by the ratio of light, especially at sunrise and sunset, and as discussed in the present, the depth of the water alters the wavelength relationship of light.

Thus, in one embodiment, the depth of an open raceway-type pond is not uniform, resulting in a change in exposure to light both in terms of ratio of light intensity and wavelength of light and / or a change in the gas exchange rate within the section does not uniform of the raceway type pond. In one embodiment, the difference between the depth in the adjacent channels is 0.05-1.0 m, for example 0.05 m, 0.10 m, 0.2 m, 0.3 m, 0.4 m, 0.5 m, 0.6 m, 0.7 m, 0.8 m, 0.9 m 1.0 m, For example, one channel has a depth of, 2-0.5 m, while the adjacent channel has a depth of 0.8-1.0 m. Alternatively, there need not be difference in depth between adjacent channels, but there could be along the length of an individual channel or between channels that are 1-10, e.g. 2-9, 3-8, 4-7 or 5-6 channels away, or specifically, at 2, 3, 4, 5, 6, 7, 8, 9 or 10 distance channels.

As described herein, the raceway ponds of the present invention may be coated. In one embodiment, covered and open raceway ponds are covered with a waterproof material. If seawater is used to grow the algae, a clay coating can be used to prevent the intrusion of salt water into the soil. In one embodiment, a waterproof plastic coating is used in place of or in addition to a clay coating. In a particular embodiment, each raceway-type pond is coated with 10-20 cm of clay and 2-5 cm, for example, about 3 cm or specifically 36 mm, of a robust synthetic coating such as HDPE.

In one embodiment, the color of the pond liners can also be chosen to alter the wavelength ratio of the light experienced by the algae as they reflect the bottom of the ponds. The color of the coating can selectively increase or decrease the exposure of water to light under red water (630-680 nm), far red (700-750 nm) and / or blue (400-450 nm). In one embodiment, the coating selectively increases the exposure of light under red water (630-680 nm), far red (700-750 nm) and / or blue (400-450 nm) by 10-90%, 20- 80%, 30-70%, or 40-60%. For example, the coating can selectively absorb up to 10, 20, 30, 40, 50, 60, 70, 80, 90 orl00% of incident light under red water (630-680 nm), far red (700-750 nm) ) and / or blue (400-450 nm) before the remaining light is reflected back through the algae's growing environment.

In a second embodiment, the coating is white to reflect the background light and maximize the light available for photosynthesis and encourage the growth of the algae especially in low density crops and / or shallow cultures lower than 30 cm depth , where light will penetrate the depth of the medium. Black coatings can be used to deliberately increase the temperature of the culture medium, for example, for the accelerated growth of Dunaliella salina in brine for desalination.

Different coating colors can be used to induce different physiological effects in algae. For example, the coating can be completely red, blue or green. Alternatively, a single stage in the sequence of the ponds may be blue, for example at the point where the seed algae growth has been synchronized in the initial covered ponds, to reinforce the synchronization of cell growth and increase the growth before cell division, just before the cells are introduced into the open culture ponds so that multiple divisions occur in the culture pond. Similarly, the culture pond can be coated in blue to stimulate the migration of chloroplasts outside the cells, to promote maximum photosynthesis (Kraml &Hermann, 1991; Furukawa et al., 1998).

The series of raceway ponds In one embodiment, the present invention provides a series of raceway ponds connected in stages. Raceway ponds are connected in such a way that they allow water and algae to pass directly between raceway ponds in successive stages of the series. However, the connection between raceway ponds may be closed and each raceway-type pond may be an environment of isolated crop. The flow of water and algae between successive stages in the series is unidirectional, that is, the passage of algae and water through the connected series of raceway type ponds is one way and algae and water are never recirculated .

In a specific embodiment of the invention, the series of connected raceway-type ponds of the present invention comprises, first, one or more stages of covered raceway ponds and, secondly, one or more stages of open raceway-type ponds. The designations of "first" one or more stages of covered raceway ponds and "second" one or more stages of open raceway ponds indicate that within the series of connected ponds, stages comprising covered raceway ponds will always come before the stages that comprise open raceway ponds. Put another way, an open raceway type pond will never be succeeded by a covered raceway-type pond.

The present inventor has found that in order to maintain the algae in the exponential growth phase, the algae must be diluted successively in order to maintain a low algal cell density, thus mimicking the natural flowering conditions of the algae and maintaining the cellular physiology that promotes rapid growth. In a preferred embodiment, nutrients are not added supplementary during the initial stages of growth. For example, the cellular density of the algae can be kept below 200,000 cells / ml, for the initial stages of growth, where the initial stages are two-thirds to three-quarters of the generations of algae growth, through repeated dilutions for maintain exponential and synchronized growth without the addition of supplementary nutrients. "Supplemental nutrients" refers to nutrients that are additional to those already present in the algae culture medium. Because supplemental nutrients are not essential during these initial stages of growth, the cost of this method of algae culture is significantly less than that associated with traditional algal culture methods.

Maintaining exponential and synchronized growth in the initial stages of algae culture is preferably achieved without the addition of supplementary nutrients. However, supplementary nutrients can be added in the desired cases. A computational model, as described herein, can be used to determine when nutrient supplementation may be desired. At the end of the growing cycles, for example in the last quarter to third of the generations of algae, supplemental nutrients can be added to increase the cellular density of the algae before harvest.

Successive dilution can be achieved in two ways.

In the first form, dilution is achieved by increasing the collective volume of raceway type ponds in each stage of the series. This increase in collective volume can be achieved by increasing the volume of individual raceway ponds at each successive stage of the series and / or by increasing the number of raceway ponds at each successive stage of the series. Thus, at each successive stage in the series of raceway type ponds of the present invention, each individual raceway type pond has a volume greater than the volume of the individual raceway type ponds of the previous stage in the series; and / or each raceway pond is immediately succeeded by a greater number of raceway ponds, where the collective volume of the raceway ponds at any given stage exceeds the collective volume of the raceway ponds of the previous stage.

In the second form, dilution is achieved by increasing the volume in stages to a maximum volume within a pond, before the water and algae are transferred to a larger subsequent pond. In the mode summarized in the model and table below, each pond is initially filled to a depth of 0.25 m where the cells complete a growth cycle. When it is time to double the volume of the water, to allow the cells grow at a low biomass with natural nutrients, the depth of the same pond is increased to 0.5 m. After the second growth cycle is complete within the pond, the total volume of the two division stages and the seawater are transferred to the larger back pond. In other modalities, the pond is filled, in sequential stages, at 0.25 m, 0.5 m, 0.75 m, and 1 m depth in four sequential dilutions "inside the pond" before it is transferred to the next larger pond.

This "stacking" of ponds is only possible due to the relatively low biomass (or low cellular concentration) of the algae compared to other farming systems. In other commercial algal culture systems (where cells are grown to a density that is between 1,000 - 2,000 mg Chl to nr3), high cell density results in gas exchange and autoshaping limitations. However, in this method, none of these are critical problems, because the cell densities are significantly lower for the first 12-16 growth cycles resulting in 50, 100, 200, 300, 400, 500 mg Chl to nr3). At these concentrations of Chl, ponds can operate without cell shading and the natural ability of seawater to absorb and regulate gases as well as exchange gases with the atmosphere allows cell growth.

Timing the dilution of algae to coincide with a particular time of day or a particular point in the cell cycle or a particular physiological state also mimics the formation of natural flowering. Timing of light intensity alteration, light wavelength ratio, nutrient availability and concentration, nutrient balance, dissolved gas concentration and / or dissolved gas ratio can be used to induce and / or maintain Synchronous cell growth and cell division.

In the context of this invention, the volume of a raceway type pond is defined as its capacity (ie, the volume of fluid that a raceway type pond is capable of sustaining) more than the volume of fluid actually sustained in the raceway type pond at any given moment. When the algae and water are transferred from a raceway-type pond (covered or open) in one stage of the series to one or more raceway ponds (covered or open) in the next stage of the series, either seaweed and The water is transferred to a single raceway pond with a larger volume or the algae and water are divided among a number of raceway ponds with a larger collective volume. Each transfer of the algae and water from one stage of the series to the next stage in the series thus involves dilution of the algae or is preceded by dilution of the algae in the current stage of the ponds.

