. Design .

Background

Global climate change caused by excessive release of carbon dioxide (CO₂) is one of the major challenges around the world. As the world's largest carbon absorbers on land, forests are crucial to the balance of the global environment. However, given the fact that there is a considerable part of the waste paper resources which are not fully used in the paper industry, the amount of deforestation for commercial purpose has been aggravated virtually. The biggest difficulty for recycling waste paper is how to remove various inks contained in waste paper efficiently and eco-friendly. Therefore, we decided to explore innovative ways of paper recycling to improve recycling efficiency and reduce pollution, thereby reducing deforestation and mitigating the climate crisis. See more details in our Project Description.

Solutions

1. Enzymatic Deinking

Deinking of waste paper is to destroy the adhesion of the ink to the fiber through a certain method, according to the composition and characteristics of the ink. In that case, the surface tension of the ink on the waste paper is reduced, resulting in the effects of wetting, penetration, emulsification and dispersion. At the same time, the connection between the ink and the fiber is broken, thus the printed ink is separated from the pulp fiber (1). Recently, the waste paper recycling industry has used various chemicals for deinking, including sodium hydroxide (NaOH), sodium carbonate (Na₂CO₃), magnesium sulphate (MgSO₄) and ethylene diamine tetraacetic acid (EDTA) (2), which results in an alkaline circumstance that will adversely affect the quality of the recycled pulp fibers and cause a certain degree of pollution to the eco-environment as well. However, studies have shown that deinking using enzymes, the biological catalysts, can not only improve the strength of paper, but also reduce the pollution of waste water (3). Therefore, for the purpose of improving the performance of paper and achieving environmental sustainability, we decide to obtain the deinking enzymes by autolysis methods or extracellular secretion from the engineered bacteria, and use these enzymes to replace traditional chemical reagents for deinking, while finally use limonene to extract ink for separation from pulp.

1.1 Deinking System

The deinking system of our REPARO project is a biological enzyme system of high efficiency and stability. To achieve efficient pulp deinking, we selected several enzymes as potential deinking agents, such as cellulase and laccase, to break down ink binder and remove the ink (Figure 1).

Cellulase

Cellulase belongs to the family of hydrolases, which can catalyze the hydrolysis of cellulose and its derivatives, changing the fiber surface or the bonds in the surrounding ink to break the cellulose molecules and promote the separation of ink from the surface of the paper fiber (4). We used the cellulase EG5C of Bacillus subtilis , which can bind closely and quickly to the pulp fiber via its specific cellulose binding module (CBM), contributing to the hydrolyzation of pulp fiber for promoting the deinking effects (5).

Monooxygenase

Monooxygenase participates in a process known as 'oxygen activation' whereby molecular dioxygen (O₂) is bound to, ultimately generating a released reactive oxygen (6). This reactive oxygen species (ROS) causes alkaline breakage of conjugated side chains of lignin and other colored substances such as azo dyes through nucleophilic reaction to achieve the purpose of bleaching (7). We investigated several monooxygenases, including OleT_JE from Jeotgalicoccus sp. ATCC 8456 (8), CYP199A4 from Rhodopseudomonas palustris (9) and SfmD from Streptomyces lavendulae NRRL 11002 (10) to determine the best for deinking of the waste paper.

Laccase

As a lignin-degrading enzyme (11), laccase can degrade the remaining lignin in the fibers on the surface of the pulp through oxidation, weakening the binding between the surface microfibers to promote the separation of ink (12). Besides, laccase can also degrade aniline substances and azo dyes, which will assist the process of deinking. We decided to introduce the laccase from Rheinheimera sp., which was reported to significantly improve the brightness, break length, tear coefficient and burst coefficient of the processed paper (13).

1.2 Ink Separation

Currently in industry, deinking flotation is the preferred method for removing printed ink from paper during the recycling process. The process is based on the difference between hydrophilic and hydrophobic inks, in which the latter can be removed from the pulp suspension by adhering to bubbles (14). However, this method will cause a certain loss of fiber, thus lowering the recovery rate. Therefore, we decided to implement extraction via a certain hydrophobic solvent rather than floatation during the deinking process. We chose limonene, a solvent that can be biosynthesized (15), from a range of candidates to extract the ink in the pulp after enzymatic deinking (Figure 2), overcoming the shortage of flotation method that carries away a large amount of pulp fibers so as to minimize the loss of fibers.

