Mixing Gram-Positive, Gram-Negative Bacteria, and Fungi in Liquid and Powder Products – What Really Happens?

March - 2025

In part one of this blog series, we explored why combining bacilli and fungi in a single product is not always the best idea. But what happens when you take it a step further and mix Gram-positive bacteria, Gram-negative bacteria, and fungi in a liquid formulation? And does the situation change if the product is in powder form?

To fully understand the interactions between these microorganisms, let's first look at the differences between Gram-positive and Gram-negative bacteria, and then explore the effects of their coexistence in different product formulations.

The Difference Between Gram-Positive and Gram-Negative Bacteria

The terms Gram-positive and Gram-negative refer to how bacteria react to the Gram staining test, which is based on differences in their cell wall structure.

🦠 Gram-Positive Bacteria

✔ Thick peptidoglycan layer in their cell wall, making them more resistant to physical stress.

✔ Stain purple in a Gram test.

✔ Generally more resistant to desiccation (drying out) and environmental stress.

✔ Often produce spores (e.g., Bacillus species), which allow them to survive harsh conditions.

✔ More susceptible to certain antibiotics that target peptidoglycan synthesis (like penicillin).

Examples: Bacillus, Lactobacillus, Staphylococcus

🦠 Gram-Negative Bacteria

✔ Thin peptidoglycan layer, surrounded by an outer membrane containing lipopolysaccharides (LPS).

✔ Stain pink in a Gram test.

✔ More sensitive to environmental stress but more resistant to antibiotics due to their outer membrane.

✔ Tend to thrive in moist environments but don’t form spores like many Gram-positive bacteria.

✔ Can release endotoxins (from their LPS layer) when they die, potentially leading to contamination issues.

Examples: Escherichia coli, Pseudomonas, Salmonella

What Happens When You Mix These Microbes in a Liquid Product?

A liquid formulation provides a rich environment for microbial activity. However, combining Gram-positive, Gram-negative bacteria, and fungi in a single liquid medium presents several challenges:

1️⃣ Competition for Nutrients

Each microorganism type has its own nutritional preferences, leading to competition:

• Gram-negative bacteria often grow faster in nutrient-rich environments, potentially outcompeting Gram-positive bacteria and fungi.

• Fungi break down complex organic matter, but if bacteria consume the simple sugars first, fungi might struggle to grow.

2️⃣ pH Imbalances

Different microbes produce different metabolic byproducts:

• Lactic acid bacteria (Gram-positive, like Lactobacillus) lower the pH, making it harder for other bacteria and fungi to thrive.

• Gram-negative bacteria (e.g., Pseudomonas) may produce alkaline compounds, counteracting acidity but affecting microbial balance.

• Fungi generally prefer slightly acidic conditions, so an unstable pH can inhibit their growth.

3️⃣ Toxin Production & Contamination Risks

• When Gram-negative bacteria die, they release endotoxins from their cell walls, which can be harmful or affect product stability.

• Some fungi produce mycotoxins, which can accumulate if bacteria don’t suppress their growth.

• The presence of both bacteria and fungi increases the risk of biofilm formation, where microbes stick together and become harder to control.

4️⃣ Preservation and Stability Issues

• Gram-negative bacteria are more prone to die-off in certain liquid conditions, leading to spoilage.

• Fungi can form visible colonies or even unwanted mold in liquid suspension.

• Gram-positive spore-forming bacteria (like Bacillus) might dominate the mix because they can survive in harsher conditions than the others.

Bottom Line for Liquid Products:

🔹 Shorter shelf life due to microbial interactions.

🔹 Risk of pH shifts, biofilm formation, and toxin release.

🔹 Possible uneven survival of microbes, making the product inconsistent.

What Happens in a Powdered Product?

When a microbial product is formulated in powder form, the dynamics change drastically because microbes are often in a dormant state due to lack of moisture. However, some challenges still arise:

1️⃣ Survival Differences in Dry Conditions

• Gram-positive bacteria (e.g., Bacillus) can form spores and survive extreme dryness for long periods.

• Gram-negative bacteria do not form spores and are more vulnerable to dehydration, reducing their viability in powder form.

• Fungi often struggle in completely dry conditions unless they are formulated as dry spores or freeze-dried cells.

2️⃣ Reactivation Upon Moisture Exposure

Once the powder is rehydrated (e.g., when mixed into water or a growing medium):

• Gram-positive bacteria (spores) activate quickly and start multiplying.

• Gram-negative bacteria may struggle if they were weakened during drying.

• Fungi take longer to establish because they need more stable moisture levels.

3️⃣ Stability and Shelf Life

• Powder formulations generally have a longer shelf life because microbes remain inactive.

• If stored in proper conditions (dry, cool, low oxygen), most microbes survive well.

• However, when moisture enters the packaging, unwanted microbial interactions can still occur.

  
  

Bottom Line for Powdered Products:

✅ Longer shelf life due to microbial dormancy.

✅ Less competition because microbes are inactive until rehydration.

❌ Potential viability loss for Gram-negative bacteria and fungi.

❌ Moisture contamination can trigger unwanted microbial growth.

Final Thoughts – Should You Mix These Microbes in One Product?

When developing a microbial product, it’s crucial to consider how different organisms interact in both liquid and dry environments.

🧪 For liquid formulations:

⚠️ High risk of nutrient competition, pH imbalance, toxin production, and biofilm formation.

✅ Best suited for single-microbe solutions or carefully balanced probiotic blends.

🏜️ For powder formulations:

✅ Better stability as microbes remain dormant.

⚠️ Gram-negative bacteria and non-spore-forming fungi may struggle to survive long-term.

🔬 The best approach? If you need multiple microbial strains, consider separate formulations or staged applications instead of forcing them into a single product.