AU2018101956A4 - Biofertiliser composition - Google Patents

Abstract

A biofertiliser composition comprising a living microalgae component consisting of a live microalga of Chlorella spp. and a live microalga of Anabaena spp.

Description

TECHNICAL FIELD

The present disclosure relates to a biofertiliser composition and to methods for the preparation of the composition. The invention also relates to a method of fertilising a plant with the biofertiliser composition.

BACKGROUND

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

One of the major issues of global concern is food security since rising populations and restricted lands under cultivation require ever-increasing agricultural production. Intensive agricultural practices require constant input of agrochemicals such as synthetic, chemical fertilisers to increase yield. However, commercial phosphate fertilisers contain small amounts of heavy-metal contaminants as these are minor constituents in their main ingredient, phosphate rock. Animal manures and sewage sludges are the base for many organic fertilisers and these may also contain heavymetal contaminants. These heavy metals may accumulate in soil with repeated fertiliser applications (Mortvedt, 1995) Heavy metals in the soil may be absorbed by plants and affect the quality of plants and agricultural products.

Application of chemical fertilisers adversely affects the dynamic equilibrium of soil and affects agro-biodiversity by destroying non-target soil microorganisms. A reduction in the quantity and activity of the soil microorganisms reduces the conversion of organic matter in the soil, the decomposition of minerals and the degradation of toxic) substances. As a result, long-term application of chemical fertilisers aggravates the acidification of the soil, aggravates soil compaction, inhibits plant root development, and seriously reduces plant stress resistance.

Moreover, the production cost of chemical fertilisers is high, so poor farmers in less developed nations cannot afford to use them, or at least to use desired the quantity.

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Furthermore, continued use of chemical fertilisers leads to soil erosion and degradation of local ecosystems. In turn, this can lead to an increase in pest infestation and diseases and enhanced water demand, paradoxically causing a decrease in crop productivity.

Biofertilisers have emerged as the best alternative to chemical fertilisers (Chatterjee et al, 2017). A biofertiliser comprises living microorganisms, which on application colonise the rhizosphere, thus promoting growth by accelerating the availability of primary nutrients to the host plant. Biofertilisers comprise microorganisms, including bacteria, fungi, and cyanobacteria as well as their metabolites that are capable of 10 enhancing soil, crop growth, and yield. Bacteria useful as biofertilisers include symbiotic nitrogen fixers like Rhizobium spp. associated with leguminous crops and non-symbiotic free-living nitrogen fixers like Azotobacter, which can be applied to crops like maize, wheat, cotton, mustard, potato, and other vegetable crops. Phosphate-solubilising bacteria such as Pantoea agglomerans strain P5 and 15 Pseudomonas putida strain P13 can solubilise insoluble phosphate from organic and inorganic sources.

Cyanobacteria, previously known as blue-green algae, can fix atmospheric nitrogen and convert it into a form in which it is available for plant growth. Cyanobacteria such as those of Nostoc spp., Anabaena spp., Tolypothrix spp. and Aulosira spp. are used 20 in rice paddy fields (Sahu et al, 2012). Algal extracts and harvested algal biomass, generally from the macroalgae, are considered good fertilisers since they introduce mineral substances, amino acids, vitamins, and plant growth regulators including auxins, cytokinin and gibberellins to the soil (Hamed, et al, 2018).However, the benefit is limited since only the materials available in the extract or in the tissue of the 25 harvested algae as it decomposes is available to the plant. The present invention seeks to provide a biofertiliser containing live microalgae which is storage stable and which is effective in promoting growth of a plant to which it is applied.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a biofertiliser composition comprising a living microalgae component consisting of a live microalga of Chlorella spp. and a live microalga of Anabaena spp.

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In another aspect, the present invention provides a biofertiliser composition comprising a living microalgae component consisting of a live microalga of Chlorella spp. and a live microalga of Anabaena spp. in a ratio from 1.0:0.2 to 1.0:5.0.

In another aspect, the invention provides a biofertiliser composition comprising a living microalgae component consisting of a live microalga of Chlorella spp. and a live microalga of Anabaena spp. and nutrients for microalgae comprising a source of any one or more of iron, bicarbonate, calcium, magnesium, nitrate, phosphorous, manganese, zinc, copper, molybdenum and borate.

In an embodiment the live microalga of Chlorella spp. is Chlorella pyrenoidosa.

In an embodiment the live microalga of Anabaena spp. is Anabaena azotica.

In another aspect, the invention provides Chlorella pyrenoidosa deposited at China General Microbiological Culture Collection Center (CGMCC) under Accession No 16726 on 9 November 2018.

In another aspect, the invention provides Anabaena azotica deposited China General 20 Microbiological Culture Collection Center (CGMCC) with Accession No 16725 on 9 November 2018.

In another aspect, the invention provides a method of fertilising a plant comprising applying a biofertiliser composition comprising a living microalgae component 25 consisting of a live microalga of Chlorella spp. and a live microalga of Anabaena spp., and, optionally, an agriculturally acceptable carrier, to the plant and/or to soil surrounding the plant.

In another aspect, the invention provides a method of fertilising a plant comprising applying a biofertiliser composition comprising a living microalgae component consisting of a live microalga of Chlorella spp. and a live microalga of Anabaena spp. in a ratio from 1.0:0.2 to 1.0:5.0, and, optionally, an agriculturally acceptable carrier, to the plant and/or to soil surrounding the plant.

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In another aspect, the invention provides a method of fertilising a plant comprising applying a biofertiliser composition comprising a live microalga of Chlorella spp. and a live microalga of Anabaena spp. and a culture medium comprising nutrients for microalgae comprising a source of any one or more of iron, bicarbonate, calcium, 5 magnesium, nitrate, phosphorous, manganese, zinc, copper, molybdenum and borate to the plant and/or to soil surrounding the plant.

In another aspect, the invention provides a method of preparing a biofertiliser, comprising the steps of:

a) providing a live microalga of Chlorella spp.;

b) providing a live microalga of Anabaena spp.;

c) culturing the microalga of Chlorella spp.;

d) culturing the microalga of Anabaena spp.;

e) combining the Chlorella spp. culture and the Anabaena spp. culture.

In an embodiment, the Chlorella spp. culture and the Anabaena spp. culture are combined to provide a ratio of Chlorella to Ababaena of 1.0:0.2 to 1.0:5.0.

In an embodiment culturing takes place in a culture medium comprising nutrients for 20 microalgae comprising a source of any one or more of iron, bicarbonate, calcium, magnesium, nitrate, phosphorous, manganese, zinc, copper, molybdenum and borate.

DETAILED DESCRIPTION

Microalgae are autotrophic plants that are widely distributed in terrestrial soils, rivers and lakes, and have high photosynthesis utilization. The present invention relates to the use of living microalgae as a biofertiliser. The biofertiliser is effective in improving soil condition, supplementing natural nitrogen, activating soil trace elements, improving plant quality and resistance to overcome or ameliorate pest infestations and 30 diseases in the plant.

In particular, the invention provides a biofertiliser composition comprising a living microalgae component consisting of live Chlorella pyrenoidosa and live Anabaena azotica.

