Team:UESTC-China/Proof Of Concept
Overview
At present, there is not yet an efficient, environment-friendly, and suitable way of waste paper disposal for various walks of life (especially for office staff). As China Paper Yearbook 2020 gives this data: the recycling rate of waste paper in China
is 46.5% in 2020, while the utilization rate is 54.9%, which is much lower than we expect. In fact, recycling one ton of paper will save 17 trees, 3 cubic meters of landfill space and reduce water pollution by 35%. So it is really
meaningful to spare no effort to solve this problem. So we designed Deinker to realize the in-situ regeneration of office waste paper. For months, UESTC-China is working on this issue hard and have finally
proved the feasibility of paper deinking and initially realize this CONCEPT of the 'Deinker' in the real world.
Bio-Engineering
To achieve the purpose of using enzymes to replace traditional physical and chemical deinking methods, we have tried four different kind of enzymes. Such a variety of enzymes are rarely used in the meantime. After monthes hard working, we found that endoglucanase
EGL7(BBa_K3819003), xylanase XynB(BBa_K3819062), and lipase Lipase(BBa_K3819128) can allow biodeinking to occur, by helping the ink detach from the waste paper and decomposing hazardous substance.
To be specific, EGL7 can cut the surface fibers to separate the printed text from paper; XynB can remove ink and reduce the damage to paper through the complementary and synergistic effect of cellulase; Lipase can degrade the debris of ink which contains
some lipids linked materials, to detach ink from the fiber, to achieve the purpose of deinking. (Click here for more details.) Through Dot-Blot, Western-Blot
and enzyme activity definition, their expression and function have successfully been proved.
1.Dot-Blot
By adding different tags on the C-terminal of target genes, which could combine with specific antibodies and show their expression by substrate chemiluminescence, UESTC-China could easily tell the presence of proteins in fermentation broth(Figure 1). These
proteins were emitted directly into supernatants.
Figure1. The Dot-Blot results of recombined GS115 fermentation broth. All samples were gained after fermentation in YPD for 96h. EGL7(His-Tag), XynB(His-Tag) and Lipase(Myc-Tag) were tested by adding 2ul fermentation supernatant
on NC membrane and exposing 60s.
2.Enzyme Activity Assay
After fermentation in YPD for 3~4 days, we tested enzymes' activities.
DNS method is widely used in polysaccharide hydrolase mediated reducing sugar systems, and the color of reaction system is proportional to the reducing sugar content. For endoglucanase EGL7 and xylanase, which could digest cellulose
and xylan respectively and produce reducing ends, the color of reaction system would be darker than control, in which DNS was added before reaction to inactivate enzyme.
Figure2. The DNS results of EGL7 and XynB reflects enzymes' activity. Both solutions were gained after 96h of fermentation. (a)EGL7 reaction results. 2ml fermentation supernatant reacted with 2ml 0.8%(w/v)sodium carboxymethyl
cellulose (CMC) for 30min, then added 5ml DNS to shut down the process. During boiling in water bath, DNS combined with reducing sugar and produced dark brown, while control just shown unobvious color change.(b)XynB reaction results.
Except changing substrate from CMC to 1% Xylan solution and using ammonium sulfate deposition method to gain concentrated protein, other procedures are same as EGL7's activity defining procedure.
Lipase activity can be measured by Alkali Titration. Olive oil emulsified with polyvinyl alcohol(PVA) by high-speed homogenizer was digested by Lipase and produced fatty acid. Then add two drops of phenolphthanlein incicator and use 100mM NaOH solution
to titrate until reaction systems turn red and maintain more than 30s. Heat inactivated Lipase solution was been used in controls.
Figure3. Alkali Titration results of Lipase. 1ml concentrated protein solution mixed with 5ml 50mM, PH=5.0 phosphate buffer and 4ml emulsion, then reacted at 40℃ for 15min. In the experiment system. Produced fatty acid neutralizes
100mM NaOH solution so experiment system's color was stable during titration, while system turn red directly in control.
