Method for identifying and isolating bioactive compounds from seaweed extract
阅读说明：本技术 从海藻提取物中鉴定和分离生物活性化合物的方法 (Method for identifying and isolating bioactive compounds from seaweed extract ) 是由 席琳·科南 菲利普·波汀 安妮·吉博伊 萨曼莎·贝丝 让－玛丽·朱伯特 于 2018-12-17 设计创作，主要内容包括：本发明涉及一种分离和纯化从海藻获得的提取物中的生物活性化合物的方法。该方法包括以下步骤：(a)使所述提取物循环通过具有合适的截留分子量的超滤膜；(b)收集来自所述提取物的滤液,以获得第一滤液级分和渗余物；和(c)冲洗所述渗余物以获得一种或多种另外的滤液级分。然后可以评估第一滤液级分和另外的滤液级分的生物活性以确定它们对植物生长的功效。还描述了分离自藻类物种的一种或多种生物活性分子,其中所述一种或多种生物活性分子具有在约0.15kDa至约1.0kDa范围内的分子量并且能够增强或改善植物生长。(The present invention relates to a method for the isolation and purification of biologically active compounds in extracts obtained from seaweed. The method comprises the following steps: (a) circulating the extract through an ultrafiltration membrane having a suitable molecular weight cut-off; (b) collecting the filtrate from the extract to obtain a first filtrate fraction and a retentate; and (c) rinsing the retentate to obtain one or more further filtrate fractions. The biological activity of the first filtrate fraction and the additional filtrate fraction can then be evaluated to determine their efficacy on plant growth. Also described are one or more bioactive molecules isolated from an algal species, wherein the one or more bioactive molecules have a molecular weight in the range of about 0.15kDa to about 1.0kDa and are capable of enhancing or improving plant growth.)
1. One or more bioactive molecules isolated from an algal species, the one or more bioactive molecules having a molecular weight in a range of about 0.15kDa to about 1.0 kDa.
2. The one or more bioactive molecules of any of the preceding claims, wherein the one or more bioactive molecules is capable of improving plant growth.
3. The one or more bioactive molecules of any one of the preceding claims, wherein the algal species is a brown algae species.
4. The one or more bioactive molecules of any of the preceding claims, wherein the brown algae comprises an algal species selected from the group comprising: ascophyllum nodosum, Fucusticus, Sargassum (Sargassum sp), and combinations comprising one or more of the foregoing.
5. The one or more bioactive molecules of any of the preceding claims, wherein the one or more bioactive molecules does not comprise a sulfated polysaccharide or laminarin.
6. A method of improving plant growth, the method comprising the step of applying a composition comprising one or more isolated bioactive molecules of any of claims 1 to 5 to at least one of soil, a plant, or a seed.
7. The method of claim 6, wherein improving plant growth comprises at least one of: promoting seed germination, stimulating root development, prolonging vegetative stage, increasing production stage or increasing harvest stage.
8. A method for the isolation and purification of biologically active compounds in an extract obtained from seaweed, said method comprising the steps of:
a) circulating the extract through an ultrafiltration membrane having a suitable molecular weight cut-off;
b) collecting the filtrate from the extract to obtain a first filtrate fraction and a retentate; and
c) flushing the retentate to obtain one or more additional filtrate fractions.
9. The method of claim 8 further comprising the step of assessing the biological activity of the first filtrate fraction and the additional filtrate fraction to determine their efficacy on plant growth.
10. The method of claim 9, wherein the efficacy on plant growth comprises at least one of: promoting seed germination, stimulating root development, prolonging vegetative stage, increasing production stage or increasing harvest stage.
11. The method of any one of claims 8 to 10, wherein the extract is produced from a brown algae species.
12. The method of any one of claims 8 to 11, wherein the extract is obtained from Ascophyllum nodosum (Ascophyllum nodosum), Fucus vesiculosus (Fucus vesiculosus), Sargassum sp.
13. The process according to any one of claims 8 to 12, wherein the retentate comprises active molecules selected from the group consisting of sulfated polysaccharides and laminarin, and
wherein the active molecule is capable of alleviating abiotic stress in a crop.
14. The method of any one of claims 8-13, wherein the first filtrate comprises biologically active molecules having a molecular weight in the range of about 0.15kDa to about 1.0 kDa.