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The coccoid green microalgae genus Chlorella is one of the most important commercial microalgae. The world production was up to 2,000 tons dry weight in 2005 (Spolaore et al. 2006). The main market for Chlorella is human nutrition, the dried 5 biomass being processed as powder, capsules or tablets (Gors et al. 2010). The genus includes the species Chlorella autotrophica, Chlorella lewini Chlorella coloniales, Chlorella minutissima, Chlorella pituita, Chlorella pyrenoidosa, Chlorella pulchelloides, Chlorella rotunda, Chlorella singularis, chlorella sorokiniana, Chlorella variabilis, Chlorella volutis and Chlorella vulgaris. Chlorella pyrenoidosa is widely distributed. It 10 is a green alga with a prominent pyrenoid structure in its chloroplasts. It has a capability for photosynthesis greater than that of most other plants. It can increase the dissolved oxygen in water and degrade ammonia present in the water in which it grows.

Anabaena is a genus of filamentous cyanobacteria that exist as plankton. They are known for their nitrogen fixing ability. The genus includes the species:

A. aequalis

A. affinis

A. angstumalis angstumalis

A. angstumalis marchita

A. aphanizomendoides

A. azotica

A. azollae

A. bornetiana

A. catenula

A. cedrorum

A. circinalis

A. confervoides

A. constricta

A. cyanobacterium

A. cycadeae

A. cylindrica

A. echinispora

A. felisii

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A. flos-aquae flos-aquae

A. flos-aquae minor

A. flos-aquae treleasei

A. helicoidea

A. inaequalis

A. lapponica

A. laxa

A. lemmermannii

A. levanderi

A. limnetica

A. macrospora macrospora

A. macrospora robusta

A. monticulosa

A. nostoc

A. oscillarioides

A. planctonica

A. raciborskii

A. scheremetievi

A. sphaerica

A. spiroides crassa

A. spiroides spiroides

A. subcylindrica

A. torulosa

A. unispora

A. variabilis

A. verrucosa

A. viguieri

A. wisconsinense

A. zierlingii.

Anabaena azotica is a nitrogen-fixing cyanobacterium with high nitrogen fixation and decomposition functions. In the biofertiliser of the present invention, Chlorella spp. and Anabaena spp. are present in a living state and in a ratio which ensures that there is good compatibility between them. Typically, these organisms are present in a ratio

2018101956 05 Dec 2018 from 1.0:0.2 to 1.0:5.0.

In an embodiment the ratio of Chlorella spp. to Anabaena spp. is from 1.0:0.5 to 5 1.0:2.0.

In an embodiment the ratio of Chlorella spp. to Anabaena spp. is from 1.0:0.8 to 1.0:1.2.

In an embodiment the ratio of Chlorella pyrenoidosa to Anabaena azotica is from 10 1.0:0.2 to 1.0:1.5.0.

In an embodiment the ratio of Chlorella pyrenoidosa to Anabaena azotica is from 1.0:0.5 to 1.0:2.0.

In an embodiment the ratio of Chlorella pyrenoidosa to Anabaena azotica is from 1.0:0.8 to 1.0:1.2.

Typically, compositions in accordance with the present invention comprise greater than 10⁶ cells permL of culture medium. Advantageously the shelflife of compositions 20 in accordance with the present invention is greater than 18 months when the biofertilser is packaged and stored at a temperature from 5°C to 30°C.

Any of the various strains of Chlorella pyrenoidosa and Anabaena azotica may be used. The organisms may be collected from a soil sample or taken from a culture of 25 organisms collected earlier. If taken from a soil sample, a small amount of soil is taken, then diluted with sterilized water. Drops of the diluted soil sample or a sample taken from a collection are viewed under a microscope to pick up a single cell or few cells with a relatively large cell diameter and/or with rich and bright colour and/or with clear cell walls. In this way the strongest and healthiest cells are selected for culture. The 30 single cell or the cells selected are then introduced to a culture medium.

In an embodiment the Chlorella pyrenoidosa strain is a strain deposited at China

General Microbiological Culture Collection Center (CGMCC) under Accession No

16726 on 9 November 2018.

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In an embodiment the Anabaena azotica is a strain deposited China General

Microbiological Culture Collection Center (CGMCC) with Accession No 16725 on 9

November 2018.

In an embodiment the living microalgae component is provided as a culture in a culture medium. The culture medium may comprise any suitable minerals that can provide nutrition to microalgae.

In an embodiment the culture medium comprises nutrients for microalgae comprising 10 a source of any one or more of iron, bicarbonate, calcium, magnesium, nitrate, phosphorous, manganese, zinc, copper, molybdenum and borate. It will be appreciated that any combination of these nutrients may be present, depending on the requirements of the microalgae. Advantageously, all are present in the culture medium. Other nutrients such as potassium, sodium and chloride may be present, 15 often as counterions, but there may also be sources of these in the culture medium.

In an embodiment the calcium source comprises one or more compound selected from the group consisting of calcium chloride (CaCk); calcium carbonate (CaCCh); monocalcium phosphate, monohydrate (Ca(H2P04)2.H20); dicalcium phosphate, 20 anhydrous (CaHPCL); dicalcium phosphate, dihydrate (CaHP04.2H20); tricalcium phosphate (Ca3(P04.)2) and calcium sulphate (CaSCh); or bonemeal, oyster shell grit; or ground limestone (CaCCE).

In an embodiment the copper source comprises one or more compounds selected 25 from the group consisting of copper sulphate (CuSCh); copper sulphate, pentahydrate (CUSO4.5H2O); copper chloride (CuCh); copper oxide (CuO); and copper (II) hydroxide (Cu(OH)2).

In an embodiment the iron source comprises one or more compounds selected from 30 the group consisting of ferrous sulphate, heptahydrate (FeS04.7H2O); ferrous (II) carbonate (FeCOs); and ferrous oxide (FeO).

In an embodiment the magnesium source comprises one or more compounds selected from the group consisting of magnesium chloride (MgCh); magnesium oxide (MgO);

2018101956 05 Dec 2018 magnesium carbonate (MgCCh); dimagnesium phosphate, trihydrate (MgHP04,3H20);

magnesium sulphate (MgSCM); and magnesium sulphate, heptahydrate (MgS0₄.7H₂0).

In an embodiment the manganese source comprises one or more compounds selected from the group consisting of manganese oxide (MnO); manganese dioxide (MnCh); manganese carbonate (MnCCh); manganese chloride, tetrahydrate (MnCl2.4H20); manganese sulphate (MnSCh); manganese sulphate, hydrate (MnSCU.hkO); and manganese sulphate, tetrahydrate (MnS04.4H20).

In an embodiment the molybdenum source comprises one or more compounds selected from the group consisting of sodium molybdate, dihydrate (Na2Mo4.2H2O) and sodium molybdate, pentahydrate (Na2M04.5H20).

