WO2014072962A1

WO2014072962A1 - A temporary immersion bioreactor method and relative product

Info

Publication number: WO2014072962A1
Application number: PCT/IB2013/060083
Authority: WO
Inventors: Ariel Domingo ARENCIBIA RODRIGUEZ; Laura V ARRU
Original assignee: Universidad Católica Del Maule
Priority date: 2012-11-12
Filing date: 2013-11-12
Publication date: 2014-05-15
Also published as: BR112015010813B1; BR112015010813A2; CL2015001280A1; ITRE20120080A1

Abstract

A temporary immersion bioreactor method and a product obtained therefrom, the method comprising steps of: a. inserting one or more plants (200) capable of enacting photosynthesis into a container (110); b. inserting a liquid medium (205) into the container (110), such as to immerse the plants (200), leaving the plants (200) immersed for a period of time such as to multiply the plants and grow vegetable biomass; c. removing the liquid medium (205) from the container (110) and leaving the plants (200) in an air atmosphere with added carbon dioxide for a further period of time; d. repeating steps b. and c. in alternation for a predefined total period of time, exposing the plants (200) to a photosynthetically-active radiation.

Description

A TEMPORARY IMMERSION BIOREACTOR METHOD AND RELATIVE PRODUCT

DESCRIPTION

The present invention is in general a temporary immersion bioreactor method (TIB) . More specifically, the present invention is a temporary immersion bioreactor method for a production of a natural phytocomplex. As is known, a main objective of contemporary agricultural production is to increase crop yields while reducing costs. To attain this objective, efforts are generally made to intervene on all abiotic and biotic environmental factors that might harm crops and reduce yields.

Abiotic factors are the non-living components of an ecosystem, for example light, medium (surface and subsurface) , rock, water, air, nitrogen content, climatic factors, etc. The problems caused by this class of environmental factors can be partially controlled and resolved by humans, using fertilizers, controlled irrigation, artificial illumination, greenhouses or nurseries, which artificially create optimum conditions for development of plants.

The biotic factors, also known as biological factors, include all living species that compromise the development of cultivated plants. Biotic factors can be macroscopic (for example, herbivorous insects) or microscopic pathogens that cause diseases (for example, viruses or bacteria) . Pathogens propagate rapidly in crops (in particular in crops of low biodiversity) , infesting and weakening large numbers of plants and reducing agricultural yields.

Critical stages exist in agricultural production, when plants are more vulnerable to diseases caused by pathogens. One of these critical stages is, for example, a transplanting step, when plants germinated and grown under cover, for example in greenhouses or nurseries, are planted in open ground, where it is no longer possible to control weather conditions and, in particular, the proliferation and spreading of pathogens.

Consequently, biotic factors are generally more difficult to control compared to abiotic factors, and are the main cause for reductions in agricultural yields.

In an effort to resolve these types of problems, known-type methods involve a use of phytosanitary products of chemical origin (widely available on the market) , including, for example phytopharmaceutical drugs, agro-drugs, and pesticides, which are synthesised and used in agriculture to treat specific plant diseases.

These chemical products act directly on the disease vectors and effectively reduce the infective capacity of pathogens, but can also produce significant side-effects, including, for example, an accumulation of toxic residues in the ground, toxicity for humans, marked environmental impact, and induction of tolerance phenomena in pathogens. As a consequence, in recent years there has been a considerable increase in interest in, and commercial availability of, ecological phytosanitary products of natural origin, utilized principally in the so-called organic agriculture sector, in particular in the horticulture and fruit and vegetables sector.

On the basis of these observations, an aim of the present invention is to provide an eco-compatible method to obtain a natural phytocomplex capable of increasing the intrinsic resistance of plants to micro-organisms.

A further aim of the present invention is to achieve the abovementioned aim by way of a simple, rational, and relatively economic solution.

These and other aims are attained by the characteristics of the invention as described in the independent claims. The dependent claims describe preferred and/or particularly advantageous aspects of the invention.

In particular, an embodiment of the present invention provides an innovative temporary immersion bioreactor method.

Conventionally, temporary immersion bioreactor (TIB) technology began as a micropropogation technique which, utilizing modern test-tube cultivation methods for vegetable cells and tissues, permits the production of seedlings free of pathogens, intended for subsequent ground planting.

Compared to these conventional techniques, the temporary immersion bioreactor method of the present invention differs essentially in that it is not applied to cell cultures, but to entire plants, which are provided with the necessary conditions for photosynthesis.

