White Paper: Production of Cannabinoids using biotechnology and synthetic chemistry as a path to sustainability

Introduction

Sustainable production methods for cannabinoids will become increasingly important as the demand for consumer products utilising these compounds increases in response to global regulatory relaxation of their use. Primarily driven by increasing demand for CBD for health and wellness purposes, the total global CBD market is estimated to reach USD 47.22bn by 2028, up from USD 4.9bn in 2021, at a compound annual growth rate (CAGR) of 21.3% [35]. The growing popularity is evidenced by the UK market (which according to the Association for the Cannabinoid Industry (ACI) has become the world’s second-largest consumer cannabinoids market after the US) spending more on cannabinoid extracts domestically than vitamin B and C combined, indicating the growing popularity of cannabinoids in wellness [36]. Elsewhere, the number of use cases in FMCG for cannabinoids is continuing to climb as the global CBD skincare market totted up a net worth of $710m in 2018 and is projected to surge to $959m by 2024 according to Prohibition Partners’ Disrupting Beauty report [37].

Until recently, access to cannabinoids has been restricted to the cultivation of Cannabis sativa and extraction therefrom, a labour and energy intensive process; and akin to other crop productions systems the limitations are being realized as cannabinoid demand is commercialized. High yield variability, microbial and chemical contamination and crop-loss are all issues that plague Cannabis sativa. Furthermore, this type of agriculture has a negative environmental impact as it uses large amounts of land, water, fertilizers and pesticides, and has a high carbon footprint (electrical usage). For instance, the fermentation process in the CBD production used by Willow Biosciences Inc, a biotechnology company producing ultra-pure, biosynthetically produced CBG compounds supplied to Cellular Goods, enables resource efficient and sustainable growth. Willow Biosciences Inc has found that outdoor Carbon Dioxide (CO2) emissions are 44 times higher than by fermentation, and warehouse agricultural production produces 527 times greater CO2 emissions per one kilogram of CBD produced.

Although these challenges have been marginally managed through technological controls (advanced agronomy, green houses and indoor cultivation methods), the production capabilities of Cannabis sativa have shown diminishing economic returns, particularly in the case of extracting / producing minor cannabinoids. With cannabis sativa, approximately 90% of the plant’s mass ends up as botanical waste, the culmination of an essential process of growth and maturation where the plant expends a great deal of energy and resources. Cannabinoids collectively make up just 2-5% of the whole plant structure by mass (flower, stem and roots) [7]. Recovery of all the produced molecules (cannabinoids) from the plant is impossible as these elements need to be extracted, which yields complex, variable mixtures of the molecules of interest. Isolation and further separation and purification of these molecules is challenging, largely due to their relatively similar chemical structure.

Biotechnology and synthetic chemistry have provided us with the tools and processes necessary to produce cannabinoids at commercial scale with significantly less of the associated environmental drawbacks and waste. Focusing on the production of cannabinoids through microorganisms has the prospect of not only improved production economics, but most importantly, improved environmental sustainability. Although this technology is still relatively young and further improvements are realized with each iteration, notably in recovery efficiency and multi-fold increases in cannabinoid titres (in biotherapeutic manufacture, titer determination is the measurement of the concentration of the target protein in the fermentation broth). These critical metrics will help establish biosynthesis as an alternative to existing and established agricultural-based production. Biosynthesis uses less energy, is less resource intensive, and has further benefits of steady supply chains and a higher purity, a superior product.

Figure 2., below illustrates the general production scheme and metrics to produce 1000 kg of Cannabigerol (CBG). Key take aways from Figure 2., are the cultivation / fermentation durations and the production yields; biosynthetic processes providing a clear advantage in the production speed (stable supply) and the reduced production footprint. The biosynthesis process illustrated can readily be achieved in a building footprint of less than 1 acre.

As Figure 2., above demonstrates, biotechnology and synthetic chemistry allow cannabinoids to be produced at a commercial scale with increased cost-efficiency and significantly decreased environmental drawbacks compared with agricultural production. To produce 1,000kg of CBG, field-based production requires 208% more time.