In an embodiment the phosphorous source comprises one or more compounds selected from the group consisting of monocalcium phosphate, monohydrate (Ca(H2PO4)2.H2O): dicalcium phosphate, anhydrous (CaHPCU); dicalcium phosphate, dihydrate (CaHPO4.2H2O) tricalcium phosphate (Ca3PO4)2); potassium orthophosphate (K2HPO4): potassium dihydrogen orthophosphate (KH2PO4); sodium 20 hydrogen orthophosphate (Na2HPO4); sodium dihydrogen orthophosphate, hydrate (NaH2PO4.H2O); sodium dihydrogen orthosphosphate, dihydrate (NaH2PO4.2H2O); dimagnesium phosphate, trihydrate (MgHPO4.3H2O); and rock phosphate (CasFOi2P3).

In an embodiment the zinc source comprises one or more compounds selected from the group consisting of zinc carbonate (ZnCO3); zinc chloride (ZnCI) zinc oxide (ZnO); zinc sulphate (ZnSCU); zinc sulphate, hydrate (ZnSCU.hkO); and zinc sulphate heptahydrate (ZnSO₄.7H₂O).

In an embodiment the sodium source, if provided separately, comprises one or more compounds selected from the group consisting of sodium chloride (NaCI); sodium bicarbonate (NaHCOs); and sodium sulphate (Na2SO4).

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In an embodiment the potassium source, if provided separately, comprises one or more compounds selected from the group consisting of potassium chloride (KCI); potassium carbonate (K2CO3); potassium bicarbonate (KHCO3); potassium acetate (KC2H3O2): potassium orthophosphate (K3PO4); and potassium sulphate (K2SO4).

In an embodiment the chloride source, if provided separately, comprises one or more compounds selected from the group consisting of sodium chloride (NaCl) and potassium chloride (KCI).

In an embodiment the culture medium further comprises at least one chelation agent. In an embodiment the chelation agent comprises one or more compounds selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (PTDA), N- (hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), ethylenediamine-N,N'-bis (EDDHA), nitrilotriacetic acid (NTA), ethylenediamine-N,N'-disuccinic acid (EDDS), iminodisuccinic acid (IDS), methylglycinediacetic acid (MGDA), glutamic acid diacetic acid (GLDA), ethylenediamine-N,N'-diglutaric acid (EDDG), ethylenediamine-N,N'-dimalonic acid (EDDM), hydrodesulfurization (HDS), 2-hydroxyethyliminodiacetic acid (HEIDA), and (2,6-pyridine dicarboxylic acid).

In an embodiment, the culture medium further comprises a buffering agent. In an embodiment the buffering agent comprises one or more compounds selected from the group consisting of citrate salts, acetate salts, histidine salts, succinate salts, malate salts, phosphate salts or lactate salts, and/or the respective free acids or bases 25 thereof, as well as mixtures of the various salts and/or acids or bases thereof. The term mixture here covers both mixtures of different salts of the same acid, such as, for example, mixtures of different citrate salts, and mixtures of salts of different acids, such as, for example, mixtures of citrate and acetate salts. The buffer preferably consists of one or more citrate salt(s) and/or the free acid thereof (for example citric 30 acid, citric acid monohydrate, trisodium citrate dihydrate, tripotassium citrate monohydrate), typically citric acid.

In an embodiment, the culture medium further comprises water, typically deionized water.

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In an embodiment the culture medium comprises: 2-6 mg/L disodium edetate, 5-13 mg/L citric acid, 0.05-0.15 mg/L FeSCM .7H2O, 0.15-0.35 g/L NaHCO3, 0.03-0.1 g/L

CaCI₂, 0.05 -0.2 g/L MgSO₄.7H₂O, 1-3.5 g/L NaNOs, 0.05-0.15 g/L K2HPO4, 0.2-1 g/L

KH2PO4, 1.5-2.5 mg/L MnCl2.4H₂O, 0.02-0.05 mg/L ZnSO4.7H₂O, 0.05-0.1 mg/L

CUSO4 5H2O, 0.02-0.05 mg/L NaMo4.2H2O and 2-4 mg/L HsBChin aqueous solution.

The compositions of the present invention may also comprise one or more other agriculturally acceptable component. Examples of such components include additional 10 nutrient material, crop protection products, fertilisers, emulsifiers, thickeners, anticaking agents, suspension agents, dispersion agents, carriers or excipients and wetting agents, provided that they are not incompatible with survival of the living microalgae.

In an embodiment the composition comprises an agriculturally acceptable carrier. Suitable carriers should not be phytotoxic and should not be toxic to algae. Biofertiliser compositions can be designed for application directly to plants or their locus or can be concentrates or formulations which are normally diluted with additional carriers and adjuvants before application. They may include inert or active components and can be 20 liquids such as, for example, suspensions of the microalgae or solids, such as, for example, dusts, granules, water dispersible granules, carrying the microalgae. Suitable agricultural carriers useful in preparing agricultural compositions of the present invention are well known to those skilled in the art. Water is generally the carrier of choice for liquid formulations. Suitable solid carriers include talc, pyrophyllite 25 clay, silica, attapulgus clay, kieselguhr, chalk, diatomaceous earth, lime, calcium carbonate, bentonite clay, Fuller's earth, cotton seed hulls, wheat flour, soybean flour, pumice, wood flour, walnut shell flour, lignin, and the like.

In an embodiment the agriculturally acceptable carrier is water.

In an embodiment the agriculturally acceptable carrier is de-ionized water.

In an embodiment the composition further comprises crop protection productions, provided that the crop protection product can be applied to a plant without detriment

2018101956 05 Dec 2018 to the plant and is not toxic to algae. Examples of crop protection products include plant and seaweed extracts, plant growth regulators, metabolic stimulating agents, extracts from humus such as fulvic acid, insecticides, fungicides and the like.

While not wishing to be bound by theory, it is believed that microalgae can produce polysaccharides, proteins and other nutrients for plants and provide these to the plant, as well as mobilizing nutrients such as phosphorous and iron. In addition, the composition of the present invention fixes nitrogen. Accordingly, the living microalgae of the present invention can be used as biological fertilisers to replace certain quantity 10 of chemical fertilisers in order to promote plant growth.

In an embodiment the composition further comprises fertilisers.

In an embodiment the composition further comprises liquid fertilisers or solid fertilisers.

In an embodiment the composition further comprises particulate fertiliser such as ammonium nitrate, urea and the like.

The present invention also provides a method of preparing a biofertiliser, comprising the steps of:

a) providing a living microalga, the microalga being of the species Chlorella pyrenoidosa;

b) providing a living microalga, the microalga being of the species Anabaena azotica;

c) culturing the Chlorella pyrenoidosa;

d) culturing the Anabaena azotica;

e) combining the Chlorella pyrenoidosa culture and the Anabaena azotica culture in a ratio of 1.0:0.2 to 1.0:5.0.

In an embodiment, step (c) further comprises expanding the Chlorella pyrenoidosa culture.

In an embodiment, step (d) further comprises expanding the Anabaena azotica culture.

Collectively, the steps of culturing Chlorella and expanding the culture and culturing Anabaena and expanding the culture to obtain two algal cultures is referred to herein

2018101956 05 Dec 2018 as step S1. The step of mixing the two algal cultures to obtain a biofertilizer composition is referred to herein as step S2.

