Team:Edinburgh/Description

The SuperGrinder






Description The Waste Problem Cellulose Keratin Chitin PET

The SuperGrinder: value from waste

We aim to revolutionise the extraction of valuable materials from the most common recalcitrant waste polymers, including cellulose, keratin, chitin and PET. These are all resistant to natural biodegradation but hold great potential for integration to the circular economy. Current treatments are often unsustainable, requiring harmful chemicals, high energy and resulting in downcycling of materials to lower value products.

Our team seeks to combine physical and enzymatic treatment of these polymers by constructing a ‘Super Grinder’, featuring silica-tagged enzymes immobilised on silica beads. These enzyme-laden silica beads are used to grind the substrate, both contributing to the physical comminution of the waste material and (theoretically) increasing enzyme accessibility to the substrate surface to improve degradation efficiency above that of a standard bioreactor. Immobilisation may have the added benefits of improving enzyme stability and recyclability, and our design operates at ambient temperatures, decreasing its carbon footprint and cost. We also aim to improve enzyme activity using directed evolution techniques.


Project aims

The Waste Problem

To many of us, waste management is a hidden problem: “out of sight, out of mind”. However, future generations will not be so privileged. The production of solid waste is becoming a growing burden to our environment and societies, and this is only increasing; estimates suggest that “peak waste” will not be reached until after the end of the century[1]. The re-utilisation of waste from industrial, agricultural, and commercial sources for new applications in a circular economy will therefore be necessary if the UN 2050 Climate Goals are to be met[2].

As a team we focussed on enzymatic degradation of insoluble polymers, including cellulose, keratin, chitin and PET. These resources are abundantly produced, but because of the insoluble nature of these materials, current processes for using them are expensive and inefficient, leaving much room for improvement using synthetic biology approaches. The “SuperGrinder” platform that we developed is not limited to these example applications and could also be extended to treatment of alternative waste resources with future work.

Cellulose

Lignocellulosic biomass is the most common polysaccharide on the planet [3]. It is abundant in agricultural and domestic waste streams, and it is estimated that 1 billion tonnes will be made available annually within the European Union[4]. However, in its polymeric form it is of little value due to its complex crystalline structure which has evolved to specifically withstand enzymatic and microbial attack[3]. Typically waste cellulosic material is incinerated or composted, however this wastes valuable glucose monomers that could be upcycled to feed into a sustainable sugar bioeconomy[5]. Glucose released from sources such as waste paper, crop residues and non-food biomass can be used as a substrate for microbial fermentation to produce a wide variety of biofuels (such as bioethanol) and a range of renewable chemicals.

A variety of cellulases were investigated in our study for cellulose degradation. Exoglucanases hydrolyse β-1,4-glucosidic bonds from the non-reducing end of cellulose and endoglucanases cleave internal β-1,4-glucosidic bonds within the polymeric strand, exposing more ends for exoglucanase action. For optimal cellulose degradation, a combination of exoglucanase, endoglucanase and additional complementary activities such as beta-glucosidase and endoxylanase activities (for endohydrolysis of β-D-xylosidic linkages of xylan, another important polysaccharide found in agricultural waste) should be used.

We studied the enzymes detailed below:

Enzyme

Enzymatic action

Source organism

Cex

Exoglucanase, endoxylanase

Cellulomonas fimi

Chu2268

Exoglucanase

Cytophaga Hutchinsonii

CenA

Endoglucanase

Cellulomonas fimi

Keratin

Keratin-rich animal by-products are highly abundant wastes. The poultry industry produces an estimated 40 million tonnes of feather waste annually and there are few natural uses for these in their raw form. As a result, most are currently incinerated or disposed of in landfill[6].