15. The method of any one of claims 8 to 14, wherein the ultrafiltration membrane has a molecular weight cut-off of less than 3 kDa.
16. The method of any one of claims 8-15, wherein the ultrafiltration membrane has a molecular weight cut-off of less than 2 kDa.
17. The method of any one of claims 8-16, wherein the ultrafiltration membrane has a molecular weight cut-off of less than 1 kDa.
Technical Field
The present invention relates generally to a process for the purification and isolation of biologically active compounds responsible for plant growth stimulation from seaweed extracts.
Background
One of the challenges in modern agriculture is to address the social need for sustainability, quality and safety of agricultural production and to adapt it to the world population growth by improving yield and crop tolerance to changing environments.
Biostimulants can be used to improve plant nutrition, affecting yield and quality parameters. Plant biostimulators generally belong to one of these categories, namely hormone-containing products, plant extract-based products, micronutrient-based products, amino acid-containing products and humic acid-containing products, but may not be strictly limited to these categories. Plant biostimulators are used to treat crops in commercial settings for their ability to increase growth rate, increase stress tolerance, increase photosynthetic rate, and increase disease tolerance. Plant biostimulators are generally thought to act by up-regulating or down-regulating key biological pathway genes.
Plant biostimulators contain one or more substances and/or microorganisms whose function, when applied to plants or rhizospheres, is to stimulate natural processes to enhance/benefit nutrient absorption, nutrient efficiency, tolerance to abiotic stress and crop quality, as defined by the European Biostimulator Industry Council (EBIC). Biostimulants have no direct effect on pests and therefore do not belong to the regulatory framework of pesticides.
Biostimulants are available in a variety of formulations and in a variety of ingredients, but are generally classified based on their source and content. These groups include Humic Substances (HS) and Amino Acid Containing Products (AACP).
Biostimulants are available in a variety of formulations and in a variety of ingredients, but are generally divided into seven major groups depending on their source and content. These groups include humic substances (humic and fulvic acids, protein hydrolysates and other N-containing compounds, seaweed extracts and botanicals, chitosan and other biopolymers, inorganic compounds, beneficial fungi and beneficial bacteria.
Despite the large quantities of fertilizers and plant bio-stimulants commercially available, there remains a need for improved products that can meet a variety of needs. Thus, there is a need for new products and methods for improving plant growth response and development.
Seaweed and seaweed-derived products have been widely used in crop production systems due to the presence of many plant growth stimulating compounds in these products. Thus, the biostimulating potential of many of these products has not been fully exploited due to the lack of scientific data on the growth factors present in seaweeds and their various modes of action in affecting plant growth.
Although the physiological role of seaweed extracts in plant defense and plant growth has been studied, little is known about the specific bioactive compounds responsible for these plant stimulators in seaweed extracts. It would be desirable to accelerate the identification of these bioactive components in algae, including, for example, brown algae extracts.
Phaeophyceae (Phaeophyceae) or brown algae is a large group of mainly marine multicellular algae, including various types of algae located in two hemispheric waters. They play an important role in the marine environment as food and as habitat. Many brown algae, such as members of the order Phaeophyta (Fucales), typically grow along rocky coasts. Over 1500 species of brown algae are known worldwide. Some species, such as Ascophyllum nodosum, are important in commercial use and also have environmental impact.
Michailovna et al, U.S. patent No. 7,611,716, describes a method of treating seaweed to obtain in a single process an extract containing acidic and neutral polysaccharides and an extract containing low molecular weight bioactive compounds useful in the pharmaceutical, food, fragrance and cosmetic industries. However, this reference only describes methods for processing seaweed and does not provide any method for identifying potential plant bio-stimulant compounds contained therein.
United states patent No. 3,856,569 to Strong et al describes a method of purifying and concentrating a desired polysaccharide, such as carrageenan or alginate, from an aqueous solution derived from seaweed (Rhodophyceae) and brown algae (Phaeophyceae) by subjecting the solution to ultrafiltration. However, this reference again provides only one method of treating the seaweed and does not provide any method of identifying the bio-stimulant compound contained therein.
Due to the increasing demand for organic, environmentally friendly and non-hazardous products for human health, there is an increasing demand for natural bio-stimulants. Furthermore, there is a need for biostimulators that are similar or more effective than conventional biostimulators that have been used. Furthermore, there remains a need in the art for improved methods of isolating and purifying biostimulant compounds, including extracts from seaweed.
