WO2017179057A1

WO2017179057A1 - Cultivation of wolffia plants

Info

Publication number: WO2017179057A1
Application number: PCT/IL2017/050444
Authority: WO
Inventors: Marvin Edelman; Barak Cohen
Original assignee: Yeda Research And Development Co. Ltd.
Priority date: 2016-04-13
Filing date: 2017-04-12
Publication date: 2017-10-19
Also published as: IL245102A0

Abstract

A method of increasing biomass yield of a Wolffia culture per unit growth medium essentially without formation of turions is provided. The method comprising subjecting the culture to volumetric cultivation conditions such that the Wolffia plant of the Wolffia culture grows throughout a liquid column of the culture.

Description

CULTIVATION OF WOLFFIA PLANTS

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to cultivation of

Woljfia plants.

Woljfia are minute monocotyledonous plants of the family Lemnaceae described in details in Landolt E. (1986) The family of Lemnaceae - A monographic study, Vol 1. Veroeffentlichungen des Geobotanischen Institutes der ETH, Stiftung Ruebel, Zurich, pp. 566; and Landolt E, Kandeler R. (1987); The family of Lemnaceae - A monographic study, Vol 2. Veroeffentlichungen des Geobotanischen Institutes der ETH, Stiftung Ruebel, Zurich, pp. 638. Briefly, they measure 0.4 mm to 2.5 mm in size. The majority of Lemnaceae species grow on the surface of fresh water bodies. A few species of Lemna (L. trisulca, L. tenera, and L. valdiviana) and most species of Wolffiella can grow submerged. All known species of the genus Woljfia are gibbous and float unattached on fresh water surfaces. Under adverse conditions (crowding or other stresses) some Woljfia species, such as W. globosa, form true turions and sink to the bottom of the water column in a non-growing, hibernational state.

Woljfia globosa has a severely reduced anatomy; it is essentially a rootless thalus. While the species is capable of flowering, it normally grows strictly vegetatively. Daughter plants arise by budding, yielding genetically uniform clones. Under permissive conditions, vegetative log phase growth is nearly exponential, resulting in biomass doubling times of approximately 2-3 days. A high percentage of the plant solute is protein. The plant is likewise rich in vitamins and minerals, and is edible by domesticated animals and man. These characteristics position W. globosa as an attractive plant for agro technological exploitation.

Commercial growth of Lemnaceae plants is fraught with difficulties: water ecosystems are prone to microorganismal contamination and there is little industrial experience for guidance. It is commonly held that Lemnaceae are only able to utilize nutrients from the upper water layers. Essentially, as practiced today, growth of Woljfia is limited to two dimensions at or near the surface Ruekaewma et al. 2015 Songklanakarin J. Sci. Tecnol. 37(5):575-580; pool depth figures almost exclusively as a sink for nutrients. SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of increasing biomass yield of a Woljfia culture per unit growth medium essentially without formation of turions, the method comprising subjecting the culture to volumetric cultivation conditions such that the Wolffia plant of the Woljfia culture grows throughout a liquid column of the culture, wherein the conditions are selected from the group consisting of:

(i) air flow above 100 L/hour (h);

(ii) light regimen above 12 h per 24 h period; and

(iii) light regimen comprising light intensity lower than 250 μιηο1/ιη²/8 , thereby increasing the biomass yield of the culture.

According to some embodiments of the invention, the conditions comprise:

(i) air flow above 100 L/hour (h); and

(ii) light regimen above 12 h per 24 h period.

According to some embodiments of the invention, the conditions comprise:

(i) air flow above 100 L/hour (h); and

(ii) light regimen comprising light intensity lower than 250 μιηο1/ιη²/8

According to some embodiments of the invention, the conditions comprise:

(i) light regimen above 12 h per 24 h period; and

(ii) light regimen comprising light intensity lower than 250 μιηο1/ιη²/8

According to some embodiments of the invention, the conditions comprise:

(i) air flow above 100 L/hour (h);

(ii) light regimen above 12 h per 24 h period; and

(iii) light regimen comprising light intensity lower than 250 μιηο1/ιη²/8

According to some embodiments of the invention, the conditions comprise:

(i) air flow above 400 L/hour (h);

(ii) light regimen above 18 h per 24 h period; and/or

(iii) light regimen comprising light intensity lower than 100 μιηο1/ιη²/8.

According to an aspect of some embodiments of the present invention there is provided a method of increasing biomass yield of a Woljfia culture per unit growth medium, the method comprising subjecting the culture to volumetric cultivation such that the Wolffia plant of the Woljfia culture grows in a submerged state, thereby increasing the biomass yield of the culture.

According to an aspect of some embodiments of the present invention there is provided a Woljfia plant capable of growing in a submerged state.

According to some embodiments of the invention, the plant is capable of growing in the submerged state under static conditions for at least 10 generations.

According to some embodiments of the invention, the plant has a starch content that is at least 25 % greater than that of wild type grown in the same conditions.

According to some embodiments of the invention, the volumetric cultivation is by agitation of the plant throughout the liquid column.

According to some embodiments of the invention, the agitation is effected by bubbling.

According to some embodiments of the invention, bubbles of the bubbling are of a diameter of 0.1 mm to 50 cm.

According to some embodiments of the invention, a rate of the bubbling comprises 1 to 10,000 bubbles per second.

According to some embodiments of the invention, the bubbling is with air enriched with C0₂.

According to some embodiments of the invention, the C0₂ is comprised at a concentration of 0.05 to 15 %.

According to some embodiments of the invention, the bubbling is with air enriched with oxygen_.

According to some embodiments of the invention, the oxygen is comprised at a concentration of up to 28 %.

According to some embodiments of the invention, the agitation is further effected by spinning, rocking or rotating.

According to some embodiments of the invention, the increasing biomass yield results in an increase of at least 0.2 fold in standing-area density as compared to that in surface growth under the same conditions without the volumetric cultivation.

According to some embodiments of the invention, the increasing biomass yield results in an increase of at least 0.2 fold in biomass weight as compared to that in surface growth under the same conditions without the volumetric cultivation. According to some embodiments of the invention, the submerged state of the plant is also characterized by a dividing phenotype and green color throughout the volumetric cultivation.

According to some embodiments of the invention, the agitation results in contact of the plant with an air surface.

According to some embodiments of the invention, the Woljfia plant is Woljfia globosa.

According to some embodiments of the invention, the Woljfia plant is Woljfia australiana.

According to some embodiments of the invention, the Woljfia plant is the plant of any one of claims 2-3.

According to some embodiments of the invention, the volumetric cultivation is for 2 days to 4 weeks.

According to some embodiments of the invention, the volumetric cultivation is effected in a container having an internal fillable volume of 1 ml to 3,000,000 liters.

According to some embodiments of the invention, the plant is a transgenic plant.

According to some embodiments of the invention, the plant is a non-transgenic plant.

According to an aspect of some embodiments of the present invention there is provided a harvested material of a Woljfia plant grown according to the method as described herein.

According to some embodiments of the invention, the harvested material comprises a ratio of floating Wollfia plants to submerged Wollfia plants which is smaller than that found by surface growth in the logarithmic state.

According to an aspect of some embodiments of the present invention there is provided a method of producing feedstock, the method comprising:

(a) growing Woflfia plants according to the method as described herein; and

(b) harvesting the Woljfia plants.

According to an aspect of some embodiments of the present invention there is provided a method of producing a biofuel, the method comprising:

(a) growing Woljfia plants according to the method as described herein;

(b) harvesting the Woflfia plants; and (c) processing the biofuel from the harvested Woljfia plants.

According to some embodiments of the invention, the biofuel is selected from the group consisting of ethanol, butanol and biogas.

According to an aspect of some embodiments of the present invention there is provided a method of bioremediation, the method comprising growing Woljfia plants as described herein in the presence of a pollutant in a contaminated site.

According to some embodiments of the invention, the pollutant is selected from the group consisting of phosphorus, nitrogen, heavy metal, cyanotoxin, sex steroid and corticosteroid.

According to an aspect of some embodiments of the present invention there is provided a method of extracting a protein or metabolite of interest, the method comprising:

(a) growing Woflfia plants according to the method as described herein;

(b) harvesting the Woljfia plants; and

(c) extracting the protein or metabolite of interest from the harvested Woljfia plants.

