Overview

As a recyclable resource rich in nutrients such as amino acids and sugars, the spent coffee ground (SCG) holds significant potential for producing valuable organic fertilizers. Residual caffeine, however, inhibits plant growth due to its phytotoxicity, substantially limiting the application of SCG in biofertilizer production. Conventional decaffeination methods suffer from high costs and environmental concerns. Here, we genetically engineered Escherichia coli (E.coli) to integrate genes involved in caffeine degradation, aiming to remove the residual caffeine from SGG during fermentation while producing valuable biofertilizers. Our project provides an eco-friendly and cost-effective solution for the recycling of SCG. The safety and scalability of the engineered E.coli ensure its broad applicability in agriculture, horticulture, and environmental protection.

The issue and the challenges

As high school students passionate about biology and environmental sustainability, we often discuss how to reduce waste and repurpose resources in our school and local communities, and we are inspired to tackle a real-world problem that intersects daily life with scientific innovation.

Coffee is a widely consumed beverage with annual worldwide consumption exceeding 10 million tons, which leads to a generation of approximately 6~8 million tons of spent coffee grounds (SCG) as waste (Campos-Vega et al., 2015). SCG is rich in organic matter, containing cellulose, hemicellulose, lignin, as well as essential nutrients such as nitrogen, phosphorus, and potassium, making it a potential high-quality raw material for organic biofertilizers (Mussatto et al., 2011; Pujol et al., 2013). However, through our human practice (HP), we noticed that over 90% of citizens are not very familiar with SCG recycling. The majority of SCG is landfilled or incinerated, which not only wastes its value, but also emits greenhouse gases, exacerbating environmental pollution (Karmee, 2018). This sparked our curiosity: could we transform the common waste into beneficial biofertilizer?

Through discussions with our teachers, we learned a truth most (82.24%, through our HP) people don’t know, that caffeine (1,3,7-Trimethylxanthine, C8H10N4O2), remaining in SCG, is a natural phytotoxin that can inhibit plant development by disrupting cell division and root growth (Ashihara et al., 2008). For example, field experiments revealed 10% (v/v) espresso SCG directly composted in the soil substantially decreases germination rates and production yields in crops such as lettuce and tomatoes (Cervera-Mata et al., 2020; Cruz et al., 2015). Therefore, effective decaffeination is an essential step prior to converting SCG into biofertilizer (See Figure 1). Motivated by the desire to create an eco-friendly and scalable solution, we hoped to apply our knowledge in synthetic biology to design a bacterial system that could detoxify SCG, to turn waste into growth, pollution into possibility.

Figure 1. The idea of recycling SCG. Our idea introduces a solution: to detoxify SCG by making the phytotoxic caffeine a neutral molecule. This process transforms SCG from a harmful waste product into a high-value, plant-safe organic fertilizer. Some of the visual elements in this figure are generated by AI.

Current solutions & Problems

Currently, conventional decaffeination primarily relies on physical or chemical methods, including solvent extraction and supercritical CO₂ extraction (SC-CO2) (See Table 1). The fact that the caffeine molecule is highly soluble in organic solvents allows for its extraction using solvents such as dichloromethane or ethyl acetate. This method can dissolve and remove up to 90% of caffeine, but it may lead to loss of other nutrients and residue of chemical solvents (Al-Dhabi et al., 2017). In contrast, SC-CO2 utilizes supercritical CO₂ under high pressure (73~300 atm) and moderate temperature (31~50℃) to selectively remove caffeine from SCG without chemical residues. Notably, this caffeine-specific method is commonly used in decaf beverage industries. It requires expensive high-pressure equipment, which restricts its application in the biofertilizer industry (Saldaña et al., 2002). DIY composting is an inexpensive and eco-friendly way to break down caffeine on a small scale at home, yet the fermentation is slow (several months) and insufficient, resulting in an unstable fertilizer quality (Santos et al., 2017).

Table 1. Conventional decaffeination

Method	Advantage	Disadvantage
Solvent extraction	Remove 90% of caffeine	Chemical solvent residues.Loss of other nutrients.
SC-CO2	High selectivity.	High costs (equipment and operation).
Home composting	inexpensive and eco-friendly	Slow and insufficient fermentation.

Therefore, the above-mentioned methods are not suitable for the biofertilizer industry. We are seeking a new solution!

Previous iGEM project

Previous iGEM projects have also explored bacterial systems for caffeine metabolism, providing valuable insights for our project. The 2015 Austin_UTexas team (#1627) developed a synthetic riboswitch in E.coli that responded to caffeine, showcasing the potential for designing caffeine-sensitive genetic circuits. Meanwhile, the 2024 NEFU-China team (#5313) successfully expressed the ndmABCDE gene cluster in E.coli to degrade caffeine, demonstrating the feasibility of this pathway in a heterologous host. Their work not only confirmed the functionality of key ndm genes but also established a solid technical foundation for microbial caffeine degradation. Building upon these pioneering efforts, we aimed to optimize the system by designing a modular expression strategy to enhance the efficiency and stability of the complete caffeine degradation pathway in E.coli.

