The cannabis industry continues to experience growth, and with that growth comes added demands on growers as well as evolving regulations when it comes to testing, product labeling, and more. 

To conform to these regulations, cultivators are increasingly turning to microbial decontamination technologies and assessing which options are available — and that assessment has led to greater awareness of the effectiveness of ozone treatment for cleaner cannabis. 

Understanding the current cannabis market landscape 

The cannabis industry faces many external forces that challenge its cultivators and their ability to succeed in an increasingly saturated market. Research conducted in 2022 shows that increasing competition, declining prices, and high failure rates are just some of the many reasons why operators in the industry face difficulties turning a profit. 

Each harvest is critical, and some companies are one failed harvest away from potentially losing their business. Internal quality challenges such as pest control management, high microbial loads, and failure to meet target cannabinoid content affect not only the harvest yield, but also the quantity of usable and sellable product. While some cultivators have opted to cut their losses and move on to less risky endeavors, those who are truly passionate about the industry and their product strive to offer the best quality to their customers.

Unfortunately, current strategies for failed products involve either destroying contaminated batches, off-loading quality harvest to extractors at bottom dollar prices, or mitigating microbial contamination using irradiation that rids cannabis of its medicinal and artisanal quality. Nationally, the cannabis market could generate more profit by adopting treatment technologies that ultimately give them more control over their product quality by maintaining regulatory compliance prior to testing and avoiding the penalties and financial losses associated with inferior or unusable products.

Ozone as a viable microbial-control step strategy

Ozone treatment as a viable kill-step method is widely used in aqueous settings, most notably as a tertiary treatment step in advanced municipal drinking water treatment plants all over North

America. Ozone, chemical formula O3, is a molecule composed of three oxygens, which can easily be generated using an ozone generator. This converts the naturally occurring oxygen in the air into ozone, and when injected into an environment, it will begin to immediately interact and oxidize molecules, including organic materials like the cell membranes of microorganisms. This oxidizing effect causes cell membrane disruption which leads to lysis and cell death, the mode of action behind microbial remediation using ozone as a treatment method.

Figure 1. Ozone molecules react with the cell membrane of microorganisms resulting in cell death by changing the molecular structure and function of the molecule, for example, by breaking double bonds in the lipid bilayer.

WillowPure systems use ozone disinfection principles in practical application to reduce microbial contamination on cannabis products by gently exposing post-harvest material to gaseous ozone for a prescribed duration, ranging from four to six hours on average. In the following table, cultivars were tested at various treatment intervals (2, 3, 4, and 6 hours) to show their corresponding percent kill on Total Yeast and Mold (TYM), which include persistent humidity-loving Aspergillus mold species capable of producing dangerous mycotoxins. Pre- and Post-Treatment CFUs were compared to calculate the kill percentage, which can be expressed as a log reduction. CFU/g or “Colony Forming Units per gram” are the enumeration of microorganisms that are able to form a colony in one gram of sample; a single cell of bacteria that grows into one colony of bacteria which can be seen by the naked eye.

WillowPure 360 Case Study Results

Table 1 contains data from one participant cultivator utilizing a WP360 system. This study shows

consistent percent kill of over 99% TYM for trials two hours and longer:

Table 1. Anonymized average data comprised of 34 WillowPure 360 runs from a participant cultivator showing percent kill of over 99% for exposures above one hour, conducted by independent third-party laboratories.

Typically, it is recommended to expose cannabis products for four to six hours to ensure a complete permeation of ozone throughout the entire bulk of the product. This is especially important if the initial contamination levels are unknown, or if the product is already known to be contaminated. More exposure time is also recommended for a product that is wet, dense, old, exposed to unclean environments and any other properties that would suggest that the product likely harbors more microorganisms than is typical.

Log Reduction in a Nutshell

In order to describe kill-step efficacy in microbial populations, the concept of log reduction in microbiology applies. Simply put, one “log reduction” decreases the microbial population by a factor of ten. 10,000 CFU reduced by a factor of 10 is 1,000 CFU (1 log reduction). A subsequent 1 log reduction would reduce 1,000 CFU to 100 CFU (a total 2 log reduction from the initial 10,000 CFU), and so forth. To calculate the log reduction, we can use the formula:

Log reduction = log10(Initial count / Final count)

This can also be expressed as percent kill. For example, a 99.999% kill is a 5-log reduction, shown in Table 2 below:

Table 2. An initial 10,000 CFU reduces to 1,000 CFUs at a 1-log reduction, 100 CFUs at a 2-log reduction and so forth.

WillowPure360 Average Results

With a more robust data set from 20 independent cultivators and 184 raw data points, Figure 2

shows that generally, on average, a two-hour run results in a 1.1-log reduction (approximately 92% kill), a four-hour run results in just under 1.4-log reduction (approximately 96% kill), and a six-hour run approaches a log reduction of 1.9 (approximately 98% kill).

Figure 2. Anonymized data from 20 cultivators consisting of 184 raw data points, showing total yeast and mold deactivation at two, four, and six hours of exposure time, and expressed as percent kill.

In Table 1, we see that the cultivator started with a Total Yeast and Mold count of 150,001 for

cultivar C, run time of four hours. This is the declared quantitative value of “TMTC” or “too many to count” in 3rd party laboratories. After four hours, this number was reduced to 50 CFUs, which outperformed the average reductions seen in Figure 2. The Cultivator in Table 1 achieved an approximate 3.5 log reduction, compared to the average 1.4 log reduction seen in Figure 2.

These better-than-average results can be attributed to proper environmental conditions such as

optimal relative humidity, optimal water activity, and the application of Good Manufacturing Practices (GMP) principles to production and post-production processes. GMP practices in cultivation tend to mitigate microbial loads, especially in controlling Aspergillus species, molds that are extremely difficult to control and eradicate once present in the facility. As the proverb goes: “Good in, good out.” Company ethos and culture regarding quality will be reflected in the results, which trickles down to the bottom line. GMP principles as well as microbial loads and percent kill as a function of time will be amply discussed in subsequent white papers.

Key Highlights From Our WillowPure Ozone Treatment Research

  • Results from 20 cultivators show average log reduction ranges from 1.1 to 1.9 (92% to 98% kill) over two to six-hour ozone exposures.
  • Proper environmental conditions and adherence to GMP principles contribute to better-than-average results, reflecting the importance of quality ethos in cultivation practices.
  • Willow Scientific microbial partnership program, which applies agricultural best practices and microbial insights, is also a contributing factor to successful and consistent high microbial kill using the WillowPure 360.
  • One study found the WillowPure 360 to consistently kill over 99% of Total Yeast and Mold (TYM) for trials two hours and longer.
  • Kill percent increases with longer ozone exposure, emphasizing time as a critical factor.

Experience Cleaner Cannabis Through WillowPure Systems Are you assessing which decontamination technologies options are available to you? Do you think your cultivation space could benefit from ozone treatment as a microbial-control step? Visit our WillowPure product page to learn more about the advantages of this cutting-edge system for cultivators, or contact one of our experts today to chat further!