RoSynth Project Implementation Plan

The co-culture system of yeast and bacteria on a “mega-plate” Petri dish by a duo-channel 3D bioprinting handling robotic arm which satisfies these customers’ needs. The robot arm is able to help print longer bioink strains and therefore more yeast and bacteria can react. Additionally, the Petri dish size is customizable and hence customizable amount of chemicals as requested, ensuring sustainable and efficient industrial operations.

Main components of this novel machine designed for industrial production:

• Robotic Arms: Able to extend and stretch to distant locations and transfer the bioink precisely and accurately
• Dual-Channel: A motor is incorporated in the robot arm which can drive the channels for alternating bioinks (printing bacteria and yeast as needed)
• Movable Base: The liquid handling robot’s base is movable, further enhancing its flexibility to reach distant locations for printing. It will also allow better space utilization and the machine will be able to work at different stations and carry multiple printing tasks.
• Pipette Tips: Printing nozzles are disposable after use, this avoids possible contamination when transferring the liquid
• UV Light Implemented at the Wall of the Mega Parallel Culture Plate: The UV light will be used to illuminate the plate and the pipette tips at the beginning of each printing to kill all the bacteria and viruses and avoid bio-contamination.

We will apply The Good Manufacturing Practices (GMP) requirements by the United States Food and Drug Administration throughout our project, encompassing a comprehensive set of standards aimed at ensuring the quality and safety of the biomanufacturing of rosmarinic acid. We will maintain high level of cleanliness within the manufacturing facility, utilizing suitable materials such as the safety and durability of our 3D bioprinter for the small-scaled chemical production and the robotic arm for upscaling throughout the production process, establishing clear and well-defined roles and responsibilities among personnel [17]. These GMP guidelines collectively promote the production of safe, high-quality goods while minimizing potential risks and hazards to both consumers and the manufacturing process for our project.

Extraction

The extraction process of rosmarinic acid produced by 3D-bioprinted microbes involves dissolving the bioactive compound from the bioprinted hydrogel incubation culture media. Typically, solvents such as ethanol or methanol are used to dissolve rosmarinic acid from biological matrices [18]. The extraction solution is then subjected to techniques such as maceration, sonication, or Soxhlet extraction to facilitate the transfer of rosmarinic acid (RA) into the solvent [18]. After extraction, the solvent is usually evaporated under controlled conditions to obtain a concentrated rosmarinic acid extract and the rosmarinic acid will be validated by HPLC as we discussed with Dr. Elizabeth Onderko from Capra Biosciences. This extract can then be further purified and processed to produce a high-quality, purified form of rosmarinic acid suitable for a variety of applications, including pharmaceuticals, cosmetics and dietary supplements. The extraction process is a critical step in obtaining rosmarinic acid from 3D bioprinting systems for subsequent use in various industries.

Additionally, a simpler, cheaper and more environmentally friendly extraction process could be dissolving the bio-ink by competing away the cross-linking calcium ions with sodium citrate since we are using alginate for the bio-ink. One nice feature of the bioprinted microbes is that it allows the biomass to be saved and repeatedly used for several batches of RA production. If the RA will diffuse out of the bio-ink by itself then it will be much easier to collect the RA that diffused into the media instead of dissolving the bioink - this will avoid solvent usage and loss of biomass. The diffusion process could also be sped up by pumping or sucking media through the bioink.

Purification and Storage

The purification process of rosmarinic acid produced by 3D bioprinters typically begins with the extraction of the compound from the bioprinted bioink stack. The extract may contain impurities such as other microbial metabolites, bacteria/yeast cell debris, or solvents used in the extraction process. To purify rosmarinic acid from the culture medium, the extract can be subjected to various techniques, including solvent extraction or chromatography, depending on the specific impurities present, and suitably dried in vacuum until approximately one third of its volume.

These methods are designed to separate rosmarinic acid from contaminants and produce high-purity compounds suitable for various industrial applications. Purification is critical to ensure the quality, safety, and effectiveness of the rosmarinic acid extracted after the 3D bioprinting and incubation process.

Distribution

Since we choose to solve the local problem first and aim for the market in the Great Rochester Area, our production process will take place in Rochester, NY, and sell in the local area.

Traditional Value Chain

• Lots of shipping! High cost and emissions, products can degrade in transit
• Extensive upstream processes are required to procure and refine production materials, and value is lost along the way for rural farmers and laboratory suppliers as they pay others to manage their goods. For example, India’s Medical and Aromatic Plant trade has a lot of MAP and the harvesting is done by those in poor rural communities but that value tends to be added externally and not to these communities themselves [19]
• Major value addition is in manufacturing (extraction or synthesis from raw materials)
• Lots of transactions in supply chain which means larger cost for final suppliers, higher price of product for consumer without higher value of product
• Current sourcing methods for raw plant materials are both unsustainable and do not guarantee a standard of quality, which can increase the cost of extraction and impact value of final product

Cosmetics Industry:

• Skin care product ingredients and quantities are customizable with the exact formulation ingredients.
• Bioavailability for the skin delivery by traditional topical formulations is less than 2% of each dose applied [20]. The 3D biomanufacturing approach has several benefits when compared to conventional manufacturing methods, including the ability for one-step fabrication and customization [21]. In addition, the utilization of 3D printed chemicals has demonstrated promise in enhancing the effectiveness of skin delivery and encouraging adherence among users [22]
• Our system enables printing complex compound ingredients and allows for anti-aging and antioxidant creams [5] that could be customized for different people.
• Branding 3D printed skincare ingredient methods through co-culture could make us stand us out among the competitors and differentiate ourselves from the market competition.