Biosynthetic processes at production scale

Microbial fermentation

Biosynthesis is a broad term encompassing microbial fermentation, genetic engineering and molecular biology all leveraged to create modified microorganisms with the capability to produce an organism with novel traits and alternative access to bioproducts. These candidate organisms are then transferred into the care of fermentation scientists, who test out the enabling cellular machinery in real-world conditions. These experts create the physical environment for the organisms to grow. Fermentation is considered the obvious technology for the effective conversion of renewable, sustainable feedstocks into desirable secondary products. Fermentation has been shown to be well suited to the conversion of sugars and vegetable oils [8]. There are several examples demonstrating the potential of fermentation to utilize a broad range of carbon sources as feedstocks: from simple gases like biomethane and hydrogen; vegetable oils; simple and complex sugars. The organism and fermentation process must be able to use and convert a specific carbon feedstock while also directly yielding specific products of commercial interest [9-11]. Employing biotechnological methods, the resulting metabolites can be produced in relatively high conversion yields and titres [11]. There are some outstanding examples where the fermentation process has been implemented in industry through metabolic engineering: in Escherichia coli and Saccharomyces cerevisiae; the production of 1,4-butanediol [12,13]; and succinic acid [14]. Lee et.al., [15] presented an informative “map” of chemicals, and the routes to synthesize them using fermentation. The speed and precision of biotechnological advances is staggering; however, barriers remain, largely due to cellular constraints such as uptake and export rates, and the need to balance energy and redox, as well as growth constraints. Carbon feedstocks need to be directed in a balanced ratio between cell biomass and desired product, the two are closely tied and different depending on the microbial bio-factory employed (bacterial vs yeast). There are cases where this balance or trade-off does not exist, because either the theoretical yield or the productivity is too low. These are central metrics and significant efforts are invested into analyzing the controlling factors, leading to a reduced number of potential chemicals that can be produced via biosynthetic routes [16,17]. Significant enhancements in biotechnological production are becoming realities using leading computational methods in metabolic engineering [18]. With these technological leaps, challenges that seem insurmountable today may very well be minor issues in the near future.

Binarity of bioprocesses

Upstream

In every bioprocess there are two fundamental elements, up-stream or fermentation, mainly driven by biology, followed by down-stream or isolation and purification, governed by physical chemistry. Process requirements include the requisite need for a bio-based feedstock, a stable biological conversion technology, as well as downstream product recovery schemes falling within the framework of “green” or sustainable chemistry. For a fermentation production process to be sustainable, the choice of feedstock is key. For bulk-chemical production, the feedstock can contribute somewhere between 70 to 80 % of the up-stream production cost. The use of a sustainable feedstock is an essential pre-requisite for a bulk product (including cannabinoids) production process as it largely dominates the environmental profile of these processes [19]. In defining what a sustainable feedstock looks like, it must meet the basic requirements of the triad shown in Figure 3. The feedstock must be:

1. Renewable: to ensure that CO2 release upon product end-of-life will be re-incorporated in growth to produce future feedstock [20].
2. Inexpensive: competitive as the cost of feedstock is a dominant factor in the process.
3. Available: not to compete with food chains and will not lead to price variations as a result of market disturbance [21].

The majority of fermentation processes use refined glucose or sucrose as the feedstock because the organism bio-factories can efficiently use it [22], with glucose being the universal carbon source for modern industrial fermentation processes. As the number of fermentation processes increase in both number and scale, availability of refined glucose (or sucrose) will begin to be stressed; even though it is renewable and inexpensive, and it comes from a wide variety of sources (sugar beets, sugarcane) but too high of an industrial demand will detract from the food chain supply. When demand exceeds supply, prices rise, and the concept of sustainability becomes stressed. Thus, alternative, non-food sources of glucose are being explored, with significant work and investment in extraction from waste lignocellulosic material [23]. There are innovative processes being developed using alternative feedstocks for example the use of bio-methanol for producing single cell proteins [24]; however, such examples are limited and only represent a fraction of the total existing fermentation processes of any given bioproduct. Regionality also plays a decisive role as there may be a bias towards one feedstock over another, the most notable example is South American ethanol producers using the abundantly available sucrose in the region opposed to a refined glucose.

