Implementation

Every year, millions of tons of shrimp shells, crab exoskeletons, and insect remains are discarded across the globe. These shells are more than refuse: they are natural reservoirs of chitin, the second most abundant biopolymer on Earth after cellulose. Hard, resilient, and chemically complex, chitin has long resisted efficient recycling, leaving industries with mountains of waste and farmers with growing needs for sustainable soil enrichment.

Our vision with Chitinator et al. is to transform this narrative. By harnessing engineered microbes as living factories, we aim to convert chitin-rich waste into a bioactivator that nourishes the soil, boosts crop yields, and supports a circular bioeconomy.

The core of our approach lies in the design of a fusion enzyme, bringing together the complementary powers of an endochitinase and an exochitinase. Connected by a flexible linker, these domains work in synergy: the endochitinase cuts randomly within the polymer chain, creating new ends, while the exochitinase trims the chain progressively from these ends. Together, they dismantle chitin far more efficiently than either could alone.

We begin with E. coli as a screening platform, using antibiotic-resistant plasmids to confirm expression, secretion potential, and enzymatic activity through assays on colloidal chitin plates and zymography. Once activity is validated, the construct is transferred to Bacillus subtilis 168, a GRAS organism with a natural ability to secrete proteins directly into the environment. At this stage, selection still relies on antibiotic markers , but the long-term vision is clear: plasmid systems without resistance genes, suitable for industrial and environmental use.

Laboratory success is only the beginning. To bring Chitinator et al. to life beyond the bench, we envision large-scale production in bioreactors. These controlled vessels maintain optimal pH, temperature, oxygen, and nutrient levels, allowing B. subtilis to grow and continuously secrete chitin degrading enzymes.

But the story begins even earlier with the pre-treatment of chitinous waste. Shrimp and crab shells undergo washing, deproteinization (removal of residual proteins), and demineralization (elimination of calcium carbonate). After drying and grinding, the shells are reduced to fine chitin powder, the perfect substrate for our engineered bacteria.

When this powder enters the bioreactor, it meets billions of B. subtilis cells releasing fusion chitinases. Bonds are broken, polymers unravel, and chitin is reduced into N- acetylglucosamine (GlcNAc) and other soluble oligosaccharides. What once was an intractable waste product becomes a nutrient-rich solution with high potential in agriculture.

After degradation, the bioreactor broth undergoes filtration to remove bacterial biomass and residual solids. What remains is a concentrated liquid enriched with soluble sugars, nitrogenous compounds, and bioactive fragments released from chitin breakdown.

Instead of being converted into multiple formats, our vision focuses on one clear and practical form: a liquid bioactivator. This liquid is directly usable in agriculture, designed to be sprayed onto crops after the application of conventional base fertilizers.

In garlic cultivation, for example, chemical fertilization provides the essential macronutrients early in the growth cycle. Our liquid bioactivator would then be applied as a foliar spray, coating the leaves and penetrating the plant through natural pores and cuticles. This allows the bioactive molecules to act as growth enhancers and stress protectants, while at the same time enriching the rhizosphere as excess solution drips into the soil.

By combining standard fertilization with a biological activator, farmers could benefit from stronger plant development, improved yield, and a more sustainable farming practice that reintroduces nutrients derived from waste back into the cycle of cultivation.

The true test of Chitinator et al. is not inside the laboratory but in the fields. Imagine garlic crops in Thrace and Thessaly or tomato greenhouses in Crete and beyond. Farmers apply our liquid bioactivator as a foliar spray after their base fertilization, not to replace nutrients, but to unlock the plant’s own strength.

The impact of this application goes beyond simple soil enrichment. Chitin-derived molecules are known to act as elicitors, triggering the plant’s natural defense mechanisms. In practice, this means:

• Plant immunity activation: stimulation of the plant’s innate immune system, making it more resistant to fungal pathogens and other common crop diseases.
• Stress tolerance: improved resilience under abiotic stresses such as drought, salinity, or heat.
• Healthier growth: stronger plants, greener leaves, and more uniform development, leading to higher quality harvests.
• Reduced chemical inputs: by enhancing natural defenses, farmers can decrease their reliance on fungicides and other crop protection chemicals, lowering both costs and environmental burden.

By reintroducing bioactive fragments derived from shrimp shells into the farming cycle, Chitinator et al. turns agricultural practice into a story of resilience: from waste discarded at sea, to crops that stand stronger against the challenges of the field.

Effectiveness will be rigorously evaluated at multiple levels:

• Soil chemistry: quantifying carbon, nitrogen, and phosphorus before and after application.
• Plant physiology: monitoring biomass, growth rate, and stress markers in garlic, tomato, and other key crops.
• Field trials: comparing treated and untreated plots over multiple growing seasons, across different soil types and climates.

These experiments will not only validate the product’s efficiency but also refine its dosage, application methods, and cost-effectiveness for farmers.

While our journey begins within iGEM, it will not end there. Collaborations with universities, research institutes, and agricultural partners will allow us to scale beyond the laboratory. One can envision bioreactors installed near fishing harbors, continuously processing tons of discarded shells into high-value bioactivators for nearby fields.

The potential reaches even further: a global network of waste valorization facilities, turning one of the world’s most abundant biopolymers into a cornerstone of sustainable farming.

Chitinator et al. is more than a project. It is a story of transformation , where waste becomes resource, the ocean meets the soil, and biology bridges the gap between sustainability and necessity.

1. Schumann W. Production of recombinant proteins in Bacillus subtilis. Adv Appl Microbiol. 2019;106:1–21.

2. Westers L, Westers H, Quax WJ. Bacillus subtilis as cell factory for pharmaceutical proteins: A biotechnological approach to optimize the host organism. Biochim Biophys Acta Mol Cell Res. 2004;1694(1–3):299–310.

3. Zhang X, Zhang Y, Liu H. Engineering Bacillus subtilis for the production of industrial enzymes and chemicals. Biotechnol Adv. 2016;34(5):798–805.

4. Harwood CR, Cranenburgh R. Bacillus protein secretion: An unfolding story. Trends Microbiol. 2008;16(2):73–79.

5. Chen J, Zhang W, Wang S. High-cell-density fermentation of Bacillus subtilis for enhanced production of recombinant enzymes. J Ind Microbiol Biotechnol. 2019;46(7):939–950.