According to some embodiments of the invention, the protein is endogenous to the Woljfia plant.

According to some embodiments of the invention, the protein is a recombinant protein.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

FIG. 1 shows growth of Woljfia globosa after NMU treatment, pictured after 20 vegetative doublings;

FIG. 2 shows the difference between wild type (WT) Woljfia globosa true turions and sunken NMU-treated Woljfia globosa plants;

FIGs. 3A-B show the increase in biomass of Woljfia globosa per unit of growth medium. Figure 3A - Growth vessel at the start of the experiment; Figure 3B - Growth after 6 days.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method of cultivation of a Woljfia plant for increasing biomass yield.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Commercial growth of Lemnaceae plants is fraught with difficulties: water ecosystems are prone to microorganismal contamination and there is little industrial experience for guidance. It is commonly held that Lemnaceae are only able to utilize nutrients from the upper water layers. Essentially, as practiced today, growth of Woljfia is limited to two dimensions at or near the surface, whereby a major limiting factor is space.

Whilst reducing the present invention to practice, the present inventors were able to generate Woljfia plants that grow submerged at the bottom of the liquid column, as well as, independently, identify growth conditions which allow the growth of wild-type Woljfia throughout the liquid column, thereby mitigating the limitation of growth at or near the surface.

As is illustrated hereinbelow and in the Examples section which follows, the present inventors showed that vigorous bubbling such that agitates the plants throughout the liquid column (volumetric growth) causes a far higher increase in standing density area (Example 5) as compared to that obtained by surface growth. Bubbling of air is critical for biomass accumulation, as bubbling of gas other than air, or C0₂/oxygen- enriched air, failed to support volumetric growth (Example 2). The advantage of volumetric growth compared to static growth at or near the surface was manifested by both biomass fold increase and biomass doubling time (Example 3) as well as in standing density area at late log phase (Example 7). Plants subjected to bubbling retain a green, dividing and a healthy looking phenotype (Example 4, Example 6). Even at late log phase, a green and healthy phenotype is evident for both floaters and sinkers. No true turions are evident (Example 7). Compared to floaters, sinkers are endowed with higher starch content. The results were substantiated on various Wolffia globosa strains and other Wolffia species (Examples 10-11). The present inventors were also able to mutate wild type Wolffia to generate Wolffia plants which retain a stable submerged (sunken) phenotype through a number of generations, in the absence of bubbling without a significant increase in true turions (Examples 13-14).

Without being bound by theory, it is suggested that there are two, somewhat opposing factors involved in volumetric growth of normally surface growing Wolffia plants: one is contact with an air surface, the other is an applied method of agitation, shaking, etc., which manages to submerge the floating plants and keeps them moving about throughout the liquid column. The simultaneous application of both of these vectors is crucial for volumetric growth.

Thus according to an aspect of the invention there is provided a method of increasing biomass yield of a Wolffia culture per unit growth medium, the method comprising subjecting the culture to volumetric cultivation such that the plant of the Wolffia culture grows in a submerged state, thereby increasing the biomass yield of the Wolffia culture.

According to another aspect there is provided a method of increasing biomass yield of a Wolffia culture per unit growth medium essentially without formation of turions, the method comprising subjecting the culture to volumetric cultivation conditions such that the Wolffia plant of the Wolffia culture grows throughout a liquid column of the culture, wherein said conditions are selected from the group consisting of:

(i) air flow above 100 L/hour (h);

(ii) light regimen above 12 h per 24 h period; and (iii) light regimen comprising light intensity lower than 250 μιηο1/ιη²/8 , thereby increasing the biomass yield of the culture.

According to some embodiments the conditions comprise:

(i) air flow above 100 L/hour (h); and

(ii) light regimen above 12 h per 24 h period.

According to some embodiments the conditions comprise:

(i) air flow above 100 L/hour (h); and

(ii) light regimen comprising light intensity lower than 250 μιηο1/ιη²/8.

According to some embodiments the conditions comprise:

(i) light regimen above 12 h per 24 h period; and

According to some embodiments the conditions comprise:

(i) air flow above 100 L/hour (h);

(ii) light regimen above 12 h per 24 h period; and

(iii) light regimen comprising light intensity lower than 250 μιηο1/ιη²/8.

According to some embodiments the conditions comprise:

(i) air flow above 400 L/hour (h);

(ii) light regimen above 18 h per 24 h period; and/or

Further conditions are described hereinbelow in much details.

As used herein the term "Woljjia" refers to a genus of 9-11 species of the Lemnaceae family. Woljjia species natively grow at or near the surface, and are also referred to herein as "wild-type" or "floaters".

Examples of Woljjia species that can be used in accordance with the present teachings include, but are not limited to, Woljjia angusta, Woljjia arrhiza, Woljjia australiana, Woljjia borealis, Woljjia brasiliensis, Woljjia columbiana, Woljjia cylindracea, Woljjia elongata, Woljjia globosa, Woljjia microscopica and Woljjia neglecta, wherein each possibility represents a separate embodiment.

According to a specific embodiment, the Woljjia plant is Woljjia globosa.

According to a specific embodiment, the Woljjia plant is Woljjia globosa var. Noam or Woljjia globosa strain 9331.

According to a specific embodiment, the Woljjia plant is not Woljjia arrhiza. According to a specific embodiment, the Woljfia plant is Woljfia australiana. According to a specific embodiment, the Woljfia plant is Woljfia australiana strain 7211.

According to a specific embodiment, the Woljfia plant is a wild-type plant.

According to a specific embodiment, the Woljfia plant is a mutated plant.

As used herein the term "mutated plant" refers to any Woljfia plant which exhibits an agriculturally valuable or commercially valuable trait. Examples include but are not limited to increased biomass/yield, carbohydrate (starch)/protein content, biotic stress tolerance, abiotic stress tolerance and the like.

According to a specific embodiment, the mutated plant is capable of growing in a submerged state.

As used herein the term "growing" refers to biomass increase by daughter plant emergence by a process of budding. The mutated plant grows in a sunken state, as opposed to wild type Woljfia that may be found in a sunken state when stressed but only as a non-growing true turion (in hibernation).

As used herein "turion" refers to a dormant state of Wolffia incapable of biomass increase even in the presence of a carbon source.

As used herein "essentially without the formation of turions" means that over 90 %, with a range of 80% to 98%, of the biomass in the culture is in a stage of vegetative growth.

As used herein "submerged" refers to the presence of plants in the culture growing anywhere throughout the liquid column that is not at the liquid surface. "Sunken" or "sinker" refers to growing plants which sink to the bottom of the culture vessel when the culture is not subjected to agitation.

According to a specific embodiment, the submerged state of the plant is also characterized by a dividing phenotype and green color throughout said volumetric cultivation.

Submerged Woljfia can occur spontaneously, as a result of volumetric growth, such that in the absence of bubbling, the Woljfia plant retains its submerged phenotype. Such submerged phenotypes are typically transient, retaining their submerged phenotype for e.g., at least 5 hours or 10 hours or 24 hours following cessation of bubbling, after which they rise in the growth column and float. Alternatively, submerged Woljfia can be obtained following exposure to a mutagen or stress and selection for the desired phenotype (i.e., growth in submerged state).

Examples of stress conditions which can be used according to some embodiments of the invention include, but are not limited to, chemical mutagenesis by N-Nitroso-N-methlyurea (NMU) and ionic shock treatment. Using these conditions, the present inventors were able to obtain lines NE3, and NE10 obtained following incubation with 10 mM NMU in 7% glacial acetic acid, final concentration, for 60 minutes.

Examples of mutagens which can be used in accordance with the present teachings include, but are not limited to gamma radiation, UV radiation alkylating agents such as NEU, EMS, NMU and the like. The skilled artisan will know which agent to select. Guidelines for plant mutagenesis are provided in K Lindsey Plant Tissue Culture Manual - Supplement 7: Fundamentals and Applications, 1991.

According to a specific embodiment, the submerged Woljfia comprises a leaky mutation.

According to a specific embodiment, the plant is capable of growing in a submerged state under static conditions for at least 50 e.g., at least 100, generations (e.g., up to at least 200 days).

