Recombinant production of various enzymes for sutilisation by IPE
Partner IPE is very experienced in enhancing the heterologous expression of various enzymes using Bacillus species. The genes coding for plastic degradation enzyme provided by other partners will be expressed in various Bacillus strains including the commercially available Bacillus subtilis ATCC6051. Using these strains, IPE will optimise the cultivation conditions in bioreactors. New fermentation strategies will be applied to enhance the enzyme expression levels. Enzyme yields and specific activities of each enzyme will be determined using the specific assays towards respective polymer types. Finally, single strains will be cultivated together in mixed cultures to produce specific enzyme cocktails in one-pot.

Mixed enzymatic plastic degradation (IPE)
Using the recombinant enzyme produced, IPE will scale up the mixed enzyme reactor developed by partner UG to up to 10 L for an optimised degradation of polyester mixtures at a desired thermophilic condition of up to 75°C. Further up-scaling of enzyme production until 250 L is possible. The optimised micro-reactor reaction will be scaled-up to higher scales by IPE and thus enable the extension of reaction conditions validated for an efficient degradation of plastic waste consisting of mixed polymer types at an industrial relevant scale.

Comprehensive analytics for hydrolysed plastics by IPE
To analyse plastic degradation products and evaluate plastic mixtures, analytical methods are key. The purpose of this subtask is to develop suitable methods for the analysis of hydrolysed mixed plastics. Plastic degradation products include oligomers and fully hydrolysed monomers, as well as not degraded solids. We have an array of analytical methods and tools including HPLC-MS, GC-MS, FITR, TGA, SEM, and NMR that will be utilised for the qualitative and quantitative analysis of plastic hydrolysis mixtures (Jia et al. 2010, Jia et al. 2011). With the established methods, the plastic hydrolysates from WP2 will be characterised in detail.

Separation of aromatic monomers by IPE
The purpose of this subtask is to establish a method that allows selective separation of aromatic monomers for subsequent PHA synthesis in Task 5.04. The aromatic monomers either originate from mixed plastic degradation, mainly TA from PET, or from de novo synthesis in Task 5.06. According to the physicochemical properties of the aromatic monomers and the other constituents of the mixed plastic hydrolysate, a suitable separation method will be chosen. We have at hand

everal separation methods such as adsorption, extraction, and filtration (Li et al. 2013, Li et al. 2014, Yu et al. 2015, Li et al. 2019). After establishing a method for aromatic separation, we will continue to work on the separation process to increase the separation efficiency.

Enzyme immobilisation for enhanced degradation of mixed plastic oligomers (IPE)
To increase the rate of plastic degradation from long chain polymers, via short oligomers to monomers, and to reduce enzyme costs, we will develop immobilisation techniques for oligomer hydrolases. Specifically, the plastic degradation enzymes of MIX-UP (Task 2.01, Task 2.02, and Task 2.03) will be tested for performance on respective plastic oligomers. Again, for PET, dedicated dimer hydrolases are described (Liu et al. 2018). The MIX-UP enzymes and enzymes from literature with high oligomer hydrolysis activity will be immobilised onto magnetic material to form multi-enzyme surfaces. We have established a range of technologies for protein immobilisation that can be tested. These include 1) magnetic particles functionalized with glutaraldehyde and hexamethylene diamin, to form different length arms for enzyme immobilisation, 2) magnetic graphene oxide functionalized with Na,Na-Bis(carboxymethyl)-L-lysine hydrate (NTA-NH₂) to produce MGO-NTA nano-sheets, 3) material with silica for enzyme binding through covalent bonding (Liu et al. 2004, Zhou et al. 2015), and 4) particles of graphene oxide where the functional group on the graphene oxide surface interacts with enzymes. Adsorption capacities of the enzymes with different support materials will be determined. The enzyme stabilities will be studied at different pH, temperatures, and substrates. Storage stability, kinetic parameters, and catalytic and specific activities of the immobilised enzymes will be investigated. The re-usability of the immobilised enzymes for at least ten cycles of mixed plastic degradation will be estimated. The immobilised enzymes will be used for mixed enzymatic plastic degradation in Subtask 2.07.2.

Design of a sustainable pilot system for mixed plastic degradation (IPE)
In order to make the next move after validation of chosen process steps in industrial environments (Task 8.06) we will consolidate all that we learned in MIX-UP to design a pilot plant involving all relevant steps from protein production to the final valorisation. This will be done based on the optimised process steps developed in the single WPs and the final validation.
Pilot plant design is an important part of process engineering project (Zhang et al. 2016, Zhang et al. 2017). The aim of this task is to develop a complete concept of a

pilot plant for the production of PHA based on mixed plastic waste. The capacity of the pilot plant will be designed for 50 kg plastic waste per batch. The whole system includes all necessary utilities (e.g., water supply and drainage, gas supply, environmental protection etc.), control and feedback regulations (such as the control of temperature, pH, and agitation), and the production part, which can be divided into 5 systems (see also Fig. 2).

1. The pre-treatment of plastic waste: The pre-treatment of the mixed plastic waste is necessary as it will make the degradation more efficient. We will include mechanical treatment such as smashing and rinsing and other treatments such as UV light and chemical treatment. The control of treatment conditions such as temperature and pressure will be considered.
2. The enzyme production stage: yields enzyme cocktails of native and recombinant enzymes. Control of parameters such as temperature and pH value will be included.
3. The enzymatic hydrolysis: This is the stage where mixed plastic waste enters the workflow. The biodegradation reactors for mixed plastics is one of the major equipment pieces, with online-detection systems such as ReactIR and control systems for controlling the temperature and pH. The treatment capacity will be designed for 50 kg mixed plastic per batch. The optimised results of WP2 and WP4 will be used here.
4. The fermentation system using mixed cultures: In this system, the plastic monomers will be converted into PHA using defined mixed bacterial cultures in a fermenter. The online-detection system, control system (such as temperature and pH) will be designed. The optimised results of WP5 will be used here.
5. The separation and purification system: The products or by-products can be purified in this part. It is not only used for preparing the final products, separation will also play a role in other parts.

The calculations will feature mass and energy balances based on the results of WP2, WP4, and WP5. The environmental impact will be analysed including the treatment of waste gas, solid residue, and wastewater. The economic appraisal will be done by estimating the investment for equipment, material and environmental protection. The cost of production will be calculated based on the prices of the substrates and utilities. The period until the break-even-point is reached will be calculated including the estimate of time per batch.