POWRHOUSE R&D EXPERIMENT SERIES, PART 1: CFU Count Using Serial Dilution and Nanobubble DO Treatment

Jeremy Pfeiffer CIO, Kairospace Technologies Inc | PowrHouse R&D Affiliate jeremy@kairospacetech.com

Kevin Crouch VP of Cultivation – PowrHouse
kevin@powr.house 

Objective 

This study evaluated the impact of dissolved oxygen (DO) levels on microbial growth by measuring colony-forming units (CFUs) in culture solutions. Three experimental groups, each exposed to a different DO level, were compared to a control group with no additional oxygen. The experiment was conducted at PowrHouse in Sacramento, CA, focusing on synganic cannabis cultivation—an approach that integrates synthetic fertilizer salts with organic beneficial microbe inoculants to enhance the efficiency of irrigation and foliar feed applications, plant health, and nutrient absorption. 


Materials and Methods

Materials

  • PowrHouse inoculant solution – GreenGro MadRoots All In One + Green Aminos 
  • Sterile saline or buffer (for dilution)
  • Nutrient agar plates
  • Sterile pipettes or micropipettes
  • Sterile dilution tubes (5 mL tubes)
  • Sterile spreader or inoculating loop
  • Incubator (set at 37°C)
  • Dissolved oxygen (DO) nanobubble treatment system (Kairospace AGPACK 40)
  • OpenCFU AI software for automated colony counting

Experimental Setup

Test Groups

  • Control Group: No additional oxygen treatment

  • Test Group 1: High dissolved oxygen (24 ppm DO)

  • Test Group 2: Medium dissolved oxygen (12 ppm DO)

Serial Dilution Procedure

  • Preparation of Serial Dilutions
    • 1 mL of culture solution was added to 9 mL of sterile saline to create a 10⁻¹ (5⁻¹) dilution.

    • 1 mL of the 10⁻¹ dilution was transferred to 9 mL sterile saline to make a 10⁻² (5⁻²) dilution.

    • The process was repeated for additional dilutions (10⁻³ etc.) as needed.

  • Plating the Dilutions
    • 0.1 mL of each dilution was spread onto nutrient agar plates.

    • Plates were incubated at 37°C for 24-48 hours.

  • Colony Counting and CFU Calculation
    • Colonies were counted from plates with colony counts within the 30-300 range.

    • CFU/mL was calculated using the equation:


Results and Calculations

Observed Colony Counts

Group Dissolved Oxygen (ppm) pH EC CFU Count Dilution Used
Control 8 ppm 5.6 3.2 91 10⁻²
Test Group 1 24 ppm 5.6 3.2 127 10⁻²
Test Group 2 12 ppm 5.6 3.2 242 10⁻²

CFU/mL Calculation

For each group, using dilution factor = 10² and plated volume = 0.1 mL:


Analysis and Observations

Test Group 1 (High DO – 24 ppm) The microbial population exhibited a CFU count of 127,000, indicating substantial growth but with a predominance of medium-to-large colonies. This suggests that while oxygen availability supports microbial proliferation, excessively high DO levels may create oxidative stress that selectively favors oxygen-tolerant species while potentially limiting obligate anaerobes. This could explain the moderate CFU count compared to the medium DO group, where microbial diversity was more pronounced. Future studies should examine whether oxidative stress markers (e.g., catalase activity or ROS production) influence species composition at high DO levels.

Test Group 2 (Medium DO – 12 ppm) This group had the highest CFU count (242,000) and the most diverse colony sizes, suggesting that a moderate DO level optimally supports a balanced microbial community. The presence of small, medium, and large colonies implies that both fast-growing and slow-growing species thrive under these conditions. The diversity suggests that this DO range optimizes microbial metabolic efficiency by providing sufficient oxygen without imposing oxidative stress or selectively excluding obligate anaerobes. These findings indicate that 12 ppm DO may be the most effective condition for hydroponic microbial inoculation, promoting both microbial growth and functional diversity essential for plant nutrient uptake. 

Control Group (Ambient DO – 8 ppm) The control group exhibited the lowest CFU count (91,000) with predominantly large colonies, suggesting that lower DO conditions may favor slower-growing, potentially more stress-tolerant or anaerobic species. The absence of smaller, fast-growing colonies indicates that lower oxygen availability might limit the expansion of facultative aerobes, leading to a microbial community dominated by species that are either oxygen-resistant or metabolically slower. This observation highlights the potential for DO levels to influence not just growth rates but also shifts in microbial community structure, which could have implications for nutrient solubilization and biofilm formation in hydroponic systems.