In an embodiment the medium solution for expanding culture of step S1 comprises an aqueous solution of: 2-6 mg/L disodium edetate, 5-13 mg/L citric acid, 0.05-0.15 mg/L FeSCU .7H₂O, 0.15-0.35 g/L NaHCOs, 0.03-0.1 g/ L CaCI₂, 0.05 -0.2 g/L MgSCU .7H₂O, 1-3.5 g/L NaNOs, 0.05-0.15 g/L K₂HPO₄, 0.2-1 g/L KH₂PO₄, 1.5-2.5 mg/L MnCI₂.4H₂O, 0.02-0.05 mg/L ZnSO₄.7H₂O, 0.05-0.1 mg/L CuSO₄-5H₂O, 0.02-0.05 mg/L NaMo4.2H₂O and 2-4 mg/L H3BO3.

In an embodiment the medium solution for expanding culture of step S1 comprises an aqueous solution of: 2.5-5 mg/ L disodium edetate, 6-12 mg/L citric acid, about 0.062 mg/ L FeSO₄ .7H₂O, 0.162-0.324 g/ L NaHCOs, 0.036-0.072 g/ L CaCI₂, 0.075 -0.15 g/L MgSO₄ .7H₂O, 15-3.0 g/L NaNOs, 0.06-0.12 g/L K₂HPO₄, 0.315-0.63 g/L KH₂PO₄, 15 and 1-2ml of As solution. As solution consists of 2.5 g/L MnSO4.7H₂O, 222 mg/L

ZnSO₄.7H₂O, 79 mg/L CuSO₄-5H₂O, 21 mg/L NaMo₄.2H₂O and 2.86 g/L H3BO3.

The medium solution provides nutrient elements for the microalgae, promoting healthy growth and rapid propagation of the microalgae, improving activity of the microalgae, 20 and thereby improving the efficacy of the biofertiliser.

In an embodiment, the expansion and cultivation process of step S1 comprises selecting and isolating single microalgae cells, then grading and expanding the single microalgae cells until the number of microalgae cells is >10⁶ /ml in each stage of 25 expansion and cultivation, then entering the next stage to expand the cultivation process. Viability of the microalgae in the culture is enhanced when the cells have a biological density of > 10⁶cells/mL, and, in an embodiment, the cells can continue to multiply as a graded expansion.

In an embodiment a graded expansion is undertaken wherein a single microalga is cultured in a 15 mL medium solution for 10-15 days, whereafter 15 mL of algae liquid is obtained, and inoculated into 85 mL of the medium solution for further cultivation (10-15) days to obtain 100 mL of algae solution. The 100mL of algae solution is transferred to a vessel containing 0.9L of medium solution for further cultivation (1014

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15) days to obtain 1L algae solution. In turn, this is transferred to a vessel containing

4L of medium solution for further cultivation (10-15) days to obtain 5L algae liquid, which is transferred to 13 L of medium solution for further cultivation (10-15) days to obtain 18 L of algae liquid, which is transferred to 782 L of medium solution for further cultivation (10-15) days to obtain 800 L of algae solution.

The culture obtained has suitably high biomass and high microalgae activity. The person skilled in the art will be able to determine appropriate conditions under which the culture may be performed. In particular, the person skilled in the art can establish 10 the light intensity and culture temperature needed to promote the growth and reproduction of microalgae. Typically, during the expansion of the step S1, the light intensity is 2300-2700 lux. Typically, during the expansion of the step S1, the culture temperature is 25-27 °C. In an embodiment, in the expansion and cultivation process of step S1, purified air is continuously introduced into the algae liquid. The person 15 skilled in the art can determine the parameters for introduction of air to the culture medium. Typically, the air pressure is 0.14-0.6 MPa. In an embodiment an inert gas such as CO2 may be introduced intermittently. Typically, the pressure is 0.14-0.6 MPa, the ventilation interval is 3-5 h, and the duration of each inert gas introduction is 20-50 min. A process with continuous introduction of air and periodic introduction of an inert 20 gas promotes the growth and reproduction of microalgae cells, but also acts to stir the algae liquid so that the microalgae are evenly distributed in the algae liquid. Accordingly, the uniformity and consistency of the growth and reproduction of the microalgae is improved.

In an embodiment, the method further comprises the step of adding an agriculturally acceptable carrier.

The biofertiliser of the present invention solution of the present invention may be applied to a plant and/or to the rhizosphere to enhance rhizosphere processes and so 30 fertilise the plant.

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As used herein, the term “biofertiliser” refers to a preparation containing living cells or latent cells of efficient strains of microorganisms that help crop plants' uptake of nutrients by their interactions in the rhizosphere when applied through seed or soil.

As used herein, the term “rhizosphere” refers to the zone of soil surrounding a plant root where the biology and chemistry of the soil are influenced by the root. The rhizosphere contains many bacteria and other microorganisms that feed on sloughedoff plant cells and the proteins and sugars released by plant roots. The rhizosphere plays an important role in regulating soil organic matter decomposition and nutrient 10 cycling.

As used herein, the term “rhizosphere processes” refers to processes that take place in the rhizosphere. Rhizosphere processes are largely controlled by or directly influenced by plant roots. These processes include exudation of soluble compounds, 15 water uptake, nutrient mobilization by roots and microorganisms, rhizospheremediated soil organic matter decomposition, and the subsequent release of CO2 through respiration.

As used herein, a “living” cell is a cell which possesses all the characteristics of life: 20 metabolism, heredity, reproduction, response to stimuli, and homeostasis. Therefore, a significant proportion of the algae used in the current invention will possess all the characteristics of life of an algal cell. Moreover, the organisms will survive following application to the soil and multiply within the rhizosphere.

As used herein, the term “microalgae” or the singular “microalga” refers to microscopic algae, typically found in freshwater and marine systems, living in both the water column and sediment, and also includes cyanobacteria.

While not wishing to be bound by theory, it is believed that algae in accordance with 30 the present invention, in addition to occupying the rhizosphere, may colonise air spaces within the plant root, and can also colonise air spaces elsewhere in the plant such as in the leaf. In the rhizosphere microalgae envelops plant roots and form a symbiotic interaction with plant roots, fixing nitrogen from the air and supplying nitrogen in available form and other nutrients to plant root. Microalgae can not only

2018101956 05 Dec 2018 survive and multiply on the surface of plant roots, but also enter the interior of plants, survive in the roots, stems, leaves and fruits of plants, and provide plants with nutrients. Once established in the rhizosphere, the microalgae cells multiply, absorbing carbon dioxide and releasing a large amount of oxygen, which creates a 5 good environment for aerobic microorganisms in the soil. Thus, the addition of the biofertiliser of the present invention activates all kinds of indigenous microbes in the soil and improves the soil microecological environment. In addition, microalgae propagate and develop as they are autotrophic. Their metabolism is such that they are involved in adsorption, degradation, transformation and solidification of pollutants such 10 as chemical residues and heavy metal residues in soil. The organic matter that plants and animals and humans cannot decompose can be decomposed in soil treated with the microalgae into inorganic matter, water and nitrogen dioxide, thus changing the soil structure, regulating soil pH value, improving soil fertility. Microalgae can also decompose and dissolve insoluble phosphorus and potassium, and activate insoluble 15 calcium, magnesium or sulfur elements into a plant absorbable available state. The biostimulants produced by the microalgae provide nutrients for crops to stimulate growth, control or inhibit the activities of plant pathogens, enhance the stress resistance of crops and block heavy metals entering the plant through the plant roots so as to produce safe and healthy food for humans. In addition, compared with the 20 existing chemical fertilisers, the application of microalgae does not cause the enrichment of heavy metals in the soil and effectively reduces the absorption of heavy metals by plants, thereby improving plant growth and the quality or agricultural products. Furthermore, it does not reduce the quantity and activity of soil microorganisms, nor does it affect the transformation of soil microorganisms into 25 organic matter in the soil, the decomposition of minerals and the degradation of toxic substances, nor will it cause acidification and compaction of the soil.

