Cannabis Microbial Testing Research Paper
I would like to propose a comprehensive review of the existing cannabis microbial testing policies. While these regulations aim to ensure the safety and quality of cannabis products, it is important to recognize their limitations and the areas where they may be improved.
By initiating this review, we can identify and address the shortcomings of the current microbial testing framework. This will allow us to develop more effective and robust policies that align with the latest scientific advancements and industry best practices. Such an endeavor demonstrates our commitment to continuous improvement and our dedication to upholding the highest standards in the cannabis industry.
With that said, My name is Alexa and I am the Director of Cultivation for Hypha, a state-licensed cannabis cultivation facility located in Kalkaska, Michigan. I hold certifications in soil microbiology and microscopy. I am also a co-founder of Camp Compost, an educational festival that celebrates the regeneration of soil health through composting and microscopic life.
At Hypha, we employ a living soil ecosystem approach to cultivate our cannabis. This method mimics nature in an indoor setting, aiming to provide the cannabis plants with the conditions they would experience in nature. Our facility is committed to sustainable and regenerative practices, such as reusing soil and recycling condensate water from AC units for irrigation. The cornerstone of our living soil ecosystem is our compost, which is made from our cannabis "waste" products. It plays a vital role in maintaining soil fertility and the overall health of our plants.
Hypha has been operating for four years, and despite the prevalence of microorganisms in our growing environment, most of our products successfully pass state testing. However, like many other cultivation facilities, we have faced intermittent challenges with aspergillus PCR tests. We have conducted many rounds of research and development testing in collaboration with Cambium Analytica, a laboratory in Traverse City, to identify the cause of our aspergillus test failures. Unfortunately, the data collected from these tests has been inconclusive and has not pointed to any specific flaws in our processes.
While cannabis microbial testing may seem to provide us with adequate information regarding the safety of cannabis, Several studies have highlighted these limitations and shortcomings in the current testing methods.
For example, A peer-reviewed study from McKernan published in 2016 [3] highlights this lack of selectivity via metagenomic analysis of the plates used to test cannabis. Specifically, total yeast and molds (TYM) counts are most affected by this lack of selectivity—in some cases, the reads from the plates surveyed displayed up to 60% bacterial growth. This lack of specificity puts cultivators in a difficult spot as many of their crops will fail for what are often completely benign bacterial communities that can be beneficial for staving off more harmful microbial contaminants. Another study [7] on the use of various growth mediums in plating methods showed that TYM numbers sometimes align from method to method, it is more common that the results will vary drastically, sometimes by more than 50%. This is one of the main pitfalls that plate count testing methodologies face. The variation in results obtained from different plating methods further emphasizes the need for testing protocols that can account for the diverse nature of cannabis matrices.
It is important to acknowledge these shortcomings and address them to improve the reliability and effectiveness of cannabis microbial testing. Currently, in Michigan aspergillus is required to be tested for using polymerase chain reaction (PCR), which can detect any aspergillus DNA. Consequently, a single aspergillus spore could cause cannabis products to fail state testing.
Due to the omnipresence of microorganisms in growing environments cultivators chose to sterilize their products using chemicals like ozone, hydrogen peroxide, chlorine dioxide or other fungicides. However, chemical sterilization is not always effective in passing tests, leading many cultivators to remediate their products using radiation, such as X-rays, gamma rays, or radio waves, as it has proven to be the most reliable method for passing state tests.
Interestingly, research has shown [13] that due to the high sensitivity of PCR testing, genetic material from Aspergillus fungi can remain detectable in flower samples even after radiation treatments.
It is crucial to acknowledge that Aspergillus is an organism that exists ubiquitously in the atmosphere and can be found in soil, water, and air. In fact, the Centers for Disease Control and Prevention (CDC) website states that it is virtually impossible to completely avoid breathing in some Aspergillus spores, and most individuals unknowingly inhale several hundred spores every day, which are generally eliminated by the immune system.
In addition, Multiple studies have identified aspergillus as a common endophyte in the cannabis plant tissue. For instance, a study published December 2012 [1] investigated fungal endophytes associated with different parts of Cannabis, and revealed various species colonizing the plant. Aspergillus was found to be the most frequently occurring genus, with species such as Aspergillus niger and Aspergillus flavus.
