# Future-proof agriculture: Calcium Alginate Beads

## Contents

1. Abstract

2. Introduction

3. How do you make the calcium alginate bead?

4. How do slow-release calcium alginate beads work?

5. Advantages of the slow discharge of biofertilizers

6. Behaviour of bead in soil

7. Bead production: analysis of the method

8. Suggested biofertilizer composition

10. Conclusion

11. References

# Abstract

In order to ensure global food security, we must optimise the use of fertilizers in soil to improve crop yield. Scientists have attempted to do so by replacing the chemicals in fertilizers with more efficient microorganisms to create biofertilizers. Although this process can significantly improve agricultural yield, fertilizer runoff continues to limit optimal crop yield.

To combat fertilizer runoff, this paper proposes a new biodegradable mechanism, calcium alginate beads, which release nutrients gradually, thus avoiding fertilizer runoff. Calcium alginate beads are made of sodium alginate and calcium chloride. The instant formation of a bead when sodium alginate is added to calcium chloride makes the process time, equipment and energy efficient. It is important to emphasize that the bead itself is merely the slow release mechanism, and that depending on what is added, the bead can be tailored to different types of soil[1].

The product outlined in the article is a novel idea. However, in order to analyse how this product would interact with the real environment, I have reviewed results from experiments conducted by other researchers. I do not claim ownership of any of the below experiments.

Nowadays, overpopulation and ineffective fertilizers are rapidly damaging food availability. This paper suggests that calcium alginate beads are an effective, economical and easy solution to ensure food security and indirectly combat malnutrition and climate change. From a commercial perspective, as these problems worsen, the demand for calcium alginate beads which release biofertilizers will only increase.

# Introduction

For the past decades, fertilizers have been used to enhance plant growth and yield. Some people consider this to be an easy, effective and economic method to solve a world-threatening agricultural problem: food insecurity.

However, the excessive use of conventional fertilizers is becoming increasingly problematic as it negatively affects the environment, human health and endangers food security:

• Fertilizer runoff into oceans or other water sources results in [2]:
• Eutrophication, leading to the release of neuro and hepatotoxins which increases both animal and human mortality if they make their way up the food chain;
• A decrease in aquatic wildlife biodiversity, which disrupts food chains for humans and other organisms;
• Several life threatening diseases that include methemoglobinemia (blue baby syndrome), cancer, adverse reproductive conditions (especially neural tube defects), diabetes and thyroid complications if the nitrogen contaminated water is used as drinking water in Less Economically Developed Countries (LEDCs);
• Enhanced nutrient content in soil increases competition between microorganisms, reducing their biodiversity, and can result in root burn in plants caused by overabundance of soluble salts in the soil [3].
• Accumulation of certain nutrients in soil can have other unforeseen damaging effects that we are yet to discover [3].

The use of conventional fertilizers is inducing the opposite effect that was intentionally planned. Therefore, researchers have increasingly shifted their focus to the production of biofertilizers containing microorganisms. Biofertilizers have positive effects on soil biota, heavy metal contamination control, mycorrhizae, rhizosphere nourishment, and disease resistance [9].

 This paper certainly encourages the use of biofertilizers. However, the main objective is to develop an innovative agricultural technique which optimises the use of biofertilizers by a slow release mechanism: calcium alginate beads.

# How do you make the calcium alginate bead?

Using results from previous experiments, I analyze sections of different methods used in these experiments and merged these points to produce my own, original summary of a novel method:

Dropping a solution of sodium alginate, starch, fulvic acid and biofertilizer into a separate calcium chloride solution using a pipette instantaneously forms a calcium alginate capsular membrane due to the cross-linking of calcium cations with alginate [6][7]. In order to remove any surface chemicals from the bead, it is washed twice in cold water. The subsequent drying of the beads provides protective benefits and increases shelf-life dramatically.

Depending on the customer’s requirements, the composition of the biofertilizer inside the bead can be altered. For example, I suggest that nitrogen-fixing endophytes, melanised endophytic fungi and phosphate-solubilizing bacteria are added to the mix for several reasons, as highlighted in Section 8 of this article. This combination would be beneficial for countries where soil biota, mycorrhizae and ammonia (NH3) concentrations in soil are poor and food security issues are high in the agenda. In addition, these specific microbes would increase carbon sequestration in soil organic matter, thus reducing greenhouse gas emissions. Ultimately, reducing the effects of global warming would have collateral improvements on crop yields.