3.Western-Blot
Although our chassis, GS115, has a much lower glycosylation level than saccharomyces cerevisiae, we still doubt if glycosylation would interfere our enzyme's structure and activity.
Take XynB as a example, due to low protein concentration in fermentation broth, some bands are not clear enough to be found by SDS-PAGE, so we choose Western-Blot to identify XynB' expression and size.
Figure4. The western blot results of recombined GS115 fermentation broth. Control is GS115’s concentrated fermentation supernatant. XynB, which is 43.4KDa. Line 1, 2, 3 refer to sediment, supernatant and concentrated supernatant.
De-inking Efficiency Validation
In the deinking effect verification experiment, we conducted a single-enzyme experiment, and the results showed that the residual ink spots on the paper were significantly reduced after the action of active cellulase, xylanase, and lipase.
This part of the experiment shows that biological enzymes such as cellulase and xylanase have significant effects in deinking. In addition, although lipase has the effect of deinking, the results of enzyme synergistic experiments show
that the synergistic effect of cellulase and xylanase is the best. So we insisted on our first design, in which only cellulase and xylanase are fused with dockerin and would dock with Scaffoldin and form Cellusome.
1.Cellulase
Residual ink significantly decreases(p＜0.01)when treated time is more than 20min and less than 40min(Figure 5 left). The least residual ink is on 40min treatment, we can see in Figure 5 right that the mean of residual ink shift left which
means the residual ink decreases.
Figure5. Results of Cellulase deinking efficiency. The left table refers to Deinking Efficiency of 1mg/ml business cellulase at different times. The right table refers to Residual ink distribution when treated with 1mg/ml cellulase
in 40min. Inactive business cellulase (Cel-) or active cellulase (Cel+) .
2.Xylanase
Residual ink significantly decreases(p＜0.01)when treated time is more than 1min and less than 40min. The least residual ink is on 30min treatment, and the mean of residual ink shift left which means the residual ink decreases. We also
detected the lignin content before and after treatment with xylanase, the result showed that lignin content dramatically decreased after treating with xylanase in 30min as expected (see in Fig.6).
Figure6. Results of xylanase deinking efficiency. The top table refers to deinking Efficiency of business 0.5mg/ml xylanase at different times; The middle table refers to residual ink distribution when treated with 0.5mg/ml
xylanase in 30min; The bottom table refers to ligin content after treated with 0.5mg/ml xylanase in 30min. Inactive business xylanase (Xyn-) or active cellulase (Xyn+) .
3.Lipase
Lipase can degrade ester content in ink toner, so that the ink can dissolve from the surface of the paper. We can see in Figure 7 that residual ink significantly decreases(p＜0.01)when treated time is more than 30min and less than 40min.
Figure7. Deinking Efficiency of business lipase at different times. Inactive business lipase (Lip-) or active cellulase (Lip+).
4.Multi-enzymes
In addition to the single-enzyme experiment, we also conducted a multi-enzyme synergy experiment. In this experiment, we tested the proportion of synergistic enzymes based on the possibility of the cellosome we designed. The results showed
that the proportion of enzymes with the least residual ink dots when cellulase: xylanase=2:1.
Figure8. Deinking efficiency of multiple business enzymes in 30min or 40min. Cel means we used 1mg/ml cellulase solution to treat the papers. Cel-Xyn means we used 1mg/ml cellulase and 0.5mg/ml xylanase solution to treat the
papers. Cel-Xyn-Lac means we used 1mg/ml cellulase, 0.5mg/ml xylanase, 1mg/ml laccase solution to treat the papers. Cel-Xyn-Lac-Lip means we used 1mg/ml cellulase, 0.5mg/ml xylanase, 1mg/ml laccase and 1mg/ml lipase solution to treat
the papers.
Modeling
Modeling is closely related to our experiments as the theoretical basis.
In order to test our enzyme deinking efficiency, we proposed a statistical method of ink residue count, which helped us to determine simply and directly how much residual ink there is on the paper.
