Aquatic plants (Azolla filiculoides, Azolla pinnata, Landoltia punctata) and protists, thraustochytrids as renewable and sustainable sources of protein, phenolic, carotenoids and omega-3 fatty acids
Creators: name: TRAN, Thi Linh Nham
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310806
subject: Plant physiology
classificationCode: 410303
subject: Bioremediation

description: Food demand is increasing, and persistent climate change is prompting scientists to search for new sources of food and food supplements. Omega-3, carotenoids, squalene, proteins and a large spectrum of phenolic compounds are valuable biomolecular components that bring benefits to human health. Therefore, the demand for these biomolecules is on the rise. The original sources of these biomolecules have predominantly been animals, fish, and terrestrial plants. However, there are many challenges associated with these, such as a lack of land for animals and terrestrial plants due to industrialisation, rising sea levels, desertification, urbanisation, and pollution. These issues are triggering an intensive search targeting the use of marine and aquatic plant biomass for the production of these biological chemicals.

Due to associated high growth rates, biomass yield, the ability to grow on marginal lands and the valuable biomolecules present in aquatic plants (e.g., Azolla and duckweed species) and in marine protists (e.g., thraustochytrids), these species were targeted as a renewable and sustainable source for the production of proteins, phenolic compounds, omega-3, carotenoids, and squalene. The current research contained three main parts: The first part was designed to assess the growth and productivity of two Azolla species (A. filiculoides, A. pinnata), a duckweed representative (L. punctata) and two thraustochytrid species (MAN 65 and MAN 70). The second part focused on the assessment of biomass content and composition of the lipids, oil bodies, carotenoids, squalene and proteins. In the third part, the extracted protein was used to make complex coacervation with polysaccharides (chitosan/alginate). The interaction of protein and polysaccharides (and their characteristics) was measured to determine the optimal protein and pH ratio for complex coacervation. 

Nitrogen free in SH ½ medium was found to represent an optimum culture medium for the growth and protein production for Azolla (A. pinnata and A. filiculoides) ( growth rate is up to 0.06g/day DW and protein accumulation reach up to 28% in dry biomass).  The commercial medium was noted that the optimum culture medium for the growth and protein production of L. punctata (up to 0.03g day DW, protein yield reached 35% in dry weight). However, the yield of the extracted protein was only 5-8%, and the level of protein content in extracted protein was only 65%. Further, the solubility of the extracted protein was low. These characteristics were used to predict that the coacervation between protein from L. punctata and polysaccharide would not be efficient or economically viable. 

Stress condition (nutrient delete and direct sunlight in the summertime in Australia) led to the rise more than a two-fold increase in total production of phenolic compound and their representatives, anthocyanins (up to 18 times), flavonoids (up to 4.7-times), and condensed tannins (up to 2.7 times) in A. pinnata and A. filiculoides. An increase in total carbohydrates was also observed. However, the level of total proteins and lipids decreased. The stress condition showed no impact on the fatty-acid profiles in the Azolla species, while it impacted both species' protein production and the synthesis of alanine, arginine, leucine, tyrosine, valine and methionine amino-acid. 
In the investigation of thraustochytrids, the comprehensive characteristics of two strains (MAN 65 and MAN 70) were assessed, namely, the productivity of growth with different carbon sources; and the accumulation of added-value chemicals, such as lipids, proteins, carotenoids, squalene in biomass and de-oiled cakes, supernatants, extracellular polysaccharide matrices, and recovered oil body. Glycerol and glucose were recognised as optimal carbon sources for lipid production, where in both strains, lipid accumulation reached 45% DW and more than 6g/L DW. However, glucose was identified as the best carbon source for DHA and squalene production. Glucose and fructose have been shown to be optimal carbon sources for carotenoid production. Additionally, the incubation time was also considered as having obtained the highest yield of these valuable biomolecules. Day 5 was an optimal time for the highest lipid production. The highest carotenoid production was recorded on day 7. Day 3 and 5 were noted for being the optimal times to harvest biomass towards obtaining the highest squalene (up to 13 mg/g DW) and protein accumulation. Thus, to obtain the highest target biomolecule, it is necessary to consider the carbon source used in the culture and the incubation time. 

The lipid extracted from dry biomass reached up to 40% DW. It was found to be rich in polyunsaturated fatty acids (up to 80% of total fatty acids), represented mainly by docosahexaenoic acid (75% of polyunsaturated fatty acids). The squalene and carotenoid accumulation in the cells reached 13 mg/g DW and 72 µg/g DW, respectively. The high concentration of protein in the biomass was shown for both strains. It was approximately 29% DW in biomass and 2.7 g/L in supernatants. A new and easy way was described for the extraction of oil bodies under high pH conditions. 

A protein extracted from the biomass of de-oiled thraustochytrids reached 91.64 % (w/w) protein content with traces of ash (up to 3%, w/w) and lipid (up to 3%, w/w). The isoelectric point of the extracted protein was observed at pH 4. The most abundant amino acids found in the proteins were aspartic acid, glutamic acid, histidine, and arginine. The denaturation temperature of the proteins ranged from 167.8°C to174.5°C, indicating their high thermal stability. The proteins showed high emulsion activity (784.1 m2/g) and emulsion stability (209.9 mins) indices. 

Finally, an examination of thraustochytrid protein isolate (TPI) and polysaccharide reaction showed that the complex coacervation of TPI and alginate formed at a pH of 2.5, while TPI and chitosan formed complex coacervates at a pH of 6.5 with a TPI - to - chitosan ratio of 2:1. The thermal stability of TPI and chitosan complex coacervates was identified as higher than that of TPI and chitosan only.  

This study provides important information on culturing Azolla (A. filiculoides and A. pinnata), duckweed (L. punctata) and thraustochytrids. It also represents a comprehensive analysis of two Azolla species (A. filiculoides and A. pinnata), one duckweed species (L. punctata) and two new thraustochytrids, all of which are capable of producing valuable biomolecules.