More particularly, the temporary immersion bioreactor method of the present invention comprises steps of:

a. placing one or more plants capable of photosynthesis inside a container, preferably a sterile transparent container,

b. introducing a liquid medium into the container, such as to immerse the plants completely or at least a portion thereof, for example the leaves or roots (if present) thereof, and leaving the plants immersed for a predefined period of time,

c. removing the liquid medium from the container and leaving the plants in an air atmosphere with added carbon dioxide for a further period of time, and then

d. repeating steps b. and c. in alternation for a given period of time, exposing the plants to photosynthetically active radiation (PAR) .

It should be noted that "liquid medium" here refers to a liquid or semi- liquid containing water and nutrient substances, including mineral salts, plant growth hormone, and saccharose. Photosynthetically active radiation is defined as a luminous radiation, typically within the spectral interval (waveband) of solar radiation, utilizable by photosynthetic organisms in a photosynthesis process.

An outcome of the method of the invention is that the plants release metabolites and/or other biologically active compounds comprising components of leaf and/or root exudates into the liquid medium in which they are immersed. These compounds include, in particular, phenols, isoflavonoids, indolines, tannins, phytosterols, polysaccharides, sesquiterpenes, alkaloids, and vitamins, each of which can provide a high level of antimicrobial and/or immunomodulating action.

The liquid medium enriched with these compounds is a natural and ecologically-sustainable fluid phytocomplex, offering effective, phytoprotective, fortifying, and biostimulating properties.

In particular, in vivo tests in which this phytocomplex was sprayed directly onto ground cultivated plants, specifically onto tomato plants, demonstrated the capacity of the phytocomplex to induce various grades of resistance in the plants to pathogens, reducing mortality of the plants, in particular during the transfer step from nursery to field.

Other advantages of the phytocomplex include:

- a direct action on plants rather than on disease vectors, in contrast to many traditional products available on the market,

- no effects of bioaccumulation or phytotoxicity,

- no impact on the environment or user,

- no residues,

- does not induce resistance in pathogens,

- does not stress the plant in any way following application,

- the composition is appropriate for use in organic agriculture, in full compliance with the relevant EC regulations.

Controlled environment tests have also demonstrated the effectiveness of the present phytocomplex as an antibacterial agent, such that the complex is usable not only in agriculture but also in other sectors, for example in the foodstuffs packaging sector.

In the light of these discoveries, after termination of the temporary immersion bioreactor method, the liquid cultivation medium can be collected and possibly filtered and/or concentrated, and finally commercialized as a biologically active liquid product for antibacterial and/or phytosanitary applications, for example as a generic tonic for cultivated plants.

Furthermore, the plants directly used in the temporary immersion bioreactor method can subsequently be sold for ground planting.

In this way, the invention advantageously configures a closed circuit process, producing no waste products and only products of commercial value.

In this respect, in a preferred aspect of the invention the plants directly used in the present temporary immersion bioreactor method belong to a genus selected from among: Rubus spp. (plant genus belonging to the Rosaceae family, including the raspberry) , Vaccinium (plant genus belonging to the Ericaceae family, including bilberries) , Aristotelia chilensis, Eucalyptus spp., Saccharum spp. (e.g. sugarcane).

The choice of plants belonging to these genera is advantageous both because they provide a very effective phytocomplex, and because these plants are widely utilized and are consequently of high commercial value. In a further preferred aspect of the invention, a quantity of 10 to 20 plants are inserted into the containers described herein above for every litre of liquid medium.

This arrangement is advantageous for increasing the yield of the temporary immersion bioreactor method.

In a further aspect of the invention the liquid medium wherein the plants are immersed contains a quantity of saccharose less than 30 g/litre. This relatively low quantity of saccharose is advantageous in that the plants, enacting photosynthesis, release additional metabolites into the liquid medium.

In a further preferred aspect of the invention, the duration of each step b., this being the lapse of time wherein the plants are left immersed in the liquid medium, is between 1 and 10 minutes.

The duration of each step c, this being the lapse of time wherein the plants are left in an atmosphere of air and carbon dioxide, is preferably between 2 and 8 hours.

The total period of time wherein steps b. and c. are alternately repeated is preferably between 15 and 25 days.

All these aspects of the invention offer the advantage of permitting effective growth of the plants and obtaining adequate enrichment of the liquid medium.

