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Nitrogen for Bacillus thuringiensis, Bradyrhizobium japonicum and Bacillus amyloliquefaciens

These microorganisms are three of the most widely used microbial inoculants in the world.

Bradyrhizobium japonicum fixes nitrogen and has a highly specific symbiosis with soybeans.

Bacillus thuringiensis is used not only in agriculture but also to control mosquitoes and prevent malaria.

Bacillus amyloliquefaciens can function as a biopesticide and a biofertilizer. It produces antifungal compounds, and some strains produce chitinases, which degrade fungal cell walls. In addition, B. amyloliquefaciens helps to stimulate plant growth. One of its features that has resulted in such large-scale use is its resistance to many fungicides and other pesticides.

The market for these microorganisms is rapidly growing.

Eighty percent of the soybeans in Brazil are treated with B. japonicum. This is highly significant for the sale of this microorganism, since Brazil produces nearly 136 million tons of soybeans/year (CONAB survey). The size of its production area for soybeans is increasing.

The production of seeds that are pre-inoculated with B. japonicum has increased by 30% in 10 years and provides a ready market to enhance the sales of this biostimulant. The global revenue from B. japonicum sales is 209.5 M€.

The global market for B. thuringiensis was $226.4 million USD in 2020 and is expected to reach $327.6 million USD by 2026 (360 Research Reports). The compounded annual growth rate (CAGR) is expected to be 6.3% between 2019 and 2025 (QYR Research).

The existence of highly optimized fermentations is part of the reason why B. thuringiensis  can be produced in such large quantities for its insecticidal endotoxins.

B. amyloliquefaciens is one of the most prominently used PGPR and a significant part of the biopesticide market, which has a CAGR of between 10 and 20%.


Goals of fermentation processes

The gold standard of the fermentation of biostimulants and biocontrols is to have a large amount of living and active biomass that remains viable and stable in the field.

However, CFUs are not always the best metric, particularly for microbes that produce spores or metabolites like endotoxins. The production of the B. thuringiensis endotoxin is critical for the successful use of this microorganism. These toxins are formed after the exponential growth phase of vegetative cell cycle as a result of nutrient depletion and cell accumulation. The cultures must have a high rate of sporulation to produce these toxins.

Many biopesticides like B. amyloliquefaciens are highly effective at controlling plant diseases because of the array of metabolites they produce, including antibiotics and siderophores. Siderophores are compounds that sequester iron in the soil, so that B. amyloliquefaciens can use it, but it is unavailable to other organisms like pathogenic bacteria and fungi. In cases such as these, not only high biomass is important. The cultures must have suitable vegetative cells to efficiently synthesize the antifungal compounds.

Nitrogen compounds are essential for industrial fermentation processes.

Bacteria require a substantial amount of nitrogen to thrive. Nitrogen is essential for the production of amino acids, proteins, RNA and DNA, and a number of growth factors. It also provides the cells with energy during the fermentation process.

The type of nitrogen used in fermentations can be pivotal to the success of the microbe when it is inoculated on plants or in soil. For example, high concentrations of nitrate or ammonium inhibit nitrogen fixation by B. japonicum.

There are numerous sources of nitrogen, some better suited for particular microbes than others. Nitrogenous compounds are either inorganic or organic. Inorganic nitrogen includes ammonium, nitrates, and urea.

Organic sources can be derived from plants, including cottonseed, peanut, and soybean flour, wheat gluten, and corn pulp and steep. It can also be derived from animals, including fish meal, peptone, and meat extract.

Yeast provide additional sources of organic nitrogen that can be provided in the form of yeast extract and yeast peptone. Yeast-based nutrients provide a quality source of nitrogen that is composed of a number of compounds, including proteins, amino acids, and vitamins and minerals like B-vitamins. Its rich array of nitrogenous compounds enhances the likelihood that microbes will thrive during fermentation and subsequent use in the field.


Use of optimal nitrogen sources can lower the cost of fermentation

It can be expensive to conduct fermentations on a large-scale, and this is a particular problem with the production of B. thuringiensis in developing countries. Therefore, a substantial amount of research has been conducted on optimizing fermentations with price in mind.

Choosing the best nitrogen source is a key step in optimizing the fermentation to improve the success of biofertilization and biocontrol. In an attempt to lower costs, some scientists have turned to inorganic sources of nitrogen, since they are much less expensive. However, these forms of nitrogen are often unable to meet the nutritional needs of highly demanding microbes.

Many researchers have found that using organic sources of nitrogen improved the success of their fermentation of microbial inoculants like B. thuringiensis and B. amyloliquefaciens.

In particular, research by Içgen et al. demonstrated the benefits of fermenting B. thuringiensis with organic sources of nitrogen. This microbe only produces toxins when the cultures are sporulating. This study examined the effects of organic forms of nitrogen, such as peptone, glutamate, soybean flour, and casamino acids on growth, sporulation, and toxin production. The inorganic forms of nitrogen included ammonium sulphate, ammonium chloride, potassium nitrate, ammonium nitrate, urea, and NH4H2PO4.

Except for  NH4H2PO4, cells grown with inorganic nitrogen had very low rates of sporulation. In contrast, those grown with peptone produced three-fold higher numbers of spores than those grown on NH4H2PO4. These cultures also produced optimal amounts of toxin and had a high cell density. The combination of glutamate with a small amount of yeast extract was the second most effective with sporulation rates that increased by 60%.

While it may seem like a good idea to save money by providing mineral nitrogen instead of organic nitrogen, this can reduce the efficiency of the biopesticides and biostimulants and add to costs over the long-term.

While some microbiologists favor plant-based sources of organic nitrogen for their fermentations of biostimulants and biocontrols, the use of high quality N such as yeast-based nutrients can be a sound investment.

Yeast-based nitrogen sources can improve the fermentation of biostimulants and biocontrols.

While some microbiologists favor plant-based sources of organic nitrogen for their fermentations of biostimulants and biocontrols, the use of high quality N such as yeast-based nutrients can be a sound investment.

Procelys has used its decades of expertise in developing nitrogen sources for fermentation to develop new yeast-based nutrient sources known as LyCel®. These nutrients enable the production of high-quality and highly viable metabolically active biostimulants and biocontrols that are extremely effective in the field.

When you purchase LyCel® from Procelys, you benefit from our track record of helping customers optimize the fermentations of an array of microbial strains. This can range from setting up pilot experiments to scaling up production to a large scale.