# How do slow-release calcium alginate beads work?

Bacteria and fungi both inside and outside the bead use starch and alginate polymers within the matrix as a carbon source. This not only improves microbial development but also allows for the degradation of the alginate layers, releasing any entrapped microbes into the soil [5]. Experimental results showed that it took three to four months for the beads to be completely disintegrated. Therefore, the slow biodegradability of the beads allows for a slow microbial release, long-term supply to plants and more protection to the encapsulated bacteria.

Moreover, by changing the bead size, shape and hardness, diffusivity and biodegradability properties can be altered and adjusted to the customer’s requirements. This means the beads can be adapted to various environments, which makes them more attractive compared to non-encapsulated biofertilizers.

# Advantages of the slow discharge of biofertilizers

The natural components of the calcium alginate bead provide no toxicity to the environment as it is made of sodium alginate, calcium chloride, starch, fulvic acid and microbes. Producing calcium alginate beads is not expensive and most of its components are easily accessible. However, dried, chopped seaweed, necessary to make sodium alginate, might need to be imported by landlocked countries.

The use of these beads as a slow release mechanism reduces the amount of biofertilizer wasted in the soil, thus making their use more economical, environmentally-friendly and sustainable.

Drying the beads also increases shelf-life by reducing cellular metabolism while conferring 92.6% protective benefits from UV radiation and other soil stresses to the immobilized microbes [6]. Therefore, as the ozone layer is being destroyed and the sun increasingly damages soil biota, the demand for these beads will only increase in the future. Furthermore, immobilized microorganisms can deal with a greater variety of soil temperatures compared to free microorganisms, making them resistant to global warming effects.

Research has indicated that alginate encapsulated biofertilizers enhance microbial activity and improve plant yield better than free biofertilizers while used in the same salty environment [1]. This promises an exciting future for alginate beads in saline soils all over the world.

Last but not the least, the use of calcium alginate beads could potentially be the solution to the poor crop growth, quality and yield that populations in LEDCs are constantly challenged with.

# Behaviour of bead in soil

It is important to assess how soil properties affect bead efficiency.

1. Effect of soil salinity on calcium alginate bead release

We have already established that the slow release of encapsulated fertilizer is more efficient than adding fertilizers directly to the soil. However, it remains to be seen how the bead behaviour changes in different soil salinities.

Whilst a saline environment has negative effects on plant growth, this does not affect the viability of the bead. Experimental research where microbial fertilizer was encapsulated in calcium alginate beads, demonstrated that optimum release of the biofertilizer was obtained when immersed in salt for three weeks – content of microbes in the alginate beads changed from 1014 cfu/g to 4 cfu/g [5].

Therefore, any excess NaCl produced by the mixing of sodium alginate and CaCl2 in the encapsulation process would not affect bead efficiency.

The success of calcium alginate beads under these salty conditions demonstrates how adaptable they are to different environments all around the world [5].

• b. Effect of micro-environmental pH on calcium alginate bead release

As illustrated in Figure 1, acidic conditions weakened inter- and intra electrostatic repulsions between alginate polymers which led to an oblate structure. The larger surface area resulted in faster diffusion of particles through the capsule, defeating the aim of the slow-release mechanism, leading to more biofertilizer waste and lower plant yield. This effect was also induced by lower Ca2+ concentrations which will be addressed below.

However, the beads in Figure 1 at a pH of 4 still maintained their structure. This leads to the conclusion that the beads can handle a certain amount of acidity and that only extreme soil acidity will hinder the optimal usage of these alginate beads

In addition to this, Figure 1 demonstrates that high alkalinity does not impact the shape of the beads. Since alkaline soil is most common in countries with semiarid and arid climates, such as those in the African continent, these countries would benefit significantly from the use of calcium alginate beads [6].