According to a specific embodiment, the sunken plant has a starch content that is at least twice that of wild type growing statically on the surface.

Using such conditions, the present inventors were able to obtain the sunken NE3 and NE10 lines.

According to a specific embodiment, the submerged plant is larger than the plant grown in a floating state, it actively grows and multiplies in the submerged state.

Any of the plants used according to the present teachings can be transgenic or non-transgenic plants. The transgene may be such that imparts the plant with a commercial (e.g., recombinant pharmaceutical) and/or growth advantage. Methods of stably or transiently transforming Woljfia plants are well known in the art. See for instance 20120258491.

As used herein, the term "biomass" refers to the amount of tissue (e.g., measured in grams of plant fresh weight with excess liquid blotted away), cells, metabolites, proteins, lipids produced from the plant in a growing batch, which could also determine or affect the plant yield or the yield per growing area or in this case per unit growth medium. An increase in plant biomass can be in the whole plant or in parts thereof such as harvestable parts. Thus, for example, harvestable plant material can include the plant fibers, proteins, polysaccharides etc., typically obtained following processing of the plant.

In some further embodiments, the plant can be a whole plant or a plant part or a particulate plant material. "Whole" or "essentially intact" plant is to be understood to encompass a plant with its original whole cellular skeletal structure, namely, whole cells (viable or non-viable) without applying any crushing, grinding, powdering etc., of the plant or of at least the plant's fronds; while the term "plant part" or "particulate plant material" or "pieces of plant" is to be understood as referring to a plant after being subjected to at least one processing step that resulted in the disruption of the cellular structure of the plant, for instance, grinding, crushing or subjecting the plant to shear forces, as well subjecting to extraction processes. In some embodiments, the particular plant material encompasses one or more of whole plant cells, fractionated cells and combination of same.

In some further embodiments, the harvested plant material is obtained from fresh, partially dried plant material or essentially fully dried plant material. The harvested material may be whole plant material or processed plant material, e.g. where the cells structure was disrupted.

As used herein "volumetric cultivation" or "volumetric growth" refers to growing the plant in a submerged state, such as throughout the liquid column or in other words while exploiting the volume of the container and not just the surface for growth.

Throughout the liquid column refers to growth from surface to bottom +/- 10 % of the growth vessel (e.g., container, pond, raceway).

According to a specific embodiment volumetric growth is up to 5 meters deep, up to 4 meters deep, up to 3 meters deep, up to 2 meters deep, up to 1.8 meters deep, up to 1.6 meters deep, up to 1.3 meters deep, up to 1.1 meters deep, up to 0.9 meters deep, up to 0.7 meters deep, up to 0.5 meters deep, wherein each possibility represents a separate embodiment. The depth will depend on the desired production scale. According to a specific embodiment, the volumetric growth is 0.1-5 meters deep, is 0.1-4 meters deep, is 0.1-3 meters deep, is 0.1-2 meters deep, is 0.1-1.8 meters deep, is 0.1-1.5 meters deep, is 0.1-1.2 meters deep, is 0.1-1 meter deep, is 0.1-0.8 meters deep, is 0.1-0.6 meters deep, is 0.1-0.4 meters deep, wherein each possibility represents a separate embodiment. The depth will depend on the desired production scale.

According to a specific embodiment, the volumetric growth is limited by the photosynthetic depth, which is especially relevant under natural lighting.

In other cases, artificial illumination can be placed externally surrounding the liquid column or internally (e.g., with waterproof LED illumination) throughout the liquid column.

According to a specific embodiment, the volumetric cultivation is effected by bubbling through the liquid in such a manner that the plant grows in a submerged state. As mentioned hereinabove, the present inventors have realized that air is critical for volumetric growth of Wolffia.

Basically compressed air is introduced into the culturing medium in a manner that provides turbulence and continuous movement of the plant.

Any of numerous bubbling means (aerating device) known in the art can be used to generate bubbles e.g., pipetter, air stone, diffuser, air filters equipped with an air lift, and the like. Any of these methods is selected or calibrated specifically to generate bubbles that are effective in maintaining the plant in motion so as to leave them in a submerged state throughout the growing period. Naturally, the size of the bubbles will grow as the pressure drops along the liquid column in line with Boyle's law. Hence, the positioning of the means of bubbling along the liquid column affects the size of the bubbles. According to a specific embodiment, a single means of bubbling is placed in the container. According to another specific embodiment, a plurality of bubbling means are placed in the container. These can be placed anywhere along the liquid column (at the same depth or at different depths).

According to a specific embodiment, the air flow imparting said turbulence is 100-2000 L/h. According to a specific embodiment, the air flow is 100-1500 L/h. According to a specific embodiment, the air flow is 100-1400 L/h. According to a specific embodiment, the air flow is 100-1300 L/h. According to a specific embodiment, the air flow is 100-1100 L/h. According to a specific embodiment, the air flow is 100- 1000 L/h. According to a specific embodiment, the air flow is 200-1500 L/h. According to a specific embodiment, the air flow is 100-900 L/h. According to a specific embodiment, the air flow is 100-800 L/h. According to a specific embodiment, the air flow is 100-700 L/h. According to a specific embodiment, the air flow is 100-600 L/h. According to a specific embodiment, the air flow is 100-500 L/h. According to a specific embodiment, the air flow is 100-400 L/h. According to a specific embodiment, the air flow is 200-600 L/h. According to a specific embodiment, the air flow is 200-500 L/h. According to a specific embodiment, the air flow is 100-400 L/h. According to a specific embodiment, the air flow is 200-800 L/h. According to a specific embodiment, the air flow is 300-600 L/h. According to a specific embodiment, the air flow is 300-700 L/h. According to a specific embodiment, the air flow is 400-1000 L/h. According to a specific embodiment, the air flow is 400-900 L/h. According to a specific embodiment, the air flow is 400-800 L/h. According to a specific embodiment, the air flow is 400-700 L/h. According to a specific embodiment, the air flow is 400-600 L/h, wherein each possibility represents a separate embodiment.

According to a specific embodiment, bubbles of said bubbling are of a diameter of 0.1 mm to 50 cm (as always upon release from the bubble source). According to a specific embodiment the bubbles are of a diameter of 0.2 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.25 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.3 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.35 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.4 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.45 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.5 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.55 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.6 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.65 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.7 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.75 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.8 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.85 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.9 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 0.95 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 1 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 2 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 3 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 4 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 5 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 6 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 7 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 8 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 9 mm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 1 cm to 50 cm, wherein each possibility represents a separate embodiment.

For large scale production, according to some embodiment, bubbles of said bubbling are of a diameter of 10 cm to 50 cm (as always upon release from the bubble source). According to a specific embodiment the bubbles are of a diameter of 15 cm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 20 cm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 25 cm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 30 cm to 50 cm. According to a specific embodiment the bubbles are of a diameter of 40 cm to 50 cm, wherein each possibility represents a separate embodiment.

A terminal upper diameter when reaching the surface may be defined as the entire diameter of the growth vessel (in a round vessel). Thus, up to 20 %, 30 %, 40 %, 60 %, 70 %, 80 %, 90 % or even 100 % of the surface width/diameter of the container (vessel), wherein each possibility represents a separate embodiment.

A specific embodiment of the invention, relates to bubbles which are 2-10 mm in diameter, 3-10 mm in diameter, 4-10 mm in diameter, 5-10 mm in diameter, 6-10 mm in diameter, 7-10 mm in diameter, 8-10 mm in diameter. A specific embodiment of the invention, relates to bubbles which are 2-9 mm in diameter, 2-8 mm in diameter, 2-7 mm in diameter, 2-6 mm in diameter, 2-5 mm in diameter, 2-4 mm in diameter, 2-3 mm in diameter, wherein each possibility represents a separate embodiment. According to a specific embodiment, the rate of the bubbling comprises 1 to 10,000 bubbles per second (meaning per bubbling outlet). According to a specific embodiment, the rate of the bubbling comprises 100 to 10,000 bubbles per second, 200 to 10,000 bubbles per second, 300 to 10,000 bubbles per second, 400 to 10,000 bubbles per second, 500 to 10,000 bubbles per second, 600 to 10,000 bubbles per second, 700 to 10,000 bubbles per second, 800 to 10,000 bubbles per second, 900 to 10,000 bubbles per second, 1000 to 10,000 bubbles per second, 2000 to 10,000 bubbles per second, 3000 to 10,000 bubbles per second, 4000 to 10,000 bubbles per second, 5000 to 10,000 bubbles per second, 100 to 5,000 bubbles per second, 100 to 4,000 bubbles per second, 100 to 3,000 bubbles per second, 100 to 2,000 bubbles per second, 100 to 1,000 bubbles per second, 100 to 500 bubbles per second, 100 to 400 bubbles per second, 100 to 300 bubbles per second, 100 to 200 bubbles per second, wherein each possibility represents a separate embodiment.