In a modality, when the algae and the water of a stage in the series is used to seed the raceway type pond or raceways type tanks of the next stage, it is transferred to the raceway type pond or raceways of greater volume in the next stage of the series. Water is added to, or already present in, raceway-type ponds to be planted, so that the final volume of fluid within raceway ponds after sowing is equal to their capacity. This step of sowing results in the algae being diluted and thus a low cell density of the algae can be maintained. Maintaining this constant low algae cell density avoids the problems associated with traditional high-density algal culture, such as quorum sensing, biofilm formation and other algae responses to stress (often unpredictable).

In an alternative mode, the algae are diluted before being transferred to the next pond stage. In this modality, fresh water is added to the current state of the ponds to dilute the algae.

The successive dilution of the algae in each sowing step should not be considered to mean that the cellular density of the algae in each stage of the series is successively reduced. Because the algae multiplies quickly, in spite of the successive dilutions in each step of sowing, the approximate cellular density of the algae in each stage of the raceway ponds passing through the series can increase, decrease or stay the same.

In one embodiment, one or more environmental parameters altered to stimulate flowering formation conditions can be altered based on the predictions of a computational model. For example, one or more of the nutrient concentration, nutrient balance, water temperature, algae cell density, dissolved gas concentration and / or dissolved gas ratio can be alternated based on the predictions of a computational model. These predictions can then be used to alter one or more of the environmental parameters to maintain synchronization in the methods of the present invention.

If a computational model is used to predict the concentration of nutrients and balance required, it can be configured to take into account the natural abundances of nutrients in fresh seawater. In this way, the computational model can be used to calculate the dilution rate for fresh sea water that would provide the desired concentration and balance of nutrients.

A computational model can be used to determine the optimal operation of the pond in terms of the rate of dilution, depth of the pond and addition of nutrients. This determination is based on the predictions of the computational model, where the specific growth and nutrient intake rates of a particular algae species are entered into the model.

A computational model can include cell growth rate, nutrient requirements, pond volume, dilution rate and initial cell load and can be used to guide the dilution rate, the number of cell division cycles required and the nutrient requirements for final stages of cultivation.

One of the computational models, described in the examples herein, is based on the documented nutrient conversion rates for a given cellular load (Pitcher et al., 1996, 1998; Fawcett et al., 2008) in the environment of local production This model calculates the nutrient demand of the cells and matches this demand with the available nutrients in deep unmodified seawater. When the cell density increases beyond the level that can be sustained with unmodified seawater, the model calculates both the demand for additional nutrients and the stages of cell division in which these nutrients need to be added. An operational modality, taken from the model, is that cells that grow at a division rate of 0.9 per day, an initial load of 25,000 cells per ml of the inoculants and a dilution rate of 110% per day requires approximately $ 70 of nitrate supplement per ton of algae biomass produced.

In addition to using this populated model with operational data to advantageously guide the demand for nutrients (eg, nitrate), it can be used to stimulate exchanges between land use (more land requires less nutrients) versus the addition of nutrients ( more nutrients result in a higher production cost).

This model can be used to describe a large order of operational conditions, including cell division rates that vary from 5 times per day to one division every 5 days; initial loading in the inoculants from 1,000 cells per ml to 10,000,000 cells per ml; dilution rates of 300% per crop cycle to 10% per crop cycle: and cell division stages from 1 division to 50 divisions.

Another critical advantage of the model is that it allows the administration of doses of nitrate and other nutrients in each cell division during the productive phase of growth. In a system driven by the model, external nutrients are added to fresh seawater at a rate specified by the model, to ensure that the cells are not limited in nutrients, while maintaining the rhythmic nature of the oscillation of the cell population growth. One advantage of doing this is that the maximum rate of growth and the maximum quantum yield of photosynthesis (or optimal exploitation of sunlight) occurs during periods of synchronization.

Another advantage of the model is that it predicts the demand for nutrients in each cell division and in each stage of production, allowing the administration of controlled doses of nutrients of 0.1 - 1,000,000 moles of nitrate per cell division, event of dilution of seawater or pond. The model is a valuable operational tool for evaluating the tradeoffs between land costs and nutrient costs. In this method, the algae cells are cultured by the initial 12-16 cycles of culture with natural seawater and the nutrients present in this seawater. This allows the scaling of algae biomass to volumes where commercial exploitation becomes attractive. The counterpart is that it requires more land than high density algae farming methods require per unit of biomass produced for these growing cycles. Use the model to identify the minimum necessary soil, while the "stacking" or repeated filling of the ponds in the initial cultivation phases in combination with algal blooming physiology resulting in the efficient exploitation of nutrients and light solar, the model allows a calculation to operate the ponds with the maximum use of natural nutrients and the minimum use of land, to achieve an "ideal point" where resources are used more efficiently.

In a specific modality, there are at least two stages of raceway-type ponds covered in the series of connected raceway ponds. For example, there may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 stages of covered raceway ponds. In one mode, there are 2-10 stages of covered raceway ponds, 4-8 stages of covered raceway ponds or 5 stages of covered raceway ponds. In a preferred embodiment, covered raceway ponds are preferably covered greenhouses and are connected in linear succession, where there is a covered raceway-type pond at each stage in the series of covered raceway-type ponds. In this modality, each covered raceway type pond is at least 2 times, for example 2 to 5 times, the volume of the covered raceway-type pond of the previous stage in the series. In a specific modality, each covered raceway type pond is 2, 3, 4 or 5 times the volume of the covered raceway-type pond of the previous stage in the series.

In a specific additional modality, there are, at least, two stages of open raceway ponds in the series of connected raceway ponds. For example, there may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 stages of ponds Open raceway type. In one modality, there are 2-10 stages of open raceway ponds, 4-6 stages of open raceway ponds or 5 stages of open raceway ponds. In a specific modality, there are more open raceway ponds than closed raceway ponds in the series.

In a particular embodiment, the number of open raceway ponds in each stage increases at each successive stage in the series. In this modality, each raceway-type pond is connected to two or more open raceway ponds in the next stage in the series and the collective volume of each of the two or more open raceway ponds exceeds the volume of the raceway-type pond in the previous stage. Therefore, in this particular modality, algae and water in an open raceway type pond is diluted in two or more open raceway ponds when algae and water are transferred between stages in the series.

In a specific modality, the number of raceway ponds in each stage of the series is doubled. Therefore, in this modality, the number of raceway ponds in each stage in the series increases exponentially. For example, the number of open raceway ponds in each stage increases as shown below: 1, 2, 4, 8, 16, 32, 64, 128. In this modality, as the number of Open raceway ponds at each stage increase, so does the volume of each individual raceway type pond. In a preferred embodiment, the volume of the individual open raceway ponds in one stage is at least twice, preferably four times, the volume of the individual open raceway ponds in the previous stage. In an alternative mode, the volume of individual open raceway ponds in each stage in the series remains the same, although the collective volume of raceway ponds increases with each stage as the number of raceway ponds increases.

Algae and water can be transferred between raceway ponds in successive stages of the series without any external force, for example, can be transferred under the influence of gravity. However, algae and water, sometimes when the local topography does not allow the use of gravity transfers, will be pumped from one raceway-type pond to another, using any appropriate means of pumping.

Algae life cycle management and algae flow The algae can be grown in sea water, hypersaline water, brine for dealinization, brackish water, sewage or fresh water. The choice of water for the culture medium will depend on the algae that are cultivated. The seaweed they will grow in the water that replicates their natural cultivation environment.

The algae can be grown in seawater in order to replicate the conditions under which the natural blooms of oceanic algae occur. Seawater, in particular, deep coastal seawater, is rich in nutrients and can be used not only as the medium in which algae are grown, but also as a way to provide nutrients to algae. When seawater is used, seawater can be seawater that is pumped from the sea under the depth of mixed layer, nutrielin, pinocline or euphotic zone depending on the local hydrography. Water from this depth is used to increase the nutrient content of water; and reduce the burden of contaminants such as competing organisms, predators and viruses and other biological matter that could interfere with the cultivation of algae; and reduce allelopathic toxins or toxins excreted by other algae or organisms such as dimethylsulfide that could interfere with the regulation of cell physiology or algae culture.

