Patent Application
20130130909
This invention relates to biochemistry, and more specifically, to algal cultivation.
Provided herein are exemplary methods for controlling a density of algae growing in an aquatic environment. Some exemplary methods include applying an effective amount of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to the density of algae growing in the aquatic environment, wherein the algae includes genus Nannochloropsis. The algae may also include algae of genus Tetraselmis and/or genus Chlorella. Applying the effective amount may result in an approximate concentration of between 100 nanomolar to 1500 nanomolar DCMU in the aquatic environment. Further, the aquatic environment may include seawater, freshwater, or mixtures thereof.
Further exemplary methods for controlling a density of algae growing in an aquatic environment may include applying an effective amount of DCMU to the density of algae growing in the aquatic environment, wherein the algae includes genus Nannochloropsis, and wherein applying the effective amount results in an approximate concentration of between 100 nanomolar to 1500 nanomolar DCMU in the aquatic environment, and wherein the effective amount inhibits Nannochloropsis growth by no more than approximately twenty percent.
Other exemplary methods for controlling a density of algae growing in an aquatic environment may include applying an effective amount of DCMU to the density of algae growing in the aquatic environment, wherein the algae includes genus Nannochloropsis, and wherein applying the effective amount results in an approximate concentration of between 0.38 micromolar to 1.55 micromolar DCMU in the aquatic environment, and wherein the effective amount inhibits Nannochloropsis growth by no more than approximately twenty percent, and wherein the effective amount of DCMU inhibits Tetraselmis growth by greater than approximately 75 percent.
As evidenced herein, the inventors have discovered and innovated systems and methods to exploit the high resistance of algal genus Nannochloropsis to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (“DCMU”). They have invented systems and methods for keeping DCMU sensitive invasive phototrophs out of various growth systems. Additionally, the inventors have quantified the extent to which cultures are contaminated with invasive algal species, including demonstrating that there is a linear relationship between the fraction of invasive species in a culture and the fluorescence rise under low actinic irradiance in the presence of low levels of DCMU. Further, the inventors have identified the genetic basis for the higher degree of tolerance of Nannochloropsis to DCMU.
3-(3,4-dichlorophenyl)-1,1-dimethylurea (“DCMU”) is a broad range plant herbicide effective against most known oxygenic phototrophs. Its mode of action is to inhibit photosynthetic electron transport by binding to the QB binding pocket on photosystem II (“PSII”). Once bound, DCMU prevents the binding of plastoquinone, thereby preventing electron transport away from PSII.
Typically, DCMU is instantaneously efficacious at concentrations in the 0.1 micromolar to 50 micromolar range. The inventors herein discovered that algae of the genus Nannochloropsis, however, generally requires higher concentrations and relatively lengthy incubation periods for the effects of DCMU to take place. At a concentration of 100 nanomolar, Nannochloropsis appears to be completely resistant to DCMU following a 24 hour incubation period.
In Chlorella, which the inventors isolated from an invaded pond at a Mexico field site, DCMU had a strong herbicidal effect at a concentration of 100 nanomolar. This is demonstrated in
At step 510, the aquatic environment or algae cultivation system is inoculated with Nannochloropsis (note: step 510 may be skipped if Nannochloropsis is already present, e.g., an existing pond, vessel, photobioreactor, etc. with Nannochloropsis). According to various exemplary embodiments, the algae cultivation system may be an open pond, a closed pond and/or a photobioreactor. Further, the Nannochloropsis culture may comprise one or more strains of the genus Nannochloropsis. Outdoor Nannochloropsis cultures may be started with the addition of an initial, small amount of pure unialgal (virtually free from unwanted contaminant organisms) Nannochloropsis. Such an inoculum may be generated in a controlled environment, such as in a laboratory or in a closed system.
At step 515, the Nannochloropsis is grown in the algae cultivation system. According to various embodiments, the Nannochloropsis culture may require light (natural or artificially supplied) for growth, as well as nutrients. Other parameters such as pH should be within acceptable ranges. The basic elements typically required for Nannochloropsis growth may include carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorous, potassium, magnesium, iron and traces of several other elements.
