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Key Challenges in Bio-Agriculture Fermentation

Optimal sporulated biomass production, high productivity, robustness & environmentally friendly raw material

Global crop losses from plant pests and diseases can be as high as 31-42% a year. As stakeholders and consumers become increasingly aware of the health and environmental risks of synthetic pesticides and the soil degradation, they are increasingly turning to the use of Bio Agricultural products. This is in order to control pests and pathogens and stimulate the growth of plants.

Not only are these microbes safer and better for the environment; they can be wise economic choices. The benefit/cost ratio for chemical control is 2:1, while it is 20:1 for biological control.

Market Share is dramatically increasing

As a potential alternative to hazardous chemical pesticides, agriculturally important microorganisms have gained popularity throughout the globe.

The global biocontrol market reached $4.4 billion USD in 2019. It is projected to increase to $10.63 billion by 2027.

Biofertilizers are being increasingly used worldwide as the deterioration of soil structure from soil intensively treated with chemical fertilizers continues to inhibit yields. The global biofertilizer market was $1.49 billion USD in 2019 and is expected to reach $3.28 billion by 2027.

Biostimulants were a $1.49 billion industry in 2016 and are expected to grow at a compound annual growth rate of 10.2% from 2017 to 2025.

Farmers and Agricultural companies have a choice of types of microbial inoculants

Organisms such as Bacillus that produce spores are ideal subjects to use in the field because they can frequently inhibit both bacteria and fungi, remain viable for long periods and can tolerate harsh conditions like heat, desiccation, chemical disinfectants, and freezing.

Biostimulants

A wide variety of microorganisms and substances are used to enhance the growth of plants. This category includes fungi, bacteria, plant extracts, and several soil components, such as humic and fulvic acid.

Plant growth promoting rhizobacteria (PGPR) are a major category of biostimulants. They usually live on plant roots or in the lush area around plant roots called the rhizosphere. These bacteria enhance plant growth in several manners:

  • producing hormones
  • directly antagonizing pathogens
  • stimulating plant resistance to pathogens
  • helping symbiotic organisms get established

Prominent PGPR include Pseudomonas fluorescens and Azospirillum. These bacteria use nutrients rapidly and outcompete potential pathogens. In addition, they have multiple ways of inducing the growth of plants and stimulating the plants to ramp up their natural defenses against pathogens.

Biofertilizers

Both fungi and bacteria can function as biofertilizers.

The ability of symbiotic bacteria to fix nitrogen on legumes has led to their use as inoculants for centuries. These microorganisms produce nitrogenase, which converts atmospheric N into ammonium. This is a critical reaction for life on earth, and 60-80% of the total N fixed in nature comes from symbiotic N fixation.

Many symbiotic bacteria are species of Rhizobium. One particularly important biofertilizer is Bradyrhizobium japonicum, which specifically fixes N with soybeans. It has been estimated that soybeans require up to 80 g of assimilable N to produce a ton of grain.

Mycorrhizae are symbiotic fungi that grow on the roots of most plants. They provide phosphorous and water to plants and increase the ability of plants to obtain P and water by 10-fold. By colonizing the roots, they also protect the plants against pathogens.

Biocontrols

Most biocontrols are microbes  and can comprise fungi, bacteria, and nematodes that can control plant diseases and insect infestations. These types of organisms are also considered to be biopesticides.

The most commonly used biocontrol agent is Bacillus thuringiensis (Bt). It comprises over 90% of the commercially available biocontrols.

Not only is Bt certified organic, it has a broad use for controlling problem insects that are agricultural or household pests. Caterpillars, beetles, and flies can all be controlled by various strains of Bt. Since Bt is organic, it can be safely used within households to control various pests.

One highly desirable feature of Bt is that it is highly specific and nontoxic to humans and non-target organisms.

Bt is critical as a preventative treatment for malaria. The WHO estimates that there were 229 million cases of malaria in 2019. Pesticides used to control it include DDT (Dichlorodiphényltrichloroéthane), which is bad for the environment, and chlorpyrifos, which causes brain damage in children.

These bacteria are considered to be safe for humans, particularly compared with chlorpyrifos, which is banned in California.

Another benefit of using Bt to control mosquitoes compared with pesticides is that the risk of mosquitoes developing resistance to this microbe is very low.

Additional biocontrols used to control insects include:

  • entomopathogenic fungi Cordyceps and Beauveria bassiana
  • beneficial nematodes (often applied to the soil)

While these microbes have an excellent record of being nontoxic to humans and other mammals, there is always concern that they be thoroughly vetted to thoroughly ensure that they truly are safe to use on crops that will be consumed.