Nutraceutical/Pharmaceutical Industry:

• We could personalize the ingredients needed by different stakeholders.
• Research has shown rosmarinic acid can prevent tumorigenesis due to its antioxidative and anti-inflammatory properties [23]. Rosmarinic acid can inhibit the growth of tumor cells and therefore, can be widely used in tumor therapies [23]. Additionally, the 3D bioprinted chemicals have more enhanced properties compared to the traditional method.

Research Labs/ Healthcare Research & Development Sector:

• From our interview with Dr. Nelson, he was particularly interested in our customizable dual-channel 3D bioprinter and mentioned that such device has a great market potential to produce different chemicals needed by different research groups.

RoSynth values sustainability as a crucial part of our project implementation plan, and this project will contribute to the Sustainable Development Goals [24] as described below:

We are aiming to solve local problems by providing a stable, efficient, and localized source of chemicals for local people to use and thus minimize the amount of transportation with reduced greenhouse gas emission. Also, our bioinks are biodegradable according to our deformation tests, and our co-culture system is independent of geographical and climate restrictions, enhancing agricultural sustainability. We also raised public awareness about climate change by different outreach events and IHP interviews with different stakeholders. RoSynth’s project is well aligned with Sustainable Development Goal 13 by mitigating climate change, increasing bioproduction, and further advancing e climate resilience for the botanical market.

Furthermore, Ms. Maria Julia Oliva, from union of Ethical Biotrade, mentioned in an interview that the project satisfies SDG 15 and 17 by providing an alternative innovative solution promoting the biodiversity conservation and contribute to the preservation of ecosystems especially the fragile mediterranean ecosystems [25] and engaging interdisciplinary research taking different stakeholders’ opinions such as academic institutions, industries, and non-profit agencies for developing the project.

Why are our project a good fit for Sustainable Development Goal 13?

Sustainable Development Goal 13 (SDG 13) “Climate Action” focuses on tackling climate change by taking measures to reduce greenhouse gas emissions, adapt to the RoSynth’s impacts and promote international cooperation [24]. Our project specifically targets reducing emissions by ensuring minimal transportation during the distribution process. In addition, we have implemented climate adaptation measures to protect against climate-related disruptions and further stabilize supply chains. By proactively raising environmental awareness and conducting education activities,we help build public understanding and support for our project.

Why are our project a good fit for Sustainable Development Goal 15?

Sustainable Development Goal 15 (SDG 15) is called “Life on land” which aims to protect, restore, and sustainably manage terrestrial ecosystems and mitigate biodiversity [24]. By providing our biomanufacturing solution to produce plant-derived chemicals locally, we help prevent unregulated agriculture practices and harvesting, land degradation, and protect biodiversity. Our project underlines its commitment to protecting life on land to ensure the well-being of terrestrial ecosystems and the species that inhabit them.

Why are our project a good fit for Sustainable Development Goal 17?

Sustainable Development Goal 17 (SDG 17), “Partnership on the Goals,” emphasizes the critical role of collaboration and cooperation in achieving all other SDGs and calls for strengthening global partnerships, strengthening resource mobilization, and promoting knowledge sharing to address the multifaceted challenges of sustainable development [24]. Aligned with SDG 17, our projects actively seek partnerships and collaborations with a variety of stakeholders throughout our project development, including local communities, academia, NGOs, and different businesses, to maximize RoSynth’s positive impact. Through these partnerships, we draw on diverse expertise, resources, and networks to ensure a more comprehensive and effective approach to sustainable development and our project in general.

We made impact analysis a major consideration throughout the project. Our printing system will lead to a shift in demand from traditional farming methods and long shipping times to simple and convenient steps. In the long term, this will result in significant growth for the greater Rochester economy and the U.S. economy. Additionally, since people can customize the chemicals they want to produce and the quantities they want to produce, companies, organizations, and individuals will need to wait less and save more money through less shipping after production lines are optimized.

Indirect impacts include greenhouse gas emissions from the production and transportation of products, as well as over-harvesting of wild populations or unregulated agricultural practices. In addition, the long-term effects of exposure to the engineered strains during production, transportation, or the environment are also indirect effects of our products. The impact of yeast and bacteria escaping from laboratory environments on healthy individuals is currently unknown, but this is unlikely under strict safety and security guidelines. Our goal is to adequately reduce the release of microorganisms into the environment by establishing policies and regulations that, as previously stated, discourage dual uses of items wherever possible.

Market Expansion:

After the 3D bioprinting system is launched in industry and the optimized chemicals start entering the market in North America, we aim to provide our solutions to Asia-Pacific and Europe, the major botanical ingredients markets especially for personal care and cosmetic industries [5]. We will gradually expand our market to the globe while supporting human rights and fully taking bioethical, environmental, and socio-economical factors into consideration during our development. According to the Frost & Sullivan report on revenue forecast by region 2020-2030 [5], the market value of botanical ingredients is $1,424.5 million in Asia-Pacific, $743.1 million in North America and $461.5 million in Europe by 2030.

Since the cost for the first market entry is typically very high and we need enough revenue to recover the cost, we decided to focus on producing on a small scale locally first to manifest the success in the short-term and access other markets in the long-term.

To increase our product accessibility on the global scale, we should lower our prices in the low-income countries by developing our manufacturing sectors in those areas which would minimize the costs such as employment, transportation-related costs, and increase the convenience to access the local market.

After manifesting our success regionally, RoSynth Biotechnologies will continuously develop our Research & Development sector and collaborate with different field experts and researchers to explore other potential applications for the bio-manufacturing method we developed whenever a value proposition for applications emerges.