Accordingly, the present invention is also concerned with a method of fertilizing a plant comprising applying a biofertiliser composition comprising a living microalgae 30 component consisting of live Chlorella pyrenoidosa and live Anabaena azotica.

Typically, the biofertiliser is applied following plant germination. Advantageously it is applied as soon as possible after plant germination

2018101956 05 Dec 2018

The method may further comprise applying one or more chemical fertilisers, and this may be together with the biofertiliser (either as a part of the composition or concurrently with, or immediately following, application of the biofertilizer) or at a different point in time. In the latter case, an initial application of a chemical fertilizer 5 may occur before or after the application of biofertilizer. Typically, the or each chemical fertiliser is applied periodically throughout the life of the plant, and generally at the application rate typical for the product.

In an embodiment a composition in accordance with the present invention is applied 10 at a rate of from 2 to 5 litres per hectare. The composition is applied by methods that will be well understood by the person skilled in the art. Advantageously the composition is agitated prior to application as the microorganisms it contains may settle. Typically, the composition is applied by spraying, such as by a boom sprayer or aerial spraying, or by introduction into an irrigation system (so called “fertigation”).

Typical application rates are as follows:

DILUTION RATES (ALGAE: Water ratio)

ALGAE AND SOIL 1:25 TO 1:50

CROP	APPLICATION TIMING	RATES
APPLES/PEARS	At post flowering then every 4-6 weeks up to harvest. Apply post harvest whilst leaves actively growing.	3 - 4 l/ha through trickle or fertigation into root zone.
BRASSICAS	Apply from 4 true leaf stage every 3-4 weeks.	3 l/ha fertigation or boom spray.
CANOLA	At 2-4 true leaf stage or at flower initiation.	3 l/ha fertigation, boom or aerial spray.
CEREALS	At 2-4 true leaf stage or at tillering with post emergent herbicide.	3 l/ha fertigation. Boom or aerial spray.
CITRUS	1st application at start of growth flush. 2nd just post flowering. Then every 6-8 weeks up to harvest.	4-5 l/ha trickle or fertigation into root zone.
CUCURBITS	1st apply at 6 leaf stage. 2nd just before flowering. Then every 2-3 weeks up to harvest.	3 l/ha with fertigation, boom or aerial spray.

2018101956 05 Dec 2018

DAIRY/BEEF	Apply with 12 weekly base fertiliser application or at start of first growth flush in Spring and Autumn.	4-5 l/ha fertigation, boom or aerial spray.
FIELD BRASSICAS	At 2-4 true leaf stage.	4 l/ha fertigation, boom or aerial spray.
GRAIN LEGUMES	At 2-4 weeks post emergence.	4 l/ha fertigation, boom or aerial spray.
GRAPES	1st application 1-2 weeks post bud burst. 2nd just before flowering. Follow up every 21 days.	4 l/ha fertigation or turbo mist sprayer.
HAY & SILAGE	Apply at closing up with 30% reduced base granular fertilizer.	4-5 l/ha fertigation, boom or aerial spray.
LEAFY VEGETABLES	Apply every 2-3 weeks from 6 leaf stage.	3-4 l/ha fertigation or boom spray.
LUCERNE	Apply post grazing or harvesting with approx 150mm new growth flush.	4-5 l/ha fertigation, boom or aerial spray.
MELONS	1st apply at 4-6 leaf stage. 2nd just prior to flowering. Then every 2-3 weeks.	4-5 l/ha fertigation, boom or aerial spray.
NURSERY PLANTS	Apply to potting mix once roots have established.	10-20ml/l in backpack or 3 l/ha through fertigation.
OLIVES	Apply every 3 weeks Sept-Dec from bud burst. Then every 3 weeks from fruit formation to harvest.	4 l/ha trickle or fertigation into root zone.
STONEFRUIT	1st apply prior to bud burst. 2nd application just post petal fall. Then every 4 weeks from fruit formation to harvest. Apply post harvest while still active leaf growth.	4 l/ha trickle or fertigation into root zone.
SWEETCORN	1st apply at 4-6 leaf stage. 2nd application at early tassel formation.	4 l/ha fertigation, boom or aerial spray.
TOMATOES	1st application at first true leaf stage. 2nd application at 6 leaf stage. Then every 2-3 weeks from 1st flowering.	3 l/ha fertigation, boom or aerial spray.

Hereinafter, embodiments of the invention are illustrated in more detail with reference to the following examples. However, the present disclosure is not limited thereto. Furthermore, what is not described in this disclosure may be sufficiently understood 5 by those who have knowledge in this field and will not be illustrated herein.

Examples

2018101956 05 Dec 2018

The algae of Chlorella pyrenoidosa and Anabaena azotica in the following examples are available from the China General Microbiological Culture Collection Center (CGMCC) under Accession Nos 16726 and 16725, respectively, having been deposited there on 9 November 2018.

Example 1

An active microalgae nutritional remediation solution useful as a biofertiliser was prepared using a method comprising the following steps S1 and S2:

involved culturing and expanding the green algae Chlorella pyrenoidosa and the nitrogen-fixing cyanobacteria Anabaena azotica to obtain two algae liquids. The expansion and cultivation process of the two algae liquids included: (a) selecting and isolating single microalgae cells under the microscope (b) culturing the single microalgae cells in 15ml_ medium solution for 10 days, obtaining 15ml_ algae liquid (c) transferring to 85ml_ medium solution (d) continuing to culture for 10 days, obtaining

100mL of algae liquid (e) transferring to 0.9L medium solution and continuing to culture for 10 days, obtaining 1L algae liquid (f) transferring to 4L medium solution and continuing to culture for 10 days to obtain 5L (g) transferring the algae liquid to a 13 L medium solution and culturing for 10 days to obtain 18 L of algae liquid and (h) transferring to a 782 L medium solution for a further 10 days to obtain 800 L of algae solution. At the end of each of the above stages of expansion, the number of microalgae active cells was > 10⁶ cells/mL, and the number of microalgae living cells in the finally obtained algae solution was > 10⁶ cells /mL.