Seawater can also be used to control the temperature of the algal culture environment. For example, for algae that make nocturnal migrations to the bottom of the euphotic zone to access nutrients more abundant, they also experience a regular temperature cycle where they are colder at night when they are absorbing nutrients and warmer during the day when they are absorbing sunlight near the surface. To imitate these conditions, cold and fresh seawater can be added to the culture medium warmed by the sun in the ponds regularly to induce a temperature fluctuation similar to those experienced during the diurnal migration of the algae. This is also important for the maintenance of cell culture synchronization.

Once the seawater has circulated through the series of connected raceway ponds, it is clean of algae and is returned to the surface water of the ocean, downstream, at a distance from the water intake to avoid entry of the water. water that has already been used for the cultivation of algae.

In one embodiment according to the present invention, the algae are first cultured in at least one stage of the covered raceway type ponds. The covered raceway ponds allow the control of the algae culture environment. Water used to fill covered raceway ponds can be filtered or otherwise treated to remove competing or harmful organisms before introducing them into covered raceway ponds.

In one mode, greenhouses are used to cover the raceway ponds and as a result, the environment of the algae is maintained at a higher temperature than the ambient temperature. In this mode, the temperature inside covered raceway ponds is between 25 ° C and 45 ° C, for example, between 38 ° C and 40 ° C. This will essentially inactivate organisms acclimated at temperatures of 4 ° C - 10 ° C when they are pumped from the depths of the deep sea. Advantageously, covered raceway ponds are less susceptible to contamination, either by bacteria or viruses, or by potentially competing organisms. The relatively controlled environment of the covered raceway type pond promotes the transition of the algae to the exponential growth phase.

As the algae's cell density increases, the algae are diluted successively, preferably by being transferred between stages in the series of covered raceway ponds to raceway-type ponds covered in successively larger volume. This successive dilution maintains a relatively low cell density of the algae, for example, between 100,000 cells / ml and 2,000,000 cells / ml, for example about 350,000 cells / ml. Each transfer of the algae to seed a covered raceway type pond in the next stage of the series (ie, the successive dilution of the algae) is programmed to match the growth rate of the algae. In a modality, the algae reside in each stage of the covered raceway ponds for 2 hours, 3 hours, 5 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 5 days or 10 days. In one embodiment, the algae reside in each covered raceway-type pond for two days, before being transferred to a larger-volume raceway-type pond. In one embodiment, the algae remain in a covered raceway-type pond for a sufficient time for the algae cell population to at least double, for example 2 hours, 4 hours, 12 hours, 24 hours, 36 hours or 48 hours. In a specific modality, the algae remain in a covered raceway-type pond for 24 hours before being transferred to the next stage in the series.

Once the algae enter the exponential growth phase or when sufficient amounts of algae have been grown, the algae are transferred to the first stage of open raceway ponds. In one embodiment, algae and water are transferred in volumes of 1,000 to 3,000,000 1, for example, 360,0001 to 720,0001, to the first stage of open raceway ponds. The transfer of a relatively large boll of algae is intended to seed the open raceway ponds to populate the culture environment with a large excess of several orders of magnitude of the algae product in relation to any surviving organism that was within the source water used in the open raceway type pond, thus establishing a robust population.

Preferably, the algae are diluted with fresh seawater advanced in the afternoon on a first day, before, at or around sunset (so that the algae have already completed a cell cycle), but before sunrise or sunrise the second day. The dilution of the algae is done either to maximize the exposure of nutrients and control the light signaling in the morning, for example for flagellates or algae that fix nitrogen, or in the afternoon for fast-growing algae such as diatoms. By timing the dilution in this way, the algae have time to acclimate to their new growing environment in the next stage of the raceway type ponds and continue the exponential growth. Timing also reinforces the synchronous growth of the algal cell population.

The algae can be diluted by introducing additional water in the current stage of ponds, increasing the volume of water contained within that stage. Alternatively, the algae can be diluted by transferring the algae to the next stage of the ponds and mixing the algae with water already present in the ponds.

The algae in an open raceway type pond are diluted successively by increasing the volume or number of open raceway ponds in each stage of the series.

This serial dilution maintains algae at a cell density low enough to support exponential growth. In one embodiment, the algae are successively diluted to maintain a cell density of 50,000 cells / ml at 100,000 cells / ml, eg, 200,000 cells / ml at 250,000 cells / ml. This approach is completely different from current methods for growing algae where algae are grown for artificially high densities, often achieving cell densities of more than 1 million cells / ml.

In one embodiment, the algae reside in each stage of the covered raceway ponds for 2 hours, 3 hours, 5 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 5 days or 10 days before being transferred to the next stage of the raceway type ponds in the series. In a specific modality, the algae remain in a raceway-type pond for 48 hours before being transferred to the next stage of the raceway type ponds in the series. In one embodiment, the algae remain in a raceway-type pond for a sufficient time for the algal cell population to at least double, that is, for a round of cell division to occur. In a specific modality, the algae remain in a raceway-type pond for a sufficient time for one, two, three, four or five rounds of cell division to occur. This can be, for example, 2 hours, 4 hours, 12 hours, 24 hours, 36 hours, 48 hours or 72 hours.

This successive dilution of algae maintains a low cell density that advantageously maintains the exponential growth phase, thus increasing productivity. In addition, problems associated with high-density algal culture methods such as the use of traditional raceway ponds and PRBs are avoided.

With each successive dilution of the algae, the water is added to, and / or already present in, the covered and open raceway type ponds in the next stage in the series. After the transfer of algae and water from one stage to seed the next stage in the series, the volume of fluid within each covered and open raceway type pond is equal to its capacity. In one embodiment, the entire volume of water in an open and / or covered raceway type pond is replaced every 2 hours to 8 days, or every 4 hours to 8 days, preferably, every 24 hours to 72 hours. In a preferred embodiment, the volume of water passing through the series of covered and open raceway-type ponds connected in 24 hours is greater than 20-50% of the entire volume of the open and covered connected raceway-type pond series. , for example, greater than 30% of the entire volume of the series of raceway-type ponds connected open and covered, for example, greater than 50% for both the sowing ponds as a crop. This water exchange rate is much higher than in traditional raceway type ponds, where the water replacement rate is usually around 0 - 20% in 24 hours, in order to match the growth rate of the cells and the rate of evaporation.

This high volume of water exchange has several advantages. In traditional raceway ponds, a high algae cell density is maintained and any contamination can potentially make the entire raceway pond become non-harvestable. However, the series of raceway ponds of the present invention is inherently resilient, since small degrees of contamination do not matter because all contaminants are inevitably cleared from the series of ponds and none of the seaweed products is reintroduced into the pond. series of raceway ponds.

Preferably, seawater is used to grow the algae, since the high volume of seawater exchange has particular advantages. First, seawater can be used to manage the temperature of the algae's growing medium, because significant volumes, for example 75% of the volume of a raceway-type pond, of deep, cool and cold seawater are added to each raceway-type pond to be seeded from algae grown in raceway ponds in the previous stage in the series. This addition of cold water mimics the vertical migration of algae from water surfaces to the cooler, nutrient-rich water in natural oceanic algal blooms and the repeated cyclic temperature fluctuation contributes to the recreation of the environment that promotes flowering.

The use of seawater also provides algae with a physiological excess of nutrients, including nitrate, phosphate, trace elements and bicarbonate. Unlike traditional raceway-type systems that require constant and careful additional nutrients for a population of static algae, the addition of seawater is simple and relatively inexpensive. When available, the cultivation of algae in nutrient-rich desalinated brine can be used to stop potentially competitive organisms that are incompatible with the hypersaline environment.

In addition, the regular flow of seawater through the raceway ponds act to regulate the bicarbonate ionic concentration and pH of the algae culture medium. Traditional raceway ponds and PBRs require active management of pH changes since high algae cell densities rapidly consume minerals, particularly bicarbonate. A regular flow of fresh sea water provides a fresh regulation capacity and avoids extremes of pH that promotes the growth of algae to avoid algae stress. The use of fresh seawater as a source of nutrients and pH regulator in this way reduces the capital expenditure required to control the algae growth medium within the raceway ponds, drastically reducing the operational cost of the large-scale algae farm.