Aspergillus holds significant importance both in environmental and industrial contexts. Healthy soil depends on the presence of aspergillus, as this microorganism plays a crucial role in the cycling of carbon and nitrogen. Its enzymes have widespread applications in various industries. For instance, Aspergillus niger is harnessed to manufacture citric acid, a key ingredient used in beverages, food products, detergents, cosmetics, and pharmaceuticals. Meanwhile, Aspergillus terreus produces a valuable secondary metabolite called lovastatin, a potent medication for reducing blood cholesterol levels in both humans and animals. Additionally, Aspergillus oryzae, also known as kōji mold, has been employed by Asian cultures since 300 BC to craft soy sauce, miso, and sake, showcasing its historical significance in culinary traditions. [17]
A Report on the American Academy of Microbiology Colloquium held in Washington, DC, on October 18, 2017,[11] presented research indicating that humans inhale anywhere between 1000 to 10 billion mold spores daily. As the majority of cannabis products in Michigan are now sold for recreational purposes, it is crucial to acknowledge that the presence of Aspergillus spores or other mold spores within the product has minimal impact on the vast majority of consumers. Such spores are no more harmful than the ones present in the air we breathe.
This broader context highlights the need to carefully consider the potential risks associated with Aspergillus contamination in cannabis. Particularly in relation to specific populations such as immunocompromised individuals, while also acknowledging the inherent ubiquity of Aspergillus in our environment and the relative impact on the general population.
The stringent testing standards imposed upon cannabis growers contrast with the limited amount of research available concerning the actual impact of cannabis contaminants on human health. A study on the effect of cannabis contaminates published June 2018 [5] noted that establishing a direct causal relationship between cannabis and its potential consequences presents challenges due to various factors, including the concurrent use of tobacco, alcohol, and other substances by cannabis users. Moreover, the time delay between consumption and diagnosis further complicates the matter. Additionally, the scarcity of adequately powered and well-designed epidemiological or experimental studies examining the effects of contaminants on humans adds to the complexity. It is essential to recognize that harmful toxins exist in various aspects of our environment, including food, water, and air, which contribute to human diseases. Consequently, designing an experiment that unequivocally links cannabis contaminants to human diseases would be challenging.
Despite the need to protect consumers from excessive amounts of microbial contamination, growing plants in completely sterile environments is neither feasible nor beneficial.
The fertility of our soil in a living soil growing system relies on the presence of microorganisms. It is fascinating to note a mere tablespoon of healthy soil contains a greater number of microorganisms than the total human population on Earth. Until the work of Dr. Eline Ingham, the significance of these microorganisms in our soils remained largely unknown. Dr. Ingham's research revealed that plants allocate approximately 50% of their energy towards attracting microorganisms to their root zones, as these microorganisms facilitate nutrient cycling. This process occurs as bacteria, fungi, and other decomposers break down soil particles and organic matter and release plant-available forms of nutrients into the soil solution. This is just one of the remarkable functions of microorganisms, and ongoing research continues to uncover the dynamic and complex interactions between plants, humans, and microorganisms.
In a study published in March 2020, [8] researchers explored the role of endophytes in the production of secondary metabolites in cannabis. These endophytes are microorganisms that reside within plant tissue and produce biologically active compounds that mimic or contribute to the effects of the host plant's own metabolites, thereby influencing the production of cannabinoids, terpenes, flavonoids, and phytohormones. Understanding the microbial partnerships with cannabis is imperative, as it has the potential to enhance agricultural practices, improve plant fitness, and increase cannabinoid yields, and even provide potential health benefits to humans through the active metabolites of microbial endophytes. This research underscores the need for further studies on cannabis endophytes and their biological properties.
Similarly, a pioneering study conducted on tomatoes [9] revealed that the microbiome within glandular trichomes, specialized plant structures, differs significantly from other parts of the plant. This unique community of microorganisms also plays a role in the production of secondary metabolites. The metabolites released by these trichomes shape a distinct microbial community, potentially enriching beneficial taxa while acting as a defense mechanism against plant pathogens. These findings contribute to our understanding of the plant microbiome and emphasize the importance of micro-niches in structuring bacterial communities within the phyllosphere of plants.