Figure 1: Comparison of alginate particle profile versus combinations of Ca2+ concentration and pH value in a 0.8% alginate solution. Scale bar = 1 mm. [6]

• c. Effect of temperature on immobilized cells and on calcium alginate bead release

Calcium alginate polymers have excellent thermal properties. Figure 2 shows that the surface of calcium alginate beads remains relatively smooth until temperatures are raised to 500°C (indicating the beginning of its decomposition into CaCO3 and Ca(OH)2). At temperatures above 500°C, the matrix structure is distorted, leading to increased permeability. Therefore, rising temperatures due to climate change will not have a significant impact on the efficiency of bead release, meaning that the demand for these temperature-withstanding beads will only increase in the future.

An experiment conducted using dark septate root endophytes to control seedling survival, growth and disease intensity concluded that elevated temperatures did not significantly endophytic colonization and its influence on the seedlings [8].  [8]

• d. Effect of bead size on microbial release in soil

First of all, it should be borne in mind that the size of the bead is directly related to its efficiency. As illustrated by Figure 3, a diameter of 2 mm allows for optimal activity of the immobilized microbes due to its optimal pore size that allows for sufficient nutrient diffusion within the matrix [5]. In fact, cellular death increases as bead diameter increases or decreases away from the optimum. Moreover, smaller beads have the advantage that they can be located closer to plant cells, reducing competition with other soil biota and improving symbiotic relationships between the endophytes and plants.

Figure 3: Effect of bead size on relative activity of immobilized microbes [6].

Furthermore, an experiment focusing on the bead size in different soil humidity showed that the capsule’s diameter increased rapidly from 4 mm to 4.7 mm, regardless of the soil humidity. Figure 4 shows that bead size remains constant after 20 hours. This shows that small bead sizes only require very small amounts of water to sustain microbial release [6].

Figure 4: change in diameter of bead after hours of storage [6]

# Bead production: analysis of the method

The composition of alginate matrices can vary. By using different biofertilizer mixtures, particle sizes and concentrations of fulvic acid, starch, sodium alginate and calcium chloride, the polymer’s properties can be adjusted for specific applications. However, this paper recommends the following conditions for optimal bead efficiency.

1. The use of calcium chloride
• Internal vs external calcium sources

Calcium chloride is an easily accessible, non-toxic salt that improves soil structure and permeability while providing the potential to combine with other elements to reduce heavy metal contamination.

By using different calcium sources the gelation mechanism of polymer is changed, leading to different bead properties. When sodium alginate solution is dropped into an external calcium source, Ca2+ forms cross-links with the alginate polymers progressively from the surface to the interior of the bead. Internal gelation involves an extra step where sodium alginate is mixed with an interior CaCO3 source before being dropped into an acidic solution. This extra step requires more equipment and resources, thus being less environmentally-friendly and less economical.

Moreover, from Figure 5 it was concluded that external gelation methods produce more regular-sized and uniform beads with lower permeability. The viability of the alginate beads is also higher when prepared via external gelation (due to a 3x stronger matrix), making them more durable allowing for a longer shelf-life. Therefore, external gelation methods are favoured to provide optimum slow release characteristics of beads and greater efficiency in soil [7].

(a)                                                                                 (b)

Figure 5: High-resolution transmission electron microscope images of the outer surface of beads by internal gelation (a) and external gelation (b) [7].

• Different calcium salts

Since calcium chloride reaches a gel strength plateau the fastest (at 100 s) when compared to calcium lactate (at 500 s) and calcium gluconate (at 2000 s), calcium chloride is the best external calcium source as the beads can be produced faster resulting in a lower energy demand of the process. This increases the sustainability and decreases the cost of the process.

Furthermore, as a result of its lower affinity in alginate intermolecular cross-linking, using Ba2+ as a cross-linker instead of Ca2+ results in a softer, more fragile bead, which has higher permeability, and a shorter shelf-life. Therefore, using Ca2+ cross-linkers is favourable, cheaper and more environmentally friendly [14].

• Calcium chloride concentrations

As can be seen in Figure 6, calcium chloride at a 0.2 M concentration provided more stable beads with maximum percent immobilization yield. The percent immobilization of cells decreased when concentration of calcium chloride increased or decreased beyond optimum level. For example, higher concentrations lead to harder beads and lower permeability which decreases the diffusion and immobilization yield of microbes.