Down-stream

The recent evolution of technology-augmented molecular biology is occurring at unprecedented rates, enabling major advances in upstream processes, which are leading to dramatic improvements in biologically diverse products, cell culture titers, and overall productivity. At the same time, this evolution places increased strain on existing downstream bioprocessing technology, where product recovery schemes are dominated by mature unit operations that have not seen the same rate of advancement as the biological front-end. Product recovery processes makes up a substantial cost of the overall production either through the cost of the process itself or the purification performance, both impacting the amount of actual recovered material. Recovery and purification of fermentation products (small molecules) in general is the costliest unit operation, typically 40-60% of the overall process. Figure 4 is presented showing the general steps of a typical bioprocess [25].

Bulk-chemicals produced via fermentation are reasonably soluble in water and the technologies and approaches for recovery have been developed and matured to work efficiently. Cannabinoids on the other hand have limited to no water solubility and are thermally unstable, thus creating a novel challenge to production and recovery. Approaches taken on the upstream to compensate, tend to cascade down-stream creating purification complexity, novel methods and approaches are co-evolving; the fact remains that this is the major current hurdle in realizing direct, cost competitive biosynthetic production of cannabinoids as compared to conventional agriculture. This issue is also present when processing agriculturally sourced cannabinoids. It is, however, somewhat minimized as plant-based cannabinoids are most commonly brought to an 80-85% level of purity and in the form of “distillates” which are a far cry from 100% pure. The exception does lie with easily crystallizable molecules such as CBD and CBG, hence the abundance. To approach high purity, efficiencies must be high and closely matched at both the upstream and down-stream phases to optimize economic return. Agricultural derived cannabinoids start off as complex mixture, comprised of hundreds of compounds impacting the purity and quality of the final purified cannabinoid, with the main “bad-players” being trace pesticides and the presence of the intoxicating THC, which is not present through biosynthetic production routes.

Isolation and recovery of fermentation products in general, including cannabinoids, is essential as “products” tend to be single components brought to high purity through several processing steps, the more steps the more cost and loss. This consequently makes it more expensive than the fermentation process itself and the node with the greatest economic challenge. For the recovery of small molecules (cannabinoids fall into this classification), distillation methods are of high importance; several variations are available, and much knowledge exists. Distillation is an effective go-to process where energy is cheap and for many processes this operation will remain dominant. Distillation can rarely be applied directly to fermentation cultures; there must be a preceding separation of the biological component. Cell separation is achieved either by filtration (usually microfiltration, but dependent upon cell morphology) or centrifugation to clarify the liquid culture. The liquid portion of the fermentation broth contains the actives of interest (cannabinoids) and other fermentation products, the selective removal from this volume is the first step of purification. Cannabinoids are extracted into an organic solvent, phase separated and the cannabinoids are removed and concentrated by either solid or liquid phase chromatographic processes. Reactive extraction [26] and crystallization are technologies that are applied as a last step to obtain the cannabinoid in purity exceeding 95%.

Aspects of sustainable production

Industrialization and the exponential increase in the worldwide human population are drastically affecting the living standard and availability of resources. These challenges have resulted in continuous depletion of natural sources and pose many problems, such as energy crises, global climate change and food shortages [27]. Sustainability and sustainable development are a balancing act between competing needs - our need to move forward technologically and economically, and the need to protect the environment in which we and others share, and it has become of paramount importance to develop clean and sustainable sources to produce fuels, commodity chemicals, food ingredients and pharmaceuticals. In this context, the utilization of natural substances demands sustainable approaches to balance the use of environmental resources, economic growth, and social responsibility. Biotechnology and chemical synthesis encompass key technologies, such as fermentation, bio-catalysis, semi-synthesis and full synthesis represent a sustainable approach to the production of various biobased products [28, 29].