The required nutrients for Nannochloropsis growth may be contained in the water, supplied subsequently in dilution waters, or supplied independently of the dilution waters, in a concentration sufficient to allow Nannochloropsis to grow and reach a desired final density. The amount of nutrients needed to yield a prescribed Nannochloropsis density may be determined by the cell quota for that nutrient. That is, by the percent of the algal dry mass that is comprised of the element contained in the nutrient. The inverse of the cell quota is called the algae growth potential for that nutrient or element. For instance, if the desired final density is 1 gram/liter and the Nannochloropsis strain under consideration contains ten percent (10%) nitrogen in its biomass (i.e., a cell quota of 0.1), then the initial concentration of the atomic nitrogen in the culture should be at least 0.1 gram/liter. The same calculation may be performed for all nutrients to establish their initial concentration in the culture.
In various embodiments, a wide variety of systems utilized for the mass culturing of algae may be optimized for Nannochloropsis growth. The time-averaged light intensity to which Nannochloropsis may be exposed may be adjusted by changes in the mixing intensity and in the optical depth of the apparatus. In panel-shaped modular photobioreactors, the latter may be performed by controlling the distance between two consecutive panels. On the other hand, the optical depth in open ponds may be the depth of the pond. Similarly, the temperature in closed photobioreactors may be precisely controlled by means of indirect heat exchange. In open ponds, the temperature may be controlled by adjusting culture depth. After two to ten days, Nannochloropsis may reach a productive operating density depending on light intensity, temperature, and the starting inoculum size.
Once the Nannochloropsis is grown to a desired density, according to some embodiments, it may either be removed (and a new culture may be started with a new inoculum), or it may be diluted according to a prescribed schedule or rate. In the first case, culturing may be performed in a batch mode and may require frequent re-inoculation. In the latter case, culturing may be performed in a continuous or a semi-continuous fashion, depending on the way the dilution is performed. For example, assuming that the desired dilution rate is fifty percent (50%) per day of the culture volume, culture dilution may take place in one or more of several techniques. Culture dilution may take place continuously over the day (or over part of the day) at a constant or at a variable rate. Culture dilution may alternatively take place semi-continuously once a day (i.e., fifty percent (50%) of the culture is removed and replaced with a new growth medium in a short period of time every day); semi-continuously twice a day (i.e., twenty-five percent (25%) of the culture is removed each time at two different times every day); or semi-continuously at any other desired frequency over the day. In some embodiments, culture dilution may comprise removing the Nannochloropsis culture medium from the growth system—whether this is in an open pond or in a closed photobioreactor—and replacing this portion with fresh medium, which may contain all of the nutrients in the quantity sufficient for the growth of the Nannochloropsis between two consecutive dilutions.
At step 520, after the algae cultivation system is inoculated with Nannochloropsis and/or the Nannochloropsis is grown to a desired density, the algae cultivation system may be observed (e.g., visually with a naked eye, microscopically, and/or analytically, including the taking and analysis of samples). Such observations or sampling may take place every minute, hourly, daily, every other day, three times a week, weekly, and/or on any other suitable basis. In connection with this process, one or more determinations may be made as to a relative level or amount of predators and/or invaders in comparison to an actual and/or desired density or dominance of Nannochloropsis.
At step 525, a determination is made whether Nannochloropsis dominance in the algae cultivation system is being challenged by predators and/or invaders. Based upon this determination, a decision may be made whether to apply an effective amount of DCMU. If the level or amount of predators and/or invaders is less than a prescribed level, the algae cultivation system may continue to be observed without the application of DCMU.