In light of the above, it is recommended to use external gelation methods with Ca2+ cross-linkers at a concentration of 0.2M. However, it is possible to control the diffusivity of the biofertilizer by altering the above conditions, making the alginate beads more popular in the market by meeting each customer’s requirements [14]. [14]

1. Effect of pH on bead production

The pH controls the gel formation process by regulating the dissociation of alginate and the cross-linking of the calcium cations. As shown by Figure 7, the volume of the beads during production was smaller at a pH lower than 4 due to lower repulsion between alginate chains. This led to lower permeability, stronger matrix structures and slower release diffusivity. At a pH of above 10 the bead volumes increased due to more repulsion between alginate chains, while at a pH of 12 the beads dissociated. It can therefore be concluded that alginate bead formation is most stable, feasible and efficient using a pH between 4 and 10 [8].

[8]

c. The effect of curing time on immobilization yield of microbes

Figure 8 shows that maximum immobilization of cells occurred when curing time of alginate beads was kept to 90 minutes. This is because the probability of cross-linkage formation between Ca2+ and the alginate matrix increases with time. However, fragile beads are formed when curing times deviate from the optimum, leading to reduced immobilization yield and more microbial leakage. The low energy and money demand of the curing process allows for relatively fast bulk production [14].

d. Extraction of sodium alginate from brown seaweed

Alginate is present in the cell walls of brown seaweed as the calcium, magnesium and sodium salts of alginic acid. Since calcium and magnesium salts do not dissolve in water, it is possible to extract the insoluble salts and convert them into sodium alginate.

For maximum yield, the brown seaweed should be collected during high tide times: mostly between 4-6 pm and March-April. This is because during low tides, coral reefs and sea urchins were exposed and their sharp tips would make collection procedures more dangerous for both humans and the unprotected wildlife [11].

Method for sodium alginate extraction:

1. Clean seaweed sample by washing with fresh water to remove salts, sand, shells and unwanted substances.
2. Treat with 0.3% aqueous HCl for 30 minutes to break down the outer cellulose of cells and expose the alginate.
3. Chop samples into small pieces using a shredding machine to increase surface area.
4. Stir with 12% aqueous Na2CO3 (pH 10-11) for 15 minutes. As a result, a condensed suspension is formed.
5. Discard the bottom reddish-coloured liquid by draining.
6. Add 5% HCl to precipitate the calcium salts.
7. Wash the treated sample with water to remove excess chemicals.
8. Treat the alginic acid with Na2CO3 to produce sodium alginate.

Analysis of the method:

Conventional methods to extract sodium alginate involve the removal of residual cellulose and starch by using filter aids in a filtration process [11]. However, vast amounts of water are needed before the viscous gel can be filtered. Moreover, using filter aids is very expensive and would raise the cost of calcium alginate beads. Therefore, the method proposed above is aimed to incorporate starch within the sodium alginate. The benefits of starch are explained in another section below.

After sodium alginate is obtained, other methods suggest heating the sodium alginate solution at 500˚C for 6 hours in order to turn the gel into powder, for easier transportation. However, heating at such high temperatures would have disadvantages that cannot be disregarded, such as the overuse of fossil fuels and its impact on the environment. On balance, transporting the liquid alginate is more beneficial than converting it into powder [11].