Disclosure of Invention
The object of the present invention is to identify and purify substances which can be used as plant biostimulants.
It is another object of the present invention to identify and purify various seaweed extracts for the biologically active compounds responsible for stimulating plant growth.
To this end, in one embodiment, the present invention relates generally to one or more bioactive molecules isolated from an algal species, the one or more bioactive molecules having a molecular weight in the range of about 0.15kDa to about 1.0 kDa.
In another embodiment, the present invention relates generally to a method for isolating and purifying biologically active compounds from an extract obtained from seaweed, the method comprising the steps of:
a) circulating the extract through an ultrafiltration membrane having a suitable molecular weight cut-off;
b) collecting the filtrate from the seaweed extract to obtain a first filtrate fraction and a retentate; and
c) flushing the retentate to obtain one or more additional filtrate fractions.
Drawings
For a more complete understanding of the present invention, reference is made to the following description, taken in conjunction with the accompanying drawings, in which:
figures 1a and 1b depict Size Exclusion Chromatography (SEC) fractionation on filtered RM-3496 extract and chromatograms of standards injected on SEC to assess the average molecular weight of molecules present in the different fractions.
Fig. 2 depicts a box plot showing the efficacy of SEC fractionation of RM-3496 on lettuce.
FIGS. 3a and 3b depict boxplots showing the aerial and root fresh weights of in vitro cultures of Arabidopsis thaliana (Arabidopsis thaliana) treated with the F3 fraction compared to untreated controls, RM-3496 extract and reconstituted RM-3496 extract.
Fig. 4 depicts a view of an ultrafiltration method according to an aspect of the present invention.
Fig. 5 depicts a box plot showing the aerial fresh weight of lettuce treated with various ultrafiltration fractions, retentate and extract.
FIG. 6 depicts a box plot showing the fresh weight of the aerial parts of wheat treated with various ultrafiltration fractions, retentate and extracts.
Detailed Description
Plant "biostimulant" refers to an organic material containing one or more substances and/or microorganisms that function to stimulate natural processes to enhance/benefit nutrient absorption, nutrient efficiency, tolerance to abiotic stress, and crop quality when applied to a plant or rhizosphere.
When introducing elements of the present invention or the preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.
As used herein, the term "about" refers to a measurable value, such as a parameter, amount, duration, etc., and is intended to include variations of +/-15% or less, preferably +/-10% or less, more preferably +/-5% or less, even more preferably +/-1% or less, and still more preferably +/-0.1% or less of the specifically recited value, so long as such variations are suitable for carrying out the invention described herein. Further, it is to be understood that the value to which the modifier "about" refers is itself specifically disclosed herein.
The present invention describes a method for the purification and isolation of bio-stimulant compounds from seaweed-derived extracts capable of increasing the growth rate and yield of a wide range of crops.
As described herein, in one embodiment, the present invention provides a method for purifying biologically active compounds responsible for stimulating plant growth in seaweed extracts by metabolomic profiling of the seaweed extracts.
Thus, in one embodiment, the present invention relates generally to one or more bioactive molecules isolated from an algal species, the one or more bioactive molecules having a molecular weight in the range of about 0.15kDa to about 1.0 kDa. The one or more bioactive molecules are bioactive molecules capable of improving plant growth. In one embodiment, the algal species is a brown algae species. The brown algae may comprise an algal species selected from the group comprising: ascophyllum nodosum, Fucus vesiculosus, Sargassum (Sargassum sp), and combinations comprising one or more of the foregoing. In one embodiment, the one or more bioactive molecules do not comprise sulfated polysaccharides or laminarin.
The present invention also relates generally to a method of improving plant growth, comprising the step of applying a composition comprising an isolated one or more bioactive molecules to at least one of soil, a plant, or a seed. Improving plant growth comprises at least one of: promoting seed germination, stimulating root development, prolonging vegetative stage, increasing production stage or increasing harvest stage.
The present invention also relates generally to a method for the isolation and purification of biologically active compounds in an extract obtained from seaweed, comprising the steps of:
a) circulating the extract through an ultrafiltration membrane having a suitable molecular weight cut-off;
b) collecting the filtrate from the extract to obtain a first filtrate fraction and a retentate; and
c) flushing the retentate to obtain one or more additional filtrate fractions.