According to a specific embodiment, the rate of the bubbling comprises 100 to 200 bubbles per second.

As mentioned, the present inventors have uncovered that bubbling with air (oxygen/C0₂) is critical for volumetric growth.

Hence, according to a specific embodiment, bubbling is effected with air.

According to another specific embodiment, bubbling is effected with C0₂ enriched air.

According to a specific embodiment, C0₂ is comprised at a concentration of 0.05 to 15 %. According to a specific embodiment, C0₂ is comprised at a concentration of 0.05 to 12 %. According to a specific embodiment, C0₂ is comprised at a concentration of 0.05 to 10 %. According to a specific embodiment, C0₂ is comprised at a concentration of 0.05 to 8 %. According to a specific embodiment, C0₂ is comprised at a concentration of 0.05 to 6 %. According to a specific embodiment, C0₂ is comprised at a concentration of 0.05 to 5 %. According to a specific embodiment, C0₂ is comprised at a concentration of 0.05 to 4 %. According to a specific embodiment, C0₂ is comprised at a concentration of 1 to 10 %. According to a specific embodiment, C0₂ is comprised at a concentration of 1 to 8 %. According to a specific embodiment, C0₂ is comprised at a concentration of 1 to 6 %. According to a specific embodiment, C0₂ is comprised at a concentration of 1 to 5 %. According to a specific embodiment, C0₂ is comprised at a concentration of 1 to 4 %. According to a specific embodiment, CO₂ is comprised at a concentration of 1 to 3 %. According to a specific embodiment, C0₂ is comprised at a concentration of 2 to 4 %, wherein each possibility represents a separate embodiment.

According to another specific embodiment, bubbling is effected with oxygen enriched air.

According to a specific embodiment, oxygen is comprised in said air at a concentration of up to 28 % %.

According to a specific embodiment, oxygen is comprised in said air at a concentration of up to 26 % %.

According to a specific embodiment, oxygen is comprised in said air at a concentration of up to 24% %.

The present inventors recorded a specific embodiment in slow-motion filming (not shown) at 240 frames per second, 10-20 air bubbles were counted to emit from the 1 mm pipette tip bore per second. At a normal-motion video speed of 30 frames per second this number is increased by a factor of eight and translates to 80-160 bubbles per second. The bubbles rapidly coalesce and grow in size from a diameter of 2-4 mm at a height of 2 mm from the 1 mm bore of the Pasteur pipette tip near the tube bottom to bubbles of 5-7 mm diameter at a height of 70-90 mm from tube bottom. A total of 10- 15 coalesced bubbles are simultaneously rising in the tube at any given time. It takes from 0.25 to 0.35 sec for a bubble to rise the full 90 mm in the tube at a normal viewing speed of 30 fps, wherein each possibility represents a separate embodiment.

In another recorded slow motion filming the bubble diameter is 15-20 mm at a height of 70-90 mm from tube bottom.

According to a specific embodiment, bubbling is effected such that the plant becomes in contact with air surface (which is not the air surface of the culture, but of the bubbles).

To ensure sufficient agitation of the plants and prevent their floating at the surface the plants are subjected to agitation by any agitating means which spin, cycle, rock or rotate the culture and plants therein.

The present teachings avoid agitation of the medium alone without movement of the plants in the liquid column. Culturing of the plant is effected using methods which are well known in the art, taking into consideration the lighting, nutritional and temperature conditions (e.g., 30 °C or below 30 °C), which are necessary for plant growth, be it optimal or sub-optimal.

In some embodiments, the plants are grown as a monoculture. In other embodiments the plants are grown in a mixed culture with other plants of the same species (e.g., floaters and sinkers) or strains or different species. In still other embodiments, plants are grown as part of a complex ecosystem that comprises one or more additional animal, plant or prokaryote. In yet another embodiment, the plants are grown in an axenic culture.

In some embodiments, the plants are grown in closed systems (while allowing for escape of forced air bubbles from the growth container).

In some embodiments, the plant are grown under sterile conditions.

In some embodiments, the plants are grown in open systems.

In some embodiments, the plants are grown in direct exposure to sunlight. In other embodiments the plants are grown in indirect light.

According to specific embodiments, the light intensity is 10-350 μΕ /m /s. According to specific embodiments, the light intensity is 10-250 μΕ /m /s. According to specific embodiments, the light intensity is 10-150 μΕ /m /s. According to specific embodiments, the light intensity is 10-100 μΕ /m /s. According to specific embodiments, the light intensity is 10-50 μΕ /m /s. According to specific embodiments, the light intensity is 20-100 μΕ /m /s. According to specific embodiments, the light intensity is 40-100 μΕ /m /s. According to specific embodiments, the light intensity is 30-100 μΕ /m /s. According to specific embodiments, the light intensity is 40-250 μΕ

2 2

/m /s. According to specific embodiments, the light intensity is 40-150 μΕ /m /s, wherein each possibility represents a separate embodiment.

According to specific embodiments, the light regime is above 12 h per 24 h period. According to specific embodiments, the light regime is above 13 h per 24 h period. According to specific embodiments, the light regime is above 14 h per 24 h period. According to specific embodiments, the light regime is above 15 h per 24 h period. According to specific embodiments, the light regime is above 16 h per 24 h period. According to specific embodiments, the light regime is above 17 h per 24 h period. According to specific embodiments, the light regime is above 18 h per 24 h period. According to specific embodiments, the light regime is above 19 h per 24 h period. According to specific embodiments, the light regime is above 20 h per 24 h period. According to specific embodiments, the light regime is above 21 h per 24 h period. According to specific embodiments, the light regime is above 22 h per 24 h period. According to specific embodiments, the light regime is above 23 h per 24 h period. According to specific embodiments, the light regime is constant lighting, wherein each possibility represents a separate embodiment.

Other conditions can be selected and/or varied to support rapid growth, desirable protein profiles and/or carbohydrate production, and the like. In yet another embodiment, the plants are grown under artificial lighting.

In some embodiments, the nitrogen source used to promote the growth of the plants is comprised of animal waste, such as cow dung or pig waste and the like. In other embodiments, the nitrogen source is urea. In still other embodiments, the nitrogen source is biogas plant slurry.

The culture can be fitted with heating elements and/or a cooling system in order to regulate the temperature of the growing plants.

Using volumetric growth, the present inventors were able to increase the biomass yield of the Woljfia plants significantly over that obtained when growing a floating culture.

Thus, according to a specific embodiment, increasing biomass yield results in an increase of at least 0.2 (e.g., 0.2-9, 1.2-9, 1.5-9, 2-9, 2.5-9, 3-9, 4-9, 5-9) fold in standing-area density as compared to that in surface growth under the same conditions without said volumetric cultivation.

According to an alternative or an additional embodiment, increasing biomass yield results in an increase of at least 0.2 (e.g., 0.2-500, 1.5-500, 5-500, 10-500, 50-500, 100-500, 200-500, 300-500) fold in biomass weight as compared to that in surface growth under the same conditions without said volumetric cultivation.

The culture is typically kept in log phase growth until harvested. Operationally, stationary phase starts when crowding occurs. (The population growth curve for Lemnaceae is generally like that for microorganisms such as E. coli.). Thus, for example, the culture is grown volumetrically for 2-4 days (a single doubling) to 2-4 weeks (4-14 doublings) before harvesting or crowding brings biomass increase to a halt. Large scale settings for Woljfia can operate within about the same time frames.

The timing much depends on the intended use and the scale of the setting.

According to the invention, the incubation may be any type of culturing known in the art, such as batch culture, fed batch culture and continuous culture.