c. The effect of sodium alginate concentration on microbial immobilization

Since alginates are natural polysaccharides and non-toxic, they have been approved for human use by the US Food and Drug Administration and have no effect on the environment [10]. Their availability at low cost (US$2 per kg) means that they are more accessible to LEDCs compared to other expensive conventional fertilizing techniques. As shown by Figure 9, research indicates that a 4.0% of sodium alginate concentration in bead production allows for optimum microbial immobilization [5]. As the concentration of sodium alginate increases or decreases from maximum levels, immobilization efﬁciency decreases. High concentrations of alginate lead to more diffusion barriers, thus causing hindrance for microbes to move through the bead. Lower concentrations of alginate result in the softer and more fragile beads, where microbial leakage may occur. Figure 9: % Sodium alginate composition effect on immobilization yield of microbes [5]. Since 4% concentration involves a relatively low amount of sodium alginate, it is not very expensive for landlocked countries to import this from coastal seaweed sites. d. Benefits of adding starch in calcium alginate beads Starch, an inexpensive and ubiquitous biopolymer, can be incorporated in the alginate matrix. Firstly, starch provides protective benefits to the alginate bead and microbes. Microscopic observations showing bacteria at the surface of starch granules due cellular adhesion confirms this. Moreover, starch improves the compressive strength of the beads from 5.54 Mpa to 5.62 Mpa which increases shelf-life. The increase in storage period can also be explained by the fact that starch decreased bead water content by 65% and improved the bead drying process [6]. This increase in water retention results in lower water requirements during bead production and ultimately lower prices. Secondly, encapsulated microorganisms can use the starch as a nutrient while attached to the granules, which improves the development of the cells in the bead and soil as well as the biodegradability of the beads [15]. Moreover, adding 0.4% starch significantly optimized immobilization. For example, starch allowed a 74% increase in yeast encapsulation and a more uniform cell distribution [15]. e. Use of fulvic acid and effect on other bead components Humic substances are formed through the microbial degradation of plant material. Humic and fulvic acids are the most common compounds of soil humic substances. It is suggested to add fulvic acid instead of humic acid, because unlike humic acid, fulvic acid is non-toxic to the environment and improves overall health whilst also enhancing soil and plant biodiversity [19]. Fulvic acid-containing plants have positive effects on endurance, testosterone production, detoxification processes, muscle adaptation and nutrient absorption. For example, one single fulvic acid molecule is capable of transporting over 60 minerals and other nutrients directly into cells [20]. By including fulvic acid into the bioformulation, the beads will become more attractive to nations all over the world by mitigating malnutrition and low crop quality simultaneously. • Extraction of fulvic acid Pyrophosphate acid (pH 2–4) can be used as an extractant for soil fulvic acids at room temperature. After treatment with the extractant solution, low-ash fulvic acids are obtained at a high yield. Due to the abundance of organic matter in soil, fulvic acid is easy to find and thus relatively cheap ($7 per litre) [16]. Moreover, it prevents the need for fulvic acid transportation to different countries which would consecutively increase greenhouse emissions.

• Effect of fulvic acid on soil

Fulvic acid has the ability to photosensitize chemical reactions. Moreover, the acid can enhance or retard the uptake of compounds by plants since its small size allows it to carry vital nutrients through the cell membrane. Increasing the soil nitrogen, phosphorus, potassium, and organic matter content is another positive impact that fulvic acid has on plant rhizospheres [21].

Furthermore, an experiment focusing on the effect of fulvic acid on soil biota demonstrated that fulvic acid encourages the increase of Firmicute bacteria and Basidiomycota and Mortierella fungi, which have all been reported to improve plant growth. On the other hand, the experiment showed that fulvic acid led to a decrease in Ascomycota fungi and other related genera that are considered pathogenic to plants [21].

Another benefit of adding fulvic acid to my bioformulation is it’s chelation with cations and control of heavy metal contamination. The effects of heavy metal contamination (such as severe toxicity to plants and humans who consume those crops) are well known for a long time. The high electronegativity and affinity towards cations allows the reduction of heavy metals. For example, fulvic acid has been proven to remove 57.28% of Cu, 15.49% of Pb and 2.65% of Cd from contaminated soil [16]. Furthermore, soil pH did not hinder the efficacy of fulvic acid when recuing heavy metal content: 20°C allowed for optimum cation immobilization under acidic or neutral environments, while 30°C allowed for the same efficiency under alkaline environments. Since these temperatures fall within the range of climate variations in most countries, a formulation containing fulvic acid would be beneficial regardless of the climate.

In addition to this, fulvic acid can mitigate CO2 emissions from the agricultural industry by encouraging plant biomass production and improving biological stability of soil organic matter. The use of fulvic acid therefore allows the agricultural sector to be more sustainable and carbon neutral [16].

• Effect of fulvic acid on alginate bead properties

Fulvic acid enhances oxygenation and the access to nutrients of encapsulated microorganisms, thus facilitating cellular metabolism within the beads.

Moreover, an experiment which investigated the release properties of fulvic acid-enriched alginate beads containing Bacillus megaterium revealed that the bead released the cells in a steady and constant manner for 1 week while promoting lettuce growth [13].