1.3 Enzyme Obtainment

It is relatively easy to produce the target protein (enzyme) from engineered bacteria in the laboratory and purify it, however in industry, the additional purification process will greatly increase the cost of paper recycling. To bring our REPARO solutions closer to the industrial stage, two methods for obtaining target proteins from engineered bacteria without the need of purification have been proposed: one is to construct an autolytic system to induce the lysis of engineered bacteria and release the intracellularly produced target protein, the other is to harness the bacterial secretion machine (signal peptide-dependent pathway) to secrete the target protein into the extracellular circumstance.

Autolytic System

It is reported that the E. coli autolytic system FLSA (FhuD-lysozyme-SsrA mediated autolytic system) (Figure 3) can efficiently release target proteins from cells, so we chose to use this system to realize the release of deinking enzymes after production (16). This system integrates the Sec-Tat dual pathway signal peptide FhuD, T7 lysozyme, and E. coli ClpX/P-SsrA protein degradation machinery for effective cell lysis without disturbing recombinant protein production. First, N-terminal FhuD signal peptide guides the translocation of the folded T7 lysozyme to access the peptidoglycan layer to confer the cell lytic effect. Second, the C-terminal SsrA tag helps to eliminate cell toxicity by degrading the leaky expression of cytoplasmic T7 lysozyme, which can reduce the growth burden caused by T7 lysozyme leaky expression.

Signal Peptide

Since the supernatant of bacterial culture could be used directly, extracellular secretion of target protein rather than undergoing cell lysis after a certain induction offers a possibility to simplify the process of obtaining target enzymes. Therefore, we also proposed a design to secrete enzymes out of the engineered bacteria via signal peptides (Figure 4). Given the previous successful cases of using LMT signal peptide (XMU-China 2021, XMU-China 2023), we decided to compare the performance of this sequence we discovered before with another seven commonly used signal peptide sequences then choose the best one to assist the secretion of deinking enzymes (See more details in our Model page).

2. Harmless Treatment of Wastewater

After enzymatic deinking, many chemical bonds between ink and paper are destroyed, which leads ink to enter the deinking wastewater. However, there are a variety of soluble heavy metals in the ink that are not destroyed in the recycling process of waste paper (17), including lead (Pb), chromium (Cr), cadmium (Cd) and mercury (Hg). As a result, the deinking wastewater cannot be discharged directly, and a demand for removal of heavy metal ions has been placed on the harmless treatment of wastewater. In order to remove heavy metals from wastewater, we decided to use metallothioneins (MTs) to bind them via the strategy of surface display or co-expression with the transporter of specific metal ions (Figure 5).

2.1 Metallothionein

Metallothioneins are a class of low molecular weight and cysteine-rich metal-binding proteins, while one metallothionein can bind 6-9 heavy metal ions. Common metallothioneins can bind divalent heavy metal ions generally, but lack the function of treating hexavalent chromium (Cr₂O₇^2-). However, human metallothionein Mt2A and MT3 have adsorption capacity for both common metal ions and Cr₂O₇^2- ions (18), which can be heterogeneously expressed in engineered bacteria for wastewater treatment. Therefore, for distinct treatment situations, we decided to construct engineered bacteria to express metallothioneins in two different patterns: one is to display the metallothioneins on the surface of engineered bacteria, while the other is to express the metallothioneins intracellularly accompanied with the transporter of heavy metal ions.

Surface Display

It was reported that displaying metallothioneins on the surface of engineered bacteria could improve the efficiency of adsorbing heavy metal ions (19), which might be attributed to the expanded contact area with the metal ions. Additionally, this strategy offers certain portability to subsequent treatment of engineered bacteria to recycle heavy metals. Based on these, we chose the classic INPNC surface display system, which has been proven to display various proteins efficiently (20), to anchor metallothioneins (MT2A and MT3) on the surface of engineered bacteria.

