3 Key Steps in Gram-negative Bacteria Formulation

April 18,2024

Continued efforts to decrease pesticide and fertilizer use while maintaining and increasing agricultural productivity have created a need for novel approaches in crop production. Biocontrol agents (BCAs) are at the forefront of this new wave, with microbial products as the most promising allies. Specifically, the use of bacteria as ag input products has been gaining increasing attention from large ag input producers, with Bacillus strains being the most widely used microbial BCAs globally. This isn’t surprising, as these Gram-positive bacteria offer a wide range of benefits and are comparatively easy to produce, stabilize and formulate.

However, there are many bacteria that offer incredible benefits and solutions for agriculture that are not as easy to adapt into a ready-to-use product. Gram-negative bacteria belong to this group, with their distinctive features and high sensitivity increasing the level of processing complexity.

This article explores this complexity through three key steps in Gram-negative bacteria formulation; but before diving into the technicalities, let’s have a round of introductions – why are these bacteria so distinctive and why are they significant for agriculture?

What makes Gram-negative bacteria so special?

Gram-negative bacteria are a diverse group of microorganisms characterized by their specific cell wall structure. Unlike Gram-positive bacteria, they possess a thin peptidoglycan layer surrounded by an outer membrane containing lipopolysaccharides (LPS).

This fundamental structural difference influences various aspects of their behavior and interaction with the environment:

  • Outer membrane: The presence of an Outer membrane provides Gram-negative bacteria with additional protection against environmental stressors and antimicrobial agents.
  • Lipopolysaccharides (LPS): LPS molecules play a crucial role in the pathogenicity of Gram-negative bacteria and serve as potent immunostimulants in plant-microbe interactions.
  • Porins: These protein channels facilitate the transport of molecules across the Outer membrane, contributing to nutrient uptake and environmental adaptation.
  • Resistance mechanisms: Gram-negative bacteria often possess efflux pumps and enzymatic systems that confer resistance to antibiotics and other stress-inducing agents.

Benefits of Gram-negative bacteria in agriculture

Gram-negative bacteria offer numerous advantages in agricultural applications, including:

  • Nitrogen fixation: Certain species of Gram-negative bacteria, such as Azotobacter and Rhizobium, have the ability to fix atmospheric nitrogen, thereby promoting soil fertility and plant growth.
  • Disease suppression: Some Gram-negative bacteria, like Pseudomonas, exhibit antagonistic activity against plant pathogens, helping to suppress diseases and enhance crop health.
  • Nutrient mobilization: Some Gram-negative bacteria produce enzymes that solubilize phosphorus and other essential nutrients, making them more accessible to plants.
  • Bioremediation: Certain Gram-negative bacteria have the capacity to degrade pollutants and toxins in the soil, contributing to environmental health and sustainability.

Most significant genera of Gram-negative bacteria for agricultural applications include Azotobacter, Burkholderia, Pantoea, Pseudomonas, Rhizobium, Serratia.

Gram-negative bacteria formulation as a key for product efficacy

Before Gram-negative bacteria are ready to deliver their benefits to crops, they need to be processed and transformed into a form that can be easily packed, stored, distributed and applied. The gap between production and ready-to-use products is formulation, and it is a pivotal process in ensuring stable shelf life, stability and efficacy of microbial products.

The Gram-negative bacteria formulation composes of three key steps – stabilization, encapsulation and drying. The goal of each is to prolong shelf life and viability of bacteria, but the how’s are a bit different.

Step 1: Stabilization

Stabilization ensures that microbial products maintain their viability, functionality, and efficacy over time, despite various environmental stresses encountered during storage, handling, and application. Stabilization techniques are essential to preserve the integrity of microbial cells and protect them from factors that can compromise their viability.

The sensitive nature and specific cell wall structure of Gram-negative bacteria come with considerable limitations in stabilization:

  • Sensitivity to environmental stressors: Gram-negative bacteria are more susceptible to environmental stressors such as temperature fluctuations, pH changes, and desiccation, which can compromise their viability and efficacy.
  • Outer membrane permeability: The presence of an outer membrane restricts the entry of stabilizing agents and nutrients.
  • Compatibility with stabilizing agents: Certain stabilizing agents may interact adversely with Gram-negative bacteria, jeopardizing their viability.

Selection of appropriate stabilizing agents within these limitations is challenging, but critical for long-term viability of Gram-negative bacteria. Depending on the specifics of the bacterial strain and application route, addition of stabilizing agents might include cryoprotectants, surfactants, and protective polymers.

Step 2: Encapsulation

Encapsulation in the formulation of microbes is a technique used to enclose beneficial microorganisms within protective matrices or coatings. This process provides a physical barrier that shields the microbial cells from environmental stresses, prolongs their shelf-life and eases their application.

Several encapsulation techniques have been adapted for encapsulating Gram-negative bacteria, including:

  • Ionic gelation: Utilizing polymers like alginate and calcium ions to form crosslinked gel beads encapsulating bacteria.
  • Emulsification: Creating emulsions of bacterial suspensions within a polymer matrix, followed by solidification to form microcapsules.
  • Electrospraying: Electrospraying bacterial suspensions mixed with encapsulation materials to generate microspheres or nanoparticles.
  • Layer-by-layer assembly: Sequential deposition of polyelectrolyte layers onto bacterial cells to form multilayered coatings, providing protection and stability.

Note: Careful selection of the encapsulation technique and materials is crucial, as certain processes and substances can negatively affect the long-term viability of Gram-negative bacterial cells.

Step 3: Drying

Removing excess water from the encapsulated bacterial cells ensures stable shelf-life, viability and functionality of the end-product. However, there is a fine line in this process – removing enough to ensure optimal performance of the bacteria, while keeping enough for them to survive. Bacteria are living organisms after all, and they need water to survive.

Different drying techniques can be employed for Gram-negative bacteria, and the three most commonly used ones include freeze-drying, spray drying, and fluid bed drying. Which one will be employed depends on the specifics of the bacterial strain and added stabilization agents.

  • Freeze drying – involves freezing the bacterial suspension followed by sublimation of ice under reduced pressure, resulting in dried bacterial pellets or powders. The process lasts longer and requires specific machinery. Suitable for heat-sensitive Gram-negative bacteria.
  • Spray drying – involves atomizing the formulation into droplets, which are then dried using hot air, leading to the formation of dry powder particles. Rapid process suitable for large scale applications, but with increased risk of heat stress and reduced bacterial viability.
  • Fluid bed drying – suspends the bacterial particles in a stream of hot air or gas, promoting efficient moisture removal and yielding dried bacterial granules or powders. Rapid process suitable for large scale applications, but with increased risk of mechanical stress and reduced bacterial viability.

Note: The optimal drying technique will cause minimal damage to bacterial cells while effectively removing excess water.

Costs?

Successful Gram-negative bacteria formulation is not a small challenge to take on. Aside from the great level of expertise necessary to select the right techniques and agents, this process also requires specific equipment and input materials that can be quite expensive, especially at industrial scale. Developing a cost-effective and scalable formulation technique for Gram-negative bacteria is crucial for their wider adoption in agriculture and other industries.

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