With the development of society and technological advancement, the continuous increase in the production and consumption of paper waste poses a severe threat to the global environment. The fashion industry, which relies heavily on textiles and printing processes, is one of the major contributors to environmental pollution, particularly through wastewater discharge. In recent years, environmental issues have gained increasing attention, leading to a growing number of disputes and challenges in this sector. This has left the textile and printing industry grappling with numerous problems, including a decrease in orders and the closure of many businesses. Finding environmentally-friendly solutions for paper and wastewater management, promoting resource recycling, or even transforming it into a novel industry has become a global focal point of concern.

The composition of textile printing and dyeing wastewater includes dyes, pulp, additives, oils, acidity or alkalinity, fiber impurities, sand particles, and inorganic salts, among other components. In wastewater treatment, textile printing and dyeing factories often employ a combination of physical and biochemical processes. Physical treatment primarily aims to remove suspended solids and solid impurities such as pigments, including processes like sedimentation, filtration, and adsorption. Chemical treatment, on the other hand, involves adding chemicals to alter the pH or redox properties, causing harmful substances to undergo chemical reactions and either precipitate or become soluble, ultimately purifying the water quality.However, textile printing and dyeing wastewater has limited biodegradability, and traditional biological methods alone cannot ensure compliance with standards, often leading to high treatment costs.

Therefore, we are committed to developing an environmentally friendly technology capable of treating both paper waste and textile printing and dyeing wastewater. Through the engineered modification of Escherichia coli, we aim to enable it to break down cellulose in paper waste and utilize the resulting substances to produce bacterial cellulose, which can be used in the treatment of textile printing and dyeing wastewater. We hope that this technology can change the current landscape of paper recycling and wastewater treatment, offering an innovative solution to address global environmental issues.

Escherichia coli is a common bacterium that is widely found in the natural environment. E. coli is easy to cultivate and has a relatively complete and versatile genome. Therefore, it is frequently used in genetic expression studies. Researchers often insert foreign genes into expression vectors of E. coli and then transform these vectors into E. coli cells to achieve efficient expression of the foreign genes, yielding a large quantity of the target protein. Additionally, E. coli's genome contains multiple restriction enzyme recognition sites, allowing for gene cloning by inserting foreign DNA fragments into the E. coli chromosome.
In summary, to facilitate genetic manipulation and obtain a sufficient amount of the target protein, we have chosen to use Escherichia coli as our host microorganism.

Our project consists of two distinct systems. The Plant Cellulose Hydrolysis System is responsible for degrading plant cellulose, while the Bacterial Cellulose Producing System utilizes the glucose generated by the former system to produce bacterial cellulose

It is an abstract process to degrade the Plant cellulose. In general, we insert 3 genes into E. coli's plasmid: cex, cen and cep94A. The cex gene and cen gene encode exoglucanase and endo-1,4-β-D-glucanohydrolase respectively, and the cellulose can degrade into several cellobiose under the combined action of these two enzymes. Then cellobiose-phosphorylase encoded by the cep94A gene further hydrolyze the cellobiose into glucose.
The glucose will be used as the energy source of the bacterial cellulose producing system, because E.coli can gain energy from these small biological molecules.

  Komagataeibacter xylinus is a bacterium known for its ability to produce bacterial cellulose. It was first described in 1886 by Adrian John Brown, who discovered it while studying fermentation: it is the only bacterium whose operons can be extracted and genetically manipulated to be added to E. coli to produce bacterial cellulose.
  Now, let's take a closer look at this operon.
  Cellulose synthesis operon mainly has 2 units, Acetobacter cellulose synthesis operon and BC synthesis operon
  Acetobacter cellulose synthesis operon generally has 4 subunits, acsAB, acsC and acsD：
- acsAB encode a single polypeptide that has both substrate binding and activator-binding regions. 
- acsC encodes proteins that are similar to the proteins involved in membrane channels or pore formation, which suggests that acsC/bcsC is responsible for the formation of pores to secrete cellulose. 
- acsD controls the crystallization of cellulose into nanofibrils. acsD could provide passageways for extruding glucan chains. 

In the project, we designed a bacterial cellulose production system and a plant cellulose decomposition system. The plant cellulose decomposition system will use glucose, the decomposition product of plant fiber, as the power source of bacterial cellulose production to ensure the successful production of bacterial cellulose under the cooperation of the two systems.

Our target users are wastewater treatment facilities in the textile printing and papermaking industries, both of which face significant wastewater treatment challenges.