Transporter

Even though better performance on absorbing various heavy metal ions was observed when surface displayed, the metallothioneins showed low efficiency on absorbing Cd²⁺ (21). To tackle this challenge, the metallothioneins are designed to be expressed intracellularly rather than displayed on the surface of the engineered bacteria, while a Cd²⁺ transporter will be co-expressed in the same bacteria to transport Cd²⁺ into the cells, thus achieving the bioremediation of Cd²⁺ (22).

MntA, an endogenous protein in Lactobacillus plantarum , plays the role in transporting Mn²⁺ and Cd²⁺ into the bacteria, whose transporting efficiency for Cd²⁺ will not be interfered by other metal ions except Mn²⁺ (reduced to 20% of the original) (23). Given the extremely low concentration of Mn²⁺ in wastewater and the presence of many other heavy metal ions (17), we decided to overexpress this transporter to meet our needs.

2.2 Biosafety

There is a potential risk that the engineered bacteria might escape into the opening environment since they are involved in the situations to be put directly into the pulp for use (See more details in our Proposed Implementation page), which will pose a threat to the environment and biodiversity indeed once unwanted survival and accumulation of the engineered bacteria happens (24). Considering biosafety, we designed a kill switch for biocontainment of the engineered bacteria. For not adding extra chemicals (such as various inducers) into the wastewater, we decided to design a light-triggered kill switch to realize the "suicide" of the engineered bacteria: after deinking in a dark circumstance, the inbuilt kill switch will be turned to the “ON” state via being exposed to the light illumination of specific wavelength so as to achieve the goal of biocontainment.

At present, many optogenetic systems that have been developed in prokaryotes mainly respond to the wavelength of blue light rather than other colors of light in the visible spectrum (25). Besides, these blue light responding-optogenetic systems generally utilize FMN or FAD as the chromophore of photosensor, which can be synthesized by the bacteria naturally (26). Taking this advantage, no additional chromophore synthase genes need to be cloned and introduced into the engineered bacteria, which remains more space for the functional exogenous genes in a single cell. For these reasons, we decided to select a suitable system that responds to the blue light to build the kill switch. Finally, the blue light-induced eLightOn system was chosen: in the dark, LexRO dimerizes and binds to the promoter pColE408 to inhibit its transcription; while under blue light illumination, the LexRO dimer dissociates from the promoter and thus initiates the expression of gene downstream (27). The eLightOn system showed a better performance over the other existing single-component bacterial light-activated expression systems, with benefits including a high ON/OFF ratio of >500-fold, a high activation level, fast activation kinetics, and/or good adaptability to different promoters and E. coli strains (27).

ccdB, a gene encoding the toxin that interferes with the activity of DNA gyrase (28), is placed under the control of promoter pColE408 to confer the killing effect as a blue light-induced pattern. In addition, to reduce the growth burden caused by the potential leaky expression of toxin, an antitoxin gene ccdA is introduced as well, which is designed to be controlled by a weak constitutive engineering promoter p2)-114v (Figure 6A). And therefore, once the expression level of CcdB upon blue light illumination exceeds that of CcdA, the engineered bacteria will be killed due to the toxicity of CcdB (Figure 6B).

Future Expectations

In daily life, people's needs for handwriting erasure are extensive and diverse. However, our current design is mainly focused on the deinking of pulp in the printing plant, so we hope to develop a product that can easily remove handwriting in daily life according to the deinking principle referred before.

The ink used in daily handwriting is mainly composed of hydrophobic pigments and binders, so using a better hydrophobic solvent may be possible to achieve the removal of handwriting. Our experiments showed that the microemulsion consisting of surfactant sodium dodecyl sulfate (SDS) and limonene had a good effect on dissolving ink and removed the handwriting to a certain extent. Considering environmental protection, we hope to choose rhamnolipid, a kind of surfactant that can be biosynthesized and easily degraded (15), accompanied with limonene to form microemulsion to achieve handwriting removal (Figure 7).

Additionally, for improving the recycling ability of the microemulsion, we intend to utilize adsorbents to adsorb the ink that is dissolved in the microemulsion after being removed from the paper. The adsorption materials containing chitosan as the main component can adsorb ink molecules through both physical and chemical effects while having the advantages of low toxicity, excellent biocompatibility and low cost over the traditional polymer hydrogel (29). Therefore, this kind of material is chosen as the adsorbent conferring the reusability for the microemulsion, thus assisting the removal of daily handwriting.

References

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