Incubator, serial dilutions and corresponding agar plate samples. 

Serial dilution plating and digital image analysis provided statistically significant results, supporting the hypothesis that dissolved oxygen impacts microbial proliferation and colony morphology.

OpenCFU counting software 


Conclusion 

This preliminary study demonstrates that dissolved oxygen levels significantly influence microbial proliferation, colony morphology, and potentially microbial community composition. The findings are particularly relevant to synganic hydroponic cannabis cultivation, where optimizing microbial oxygen exposure can enhance plant health, root colonization, and nutrient efficiency. 

    • High DO (24 ppm) supported microbial growth but may have selectively favored oxygen-tolerant species while limiting obligate anaerobes. 

    • Medium DO (12 ppm) resulted in the most diverse and balanced microbial population, suggesting it is the optimal condition for hydroponic microbial inoculation. Specifically in the context of this study. 

    • Low DO (8 ppm) produced fewer, larger colonies, suggesting a shift toward slower-growing or stress-tolerant species. 

While CFU counts provide insight into microbial proliferation, they do not fully capture functional diversity or metabolic activity. Future studies should incorporate molecular and biochemical characterizations, including: 16S rRNA sequencing to determine species composition shifts. Metabolic assays (catalase/oxidase tests, ATP quantification) to assess microbial function. Viability stains (e.g., propidium iodide for oxidative stress assessment) to determine whether high DO induces cell damage.
These additional analyses will provide a more comprehensive understanding of how DO levels affect microbial function in hydroponic systems, optimizing conditions for enhanced plant-microbe interactions and agricultural productivity. 


Further Context: Synganic Applications and MICROBIAL Productivity in Hydroponics

Optimizing Dissolved Oxygen (DO) Levels in Inoculant Solutions

One of the primary objectives in synganic cultivation is determining the optimal level of dissolved oxygen (DO) to introduce into the inoculant solution. Typically, inoculants are aerated under an airlift system before being integrated into the irrigation process. Airlift brewing provides increased airflow, higher oxygenation, and greater agitation, which enhances microbial solubilization and promotes a more uniform microbial culture.

In this study, we explored the application of DO nanobubbles and recirculation mixing as an alternative to traditional airlift brewing. The aim is to establish a diverse microbial community in the inoculant, ensuring its full preparation before incorporation into the irrigation system. The inoculant solution must be well-balanced before being applied to soil, substrate, or used as a foliar spray to maximize its effectiveness in plant health, nutrient cycling, and disease resistance.

Further Context: Bacterial Productivity in Hydroponics 

For cannabis hydroponics, identifying productive bacteria colonies is crucial for promoting plant growth, nutrient cycling, and soil health. The following characteristics indicate that bacterial strains are efficient and prolific: 

    • Colony size and growth rate: Large colonies suggest fast growth and nutrient efficiency, while uniform growth patterns indicate adaptability to the hydroponic environment. 

    • Colony morphology: Smooth and mucoid colonies are beneficial, as they often indicate exopolysaccharide (EPS) production, crucial for biofilm formation and stable root colonization. 

    • Color and pigmentation: Pigmented colonies may produce plant-beneficial secondary metabolites, while robust, non-pigmented colonies are often highly efficient in nutrient cycling. 

    • Antagonistic activity: Colonies with clear inhibition zones suggest biocontrol potential, suppressing plant pathogens and promoting healthier root microbiomes. 

    • Resilience and stress tolerance: Strains that maintain stability across different pH, temperature, and nutrient conditions are ideal for hydroponic setups. 

By focusing on these characteristics, Bacillus species and other beneficial microbes can be optimized for hydroponic cannabis cultivation, improving nutrient solubilization, plant growth, and pathogen suppression in a synganic system. 

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POWRHOUSE R&D EXPERIMENT SERIES, PART 1: CFU Count Using Serial Dilution and Nanobubble DO Treatment

This study evaluated the impact of dissolved oxygen (DO) levels on microbial growth by measuring colony-forming units (CFUs) in culture solutions. Three experimental groups, each exposed to a different DO level, were compared to a control group with no additional oxygen. The experiment was conducted at PowrHouse in Sacramento, CA, focusing on synganic cannabis cultivation—an approach that integrates synthetic fertilizer salts with organic beneficial microbes to enhance the efficiency of irrigation and foliar feed applications, plant health, and nutrient absorption. 

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