At step 530, if the level or amount of predators and/or invaders exceeds an actual or desired level, an effective amount of DCMU may be applied to the density of algae growing in the algae cultivation system. One exemplary method is applying an effective amount of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to the density of algae growing in the aquatic environment, wherein the algae includes genus Nannochloropsis. The algae may also include algae of genus Tetraselmis and/or genus Chlorella. Applying the effective amount may result in an approximate concentration of between 100 nanomolar to 1500 nanomolar DCMU in the aquatic environment. Further, the aquatic environment may include seawater, freshwater, or mixtures thereof.
Further exemplary methods for controlling a density of algae growing in an aquatic environment may include applying an effective amount of DCMU to the density of algae growing in the aquatic environment, wherein the algae includes genus Nannochloropsis, and wherein applying the effective amount results in an approximate concentration of between 100 nanomolar to 1500 nanomolar DCMU in the aquatic environment, and wherein the effective amount inhibits Nannochloropsis growth by no more than approximately twenty percent.
Other exemplary methods for controlling a density of algae growing in an aquatic environment may include applying an effective amount of DCMU to the density of algae growing in the aquatic environment, wherein the algae includes genus Nannochloropsis, and wherein applying the effective amount results in an approximate concentration of between 0.38 micromolar to 1.55 micromolar DCMU in the aquatic environment, and wherein the effective amount inhibits Nannochloropsis growth by no more than approximately twenty percent, and wherein the effective amount of DCMU inhibits Tetraselmis growth by greater than approximately 75 percent.
Generally, if the density or dominance of Nannochloropsis increases, while the presence of the predators and/or invaders decreases, one may assume the application of DCMU was effective (i.e. an effective protocol).
Various embodiments may include a system for applying an effective amount of DCMU to a density of algae growing in an aquatic environment. The system may include a communications interface, a computer readable storage medium, and a processor. The computer readable storage medium may further comprise instructions for execution by the processor. The instructions for execution by the processor cause the processor to apply an effective amount of DCMU to a density of algae growing in an algae cultivation system. The processor may execute other instructions described herein and remain within the scope of contemplated embodiments.
Another embodiment may include a computer readable storage medium having a computer readable code for operating a computer to apply an effective amount of DCMU to a density of algae growing in an algae cultivation system. Examples of computer readable storage medium may include discs, memory cards, servers and/or computer discs. Instructions may be retrieved and executed by a processor. Some examples of instructions include software, program code, and firmware. Instructions are generally operational when executed by the processor to direct the processor to operate in accord with embodiments of the invention. Although various modules may be configured to perform some or all of the various steps described herein, fewer or more modules may be provided and still fall within the scope of various embodiments.
Consequently, as little as 100 grams, and as much as 400 grams, were needed. The commercial DCMU source was 90% DCMU by mass, so 90 grams and 360 grams effective amounts of DCMU (M.W. 233 amu) in 1 million liters of volume converts to DCMU concentrations of 0.38 micromolar and 1.55 micromolar, respectively.
Note “D” denotes the dumping of the contents of the pond.
The systems and methods herein may utilize DCMU in open pond systems or in closed systems such as vessels to kill or otherwise inhibit photosynthetic organisms other than Nannochloropsis. Additionally, DCMU may be utilized in conjunction with fluorescence imagining and/or detection to quantify and/or determine contamination in Nannochloropsis cultures. DCMU may also be utilized to select against photosynthetic organisms other than Nannochloropsis, so that Nannochloropsis cultures can be established from water samples containing a plurality of algal species. Further, the amino acids of positions 227-231 of the Nannochloropsis D1 protein may be changed to the evolutionarily conserved sequence to improve growth of Nannochloropsis. Finally, by changing the amino acids of a phototrophic organism's D1 protein to match the evolved moiety changes in Nannochloropsis, a photosynthetic organism may acquire heightened resistance to DCMU.
While various embodiments have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of a preferred embodiment should not be limited by any of the above-described exemplary embodiments.
The present application claims the benefit and priority of U.S. Provisional Patent Application Ser. No. 61/494,330 filed on Jun. 7, 2011, titled “DCMU Resistance in Nannochloropsis,” which is hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61494330 | Jun 2011 | US |