Our solution

Our project aimed to address the challenges of caffeine phytotoxicity in SCG through designing a biological decaffeination system. The enzymes, N-demethylases (Ndm; including NdmA, NdmB, NdmC, NdmD and NdmE), encoded by Pseudomonas putida (P.putida) can degrade phytotoxic caffeine into neutral xanthine via sequential demethylations (Summers et al., 2012; Summers et al., 2011; Summers et al., 2013) (See Figure 2). Due to the unsuitability of P.putida for fermentation, we intended to express these ndm genes in Escherichia coli (E.coli), our chassis organism, through a synthetic biology method (Kallio et al., 2014).

Figure 2. N-demethylases-dependent sequential demethylation. Phytotoxic caffeine is degraded through the stepwise removal of methyl groups by the specific enzymes NdmA, NdmB, NdmC, NdmD, and NdmE, originally identified in P.putida. This enzymatic pathway converts caffeine into theobromine, then to paraxanthine, and finally to non-toxic xanthine, effectively eliminating its plant-growth inhibitory effects. Our project involves expressing these ndm genes in E.coli to enable biological decaffeination of SCG. The caffeine-xanthine metabolic pathway is adapted from a previous review (Summers et al., 2015).

To this end, we integrated ndm genes into the pET-28a expression vector (See Figure 3). The coding sequences of the ndm genes were synthesized by a biotechnology company, followed by amplification of these target fragments by polymerase chain reaction (PCR). A double restriction enzyme digestion was applied to linearize pET-28a vector and leave the corresponding sticky ends on the fragments. Subsequently, we ligated the fragments to the vector with DNA ligase and transformed the ligation products in E.coli DH5α to construct pET-28a-Ndm plasmids. Finally, we transformed these plasmids into E.coli BL21, to obtain the engineered bacteria we desired.

Figure 3. Schematic of the molecular cloning procedure to construct the engineered E.coli. The process began with the amplification of ndm genes by PCR using synthetic templates. The amplified ndm fragments and pET-28a expression vector were digested and then ligated. The final constructs were transformed into E.coli, yielding our engineered strains capable of caffeine degradation.

After the engineered bacteria fully grow in the SCG fermenter, the inducer IPTG (Isopropyl-beta-D-thiogalactopyranoside) is added to switch on the transcription of the ndm genes. To optimize the fermentation and caffeine degradation, we also probed for:

​ 1) best fermentation temperature and endpoint

​ 2) the best concentrations of nutrients for bacterial growth and caffeine degradation

Certainly, we will never stop there! We also connected our lab work with real-world needs through our extensive HP. We interviewed with coffee chains like Starbucks and Manner who expressed strong interest in sustainable, low-cost recycling options. Expert advice from Dr. Tong Zhou helped us refine our strategy, suggesting a focus on the home gardening market first and cost-effective production. Conversations with industrial partners like Paques and Tian Ren Xue Bio-Tech provided important awareness of scaling challenges and regulatory requirements. This collective feedback led us to iterate our project design, incorporating a robust bio-containment strategy, planning for post-fermentation sterilization, and adopting a phased rollout approach from greenhouses to open fields.

Perspective

Based on the principles of bioprocess engineering, our benchtop caffeine bio-reactor is designed to provide a safe and efficient environment for caffeine degradation. The core design philosophy centers on stability, controllability, and observability, achieved through a double-layer borosilicate glass structure with a water jacket for precise thermal regulation (±0.3 °C), a combined microporous aeration and mechanical stirring system to optimize oxygen mass transfer, and full visual access for real-time monitoring. The system also ensures a sterile environment via UV sterilization and robust sealing, while modular components support maintenance, scalability, and reproducible operation, making it an ideal platform for the Ndm enzyme-catalyzed detoxification of spent coffee grounds. This bioreactor enables local cafes and communities to directly convert daily coffee waste into safe, nutrient-rich biofertilizer, reducing disposal costs and supporting urban gardening. In schools and research labs, it serves as an accessible platform for demonstrating synthetic biology and enzymatic degradation processes. For future applications, a compact version could allow home gardeners to efficiently transform kitchen scraps into organic fertilizer. By bridging laboratory innovation with real-world implementation, our reactor provides a practical, eco-friendly solution for circular bioeconomy at a local scale.

Our future work will focus on scaling up the fermentation process, validating the safety and efficacy of the resulting biofertilizer in field trials, and exploring the potential for co-production of other valuable compounds (e.g., bioplastics and biofuels) from SCG. By transforming a waste product into a resource, our approach aligns with circular economy principles and contributes to sustainable development goals in agriculture and environmental protection.

References

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