Since fulvic acid is already found abundantly in soils and continuously in contact with diverse soil biota, the acid will also not have a significant effect on the immobilized microbes while encapsulated.

# Suggested biofertilizer composition

The effect of the biofertilizer formulation on the soil, plant and environment depends on its microbial constituents. The biofertilizer proposed below (containing nitrogen-fixing endophytes, melanised endophytic fungi and phosphate-solubilizing bacteria) has the following unique properties:

1. Nitrogen-fixing endophytes

Nitrogen-fixing endophytes live in a symbiotic relationship with plants where they provide NH3 to the host in return for nutrients. Since these microbes are sheltered inside plant tissue, they face less competition compared to other endophytes when reducing atmospheric N2 to NH3 [9]. Moreover, the partial pressure of oxygen inside the plant tissue also optimises efficient nitrogen-fixation. Due to their close proximity with plant cells, these endophytes can make the fixed nitrogen directly available to plants. They also induce systemic tolerance and resistance via the production of 1-aminocyclopropane-1-carboxylase deaminase [9]. In addition to this, the production of phytohormones and siderophores by these endophytes makes them suitable to maximise crop development.

1. Melanised endophytic fungi

These wispy, dark webs of fungi live inside plant roots and also exchange nutrients for carbon in a symbiotic relationship. Experimental results show that these endophytes are capable of taking the carbon that a plant obtains after photosynthesis and storing it in complex compounds in the soil that last for thousands of years. This is achieved by storing the carbon in melanin strands (carbon-rich organic compounds inside soil clumps) to protect it from degradative forces such as oxygen and other microbes. The breakdown of carbon and its release back into the atmosphere via the carbon cycle is thus prevented. This allows for fast sequestration of significant amounts of carbon in the soil. As a result of this, organic matter in the soil is improved (improving soil biota and plant growth) and excessive CO2 gases in air are reduced [9].

1. Phosphate solubilizing bacteria (PSB)

PSB have the ability to dissolve phosphate compounds, thus increasing phosphate concentration in soil. Higher phosphate levels improves crop yield (due to it’s indispensability in healthy plant growth), enhances nutrient availability for soil biota and reduces heavy metal concentration due to cation immobilization in compounds. Experimental results show that PSB increased the soil phosphate content from 12.28 mg/kg to 17.30 mg/kg, thereby enhancing the immobilization rate of Pb and Cd from 69.95% to 80.76% and from 28.38% to 30.81% respectively. Microcalorimetric analysis also demonstrated that PSB significantly improved soil mycorrhizal percent from 25.26% to 31.80%, probably due to the decrease in heavy metals and increased phosphate content. Numerous studies have shown that PSB are a cost-effective and environmentally-friendly strategy to reduce heavy metal concentration and simultaneously improve soil biota and crop quality. Enhanced crop quality further reduces human health problems caused by toxicities in harvests [15].

Dry formulations are extremely attractive from commercial and agricultural perspectives due to longer survival of bacteria. By reducing the water content of the beads, enzymatic activity and cell metabolism decreases, which increases the shelf-life of such dry formulations from 1 to 14 years [14].

Since this shelf-life is much longer compared to other non-encapsulated biofertilizers, competition on the market is reduced. Upon contact with high moisture levels in soil, the microorganisms are activated and their diffusion out of the slowly-degrading matrix begins. For example, an experiment demonstrated that ectomycorrhizal inoculum immobilized in calcium alginate beads remained 100% viable after 18 months in a NaCl solution at 8°C [11]. Moreover, drying the beads at 24°C for 24 hours, not only improves long-term viability but also provides improved UV radiation protection (7.2% protection) compared to non-encapsulated biofertilizers, which further increases microbial viability and yield after long periods of time [4].

# Conclusion

Biofertilizers encapsulated in calcium alginate beads is an innovative agricultural technique which optimises the use of microbial inoculants through a slow release mechanism.

Although the composition of the biofertilizer can be adjusted to different soil and customer requirements, the advantages of using nitrogen-fixing endophytes, melanised endophytic fungi and phosphate-solubilizing bacteria have been outlined throughout this paper. Their ability to capture carbon in the long term, has a positive effect on the environment and plant growth, addressing the interconnected issues of global warming and food security.

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