In a further preferred aspect of the invention, the carbon dioxide added during step c. is introduced in a quantity between 0.1% and 0.9 % relative to the quantity of air.

The photosynthetically-active radiation to which the plants are exposed during the method of the bioreactor preferably is of a luminous intensity comprised between 30 and 120 uM rnT² s^_1 (micromoles per square meter per second) .

Furthermore, the photosynthetically active radiation preferably comprises natural light.

These aspects of the invention offer the advantage of rendering the photosynthesis of the plants particularly effective in a controlled environment .

In a further aspect of the invention, during the temporary immersion bioreactor method, the plants can be stimulated such as to induce the plants to produce and release an increased quantity of metabolites and/or other biologically active compounds.

In particular, in this aspect of the invention the plants can be exposed to ultraviolet light and/or treated with hydrogen peroxide (oxygenated water) , and/or treated with BABA (β-Aminobutyric acid) .

An alternative embodiment of the present invention is the liquid product obtained from the temporary immersion bioreactor method as described herein above, including the steps of:

a. inserting one or more plants capable of photosynthesis inside a container,

b. inserting a liquid medium into the container, such as to immerse the plants completely, or at least a portion thereof comprising for example the leaves or roots (if present) , and leaving the plants immersed for a certain period of time,

c. removing the liquid medium from the container and exposing the plants to an air atmosphere including added carbon dioxide for a further period of time,

d. repeating the steps b. and c. in alternation for a predefined total period of time, and exposing the plants to a photosynthetically active radiation.

This embodiment of the invention offers the same advantages described herein above, in particular that of providing a fluid product of natural origin and ecologically sustainable, exhibiting effective phytoprotective, fortifying, and biostimulating properties.

Further characteristics and advantages of the invention will better emerge from the detailed description made herein, provided by way of non- limiting example in the accompanying figures of the drawings. Figure 1 schematically illustrates an apparatus for the execution of a temporary immersion bioreactor method, during a dry step.

Figure 2 illustrates the apparatus of figure 1 during an immersion step.

Figures 1 and 2 illustrate an apparatus 100 for the execution of a temporary immersion bioreactor (TIB) process.

The apparatus 100 comprises at least a pair of sterile containers, including a first container 105 and a second container 110. The containers 105 and 110 are made of transparent material, for example a plastic material, and are hermetically closed using a respective closing element 115.

A plurality of relatively young complete plants 200 is inserted into the container 110. The plants 200 must be sufficiently developed such as to be capable of photosynthesis. In particular, the plants 200 must exhibit leaves. Preferably, the plants 200 must also be without roots such as to be capable of multiplication and to exhibit greater vegetable biomass, which is the component that produces more natural metabolites through photosynthesis. Preferably, the plants 200 must also be pathogen free. For this reason, the plants 200 may be previously obtained by micropropogation of sterile meristems in a controlled environment.

In a preferred aspect of the invention, the plants 200 can belong to a genus selected from among: Rubus spp. (plant genus belonging to the Rosaceae family, including the raspberry) , Vaccinium (plant genus belonging to the Ericaceae family, including bilberries) , Aristotelia chilensis, Eucalyptus spp., Saccharum spp. (including sugar cane).

A liquid medium or growth medium 205, this being a liquid solution containing water and nutritional substances for the plants 200, including for example mineral salts, is inserted into the other container 105. For example, the liquid medium 205 may be a water solution containing a salt of known type commercialized under the name "MS salt", to which other substances serving to regulate the multiplication of the plants 200 can be added. The liquid medium 205 also contains a certain quantity of saccharose. In particular, the saccharose is preferably present in a quantity inferior to 30 grams per litre of solution, for example between 5 and 25 g/litre. The pH of the liquid medium 205 is preferably basic, for example exhibiting a pH of approximately 5.7.

The total quantity of liquid medium 205 in the container 105 is proportional to the quantity of plants 200 in the container 110. Preferably, the quantity of liquid medium 205 is equal to about 1 litre for every 15 to 20 plants 200.

In a first step of the process, the liquid medium 205 contained in the container 105, is inserted into the container 110, such as to completely immerse the plants 200, or at least the leaves of the plants 200 (see figure 2) .

The transfer of liquid medium 205 can be achieved using a pipe 120 connecting the two containers 105 and 110, and a pump 125 located at a point along the pipe 120, such as to draw the liquid medium 205 from container 105 and deposit it in container 110. Naturally, this transfer could be achieved in numerous other different ways.