Feathers are rich in protein, containing 90% keratin by weight[7]. Amino acids released from keratin could be utilised in cell culture media to improve the life-cycle sustainability of biotechnological processes dependent on microbial cultures, or used as nitrogen-rich fertiliser or as a nutritional supplement for livestock. However, feather keratin has an extremely tough filament-matrix structure that poses a technical challenge to accessing the potential value. Dimeric β-sandwiches assemble to form a repeating helical β-filament, strengthened by intrachain and interchain bonding (e.g. crosslinked by strong disulphide bonds, hydrogen bonds, ionic bonds and hydrophobic interactions). These filaments are further embedded in a matrix of keratin-associated proteins. Altogether this makes feathers extremely robust and difficult to degrade[8,9]. Currently used physicochemical processes for keratin hydrolysis are energy-intensive and the harsh conditions result in amino acid degradation to non-nutritive forms. Therefore an enzymatic conversion operating at ambient temperatures and pressures can be highly beneficial.

Although keratin is resistant to most standard proteases, specialised keratin-degrading enzymes (termed “keratinases”) can convert keratin substrates to soluble amino acids without affecting amino acid integrity. We focussed on two keratinases: KerA from Bacillus licheniformis, and MtaKer from Meiothermus taiwanensis.

Chitin

There is an abundance of global crustacean waste which is currently underutilised. Global lobster, shrimp, and crab fishing produces 6 to 8 million tonnes of biowaste every year [10]. Scotland’s fishing industry contributes substantially to global crustacean waste with 38,000 tonnes of crustaceans being caught in 2019 by Scottish vessels[11].

Crustacean shells are rich sources of chitin, which is the second most abundant polysaccharide on Earth (after cellulose). Chitin polymers are made up of N-acetyl-2-amino-2-deoxy-D-glucose (GlcNAc) monomers. Chitin can be used in a variety of medical, agricultural and biomaterial applications owing to it being non-toxic, biodegradable, and biocompatible. When over 50% of the chitin GlcNAc units are deacetylated chitin is converted to its deacetylated derivative chitosan (Figure), which has more industrial applications due to having a much higher solubility[12].

Crustacean shells are composed of 20-30% chitin, 30-40% protein, and 30-50% minerals (predominantly CaCO3). Extraction of chitin therefore requires two stages: demineralisation and deproteination. These steps can be achieved chemically or biologically. Chemical extraction is expensive and environmentally damaging due to the use of strong chemicals and use of high temperatures. Alternatively, biological extraction offers a greener process yielding higher quality chitin, however, it is currently industrially limited due to slow production rates[13]. Both extraction methods require pre-treatment of the shells to grind them for increased surface area, which is where our SuperGrinder concept can improve the process. We subjected Subtilisin E to directed evolution to increase the efficiency of the deproteination stage. Chitin deacetylase from Colletotrichum lindemuthianum hydrolyses the acetamido group in the N-acetylglucosamine units of chitin to produce chitosan and chitooligosaccharides, releasing acetate.

PET

Every minute, 1 million plastic drinking bottles are purchased worldwide, while approximately 5 trillion single-use plastic bags are used annually[14]. Polyethylene terephthalate (PET) is the most major contributor to the global plastic pollution problem, with a global production of 70 million tonnes for use in packaging and textiles[15]. However the resistance to degradation of plastics can cause major environmental problems. Less than 30% is currently recycled[16], and disposal of the remaining portion causes contamination of habitats, filling of landfills, pollution from incineration (releasing toxic fumes), or toxicity to ecosystems due to its natural hydrophobicity creating adsorption sites for organic pollutants and heavy metals[17].

PET hydrolase (PETase) and MHET hydrolase (MHETase) enzymes were isolated from Ideonella sakaiensis 201-F6 and shown to work in synergy to degrade PET to monomers terephthalic acid and ethylene glycol[18]. We used a modified double mutant PETase (S238F/ W159H) reported to have higher capacity for PET breakdown than wild-type PETases due to changes in interaction with the PET substrate at the modified active site residues[19]. This modification was inspired by cutinase enzyme structures. Leaf-branch compost cutinase (LCC) is another enzyme with reported activity against PET. We used the mutated version LCC-WCCG (F243W/ D238C/ S283C/ Y127G) with increased thermostability in conjunction with retention of specific activity, which was previously shown to achieve 85% conversion of PET in 15 hours, compared to the wild-type LCC reaching only 53% conversion of PET in 20 hours[15].

References
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