. Implementation .

Overview

Forests play a crucial role in carbon sequestration, which helps slow global warming and mitigate the climate crisis. However, official data indicated that 129.65 million tons of paper products were produced in 2023, which accounts for 39.5% of total timber consumption in China. Thus, improving the efficiency of paper recycling and deinking can help save timber, protect forests, and alleviate the climate crisis.

The current deinking methods demand a substantial amount of toxic chemicals. It further results in the release of poisonous organic agents and heavy metals that may cause environmental contamination, which are the critical bottlenecks in improving the utilization rate of recycled pulp and realizing the greening of the paper production process. We design an alternative solution based on synthetic biology (see Design for details) with higher deinking efficiency and lower cost, facilitating the recycling of paper. Our project also provides a new way for the green transformation of related industries, proposing a new pathway to address the climate crisis.

Who Are the End Users?

Our project can be of great benefit to recycled paper enterprises and researchers.

1. Recycled Paper Enterprise

Our project primarily targets the paper enterprise that uses waste paper as raw material. Currently, paper production is over-dependent on forests, which may lead to the forest resource crash and deteriorate climate crisis. The current waste paper recycling route still faces with many challenges, such as high cost, environmental pollution, and health risks (please refer to IHP for more information). Our project provided paper enterprise with a standard operating procedure, in which the chemical deinking process was replaced by the synthetic biology method. This innovative approach not only addresses the current challenges but also offers economical, sustainable, and safe waste paper recycling. In the future, the paper enterprise can use our technological process directly, reaping these benefits.

2. Researchers

Our project developed an engineered strains which was used in industrial scale. So, we must take the cost into consideration that abandon the complex protein purification steps and inducible system. Instead, we designed a system using signal peptides to secrete enzymes extracellularly regulated by constitutive promoters. We systematically investigate the secretory performance between different signal peptides, such as Algen, Aly01, OmpA, PelB, TorA, YebF, and LMT, in which LMT was discovered in 2021 by our team (XMU-China 2021, XMU-China 2023). Among them, the LMT signal peptide (see Model and Parts for details) showed the best performance in secreting proteins extracellularly in E. coli BL21 (DE3). Beyond that, we also investigated the combinations of constitutive promoter and LMT and selected the best one. This can be an invaluable platform for research groups (and iGEM teams) around the world to develop more and more engineered strains with secretion functions and to skip expensive protein purification procedures.

Moreover, we have established a method for rapidly screening enzymes with high deinking efficiency, enabling us to swiftly identify targeted modification sites (see Part Collection for details). By integrating our efficient secretion system using the LMT signal peptide and optimized Anderson promoters, this comprehensive process is directly applicable by researchers for similar applications and developments. It provides new insights into enzyme engineering and synthetic biology, facilitating the production of high-quality recycled paper. Our parts collection serves as a valuable resource for future iGEM teams and researchers, expediting the efficient production and modification of target proteins.

Usage

1. Deinking

We employ a groundbreaking approach, harnessing the power of engineered E. coli BL21 (DE3) to express a variety of enzymes such as cellulase, monooxygenase, and laccase. These enzymes, with their diverse catalytic functions, enable high-efficiency deinking. Coupled with the signal peptide, the enzymes can be secreted extracellularly, eliminating the need for complex and costly protein purification operations. The supernatant, rich in LMT-CYP199A4 T253E, enhances the two-phase separation system, effectively separating ink from pulp.

2. Extraction

Currently, deinking flotation is the preferred method for removing printed ink from paper during the recycling process in industry. Unfortunately, this method will cause an inevitable loss of fiber, thus lowering the recovery rate. Therefore, we develop the extraction method to remove the ink in the pulp after enzymatic deinking, increasing overall efficiency and reducing fiber loss. Limonene, a hydrophobic solvent that can be biosynthesized (1), effectively enhances ink separation efficiency, making the paper recycling process more environmentally friendly and efficient. So, we developed an alternate routing that extracted the ink in the pulp, overcoming the shortage of flotation method that carries away a large number of pulp fibers to minimize the loss of fibers.

3. Recovery of Heavy Metals

To address heavy metal in the wastewater during deinking, we engineered E. coli BL21 (DE3) with heavy metal ion adsorption capabilities (see Design for details). We cultured and collected the engineered strains and introduced them into the wastewater treatment pool. After a certain period, heavy metal ions such as Cd²⁺, Cr₂O₇^2-, and most divalent metal cations are enriched in the engineered bacteria. The bacteria, which settle to the bottom of the pool, are collected for further treatment and recovery of the heavy metals.

Safety

1. Kill Switch

To prevent engineered bacteria from escaping into the open environment and causing biosafety issues, we designed a blue light-activated kill switch. The switch is activated when exposed to blue light, triggering bacterial lysis and death. We treat the effluent with blue light to ensure complete elimination of the bacteria. This measure further ensures environmental safety by preventing uncontrolled bacterial spread.

2. Risk Assessment

Given the use of engineered bacteria in our project, there are potential risks to public health and the environment. Therefore, before we implement our devices outside the laboratory, we conduct extensive testing to ensure the safety of our engineered bacteria for humans and the environment. This thorough risk assessment is a crucial part of our process, ensuring that safety is always a top priority.

It is important to note that China has enacted a biosecurity law, which came into effect on April 15, 2021, and was updated in April 2024. This law establishes systems for biosecurity risk prevention and control, including risk monitoring and early warning, risk investigation and assessment, and information sharing. It also includes provisions to prevent and respond to specific biosecurity risks, such as major emerging infectious diseases, epidemics, and sudden outbreaks, and biotechnology research, development, and application. To ensure that our activities are in compliance with the law, we will be applying for the necessary permits and approvals if we proceed with our proposed implementation.

Future Plan

1. Patent Protection

Our technology has reached a maturity level that allows us to pursue patent protection for two key innovations: the enzyme secretion process using extracellular methods and the limonene extraction technique. These unique processes, which are at the forefront of our industry, will be safeguarded by patents, giving us a competitive edge. This protection will enable us to collaborate with industry partners and confidently expand our technological applications.

2. Competitive Analysis and Cost Evaluation

We have conducted an in-depth competitive analysis and cost evaluation. Our findings highlight significant advantages over current methods, particularly in reducing high costs, environmental pollution, and health risks (see LCA for details). Our approach offers a more sustainable and economically viable solution, which is increasingly important as industries seek greener alternatives. By addressing these critical issues, we not only position ourselves as leaders in innovative and responsible paper recycling technologies but also confidence to lead investors of our future success.

3. Scale-up Production and Market Entry

Our next step is establishing a company dedicated to scaling up production and conducting pilot and mid-scale trials. This will involve refining our processes and ensuring consistent quality and efficiency. We aim to build robust sales channels and actively promote our products at industry exhibitions to increase visibility and market penetration. Additionally, we will seek funding to enhance product performance and develop new products, ensuring our solutions remain at the forefront of the industry.

By focusing on resource reuse and sustainability, our approach significantly reduces waste and conserves natural resources. Our innovative deinking process minimizes fiber loss and enables the recycling of paper into high-quality products. This contributes to a circular economy, promoting environmental responsibility and reducing the carbon footprint of the paper industry. Our unwavering commitment to sustainable practices not only benefits the environment but also meets the growing demand for eco-friendly solutions in the market, giving stakeholders confidence in our ethical practices.

References

1. X. Kong et al., Efficient Synthesis of Limonene in Saccharomyces cerevisiae Using Combinatorial Metabolic Engineering Strategies. J. Agric. Food. Chem. 71, 7752-7764 (2023).