In a more recent study published in June 2022 [10], researchers investigated hops and cannabis and discovered that plants cultivate endosymbiotic bacteria within non-photosynthetic cells in leaves and bracts, extracting and utilizing nitrogenous nutrients from them. This research further emphasizes the diverse ways in which plants interact with microorganisms and underscores the significance of maintaining a diverse microbiome.
I present these articles to emphasize the significance of supporting a diverse microbiome in cannabis cultivation. Recent research has increasingly highlighted the importance of maintaining a balanced and varied microbial community to enhance plant health, resilience, and productivity.
It is vital to recognize that cannabis cultivation involves working with a living plant, which inherently introduces various variables associated with natural processes. As the cultivation director and co-founder of a grow utilizing a living soil ecosystem, I have witnessed firsthand the significance of microorganisms in maintaining ecosystem balance, promoting plant health, and mitigating harmful pathogens. It is essential to acknowledge that flawed microbial testing can impact growers regardless of their chosen cultivation methods, as the plant naturally harbors a microbiome. Just as humans rely on microbial partners for survival, so does the cannabis plant. As a result, it is crucial to understand the advantages of microorganisms when formulating policies for cannabis microbial testing.
Acknowledging the limitations and inconsistencies inherent in both plate count methods and molecular-based methods, it becomes evident that an alternative approach is necessary for the benefit of both consumers and cultivators.
Given the omnipresence of aspergillus and other molds in our atmosphere and the fact that aspergillus has been identified as a common cannabis endophyte the pass/fail requirement for aspergillus or any PCR mold testing is excessively strict. Thus, I propose that the state reconsiders the current aspergillus pass/fail testing requirements for cannabis as a limit should be established due to its ubiquity in our environment.
A recent white paper written by New York Ph.D researchers determined that there is a “lack of data regarding the real world harm of aspergillus spores'' and suggests “suspending aspergillus pcr testing until certain data has been collected.” Without the data proving how much aspergillus is dangerous for users, it is evident that safe cannabis products are being destroyed or remediated due to the high sensitivity of PCR testing. There is no data to show how much aspergillus is harmful to humans, once this data is established an action limit could be applied based on harmful levels. [20]
To address the potential inconsistencies and variations in microbial testing methods, Michigan could consider following the example set by states like New York [15], by implementing a labeling requirement rather than a pass/fail system for cultivators. By mandating the inclusion of total yeast and mold and aspergillus count on product labels, consumers are empowered to make informed decisions based on their preferences and needs.
This alternative approach recognizes that the presence of yeast and mold is a common occurrence in natural environments, including cannabis cultivation. Rather than imposing strict pass/fail criteria that may not fully reflect the safety or quality of the product, labeling microbial counts such as aspergillus and total yeast and mold provides transparency and allows consumers to assess the levels according to their own comfort levels and health considerations.
Implementing a labeling requirement for microbial testing can strike a balance between consumer safety and cultivator viability. It acknowledges the ubiquitous presence of yeast and mold while providing valuable information to consumers. By adopting this approach, Michigan can align itself with best practices from other states and ensure that consumers have access to accurate information regarding microbial levels in cannabis products.
It is worth considering this alternative as a potential solution to the limitations of pass/fail testing, as it embraces transparency, consumer choice, and industry viability. Further evaluation and discussions among relevant stakeholders would be valuable in determining the feasibility and effectiveness of adopting a microbial count labeling requirement in Michigan's cannabis industry.
In light of the growing recognition of the inconsistencies in microbial testing, I propose that the CRA takes the initiative to form a committee dedicated to addressing these limitations. The primary objective of this committee would be to find a balanced approach to testing that benefits both consumers and cultivators. This committee will allow us to develop more effective and adaptable policies that align with the latest scientific advancements.
By acknowledging the current testing method's limitations and actively seeking improvements, we demonstrate a steadfast commitment to ensuring the safety and quality of cannabis products.
Establishing such a committee would be a clear demonstration of our dedication to remaining at the forefront of the industry and proactively adapting to emerging challenges. By regularly evaluating and updating testing procedures, we can inspire confidence in consumers, protect public health, and foster a thriving and reputable cannabis industry. If appointed, I would gladly offer my service on this committee. Together, we can work towards creating a robust framework that safeguards the interests of all stakeholders involved in the cannabis industry.