Biosynthesis via fermentation technology is well established and is currently used to produce a plethora of useful products using renewable sources instead of fossil-based sources. The utilization of diverse renewable sources, including lignocellulosic biomass, agro-industrial residues, food wastes and wastewater from industrial plants to produce biobased products and value-added compounds through fermentation processes fulfils the economic viability of manufacturing, and greater sustainability from an environmental point of view [30]. Fermentation feedstock sources are one of the major topics in biosynthetic process sustainability. First generation feedstocks were the first crops and plants used to produce biosynthetic products, they are considered the most efficient fermentation carbon source as they require less land to grow and have a higher yield and “fermentative efficiency” than other feedstock generations.Examples of first-generation feedstock include corn, wheat, sugarcane, potato, sugar beet, rice and plant oil [31]. Nearly all industrial fermentation processes currently utilize first generation feedstocks, as the organism bio-factories developed demand such a requirement. All the current biosynthetic cannabinoid producers employ first-generation feedstocks, with some emerging cannabinoid companies exploring alternatives such as C1 (bio-methane, CO2, methanol) and second-generation feeds. Second generation feedstock refers to crops and plants that are not suitable for human or animal consumption [31]. Second generation feedstock are categorized as either non-food crops (cellulosic feedstock) or waste materials from first-generation feedstock (e.g., waste vegetable oil).

For the foreseeable future, economic and environmental sustainability will be the technology drivers. Biosynthesis will continue to be at the heart of advancements; however, a transition from large-scale fermentation process relying on living organisms to produce products will gradually give way to hybrid and/or bio-catalytic processes where the whole organism is not required but only the enzymatic machinery. Bio-catalysis is the use of biologically active components (enzymes) to facilitate transformations [32]. This process evolution advances both the economic and environmental sustainability aspects, as less fermentation biowaste is produced; processes can be made continuous (opposed to fermentation which is batch) resulting in a smaller operational footprint resulting in lower energy use. Additionally, enhanced capabilities may be tapped as the enzymes are outside of the organism where they must adhere to biological constraints. Scientific research is helping to understand the structure and functional activities of enzymes, which is in turn leading to an increase in their stability, activity, sustainability, and substrate specificity.

Conclusion

There is a significant need for sustainable bulk-chemical production, including the popularized cannabinoids, particularly given the cultural pressures of rising consumer awareness, product sustainability, climate change and environmental stewardship. Biotechnology offers opportunities to enable more sustainable production of feedstocks, bioconversion and even downstream recovery. A range of new technologies and research will need to be carried out to usher in industrial acceptance and implementation. Together with the need for creative chemical solutions and biological improvements, there is also the requirement for more process-based research. Meanwhile, developments in molecular biology appear to show no slowing, and will dominate much of the biotechnology landscape. For biotechnologies to succeed in supplanting chemical or agricultural processes, it is imperative that molecular biologists work together with bioprocess engineers to create seamless processes. Biotechnology developers will endeavor to create a bio-based manufacturing industry using microbes to produce fuels, chemicals, and medicines. Natural plant products, such as medicines, flavors, and fragrances, will continue to be challenging to extract sustainably from natural sources or to be synthesized chemically due to their complex structures. Developing and incorporating such pathways into new hosts requires finding or modifying a suitable microorganism host that will accommodate and accept the pathway, resulting in a biosynthetic route to the compound followed by scaling the process. Feedstock type and supply will likely be a large steering force, gradually shifting from first generation agricultural to fully renewable. For some chemicals, the choice may be clear, for others the presence of impurities in the feedstock may be challenging. Hybrid processes using combinations of fermentation, cellular and enzyme bio-catalysis and chemical catalysis [33], as well as leveraging the strengths of each conversion technology, will pave the way for effective, sustainable production of cannabinoids. As the technology develops, matures and the industry further recognizes its potential, the production of cannabinoids through fermentation of microorganisms will be the next frontier for biotechnology through improved production processes that prioritize product quality and environmental sustainability. The environmental benefit of using lab-made cannabinoids is evident.

