The global market of leather is rapidly increasing to meet the rising demands of high quality leather. Currently, there are two main types of traditional leather, namely animal leather and plastic faux leather, both of which have significant environmental impacts.

Figure 1. Flow chart of chrome tanning and wastes

Animal leather has a relatively longer history than any other leather materials since our ancestors started to wear animal fur as cloths at the very beginning of an entire human history. The variety of the usage of animal fur is more in the present, approximately 50% of the animal leather products is utilized to make shoes, 25% for clothing and about 25% for other products. Animal leather is currently the most prevalent type of leather in the world. Over the next few years, the animal leather market is anticipated to reach 13.1 billion dollars in 2022.

Animal leather production namely consists of washing, fleshing, tanning, and post-production. Washing removes dirt from raw hide, fleshing further cleans the raw hide, tanning to dye the hide, and post-production processes will differ based on the use of leather.

Animal leather production pollutes the environment by releasing large amounts of heavy metals such as chromium and cadmium which are toxic to the ecosystem, namely in the tanning step. One process of animal leather production is tanning which is the most risky and toxic step, since over 90% production requires the use of chromium tanning (Parvez, 2020). The amount of chromium being discharged by leather industries is about 170000 tons per year (Barik & Sivaram, 2019). Plants which are polluted by chromium will have severe decrease in seed germination and oxidative imbalances (Deckert, 2012). Chromium also affects human immune system and can cause lung cancer (Shrivastava et al., 2002).

Faux leather is mostly composed of polyvinylchloride and vinyl and it popular since the 1910s. Polyurethane and vinyl synthetic leathers are used mainly in clothing and upholstery. Weaknesses of faux leather is obvious, as the fabric breathability, moisture-wicking abilities, stretchability and prone to pilling are usually low for PVC-made Faux leather. However, the heat retention ability of faux leather is quit high and similar to that of the traditional leather. (Sewport Support Team, 2021).

As the demand for cheaper leather grows, the market size of artificial leather, a substitute for traditional leather, is expected to grow annually at a rate of 7.8% from 2021 to 2028 (Grand View Research, 2021). Prior to production, we need to prepare polyester materials to act as a base material. Polyvinylchloride will be then bound with base fabrics and textiles. At the end, the leather could be cut in different sizes.

Most artificial leathers are produced by plastics such as polyurethane and polyvinyl chloride which are harmful for the environment because they are not biodegradable (Doe, 2020). During the process of PVC leather production, polyvinylchloride with stabilizers, plasticizers and lubricants are combined while PVC might be emitted to the environment in this process and cause harmful effects (How Do Faux Leather Fabrics Compare to Real Leather?, 2016).

Figure 2. Cross section of faux leather.

Bacterial cellulose membrane (BCM) is the material we chose for the Neoleathic age. Bacterial cellulose membrane, as its name suggests, is formed by tangling secreted cellulose fibrins. We already see BCM in our daily lives, especially in the foods we eat. It is also found in nata jelly in milk teak. In the medical setting, BCM is used as a wound dressing, especially for burn patients, due to its strong water retention ability and its highly adaptable shape.

Figure 3. Kombucha SCOBY with BCM. The whitish membranes are BCM.

High-quality BCM is commonly produced by pure cultures of two genera of gram-negative acetic acid bacteria: Komagataeibacter and Gluconacetobacter. These genera of bacteria can produce BCM at an astonishing rate, more than 10g/liter of medium in less than a few days. On top of the already amazing rate, BCM can be produced at a faster rate in a symbiotic co-culture of bacteria and yeast (SCOBY), where Komagataeibacter is co-cultured with Saccharomyces cerevisiae (Gilbert, 2021). For our project, we hope to use BCM produced from SCOBY and engineer it into a leather substitute.

While BCM is a strong material, BCM strength is still a major concern. Raw BCM breaks easily when latitudinal force is applied. To turn BCM into a suitable leather substitute, we had to increase its tensile strength and softness.

Our design centers around “nets”. BCM is a net made from cellulose fibers, and we want to increase its tensile strength and softness by layering BCM with a spider silk fibroin net. The spider silk fibroins will form hydrogen bonds with other fibroins, due to the presence of multiple beta-pleated sheets in the fibroins, thus creating a hydrogen bond net in the BCM.