The method further comprises the step of assessing the biological activity of the first filtrate fraction and the further filtrate fraction to determine their efficacy on plant growth. The efficacy on plant growth may include at least one of: promoting seed germination, stimulating root development, prolonging vegetative stage, increasing production stage or increasing harvest stage. According to one embodiment, the extract is produced from brown algae species. The extract can be obtained from Ascophyllum nodosum, Fucus vesiculosus, or Sargassum (Sargassum sp.) algae.
In a preferred embodiment, the retentate comprises active molecules selected from the group consisting of sulfated polysaccharides and laminarin, which are active molecules capable of alleviating abiotic stress (such as salt excess) in crops. The first filtrate comprises biologically active molecules having a molecular weight in the range of about 0.15kDa to about 1.0 kDa.
The ultrafiltration membrane may have a molecular weight cut-off (MWCO) of less than 3kDa, preferably a MWCO of less than 2kDa, and most preferably a MWCO of less than 1 kDa.
Ascophyllum nodosum (fucoidin) is a species of fucoidan of brown algae species found in the North Atlantic ocean and has been used as a source of biostimulant for agricultural crops to improve plant growth, plant productivity and food quality.
Several purification techniques were examined to desalt and purify these seaweed extracts based on the polarity and size of the molecule. For each of the purification procedures, the purification procedure was,fractions were assayed on lettuce to ensure that the biological activity remained in the different desalted fractions or remained associated with the salt. The purification step was applied to seaweed extract (RM-2705, fromThe obtained fucoidin extract) and the non-polar purified fraction showed a high purity level. Most of the biomolecules (about 69%) were co-eluted with salt to show that the bioactive molecules were located in one of the non-polar purification fractions. Then, different grades were tested on lettuce in comparison with the whole extract. However, the results show that the biologically active molecule responsible for stimulating plant growth is present in the polar part which also contains salt. It was determined that the polar fractionation according to the molecules contained in the extracts RM-2705 and RM-3496 is not suitable for desalting and purification of seaweed extracts. Indeed, for all fractionation techniques used, the bioactive molecule remains associated with the salt.
Thus, a challenge of another method of desalinating seaweed extract while maintaining plant growth biostimulating activity is presented. Various purification techniques were investigated, including liquid-liquid extraction (LLE) using ethyl acetate, solid-phase extraction (SPE) using different adsorbents (normal phase: cyanopropyl-silica and reversed phase "XAD2 ", solid liquid extraction with butanol (SLE) and Size Exclusion Chromatography (SEC).
Furthermore, to obtain information on the stability of bioactive molecules, the thermostability of the activity of RM-2705 extract was determined on lettuce. The results show that the bioactive molecule has a certain thermal stability and that autoclaving or boiling of the treated extract increases the aerial fresh weight of lettuce.
Fractionation based on polarity appears to be inefficient in purifying bioactive molecules, and therefore fractionation based on the particle size of the seaweed extract molecules was attempted to desalt them.
Determine in addition to usingAll these techniques appear to be inefficient for this purpose, except for Size Exclusion Chromatography (SEC) on 30 resins, also known as gel filtration chromatography (GP). SEC was found to be the only effective method for desalting and purifying the seaweed filtrate.
Based on this, the seaweed extract (RM-3496) was fractionated by SEC fractionation method and the molecules were eluted according to their size (or molecular weight) as shown in fig. 1. a. Using SuperdexResins (GEHealthcare) to ensure good separation of molecules with molecular weights below 10 kDa. The smaller the molecule (i.e., the smaller the molecular weight), the more molecules are trapped in the porous beads of the gel and are eluted later. Thus, the molecular weight of the molecules decreases from the first fraction (i.e., F1) to the last fraction (i.e., F6). Fractions F1 and F2 were composed of larger molecules that flowed through the column faster than the salt, and the very low molecular weight molecules eluted in fractions F5 and F6.
To assess the molecular weight range of the molecules contained in the different fractions, a mixture of standards (0.5% w/v) was injected onto the SEC system. The chromatogram for the standard is depicted in fig. 1. b. a) Laminarin (from about 3 to about 5kDa, b) raffinose (594Da), c) sucrose (343.3Da), d) citrate (343.3Da), e) mannitol (182.2Da) and f) glycine (75.1 Da). According to these results, fraction F1 contains molecules of high molecular weight (above 4kDa), and fraction F2 contains laminarin (from about 3 to about 5kDa) that elutes between fractions F1 and F2.
