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Single-step disentanglement and fractionation of microalgal high-value products through acoustophoresis

Periodic Reporting for period 1 - AlgCoustics (Single-step disentanglement and fractionation of microalgal high-value products through acoustophoresis)

Reporting period: 2020-01-06 to 2022-01-05

Mass production of biomass-based biofuels and biocommodities is required to disrupt the current fossil-based ones. Microalgae, could play a key role in these endeavours, due to their increased photosynthesis efficiencies, independence from quality arable land and ability to uptake CO2 while growing on liquid effluents. Aquatic biomass can provide renewable energy as well as high-value molecules, which can be used in food, feed, cosmetics, biomaterials, nanostructures and pharmaceutical industries. However, in order to greatly increase the economic viability of aquatic biomass, all components need to be valorized. Unfortunately, this is not possible using current/conventional biorefinery technologies, where up to 90% of the biomass is being treated as a waste. The value of these broken-down compounds sees more than a ten-fold reduction, rendering the biorefinery economically unfeasible.
Therefore, in furtherance of developing multiproduct biorefineries, selective and economically feasible extraction and separation technologies will need to be developed and implemented. Significant microalgal cell disruption and extraction advances have been recently made by employing external fields such as lasers, ultrasonic waves and microwaves, in combination with less aggressive solvents and ionic liquids. However, the issues regarding the use of chemicals and multiple separation stages remain. Thus, we are proposing a game-changing single-step disentanglement and separation of microalgal high-value components by using acoustic waves at different frequencies allowing thus a complete process finetuning and eliminating the need for chemicals. Moreover, by including our previously-developed ultrasound disruption technology, the whole cell breakdown, extraction and separation steps could be reduced to one single process governed and finely-tuned through the employed frequency ranges.
The work performed during the implementation of this project looks at several specific key objectives, such as: i) identifying and isolating various microalgal cellular components; ii) understand the nature of microalgal cell debris generated through ultrasound disruption processes; iii) determining the optimal compound disentanglement conditions by identify the impact of different ultrasound frequencies on the change in microalgal particle size distribution; iv) determine the subsequent ultrasound fractionation potential of the extracted microalgal molecules through modelling and simulation approaches backed by experimental observations; v) design and test a proof-of-concept “lab-on-a-chip” integrated system for the simultaneous disentanglement and separation of microalgal added-value components using ultrasound field forces.
Acoustic forces in the ranges of bellow 100 kHz were successfully used by different authors to disrupt various single-cell organisms. However, most of the time the shear forces were so high that most of the cellular components were disrupted in the process as well. Recently however, I showed using Tisochrysis lutea microalgal cells that by fine-tuning the ultrasonication intensity, together with other process parameters such as cell concentration and treatment time, the cells can be mildly disrupted while keeping the rest of the components, such as the chloroplast, nucleus, starch granules and lipid droplets, intact.
In order to characterize and understand the nature of the microalgal components, these need to be completely disentangled from the rest of the cellular debris. My preliminary work on T. lutea cellular fragments has proved that even if disentangled components are obtained through a mild acoustic disruption approach, indeed a significant part of these fragments remain attached. Thus, significant work has to be done to understand how the non-covalent bounds respond to high-frequency sonication treatments and if that would be sufficient to break them in order to obtain disentangled components.
The acoustophoretic behaviour of these cellular organelles and other components is determined by their size and acoustic contrast factor, which in turn refers to mostly the particle density and compressibility. By using and extrapolating literature data, I modeled and simulated the behaviour of some of the most common microalgal cell components such as the chloroplast, nucleus, lipid droplets and starch granules. However, these results are incomplete and require further focus on accurately determine the size, density and compressibility of these cellular components in order to adequately predict their acoustic behaviour.
The acoustophoretic process is determined by a series of variables such as microchannel dimensions and geometry, sample flow rate, acoustic frequency and intensity, density of cellular components, and even fabrication technology and materials. I demonstrated in silico that using a silicon and glass microfluidic system with one inlet and three outlets, that indeed these different components could be efficiently separated into high-purity streams. However, these simulations require further investigations into additional geometries and flow rates in order to increase the separation efficiency while maintaining relatively high flow rates.
The societal contributions brought by the project is grouped in two main categories: technological and economic. The technological contributions refer to the proof-of-concept acoustic disruption, disentanglement and fractionation of microalgal cells and particles. This is, to the best of my knowledge, the first time such approach is demonstrated, with huge impact on a series of adjacent academic areas. The economic contributions, on the other hand, refer to the potential this game-changing technology has on wide-scale implementation of green chemistry and white biotechnology concepts, pillars of the circular economy model. Once successfully demonstrated under scaled-up, extended-use conditions, this novel approach will spark interest of a whole range of national and global stakeholders, bringing closer to commercialization the circular multiproduct biorefinery model. Companies such as FUMI Ingredients already expressed their interest in the idea, and they are interested in seeing the results from the perspective of implementing the technology into their existing process. More specifically, the research impacts a whole range of adjacent research fields such as down-stream biotechnology, biorefinery, microfluidics, lab-on-a-chip systems, acoustic phenomena and sonochemistry. Thus, novel directions and interesting solutions might arise in areas not directly related to microalgal biorefineries, such as targeted medicine, single-cell biochemistry and micro-bioreactors, as well as conventional biotechnology and food production. Thus, the potential scope and impact of this project are huge. The generated results are made available to stakeholders such as scientists working in adjacent fields as well as EU policy makers, through an open-access publication model, conference presentations and round-table discussions.
Disruption of T. lutea and the design and simulation of a microfluidics chip of acoustophoresis