Harvesting type and conditions will much depend on the intended use of the harvested material.

When using wild-type Woljfia, the plants float upon cessation of the agitation. A floating matt is formed and a skimming mechanism applied. The culture medium in some cases can then be recycled for further use.

Alternatively, when the harvested material is a secreted product, the medium is collected. For instance secreted biologically active polypeptide (e.g., transgene product) can be harvested from the culture medium by any conventional means known in the art and purified by chromatography, electrophoresis, dialysis, solvent-solvent extraction, and the like.

Sinker forms can be harvested through an escape valve at the bottom of the column.

The present invention also envisages a harvested material of a Woljfia plant grown as described herein.

According to a specific embodiment, the harvested material comprises a high starch content plant, as described above.

The present invention can be performed using a cultivation system (such as those used by traditional Woljfia growers, supplemented with bubbling means (aeration device(s)) as described herein and possibly agitation means for complementing the agitation formed by the bubbling means to effect volumetric growth.

According to a specific embodiment, the plant volumetrically cultivated according to the present teachings has a protein concentration above 15 % of dry matter (e.g., 25-45 %, 25-40 %, 25-35 %, 25-30 %, 20-48 %, 20-45 %, 20-40 %, 20-35 %, 20- 30 % or 25-48 %), wherein each possibility represents a separate embodiment.

Embodiments of the invention include methods of growing Woljfia that can be useful as food, feed, fuel, fertilizer, organic and/or inorganic products and/or for bioremediation. Certain embodiments provide methods for extruding proteins from wet biomass without corresponding loss of carbohydrates.

Thus, according to an aspect of the invention there is provided a method of producing feedstock, the method comprising:

(a) growing Woflfia plants according to the method described herein; and

(b) harvesting the Woljfia plants.

According to a further aspect there is provided a method of producing a biofuel, the method comprising:

(a) growing Woljfia plants (e.g., a Woljfia capable of growing in a submerged state) under volumetric growth conditions;

(b) harvesting the Woflfia plants; and

(c) processing the biofuel from the harvested Woljfia plants.

Woljfia can be utilized to produce biofuel (ethanol, butanol and biogas), which are promising alternative energy sources to minimize dependence on limited crude oil and natural gas. The advantages of using the present methodology include high rate of nutrient (nitrogen and phosphorus) uptake, high biomass yield and great potential as an alternative feedstock for the production of fuel ethanol, butanol and biogas.

There are mainly two processes affecting the accumulation of starch in Woljfia biomass: photosynthesis for starch generation and metabolism-related starch consumption. The cost of stimulating photosynthesis is relatively high based on current technologies. Considerable research efforts have been made to inhibit starch degradation. The submerged type of Woljfia as described herein are endowed with a high starch content and therefore they are particularly suitable for biofuel production.

According to an additional aspect there is provided a method of bioremediation, the method comprising growing Woljfia plants as described herein in the presence of a pollutant in a contaminated site.

Woljfia acts as a bioremediator of excess phosphorus and nitrogen through its rapid growth and uptake of these elements. Woljfia is used in Thailand to treat shrimp farm effluent. It is well accepted that Woljfia show promise for use in sustainable wastewater treatment systems. Woljfia accumulates toxic heavy metal such as lead, cadmium, chromium and arsenic as well as cyanotoxins such as microcystin. Because of its mixotrophic ability, including organotrophy (uptake of dissolved organic carbon) Woljfia also accumulates exotic molecules including sex steroids and corticosteroids found in human sewage discharge, helping to reduce their concentration and downstream consequences. At the same time such substances enhance growth of Woljfia through stimulation of DNA and RNA, and increase its production of proteins and sugars (Szamrej and Czerpak 2004).

Thus, according to a specific embodiment, the pollutant is selected from the group consisting of phosphorus, nitrogen, heavy metal, cyanotoxin, sex steroid and corticosteroid.

The harvested plants can be used as a nutritional source. High protein and carbohydrate content, fast growth rate, and ease of harvest place Woljfia as a potential major food source for humans and feed source for animals. Woljfia is estimated to produce 60 times more protein per hectare per year than soybeans. Woljfia is eaten by herbivorous fish as well as a variety of waterfowl. It is also used both as fodder for cattle, pigs and poultry and as a fertilizer because of its high phosphorus and nitrogen accumulation, in Africa, India, and Southeast Asia.

Also provided is a method of extracting a protein or metabolite of interest, the method comprising:

(a) growing Woflfia plants according to the method described herein;

(b) harvesting the Woljfia plants; and

According to a specific embodiment, the protein is endogenous to the Woljfia plant.

According to a specific embodiment, the protein is a recombinant protein.

Generally, a genetically modified Woljfia is a known expression system for producing various proteins (see U.S. Patent No. 6,040,498), including for the production of monoclonal antibodies (see U.S. Patent No. 7,632,983).

The present teachings further relate to processed products generated from the harvested plant. These include, but are not limited to biofuel, meal, energy bar, bread, cake, super absorbent polymers, plastics, chlorophyll and carotenoid based pigments and the like. As used herein the term "about" refers to ± 10 %

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

EXAMPLE 1

THE EFFECT OF BUBBLING ON BIOMASS INCREASE DURING COLUMN

GROWTH

Experimental Procedures

General - Plants were grown in Hoagland solution (Landolt & Kandeler 1987, supra) at 35-40 μΕ/ml/s, of continuous cool white fluorescent light, at 25 +2°C, in glass tubes (length, 150 mm; inner diameter, 23 mm). The tubes were covered with a layer of Parafilm, punctured to allow the insertion of a sterilized, cotton-plugged, 230 mm long- nosed Pasteur pipette. In some cases, a plastic tube cap with indentations on the inner surface to permit adequate exchange of gasses and a hole bored in the center was added above the punctured Parafilm. The pipette, connected to a forced air source stepped- down to a flow rate of 550 +50 liters air per hour, was inserted until just above the bottom of the liquid column. Several punctures with a 25 gauge hypodermic needle were made in the Parafilm cover to allow for gas flow. Vigorous bubbling of air agitated the plants so that virtually all were constantly moving about at a fast pace, promoting a continuous presence of plants throughout the liquid column. All solutions and glassware were sterilized. The forced air source was passed through a cotton filter but not sterilized.

Specific assay of Example 1.

Thirty five ml of Hoagland solution, reaching a height of 9 cm, were introduced into each of two glass columns set up as above. Woljfia globosa var. Noam plants in the amounts listed in Table 1, were introduced and air was vigorously bubbled through the growth vessel, or not, as indicated. At day seven, 5 ml of water was added to the bubbled sample to counter evaporation. After fifteen days of growth, the plants were harvested by emptying tube contents onto a 40 mm diameter, 50μιη mesh net. Excess liquid was removed by blotting on successive, sterile 90 mm diameter Whatman 1 filter circles until no wetness was obvious on the last filter. Biomass fold-increase (biomass at collection day / biomass at initial day) and biomass doubling times (www. doubling- time. com/compute.php) were then calculated (Table 1).

Table 1: Biomass accumulation in growth tubes with and without bubbling

Biomass Biomass

Plant Biomass (mg) fold doubling

increase time (d)

Day 0 Day 15

No Bubbling 91 260 2.86 9.9

Bubbling 149 897 6.02 5.8

The results show that bubbling, with the ensuing agitation of the plants throughout the growth column, produced significantly greater accumulation of biomass than static growth on the surface without bubbling.

EXAMPLE 2

THE REQUIREMENT FOR BUBBLING OF AIR ON BIOMASS INCREASE DURING VOLUMETRIC GROWTH

This experiment was set up to distinguish between bubbling per se and the gaseous content of the bubbles as a factor in volumetric growth. Woljfia globosa var Noam plants were placed in glass columns (2.3 cm diameter) or Petri plates (9 cm diameter) containing Hoagland solution and treated as in Example 1. Bubbles of gas were provided either as forced air or forced 100 % nitrogen. After an acclimatization period of 5 days, the plants were weighed and returned to their growth vessels. At this point, the experiment was initiated and growth assessed after 4 days. Weight was determined as in Example 1. Biomass fold increase and biomass doubling times were determined as in Example 1. The results are summarized in Table 2.