In order to avoid the error and contingency of judging deinking effect by human eyes, we designed a unified standard for ink on any form of paper, and established a model of "Computer Vision Completed Ink Recognition" to quantitatively count the amount of ink left on paper.
In order to verify that there was no problem with our genetic modification of deinking enzyme, we constructed the spatial structure of the protein and carried out molecular docking.
In order to avoid the error and contingency of judging deinking effect by human eyes, we designed a unified standard for ink on any form of paper, and established a model of "Computer Vision Completed Ink Recognition" to quantitatively count the amount of ink left on paper.
In order to verify that there was no problem with our genetic modification of deinking enzyme, we constructed the spatial structure of the protein and carried out molecular docking.
Figure9. The spatial structure of EGL7, EG1, XynB and scaffold.
In order to effectively plan the placement and distribution of our deinking equipment, we built a Deinkers Location Decision-making model to enrich the properties of the deinking equipment and predict the best area for its deployment.
Figure10. Distribution map of 17 kinds of POI in cities.
Hardware
We have designed a hardware adapted to deinking enzymes to accomplish deinking process. The hardware equipment consists of paper transfer part, enzyme smearing part, paper fiber separation part and paper drying part. Deinking needs to be realized through
the coordination of the above four parts and the correct manual operation. We have completed three iterations of the hardware and solved some practical problems with the use of the device. Click here for more details.
The fourth generation deinking equipment under design will be an all-round improvement on the third generation equipment. We hope that our devices will be able to realize their functions and value in the close future.
Figure11. Current device model.
Video1. Deinking are repeated from left to right.
Human Practice
The in-situ regerneration of office paper is too comprehensive to be settled without considering its relationships with society, humanities, science, safty, environmental protection and resource reuse. But we tried our best to understand this problem
and launched tailored work in four aspects: 1) pratical problems, 2) science and technology, 3) market industry and 4) policy and law.
First, we designed a stepped questionnaire to gradually understand the problems and the needs of potential users. Then we went to the producing and recycling sources of office waste paper, carried out propaganda work
and did a feasibility study of our project. We also established the Yeast Alliance, and communicated with academic frontier researchers of State key laboratory of pulp and paper engineering, in which we acquired
a lot of suggestions to iterate our work. In addition, we made business plan and participated in innovation competitions( Video2. ), where we received a lot of feedbacks from business people and relevant scholars. To meet the social demands better, we communicated with personnel in government environmental protection department.( Click here for more details)
Figure12. The logo of Yeast Alliance and Feedback from other teams.
Video2. UESTC-China at China-U.S. Young Maker Competition.
While gradually improving our program, we have also made a lot of efforts in education and collaboration. We have designed a series of activities related to waste paper recycling and saving paper to spread our project
concept. At the same time, with the philosophy that "science not silence", we have introduced synthetic biology to people of all ages in novel ways -- visual, auditory and sensory ways ( Click here for more details ). We also cooperated extensively with other iGEM teams and hosted two meet-up events, which not only helped us gain friendship, but also improve our project through communication (Click here for more details ).
Figure13. A small part of our HP works.
Figure14. The page of our meetup in Calendar.
References
[1] China Paper Association, 2020, China Paper Industry 2020 Annual Report(http://www.chinappi.org/reps/20210430094706408645.html)
[2] Toner Buzz, 2021, Facts About Paper: How Paper Affects the Environment(https://www.tonerbuzz.com/facts-about-paper/)
[3] Environment Paper Network, 2018, THE STATE OF THE GLOBAL PAPER INDUSTRY(http://environmentalppaper.org/wp-content/upload/2018/04/StateOfTheGlobalPaperIndustry2018_FullReport-Final-1.pdf)
[2] Toner Buzz, 2021, Facts About Paper: How Paper Affects the Environment(https://www.tonerbuzz.com/facts-about-paper/)
[3] Environment Paper Network, 2018, THE STATE OF THE GLOBAL PAPER INDUSTRY(http://environmentalppaper.org/wp-content/upload/2018/04/StateOfTheGlobalPaperIndustry2018_FullReport-Final-1.pdf)