Given the diurnal growth cycles of the flowering, the algae are synchronized with both light (Bruyant et al., 2005) and coordinated with nutrient absorption cycles (Levitan, et al., 2010), the depth of the ponds can be adjusted in a programmed cycle to recreate the necessary conditions to retain and reinforce this synchronization. In one embodiment, this can be achieved by increasing the pond depth of the culture ponds with fresh seawater before they are transferred to the next larger culture pond the next day. The timing of the increase in the depth of the water in the raceway type pond will depend on the species of algae.

For example, for flagellates that form bloom at the end of the highest point photosynthesis, which occurs shortly after the solar irradiation time highest point, for example, after 2 mm in the afternoon, the depth of the pond is increased by the addition of fresh seawater at a rate of 10-50 cm per hour, for example, 30 cm per hour, for 2-7 hours, for example, 5 hours to achieve a Final depth of 150cm-200cm, for example, 180cm, for sunset, which can occur at around 7mm at night. In a specific modality, when the term of the highest point of photosynthesis is at 3 pm and the sunset is at 7 mm, at 3 pm the ponds have a depth of 60 cm, at 4 PM the ponds have a depth of 90 cm, at 5 pm the ponds have a depth of 120 cm, at 6 pm the ponds have a depth of 150 cm and at 7 pm the ponds have a depth of 180 cm.

The above has several advantages. First, because the dilution of the cells coincides with the dilution with fresh seawater, new nutrients are available at the time they are most needed to support cell growth, as opposed to carbon fixation (during photosynthesis for 1-3 hours prior). Secondly, because the greatest depth of cultivation reaches about two meters at sunset time, the light relationships under red water (630-680 nm): far red (700-750 nm) and blue ( 400-450 nm): red (630-680 nm) are sufficiently altered (Ragni &; Ribera D'Alcala, 2004) to allow signaling of the light field (Lopez-Figueroa, 1992, Hughes et al., 1984; Dring 1988) similar to the conditions of real-world cultivation (as opposed to those in deep raceways). fixed). Third, the CO2 that is Excreted by the algae during the night, it accumulates and dissolves in the diluted culture medium, during the dark hours, leaving it available for resorption the next morning. Fourth, the ponds are then hydraulically loaded (at a higher elevation than the receiving pond) for unloading the following morning, after sunrise (to ensure appropriate dawn light signaling), for example at dawn ( for example, 6 AM) to be quickly drained to the subsequent cultivation pond, with a volume greater than or equal to four times the volume of the previous pond, at a pond drainage flow rate at 90 cm per hour to drain the pond and expose the cells of the algae to the maximum irradiation of light at the highest point of their photosynthetic efficiency.

This control of light signaling and addition of programmed nutrients is a key characteristic of this embodiment in the present invention, to maintain the reinforcement of the cell culture synchronization and the maintenance of the exponential growth phase.

In an alternative modality, to reinforce the synchronization in green algae, the pond may be filled after sunset, for example, at 7 mm or later and drained to the subsequent culture pond at least one hour before sunrise, for example, at 5 AM, to reduce exposure to red light (630-680 nm) and increase the relative and general exposure to blue light (400-450 nm) to retard the compromise point for cell division (after which cells can complete the cell cycle independent of light) to then change this and increase the size of the cell, which, in turn, allows two rounds of divisions the following day (Oldenhof et al, 2006).

In a third embodiment, for the cultivation of cyanobacteria, it is desirable to handle the activity of the Mehler reaction which has been shown to be independent of the cell cycle and accounts for 50-60% of the oxygen capture stimulated by light in irradiations equal to or that exceed the irradiation of culture (Calquin et al., 2004). The Mehler reaction is where photosynthesis is used for nitrogen fixation, during which the oxygen produced by PSII is reduced again after PSI. This is particularly relevant in high light desert environments where the energy of light that is available is greater than the light needed to support the maintenance of carbon fixation and cell culture. Nitrogen fixation can be used deliberately to reduce the nutrient requirement for filamentous cyanobacteria. In this mode, the ponds are kept at a constant depth and under 20-50 cm, for example 30 cm, at all times to maximize exposure to light and air for fixation of nitrogen and the harvesting of excess energy to feed this fixation, thus reducing nutrient costs.

Similarly, in a modality for the flowering flagellates, where at the end of the highest point of photosynthesis, for example, after 2 PM in the afternoon, the increase in water depth is used to disconnect the cycle of nutrients of the light cycle. As noted above, the depth of the pond increases with the addition of fresh seawater at a rate of 10-50 cm per hour, for example 30 cm per hour, for 2-7 hours, e.g. 5 hours to reach a total depth of 180 cm for sunset e.g. 7 PM at night. In a specific modality, when the term of the highest point of photosynthesis is at 2 mm and the sunset is at 7 mm, at 3 pm the ponds have a depth of 60 cm, at 4 PM the ponds have a depth of 90 cm, at 5 pm the ponds have a depth of 120 cm, at 6 pm the ponds have a depth of 150 cm and at 7 pm the ponds have a depth of 180 cm. The discontinuation of the nutrient and light cycle is achieved because in the afternoon when the cells have accumulated metabolic energy during the hours of photosynthesis highest point and are using this energy to reduce the nitrate to ammonium in the hours with less productivity of photosynthesis and in the dark (Dugdale & Goering, 1967). Here the addition of seawater at the end of the day also provides fresh nitrate for the cells to absorb, convert and exploit for protein synthesis and mimic previous migration patterns of natural flowering (Flynn &Fasham, 1997). The nutrient conversion rate can be modeled (Flynn &Fasham, 2003) to fine-tune the control of timing and duration of nutrient addition.

The carbon-to-nitrogen ratio in algal cells is an important indicator of the ability to fix new carbon, from seawater and the atmosphere, versus the existing carbon, which was excreted as a result of the metabolism of algae cells carried out in the dark and excreted seawater (Levitan et al., 2010). Carbon that is accumulated during the highest productivity of photosynthesis can be assigned to cell culture, nitrogen fixation or production of PET and other activities. Therefore, the carbon ratio can increase in relation to nitrogen during the highest point of photosynthesis and decrease when the cellular metabolism dominates. This ratio can be measured and used to ensure, for example, optimal mixing regimes that, at night, when the cells emit "old" carbon (previously absorbed), the flow rate of the ponds is reduced to 5-15 cm / sec, eg10 cm / sec, to avoid degassing and loss of dissolved CO2. Similarly, the relationship can be used to determine the timing and rate of dilution and / or transfer of the algae. For example, it is desirable to dilute the algae with fresh seawater after the highest carbon to nitrogen ratio is achieved, to allow the cells to absorb nutrients for protein and biomass synthesis and subsequent cell division.

Planting and Photobioreactors In one embodiment, the first stage of the covered raceway type ponds is sown with algae grown in a photobioreactor (PBR) or a seed pond.

A PBR achieves a highly controlled environment within the reactor to maintain a non-contaminated broth culture. In order to avoid contamination with competing organisms, bacterial or viral infection or predatory organisms that reduce the yield or availability of algae for planting, a PBR preferably uses decontaminated and pretreated seawater, filtered with membrane and sand. The exchange of gases is carefully controlled, for example when injecting or bubbling CO2 into the reactor, and removing excess O2. The addition of nutrients and the removal of waste products is also carefully controlled. Preferably, a PBR operates in a sterile environment.

A pond for sowing is an open pond or closed, preferably a closed raceway type pond, where the culture conditions for the algae can be controlled. The sowing pond is used to grow a sufficient algae population to plant the first stage of covered raceway ponds.

In one embodiment, the first stage of the covered raceway ponds is planted with algae from a PBR or seed pond in the early morning, for example, from an hour before to two hours before dawn to allow the algae to explode its new growing environment, just before they were divided in the hours before sunrise, for example, 1-2 hours before sunrise. In a particular modality, the first stage of covered raceway ponds is planted between 1-2 hours before and 1-2 hours after sunrise, for example, between 1 hour before and 2 hours after sunrise or in a specific mode When the sunrise is at 6 AM, the planting occurs between 5 AM to 8 AM. Cell division can be quantified and validated with fluorescent stains and flow cytometry. The timing of this transfer depends on and is matched with the natural light cycle to induce and maintain the synchronization of cell division. So, if the first transfer occurred at dawn on the first day, the subsequent transfer to the pond of the next stage the next day would also be at dawn. In a specific modality, the first step of sowing occurs at 6 AM and then the subsequent transfer to the pond of the next stage on the next day will be at 6 AM and the next day at 6 again.