References

^{1 Sicard, D., and Legras, J. (2011). Bread, beer, and wine: Yeast domestication in the Saccharomyces sensu stricto complex. C. R. Biol. 334, 229–236.}

^{2 Steensels J, Meersman E, Snoek T, Saels V, Verstrepen KJ. Large-scale selection and breeding to generate industrial yeasts with superior aroma production. Appl Environ Microbiol. 2014; 80(22):6965-6975.}

^{3 Henry I. Miller (2018) Genetic engineering applied to agriculture has a long row to hoe, GM Crops & Food, 9:1, 45-48}

^{4 Marasco E.K., Schmidt-Dannert C. (2007) Biosynthesis of Plant Natural Products and Characterization of Plant Biosynthetic Pathways in Recombinant Microorganisms. In: Verpoorte R., Alfermann A., Johnson T. (eds) Applications of Plant Metabolic Engineering. Springer, Dordrecht.}

^{5 Woodley J.M., Towards the sustainable production of bulk-chemicals using biotechnology. New BIOTECHNOLOGY 59 (2020) 59–64}

^{6 Becker J, Wittmann C. Advanced biotechnology: metabolically engineered cells for the bio-based production of chemicals and fuels, materials and health-care products. Angew Chem Int Ed 2015; 54:3328–50.}

^{7 Kuddus, Mohammed, Ibrahim AM Ginawi, and Awdah Al-Hazimi. "Cannabis sativa: An ancient wild edible plant of India." Emirates Journal of Food and Agriculture (2013): 736-745.}

^{8 Sanford K, Chotani G, Danielson N, Zahn JA. Scaling up of renewable chemicals. Curr Opin Biotechnol 2016; 38:112–22.}

^{9 Liu Y, Nielsen J. Recent trends in metabolic engineering of microbial chemical factories. Curr Opin Biotechnol 2019; 60:188–97.}

^{10 Liu Q, Liu Y, Chen Y, Nielsen J. Current state aromatics production using yeast: achievements and challenges. Curr Opin Biotechnol 2020; 65:65–74.}

^{11 Davy AM, Kildegaard HF, Andersen MR. Cell factory engineering. Cell Sys 2017; 4:262–75.}

^{12 Yim H, Haselbeck R, Niu W, Pujol-Baxley C, Burgard A, et al. Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nat Chem Biol 2011; 7:445–52.}

^{13 Burgard A, Burk MJ, Osterhout R, Van Dien S, Yim H. Development of a commercial scale process for production of 1,4-butanediol from sugar. Curr Opin Biotechnol 2016; 42:118–25.}

^{14 Choi S, Song H, Lim SW, Kim TY, Ahn JH, Lee JW, et al. Highly selective production of succinic acid by metabolically engineered Mannheimia succiniciproducens and its efficient purification. Biotechnol Bioeng 2016; 113:2168–77.}

^{15 Lee SY, Kim HU, Chae TU, Cho JS, Kim JW, Shin JH, et al. A comprehensive metabolic map for production of bio-based chemicals. Nat Cat 2019; 2:18–33.}

^{16 Dugar D, Stephanopoulos G. Relative potential of biosynthetic pathways for biofuels and bio-based products. Nat Biotechnol 2011; 29:1074–8.}

^{17 Straathof AJJ, Bampouli A. Potential of commodity chemicals to become bio-based according to maximum yields and petrochemical prices. Biofuels Bioprod Bioref 2017; 11:798–810.}

^{18 Von Kamp A, Klamt S. Growth-coupled overproduction is feasible for almost all metabolites in five major production organisms. Nat Comm 2017; 8:15956.}

^{19 Kümmerer K, Clark JH, Zuin VG. Rethinking chemistry for a circular economy. Science 2020; 367:369–70.}