At this point the plants 200 are left immersed in the liquid medium 205 for a predefined period of time. During the period of immersion, air can be blown into the container 110, for example through a further conduit 140, the air being made to bubble through the liquid medium 205, such as to promote a multiplication/growth of the plants 200.

In a subsequent step of the process, the liquid medium 205 is removed from the container 110 and returned to the container 105 (see figure 1) . This transfer of liquid medium 205 can be achieved using another pipe 130 connecting the two containers 110 and 105, and a further pump 135, located at a point along the pipe 130, to draw the liquid medium 205 from container 110 and deposit it in container 105. Naturally, this second transfer could be achieved in many other different ways.

At this point, carbon dioxide and possibly additional air could be introduced into the container 110, for example through the pipe 140, such that the plants 200 are exposed to an atmosphere high in carbon dioxide. The carbon dioxide is preferably injected into the container 110 in quantities between 0.1% and 0.9 % relative to the quantity of air. The plants 200 are then left dry in this atmosphere of air and carbon dioxide for a further predefined period of time.

At the end of this dry step, further immersion steps and dry steps are repeated in alternation such as to configure a so-called temporary immersion bioreactor (TIB) process.

This alternation of immersion steps and dry steps is repeated in continuation for a total period of preferably between 15 and 20 days. During this total time, the duration of each immersion step is preferably between 1 and 10 minutes, and repeated after predefined time intervals, each preferably between 2 and 8 hours and during which the plants 200 are maintained in the dry step.

While the immersion steps and dry steps are repeated in alternation as described, the plants 200 are simultaneously exposed to a photosynthetically active radiation (PAR) . A photosynthetically active radiation is generally a luminous radiation, typically in the spectral interval (waveband) of solar radiation, which the plants 200 are able to utilize for a process of photosynthesis.

The photosynthetically active radiation may originate from outside and illuminate the plants 200 passing through the transparent walls of the container 110. In particular, the photosynthetically active radiation can be natural light, or sunlight, or can be artificial light, generated for example by one or more fluorescent lamps, or a combination of artificial and natural light. The intensity of the photosynthetically active radiation is preferably between 30 and 120 uM rrf² s^_1 (micromoles per square metre per second ) .

As a consequence of the photosynthetically active radiation and the carbon dioxide present in the atmosphere in which the plants 200 are maintained during the dry steps, the temporary immersion bioreactor process described herein above induces the plants 200 to photosynthesize in controlled conditions.

In this way, during the immersion steps, the plants 200 release metabolites and/or other biologically active compounds as part of a leaf exudate into the liquid medium 205. These compounds include in particular phenols, isoflavonoids, indolines, tannins, phytosterols, polysaccharides, sesquiterpenes, alkaloids, and vitamins.

The plants 200 can be stimulated such as to promote the release of these compounds during the temporary immersion bioreactor process.

In particular, the plants 200 can be stimulated by exposing them to ultraviolet light, which can be generated using a suitable lamp to illuminate the plants 200 through the transparent walls of the container 110. The exposure to ultraviolet light can be continuous or over limited periods of time, during the immersion and/or during the dry steps. Preferably, the exposure to ultraviolet light is of a duration of between 5 and 20 minutes, and is repeated at time intervals preferably between 2 and 8 hours.

Alternatively, or in addition to stimulation using ultraviolet light, the plants 200 can be treated with hydrogen peroxide (oxygenated water) . The hydrogen peroxide can be sprayed directly onto the plants 200 during one or more dry steps, or can be added to the liquid medium 205. If added to the liquid medium 205, the quantity of hydrogen peroxide must preferably be between 5 and 50 mM (millimoles) per litre of liquid medium 205.

Alternatively or in addition to the stimulation described herein above, the plants 200 can be treated with BABA (/3-Aminobutyric acid) . Again, the BABA can be sprayed directly onto the plants 200 during one or more dry steps, or added to the liquid medium 205. If added to the liquid medium 205 the quantity of BABA must preferably be between 5 and 20 mM (millimoles) per litre of liquid medium 205.

Upon termination of the temporary immersion bioreactor procedure described herein above, the liquid medium 205 enriched with the metabolites and/or active compounds described above is a fluid phytocomplex of natural origin and ecologically sustainable that exhibits effective antibacterial, phytoprotective, fortifying, and biostimulating properties .

Consequently, the enriched liquid medium 205 can be collected, for example removed from the container 105, and marketed as a liquid antibacterial product and/or as a generic tonic for plants.

