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Dr. Zacariah Hildenbrand
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Cannabis and the Environment: Navigating the Interplay Between Genetics and Transcriptomics

By Dr. Zacariah Hildenbrand
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Dr. Zacariah Hildenbrand

It is that time of year where the holidays afford us an opportunity for rest, recuperation and introspection. Becoming a new father to a healthy baby girl and having the privilege to make a living as a scientist, fills me with an immeasurable sense of appreciation and indebtedness. I’ve also been extremely fortunate this year to spend significant time with world-renowned cannabis experts, such as Christian West, Adam Jacques and Elton Prince, whom have shared with me a tremendous wealth of their knowledge about cannabis cultivation and the development of unique cannabis genetics. Neither of these gentlemen have formal scientific training in plant genetics; however, through decades of experimentation, observation and implementation, they’ve very elegantly used alchemy and the principles of Mendelian genetics to push the boundaries of cannabis genetics, ultimately modulating the expression of specific cannabinoids and terpenes. Hearing of their successes (and failures) has triggered significant wonderment and curiosity with respect to what can be done beyond the genetic level to keep pushing the equilibrium in this new frontier of medicine.

Lighting conditions can greatly impact the expression of terpenes (and cannabinoids) in cannabis.Of course genetics are the foundation for the production of premium cannabis. Without the proper genetic code, one cannot expect the cannabis plant to express the target constituents of interest. However, what happens when you have an elite genetic code, the holy grail of cannabis nucleotides if you will, and yet your plant does not produce the therapeutic compounds that you want and/or that are reflective of that elite genetic code? This ‘loss in translation’ can be explained by transcriptomics, and more specifically, epigenetics. In order for the genetic code (DNA) to be expressed as a gene product (RNA), it must be transcribed, a process that is modulated by epigenetic processes like DNA methylation and histone modification. In other words, the methylation of the genetic code can dictate whether or not a particular segment of DNA is transcribed into RNA, and ultimately expressed in the plant. To put this into context, if the DNA code for the enzyme THCA synthase is epigenetically silenced, then no THCA synthase is produced, your cannabis cannot convert CBGA into THCA, and now you have hemp that is devoid of THC.So what is the best lighting technology to enhance the expression of terpenes? 

With all of that being said, how do we ensure that our plants thrive under favorable epigenetic conditions? The answer is the environment; and the expression of terpenes is an ideal indicator of favorable environmental conditions. While amazing anti-inflammatories, anti-oxidants and metabolic regulators for humans, terpenes are also extremely powerful anti-microbial agents that act as a robust a line of defense for the plant against bacteria and pests. So, if the threat of microbes can induce the expression of terpenes, then what about other environmental factors? I am of the opinion that the combination of increased exposure to bacteria and natural sunlight enhances the expression of terpenes in outdoor-grown cannabis compared to indoor-grown cannabis. This is strictly my opinion based off of my own qualitative observations, but the point being is that lighting conditions can greatly impact the expression of terpenes (and cannabinoids) in cannabis.

A plant in flowering under an LED fixture

So what is the best lighting technology to enhance the expression of terpenes? Do I use full spectrum lighting or specific frequencies? The answer to these questions is that we don’t fully know at this point. Thanks to the McCree curve we have a fundamental understanding of the various frequencies within the visible light spectrum (400-700nm) that are beneficial to plants, also known as Photosynthetically Active Radiation (PAR). However, little-to-no research has been conducted to determine the impacts that the rest of the electromagnetic spectrum (also categorized as ‘light’) may have on plants. As such, we do not know with 100% certainty what frequencies should be applied, and at what times in the growth cycle, to completely optimize terpene concentrations. This is not to disparage the lighting professionals out there that have significant expertise in this field; however, I’m calling for the execution of peer-reviewed experiments that would transcend the boundaries of company white papers and anecdotal claims. In my opinion, this lack of environmental data provides a real opportunity for the cannabis industry to initiate the required collaborations between cannabis geneticists, technology companies and environmental scientists. This is one field of research that I wish to pursue with tenacity and I also welcome other interested parties to join me in this data quest. Together we can better understand the environmental factors, such as lighting, that are acting as the molecular light switches at the interface of genetics and transcriptomics in cannabis.