Table 2: Comparison of surface and volumetric growth in air (containing normal amounts of oxygen and carbon dioxide) versus 100% nitrogen

Biomass

Biomass fold

Method of growth Biomass (mg) doubling time

increase (d)

(d)

Da O Day 4

Volumetric, bubbling

Glass column, Air 122 182 1.49 6.9

Glass Column, Nitrogen 92 82 0.89 no growth

Surface growth, no bubbling

Glass column, Air 122 192 1.57 6.1

Petri plate, air 242 562 2.56 3.3 The results showed that bubbling of 100 % nitrogen led to no biomass increase.

Instead, the culture lost weight, the plants shrunk in size and approximately one third of the plants turned white and died. In contrast, bubbling of air led to an increase in biomass (Table 2). EXAMPLE 3

VOLUMETRIC AND SURFACE GROWTH UNDER PERMISSIVE

CONDITIONS

Growth in air in the glass tubes (diameter, 2.3 cm) of Examples 1 and 2, either volumetrically with bubbling or on the surface with no bubbling, gave relatively lengthy biomass doubling times of 6.9 and 6.1 days, respectively, while growth in the Petri plate (diameter, 9 cm) in Example 2 gave a biomass doubling time of 3.3 days, as expected for a log phase culture. The following experiment was set up to provide permissive conditions (unstressed plants and log phase growth) for growth in the 2.3 cm diameter glass columns as well.

Woljfia globosa var. Noam plants, grown on the surface without bubbling, in the amounts listed in Table 3, were placed in 9 cm-diameter Petri plates containing 35 ml of Hoagland solution and in 9 cm-diameter beakers containing 500 ml of Hoagland solution, rising to a height of 9 cm. Woljfia globosa var. Noam plants, grown volumetrically with bubbling, in the amounts listed in Table 3, were introduced into glass columns (2.3 cm, inner diameter) containing 35 ml of Hoagland solution, rising to a height of 9 cm, as described above in Example 1. Forced air, enriched with 3 % C0₂, was bubbled vigorously through the growth solution in the glass columns as described above in Example 1. The entire experiment was conducted in a closed growth chamber; thus plants in the Petri plates and beakers were exposed to C0₂ enrichment as well. Plants in the amounts listed, were introduced at day zero and biomass collected and weighed as described in Example 1 above. Biomass fold-increase and biomass doubling time were calculated after 7, 10 and 14 days (Table 3). Media were replaced with fresh Hoagland solution every 3-4 days throughout the experiment. Water was added as needed to compensate for evaporation during bubbling.

Three replicate samples were measured for each growth vessel type at each time point. The results, summarized in Table 3, show that reproducibility was achieved in biomass doubling times for each growth procedure throughout the experiment. Biomass doubling times for surface growth were shorter by 23% than for bubbled growth throughout the experiment. However, the surface areas of the samples between the two methods of growth differed by a factor of fifteen. A 9 cm-diameter Petri plate, and a 9 cm-diameter beaker, each have a surface area of 63.6 cm, while the 2.3 cm-diameter glass column used for bubbling has a surface area of 4.2 cm. Calculating for equal surface areas and comparing fold-increase values (Table 3), indicates that, under the conditions of this experiment, from 3.9 to 9.0 times more biomass could be realized by volumetric growth versus surface growth in the beakers and Petri plates, respectively. Table 3: Comparison of surface and volumetric growth under permissive conditions

Biomass

Biomass doubling

Biomass (mg) Fold

time (d) increase

Da O Day 7 Day 10 Day 14 Ave +SE

Column 110 496 4.5 3.22

110 425 3.9 3.59

128 523 4.1 3.45

105 980 9.3 3.10

99 705 7.1 3.53

88 726 8.3 3.29

92 1640 17.8 3.37

115 1530 13.3 3.75

110 1450 13.2 3.76 3.45 +0.08

Petri 100 795 8,0 2.34

100 684 6.8 2.52

93 553 6.0 2.72

95 1225 12.9 2.71

114 2157 18.9 2.36

125 1487 11.9 2.80

141 3500 24.8 2.82

156 4490 28.8 2.68

1 Q Q 2.65 +0.07

171 3400 2.87

Beaker 119 432 3.6 3.76

110 678 6.2 2.67

108 832 7.7 2.38

100 6230 62.3 2.35

110 5950 54.1 2.43

^9 9 2.67 +0.22

91 4750 jZ.Z 2.45 EXAMPLE 4.

CONDITION OF PLANTS FOLLOWING VOLUMETRIC GROWTH WITH

BUBBLING

Plants from Example 3- Day 14 volumetrically grown with bubbling in glass tubes, and plants grown statically on the surface in beakers, were collected and observed under the binocular microscope. Upon cessation of bubbling, the plants rose rapidly (in seconds) to the surface. Virtually all plants (> 98%) were green following bubbling. Both volumetric and surface culture conditions resulted in plants that appeared healthy with many dividing forms.

EXAMPLE 5

STANDING DENSITY AREA FOLLOWING VOLUMETRIC GROWTH

After weighing of the biomass, plants from Example 3- Day 14, were returned to their respective growth vessels and their growth regimes continued, with a media change after 3 days. The biomass was measured after 6 days (i.e., on Day 20). The combined results from Tables 3 and 4 indicate that Day 14 densities were late log phase and that growth slowed severely or stopped altogether in the period from Day 14 to Day 20 (compare the biomass doubling times in Table 3 and Table 4). The change was not due to lack of nutrients, which were supplied, as indicated, but rather to crowding in the vessels.

Table 4: Determining late log phase

Plant weight (mg)

Biomass doubling time

Method of growth

period (d)

Day 14 Day 20

Column: volumetric 1640 2720 8.2

1530 2320 10.0

1450 2000 12.9

Petri plate: surface 3500 3310 stationary

4490 5750 16.8

3400 3730 44.9 Beaker: surface 6230 9670 9.5

5950 9500 8.9

4750 9600 8.7

The average standing densities per unit surface area for the late log phase cultures at Day 14 of Example 3 were calculated as follows:

Average weight (in mg) for the samples of Table 3- Day 14 / surface area of the growth vessels (4.2 cm 2 for glass tubes; 63.6 cm 2 for Petri plates; 63.6 cm 2 for beakers) xlO 4

(for transition from cm 2 to m 2 ). The results are presented in Table 5.

Table 5: Standing densities per unit surface area at late log phase

,„ , „ Plant weight Standing density

Method of growth . ,„„

^& Ave +SE ( fmg ,) area ( ,g/ .m _¾ )

Column: volumetric 1540+55 3.67

Petri plate: surface 3797+348 0.60

Beaker: surface 5643+453 0.89

It is noted that, as opposed to surface growth, as depth of column increases for volumetric growth, the standing density per surface area increases. For example, at a nominal industrial column depth of 1.8 m, the standing density per unit surface area would be 20 times that shown in Table 5, assuming similar rates of growth.

EXAMPLE 6

EFFECT OF LIGHT INTENSITY ON VOLUMETRIC GROWTH RATE

An experiment was set up to check the effects of light intensity on the rate of volumetric growth in our enclosed growth chamber. Woljfia globosa var. Noam plants were incubated in glass columns, as previously described in Example 1, with bubbling (540 liters per hour) at either 2^E/m2/sec or 42μΕ 2/8εΰ for 8 days in the presence of 3 % C0₂. Plants grown statically in beakers and Petri plates alongside of the glass columns were likewise kept at 3 % C0₂ throughout. The results are summarized in Table 5A.

Table 5A. Increased volumetric growth rates at increased light intensity.

21 μΕ 42 μΕ

Weight at Doubling Weight Weight Doubling

Vessel Weight Vessel

T=0 time at T=0 final time no. final (mg) no.

(mg) (days) (mg) (mg) (days)

1 210 2570 2.21 10 225 4950 1.79

Beaker 2 217 2550 2.25 11 229 4430 1.87

3 213 2350 2.31 12 195 3540 1.91

Avg 213 2490 2.26+0.03 216 4307 1.86+0.04

4 223 1860 2.61 13 216 1940 2.53

Petri

5 222 1340 3.09 14 204 2040 2.41 plate

6 191 1720 2.52 15 222 1980 2.53

Avg 212 1640 2.74+0.18 214 1987 2.49+0.04

7 204 1240 3.07 16 213 2240 2.36

Column¹ 8 207 1630 2.69 17 216 2320 2.34

9 205 1490 2.8 18 214 2200 2.38

Avg 205 1453 2.85+0.11 214 2253 2.36+0.01

At experiment end, small amounts of sunken fronds were present in glass column samples at 21μΕ but not at 42μΕ.

