Bioproducts From Euglena gracilis: Synthesis and Applications

In recent years, the versatile phototrophic protist Euglena gracilis has emerged as an interesting candidate for application-driven research and commercialisation, as it is an excellent source of dietary protein, pro(vitamins), lipids, and the β-1,3-glucan paramylon only found in euglenoids. From these, paramylon is already marketed as an immunostimulatory agent in nutraceuticals. Bioproducts from E. gracilis can be produced under various cultivation conditions discussed in this review, and their yields are relatively high when compared with those achieved in microalgal systems. Future challenges include achieving the economy of large-scale cultivation. Recent insights into the complex metabolism of E. gracilis have highlighted unique metabolic pathways, which could provide new leads for product enhancement by genetic modification of the organism. Also, development of molecular tools for strain improvement are emerging rapidly, making E. gracilis a noteworthy challenger for microalgae such as Chlorella spp. and their products currently on the market

Enzymatic production of soluble bioactive β-1,3-glucans from paramylon

Thesis by publication.Includes bibliographical references.1. Bioproducts from Euglena gracilis - current and future applications -- 2. Materials and methods -- 3. Assessment of open pond cultivation and biofilm formation of Euglena gracilis -- 4. Microwave pretreatment of paramylon enhances the enzymatic production of soluble β-1,3-glucans with immunostimulatory activity -- 5. Development of screening strategies for the identification of paramylon-degrading enzymes -- 6. Conclusive summary and future research directions -- Appendix.Paramylon is a high-molecular weight unbranched β-1,3-glucan polymer produced by the flagellated microalga Euglena gracilis. Paramylon and other β-1,3-glucans have been reported to have antitumor and immunostimulating activities, making them ideal candidates as nutraceuticals or adjuvants. Their bioactivity can be increased by breaking them down to shorter-chain soluble saccharides by chemical or enzymatic methods. Here, chemical, physical and enzymatic approaches were examined as individual or combined strategies to facilitate the production of soluble bioactive compounds from paramylon. A microwave method was developed for the pretreatment of paramylon granules, which otherwise are resistant to enzymatic hydrolysis. The granules were sourced commercially after attempts to produce paramylon in an open pond cultivation proved unsuccessful. The paramylon granules were hydrolysed with a selection of commercially-sourced enzymes and a dye-based assay was devised to assess the efficacy of the hydrolysis. The new microwave pretreatment resulted in improved enzyme accessibility and thereby substantially enhanced the hydrolysis of paramylon granules. The soluble β-1,3-glucans produced with this method were characterised by highperformance liquid chromatography (HPLC) and an immunological assay on mouse macrophages showed that they were bioactive. The pathway for enzymatic degradation of paramylon in E. gracilis was also explored with Escherichia coli cells transformed with an E. gracilis-derived cDNA library. Recombinants were screened for the production of E. gracilis β-1,3-glucanases using an assay which combines fluorescence-activated cell sorting (FACS) with enzyme activity tests in microtiter plates. Both methods were based on the use of the fluorogenic β-glucan analogue fluorescein di-β-D glucopyranoside. However, the identification of new enzymes was prevented probably due to the suboptimal quality of the original cDNA library. In a parallel approach, protein fractions obtained from E. gracilis were analysed using mass spectrometry and a recently published transcriptomic database. Four putative enzymes with a high amino acid sequence similarity to known β-1,3-glucanase sequences were identified based on their translated DNA sequences. One of the corresponding genes was introduced into E. coli and into Saccharomyces cerevisiae to produce a recombinant enzyme. Unfortunately, expression levels were too low for reliable enzyme activity assays and further characterisation. Finally, various commercially-available and in-house enzyme preparations were shown to degrade paramylon on a dye-based plate assay. This new source of paramylon-degrading enzymes deserves further characterisation, which unfortunately was outside the time-frame of this project but could form the basis for promising future work1 online resource (133 pages : illustrations

Bactericidal silver nanoparticles by atmospheric pressure solution plasma processing

Silver nanoparticles have applications in plasmonics, medicine, catalysis and electronics. We report a simple, cost-effective, facile and reproducible technique to synthesise silver nanoparticles via plasma-induced non-equilibrium liquid chemistry with the absence of a chemical reducing agent. Silver nanoparticles with tuneable sizes from 5.4 to 17.8 nm are synthesised and characterised using Transmission Electron Microscopy (TEM) and other analytic techniques. A mechanism for silver nanoparticle formation is also proposed. The antibacterial activity of the silver nanoparticles was investigated with gram-positive and gram-negative bacteria. The inhibition of both bacteria types was observed. This is a promising alternative method for the instant synthesis of silver nanoparticles, instead of the conventional chemical reduction route, for numerous applications.</p

Insights into amoxicillin degradation in water by non-thermal plasmas

Antibiotics have been extensively used as pharmaceuticals for diverse applications. However, their overuse and indiscriminate discharge to water systems have led to increased antibiotic levels in our aquatic environments, which poses risks to human and livestock health. Non-thermal plasma water. However, the issues of process scalability and the mechanisms towards understanding the plasma-induced degradation remain. This study addresses these issues by coupling a non-thermal plasma jet with a continuous flow reactor to reveal the effective mechanisms of amoxicillin degradation. Four industry-relevant feeding gases (nitrogen, air, argon, and oxygen), discharge voltages, and frequencies were assessed. Amoxicillin degradation efficiencies achieved using nitrogen and air were much higher compared to argon and oxygen and further improved by increasing the applied voltage and frequency. The efficiency of plasma-induced degradation depended on the interplay of hydrogen peroxide (H2O2) and nitrite (NO2−), validated by mimicked chemical solutions tests. Insights into prevailing degradation pathways were elucidated through the detection of intermediate products by advanced liquid chromatography-mass spectrometry.</p

Oxidative stress induced by plasma-activated water stimulates astaxanthin production in Phaffia rhodozyma

Astaxanthin is used extensively in the nutraceutical, aquaculture, and cosmetic industries. The current market necessitates higher astaxanthin production from Phaffia rhodozyma (P. rhodozyma) due to its higher cost compared to chemical synthesis. In this study, a bubble discharge reactor was developed to generate plasma-activated water (PAW) to produce PAW-made yeast malt (YM) medium. Due to oxidative stress induced by PAW, strains cultured in 15 and 30 min-treated PAW-made medium produced 7.9 ± 1.2 % and 12.6 ± 1.4 % more carotenoids with 15.5 ± 3.3 % and 22.1 ± 1.3 % more astaxanthin, respectively. Reactive oxygen species (ROS) assay results showed that ROS generated by plasma-water interactions elevated intracellular ROS levels. Proteomic analysis revealed increased expression of proteins involved in the cellular response to oxidative stress as well as carotenoid biosynthesis, both of which contribute to higher yields of astaxanthin. Overall, this study supports the potential of PAW to increase astaxanthin yields for industrial-scale production.</p