The ultrafiltrate obtained after ultrafiltration of the RM-2705 extract on an ultrafiltration system with a 1kDa cut-off membrane was injected into the SEC system. The chromatogram of the ultrafiltrate only shows the chromatographic peaks corresponding to those of fractions F3, F4, F5 and F6 from the SEC fractionation of the RM-2705 extract. Thus, fraction F3 contains molecules with a molecular weight of less than 1kDa to about 0.18kDa, fraction F4 contains molecules with a molecular weight of less than about 0.2kDa, such as mannitol (182.2Da), fractions F5 and F6 contain salts and molecules with very low molecular weight, such as glycine (75 Da). The NMR spectra of the different fractions confirmed the presence of sulfated polysaccharides (fucosan polymers) in the first fraction F1, while the second fraction F2 contained laminarin (from about 3 to about 5kDa) and the fraction four F4 contained mannitol (182.2 Da). The latter two fractions F5 and F6 contained very low molecular weight molecules and salts.
The different fractions obtained by SEC fractionation were tested for their plant growth stimulating activity on lettuce compared to the whole seaweed extract RM-3496. Prior to chromatographic injection, the seaweed extract was filtered and the filtered extract was also tested on lettuce to check its efficacy. The results show that bioactive molecules are found in fraction F3, as shown in figure 2. Significant activity was found in the F3 fraction containing molecules from about 0.15kDa to about 1 kDa.
The combination of different techniques for desalting the RM-2705 extract provides information about the physicochemical properties of the bioactive molecule. Specifically, it was determined that these bioactive molecules are polar and that their molecular weight ranges from about 0.15 to about 1 kDa. Thus, this information excludes fucoidan polymers from the growth-promoting bioactive polymers, which are the predominant sulfated polysaccharides in the acidic extract of Ascophyllum nodosum (Ascophyllum nodosum), laminarin (from about 3 to about 4kDa), beta-1, 3-glucan elicitor and mannitol (182Da), which is a polyol and can comprise about 8-10% of the extract on a dry weight basis.
To determine the biological activity of the purified F3 fraction, the model plant arabidopsis thaliana (arabidopsis thaliana) was used for in vitro culture and propagation several times. These tests are shown in fig. 3. a. The biological activity of the F3 fraction was confirmed, while the presence of salts in RM-3496 extract interfered with the growth of Arabidopsis under these culture conditions. However, reconstituted-RM-3496, corresponding to reconstitution of seaweed extract with SEC fractions, showed growth promoting activity. Furthermore, fraction F3 and reconstituted-RM-3496 also appeared to enhance root growth in this in vitro bioassay, as shown in figure 3. b.
Fraction F3 showed strong growth stimulating activity, whereas fractions F1 and F2 were inactive. However, during the development of the latter bioassay, it was shown that in the presence of salt (100mM sodium chloride), fraction F3 no longer had growth stimulating activity, whereas F1 and F2 showed similar effects, and the whole RM-2705 extract conferred salt tolerance. These results indicate that RM-2705 extract contains different active compounds with different modes of action, including (1) Low Molecular Weight (LMW) fractions responsible for growth stimulation, and (2) fractions containing laminarin and fucoidan, which confer stress tolerance (salt, and biotic stress tolerance).
Together, these results indicate that fractionation of RM-2705 extract can provide at least two types of products with different modes of action and thus different applications using the same raw material.
Since SEC is not transferable on an industrial scale, there is also a need to develop an alternative process capable of producing a fractionated product of seaweed extract which is capable of providing plant growth stimulating activity on a larger scale.
An alternative method of SEC is Ultrafiltration (UF), a fractionation process in which the seaweed extract is filtered through an ultrafiltration membrane with a suitable molecular weight cut-off (MWCO), in one embodiment the MWCO is 1kDa, yielding a fraction with biologically active molecules in the desired range of about 0.15kDa to 1.0 kDa. Thus, in one embodiment, the seaweed extract may be subjected to ultrafiltration using a 1kDa MWCO membrane.
In one embodiment and in the broadest sense, the present invention provides a method of purifying a bio-stimulant composition derived from a seaweed extract, the method comprising the step of performing ultrafiltration using a semi-permeable ultrafiltration membrane to separate the molecule of interest from the remaining mixture according to the molecular weight, size and shape of the molecule.