The results indicated that light intensity affects growth rates under volumetric conditions similarly to its effect in static conditions. In both cases, the fresh weight collected at the end of the experiment was almost double at the higher light intensity than at the lower one. Of note, doubling times at 42μΕ light were shortened by -17% for bubbled column growth and static beaker growth versus the times at 21μΕ light. The standing densities per unit surface area were calculated after eight days, when plants were at mid log phase growth. The average standing densities are shown for the two light intensities.

Table 5B: Standing densities per unit surface area at mid log phase growth.

21μΕ 42μΕ

Column, volumetric growth 1490 / 4.2 = 355 mg/cm² 2253/4.2=536 mg/cm²

Beaker, surface growth 2490 / 63.6 = 39 mg/cm² 4307/63.6=68 mg/cm 21 μ

Petri dish, surface growth 1640 / 63.6 = 26 mg/cm² 1987/63.6=31 mg/cm² The density values were used to calculate the fold increase per unit surface area for mid log phase Woljfia globosa var Noam plants growing volumetrically, versus statically on the surface. The results are shown in Table 5C.

Table 5C: Fold increase in standing densities per unit surface area for mid log phase Wolffi globosa var Noam plants

21μΕ 42μΕ

Column/Beaker 355/39 = 9 536/68 = 8

Column/Petri dish 355/26 = 14¹ 536/ 31 = 17¹

Nutrient levels may have reached limiting conditions at times in the Petri dishes.

The fold increases in standing density of volumetrically grown plants versus surface grown plants at mid log phase, at both light intensities, were higher than for those found for late log phase plants (compare Table 5C and Table 5).

EXAMPLE 7

DISTRIBUTION OF LATE LOG PHASE PLANTS FOLLOWING BUBBLING

During volumetric growth, as wild type Woljfia globosa var. Noam plants increased to late log phase densities and beyond, an amount of sunken plants accumulated at the bottom of the growth column upon cessation of bubbling. Day 20 plants of Example 5 were observed at collection time. The results showed that under continuous bubbling conditions, in late log phase growth, a portion of wild type plants (12-17 %) sink to the bottom of the column when bubbling is stopped, while the majority of the plants (83-88 %) float to the surface. Both plant types appear healthy with growing forms. No true turions or dead plants were observed.

EXAMPLE 8

SUBMERGED TYPE AND SURFACE TYPE PLANTS GROW TRUE TO TYPE

Sunken and floating plants from Example 5 were collected separately at Day 20, weighed and returned separately to fresh media for continued volumetric growth (designated: Day 0 and Day 10) with bubbling of air and enrichment of C0₂ as in Example 3. The results are shown in Table 6.

Table 6: Volumetric growth of floating and sinking wild type W. globosa var. Noam plants

Biomass doubling

Plant type Biomass (mg) Fold increase

time (d)

Day 0 Day 10 Ave+S.E.

Floaters 400 1990 5.0 4.32

400 1800 4.5 4.61

400 1870 4.7 4.49 4.47+0.08

Sinkers 400 1460 3.7 5.35

400 1670 4.2 4.85

400 1570 3.93 5.07 5.09+0.14

Both growing types continued to grow well to high standing area densities, with floaters growing, on average, 14% more rapidly. The two types remained true to their positioning, with floaters remaining floaters and sinkers remaining over 80% sinkers at Day 10.

EXAMPLE 9

STARCH ANALYSIS OF FLOATERS AND SINKERS

Non-growing, true turions of Woljfia globosa sink to the bottom of their growth column. Such turions are known to be rich in starch, which adds to their specific density and is complicit in their sinking. The present inventors determined the relative starch content of normal surface grown wild type plants (9 cm diameter Petri plate), and floaters and sinkers (note, sinkers are not turions) from Day 20 plants of Example 5. The results are summarized in Table 7.

Table 7: Starch analysis of wild type W. globosa var. Noam floaters and sinkers

Absorbance Volume (ml) Petri plate Floaters Sinkers

(nm)

A620 lx 0.019 0.02 0.034

2x 0.03 0.027 0.055

3x 0.039 0.037 0.087

A635 lx 0.015 0.016 0.03

2x 0.025 0.022 0.05

3x 0.034 0.032 0.08

Comparative analysis was done following the methods of Gur, A., Cohen, A., and Bravdo, B. (1969) Colorimetric method for starch determination J. Agric. Food Chem. 17: 347-351; and Morrison W.R. and Laignelet B. (1983) An improved colorimetric procedure for determining apparent and total amylose in cereal and other starches. J. Cereal Sci. 1: 9-20. For equal sample volumes at either wavelength, normal surface growers (Petri plate) and volumetric floaters gave similar absorbance levels, while sinkers showed at least twice the absorbance of either of the former.

This example is interpreted to show that volumetric growth-induced sinkers of wild type W. globosa have an increased level of starch over surface-grown plants and volumetrically-grown surface floaters. The significantly increased level of starch is likely the reason for their sunken phenotype and a signature of volumetrically produced sinker plants. As such, volumetrically-induced sinkers represent a novel form and methodology of achieving actively and rapidly growing Woljfia plants enriched in starch content that can achieve high standing density areas, a potentially attractive situation for biofuel production.

An additional test for starch was carried out by measuring glucose accumulation in day-20 plants using the protocol of Zeeman S. and Samuel Z. (Nature protocols, 1: 1342-1325, 2006). The protocol has 2 steps: starch degradation to glucose using specific starch degrading enzymes, and glucose quantification using an enzymatic assay. The results for volumetrically grown plants that rise to the surface upon stopping of bubbling (i.e., floaters) and plants growing statically on the surface in beakers were compared. The results are shown in Table 7A

Table 7A: Comparison of glucose content of volumetric floaters and beaker surface-grown day-20 plants.

Method of growth Sample Glucose

(mg/g dry weight)

Volumetric floaters 1 32.65

(column) 10.35

2 21.37

22.04

Avr + SE 21.35 + 4.55

Surface grown 3 24.99

(beaker) 28.76

4 26.71

16.17

Avr + SE 24.16 + 2.77

No statistical difference between the volumetric floaters and surface-grown plants in starch content was found, as measured by glucose concentration. These results (Table 7 A) are in agreement with the findings shown in Table 7. Both show that volumetrically derived floaters, either from mid log phase or late log phase plants, are indistinguishable from vegetative, surface-grown plants, either from beakers or Petri plates, in their percentages of starch. This distinguishes the volumetrically produced floaters shown here from a form of submerged resting plants described in Landolt 1986 (The family of Lemnaceae - A monographic study, Vol 1. Veroeffentlichungen des Geobotanischen Institutes der ETH, Stiftung Ruebel, Zurich, pp. 566), which in some species, and under static conditions, sink to the bottom of the growth vessel, grow slowly in the submerged state and have an increased content of starch with respect to vegetative surface growing plants.

EXAMPLE 10

VOLUMETRIC GROWTH OCCURS IN OTHER W. GLOBOSA STRAINS

Woljfia globosa strain 9331 (Rutgers Duckweed Stock Collaborative, Serial number 194, first isolated at Wuhan University, China) was grown with bubbling and CO₂ as in Example 3, and without bubbling in a Petri plate. The experiment was carried out in a growth chamber so that the Petri plate was also exposed to C0₂ enrichment. Media change was done after 6 days. At day 11 the plants were weighed.