Seaweed harvest In one embodiment, the method of the invention further comprises the harvest step of the algae. Traditional algae culture methods aim to maintain algae at their optimum growth stage to maintain a continuous high density of algae, from which the resulting algae are harvested continuously to maximize the use of high capital expenditure equipment. In addition, harvesting the entire water column requires that the entire crop pond production be centrifuged, which requires a large amount of energy. The present invention uses a different approach. The algae grown using the method and series of connected raceway type ponds of the present invention undergo a complete life cycle of algae by being maintained in an exponential growth phase before making the transition to the stationary growth phase, mimicking the natural progression of the algae at the end of their oceanic blooms.

In one embodiment of the present invention, the algae are not harvested continuously from the covered and open raceway ponds in their exponential growth phase, but they make the transition to stationary growth before being harvested in batches. By allowing the algae to make the transition to the stationary phase before harvest, the natural tendency of the algae to aggregate and form marine snow at this stage of their life cycle can be used, since the aggregation makes the harvest a lot. easier.

Before the harvest, in one mode, the algae are transferred from an open raceway pond to a harvest pond. In a particular embodiment, the algae are transferred after achieving a maximum density of 500,000-1,000,000 cells / ml. A harvest pond is an open raceway type pond that has been modified to slow down the growth rate of algae and to induce a change in algae from exponential growth to the stationary phase. In other words, a harvest pond is not designed to maintain the exponential growth of algae. In one modality, the total volume of the harvest ponds is 100-200% of the total volume of the final stage of the covered ponds. In one embodiment, the initial cellular density of the algae in the harvest ponds is between 1,000,000-2,000,000 cells / ml. This cellular density of algae is significantly higher in previous raceway ponds after the cells pass through one or two additional divisions, make the transition to stationary growth and slow down in their division rate.

Additional modifications can be made to the final harvest ponds in order to recreate the environmental conditions experienced by the algae during the collapse of the natural blooms, where all the culture ponds are drained to at least one or more harvest ponds that are under operation to promote the transition to a different physiological state that promotes the formation of flocs, for the harvest of algae.

Preferably, a harvest pond is deeper than the previous open raceway type pond. For example, a vintage pond may be 0.8; 1, 2, 3, 4, 5 or 10 m depth. This increase in depth reduces the exposure of the algae to solar irradiation, which in turn reduces the cultivation of the algae (thereby stimulating the algae to transition from exponential culture to the stationary phase). The algae in the harvest ponds are also deprived of nutrients in order to decelerate and finally stop the growth of these. This means that nutrients and fresh seawater are not added to it, since the volume of the harvest pond only adapts the volume of water added from the culture ponds.

The rate of agitation within the harvest pond is lower than in open and covered raceway type prior ponds. In one mode, the speed of the wheels of pallet is reduced to provide a flow rate less than 25-30 cm / sec. For example, the flow rate is reduced to approximately 10-15 cm / sec. Algae can also be induced to change from the exponential culture phase to the stationary phase by changing the color of the pond liner. In one embodiment, the lining of the harvest pond is green to induce phototaxis in flagellates (Hader and Lebert, 1998, Sineshchekov and Govorunova, 2001) to migrate upwards and induce algae to aggregate, for example for the purpose of a harvest accelerated The harvest of unicellular algae can be difficult due to its small cell size. Therefore, in one embodiment, the algae are added before being harvested, since the aggregates of the algae settle quickly and can be harvested much more easily. Aggregation can occur naturally as algae mature. E. huxleyi algae have a tendency for natural aggregation when they mature, by releasing extracellular and long organic molecules called Transparent Exopolymer Particles (TEPs). Long-chain organic precursors to TEP molecules begin to be released during exponential culture. However, when cellular senescence is established during the stationary phase, these precursor molecules begin to coagulate with cellular debris to form gels and aggregates. submicrons, which when agitated, form PET. Certain diseases such as Phaeodactylum tricornutum and cyanobacteria such as Synechococcus excrete a coating of cells that form free TEP when the parts are removed, commonly during the decline of a natural bloom. The ucosidad of the surface of the cells of diatomea can also improve the formation of free EPT. The aggregation can be further stimulated by adding flocculant and / or coagulants such as aluminas and flocculating polymers to further accelerate the formation of "sea snow". Sea snow is used to describe the continuous rain of organic detritus mostly (including dying or dead marine plants and animals, plankton, protist fecal matter, (diatoms) and inorganic dust) falling from the upper layers of the water column in the deep ocean. In the context of the following invention, marine snow includes the waste products of algae and other particles or materials which results in the aggregation of algae.

In an alternative modality, supplementary algae such as diatoms can be added to the algae to be harvested, in order to stimulate the formation of marine snow. Phaedactylum tricornutum is a diatom that produces precursors of small molecules PET. By combining 2-5% (of the total cell density of a harvest pond) of phaedactylum tricornutum with the product of algae in a harvest pond, stimulates the formation of marine snow and aggregation of algae.

In an alternative mode, 2-10% for example 6% of the algae in the exponential phase are pumped from an open raceway-type pond to a separate raceway-type pond. In a specific modality. This pond has channels that are about 200 meters long by 12 meters wide. The algae then move to the stationary phase using any of the tactics described above (such as depriving the algae of nutrients and / or reducing solar radiation). These "algae that promote the harvest" are allowed to mature for 2-5 days, for example 3 days, during which time they pass from the exponential phase to the stationary phase and begin to eliminate TEP precursors that concentrate the culture medium. of algae After 3 days, a part of the volume of the pond, for example 30%, is actively pumped to a harvest pond, which promotes the formation of more marine snow and aggregation. The volume of the pond can be pumped by any suitable means, but in a specific modality the aerated hydro-air unit (ASH) is used.

Once the algae has been added and the marine snow formed, the algae must be removed from the harvest ponds. This is usually achieved by using skimmers mechanical and flotation to remove the product of algae and sea snow from the surface of the water. The flotation techniques are preferably used to stimulate marine snow and algae to the surface of the water. In one embodiment, aggregates float rapidly to the surface using aeration of fine bubbles. This is achieved by putting long rows of fine bubble aerators along the bottom of the harvest ponds to create rows of bubble curtains that carry all the suspended particles to the surface. Alternatively, aerated hydrocyclone (ASH) can be used to produce aeration of microscopic bubbles to form bubbles that are much smaller and have a much larger surface area than the bubbles formed through the aeration of standard fine bubbles. An ASH also brings any coagulant and flocculant used in close contact with algal cells, thereby further stimulating the formation of sea snow and aggregation.

Certain species of small cell algae such as blue green algae or Dunaliella sp. They do not form aggregates quickly. An alternative modality uses predatory organisms to ingest the algae. The predatory organisms (harboring the algae product) are then easier to harvest because of their larger size. In a preferred embodiment, the microzooplankton are used for efficiently add hundreds of algae cells in a feeding cycle, before they are easily harvested themselves. For example, Noctiluca scintillians is a microzooplankton that is a predatory dinoflagellate that drags a thread of mucus through the water as it rises. It reaches diameters of half a millimeter and can ingest hundreds of algae cells in a crop cycle before it is divided.

The microzooplankton are cultured in separate ponds to the raceway ponds of the present invention and can be fed with a small percentage (1-3%) of the algae in the penultimate group of open raceway ponds in order to enter the phase of exponential culture. Prior to being introduced into the harvest ponds, the microzooplankton are fed insufficiently for a minimum of three days. In the specific case of the microzooplankton Noctiluca scintillians, this results in the dramatic increase in cell size and thus increases the harvesting capacity of the cells. Once the microzooplankton are pumped into the harvest ponds, they feed on algae for up to 12-24 hours before the predator cells divide at the point they are harvested. Another advantage of using microzooplankton is that they allow the accumulation of algae biomass while also increasing the relative protein content of the final harvested product. Calanus sp. and other copepods can also be grown separately from the algae product and introduced into the harvest ponds to accumulate the biomass that is not otherwise added.