The medium solution used includes: 2 mg/L disodium edetate, 5 mg/L citric acid, 0.05 mg/L FeSO₄.7H₂O, 0.15 g/L NaHCOs, 0.03 g/L CaCI₂, 0.05 g/L MgSO₄.7H₂O, 1 g/L

NaNOs, 0.05g/L K₂HPO₄, 0.2 g/L KH₂PO4, 1.5 mg/L MnCI₂-4H₂O, 0.02 mg/L ZnSO4.7H₂O, 0.05 mg/L CuSO4.5H₂O, 0.02 mg/L NaMo4.2H₂O, 2 mg/L H3BO3.

The light intensity used was 2300 lux and the culture temperature was 25°C. Purified air was continuously introduced into the algae liquid. The pressure of the purified gas was 0.14Mpa. FRs gas was intermittently introduced into the algae liquid, at a pressure of 0.14 MPa. The ventilation interval was 3 h, and the duration of each ventilation was 20 minutes.

involved mixing the two algae liquids to obtain the active microalgae nutrient remediation solution. The ratio of Chlorella to Anabaena is 1: 0.2 according to the

2018101956 05 Dec 2018 number of microalgae cells. Sterile water was added as required. The number of cells in the biofertiliser was 10⁶1 mL.

Example 2

A biofertiliser was prepared as in Example 1 save that the step S1 was modified. In this process the expansion and culture process of the two algae liquids included: (a) selecting and isolating single microalgae cells under the microscope (b) culturing single microalgae cells cultured in 15mL medium solution for 12 days, obtaining 15mL algae liquid (c) transferring to 85mL medium solution (d) continue to culture for 10 10 days, obtain 100mL of algae liquid (e) transferring to 0.9L medium solution and continuing to culture for 12 days, obtaining 1L algae liquid (f) transferring to 4L medium solution and continuing to culture for 12 days to obtain 5L (g) transferring the algae solution to a 13 L medium solution for a further 12 days to obtain 18 L of algae solution (h) inoculating into a 782 L medium solution for further 12 days to obtain 800 L of algae 15 solution. At the end of each of the above stages of expansion, the number of microalgae active cells was > 10⁶ cells/mL, and the number of microalgae living cells in the finally obtained algae solution was > 10⁶ cells/mL; In the expansion culture: the light intensity was 2500 lux; the culture temperature was 26 °C; purified air was continuously introduced into the algae liquid at a pressure of the 0.3 MPa; FRs gas 20 was intermittently injected into the algae liquid at a pressure of 0.3 MPa, with the ventilation interval being 4 hours and the duration of each ventilation being 35 minutes.

Example 3

A biofertiliser was prepared as in Example 1 save that the step S1was modified. The expansion and cultivation process of the two algae liquids included: (a) selecting and isolating single microalgae cells under the microscope (b) culturing single microalgae cells in 15mL medium solution for 15 days, obtaining 15mL algae liquid (c) transferring to 85mL medium solution (d) continuing to culture for 15 days, obtaining 100mL of algae liquid (e) transferring to 0.9L medium solution and continuing to culture for 15 30 days, obtaining 1L algae liquid (f) transferring to 4L medium solution and continuing to culture for 15 days to obtain 5L (g) transferring to 13 L medium solution for 15 days to obtain 18 L of algae solution (h) inoculating into 782 L medium solution for 15 days to obtain 800 L of algae solution. At the end of each of the above stages of expansion,

2018101956 05 Dec 2018 the number of microalgae active cells was >10⁶ cells/mL, and the number of microalgae active cells in the finally obtained algae solution was> 10⁶ cells/mL.

In the expansion culture: the light intensity was 2700 lux; the culture temperature was

27°C; purified air was continuously introduced into the algae liquid at a pressure of 0.6

MPa; Frs was intermittently injected into the algae at a pressure of 0.14-0.6 MPa with a ventilation interval of 5 hours and the duration of each ventilation being 50 minutes.

Example 4

A biofertiliser was prepared as in Example 2 save that the medium solution used included: disodium edetate 4 mg/L, citric acid 9 mg/L, FeSO4.7H2O 0.1 mg/L, NaHCO3 0.25 g/L, CaCI₂ 0.07g/L, MgSO₄.7H₂O 0.12g/L, NaNOs 2 g/L, K2HPO4 0.1 g/L, KH2PO4 0.6g/L, MnCl2.4H2O 2mg/L, ZnSO4.7H2O 0.03mg/L, CUSO4.5H2O 0.07mg/L, NaMo4.2H₂O 0.03mg/L, H3BO3 3mg/L.

Example 5

A biofertiliser was prepared as in Example 2 save that the medium solution used included: disodium edetate 6 mg/L, citric acid 13 mg/L, FeSO4.7H2O 0.15 mg/L, NaHCOs 0.35g/L, CaCI₂0.1g/L, MgSCUJhW 0.2g/L, NaNOs 3.5g/L, K2HPO4 0.15g/L, KH2PO4 1g/L, MnCI₂.4H₂O 2.5mg/L, ZnSO4.7H₂O 0.05mg/L, CuSO4.5H₂O 1mg/L,

NaMo4.2H2O 0.05mg/L, H3BO3 4mg/L.

Example 6

A biofertiliser was prepared as in Example 2 save that the medium solution used was sterile water.

Example 7

A biofertiliser was prepared as in Example 4 save that, in step S2, the ratio of Chlorella: Anabaena is 1: 0.5.

Example 8

A biofertiliser was prepared as in Example 4 save that, in step S2, the ratio of Chlorella: Anabaena is 1: 0.8.

Example 9

2018101956 05 Dec 2018

A biofertiliser was prepared as in Example 4 save that, in step S2, the ratio of Chlorella:

Anabaena is 1: 1.2.

Example 10

A biofertiliser was prepared as in Example 4 save that, in step S2, the ratio of Chlorella: Anabaena is 1: 2.

Example 11

A biofertiliser was prepared as in Example 4 save that, in step S2, the ratio of Chlorella: 10 Anabaena is 1: 5.

Comparative example 1

A conventional nitrogen fertiliser, urea, was used as a comparison and applied as described in the following examples.

Example 12 Survival rate and compatibility test of microalgae

Samples 1-11 of biofertiliser compositions are prepared as in Examples 1 -11, so reference “Sample 1” hereinafter refers to a sample of the product of Example 1, 20 “Sample 2” refers to a sample of the product of Example 2, and so on. The samples were stored at room temperature for 10 days, 1 month, 2 months, 3 months, and 6 months, and microscopically examined at the end ofthe relevant period. The survival rate of microalgae was calculated by the ratio ofthe number of microalgae living cells initially to the number of microalgae living cells at the time of microscopic examination. 25 The test results are shown in Table 1.