Table 8: volumetric growth Wolffia globosa strain 9331 under permissive conditions

Plant weight Biomass Biomass

Method of growth

(mg) fold increase doubling time (d)

Day 0 Day 11

Column: volumetric 100 620 2.5 4.18

Petri plate: surface 100 860 3.1 3.54

The results show that in addition to Wolffia globosa var. Noam, Wolffia globosa strain 9331 can grow volumetrically with vigorous bubbling in C0₂ enriched air. EXAMPLE 11

VOLUMETRIC GROWTH OCCURS IN OTHER WOLFFIA SPECIES

Wolffia australiana strain 7211 (Rutgers Duckweed Stock Collaborative, Serial number 208, first isolated at University of Melbourne, Australia) was grown with bubbling and C0₂, as in Example 3, and without bubbling in a Petri plate. The experiment was carried out in a growth chamber so that the Petri plate was also exposed to a C0₂ enrichment of 3 %. After an acclimatization period of 5 days, the plants were weighed and returned to their growth vessels. At this point, the experiment was initiated. Media change was done after 2 days and growth assessed after 4 days. Weight was determined as in Example 1. Biomass fold increase and biomass doubling times were determined as in Example 1.

Table 9: volumetric growth of Wolffia australiana strain 7211 under permissive conditions

Biomass fold Biomass doubling

Method of growth Biomass (mg)

increase (d) time (d)

Day 0 Day 4 Ave+S.E.

Column: Volumetric 202 382 1.89 4.35

232 452 1.95 4.15 197 352 1.79 4.78 4.43+0.19

Petri plate: Surface 342 1152 3.37 2.28

372 1472 3.96 2.02

392 1162 2.96 2.55 2.28+0.15

The results show that in addition to W. globosa var. Noam, Woljfia australiana strain 7211 can grow volumetrically with vigorous bubbling in C0₂ enriched air.

EXAMPLE 12

DESCRIPTION OF BUBBLING GROWTH

Woljfia globosa var. Noam plants were photographed in motion after 6 days of growth in order to demonstrate the level of agitation and bubbling used in all of the volumetric experiments. The plants were grown in 35 ml of Hoagland solution in glass tubes as in Example 2. Vigorous bubbling of air enriched with C0₂ agitated the plants so that virtually all were constantly moving about at a fast pace (Figure 3).

In slow-motion filming (not shown) at 240 frames per second, 10-20 air bubbles were counted to emit from the 1 mm pipette tip bore per second. At a normal-motion video speed of 30 frames per second this number is increased by a factor of eight and translates to 80-160 bubbles per second. The bubbles rapidly coalesce and grow in size from a diameter of 2-4 mm at a height of 2 mm from tube bottom to bubbles of 5-7 mm diameter at a height of 70-90 mm from tube bottom. A total of 10-15 coalesced bubbles are simultaneously rising in the tube at any given time. It takes from 0.25 to 0.35 sec for a bubble to rise the full 90 mm in the tube at a normal viewing speed of 30 fps.

EXAMPLE 13

SUNKEN, ACTIVELY-GROWING WOLFFIA GLOBOSA FOLLOWING

TREATMENT WITH NMU

Following treatment of Woljfia globosa var. Noam (WT) plants with 10 mM nitrosomethylurea (NMU) for 60 minutes, a line, called NE3, grew for many vegetative generations with 80-90% of the plants in a sunken state (Figure 1). However, by approximately 50 doublings, most new NE3 plants had returned to surface growth. This was a gradual process which occurred over generations 40 to 50. Such long-term transitory activity was characteristic of several lines following NMU (10 niM in 7 % glacial acetic acid, final concentration, for 60 minutes) or ionic shock (1 M sodium phosphate buffer, pH6.9, for 120 minutes) treatments. Other NMU treated lines, such as NE10, have stabilized at about 75 % of plants sunken after 100 vegetative generations.

It is well known that WT plants can also sink to the bottom of the water column; however, only in a hibernating form, known as a true turion. These turions are small (approximately 0.5 mm in length) and non-growing, without any increase in biomass. In contrast, sunken NE3 plants are larger (approximately 1.0 mm in length) and actively grow and multiply in the sunken state (Figure 2).

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A method of increasing biomass yield of a Wolffia culture per unit growth medium essentially without formation of turions, the method comprising subjecting the culture to volumetric cultivation conditions such that the Wolffia plant of the Wolffia culture grows throughout a liquid column of the culture, wherein said conditions are selected from the group consisting of:

(i) air flow above 100 L/hour (h);

(ii) light regimen above 12 h per 24 h period; and

2. The method of claim 1, wherein said conditions comprise:

(i) air flow above 100 L/hour (h); and

(ii) light regimen above 12 h per 24 h period.

3. The method of claim 1, wherein said conditions comprise:

(i) air flow above 100 L/hour (h); and

4. The method of claim 1, wherein said conditions comprise:

(i) light regimen above 12 h per 24 h period; and

5. The method of claim 1, wherein said conditions comprise:

(i) air flow above 100 L/hour (h);

(ii) light regimen above 12 h per 24 h period; and

6. The method of claim 1 or 5, wherein said conditions comprise:(i) air flow above 400 L/hour (h);

(ii) light regimen above 18 h per 24 h period; and/or (iii) light regimen comprising light intensity lower than 100 μιηο1/ιη²/8.

7. The method of any one of claims 1-6, wherein said volumetric cultivation is by agitation of the plant throughout the liquid column.

8. The method of any one of claims 1-3, 5-6, wherein said air flow is imparted by bubbling.

9. The method of claim 8, wherein bubbles of said bubbling are of a diameter of 0.1 mm to 50 cm.

10. The method of any one of claims 8-9, wherein said bubbling is with air enriched with C0₂.

11. The method of claim 10, wherein said C0₂ is comprised at a concentration of 0.05 to 15 %.

12. The method of any one of claims 8-11, wherein said bubbling is with air enriched with oxygen

13. The method of claim 12, wherein said oxygen is comprised at a concentration of up to 28 %.

14. The method of claim 7, wherein said agitation is further effected by spinning, rocking or rotating.

15. The method of any one of claims 1-14, wherein said increasing biomass yield results in an increase of at least 0.2 fold in standing-area density as compared to that in surface growth under the same conditions without said volumetric cultivation.

16. The method of any one of claims 1-15, wherein said increasing biomass yield results in an increase of at least 0.2 fold in biomass weight as compared to that in surface growth under the same conditions without said volumetric cultivation.

17. The method of any one of claims 1-16, wherein said submerged state of said plant is also characterized by a dividing phenotype and green color throughout said volumetric cultivation.

18. The method of any one of claims 7-17, wherein said agitation results in contact of the plant with an air surface.

19. The method of any one of claims 1-18, wherein said Woljfia plant is Wolffia globosa.

20. The method of any one of claims 1-19, wherein said Woljfia plant is Woljfia australiana.

21. The method of any one of claims 1-18, wherein said Woljfia plant is not Wolfia arhiza.

22. The method of any one of claims 1-21, wherein said volumetric cultivation is for 2 days to 4 weeks.

23. The method of any one of claims 1-22, wherein said volumetric cultivation is effected in a container having an internal fillable volume of 1 ml to 3,000,000 liters.

24. The method of any one of claims 1-23, wherein said plant is a transgenic plant.

25. The method of any one of claims 1-23, wherein said plant is a non- transgenic plant.

26. A harvested material of a Woljfia plant grown according to the method of any one of claims 1-25.

27. The harvested material of claim 26, comprising a ratio of floating Wollfia plants to submerged Wollfia plants which is smaller than that found by surface growth in the logarithmic state.

28. A method of producing feedstock, the method comprising:

(a) growing Woflfia plants according to the method of any one of claims 1-

25; and

(b) harvesting the Woljfia plants.

29. A method of producing a biofuel, the method comprising:

(a) growing Woljfia plants according to the method of any one of claims 1-

(b) harvesting the Woflfia plants; and

(c) processing the biofuel from the harvested Woljfia plants.

30. The method of claim 29, wherein said biofuel is selected from the group consisting of ethanol, butanol and biogas.

31. A method of bioremediation, the method comprising growing Woljfia plants according to any one of claims 1-25 in the presence of a pollutant in a contaminated site.

32. The method of claim 31, wherein said pollutant is selected from the group consisting of phosphorus, nitrogen, heavy metal, cyanotoxin, sex steroid and corticosteroid.

33. A method of extracting a protein or metabolite of interest, the method comprising: (a) growing Woflfia plants according to the method of any one of claims 1-

25;

(b) harvesting the Woljfia plants; and

34. The method of claim 33, wherein said protein is endogenous to the Woljfia plant.

35. The method of claim 33, wherein said protein is a recombinant protein.