^{20 Zimmerman JB, Anastas PT, Erythropel HC, Leitner W. Designing for a green chemistry future. Science 2020; 367:397–400.}

^{21 Palsson BO, Fathi-Afshar S, Rudd DF, Lightfoot EN. Biomass as a source of chemical feedstocks: an economic evaluation. Science 1981; 213:513–7.}

^{22 Nascimento VM, Fonseca GG. Effects of the carbon source and the interaction between carbon sources on the physiology of the industrial Saccharomyces cerevisiae CAT-1. Prep Biochem Biotechnol. 2020; 50(4):349-356.}

^{23 Sheldon RA. Green and sustainable manufacture of chemicals from biomass: state of the art. Green Chem 2014; 16:950–63.}

^{24 Abou-Zeid A., Abou-Zeid, Ahmed O.Baghlaf, Methanol as the Carbon Source of Production of Single-Cell Proteins (SCP-s). Zentralblatt für Mikrobiologie, 1983; 138(6); 451-464}

^{25 Kenneth P.Clapp, Andreas Castan, Eva K.Lindskog, Chapter 24 - Upstream Processing Equipment. Biopharmaceutical Processing: Development, Design, and Implementation of Manufacturing Processes. 2018:457-476}

^{26 Sushil Kumar, Shitanshu Pandey, Kailas L. Wasewar, Namık Ak, and Hasan Uslu. Reactive Extraction as an Intensifying Approach for the Recovery of Organic Acids from Aqueous Solution: A Comprehensive Review on Experimental and Theoretical Studies. Journal of Chemical & Engineering Data 2021; 66(4):1557-1573}

^{27 Fathima, Hina. (2014). Problems in Conventional Energy Sources and Subsequent shift to Green Energy.}

^{28 Hara, K.Y., Araki, M., Okai, N. et al. Development of bio-based fine chemical production through synthetic bioengineering. Microb Cell Fact 13, 173 (2014).}

^{29 David Romero-Suarez, Jay D. Keasling, Michael K. Jensen, Supplying plant natural products by yeast cell factories. Current Opinion in Green and Sustainable Chemistry 33, 100567, (2022)}

^{30 Rafael Rodrigues Philippini, Sabrina Evelin Martiniano, Avinash P. Ingle, Paulo Ricardo Franco Marcelino, Gilda Mariano Silva, Fernanda Gonçalves Barbosa, Júlio César dos Santos and Silvio Silvério da Silva, Agroindustrial Byproducts for the Generation of Biobased Products: Alternatives for Sustainable Biorefineries, Front. Energy Res., 29 (2020)}

^{31 Pobitra Halder, Kalam Azad, Shaheen Shah, Eity Sarker, Prospects and technological advancement of cellulosic bioethanol ecofuel production. Advances in Eco-Fuels for a Sustainable Environment. Woodhead Publishing Series in Energy 2019, Pages 211-236Bell, E.L., Finnigan, W., France, S.P. et al. Biocatalysis. Nat Rev Methods Primers 1, 46 (2021)}

^{32 Schwartz TJ, O’Neill BJ, Shanks BH, Dumesic JA. Bridging the chemical and biological catalysis gap: challenges and outlooks for producing sustainable chemicals. ACS Catal 2014; 4:2060–9.}

^{33 Yu, S., Liu, JJ., Yun, E.J. et al. (2018) Production of a human milk oligosaccharide 2′-fucosyllactose by metabolically engineered Saccharomyces cerevisiae. Microb Cell Fact 17, 101}

^{34 New Scientist Magazine (2022) Synthetic malaria drug could stem resistance.}

^{35 Vantage Market Research (2022) Cannabidiol (CBD) Market Projected to Reach Valuation of USD 47.22 Billion by 2028.}

^{36 Association for the Cannabinoid Industry (ACI) (2021) Market Sizing: Demand for CBD Soars During Lockdown as UK Market Now Estimated at £690 Million.}

^{37 Prohibition Partners (2020) Key Insights from The Impact Series: Disrupting Beauty.}