Total Yeast & Mold Count: What Cultivators & Business Owners Need to Know

By Parastoo Yaghmaee, PhD, Parastoo Yaghmaee, PhD
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Editor’s note: This article should serve as a foundation of knowledge for yeast and mold in cannabis. Beginning in January 2018, we will publish a series of articles focused entirely on yeast and mold, discussing topics such as TYMC testing, preventing yeast and mold in cultivation and treatment methods to reduce yeast and mold.


Cannabis stakeholders, including cultivators, extractors, brokers, distributors and consumers, have been active in the shadows for decades. With the legalization of recreational adult use in several states, and more on the way, safety of the distributed product is one of the main concerns for regulators and the public. Currently, Colorado1, Nevada and Canada2 require total yeast and mold count (TYMC) compliance testing to evaluate whether or not cannabis is safe for human consumption. As the cannabis industry matures, it is likely that TYMC or other stringent testing for yeast and mold will be adopted in the increasingly regulated medical and recreational markets.

The goal of this article is to provide general information on yeast and mold, and to explain why TYMC is an important indicator in determining cannabis safety.

Yeast & Mold

Photo credit: Steep Hill- a petri dish of mold growth from tested cannabis

Yeast and mold are members of the fungi family. Fungus, widespread in nature, can be found in the air, water, soil, vegetation and in decaying matter. The types of fungus found in different geographic regions vary based upon humidity, soil and other environmental conditions. In general, fungi can grow in a wide range of pH environments and temperatures, and can survive in harsh conditions that bacteria cannot. They are not able to produce their own food like plants, and survive by breaking down material from their surroundings into nutrients. Mold cannot thrive in an environment with limited oxygen, while yeast is able to grow with or without oxygen. Most molds, if grown for a long enough period, can be detected visually, while yeast growth is usually detected by off-flavor and fermentation.

Due to their versatility, it is rare to find a place or surface that is naturally free of fungi or their spores. Damp conditions, poor air quality and darker areas are inviting environments for yeast and mold growth.

Cannabis plants are grown in both indoor and outdoor conditions. Plants grown outdoors are exposed to wider ranges and larger populations of fungal species compared to indoor plants. However, factors such as improper watering, the type of soil and fertilizer and poor air circulation can all increase the chance of mold growth in indoor environments. Moreover, secondary contamination is a prevalent risk from human handling during harvest and trimming for both indoor and outdoor-grown cannabis. If humidity and temperature levels of drying and curing rooms are not carefully controlled, the final product could also easily develop fungi or their growth by-product.

 What is TYMC?

TYMC, or total yeast and mold count, is the number of colony forming units present per gram of product (CFU/g). A colony forming unit is the scientific means of counting and reporting the population of live bacteria or yeast and mold in a product. To determine the count, the cannabis sample is plated on a petri dish which is then incubated at a specific temperature for three to five days. During this time, the yeast and mold present will grow and reproduce. Each colony, which represents an individual or a group of yeast and mold, produces one spot on the petri dish. Each spot is considered one colony forming unit.

Why is TYMC Measured?

TYMC is an indicator of the overall cleanliness of the product’s life cycle: growing environment, processing conditions, material handling and storage facilities. Mold by itself is not considered “bad,” but having a high mold count, as measured by TYMC, is alarming and could be detrimental to both consumers and cultivators. 

Aspergillus species niger
Photo: Carlos de Paz, Flickr

The vast majority of mold and yeast present in the environment are indeed harmless, and even useful to humans. Some fungi are used commercially in production of fermented food, industrial alcohol, biodegradation of waste material and the production of antibiotics and enzymes, such as penicillin and proteases. However, certain fungi cause food spoilage and the production of mycotoxin, a fungal growth by-product that is toxic to humans and animals. Humans absorb mycotoxins through inhalation, skin contact and ingestion. Unfortunately, mycotoxins are very stable and withstand both freezing and cooking temperatures. One way to reduce mycotoxin levels in a product is to have a low TYMC.

