Alternative, green downstream routes for extraction and separation of modified polyhydroxyalkanoates obtained by bacterial fermentation

Plastics play an important role in modern societies due to their broad applicability and useful characteristics such as low cost, low density and high stability. Global plastics production exceeded 390 million tons annually by 2021. However, the majority of plastics are still produced from petroleum and crude oil. Long chain hydrocarbons are resistant to degradation and especially microplastics tend to bio-accumulate in the environment, while the production process itself is energy intensive. Therefore, there is a need to investigate production processes for greener and biodegradable plastics alternatives with competitive characteristics compared to traditional plastics and production processes.

One promising group of biodegradable bioplastics are called polyhydroxyalkanoates (PHAs). These are a family of thermoplastics polyesters that are used by prokaryotes as an intracellular carbon and energy storage. Traditionally these plastics are produced by fermentation, based on either single cell cultures or mixed microbial cultures (MMC). However, the downstream process, requires added processing steps due to the intracellular nature of the polymer. Therefore, these plastics have so far been prohibitively expensive to produce. Furthermore, the most common downstream processes used to extract and purify the polymers from the biomass require high energy expenditure and toxic solvents such as chloroform. Hence, a more sustainable production process from either MMC or pure cultures, using greener solvents, simple processing steps and low energy requirements, was needed.

Firstly, we investigated the use of enzymes for cell disruption to enable the extraction of the PHA polyhydroxybutyrate (PHB) from Pseudomonas putida biomass. An enzymatic pre-treatment step, using a hypotonic lysis buffer in combination with lysozyme, assisted in cell disruption and therefore the PHB extraction itself could potentially be more effective. Several different bio-based organic solvents were screened for extraction efficiency, with the highest yields produced by ethyl acetate. The extraction process was then optimized, which revealed that the most significant parameter impacting the extraction yield at lab scale was the solvent volume ratio applied to the biomass. Using ethyl acetate it was possible to achieve a yield comparable to chloroform, which is considered the benchmark for PHA extraction. However, as these organic solvents are still toxic (LD50 of 5620 mg/kg for ethyl acetate compared to chloroforms LD50 695 mg/kg), it was decided to try the application of a more novel type of solvents, called natural deep eutectic solvents (NADESs). These solvents are synthesized from two or more natural compounds, often terpenes and fatty acids, and form fluids at room temperature. These solvents come with the benefits of lower toxicity and safer handling than most organic solvents. Several different NADESs were synthesized for this work and screened for extraction efficiency. The most effective NADES was based on menthol and acetic acid in 1:3 molar ratio and the process parameters were further optimized to achieve high PHB yields and purity. The reasons for the different extraction efficiencies were briefly investigated, and it was speculated that solvent viscosity and polarity could have a significant impact on yields. Generally, NADESs were previously applied in extraction in an empirical manner, and the best performing NADESs were determined through trail and error. To improve the NADESs selection process it was decided to establish a more systematic approach to the design and application of NADESs for extraction. This was to be done by analysing the physicochemical characteristics of the NADESs and trying to establish correlations between these characteristics and extraction efficiency in the further work.

Secondly, the developed downstream process was adapted for the extraction of polyhydroxybutyrate/valerate (PHBV), a PHB co-polymer, from MMC biomass produced by fermentation using renewable feedstock (fruit waste). For this purpose a selection of different terpene based NADESs were synthesised and characterised. More emphasis was put on identifying possible causes for variations in extraction yield between the NADESs. Therefore, the Hansen solubility parameters (HSPs) of the different NADESs were analysed and compared to organic solvents, before extraction screening was performed. All the synthesised NADESs showed good characteristics for PHBV extraction based on the HSP analysis. Screening revealed a NADES based on the terpene thymol and the decanoic acid to have the highest extraction yields. This was consistent with global solubility determined during the HSP analysis. Furthermore, analysis of the NADESs viscosities and polarities showed some correlation to the experimentally determined extraction yields, with such analysis potentially helping with solvent selection going forward. After this analysis, the process conditions were optimised, achieving yields close to those of chloroform, without the application of high extraction temperatures. Furthermore, to make the process greener, the process loop was closed and all the solvents used could be recycled back into the process.

In summary, this study aimed to establish a more sustainable green extraction process for PHAs, with the use of less toxic bio-based organic solvents and NADESs. The novelty of this work lies in the systematic optimization of extraction conditions combined with a data driven approach to identify causes for yield and purity variations between different solvents. This should enable easier solvent selection in the future. The strategies developed in this work provide a blueprint for the effective extraction and purification of PHAs, with the use of green solvents