Legislative challenges for the use of biostimulants

No consensus has been found about the regulation of these beneficial microorganisms. There is concern about their safety, since they can be present on foods and the European Union has no specific regulations for biofertilizers.

Each country regulates them locally.

In Poland, “growth stimulators” fall under the Law on Fertilizers and Fertilization, and this can apply to biofertilizers.

Spain has no legislation for biofertilizers. However, local administrations have the right to regulate them.

Italy includes mycorrhizal fungi in a category called “Products with action on the soil” and “Products with specific action.” No GMOs are allowed to be used for making the product.

In the US, the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA) can regulate these microbes.

Manufacturers must categorize biostimulants to the EPA as either fertilizers or biopesticides depending on the claims made about them.
They are exempt from the traditional regulation of pesticides by FIFRA (The Federal Insecticide, Fungicide, and Rodenticide Act).
Bacillus amyloliquefaciens is generally regarded as safe (GRAS) by the FDA – a designation for entities that are safe to use on food.
Bacillus thuringiensis has undergone intensive studies to show that it is safe.

Challenges in the Use and Production of Microorganisms for Plant Protection

Many lab experiments have identified promising microbial candidates that do not live up to their potential in the field. A major reason for this is the difficulties of consistently producing them on a large scale and obtaining highly viable cells at the end of fermentation or later in the downstream process.

Fermentation conditions are critical for successfully producing large quantities of microbes.
Suboptimal fermentations do not produce viable products.
The first goal of successful fermentations is to produce high biomass. Secondly, the fermentation must produce high viable cell counts, since the microbes must be active in the field to interact with the plant. A third factor is the cells must survive until they can be inoculated into the field.
Spore-producing Gram-positive bacteria like B. amyloliquefaciensB. thuringiensis, and B. subtilis have their own set of challenges for fermentation.

High biomass is often not the only goal. The production of large amounts of spores is the most relevant objectives. Spores are more resistant and stable to the conditions found during fermentation, formulation, and storage. Improving sporulation rate is a way to increase the performance of the final product inoculated in fields.

Inducing the bacteria to produce spores can be a challenge as it needs to identify the appropriate chemical composition that induces sporulation.
Even quantifying the spores produced can be laborious when plate counts are used. Fluorimetry assays are convenient and time-saving.
Obtaining vegetative cells that produce antimicrobial compounds is another key goal. Many companies work with a particular strain that is known to be amenable to fermentation and then trademark it once they have designed an optimal product. Examples include:

  • Bacillus subtilis QST 713
  • Bacillus amyloliquefaciens D747

Bt proves to be yet another challenging microbe in its fermentations. The cells need to produce large amounts of a particular crystal insecticidal toxin. This microbe produces a variety of toxins – each with specificity for particular insects. The formation of different toxins can be differentially regulated by the nitrogen source.

An additional complication is that the toxic effects of the microbes toward insects differs within the same strain depending on the growth medium.

The use of viable carbon and nitrogen sources can greatly increase the success of the fermentative process.

Traditionally, selecting the appropriate C and N sources has been a process of trial and error. A study on improving the growth of Bradyrhizobium japonicum found that yeast extract had to be freshly prepared to effectively ferment this organism. The addition of fresh yeast extract almost doubled the mass productivity of the cells.

Multifactorial experiments that look at a variety of C and N sources and trace elements are difficult to conduct. Statistical analyses simplify the process.

The Plackett-Burman method  is a common analysis used to optimize fermentations. This method lets scientists examine a large number of factors and identify the ones that are important. For example, it enables the rapid screening of the variables that affect spore production, growth, and germination.  Mezghanni et al. used this technique on B. amyloliquefaciens to improve the production of antifungal compounds by 56.25%.
A particular challenge with Bt is defining the nutrition for sporulation, because the medium is no longer chemically defined after vegetative growth has been completed.

READ MORE ABOUT HOW BIOTECHNOLOGY CAN HELP TO FACE THE CHALLENGES OF SUSTAINABLE BIO-AGRICULTURE. 

 

 

One unexpected benefit of optimizing the media for fermentation is that sometimes compounds such as trace metals are not required. This was found to be the case when an experiment examined the effect of optimized media on the production of antifungal compounds by B. amyloliquefaciensTherefore, it can be cheaper to use the modified media in fermentation.

You will be best served by choosing a vendor with a successful track record of producing nutrients for fermentation. Procelys is the world’s largest producer of yeast-based nutrients. With our decades of experience in helping customers optimize their fermentation media, our technical experts can work with you to ensure a high yield of your bioagricultural product.

READ MORE ABOUT NITROGEN NEEDS FOR Bacillus thuringiensisBradyrhizobium japonicum AND Bacillus amyloliquefaciens.

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