Aspergillus flavus on culture.
Photo: Iqbal Osman, Flickr

Yeast and mold have been found to be prevalent in cannabis in both current and previous case studies. In a 2017 UC Davis study, 20 marijuana samples obtained from Northern California dispensaries were found to contain several yeast and mold species, including Cryptococcus, Mucor, Aspergillus fumigatus, Aspergillus niger, and Aspergillus flavus.3 The same results were reported in 1983, when marijuana samples collected from 14 cannabis smokers were analyzed. All of the above mold species in the 2017 study were present in 13 out of 14 marijuana samples.4

Aspergillus species niger, flavus, and fumigatus are known for aflatoxin production, a type of dangerous mycotoxin that can be lethal.5 Once a patient smokes and/or ingests cannabis with mold, the toxins and/or spores can thrive inside the lungs and body.6, 7 There are documented fatalities and complications in immunocompromised patients smoking cannabis with mold, including patients with HIV and other autoimmune diseases, as well as the elderly.8, 9, 10, 11

For this reason, regulations exist to limit the allowable TYMC counts for purposes of protecting consumer safety. At the time of writing this article, the acceptable limit for TYMC in cannabis plant material in Colorado, Nevada and Canada is ≤10,000 CFU/g. Washington state requires a mycotoxin test.12 California is looking into testing for specific Aspergillus species as a part of their requirement. As the cannabis industry continues to grow and advance, it is likely that additional states will adopt some form of TYMC testing into their regulatory testing requirements.

References:

  1. https://www.colorado.gov/pacific/sites/default/files/Complete%20Retail%20Marijuana%20Rules%20as%20of%20April%2014%202017.pdf
  2. http://laws-lois.justice.gc.ca/eng/acts/f-27/
  3. https://www.ucdmc.ucdavis.edu/publish/news/newsroom/11791
  4. Kagen SL, Kurup VP, Sohnle PG, Fink JN. 1983. Marijuana smoking and fungal sensitization. Journal of Allergy & Clinical Immunology. 71(4): 389-393.
  5. Centre for Disease control and prevention. 2004 Outbreak of Aflatoxin Poisoning – Eastern and central provinces, Kenya, Jan – July 2004. Morbidity and mortality weekly report.. Sep 3, 2004: 53(34): 790-793
  6. Cescon DW, Page AV, Richardson S, Moore MJ, Boerner S, Gold WL. 2008. Invasive pulmonary Aspergillosis associated with marijuana use in a man with colorectal cancer. Diagnosis in Oncology. 26(13): 2214-2215.
  7. Szyper-Kravits M, Lang R, Manor Y, Lahav M. 2001 Early invasive pulmonary aspergillosis in a leukemia patient linked to aspergillus contaminated marijuana smoking. Leukemia Lymphoma 42(6): 1433 – 1437.
  8. Verweii PE, Kerremans JJ, Voss A, F.G. Meis M. 2000. Fungal contamination of Tobacco and Marijuana. JAMA 2000 284(22): 2875.
  9. Ruchlemer R, Amit-Kohn M, Raveh D, Hanus L. 2015. Inhaled medicinal cannabis and the immunocompromised patient. Support Care Cancer. 23(3):819-822.
  10. McPartland JM, Pruitt PL. 1997. Medical Marijuana and its use by the immunocompromised. Alternative Therapies in Health and Medicine. 3 (3): 39-45.
  11. Hamadeh R, Ardehali A, Locksley RM, York MK. 1983. Fatal aspergillosis associated with smoking contaminated marijuana, in a marrow transplant recipient. Chest. 94(2): 432-433.
  12. http://apps.leg.wa.gov/wac/default.aspx?cite=314-55-102