Table 1 : Results of survival rate of microalgae living cells in microalgae nutrient

Remediation solution

2018101956 05 Dec 2018

Items	1 O Days	1 Month	2 Month	3 Month	6 Month
Sample 1	Chlorella	100%	98%	97%	95%	93%
Anabaena	1OO%	98%	96%	94%	93%
Sample 2	Chlorella	100%	98%	97%	96%	95%
Anabaena	1OO%	98%	96%	95%	94%
Sample 3	Chlorella	100%	98%	97%	96%	95%
Anabaena	1 00%	98%	97%	97%	95%
Sample 4	Chlorella	100%	99%	98%	97%	96%
Anabaena	100%	98%	97%	96%	95%
Sample 5	Chlorella	1OO%	98%	97%	96%	95%
Anabaena	100%	98%	97%	97%	95%
Sample 6	Chlorella	98%	93%	90%	86%	82%
Anabaena	98%	92%	89%	85%	80%
Sample 7	Chlorella	100%	99%	98%	97%	96%
Anabaena	100%	98%	97%	96%	95%
Sample 8	Chlorella	100%	99%	98%	98%	97%
Anabaena	100%	99%	98%	97%	97%
Sample 9	Chlorella	1OO%	99%	98%	97%	97%
Anabaena	100%	99%	98%	97%	97%
SamplelO	Chlorella	1OO%	99%	98%	97%	96%
Anabaena	100%	98%	98%	97%	96%
Samplel 1	Chlorella	100%	98%	98%	97%	96%
Anabaena	100%	98%	97%	96%	95%

It can be seen from Table 1 that a survival rate for Chlorella and Anabaena above 93% was achieved at 6 months for samples 1-5 and samples 7-11, which indicates that the growth rate of Chlorella and Anabaena is similar to the death rate. Accordingly, the compositions can maintain growth activity for a long time. It is apparent that the two microalgae have good compatibility, hence the compositions have a long shelf life. However, the survival rate of the microalgae in sample 6 was less; indicating use of a medium solution is preferable compared with the use of sterile water. When the ratio 10 of Chlorella to Anabaena is 1:0.8-1.2, the survival rate is higher, indicating the highest compatibility.

Example 13 Soil nitrogen test

12 identical areas of the same cotton planting area were selected, and samples 1-11 and the urea of Comparative sample 1 were each used to fertilize one of the areas in a 3-year comparative test. Each of samples 1-11 was applied at a rate of 400 mL/ 666 M² (6.01L/hectare), and urea was applied at a conventional application rate. Biofertiliser was applied only once per crop. During the period, the cotton root zone (020 20) cm soil was taken and the soil alkaline nitrogen was determined to reflect the soil nitrogen supply. The test results are shown in Table 2.

2018101956 05 Dec 2018

Table 2 : Soil alkaline nitrogen test results

Item	alkali-hydrolyzable nitrogen (mg/kg)
Before	After 1 Year	After 2 Years	After 3 years
Content	Content	Relative initial increase	Content	Relative first year increase	Content	Relative second year increase
Contrast Sample 1	49.17	50.19	1.02	51.28	1.09	52.39	1.11
Samplel	54.29	55.91	1.62	57.64	1.73	59.45	1.81
Sample 2	53.42	55.05	1.63	56.82	1.77	58.65	1.83
Sample 3	53.24	54.87	1.63	56.64	1.77	58.48	1.84
Sample 4	55.38	57.02	1.64	58.80	1.78	60.65	1.85
Sample 5	55.28	56.91	1.63	58.66	1.75	60.47	1.81
Sample 6	52.34	53.80	1.46	55.36	1.56	56.98	1.62
Contrast Sample 7	50.91	52.57	1.66	54.34	1.77	56.17	1.83
Sample 8	51.68	53.41	1.73	55.29	1.88	57.31	2.02
Sample 9	52.49	54.20	1.71	56.07	1.87	58.08	2.01
Sample 10	54.87	56.56	1.69	58.39	1.83	60.34	1.95
Sample 11	55.06	56.74	1.68	58.56	1.82	60.50	1.94

It can be seen from Table 2 that a single application of a biofertiliser in accordance with the present invention can significantly increase the content of alkali nitrogen in 5 the soil. Moreover, the rate of alkali nitrogen increase year by year is better than when the conventional nitrogen fertiliser of Comparative Example 1 is used in the absence of biofertiliser. It was also observed that soil compaction was significantly reduced.

Example 14 Reduction in nitrate and heavy metals in fruits or crops

For each fruit or crop, 12 identical areas of the same planting area were selected. Samples 1-11 and the urea of Comparative sample 1 were used as fertiliser as described in Example 13. The nitrate content is determined after the fruit or crop matures, and the living microalgae nutrient remediation liquid phase prepared by using the samples 1-11 is calculated. The results are shown in Table 3.

Table 3: Results of determination of percentage reduction in nitrate content of

2018101956 05 Dec 2018 fruits or crops

Item	Tomato	Cucumber	Cauliflower	Radish	Coie
Sample 1	36.3-38.4	45.2-48.1	18.2-20.3	17.2-21.1	44.5-47.8
Sample 2	36.6-38.7	45.7-48.4	18.4-20.6	17.6-21.2	44.9-48.3
Sample 3	36.8-38.8	45.9-48.5	18.6-20.8	17.9-21.3	45.1-48.4
Sample 4	36.1-39.5	46.2-48.8	18.8-21.0	18.1-21.5	45.2-48.5
Sample 5	36.5-38.7	45.5-48.3	18.3-20.7	17.7-21.2	45.0-48.1
Sample 6	32.3-35.2	36.8-42.7	17.2-19.3	16.1-19.7	43.1-46.2
Sample 7	37.3-39.2	46.1-48.9	18.8-21.1	18.1-21.5	45.2-48.6
Sample 8	38.4-40.6	46.8-49.2	19.6-21.5	18.7-21.9	45.9-49.1
Sample 9	38.2-40.5	46.6-49.1	19.5-21.5	18.6-21.9	45.7-48.9
Sample 10	37.9-39.7	46.4-49.1	19.2-21.2	18.4-21.7	45.5-48.7
Sample 11	37.7-39.5	46.3-49.0	19.1-21.2	18.3-21.6	45.4-48.8

It can be seen from Table 3 that the fruits and crops corresponding to sample 1-5 and sample 7-11 all showed a significant decrease in the nitrate content. The reduction in nitrate content in samples 8 and 9 was the most significant. Sample 6 shows a lesser 5 reduction in the percentage of nitrate content reduction, indicating that the use of sterile water as the medium solution is not preferred. The ratio of the number of individuals of Chlorella pyrenoidosa and Anabaena azotica has a certain effect on the amount of nitrate reduction in fruits and crops. When the above ratio is 0.8-1.2, the reduction of nitrate content in crops is greater, which indicates that this ratio of 10 Chlorella pyrenoidosa to Anabaena azotica is preferred.

Example 15 Increase in the sugar content of fruits or crops

For each fruit or crops, 12 identical areas of the same planting area were selected. Samples 1-11 and the comparative sample 1 were used to fertilise the areas as 15 described in Example 13. The sugar content was determined after the fruit or crop matured. The sugar content increase values of various fruits or crops were calculated, and the result is as follows in Table 4.