The deliberate addition of predatory organisms to accumulate and concentrate the algae in an easily harvested organism can produce an organism with an advantageous nutrition profile. For example, copepods can be used, which have a protein content of 44-53%, making them an attractive product.

Maintenance of raceway type ponds The method and series of connected raceway ponds of the present invention produce successive batches of algae contrary to the sustained and continuous algae state harvest that is traditionally used in the cultivation of algae. This means that lots of algae can be separated according to need. For example, as algae pass through the series of connected ponds, contamination from traces of air or other sources may occur, particularly in open raceway ponds. However, the series of connected raceway ponds of the present invention are intrinsically resilient because small degrees of contamination do not matter, since no algae product is reintroduced and all contaminants are eventually drawn from the system.

Furthermore, in one embodiment, the dilution of the algae through the repetition of addition of seawater also dilutes any contaminants present.

The raceway ponds in the series are connected to allow algae and water to flow from one raceway-type pond to another, but each pond can be isolated when required. This is particularly useful to allow ponds to be cleaned to remove biofilm or sediment formation. Therefore, between the lots of algae in different raceway ponds, where the algae are transferred every 2, 3, 4, 5, 6, 8, 10, 12, 24, 36 hours, 2 days, 3 days, 5 days or 10 days apart, a cleaning shift can be introduced where each raceway tank in the series is sequentially pumped dry, cleaned, for example with rotating brushes mounted on a truck and rinsed with water. You can carry out a cleaning cycle once a month and every several cleaning cycles you can enter an extra day to add a day to dry the ponds. The equipment can be cleaned with 0.0001-0.01% peroxyacetic acid before being seeded with algae from the raceway pond in the previous stage in the series and filled with fresh water. In this way, a wave of continuous cleanup can travel through all series of open ponds and raceway type covered raceways (and the harvest pond if desired). This can be programmed according to the prevalence of oceanic pollutants that enter with fresh seawater or environmental alterations such as rain or sandstorms.

Algae Preferably, according to the invention, the cultivated algae represent a unique species. This means that the vast majority of algae are of a single species, for example, 95% or more, 99% or more, 99.9% or more, 99.999% or more.

The cultivated algae will generally be a unique species selected from the group consisting of freshwater algae such as Chlorella vulgaris, Spirullina sp. o Cryptomonas ovate; safe water species such as Nannochlorpsis gaditana, Nannochloropsis oculata; marine species such as Skeletonema costatum, Chaetoceros gracilis marine diatoms, Tetraselmis sp. , Isochyrsis galbana or Rhodomonas minuta, or hypersaline species such as Dunaliella salina or isolated species of Pyrami onas sp. Although monocultures of pure nature are never found, since algae bloom is able to proliferate in complex oceanic communities, none of the algal mass culture regimes currently proposed or used in the art is designed to Cultivate more than one species in the same system. Sometimes, this happens spontaneously when local organisms pollute and displace organisms cultivated. However, because these commercial product type algae are generally chosen for their high lipid content, organisms with another lipid profile are removed. In one embodiment of the present invention, the algae combinations are grown in the same series of ponds to create an algae product with a particular protein or carbohydrate, which is not obtained from a single species of algae.

For example, a combination of Dunaliella salina with Nannochloropsis produces an algae product with improved properties related to each individual species. The species Nannochloropsis is an eustig atofito that grows rapidly and can produce a high oil content in high light, which complements the Dunaliella salina species that creates large amounts of protein and beta carotene in more saline waters towards the end of its cultivation cycle . Therefore, this combination produces an algae product with high protein and oil content. The combinations of algae can be cultivated from the beginning, for example when planting the first covered raceway pond in the series of raceway-type ponds connected with different species of algae from different sown ponds and / or PBRs. Alternatively, the separated species can be cultivated in parallel and combined 1, 2, 3, 4, 5, 6, 7 days, 1 week or 2 weeks before harvesting, to generate an environment of optimal culture for each species separately or in combination.

According to what was discussed in relation to the harvesting capacity, the algae combinations can be cultivated either together from the beginning or cultivated in parallel then mixed, in order to create a population of algae that will change the psychological state of the population and promote the formation of PET and marine snow, and become more easily harvestable. For example, Phaedactylum tricornutum can be combined with other species of algae to stimulate algae to aggregate and form marine snow.

The expert in the art will appreciate that, within the scope of the present invention, the different species of algae will require different modifications of the culture environment, in order to maintain the particular species of algae in the exponential culture rate phase. However, the person skilled in the art could easily adapt the claimed method and series of connected raceway ponds to adapt the particular species of algae being cultivated. For example, adding deep sea ice water to dilute the algae would not be suitable for algae that bloom naturally in warm brackish water such as Nannochloropsis gaditana. In this case, the seawater could be heated, diluted with fresh water and / or otherwise modified to an appropriate temperature and / or salinity by any suitable means, for example being passed through a greenhouse before being added to the pond.

Other modifications can be made without the use of inventive ability. For example, algae species that bloom in more turbulent environments may require more agitation during the exponential culture phase, which can be provided by the use of a paddle wheel and changes in the cross section of the raceway-type pond. In this scenario, other methods of agitation such as the use of a bubble aerator to mix the algae can be employed.

It would be desirable that any feature described herein in relation to one or more aspects of the invention could also be applied to any other aspect of the invention.

EXAMPLE Design of Model Figure 2 illustrates the design of a specific algae culture system, including the seawater pipe used to fill an elevated channel of seawater. This seawater channel feeds a series of raceway ponds. Each pond has a connection to at least the elevated seawater channel and to a lower downstream pond through a gate controlled pond discharge. The series of ponds begins with ponds planted small and covered to grow the inocula. Each subsequent pond has at least twice the capacity of the previous pond to maintain the totality of the previous pond volume and the equivalent volume of unused seawater. The design in figure 2 has two stages with four channels including the "return channel". A group of raceway ponds with eight channels, including the "return channel" and a group of 10 channels including the "return channels". This last group is drained in two deep and large harvest ponds with a total of four channels. The advantage of this rectangular design is that it efficiently embraces the coast along the edge of an ocean and allows each pond to have at least one contact with the elevated seawater channel and a discharge into the next lower and larger pond . This design allows the transfer of water within the complete pond system to depend on the gravity feed and requires a minimum of pipes, while allowing easier maintenance of the ponds. This design also benefits from the natural gradient often found along the coastline where the distance from the shore commonly results in a slight increase in elevation. Finally, the suction pipe is upstream and as far as possible from the discharge system to avoid the re-absorption of the already spent water sea.

Model System The next preferred embodiment is based on a series of open ponds and raceway-type covered ponds connected with dimensions plotted in Table 1.

Table 1 * all ponds are raceway type An exemplary series of ponds is plotted in Table 1. Each of stages 1-3 comprises a raceway-type pond, which is covered by a greenhouse. Each covered pond has two channels and is connected to the rear pond, although the ponds may be isolated by gates if required. Each successive raceway covered pond in the series is longer and wider than the previous covered pond, therefore it has a larger volume. In this particular modality, the volume of each covered raceway tank is four times larger than the volume of the previous pond, therefore the dilution rate of the algae is quadrupled in each transfer. In stage 3 the covered raceway-type pond is connected to the open raceway-type pond in stage 4. All covered raceway tanks of stages 1-3 have a depth of 0.3 m.

Stages 4-9 comprise open raceway ponds. Each raceway-type open pond is connected to the open raceway-type pond (s), although ponds may be insulated if required. The pond in stage 4 has two channels and is larger than the pond in stage 3. The pond in stage 5 has two channels and is larger in volume than the pond in stage 4. The pond in stage 6 It has two channels and is of greater volume than the pond in stage 5. The open raceway-type pond in stage 6 is then connected to two open raceway-type ponds in stage 7. The two open raceway-type ponds in stage 7 have equal dimensions and each open raceway type pond has four channels and a volume greater than that of the pond open raceway type in stage 6. The collective volume of the two open raceway ponds in stage 7 is four times larger than the volume of the open raceway type pond in stage 6. Therefore, the dilution rate of the algae it is quadrupled when the algae are transferred from stage 6 to stage 7. Each raceway-type open pond in stage 7 is then separately connected to two open raceway-type ponds in stage 8, giving a total of four raceway-type open ponds in stage 8. The four raceway-type open ponds in stage 8 have equal dimensions and each raceway-type open pond has eight channels and a larger volume than open-raceway type ponds in stage 7. The collective volume of the four open ponds raceway type in stage 8 is four times greater than the collective volume of open raceway type ponds in stage 7. Therefore, the dilution rate of algae is four times when the algae are transferred from stage 7 to stage 8.