As we navigate the ever-evolving landscape of cannabis cultivation, it becomes vital to maintain a commitment to refining our protocols for microbial testing. This ensures that our results are not only precise and reliable but also relevant in the context of practicality and feasibility for cultivators. Through these measures, we can instill confidence in consumers, protect public health, and foster a thriving and reputable cannabis industry.
In the interest of transparency and safeguarding consumer rights, I also propose the mandate the labeling of radiated cannabis products in Michigan. Adopting the use of a symbol, such as the randura which is already required by the FDA for irradiated food products. This provides a practical and recognizable way to inform consumers about the irradiation process.
Similar labeling requirements are already in place in states like Nevada, where consumers are provided with essential information to make informed choices about the products they consume. Transparency in labeling empowers consumers to make choices based on their preferences, needs, and concerns. It also promotes accountability and strengthens the relationship between producers and consumers. Mandating the labeling of radiated cannabis products will contribute to building trust and confidence within the cannabis industry.
Thank you for taking the time to consider these matters. Below, is a list of citations as well as other supporting documents and information.
Citations
Ajay Kumar Gautam, Mona Kant & Yogita Thakur (2013) Isolation of endophytic fungi from Cannabis sativa and study their antifungal potential, Archives of Phytopathology and Plant Protection, 46:6, 627-635, DOI: 10.1080/03235408.2012.749696
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2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4897766/
Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers
Kevin McKernan,a,1 Jessica Spangler,1 Lei Zhang,1 Vasisht Tadigotla,1 Yvonne Helbert,1 Theodore Foss,1 and Douglas Smith1
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3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5089129/
Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests
Kevin McKernan,a,1 Jessica Spangler,1 Yvonne Helbert,1 Ryan C. Lynch,1 Adrian Devitt-Lee,1 Lei Zhang,1 Wendell Orphe,1 Jason Warner,1 Theodore Foss,1 Christopher J. Hudalla,2 Matthew Silva,2 and Douglas R. Smithb,1
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Cannabis Microbial Testing Methodologies and Considerations
Kyle Boyar
DOI:10.1201/9780429274893-6
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5. https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bcp.13695
Cannabis contaminants: sources, distribution, human toxicity and pharmacologic effects
Laura M. Dryburgh, Nanthi S. Bolan, Christopher P.L. Grof, Peter Galettis, Jennifer Schneider, Catherine J. Lucas, Jennifer H. Martin
First published: 28 June 2018
https://doi.org/10.1111/bcp.13695
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6. https://www.frontiersin.org/articles/10.3389/fmicb.2023.1192035/full?utm_source=Email_to_rerev_&utm_medium=Email&utm_content=T1_11.5e5_reviewer&utm_campaign=Email_publication&journalName=Frontiers_in_Microbiology&id=1192035&fbclid=PAAaaPYXn05GblNNbLvoBIapy1-HQ6QrH0Jh-5cdBfvCc7kKxR3eW0BpV3YFo_aem_th_AaDBv9AxlAT3H2PsTo1GlU9D_pAVAl4n92ifEel85bzzIgi2Po6PSMTJSRuNJ2od9vE
ORIGINAL RESEARCH article
Front. Microbiol., 13 June 2023
Sec. Microbe and Virus Interactions with Plants
Volume 14 - 2023 | https://doi.org/10.3389/fmicb.2023.1192035
Total yeast and mold levels in high THC-containing cannabis (Cannabis sativa L.) inflorescences are influenced by genotype, environment, and pre-and post-harvest handling practices
Zamir K. Punja*, Li Ni, Samantha Lung and Liam Buir
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The Benefits and Pitfalls of Total Yeast and Mold Counts in Cannabis Labs
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7. https://pubmed.ncbi.nlm.nih.gov/32131457/
Microorganisms. 2020 Mar; 8(3): 355.