1. Paper Mill Phase:
    a. In the first tank, introduce the strain from our designed Plant Cellulose Hydrolysis System.
    b. Cultivate it for a specific duration to ensure optimal production of relevant hydrolytic enzymes.
    c. Input the paper mill wastewater into the first hardware device.
    d. Activate the ultrasonic disruption system within the first tank. Ultrasound helps break down our E. coli strain, releasing the enzymes responsible for cellulose degradation.
    e. Thoroughly mix to allow cellulose hydrolytic enzymes to efficiently degrade waste paper, yielding glucose.
2. Printing and dyeing factory Phase:
    a. Transfer all raw materials produced by the first system into the second tank.
    b. Then, introduce E. coli with the Bacterial Cellulose Producing System, which will utilize glucose from the first system to produce a substantial amount of bacterial cellulose.
    c. Once a sufficient quantity of bacterial cellulose is produced, activate the disruption system to break down the bacteria and release the bacterial cellulose.
    d. Collect the bacterial cellulose to create a compatible wastewater treatment unit (refer to hardware).
    e. In the final step, introduce textile printing wastewater into this system for effective treatment.

With regard to biological safety, our engineered strains will be contained within sealed processing tanks and undergo thorough ultrasonic disruption. This approach ensures safety, as the selected genes are environmentally benign.

[1]Sekar R, Shin HD, Chen R. Engineering Escherichia coli cells for cellobiose assimilation through a phosphorolytic mechanism. Appl Environ Microbiol. 2012;78(5):1611-1614. doi:10.1128/AEM.06693-11
[2]Iyer PR, Liu YA, Deng Y, McManus JB, Kao TH, Tien M. Processing of cellulose synthase (AcsAB) from Gluconacetobacter hansenii 23769. Arch Biochem Biophys. 2013 Jan 15;529(2):92-8. doi: 10.1016/j.abb.2012.12.002. Epub 2012 Dec 8. PMID: 23232080.
[3]Loke HK, Bennett GN, Lindahl PA. Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli. Proc Natl Acad Sci U S A. 2000 Nov 7;97(23):12530-5. doi: 10.1073/pnas.220404397. PMID: 11050160; PMCID: PMC18798.
[4]Saxena IM, Brown RM Jr. Identification of a second cellulose synthase gene (acsAII) in Acetobacter xylinum. J Bacteriol. 1995 Sep;177(18):5276-83. doi: 10.1128/jb.177.18.5276-5283.1995. PMID: 7665515; PMCID: PMC177319.
[5]Lai CY, Ng KL, Wang H, Lam CC, Wong WKR. Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli. Microbiol Insights. 2021 Jun 15;14:11786361211024637. doi: 10.1177/11786361211024637. PMID: 34188486; PMCID: PMC8209791.
[6]Iglesias Rando MR, Gorojovsky N, Zylberman V, Goldbaum FA, Craig PO. Improvement of Cellulomonas fimi endoglucanase CenA by multienzymatic display on a decameric structural scaffold. Appl Microbiol Biotechnol. 2023 Jul;107(13):4261-4274. doi: 10.1007/s00253-023-12581-6. Epub 2023 May 22. PMID: 37212884; PMCID: PMC10201518.
[7]O'Neill G, Goh SH, Warren RA, Kilburn DG, Miller RC Jr. Structure of the gene encoding the exoglucanase of Cellulomonas fimi. Gene. 1986;44(2-3):325-30. doi: 10.1016/0378-1119(86)90197-6. PMID: 3096818.
[8]Nikolova PV, Creagh AL, Duff SJ, Haynes CA. Thermostability and irreversible activity loss of exoglucanase/xylanase Cex from Cellulomonas fimi. Biochemistry. 1997 Feb 11;36(6):1381-8. doi: 10.1021/bi962367f. PMID: 9063886.
[9]Ong E, Gilkes NR, Miller RC Jr, Warren RA, Kilburn DG. The cellulose-binding domain (CBD(Cex)) of an exoglucanase from Cellulomonas fimi: production in Escherichia coli and characterization of the polypeptide. Biotechnol Bioeng. 1993 Aug 5;42(4):401-9. doi: 10.1002/bit.260420402. PMID: 18613043.
[10]Cabulong RB, Bañares AB, Nisola GM, Lee WK, Chung WJ. Enhanced glycolic acid yield through xylose and cellobiose utilization by metabolically engineered Escherichia coli. Bioprocess Biosyst Eng. 2021 Jun;44(6):1081-1091. doi: 10.1007/s00449-020-02502-6. Epub 2021 Feb 1. PMID: 33527231.