Each open raceway pond in stage 8 is then separately connected to two open raceway ponds in stage 9, giving a total of eight open raceway ponds in stage 9. The eight open raceway type ponds in stage 9 have dimensions the same and each open raceway type pond It has sixteen channels and a larger volume than the open raceway ponds in stage 8. The collective volume of the eight open raceway ponds in stage 9 is four times larger than the collective volume of open raceway ponds in the stage 8. Therefore, the dilution rate of the algae is quadrupled when the algae are transferred from stage 8 to stage 9.

The average depth of open raceway type ponds is 0.3 m. However, in order to induce a non-uniform flow rate, each pond has at least one channel that is significantly shallower than its adjacent channel. In this example, a channel is 0.3 m deep and its adjacent channel is 1 m deep. This non-uniform depth translates into a variable flow rate through the pond, thus mimicking the natural conditions of algae bloom. This non-uniformity also varies the amount of solar irradiation that the algae receive, with algae in the 0.30 m sections of the pond receiving greater solar radiation than the algae in the 1 m sections of the pond. This difference in depth mimics the natural migration of algae from the surface (to receive greater solar radiation) to the deeper water rich in nutrients, thus mimicking the physiology of flowering that keeps the algae in the exponential culture phase. Similarly, the width of the channels in the open raceway type ponds is also varied so that the ponds are not symmetrical. Thus, in each raceway-type open pond, at least one channel is wider than its adjacent channel. For example, a channel is 1.5 times the width of the adjacent channel. This variation of change in the width of the channels translates into a variable flow rate through the pond, thus mimicking the natural conditions of algal blooms.

In this inventive example, the complete volume of each open and covered raceway type pond is replaced within two days, allowing two cell divisions per pond. Each transfer of algae between the different dilution stages implies at least a fourfold dilution. Therefore, the same cell density of algae can be maintained throughout the series of ponds. Because the algae remain in each pond for approximately two days, a batch of algae will go through stages 1-9 in about 18 days. Because each transfer of the algae between the different stages involves at least a four-fold dilution, the new water used to fill a pond is 75% fresh seawater and 25% of the algae culture medium of the open pond or covered type previous raceway. This exchange and flow of the complete volume of pond culture medium is at a much higher rate than traditional raceway ponds, where the exchange of the complete volume of a pond commonly takes 8 days and may take longer.

This preferred embodiment of the invention uses fresh ocean water, from the depth of the ocean, which is pumped through a closed pipe, at a significant distance from the shore. Seawater is pumped at a significantly higher rate than in any other raceway pond, PBR or current algal culture methods. For example, to maintain the series of ponds of the preferred modality, which is approximately 100,000m2 of raceway ponds, the volume of seawater pumped through the system exceeds 37.5% of the total daily volume of the open ponds and covered type raceway The flow inside the open raceway covered ponds is maintained by one or more paddle wheels in each open and covered raceway pond. Paddle wheels are usually as wide as the width of the used raceway type pond and are located at one end of the pond to maintain a laminar flow along the length of the pond. open or covered raceway pond at a rate of around 30 cm / sec. In this preferred embodiment, the algal cells travel the length of the raceway-type pond approximately 100 cycles / day without significant shearing. At this flow rate, the oxygen that is produced by the algae can quickly balance and prevent the creation of reactive O2 species that contribute to photoinhibition (Falkowski et al, 1985). Similarly, through this active atmospheric interference, seawater within raceway ponds can absorb and dissolve significant amounts of CO2, which reduces the costs of CO2 inputs for these raceway ponds. Both the atmospheric CO2 and the bicarbonate ions provided by the regular addition of fresh seawater at each stage are important sources of C02 for algae to grow and maintain their exponential culture. Thus, in combination with the lower common cell density of algae, the overall cost to provide additional CO2 for algae can be reduced by 66% or effectively eliminated altogether.

The algae grown in this preferred embodiment is a unique species, Rhodomonas minuta. The pond covered in stage 1 is planted with cultivated algae in PBR. PBR has a separate input of decontaminated and pretreated seawater, filtered by membrane and sand, a feed of nutrients and controlled introduction of gas. Once sufficient volume of seeded algae has been produced, approximately 1,0001 of the algae culture medium at a cell density of 750,000 cells / ml is transferred to the covered raceway of the greenhouse in stage 1, where cells pass to a new culture medium for up to four days. As described above, the algae spend two days in each covered raceway pond in stages 2-4, where the algae go into their exponential culture phase. The algae then pass through the open raceway ponds of stages 5-9, where successive dilution, changes in the depth and width of the pond and other factors are modified in order to keep the algae in their exponential culture phase , imitating the psychological conditions experienced by algae in a natural oceanic bloom.

By the time the algae have reached the eight open raceway ponds of stage 9, approximately 18 days after inoculation of the covered raceway-type pond in stage 1, the algae will have reached a maximum density of 500,000-1,000,000 cells / ml and are ready to be harvested. Before being harvested, the algae are transferred from the eight open raceway ponds in stage 9 to the two one meter deep harvest ponds. Each harvest pond has two channels that They are 23 m wide and 180 m long. The collective volume of the harvest ponds is marginally greater than the collective volume of the eight open raceway ponds in stage 9 to adapt the flow fluctuations. However, there is no additional dilution of the algae when the algae are transferred from stage 9 to the harvest ponds. In this preferred embodiment, a batch of algae provides a biomass of 3,900 kg after drying the algae.

Harvest ponds are also open raceway type ponds, but they differ from open raceway ponds of stages 4-9 in various ways, in order to induce the algae to pass from the exponential to the stationary culture phase before Harvest. The harvest ponds are deeper than the open raceway type tanks of stages 4-9, in order to reduce solar irradiation, so that the algae can not maintain its exponential culture. No fresh seawater is added to the harvest ponds or additional nutrients, so the algae are deprived of nutrients and can not sustain exponential cultivation. The speed of the paddle wheels is reduced so that the flow rate of the medium of the algae is 10-15cm / sec. This reduction in the flow rate of the algae culture medium reduces algae agitation and gas exchange, thus stimulating the algae to move to the stationary phase EI The lining of the harvest ponds is green, which also has the effect of passing from the exponential phase to the stationary phase.

Before the algae are harvested, they are stimulated to aggregate to form marine snow. The reduction in the flow rate initially stimulates the algae to aggregate and in this preferred embodiment, the algae are even more stimulated for aggregation by coating the pond with a green coating and using supplementary algae to stimulate aggregation.

Results of the Model The following results were obtained using a model based on 20 dilutions in 10 ponds. This model is based on measured inputs and historical biomass production rates as determined by dry weight analysis of filtered pond water. The production is determined either using Chlorophyll a or biomass produced by surface area of the total pond system or by m3 of seawater. Given the natural inputs of nutrients (nitrate, carbonate), the model allows us to calculate the cost of the nutrients that have to be added per unit of surface area or volume of water of cultivation used and thus allows the evaluation of the exchange of the dilution rate, concentration of inoculant cells, culture rate, addition of nutrients, stacking of dilutions for each cell division within the same ponds and the concentration of cells and nutrients due to evaporation.

An initial load of 25,000 cells / ml was used at a culture rate of 0.9 cell divisions per day. The dilution rates of 140%, 130%, 120%, 110% and 100% per day of fresh seawater were used. A virtually reachable nitrate charge of 35 millimoles per m3 and a carbonate load of 2,120 millimoles per m3 were assumed for dilute seawater. An average evaporation rate of 1% per day was assumed.

For each of the results of the model, the numerical constants shown in Table 2 were used.

Table 2 For more information regarding these values, see Fawcett et al., 2008 and Pitcher et al., 1996.

Table 3: Results of the computational model for a 140% dilution per day Table 3 Table 3 (cont.) Table 3 (cont.) According to the conditions in Table 3, it can be seen that the additional nitrate required for the process is minimal and the cost of the additional nitrate is negligible. The biomass produced for this cost is 58kg.