A simple analogy for our design is that it is harder to pull apart or break two layered nets than a single net. To further strengthen our BCM, we used cellulose binding matrixes (CBMs) to bind our spider silk proteins to BCM. CBMs are artificial proteins derived from natural proteins with cellulose-binding functions, such as cellulase. There are three types of CBMs, which are CBMs, CBM1, CBM2, and CBM3. CBM1 is the smallest, whilst CBM3 is the biggest.The CBMs used throughout our project is CBM3 from Ruminiclostridium thermocellum (Protein Data Bank accession number 1NBC) (2) and CBM2 from Cellulomonas fimi (Mohammadi, 2019).

To link the spider silk net with the BCM net, we designed fused spider silk and CBM proteins. These fused proteins will have CBMs flanking the spider silk fibroin, thus connecting the spider silk with the BCM. The spider silk fibroin will also form hydrogen bonds with each another, in the gaps of cellulose fibers in BCM, creating a denser net made of two different materials. By fusing spider silk proteins and CBMs, we hope to make BCM leather a reality.

Figure 4. Schematic representing the increase in force needed to break two layered nets as opposed to one. The relative size of the arrows indicate the relative force.

Figure 5. Our visualization of the BCM net layered with spider silk fibroins.

As mentioned before, commercial methods of dyeing release massive amounts of pollution, specifically chromium III and VI. If handled inadequately, the chromium can escape into the environment, devastating the local ecosystem and making its way into humans.

To dye our leather naturally, we want to produce indigo and tyrian purple (6, 6’dibromoindigo) from tryptophan (trp), using three enzymes: flavin reductase fused with a trp-6-halogenase (Fre-SttH), tryptophanase (TnaA), and flavin-containing monooxygenase (FMO).

To produce tyrian purple Fre-SttH will first convert trp into 6-Br-trp under the presence of NaBr. Then, TnaA will convert 6-Br-Trp into indole. Finally, FMO will add modifications to 6-Br-Trp, who will simultaneously dimerize into tyrian purple with oxygen. To make indole, only TnaA and FMO are needed (trp to indole to indigo).

However, we cannot simultaneously express all three enzymes under the presence of trp. Trp will, more likely, take the shorter enzymatic pathway and be turned into indigo by TnaA and FMO, without being halogenated first by Fre-SttH. To overcome this obstacle, we seperated expression of Fre-SttH and TnaA and FMO by construction two strains of E. coli, one expressing Fre-SttH, the other expressing TnaA and FMO (Lee, 2021).

Our current design for producing dyes involves spatial separation of the Fre-SttH and TnaA & FMO. In the future, we would also like to produce tyrian purple using just one strain, using temporal separation, where we can express different enzymes by measuring the relative concentration of each substrates in the media. Our modeling team combined computer science, enzymatic rates derived from experiments, and biological sensor to model the concentration of different substrates over time.

By producing natural indigoid dyes using bacteria, we cut back on potential pollution associated with dyeing leather.

Figure 6. Schematic representing the production pathway of indigo and tyrian purple with trp-6-halogenase, TnaA, and FMO.

Our project consists mainly of three parts: 1. Producing bacterial cellulose membrane from symbiotic co-cultures of bacteria (Komagataeibacter) and yeast 2. Expressing spider silk proteins fused with cellulose binding matrixes which binds to BCM to improve its physical properties 3. Synthesizing natural dyes to dye our leather in a sustainable fashion

Furthermore, inspired by Ellis et al, we also designed and engineered yeast in SCOBY to improve BCM’s properties, such as in situ binding of aforementioned fused spider silk proteins and production of fragrance in BCM.

Figure 7. Figure abstract for the Neoleathic Age. Komagateaibacter sp. and S. cerevisiae BY4741 will produce bacteria cellulose membrane. E. coli BL21 (DE3) will produce spider silk proteins fused with cellulose binding matrixes. E. coli DH5α will produce natural pigment dyes. By combining these three components, we can produce NeoLeather.

All in all, our project aims to engineer a suitable leather substitute from BCM, guiding the industry towards a more humane and sustainable future.