2018101956 05 Dec 2018

Table 4: Fruit or crop sugar increase measurement results

Item	Apple	Peach	Kiwi fruit	Litchi	Strawberry	Beet
Sample 1	1.0-2.1	1.0-2.2	1.7-2.2	2.3-3.3	1.3-3.1	0.6-2.2
Sample 2	1.1-2.1	1.1-2.3	1.8-2.3	2.3-3.4	1.3-3.2	0.6-2.3
Sample 3	1.1-2.2	1.2-2.4	1.8-2.4	2.4-3.4	1.4-3.3	0.7-2.3
Sample 4	1.2-2.3	1.3-2.4	1.9-2.5	2.5-3.5	1.5-3.5	0.9-2.4
Sample 5	1.1-2.2	1.2-2.4	1.8-2.4	2.4-3.4	1.4-3.4	0.8-2.3
Sample 6	0.7-1.9	0.6-1.8	1.2-1.7	1.8-2.8	0.9-2.5	0.3-1.8
Sample 7	1.2-2.4	1.3-2.4	1.9-2.6	2.6-3.6	1.5-3.6	1.0-2.4
Sample 8	1.4-2.6	1.5-2.7	2.2-2.8	2.8-3.8	1.7-3.8	1.2-2.6
Sample 9	1.3-2.6	1.5-2.6	2.1-2.8	2.8-3.7	1.7-3.7	1.2-2.5
Sample 10	1.3-2.5	1.4-2.6	2.0-2.7	2.7-3.7	1.6-3.7	1.1-2.5
Sample 11	1.2-2.5	1.4-2.5	2.0-2.6	2.7-3.6	1.6-3.6	1.1-2.5
Item	Suqarcane	Manqo	watermelon	Hami melon	Dates	Grape
Sample 1	0.9-1.2	1.3-2.2	1.1-2.1	1.1-2.0	1.9-2.2	1.0-1.4
Sample 2	1.0-1.2	1.4-2.3	1.2-2.2	1.2-2.1	2.0-2.3	1.1-1.5
Sample 3	1.0-1.3	1.5-2.4	1.3-2.3	1.3-2.1	2.1-2.4	1.2-1.6
Sample 4	1.1-1.5	1.6-2.7	1.4-2.4	1.4-2.2	2.1-2.5	1.2-1.7
Sample 5	1.1-1.4	1.5-2.6	1.4-2.3	1.3-2.1	2.1-2.4	1.2-1.6
Sample 6	0.6-0.9	0.8-1.8	0.7-1.8	0.8-1.6	1.3-1.7	0.7-1.0
Sample 7	1.2-1.6	1.6-2.8	1.5-2.5	1.5-2.3	2.2-2.6	1.3-1.7
Sample 8	1.4-1.8	1.9-2.9	1.7-2.8	1.7-2.5	2.4-2.8	1.5-1.9
Sample 9	1.4-1.7	1.8-2.9	1.7-2.7	1.7-2.4	2.4-2.7	1.5-1.8
Sample 10	1.3-1.7	1.7-2.9	1.6-2.7	1.6-2.4	2.3-2.7	1.4-1.8
Sample 11	1.3-1.6	1.7-2.8	1.6-2.6	1.6-2.3	2.2-2.7	1.4-1.7

It can be seen from Table 4 that the fruits and crops of samples 1-5 and samples 7-11 all show a significant increase in the degree of sugar content. The increase in the sugar content is greatest in samples 8 and 9. While not wishing to be bound by theory, this is believed to be because the microalgae in the nutrient remediation solution can simultaneously survive and multiply on the surface of the plant roots and inside the plant, and form a symbiotic interaction with the roots of the plant, fixing nitrogen from the air, decomposing and dissolving the insoluble phosphorus and potassium, dissolving the poorly soluble calcium and magnesium and activating sulfur.

Accordingly, plants treated with the biofertiliser are provided with rich and balanced natural nutrients, promoting plant root development and promoting plant nutrient absorption. The amount of increase in the sugar content corresponding to Example 6 is less, indicating that the use of sterile water as a culture medium is not preferred. The medium solution used in other examples provides nutrients for the microalgae and promotes the healthy growth and reproduction of the microalgae. The ratio of the number of individuals of Chlorella pyrenoidosa and Anabaena azotica has a certain influence on the increase of sugar content in fruits and crops. When the above ratio is 0.8-1.2, the increase in sugar content in fruits and crops is greater, which indicates

2018101956 05 Dec 2018 that the microalgae are most compatible and mutually beneficial in this range and can provide better nutrition for plants.

In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. The term “comprises” and its 5 variations, such as “comprising” and “comprised of” is used throughout in an inclusive sense and not to the exclusion of any additional features.

It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect.

The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims appropriately interpreted by those skilled in the art.

2018101956 05 Dec 2018

REFERENCES:

References cited herein are incorporated herein by this reference and are as follows:

Algal Green Chemistry Recent Progress in Biotechnology, Edition: 1st, Chapter: Role of Algae as Biofertilizer, A. Chatterjee, S. Singh, C. Agrawal, S. Yadav,R. Rai, L.C. Ra, Publisher: Elsevier, Editors: Rajesh Prasad Rastogi, pp. 189-200

D. Sahu, I. Priyadarshani and B. Rath ClBTech Journal of Microbiology ISSN: 23193867 (Online) http://www.cibtech.org/cim.htm 2012 Vol. 1 (2-3) Jul.-Sept. & Oct.-Dec., 10 pp.20-26

Mortvedt, J.J. Fertilizer Research (1995) 43: 55.

https://doi.Org/10.1007/BF00747683

Seham M.Hamed, Amal A. Abd El-Rhman, Neveen Abdel-Raouf, Ibraheem B.M.Ibraheem Beni-Suef University Journal of Basic and Applied Sciences,Volume 7, 15 Issue 1, March 2018, Pages 104-110

Gors M, Schumann R, Hepperle D, Karsten U (2010) Quality analysis of commercial Chlorella products used as dietary supplement in human nutrition. J Appl Phycol 22: 265-276 Review of the taxonomic revision of Chlorella and conseguences for its food uses in Europe.

Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. J Biosci Bioeng 101: 87-96.

EDITORIAL NOTE

There is one page in the claims only .

Claims (5)

1. The claims defining the invention are as follows:

   1. A biofertiliser composition comprising a living microalgae component consisting of a live microalga of Chlorella spp. and a live microalga of Anabaena spp.
2. 2. A biofertiliser composition according to claim 1, wherein the ratio of Chlorella spp. to Anabaena spp. is from 1.0:0.2 to 1.0:5.0 and/or wherein the culture medium comprises nutrients for microalgae comprising a source of any one or more of iron, bicarbonate, calcium, magnesium, nitrate, phosphorous, manganese, zinc, copper, molybdenum and borate.
3. 3. A biofertiliser composition according to either preceding claim wherein the live microalga of Chlorella spp. is Chlorella pyrenoidosa and/or the live microalga of Anabaena spp. is Anabaena azotica.
4. 4. A method of fertilising a plant comprising applying a biofertiliser composition according to any preceding claim to the plant and/or to soil surrounding the plant.
5. 5. A method of preparing a biofertiliser, comprising the steps of:

   a) providing a live microalga of Chlorella spp.;

   b) providing a live microalga of Anabaena spp.;

   c) culturing the microalga of Chlorella spp.;

   d) culturing the microalga of Anabaena spp.;

   e) combining the Chlorella spp. culture and the Anabaena spp. culture.