Table 4: Results of the computational model for a dilution rate of 130% per day.

According to the conditions in table 4, it can be seen that the additional nitrate required for the process is 2.01 USD. The biomass produced for this cost is 58kg.

Table 4 Table 4 (cont.) . .

Table 4 (cont.) i | . .

Table 5: Results of the computational model for a dilution rate of 120% per day.

Table 5 Table 5 (cont.) I. .

Table 5 (cont.) According to the conditions in Table 5, it can be seen that the additional nitrate required for the process is 3.49 USD. The biomass produced for this cost is 58kg.

Table 6: Results of the computational model for a dilution rate of 110% per day.

Table 6 Table 6 (cont.) According to the conditions in table 6, it can be observed that the additional nitrate required for the process is 4.17 USD and the additional cost of carbonate for the process is 1.03 USD. The biomass produced for this cost is 58kg.

Table 7: Results of the computational model for a dilution rate of 100% per day.

Table 7 Table 7 (cont.) . .

Table 7 (cont.) According to the conditions in Table 7, it can be seen that the additional nitrate required for the process is 4.46 USD and the additional cost of the carbonate for the process is 13.34 USD. The biomass produced for this cost is 58kg.

Table 8: Results of the computational model for a dilution rate of 90% per day.

Table 8 Table 8 (cont.) . .

Table 8 (cont.) . . .

According to the conditions in Table 8, it can be seen that the additional nitrate required for the process is 4.59 USD and the additional cost of the carbonate for the process is 19.12 USD. The biomass produced for this cost is 58kg.

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Claims (32)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for culturing and synchronizing algae in a series of connected raceway ponds arranged in stages by manipulating the physiological state of the algae by altering one or more environmental parameters in order to simulate algal bloom formation conditions; characterized in that the alteration of one or more environmental parameters is programmed to induce the synchronization of cell division and the synchronization is maintained by altering one or more environmental parameters at regular intervals to retain the physiological state of choice; and wherein the alteration of one or more environmental parameters comprises the dilution of algae before and during the transfer between stages, and wherein the dilution is programmed to coincide with a particular hour of the day or a particular point in the cell cycle or a state physiological particular.

2. A method according to claim 1, characterized in that the alteration of one or more parameters Environmental programs are programmed to coincide with a specific point in the algae's cell cycle.

3. A method according to claim 1 or 2, characterized in that the one or more environmental parameters are additionally selected from the group consisting of: i. Intensity of light; ii. wavelength ratio of light; iii. concentration of nutrients; iv. nutrient balance, - V. water temperature; saw. density of algae cells; vii. concentration of dissolved gases; I viii. dissolved gases ratio.

4. A method according to claim 3, characterized in that the balance of nutrients is the ratio of carbon to nitrogen in the cells.

5. A method according to claim 3, characterized in that the dissolved gases are oxygen (O2) and carbon dioxide (CO2).

6. A method according to any of claims 1-5, characterized in that the algae are cultured in sea water, hypersaline water, brine for desalination, brackish water, waste water or fresh water.

7. A method according to claim 6, characterized in that the sea water, hypersaline water, brine for desalination, brackish water, sewage or fresh water is fresh, which has not been recovered or recirculated from any other part of the pond system after harvest or any other operation of the ponds.

8. A method according to claim 5 or 6, when dependent on claim 4, characterized in that the environmental parameter of the cellular density of the algae is altered by dilution of the algae grown using fresh water, which has not been recovered or recirculated from no other part of the pond system after the harvest or any other operation of the ponds.

9. A method according to claim 1-8, characterized in that the ratio of light intensity and / or wavelength of light is altered by changing the depth of the water in raceway ponds.

10. A method according to claim 1-9, characterized in that the raceway-type pond is coated and the ratio of light intensity and / or wavelength of the light is altered by changing the color of the raceway-type pond liner.

11. A method according to claim 1-10, characterized in that the raceway-type pond is covered and the ratio of light intensity and / or wavelength of light is altered by changing the color and / or color. opacity of the cover of a covered raceway type pond.

12. A method according to any of claims 1-11, characterized in that the concentration of nutrients, nutrient balance, water temperature, cell density of the algae, concentration of dissolved gases and / or ratio of dissolved gases is altered by the addition of fresh seawater, hypersaline water, brine for desalination, brackish water, wastewater or fresh water to the raceway type pond.

13. A method according to any of claims 1-12, characterized in that one or more of the concentration of nutrients, nutrient balance, water temperature, algae cell density, concentration of dissolved gases and / or dissolved gas ratio is altered based on the predictions of a computational model.

14. A method according to any of claims 1-13, characterized in that the optimum operation of the pond in terms of dilution rate, pond depth and nutrient addition is based on the predictions of a computational model, where the growth rates Specific and nutrient intake of a particular algae species are entered into the model.

15. A method according to any of claims 1-14, characterized in that the concentration of dissolved gases and / or the dissolved gas ratio is altered by altering the flow rate and / or by creating a turbulent flow in the raceway ponds.

16. A method according to claim 15, characterized in that the width and / or depth of an open raceway type pond is not uniform, resulting in a change in the flow rate and / or the creation of a turbulent flow through the pond type raceway

17. A method according to any of claims 1-16, characterized in that the depth of an open raceway-type pond is not uniform, resulting in a change in the intensity ratio of the light and / or wavelength ratio of the light and / or change in the concentration of dissolved gases and / or dissolved gas ratio and / or cellular density of the algae within the raceway type pond.

18. A method according to claim 17, characterized in that the alteration in the depth of the raceway type pond is programmed to coincide with a specific point in the cell cycle of the algae.

19. A method according to any of the preceding claims, characterized in that it comprises growing algae in a series of connected raceway ponds arranged in stages, where the series of connected raceway ponds comprises - first, one or more stages of raceway type ponds cutlery; Y - second, one or more stages of open raceway ponds; where in each successive stage in the series, the raceway ponds have a collective volume greater than the collective volume of the raceway ponds of the previous stage; Y where the raceway ponds at each successive stage of the series are seeded by transferring algae grown in the raceway ponds of the previous stage of the series so that the algae are diluted when transferred between stages in the series.

20. A method according to claim 19, characterized in that the series of covered raceway ponds comprises 2 to 10 stages of covered raceway ponds.

21. A method according to claim 19 or 20, characterized in that the series of open raceway ponds comprises 2 to 10 stages of open raceway ponds.

22. A method according to any of claims 19-21, characterized in that the number of raceway ponds in each stage in the series of covered raceway ponds and / or open raceway ponds is at least twice the number of raceway ponds in the previous stage in the series.

23. A method according to any of claims 19-22, characterized in that the cellular density of the algae in each covered raceway-type pond is between 1,000 cells / ml and 2,000,000 cells / ml.

24. A method according to any of claims 19-23, characterized in that the entire volume of water in each open raceway-type pond is replaced every 4 hours to 8 days.

25. A method according to any of claims 19-24, characterized in that the first covered raceway-type pond is seeded with algae grown in a photobioreactor or pond for sowing.

26. A method according to claim 25, characterized in that the covered raceway type pond is seeded with algae between the hours of sunset on a first day and the dawn the next day.

27. A method according to any of claims 19-26, characterized in that it also comprises the step of harvesting the algae.

28. A method according to claim 27, characterized in that the algae are harvested in batches.

29. A method according to claim 27 or 28, characterized in that the algae are added before being harvested.

30. A method according to claim 29, characterized in that the algae are added by: (i) the addition of flocculants and / or coagulants; I (ii) the addition of supplementary algae to stimulate aggregation; I (iii) the addition of predatory organisms.

31. A method according to any of claims 27 to 30, characterized in that before harvesting the algae, the growth rate of the algae is reduced by: (i) increasing the depth of an open raceway type pond; I (ii) the introduction of the algae in an open raceway pond with a green coating; I (iii) reducing the flow rate of the algae by reducing the speed of the paddlewheel; I (iv) the deprivation of nutrients to algae.

32. A method according to any of the preceding claims, characterized in that the cellular density of the algae is kept below 200,000 cells / ml, for the initial stages of growth, wherein the initial stages are two-thirds to three-fourths of the generations of growth of the algae, through repeated dilutions to maintain the exponential and synchronized growth without the addition of supplementary nutrients.