Published online 2020 Mar 2. doi: 10.3390/microorganisms8030355
PMCID: PMC7143057
PMID: 32131457
Cannabis Microbiome and the Role of Endophytes in Modulating the Production of Secondary Metabolites: An Overview
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8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067393/
Trichomes form genotype-specific microbial hotspots in the phyllosphere of tomato
Peter Kusstatscher,#1 Wisnu Adi Wicaksono,#1 Alessandro Bergna,2 Tomislav Cernava,1 Nick Bergau,3 Alain Tissier,3 Bettina Hause,3 and Gabriele Berg1
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9. https://pubmed.ncbi.nlm.nih.gov/35741397/
Histochemical Evidence for Nitrogen-Transfer Endosymbiosis in Non-Photosynthetic Cells of Leaves and Inflorescence Bracts of Angiosperms
by April Micci 1,*, Qiuwei Zhang 1, Xiaoqian Chang 1, Kathryn Kingsley 1, Linsey Park 1, Peerapol Chiaranunt 1, Raquele Strickland 1, Fernando Velazquez 1, Sean Lindert 1, Matthew Elmore 1, Philip L. Vines 1, Sharron Crane 2, Ivelisse Irizarry 3, Kurt P. Kowalski 4, David Johnston-Monje 5 and James F. White 1,*
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10. https://www.ncbi.nlm.nih.gov/books/NBK549988/
One Health: Fungal Pathogens of Humans, Animals, and Plants
Report on an American Academy of Microbiology Colloquium held in Washington, DC, on October 18, 2017
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BIOLOGICAL SYNTHESIS OF SILVER NANOPARTICLES FROM AQUEOUS EXTRACT OF ENDOPHYTIC FUNGUS ASPERGILLUS FUMIGATUS AND ITS ANTIBACTERIAL ACTION
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12. Use of X-ray irradiation for inactivation of Aspergillus in cannabis flower
Stephen Frink ,
Olivera Marjanovic ,
Phoi Tran ,
Yun Wang,
Weihong Guo,
Noahie Encarnacion,
Donelle Alcantara,
Bahman Moezzi,
Gordon Vrdoljak
Published: November 15, 2022
Front Pharmacol. 2016; 7: 108.
Published online 2016 Apr 27. doi: 10.3389/fphar.2016.00108
PMCID: PMC4847121
PMID: 27199751
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13. Evaluating the Effects of Gamma-Irradiation for Decontamination of Medicinal Cannabis
https://pubmed.ncbi.nlm.nih.gov/27199751/
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14. https://www.leafly.com/news/health/new-york-cannabis-testing-update
New York loosened weed testing rules for mold and yeast—Here’s why
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15. CDC - Aspergillus
https://www.cdc.gov/fungal/diseases/aspergillosis/index.html
https://www.cdc.gov/fungal/diseases/aspergillosis/causes.html
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16. Wikipedia - Aspergillus
https://en.wikipedia.org/wiki/Aspergillus_niger
https://en.wikipedia.org/wiki/Aspergillus_flavus
https://en.wikipedia.org/wiki/Aspergillus_fumigatus
https://en.wikipedia.org/wiki/Aspergillus_terreus
https://en.wikipedia.org/wiki/Aspergillus_oryzae
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17. https://www.mjverdant.com/cannabis-and-radiation/
Cannabis & Radiation: What Every Consumer Should Know
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18. https://www.royalqueenseeds.com/us/blog-killing-mould-on-medical-cannabis-is-irradiation-the-future-n1247
Killing Mould On Medical Cannabis: Is Irradiation The Future?
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19. Aspergillus sp. testing in the emerging Cannabis sativa industry in New York State
Seth Brophy, Tim Moshier, Bill Nichols, Daniela Vergara, Ph.D., Brandy Young, Ph.D.
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20. ‘In the air all the time’: Microscopic menace prompts OHA rule, worrying pot growers
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21. In Random Mold Tests at 25 Denver Dispensaries, 80 Percent Fail
By Thomas Mitchell October 30, 2019
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22. Too Many Mouldy Joints – Marijuana and Chronic Pulmonary Aspergillosis
Yousef Gargani,1 Paul Bishop,2 and David W. Denning1
2011; 3(1): e2011005.
Published online 2011 Jan 14. doi: 10.4084/MJHID.2011.005
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3103256/#b15-mjhid-3-e2011005