In particular, the enriched liquid medium 205 can be marketed in an unprocessed state, or after processing, or after undergoing one or more filtering and/or concentration steps.

The plants 200 can be sold for ground planting.

A specific example of the temporary immersion bioreactor process described herein above can be realized using containers 105 and 110 of transparent plastic and both of a capacity of 10 litres.

5 litres of a liquid medium 205, comprising a water solution of pH 5.7 and containing "MS salts", germination regulation substances, and a quantity between 5 and 25 g/litre of saccharose, are inserted into each container 105.

Raspberry plants 200 are inserted into each container 110, in a quantity of approximately 10 to 20 plants per litre of liquid medium 205 in the container 105.

The temporary immersion bioreactor process involves the raspberry plants 200 undergoing immersion steps of a duration of 3 minutes, repeated every 3 hours. Between immersion steps the raspberry plants 200 undergo a dry step during which they are exposed to an atmosphere containing air with carbon dioxide added at a pressure of 0.5 %. The alternation of immersion and dry steps is repeated continuously for a total process period of approximately 20 days.

During the bioreactor process, the plants 200 are maintained at a temperature of approximately 27°C and exposed to a photosynthetically active radiation comprising a combination of natural light and light produced by cold fluorescent lamps, the lamps generating a light intensity of 100 uM rrf² s^_1.

During the bioreactor process, the raspberry plants 200 are also stimulated with hydrogen peroxide, which is added to the liquid medium 205 in a quantity between 5 and 10 mM (millimoles) per litre of solution. Analyses of gaseous exchange and fluorescence of the chlorophyll, conducted with appropriate measuring devices, including a commercially available CIRAS-2, demonstrate that during the bioreactor process described herein above, the raspberry plants 200 effectively enact photosynthesis .

Spectroscopic analyses of the liquid medium 205, demonstrate that at the end of the bioreactor process, the liquid medium is high in phenols of the types found in leaf exudate of raspberry plants 200. Obviously a technical expert in the sector could introduce numerous modifications of a practical-technical nature to the process described herein above, without forsaking the scope of the invention as claimed below.

Claims

1. A temporary immersion bioreactor method, comprising the steps of: a. inserting one or more plants (200) capable of photosynthesis into a container (110) ,

b. inserting into the container (110) a liquid medium (205), such as to immerse the plants (200) continuously for a period of time,

c. removing the liquid medium (205) from the container (110) and leaving the plants (200) in an air atmosphere with added carbon dioxide for a further period of time,

d. repeating steps b. and c. in alternation for a predefined total period of time, and exposing the plants (200) to a photosynthetically active radiation.

2. The method of claim 1, wherein the plants (200) belong to a species selected from among: Rubus spp., Vaccinium, Aristotelia chilensis, Eucalyptus spp. , Saccharum spp.

3. The method of any one of the preceding claims, wherein a quantity of between 10 and 20 plants (200) for every litre of liquid medium (205) is inserted into the container (110) .

4 . The method of any one of the preceding claims, wherein the liquid medium (205) contains a quantity of saccharose of less than 30 g/litre.

5. The method of any one of the preceding claims, wherein the period of time that the plants (200) are left immersed in the liquid medium (205) is of a duration of between 1 and 10 minutes.

6. The method of any one of the preceding claims, wherein the period of time that the plants (200) are left in an atmosphere of air and carbon dioxide is between 2 and 8 hours.

7. The method of any one of the preceding claims, wherein the total period of time during which steps b. and c. are repeated is between 15 and 25 days.

8. The method of any one of the preceding claims, wherein the carbon dioxide added during step c. is introduced in a quantity between 0.1 % and 0.9 % relative to the quantity of air.

9. The method of any one of the preceding claims, wherein the photosynthetically-active radiation is of luminous intensity between 30 and 120 uM rrf² s^"1.

10. The method of any one of the preceding claims, wherein the photosynthetically-active radiation comprises natural light.

11. The method of any one of the preceding claims, comprising at least one of the following steps:

- exposing the plants (200) to ultraviolet light,

- treating the plants (200) with hydrogen peroxide,

- treating the plants (200) with BABA.

12. A liquid product (205) obtained from a temporary immersion bioreactor method comprising the steps of:

a. inserting one or more plants (200) capable of photosynthesis into a container (110) ,

d. repeating steps b. and c. in alternation for a predefined total period of time, and exposing the plants (200) to a photosynthetically-active radiation.