The ultrafiltration step may be performed using an ultrafiltration unit in which the seaweed extract solution comprising between about 1% by weight and about 15% by weight of dry matter, more preferably between about 2% by weight and about 7% by weight of dry matter, is ultrafiltered using a membrane having a suitable molecular weight cut-off (MWCO). In one embodiment, the ultrafiltration method comprises tangential ultrafiltration.
The filtrate is collected for its biostimulating properties while the retentate (or concentrate) is recycled, which is separated at the end of the process for other applications. Although not generally necessary or desirable, if desired, further purification of the retentate (or concentrate) can be achieved by adding fresh water at a rate corresponding to the removal of water and molecules having a molecular weight of less than or equal to 1kDa from the ultrafiltrate.
As shown in FIG. 4, ultrafiltration may be carried out by adding a batch of the seaweed extract to the solution reservoir (1). The solution is circulated through line (2) and pump (3) to the inlet manifold (4) of the ultrafiltration unit (5). The ultrafiltration unit (5) comprises one or more cartridges arranged in parallel to provide a suitable ultrafiltration membrane area. The ultrafiltrate then leaves the ultrafiltration unit (5) via the outlet line (6) and is collected in a tank (7). The ultrafiltration concentrate leaves the ultrafiltration unit (5) via an outlet manifold (8) and is returned to the solution reservoir (1) via line (9).
The membrane comprised in the ultrafiltration unit (5) may be a polymeric or ceramic type membrane. In one embodiment, the membrane comprises a tubular ceramic membrane comprising a plurality of channels. For example, the membrane may contain from about 15 to about 50 channels, more preferably from about 19 to about 39 channels, and may have a length of from about 50 to about 150 cm. In other embodiments, spiral membranes and cross-flow membranes may also be used in the practice of the present invention. The membrane area is typically from about 0.20 to about 0.6m2More preferably between about 0.30 and 0.40m2In the meantime.
The retentate is flushed several times to remove a major portion of molecules having a molecular weight less than the cut-off value of the membrane. The ultrafiltrate contains molecules having a molecular weight less than the molecular weight of the retentate membrane. In one instance, the cut-off value is 3kDa, more preferably 2kDa, and most preferably still 1 kDa. The molecules contained in the ultrafiltrate exhibit a lower molecular weight than MWCO, e.g., about 1kDa, and are commonly referred to as metabolites. The retentate contains molecules with a molecular weight greater than the cut-off membrane, e.g., about 1 kDa. The molecules contained in the retentate exhibit high molecular weights (e.g., laminarin from about 3kDa to about 4kDa, or fucoidan and other brown algae high molecular weight biopolymers above about 30 kDa).
It has been found that all algal species from the order Fucales (Fucales) show promising activity and can be subjected to the methods described herein. These algae species include, but are not limited to, species of the families Fucaceae (Fucaceae), Sargassaceae (Sargassaceae) and Durveilleaceae. Other species from the orders Fucales and Laminariales include, but are not limited to, Ascophyllales (Ascoseirales), Ascophyllales (Asterocladales), Acidophyales (Descarastiales), Dictyophyceae (Dictyotales), Dictyophyceae (Dictyophyceae), Jatrophaphtales (Discosporangiales), Discosporangiales, Oesophyceae (Ectocarpales), Fucales (Fucales), Fucales (Fucophyllaceae), Acacia (Ishigeales), Ishigeophyceae, Laminariales (Laminariales), Nematophyceae (Nematomatales), Nematophyceae (Skophyceae), Phaeophyceae (Phaeophyceae), Euglena (Osteiella), Phaeophyceae (Oscilales), Phaeophyceae (Oscilaria), Oscilales (Oscilales), Oscilale.
Furthermore, although the invention is described and illustrated to show positive results from algal species from the order Fucus, the method is not limited to these algal species, and may also be used to separate and analyze the filtrates of any algae or other species that may be used as biostimulant to determine the biological activity of these filtrates.
As used herein in the figures, the term "filtrate" refers to the filtrate and ultrafiltrate obtained after one or more ultrafiltration by the ultrafiltration unit.
As used herein in the figures, the term "retentate" refers to the retentate without rinsing and the retentate obtained after one or more rinsing.
Detailed Description